Handbook of Biodegradable Polymers [3 ed.] 9781501519215

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Handbook of Biodegradable Polymers

Also of Interest Sustainable Polymers for Food Packaging. An Introduction Katiyar, 2019 ISBN 978-3-11-064453-1, e-ISBN 978-3-11-064803-4 Bioresorbable Polymers. Biomedical Applications Devine (Ed.), 2019 ISBN 978-3-11-064056-4, e-ISBN 978-3-11-064057-1 Polymeric Composites with Rice Hulls. An Introduction Defonseka, 2019 ISBN 978-3-11-063968-1, e-ISBN 978-3-11-064320-6

Advanced Composites Vol. 10 Biodegradable Composites. Materials, Manufacturing and Engineering Kumar, Davim (Eds.), 2019 ISBN 978-3-11-060203-6, e-ISBN 978-3-11-060369-9

Handbook of Biodegradable Polymers Edited by Catia Bastioli 3rd Edition

Editor Catia Bastioli Novamont SpA Via G.Fauser 8 28100 Novara NO Italy

ISBN 978-1-5015-1921-5 e-ISBN (PDF) 978-1-5015-1196-7 e-ISBN (EPUB) 978-1-5015-1198-1 Library of Congress Control Number: 2019953907 Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; Detailed bibliographic data are available on the Internet at http://dnb.dnb.de. © 2020 Walter de Gruyter GmbH, Berlin/Boston Cover image: Misha Kaminsky / iStock / Getty Images Plus Typesetting: Integra Software Services Pvt. Ltd. Printing and binding: CPI books GmbH, Leck www.degruyter.com

Acknowledgments I would like to thank all the contributors to this volume for their time, patience and effort in producing this book. Special and sincere thanks go to Federica Mastroianni for her extensive effort, which went beyond the handling of the considerable correspondence associated with the book and organisation of the reviewing process. Thanks are also extended to Gian Tomaso Masala for his reviewing of the chapter references. Finally, I wish to dedicate this volume to the memory of Raul Gardini, who was a pioneer of Bioeconomy, and the unwitting origin of my interest and dedication to renewable raw materials and bioplastics.

https://doi.org/10.1515/9781501511967-202

Preface ‘The great problem of packaging, which every experienced chemist knows, was well known to God Almighty, who solved it brilliantly, as he is wont to, with cellular membranes, eggshells, the multiple peel of oranges, and our own skins, because after all we too are liquids. Now, at that time there did not exist polyethylene, which would have suited me perfectly since it is flexible, light and splendidly impermeable: but it is also a bit too incorruptible, and not by chance God Almighty himself, although he is a master of polymerisation, abstained from patenting it: He does not like incorruptible things.’

I find this extract from Primo Levi’s book ‘The Periodic Table’, the best introduction to biodegradable polymers. The durability of conventional plastics is a serious environmental drawback when these materials are used in applications with little probability of recycling, when recycling happens to be too expensive, or when plastics have a high probability of contaminating the natural environment or organic waste. In these, and only in these applications, biodegradable polymers really make the difference. Biodegradable polymers must not be a simple replacement for traditional plastics. They must be used as an opportunity to redesign applications by focusing on the efficient use of resources and tending towards the elimination of waste, by transforming local issues into business opportunities and by developing a systemic vision to counterbalance the management culture that has contributed to the dissipative growth model we are now living in. The fundamental criterion needed to avoid any aggravation of this situation, and indeed to reverse the trend, is the efficient use of resources, being aware that only a type of growth which could restore its central focus on local areas, a knowledge economy, the cascading model, and the absence of waste and rejects, will lead to continuous and harmonious growth. A similar approach requires the selection of standards which have to go beyond products and towards systems. The objective should not be to maximise market volumes but to boost local regeneration from an environmental, social and economic viewpoint, promoting a cultural leap towards a system-based economy and shared trust among the different stakeholders. For this purpose, the quality rules for biodegradable polymers have to be strict and guarantee, besides compostability and biodegradability in different environments, nontoxicity of products and additives, as well as a low environmental impact throughout the life cycle, with improving targets in terms of raw material quality and renewability level, feedstock sustainability, inuse efficiency and end of life options to close the loop. Over the past 30 years, increasing effort has been dedicated to developing polymers designed to be biologically degraded in selected environmental conditions. In particular, industrial research has focused on discovering and developing biodegradable polymers that are, at the same time, easily processable, exhibit good perhttps://doi.org/10.1515/9781501511967-203

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formance and are cost-competitive (considering both internal and external costs) with conventional polymers. When bioplastics are biodegradable according to European Norms 13432 − the European reference for the technical material manufacturers, public authorities, composters, certifiers and consumers − or its equivalents the American Society for Testing and Materials D6400, or International Organization for Standardization 14855, they can, besides other disposal options, be organically recycled through composting. Such characteristics, when composting infrastructures are available, may therefore represent a significant advantage in sectors like waste collection, catering or packaging which have a high probability of being contaminated by food, or ending up in organic waste or nature: in such cases, organic recycling must be preferred to mechanical recycling. The property of a plastic to biodegrade in household compost permits its disposal in widespread composting infrastructures, at the same time optimising the quality of organic waste and maximising its diversion from landfill. The ability of a plastic to biodegrade via composting is also proof that its chemical structure is intrinsically biodegradable. Significant literature shows that the most widespread compostable bioplastics, currently available on the market, are also able to fully biodegrade in soil and even in the marine environment or through home composting. A range of standards are also available to certify the behaviour of these bioplastics in many different environments. The present volume reviews the most important achievements, the programmes and approaches of institutions, the private sector and universities to develop biodegradable polymers, and it explores their potential in depth. The volume covers: the most relevant biodegradable polymers of renewable and nonrenewable origin, the present business situation, a review of the main studies on their environmental impact and a critical analysis of the methodologies involved, the potential of new areas such as biocatalysis in the development of new renewable building blocks for biodegradable polymers, the expansion of the biorefinery concept towards integrated biorefineries, and the main policy and funding initiatives recently undertaken at the European Union (EU) level to foster the innovation capacity in Europe and to favour the market entry of innovative biobased and biodegradable products. It also takes into consideration aspects related to the biodegradation of these polymers in different environments and the related standards and case studies (including the interactions of biodegradable items with different anaerobic digestion technologies), showing their use in helping to solve specific solid waste problems. The demand for biodegradable polymers has steadily grown over the last 10 years, at an annual rate of between 20−30%, in regions where composting infrastructures are well developed and the separate collection of organic waste is well established. Wherever the separate collection of biowaste is in place (and this is an unwavering trend in the EU), all the traditional short life pollutants of organic waste (when it is with polyethylene, renewable or not) are critical, because they are not biodegradable. The organic recycling of biowaste requires plastic-free streams in order to assure high recycling rates.

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The existing link between the increasing use of biodegradable polymers and the efficient infrastructures for organic recycling and the separate collection of organic waste can be perceived as a limitation to the fast growth of this class of material. Instead it represents a unique opportunity to reconnect the solution of long-lasting environmental problems to local growth and regional regeneration putting into practice the knowledge-based economy. The Italian case study presented in the book curated by Walter Ganapini ‘Bioplastics: A Case Study of Bioeconomy in Italy’ shows how biodegradable polymers can be a powerful catalyst for the activation of local area regeneration. The book is dedicated to the Italian approach for resolving the problem of disposable carrier bags. It is all about transforming a category of waste which presents extremely critical issues (high surface area-volume ratio, large number of articles produced, the fact that the bags, if dispersed, cannot be reabsorbed into the environment, and marine pollution) into an opportunity, in order to solve an even more pressing problem; that of organic waste being sent to landfill. A small number of disposable carrier bags, if coupled with reusable bags, and made from biodegradable, compostable plastics, can be reused as valuable resources in organic waste collection. This makes them a powerful and important means of intercepting organic waste, with no expense required from local councils, helping to achieve the objective of improving the quantity and quality of organic waste: a feedstock that is important for the future development of the bioeconomy, and for the fertility and quality of soil. Being able to count on a niche market (which is already of significant size), facilitates achieving economies of scale for biodegradable polymers, resulting in increased possibilities to build integrated local biorefineries dedicated to medium-high value-added products, demonstrators and flagships required not only for biodegradable polymers but also for a range of related building blocks and agricultural chains as a whole. This will also generate new opportunities for traditional chemistry, by laying down the foundations for the redevelopment and environmental upgrading of deindustrialised chemical plants. The change in the perception of biodegradable polymers is evident by simply considering the trends from 1989 to 2012 in the fields of ‘biodegradable’ (+2,800% for scientific literature and +1,100% for the sum of World Intellectual Property Organization (WIPO) patents, European patents (EP) and United States (US) patents) and ‘biodegradable plastics’ (+1,400% for scientific literature and +4,200% for the sum of WO, EP and US patents). The opportunity to utilise renewable raw materials (RRM) in the production of some of these biodegradable polymers and to reduce the dependency on foreign petroleum resources, along with the exploitation of new functional properties in comparison with traditional plastics, has significant benefits. Besides biodegradability, the technical developments made during the research process could have significant advantages for the final consumers and could contribute to the solution of technical, economic and environmental issues in specific market areas.

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RRM as industrial feedstocks for the manufacture of chemical substances and products, such as oils from oilseed crops, starch from cereals and potatoes, and cellulose from straw and wood, as well as organic waste, have therefore been given more and more attention over the last few years. By employing physical, chemical and biochemical processes, these materials can be converted into chemical intermediates, polymers and speciality chemicals able to replace fossil feedstocks, thus implying less energy involved during production and a wider range of disposal options resulting in a lower environmental impact. Legislative attention able to properly address this issue could become a further incentive to the development of products from RRM and maximise the environmental, social and industrial benefits. Biobased products were, in fact, one of the six sectors included in the 2007 ‘Lead Market Initiative’ of the European Commission (EC), with the aim of fostering the emergence of such lead markets with high economic and societal value, focusing on areas where coordinated policymaking can speed up market development [1]. More recently, in February 2012, the EC launched a new ‘Bioeconomy Strategy’ [2], focusing resources and investments in the strategic sector of biobased products, in order to shift Europe towards a greater and more sustainable use of renewable resources. In addition, in July 2013, the EC encouraged the creation of a public-private partnership of Biobased Industries. It includes approximately 70 full members (EU large and small companies, clusters and organisations) and more than 100 associated members (universities, research and technology organisations, associations, European trade organisations and European technology platforms) from the fields of technology, industry, agriculture and forestry, with the shared commitment to invest in collaborative research, development and demonstration of biobased technologies. A supportive and coordinated European strategy for an increased market uptake would help many biobased products, among them biodegradable biopolymers, to accelerate reaching economies of scale, in order to attract investments and generate sustainable economic growth. The EU bioeconomy already has a turnover of nearly €2 trillion and employs more than 22 million people, 9% of the total employment in the EU. Each euro invested in EU-funded bioeconomy research and innovation, with a coherent and incentivising framework, is estimated to trigger €10 of added value in bioeconomy sectors by 2025 [2]. It is also estimated that this growth will be enhanced with the development of the model of integrated biorefineries for the production of high value-added products, such as biobased chemicals and materials. Biorefineries will process a variety of biomass-based feedstocks, and the necessary growth in biomass production is expected to increase the turnover and employment of the seed sector by 10%, resulting in 5,000 extra jobs [3]. The significant increase in the importance of innovative biopolymers is linked to the achievement of high-quality standards. The quality of biodegradable products is assured not only by the control of the biodegradability parameters but also by the assessment of real functionality. A biodegrad-

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able product is useless if it does not perform as a traditional product or better in terms of mechanical resistance, duration and so on. For this reason, the commitment of producers of biodegradable biopolymers in the creation of a quality network able to guarantee the standards of the product, in all the steps of the life cycle, becomes very relevant. The elaboration and diffusion of best practices in the field of organic waste collection, where the use of biodegradable compostable bags is a tool to improve the quality of the system, has for example, permitted thousands of municipalities all over Europe to implement the proposed model. In fact, it has been demonstrated that, despite the heterogeneity of anaerobic digestion technologies and processing conditions, an efficient and optimised treatment of municipal biowaste, collected with compostable bioplastic bags, allows preserving the advantages given by the bags in the collection phase and to secure the most efficient treatment of the collected feedstock enabling the highest input and minimum production of residues. The cooperation with public bodies is also a key factor in the success of biodegradable biopolymers, because the topics under discussion are strictly related to public interest, such as safety, environment and health. The implementation of appropriate environmental policies in key areas (like waste collection) can become a further incentive to the development of products from RRM and can maximise the environmental, social and industrial advantages. Together with the intensification of investment, as well as research and development actions in the biodegradable polymers sector, it would be possible to create a network of partnerships among stakeholders of the entire supply chain, from agriculture to waste management, and thereby promote new models of development towards higher levels of sustainability and cultural growth. Today, biodegradable biopolymers are available on the market, at different levels of development, and are mainly carbohydrate-based materials. Starch can be physically modified and used alone or in combination with other polymers, or it can be used as a substrate for t h e fermentation and production of polyhydroxyalkanoates or lactic acid, is then transformed into polylactic acid through standard polymerisation processes. An alternative option is represented by vegetable oil-based polymers. Despite the constant growth of the market, the land use for bioplastics currently represents just 0.006% of the global agricultural area (which means around 300,000 ha out of 5 billion ha) and it is expected to rise to 0.022% by 2016 (that is, 1.1 million ha). Meanwhile, the increase in the efficiency of feedstock and agricultural technology is continuously enhancing good agricultural practices [4]; moreover, recent trends have focused on the use of marginal lands or contaminated soils and residues. The increasing use of bioplastics has opened entirely new generations of materials with new performances in comparison with traditional plastics. The possibility offered by physically modified starch to create functionalised nanoparticles able to modify the properties of natural and synthetic rubbers and other synthetic polymers, the naturally high oxygen barrier of starch and its derivatives, and their high permeability to water vapour already offer a range of completely new solutions to the plastic industry.

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The use of RRM, however, is not by itself a guarantee of low environmental impact. Aspects such as the production processes, the technical performance and the weight of each final product, and its disposal options, have to be carefully considered along all the steps of the product’s life. The engineering of biobased materials for specific applications using life cycle analysis in a cradle-to-grave approach is therefore a critical aspect. The involvement of upstream players, that is farmers and their associations, is a very important prerequisite. In agriculture, new agronomical approaches and the development of new genotypes for nonfood applications should be taken into consideration. Agricultural crops and processes associated with lower environmental impact and lower costs are important factors in the development of new biobased products. Effort must also be made at the industrial level in order to develop less expensive and higher performance products and low- impact technologies. Policies should therefore be focused more on supporting innovation and scale up of new technologies which can create solid added value and are capable of responding to the societal challenges faced by our planet. The involvement of specific stakeholders can be achieved if a communication programme is launched and operated in parallel with industrial activities. The success of the project is very much linked to the diffusion of a new environmental awareness, at all levels: politicians, public administrators, investors, associations, customers, non-governmental organisations (NGO), citizens and society at large, all of them must be reached by specific communications, in order to initiate a comprehensive and coherent sustainable strategy, with positive effects in the local areas involved. This, in turn, must give rise to specific legislative actions in order to quantify the social and environmental benefits linked to the nonfood use of agricultural and natural raw materials, and to the bioconversion of waste materials into industrial products.

References A Lead Market Initiative for Europe − COM(2007) 860 Final, Communication from the Commission to the Council, the European Parliament, the European Economic and Social Committee and the Committee of the Regions. [2] Innovating for Sustainable Growth: A Bioeconomy for Europe − COM(2012) 60 Final, Communication from the Commission to the European Parliament, the Council, the European Economic and Social Committee and the Committee of the Regions. [3] The Bio-based Industries Vision – Accelerating Innovation and Market Uptake of Bio-based Products, Vision Document of the Bio-based Industries Consortium − European Public-Private Partnership on Bio-Based Industries. http://biconsortium.eu/sites/default/files/downloads/ BIC_BBI_Vision_web.pdf, July 2012, p. 15. [4] European Bioplastics, Bioplastics − Facts and Figures. http://en.european-bioplastics.org/ wp-content/uploads/2013/publications/EuBP_FactsFigures_bioplastics_2013.pdf, 2013, p. 5. [1]

Contents Acknowledgments  Preface 

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 VII

Maarten van der Zee 1 Methods for evaluating the biodegradability of environmentally degradable polymers   1 1.1 Introduction   1 1.2 Background   1 1.3 Defining ‘Biodegradability’   2 1.4 Mechanisms of polymer degradation   4 1.4.1 Nonbiological degradation of polymers   4 1.4.2 Biological degradation of polymers   4 1.5 Measuring the biodegradation of polymers   6 1.5.1 Enzyme assays   7 1.5.1.1 Principle   7 1.5.1.2 Applications   7 1.5.1.3 Drawbacks   8 1.5.2 Plate tests   8 1.5.2.1 Principle   8 1.5.2.2 Applications   8 1.5.2.3 Drawbacks   9 1.5.3 Respiration tests   9 1.5.3.1 Principle   9 1.5.3.2 Applications   9 1.5.3.3 Suitability   10 1.5.4 Gas (CO2 or CH4) evolution tests   10 1.5.4.1 Principle   10 1.5.4.2 Applications   11 1.5.4.3 Suitability   11 1.5.5 Radioactively labelled polymers   12 1.5.5.1 Principle and applications   12 1.5.5.2 Drawbacks   12 1.5.6 Laboratory-scale simulated accelerating environments   13 1.5.6.1 Principle   13 1.5.6.2 Applications   13 1.5.6.3 Drawbacks   14 1.5.7 Natural environments, field trials   14 1.6 Conclusions   14 References   15

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Rolf-Joachim Müller 2 Biodegradation behaviour of polymers in liquid environments   23 2.1 Introduction   23 2.2 Degradation in real liquid environments   24 2.2.1 Degradation in freshwater and marine environment   25 2.2.1.1 Polyhydroxyalkanoates   25 2.2.1.2 Synthetic polyesters   26 2.3 Degradation in laboratory tests simulating real aquatic environments   27 2.3.1 Aerobic liquid environments   27 2.3.2 Anaerobic liquid environments   30 2.4 Degradation in laboratory tests with optimised and defined liquid media   34 2.5 Standard tests for biodegradable polymers using liquid media   36 2.6 Summary   41 References   41 Fernanda Farachi, Giulia Bettas Ardisson and Francesco Degli Innocenti 3 Environmental fate and ecotoxicity assessment of biodegradable polymers   45 3.1 Introduction   45 3.2 End of life scenarios of biodegradable polymers   46 3.2.1 Biodegradation end products   46 3.2.2 Biodegradation during organic recycling   48 3.2.2.1 Industrial composting   48 3.2.2.2 Home composting   49 3.2.2.3 Anaerobic digestion   49 3.2.3 Biodegradation in soil   50 3.2.3.1 Soil texture and structure   50 3.2.3.2 Water content   50 3.2.3.3 Organic matter   50 3.2.3.4 pH   51 3.2.3.5 Temperature   51 3.2.3.6 Oxygen   51 3.2.3.7 Sunlight   51 3.3 Investigation into polymer biodegradation   51 3.3.1 Standard on industrial composting   52 3.3.2 Identification of the intermediates of polymer biodegradation  3.4 Environmental fate of biodegradation intermediates   58 3.4.1 Physico-chemical properties and behaviour of intermediates  3.4.1.1 Ready biodegradability   59 3.4.1.2 Bioconcentration factor   63

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3.4.2 3.5 3.5.1 3.5.1.1 3.5.1.2 3.5.1.3 3.5.1.4 3.5.2 3.5.2.1 3.5.2.2 3.5.2.3 3.5.2.4 3.6

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Ecotoxicological assessment based on the environmental behaviour of the intermediates   64 Ecotoxicological assessment of biodegradation intermediates   64 Aquatic toxicity   65 Bacteria   66 Algae   66 Crustacea   66 Fish   67 Terrestrial toxicity   67 Bacteria   68 Invertebrates   68 Plants   68 Vertebrates   68 Discussion and conclusions   68 References   71

Ines Fritz 4 Ecotoxicological aspects of the biodegradation process of polymers   75 4.1 Preface   75 4.2 The need for ecotoxicity analysis of biodegradable materials   76 4.3 Standards and regulations for testing biodegradable polymers   76 4.4 Detection of the influences on an ecosystem caused by the biodegradation of polymers   78 4.4.1 Potential influences of polymers after composting   80 4.4.2 Potential influences of polymers during and after biodegradation in soil and sediment   81 4.5 A short introduction to ecotoxicology   83 4.5.1 Dose-response relationships   83 4.5.2 Investigation level of ecotoxicity tests   84 4.5.3 Length of the exposure period   85 4.5.4 End-points   85 4.5.5 The difference between toxicity tests and bioassays   85 4.5.6 Ecotoxicity profile analysis   86 4.6 Recommendations and standard procedures for biotests   86 4.6.1 Bioassays with higher plant species   87 4.6.2 Bioassays with earthworms (Eisenia foetida)   91 4.6.3 Preparation of elutriates for aquatic ecotoxicity tests   91 4.6.4 Bioassays with algae   92 4.6.5 Bioassays with luminescent bacteria   94 4.6.6 Bioassays with daphnia   95 4.6.7 Biotests with higher aquatic plants   95

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4.7 4.7.1 4.7.2 4.8 4.8.1 4.8.2 4.8.3 4.8.4 4.9 4.10 4.10.1 4.10.2 4.10.3 4.10.4 4.11 4.11.1 4.11.2 4.11.3 4.11.4

 Contents

Evaluation of bioassay results obtained from samples of complex composition   96 Testing of solid samples   96 Testing of sediments   97 Special prerequisites to be considered when applying bioassays for biodegradable polymers   97 Nutrients in the sample   98 Biodegradation intermediates   99 Diversity of the microbial population   100 Humic substances   101 Evaluation of test results and limits of bioassays   103 Research results for ecotoxicity testing of biodegradable polymers   103 The relationship between chemical structure, biodegradation pathways and the formation of potentially ecotoxic metabolites   104 Ecotoxicity of polymers   104 Ecotoxic effects appearing after degradation in compost or after anaerobic digestion   105 Ecotoxic effects appearing during degradation in soil   106 Conclusion   107 Consequences of test schemes for investigations on biodegradable polymers   109 Materials intended for organic recovery   109 Materials intended for applications in the environment   109 Final statement   110 References   111

Bruno De Wilde 5 International and national norms on biodegradability and certification procedures   115 5.1 Introduction   115 5.2 Organisations for standardisation   117 5.3 Norms on biodegradation test methods   119 5.3.1 Introduction   119 5.3.2 Aquatic, aerobic biodegradation Tests   120 5.3.2.1 Based on carbon conversion (‘Sturm’ Test)   120 5.3.2.2 Based on oxygen consumption (‘MITI’ Test)   121 5.3.2.3 Other   122 5.3.3 Compost biodegradation tests   122 5.3.3.1 Controlled composting test   122 5.3.3.2 Mineral bed composting test   123 5.3.3.3 Other compost biodegradation tests   124

Contents 

5.3.4 5.3.5 5.3.6 5.3.6.1 5.3.7 5.3.8 5.4 5.4.1 5.4.2 5.4.3 5.4.4 5.5 5.5.1 5.5.2 5.5.3 5.5.4 5.5.5 5.5.6 5.5.7 5.5.8 5.6 5.6.1 5.6.2 5.6.2.1 5.6.2.2 5.6.2.3 5.6.2.4 5.6.2.5 5.6.2.6 5.6.2.7 5.6.3 5.6.3.1 5.6.3.2 5.6.4

 125 Soil biodegradation tests  Aquatic, anaerobic biodegradation tests   126 High solids, anaerobic biodegradation tests   127 Landfill simulation tests   127 Marine biodegradation tests   127 Other biodegradation tests   128 Norms on disintegration test methods   129 Introduction   129 Compost disintegration tests   129 Disintegration in water   130 Disintegration in other environments   131 Norms on specifications for degradability   131 Introduction   131 (Industrial) Compostability   132 (Home) Compostability   134 Soil biodegradability   135 Aquatic biodegradability   136 Marine biodegradability   136 Anaerobic digestion   136 Oxo-degradation   137 Certification   137 Introduction   137 (Industrial) Compostability certification systems   138 Seedling   138 OK compost   139 Biodegradable Products Institute Logo   140 Cedar Grove logo   140 GreenPla certification system   141 The Australasian seedling logo and certification system  Other certification and logo systems   141 (Home) Compostability certification systems   142 OK Compost home   142 Other systems for home compostability   143 Other biodegradability certification systems   144 References   144

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Catia Bastioli and Franco Bettarini 6 General characteristics, processability, industrial applications and market evolution of biodegradable polymers   147 6.1 General characteristics   147 6.1.1 Polymer biodegradation mechanisms   148 6.1.2 Polymer molecular size, structure and chemical composition   149

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6.1.3 6.1.4 6.1.4.1 6.1.4.2 6.1.5 6.1.5.1 6.1.5.2 6.1.5.3 6.1.5.4 6.1.5.5 6.1.6 6.2 6.2.1 6.2.2 6.2.3 6.2.4 6.3 6.3.1 6.3.2 6.3.3 6.3.4 6.4 6.5

 Contents

Biodegradable polymer classes   150 Natural biodegradable polymers   150 Starch   150 Polyhydroxyalkanoates   152 Synthetic biodegradable polymers   154 Polylactic acid and polyglycolic acid   155 Poly(ε-caprolactone)   157 Diol-Diacid aliphatic polyesters   157 Aliphatic/Aromatic copolyesters   159 Polyvinyl alcohol   161 Modified, natural biodegradable polymers  Processability   164 Extrusion   165 Film blowing and casting   166 Moulding   167 Fibre spinning   168 Industrial applications   168 Compost bags   169 Carrier bags   171 Mulch films   173 Other applications   173 Market evolution   174 Conclusions   177 References   178

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Kumar Sudesh, Yoshiharu Doi, Paolo Magistrali and Sebastia` Gestı´ Garcia 7 Polyhydroxyalkanoates   183 7.1 Introduction   183 7.2 Production of polyhydroxyalkanoates   184 7.3 The various types of polyhydroxyalkanoates   185 7.3.1 Poly(R-3-hydroxybutyrate)   185 7.3.2 Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)   187 7.3.3 Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)   189 7.3.4 Polyhydroxyalkanoates containing medium-chain-length monomers   190 7.3.5 Uncommon constituents of polyhydroxyalkanoates   194 7.4 Mechanisms of polyhydroxyalkanoate biosynthesis   194 7.4.1 Conditions that promote the biosynthesis and accumulation of polyhydroxyalkanoates in microorganisms   194 7.4.2 Carbon sources for the production of polyhydroxyalkanoates   195 7.4.3 Biochemical pathways involved in the metabolism of polyhydroxyalkanoates   197

Contents 

7.4.4 7.5 7.5.1 7.5.2 7.5.3 7.6 7.7 7.7.1 7.7.2 7.7.3 7.8

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The key enzyme of the biosynthesis of polyhydroxyalkanoates, polyhydroxyalkanoate synthase   200 Genetically modified systems and other methods for the production of polyhydroxyalkanoates   201 Recombinant escherichia coli   201 Transgenic plants   202 In vitro production of polyhydroxyalkanoates   203 Biodegradation of polyhydroxyalkanoates   203 Applications of polyhydroxyalkanoates   204 Biomedical applications   205 Industrial applications   206 Agricultural applications   207 Conclusions and outlook   207 Acknowledgements   208 References   208

Catia Bastioli, Paolo Magistrali and Sebastià Gestí Garcia 8 Starch-based technology   217 8.1 Introduction   217 8.2 Starch   218 8.3 Starch-filled plastics   221 8.4 Structural starch modifications   222 8.4.1 Starch gelatinisation and retrogradation   222 8.4.2 Starch Jet-cooking   225 8.4.3 Starch extrusion cooking   225 8.4.4 Starch destructurisation in the absence of synthetic polymers  8.4.5 Starch destructurisation in the presence of synthetic polymers  8.4.5.1 Ethylene-acrylic acid copolymer   229 8.4.5.2 Ethylene-vinyl alcohol copolymers   230 8.4.5.3 Polyvinyl alcohol   231 8.4.5.4 Aliphatic polyesters   231 8.4.5.5 Aliphatic-aromatic polyesters   232 8.4.5.6 Other polymers   232 8.4.6 Additional information on starch complexation   233 8.5 Starch-based materials on the market   237 8.6 Conclusions   238 References   239 Michel Vert 9 Lactic acid-based degradable polymers   245 9.1 Introduction   245 9.2 Main structural characteristics of lactic acid stereocopolymers 

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9.3 9.4 9.5 9.6 9.7 9.8 9.9 9.10

 Contents

Synthesis of lactic acid-based polymers   249 Main material properties   251 Degradation of lactic acid-based polymers   252 Lactic acid-based copolymers   255 Interest in the biomedical field   256 Interest as degradable polymers in the environment   256 Interest as polymers from renewable resources   257 Conclusion   257 References   258

Paolo Magistrali, Sebastià GestÍ Garcia and Tiziana Milizia 10 Biodegradable Polyesters   261 10.1 Introduction   261 10.2 Biodegradable aliphatic polyesters   262 10.2.1 Biodegradable aliphatic polyesters with a hydroxyacid repetitive unit   262 10.2.1.1 Poly(ε-caprolactone)   262 10.2.1.2 Polyhydroxyalkanoates   263 10.2.1.3 Polylactic acid   264 10.2.1.4 Polyglycolic acid   265 10.2.1.5 Long chain polyhydroxyacid   266 10.2.2 Biodegradable aliphatic polyesters with a diol/dicarboxylic acid repetitive unit   266 10.2.3 Aliphatic polyesters biodegradation   268 10.2.4 Properties of biodegradable aliphatic polyesters   269 10.3 Biodegradable aliphatic-aromatic copolyesters   270 10.3.1 Ecoflex   273 10.3.1.1 Producer/Patents: BASF AG, Germany   273 10.3.2 Origo-Bi   274 10.3.2.1 Producer/Patents: Novamont   274 10.3.3 Biocosafe 2003F   274 10.3.3.1 Producer: Zhejiang Hangzhou Xinfu Pharmaceutical Co. Ltd   274 10.3.4 S-EnPol   274 10.3.4.1 Producer: Samsung Fine Chemicals   274 10.3.5 Properties of biodegradable aliphatic-aromatic copolyesters   275 10.3.6 Biodegradation of aliphatic-aromatic copolyesters   276 10.3.6.1 Polymer-related parameters determining biodegradation   277 10.3.6.2 Degradation under composting conditions   281 10.3.6.3 Degradation in soil   283 10.3.6.4 Degradation in an aqueous environment   285

Contents 

10.3.6.5 10.3.6.6 10.4

 285 Degradation under anaerobic conditions  Fate of aromatic sequences and risk assessment   286 Renewable monomers for biodegradable polyester synthesis  References   293

Stéphane Guilbert and Bernard Cuq 11 Material formed from proteins   299 11.1 Introduction   299 11.2 Structure of material proteins   301 11.3 Protein-based materials   306 11.4 Formation of protein-based materials  11.4.1 The solvent process   312 11.4.2 The thermoplastic process   315 11.5 Properties of protein-based materials  11.6 Applications   329 References   330

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 311

 322

David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore 12 Enzyme catalysis in the synthesis of biodegradable polymers   339 12.1 Introduction   339 12.2 Polyester synthesis   340 12.2.1 Polycondensation of hydroxyacids and esters   340 12.2.2 Polymerisation of dicarboxylic acids or their activated derivatives with glycols   343 12.2.3 Ring-opening polymerisation of carbonates and other cyclic monomers   352 12.2.4 Ring-opening polymerisation and copolymerisation of lactones   358 12.3 Oxidative polymerisation of phenol and derivatives of phenol   369 12.4 Enzymatic polymerisation of polysaccharides   380 12.5 Conclusions   384 References   385 Maurizio Fieschi and Ugo Pretato 13 Environmental life cycle of biodegradable plastics   393 13.1 Introduction to life cycle thinking and assessment   393 13.2 Bioplastics and life cycle assessment   397 13.2.1 Biodegradability and compostability   398 13.2.2 Renewable origin   401 13.2.3 Optimisation potential   405 13.3 Conclusions   405 References   407

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Enzo Favoino 14 The use of biodegradable polymers for the optimisation of models for the source separation and composting of organic waste   409 14.1 Introduction   409 14.1.1 The development of composting and schemes for the source separation of biowaste in Europe: A matter of quality   410 14.2 Main drivers for composting in the European Union   410 14.2.1 Directive 99/31/EC on landfills   411 14.2.2 The waste framework directive (Directive 2008/98/EC)   411 14.2.3 Other regulatory and political drivers   412 14.3 The source separation of organic waste: Schemes and results in the south of Europe   412 14.4 ‘Biowaste’, ‘Vegetable, Garden and Fruit’, and ‘Food Waste’: Relevance of a definition on the performance of the waste management system   415 14.5 The importance of biobags   417 14.5.1 Features of ‘Biobags’: The importance of biodegradability and its costefficiency   418 14.6 Cost assessment of optimised schemes   419 14.6.1 Tools to optimise the schemes and their suitability in different situations   422 14.6.1.1 Collection frequency for residual waste   422 14.6.1.2 Diversifying the fleet of collection vehicles   423 14.7 Conclusions   424 References   425 Christian Garaffa and Francesco Degli Innocenti 15 Collection of biowaste with biodegradable and compostable plastic bags and treatment in anaerobic digestion facilities: Advantages and options for optimisation   427 15.1 Introduction   427 15.2 Current European policies regarding biowaste, renewable energy, emission reduction and resource management   428 15.3 The role of compostable plastic bags in biowaste source separation schemes   430 15.4 Compostable plastics in anaerobic digestion: Standards and performance   431 15.5 Anaerobic digestion facilities treating biowaste: Technologies, pretreatment options and management of compostable plastic bags   433 15.5.1 Combined anaerobic and aerobic versus anaerobic only processes: Pros and cons   433

Contents 

15.5.2 15.5.3 15.6 15.6.1

15.6.2

15.6.3

15.6.4 15.7

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 434 Dry and wet technologies  Different anaerobic digestion technologies and fate of compostable plastic bags   435 Case studies of anaerobic digestion facilities managing biowaste in compostable plastic bags   437 Case study 1: Wet codigestion with a hydropulper: Compostable bags can switch from disposal (Route 3) to material recovery (Route 2)   437 Case study 2: Wet digestion with screw press/mash separation: Compostable bags skipping digestion and going directly to material recovery (Route 2)   443 Case study 3: Dry plug flow digestion: Compostable bags going partly to digestion (Route 1) and partly to material recovery (Route 2)   445 Case study 4: Dry batch digestion: Compostable bags going to digestion followed by material recovery (Route 1)   448 Conclusions   450 References   452

Ramani Narayan 16 Principles, drivers, and analysis of biodegradable and biobased plastics   455 16.1 Introduction   455 16.2 Understanding biodegradability – biodegradable compostable plastics   456 16.3 Measuring and reporting biodegradability   457 16.4 International standards for biodegradability   460 16.5 Misleading claims of biodegradability   461 16.6 Environmental and health consequences   462 16.7 US Federal Trade Commission Green Guides   463 16.7.1 Degradable and biodegradable claims   463 16.7.2 Compostable claims   464 16.7.3 Renewable materials, biobased materials and biobased content   465 16.8 Biobased plastics − carbon footprint reductions using plant/ biomass carbon and value proposition   465 16.8.1 Illustrating zero material carbon footprint using basic stoichiometric calculations   467 16.8.2 Measuring biobased carbon content   468 16.8.3 Calculating and reporting biobased carbon contents   469 16.9 Example of bio polyethylene terephthalate   470

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 Contents

Summary   471 References   472

Thomas Hirth and Rainer Busch 17 Biorefineries for renewable monomers   475 17.1 Introduction   475 17.2 Biorefinery concepts   475 17.2.1 Starch and sugar biorefineries   478 17.2.2 Oilseed biorefineries   478 17.2.3 Green biorefinery   479 17.2.4 Lignocellulose biorefinery   479 17.2.5 Aquatic biorefinery   481 17.3 Monomers based on renewable raw materials  17.4 Summary and outlook   489 References   489

 482

Alessandra Perrazzelli 18 Research and development funding with the focus on biodegradable products   491 18.1 Introduction   491 18.2 Policy initiatives and plans in the field of biopolymers and their applications   492 18.2.1 The Lead Market Initiative   492 18.2.2 Key Enabling Technologies   493 18.2.3 The Innovation Union   496 18.2.4 The Bioeconomy Strategy   496 18.3 European Union-funded research on biopolymers and their applications   497 18.3.1 Why the need for European Union-funded research?   497 18.3.2 The Framework Programmes   498 18.3.3 Specific programmes with focus on biopolymers and their applications   499 18.4 The seventh Framework Programme   507 18.5 Funded projects: Biopolymers and their applications   509 18.6 The Eco-innovation initiative   510 18.7 Horizon 2020   512 18.8 Conclusions   512 References   513 Abbreviations  Index 

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1 Methods for evaluating the biodegradability of environmentally degradable polymers 1.1 Introduction This chapter presents an overview of the current knowledge on experimental methods for monitoring the biodegradability of polymeric materials. The focus is, in particular, on the biodegradation of materials under environmental conditions. Examples of in vivo degradation of polymers used in biomedical applications are not covered in detail, but have been extensively reviewed elsewhere, e.g., [1–3]. Nevertheless, it is important to realise that the degradation of polymers in the human body is also often referred to as biodegradation. A number of different aspects of assessing the potential, rate and degree of biodegradation of polymeric materials are discussed. The mechanisms of polymer degradation and erosion are reviewed, and factors affecting enzymatic and nonenzymatic degradation are briefly addressed. Particular attention is given to the various ways of measuring biodegradation, including complete mineralisation to gases (such as carbon dioxide (CO2) and methane (CH4)), water and possibly microbial biomass. Finally, some general conclusions are presented with respect to measuring the biodegradability of polymeric materials.

1.2 Background There is a worldwide research effort to develop biodegradable polymers for agricultural applications or as a waste management option for polymers in the environment. Until the end of the 20th century, most of the efforts were synthesis oriented and not much attention was paid to the identification of environmental requirements for, and testing of, biodegradable polymers. Consequently, many unsubstantiated claims of biodegradability were made, which has damaged the general acceptance. An important factor is that the term biodegradation has not been applied consistently. In the medical field of sutures, bone reconstruction and drug delivery, the term biodegradation has been used to indicate degradation into macromolecules that stay in the body but migrate (e.g., ultrahigh molecular weight (MW) polyethylene (PE) from joint prostheses), or hydrolysis into low MW molecules that are excreted from the body (bioresorption), or dissolving without modification of the MW (bioabsorption) [4, 5]. On the other hand, for environmentally degradable plastics, the https://doi.org/10.1515/9781501511967-001

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term biodegradation may mean fragmentation, loss of mechanical properties, or sometimes degradation through the action of living organisms [6]. Deterioration or loss in physical integrity is also often mistaken for biodegradation [7]. Nevertheless, it is essential to have a universally acceptable definition of biodegradability to avoid confusion as to where biodegradable polymers can be used in agriculture or fit into the overall plan of polymer waste management. Many groups and organisations have endeavoured to clearly define the terms ‘degradation’, ‘biodegradation’ and ‘biodegradability’. But there are several reasons why establishing a single definition among the international community has not been straightforward, including: – The variability of an intended definition given the different environments in which the material is to be introduced and its related impact on those environments. – The differences of opinion with respect to the scientific approach or reference points used for determining biodegradability. – The divergence of opinion concerning the policy implications of various definitions. – Challenges posed by language differences around the world. As a result, many different definitions have officially been adopted, depending on the background of the defining organisation and their particular interests. However, of more practical importance are the criteria for calling a material ‘biodegradable’. A demonstrated potential of a material to biodegrade does not say anything about the time frame in which this occurs, nor the ultimate degree of degradation. The complexity of this issue is illustrated by the following common examples. Low-density PE has been shown to biodegrade slowly to CO2 (0.35% in 2.5 years) [8] and according to some definitions can thus be called a biodegradable polymer. However, the degradation process is so slow in comparison with the application rate that accumulation in the environment will occur. The same applies for polyolefin- starch blends which rapidly lose strength, disintegrate and visually disappear if exposed to microorganisms [9–11]. This is due to utilisation of the starch component, but the polyolefin fraction will nevertheless persist in the environment. Can these materials be called ‘biodegradable’?

1.3 Defining ‘Biodegradability’ In 1992, an international workshop on biodegradability was organised to bring together experts from around the world to achieve areas of agreement on definitions, standards and testing methodologies. Participants came from manufacturers, legislative authorities, testing laboratories, environmentalists and standardisation organisations in Europe, USA and Japan. Since this fruitful meeting, there is a general agreement concerning the following key points [12]:

1 Methods for evaluating the biodegradability of environmentally degradable polymers 

–

–

–

–

 3

For all practical purposes of applying a definition, material manufactured to be biodegradable must relate to a specific disposal pathway such as composting, sewage treatment, denitrification and anaerobic sludge treatment. The rate of degradation of a material manufactured to be biodegradable has to be consistent with the disposal method and other components of the pathway into which it is introduced, such that accumulation is controlled. The ultimate end products of the aerobic biodegradation of a material manufactured to be biodegradable are CO2, water and minerals, and the intermediate products should include biomass and humic materials. (Anaerobic biodegradation was discussed in less detail by the participants). Materials must biodegrade safely and not negatively impact the disposal process or use of the end product of the disposal.

As a result, specified periods of time, specific disposal pathways and standard test methodologies were incorporated into definitions. Standardisation organisations such as the European Committee for Standardization, International Organization for Standardization (ISO) and American Society for Testing and Materials (ASTM) were consequently encouraged to rapidly develop standard biodegradation tests so these could be determined. Society further demanded nondebatable criteria for the evaluation of the suitability of polymeric materials for disposal in specific waste streams such as composting or anaerobic digestion. Biodegradability is usually just one of the essential criteria, besides ecotoxicity, effects on waste treatment processes and so on. In the following sections of this chapter, the biodegradation of polymeric materials is looked upon from the chemical perspective. The chemistry of the key degradation process is represented by Equations 1.1 and 1.2, where CPOLYMER represents either a polymer or a fragment from any of the degradation processes defined earlier. For simplicity, the polymer or fragment is considered to be composed only of carbon, hydrogen and oxygen; other elements may, of course, be incorporated in the polymer, and these would appear in an oxidised or reduced form after biodegradation depending on whether the conditions are aerobic or anaerobic, respectively: Aerobic biodegradation: CPOLYMER + O2 → CO2 + H2O + CRESIDUE + CBIOMASS

(1.1)

Anaerobic biodegradation: CPOLYMER → CO2 + CH4 + H2O + CRESIDUE + CBIOMASS

(1.2)

Complete biodegradation occurs when no residue remains and complete mineralisation is established when the original substrate, CPOLYMER in this example, is completely converted into gaseous products and salts. However, mineralisation is a very slow process under natural conditions because some of the polymer undergoing

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biodegradation will initially be turned into biomass [13, 14]. Therefore, complete biodegradation and not mineralisation is the measurable goal when assessing removal from the environment.

1.4 Mechanisms of polymer degradation When working with biodegradable materials, the obvious question is why some polymers biodegrade and others do not. To understand this, one needs to know about the mechanisms through which polymeric materials are biodegraded. Although biodegradation is usually defined as degradation caused by biological activity (especially enzymatic action), it will usually occur simultaneously with − and is sometimes even initiated by − abiotic degradation such as photodegradation and simple hydrolysis. The following section gives a brief introduction to the most important mechanisms of polymer degradation.

1.4.1 Nonbiological degradation of polymers A great number of polymers are subject to hydrolysis, such as polyesters, polyanhydrides, polyamides, polycarbonates, polyurethanes (PU), polyureas, polyacetals and polyorthoesters. Different mechanisms of hydrolysis have been extensively reviewed; not only for backbone hydrolysis, but also for the hydrolysis of pendant groups [15–17]. The necessary elements for a wide range of catalysis, such as acids and bases, cations, nucleophiles and micellar and phase transfer agents, are usually present in most environments. In contrast to enzymatic degradation, where a material is degraded gradually from the surface inwards (primarily because macromolecular enzymes cannot diffuse into the interior of the material), chemical hydrolysis of a solid material can take place throughout its cross-section, except for very hydrophobic polymers. Important features affecting chemical polymer degradation and erosion include: a) the type of chemical bond, b) the pH, c) the temperature, d) the copolymer composition and e) water uptake (hydrophilicity). These features will not be discussed here, but have been covered in detail by [4].

1.4.2 Biological degradation of polymers Polymers represent major constituents of living cells which are most important for metabolism (enzyme proteins, storage compounds), genetic information (nucleic acids) and the structure (cell wall constituents, proteins) of cells [18]. These polymers have to be degraded inside cells in order to be available for environmental changes

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 5

and to other organisms upon cell lysis. It is therefore not surprising that organisms, during many millions of years of adaptation, have developed various mechanisms to degrade naturally occurring polymers. However, for the many new and varied synthetic polymers that have found their way into the environment only in the last 70 years, these mechanisms may not as yet have been developed. There are many different degradation mechanisms that combine synergistically in nature to degrade polymers. Microbiological degradation can take place through the action of enzymes or by-products (such as acids and peroxides) secreted by microorganisms (bacteria, yeasts, fungi and so on). In addition, macroorganisms can eat and, sometimes, digest polymers and cause mechanical, chemical or enzymatic ageing [19, 20]. Two key steps occur in the microbial polymer degradation process: first, a depolymerisation or chain cleavage step, and second, mineralisation. The first step normally occurs outside the organism due to the size of the polymer chain and the insoluble nature of many of the polymers. Extracellular enzymes are responsible for this step, acting in either an endo (random cleavage on the internal linkages of the polymer chains) or exo (sequential cleavage on the terminal monomer units in the main chain) manner. Once oligomeric or monomeric fragments of a sufficiently small size are formed, they are transported into the cell where they are mineralised. At this stage the cell usually derives metabolic energy from the mineralisation process. The products of this process, apart from adenosine triphosphate (ATP), are gases (e.g., CO2, CH4, nitrogen (N2) and hydrogen (H2)), water, salts and minerals, and biomass. Many variations of this general view of the biodegradation process can occur, depending on the polymer, the organisms and the environment. Nevertheless, there will always be, at one stage or another, the involvement of enzymes. Enzymes are biological catalysts which can induce enormous (108−1020 fold) increases in reaction rates in an environment otherwise unfavourable for chemical reactions. All enzymes are proteins, i.e., polypeptides with a complex three-dimensional structure, ranging in MW from several thousand to several million g/mol. Enzyme activity is closely related to the conformational structure, which creates certain regions at the surface, forming an active site. The interaction between an enzyme and substrate takes place at the active site, leading to the chemical reaction, eventually giving a particular product. Some enzymes contain regions with absolute specificity for a given substrate while others can recognise a series of substrates. For optimal activity most enzymes must associate with cofactors, which can be of inorganic (e.g., metal ions) or organic origin (such as coenzyme A, ATP and vitamins such as riboflavin and biotin) [18]. Different enzymes can have different mechanisms of catalysis. Some enzymes change the substrate through some free radical mechanism, while others follow alternative chemical routes. When assessing the biodegradability of polymeric materials, it is important to realise that there are an enormous amount of different enzymes − each catalysing its own unique reaction on groups of substrates or on very specific chemical bonds; in some cases acting complementarily, in others synergistically.

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1.5 Measuring the biodegradation of polymers As can be imagined from the various mechanisms described above, biodegradation does not only depend on the chemistry of the polymer, but also on the presence of the biological systems involved in the process. When investigating the biodegradability of a material, the effect of the environment cannot be neglected. Microbial activity, and hence biodegradation, is influenced by: – The presence of microorganisms. – The availability of oxygen. – The amount of available water. – The temperature. – The chemical environment (pH, electrolytes and so on). In order to simplify the overall picture, the environments in which biodegradation occurs are basically divided in two: a) aerobic (with oxygen available) and b) anaerobic (no oxygen present). The availability of oxygen greatly affects the composition of the microbial community that is active in the environment, and thus its ability to biodegrade particular polymers. This division can in turn be subdivided into 1) aquatic and 2) high solids environments. Figure 1.1 schematically presents the different environments, with examples in which biodegradation may occur [21, 22].

1) Aquatic a) Aerobic

b) Anaerobic

2) High solids

– Aerobic wastewater treatment plants – Surface waters; e.g., lakes and rivers – Marine environments

– Surface soils – Organic waste composting plants – Littering

– Anaerobic wastewater treatment plants – Rumen of herbivores

– – – –

Deep-sea sediments Anaerobic sludge Anaerobic digestion/biogasification Landfill

Figure 1.1: Schematic classification of different biodegradation environments for polymers.

The high solids environments will be the most relevant for measuring environmental biodegradation of polymeric materials, since they represent the conditions during biological municipal solid waste treatment, such as composting or anaerobic digestion (biogasification). However, possible applications of biodegradable materials other than in packaging and consumer products, e.g., in fishing nets at sea, or undesirable exposure in the environment due to littering, explain the necessity of aquatic biodegradation tests. Numerous ways for the experimental assessment of polymer biodegradability have been described in the scientific literature. Because of slightly different definitions or interpretations of the term ‘biodegradability’, the different approaches are

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 7

therefore not equivalent in terms of information they provide or practical significance. Since the typical exposure environment involves incubation of a polymer substrate with microorganisms or enzymes, only a limited number of measurements are possible:those pertaining to the substrates, to the microorganisms or to the reaction products. Four common approaches available for studying biodegradation processes have been reviewed in detail by Andrady [13, 14]: – Monitoring the accumulation of biomass. – Monitoring the depletion of substrates. – Monitoring the reaction products. – Monitoring the changes in substrate properties. In the following sections, different test methods for the assessment of polymer biodegradability are presented. Measurements are usually based on one of the four approaches given above, but combinations also occur. Before choosing an assay to simulate environmental effects in an accelerated manner, it is critical to consider the closeness of fit that the assay will provide between substrate, microorganisms or enzymes, and the application or environment in which biodegradation should take place [23].

1.5.1 Enzyme assays 1.5.1.1 Principle In enzyme assays, the polymer substrate is added to a buffered or pH-controlled system, containing one or several types of purified enzymes. These assays are very useful in examining the kinetics of depolymerisation, or oligomer or monomer release from a polymer chain under different assay conditions. The method is very rapid (minutes to hours) and can give quantitative information. However, enzyme assays are not suitable to determine mineralisation rates.

1.5.1.2 Applications The type of enzyme to be used, and quantification of degradation, will depend on the polymer being screened. For example, Mochizuki and co-workers [24] studied the effects of the draw ratio of polycaprolactone fibres on enzymatic hydrolysis by lipase. The degradability of polycaprolactone fibres was monitored by dissolved organic carbon (DOC) formation and weight loss. Similar systems with lipases have been used for studying the hydrolysis of broad ranges of aliphatic polyesters [25–30], copolyesters with aromatic segments [26, 31–33] and copolyesteramides [34, 35]. Other enzymes such as α-chymotrypsin and α-trypsin have also been applied to these polymers [36, 37]. The biodegradability of polyvinyl alcohol (PVA) segments,

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with respect to block length and stereo chemical configuration, has been studied using isolated PVA-dehydrogenase [38]. Cellulolytic enzymes have been used to study the biodegradability of cellulose ester derivatives as a function of the degree of substitution and substituent size [39]. Similar work has been performed with starch esters using amylolytic enzymes such as α-amylases, β-amylases, glucoamylases and amyloglucosidases [40]. Enzymatic methods have also been used to study the biodegradability of starch plastics or packaging materials containing cellulose [41–46].

1.5.1.3 Drawbacks Caution must be used in extrapolating enzyme assays as a screening tool for different polymers since the enzymes have been paired to only one polymer. The initially selected enzymes may show significantly reduced activity towards modified polymers or different materials, even though more suitable enzymes may exist in the environment. Caution must also be used if the enzymes are not purified or appropriately stabilised or stored, since inhibition and loss of enzyme activity can occur [23].

1.5.2 Plate tests 1.5.2.1 Principle Plate tests were initially developed in order to assess the resistance of plastics to microbial degradation. Several methods have been standardised by standardisation organisations such as the ASTM and the ISO [47–49]. They are now also used to see if a polymeric material will support growth [23, 50]. The principle of the method involves placing the test material on the surface of a mineral salts agar in a Petri dish containing no additional carbon source. The test material and agar surface are sprayed with a standardised mixed inoculum of known bacteria and/or fungi. The test material is examined, after a predetermined incubation period at constant temperature, for the amount of growth on its surface and a rating is given.

1.5.2.2 Applications Potts [51] used the method in his screening of 31 commercially available polymers for biodegradability. Other studies, where the growth of either mixed or pure cultures of microorganisms is taken to be indicative of biodegradation, have been reported [6]. The validity of this type of test and the use of visual assessment alone, for all plastics,

1 Methods for evaluating the biodegradability of environmentally degradable polymers 

 9

has been questioned by Seal and Pantke [52]. They recommended that mechanical properties should be assessed to support visual observations. Microscopic examination of the surface can also give additional information. A variation of the plate test is the ‘clear zone’ technique [53], sometimes used to screen polymers for biodegradability. A fine suspension of polymer is placed in an agar gel as the sole carbon source and the test inoculum is placed in wells bored into the agar. After incubation, a clear zone around the well, detected visually or instrumentally, is indicative of utilisation of the polymer. The method has, for example, been used in the case of starch plastics [54], various polyesters [55–57] and PU [58].

1.5.2.3 Drawbacks A positive result in an agar plate test indicates that an organism can grow on the substrate, but does not mean that the polymer is biodegradable, since growth may be on contaminants, on plasticisers which are present, on oligomeric fractions still present in the polymer and so on. Therefore, these tests should be treated with caution when extrapolating the data to field situations.

1.5.3 Respiration tests 1.5.3.1 Principle Aerobic microbial activity is typically characterised by the utilisation of oxygen. Aerobic biodegradation requires oxygen for the oxidation of compounds to its mineral constituents, such as CO2, H2O, sulfur dioxide (SO2), phosphorous pentoxide (P2O5) and so on. The amount of oxygen utilised during incubation, also called the biological oxygen demand (BOD), is therefore a measure of the degree of biodegradation. Several test methods are based on measurement of the BOD, often expressed as a percentage of the theoretical oxygen demand (TOD) of the compound. The TOD, which is the theoretical amount of oxygen necessary for completely oxidising a substrate to its mineral constituents, can be calculated by considering the elemental composition and the stoichiometry of oxidation [13, 59–62] or based on experimental determination of the chemical oxygen demand (COD) [13, 63].

1.5.3.2 Applications The closed bottle BOD tests were designed to determine the biodegradability of detergents [61, 64]. These have stringent conditions due to the low level of inoculum (in the

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order of 105 microorganisms/l) and the limited amount of test substance that can be added (normally between 2 and 4 mg/l). These limitations originate from the practical requirement that the oxygen demand should not exceed half the maximum dissolved oxygen level in the water at the temperature of the test, to avoid the generation of anaerobic conditions during incubation. For nonsoluble materials, such as polymers, less stringent conditions are acceptable and alternative ways for measuring BOD were developed. Two-phase (semi) closed bottle tests enable a higher oxygen content in the flasks and permit a higher inoculum level. Higher test concentrations are also possible, encouraging higher accuracy with the direct weighing in of samples. The oxygen demand can alternatively be determined by periodically measuring the oxygen concentration in the aquatic phase by opening the flasks [60, 65–67], by measuring the change in volume or pressure in incubation flasks containing CO2-absorbing agents [59, 68, 69], or by measuring the quantity of oxygen produced (electrolytically) to maintain a constant gas volume/pressure in specialised respirometers [59, 62, 65, 66, 68].

1.5.3.3 Suitability BOD tests are sensitive and relatively simple to perform, and are therefore often used as screening tests. However, the measurement of oxygen consumption is a nonspecific, indirect measure for biodegradation and is not suitable for determining anaerobic degradation. The requirement for test materials to be the sole carbon/energy source for microorganisms in the incubation media eliminates the use of oxygen measurements in complex natural environments.

1.5.4 Gas (CO2 or CH4) evolution tests 1.5.4.1 Principle The evolution of CO2 or CH4 from a substrate represents a direct parameter of mineralisation. Therefore, gas evolution tests can be important tools in the determination of the biodegradability of polymeric materials. A number of well- known test methods have been standardised for aerobic biodegradation, such as the (modified) Sturm test [70–75] and the laboratory controlled composting test [76–79]; as well as for anaerobic biodegradation, such as the anaerobic sludge test [80, 81] and the anaerobic digestion test [82, 83]. Although the principle of these test methods is the same, they may differ in medium composition, inoculum, the way substrates are introduced, and in the technique for measuring gas evolution.

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1.5.4.2 Applications Anaerobic tests generally follow biodegradation by measuring the increase in pressure and/or volume due to gas evolution, usually in combination with gas chromatographic analysis of the gas phase [84, 85]. Most aerobic standard tests apply continuous aeration; the exit stream of air can be directly analysed continuously using a CO2 monitor (usually infrared detectors) or titrimetrically after sorption in dilute alkali. The cumulative amount of CO2 generated, expressed as a percentage of the theoretically expected value for total conversion to CO2, is a measure of the extent of mineralisation achieved. A value of 60% carbon conversion to CO2, achieved within 28 days, is generally taken to indicate ready degradability. Taking into account that in this system there will also be incorporation of carbon into the formation of biomass (growth), the 60% value for CO2 implies almost complete degradation. While this criterion is meant for water-soluble substrates, it is probably applicable to very finely divided moderately degradable polymeric materials as well [13]. Nevertheless, most standards for determining the biodegradability of plastics consider a maximum test duration of 6 months. Besides the continuously aerated systems, described above, several static respirometers have been described. Bartha and Pramer [86] describe a two-flask system; one flask, containing a mixture of soil and the substrate, is connected to another chamber holding a quantity of CO2 sorbant. Care must be taken to ensure that enough oxygen is available in the flask for biodegradation. Nevertheless, this experimental set-up and modified versions thereof have been successfully applied in the assessment of the biodegradability of polymer films and food packaging materials [87–89]. The percentage of carbon converted to biomass instead of CO2 depends on the type of polymer and the phase of degradation. Therefore, it has been suggested to regard the complete carbon balance to determine the degree of degradation [90]. This implies that besides the detection of gaseous carbon, the amount of carbon in soluble and solid products also needs to be determined. Soluble products, oligomers of different molecular size, intermediates and proteins secreted from microbial cells can be measured as COD or as DOC. Solid products, biomass, and polymer remnants require a combination of procedures to separate and detect different fractions. The protein content of the insoluble fraction is usually determined to estimate the amount of carbon converted to biomass, using the assumptions that dry biomass consists of 50% protein and that the carbon content of dry biomass is 50% [90–92].

1.5.4.3 Suitability Gas evolution tests are popular test methods because they are sensitive and relatively simple to perform. A direct measure for mineralisation is determined and

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water-soluble or insoluble polymers can be tested as films, powders or objects. Furthermore, the test conditions and inoculum can be adjusted to fit the application or environment in which biodegradation should take place. Aquatic synthetic media is usually used, but also natural seawater [93, 94] or soil samples [86, 88, 89, 95] can be applied as biodegradation environments. A prerequisite for these media is that the background CO2 evolution is limited, which excludes the application of real composting conditions. Biodegradation under composting conditions is therefore measured using an inoculum derived from matured compost with low respiration activity [76–78, 96, 97]. A drawback of using complex degradation environments, such as mature compost, is that the simultaneous characterisation of intermediate degradation products and determination of the carbon balance is difficult due to the presence of a great number of interfering compounds. To overcome this, an alternative test was developed based on an inoculated mineral bed-based matrix [98, 99].

1.5.5 Radioactively labelled polymers 1.5.5.1 Principle and applications Some materials tend to degrade very slowly under stringent test conditions without an additional source of carbon. However, if readily available sources of carbon are added, it becomes impossible to tell how much of the evolved CO2 can be attributed to decomposition of the plastic. The incorporation of radioactive 14C in synthetic polymers gives a means of distinguishing between CO2 or CH4 produced via metabolism of the polymer, and that generated by other carbon sources in the test environment. By comparison of the amount of radioactive 14CO2 or 14CH4 to the original radioactivity of the labelled polymer, it is possible to determine the percent by weight of carbon in the polymer which was mineralised during the exposure period [51, 100–102]. Collection of radioactively labelled gases or low MW products can also provide extremely sensitive and reproducible methods to assess the degradation of polymers with low susceptibility to enzymes, such as PE [8, 103] and cellulose acetates [104, 105].

1.5.5.2 Drawbacks Problems with handling the radioactively labelled materials and their disposal are issues on the down side of this method. In addition, in some cases it is difficult to synthesise the target polymer with the radioactive labels in the appropriate locations, with representative MW, or with representative morphological characteristics.

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1.5.6 Laboratory-scale simulated accelerating environments 1.5.6.1 Principle Biodegradation of a polymer material is usually associated with changes in the physical, chemical and mechanical properties of the material. It is indeed these changes, rather than the chemical reactions, which make the biodegradation process so interesting from an application point of view. These useful properties might be measured as a function of the duration of exposure to a biotic medium, to follow the consequences of the biodegradation process on material properties. Biotic media can be specifically designed at laboratory scale to mimic natural systems whilst allowing maximum control of variables such as temperature, pH, microbial community, mechanical agitation and supply of oxygen. Regulating these variables improves the reproducibility and may accelerate the degradation process. Laboratory simulations can also be used for the assessment of long-term effects, achieved by continuous dosing, on the activity and environment of the disposal system [50].

1.5.6.2 Applications The Organisation for Economic Co-operation and Development’s ‘Coupled Unit’ test [106] simulates an activated sludge sewage treatment system, but its application for polymers would be difficult as DOC is the parameter used to assess biodegradability. Krupp and Jewell [107] described well-controlled anaerobic and aerobic aquatic bioreactors to study the degradation of a range of commercially available polymer films. A relatively low loading rate of the semicontinuous reactors and a long retention time were maintained to maximise the biodegradation efficiency. Experimental set-ups have also been designed to simulate marine environments [108], soil burial conditions [108–110], composting environments [111–116], and landfill conditions [117–119] at laboratory scale, with controlled parameters such as temperature and moisture level, and a synthetic waste to provide a standardised basis for comparing the degradation kinetics of films. A wide choice of material properties can be followed during the degradation process. However, it is important to select one which is relevant to the end-use of the polymer material or provides fundamental information about the degradation process. Weight loss is a parameter frequently followed because it clearly demonstrates the disintegration of a biodegradable product [120–122]. Tensile properties are also often monitored, due to interest in the use of biodegradable plastics in packaging applications [54, 123, 124]. In those polymers where biodegradation involves a random scission of the macromolecular chains, a decrease in the average MW and a general broadening of the MW distribution provide initial evidence of a breakdown process [86, 125, 126]. However, no significant changes in material characteristics may be observed in recovered material if the mechanism of biodegradation involves

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bioerosion, i.e., enzymatic or hydrolytic cleavage at the surface. Visual examination of the surface with various microscopic techniques can also give information on the biodegradation process [115, 127–130]. Likewise, chemical and/or physical changes in the polymer may be followed by (combinations of) specific techniques such as infrared [10, 131] or ultraviolet (UV) spectroscopy [84, 132], nuclear magnetic resonance measurements [115, 126−133], X-ray diffractometry [115, 134, 135] and differential scanning calorimetry (DSC) [115, 136, 137].

1.5.6.3 Drawbacks An inherent drawback in the use of mechanical properties, weight loss, MW, or any other property which relies on the macromolecular nature of the substrate is that in spite of their sensitivity, these can only address the early stages of the biodegradation process. Furthermore, these parameters give no information on the extent of mineralisation. Especially in material blends or copolymers, the hydrolysis of one component can cause significant disintegration (and thus loss of weight and tensile properties) whereas other components may persist in the environment, even in a disintegrated form [13]. Blends of starch, poly(3-hydroxy butyrate) or poly(ε- caprolactone) with polyolefins are examples of such systems [11, 43, 138].

1.5.7 Natural environments, field trials Exposure in natural environments provides the best true measure of the environmental fate of a polymer, because these tests include a diversity of organisms and achieve a desirable natural closeness of fit between the substrate, microbial agent and the environment. However, the results of that particular exposure are only relevant to the specific environment studied, which is likely to differ substantially from many other environments. An additional problem is the timescale for this method, since the degradation process, depending on the environment, may be very slow (months to years) [23]. Moreover, little information on the degradation process can be gained other than the real time required for weight loss or total disintegration. Nevertheless, field trials in natural environments are still used to extrapolate results acquired in laboratory tests to biodegradation behaviour under realistic outdoor conditions [115, 116, 127, 139, 140].

1.6 Conclusions The overview presented above makes clear that there is no such thing as a single optimal method for determining the biodegradation of polymeric materials. First of

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all, the biodegradation of a material is not only determined by the chemical composition and corresponding physical properties, but the degradation environment, to which the material is exposed, also affects the rate and degree of biodegradation. Furthermore, the method or test to be used depends on what information is requested; especially as the biodegradation concept is very important in relation to the end of life of a material, while it could be just one aspect of health and environmental safety in other cases. It is fairly obvious, but often neglected, that one should always consider why a particular polymeric material should be (or not be) biodegradable when contemplating how to assess its biodegradability. After all, it is the intended application of the material that governs the most suitable testing environment, the parameters to be measured during exposure and the corresponding limit values. For example, investigating whether biodegradation of a plastic material designed for food packaging could facilitate undesired growth of (pathogenic) microorganisms requires a completely different approach from investigating whether its waste can be discarded via composting (i.e., whether it degrades sufficiently rapidly to be compatible with existing biowaste composting facilities). It is important to state that it will not be sufficient to ascertain macroscopic changes, such as weight loss and disintegration, or growth of microorganisms, to define a material as biodegradable because these observations may originate from a partial biodegradation or from the degradation of a component of the material itself. In order to study the real biodegradation of a material in the environment (composting, anaerobic digestion, soil and water) it is necessary to determine the mineralisation, which is the transformation of the material into: CO2, CH4 (anaerobic condition), water and new biomass. Furthermore, it is important to evaluate the eventual toxic effects that the addition of the material could have on the environment, in order to avoid introducing dangerous substances. In this way we will be sure that no harm will be caused to the environment itself. This is the same approach followed by the principal standardisation bodies with standards regarding the compostability of plastics and packaging. These standards (European Norms 13432, ISO 17088 and ASTM D6400) describe the specifications for compostable plastics and packaging.

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EN 14046:2003, Packaging − Evaluation of the Ultimate Aerobic Biodegradability of Packaging Materials under Controlled Compositing Conditions − Method by Analysis of Released Carbon Dioxide, European Committee for Standardization, Brussels, Belgium, 2003. [80] ASTM D5210-92(2007), Standard Test Method for Determining the Anaerobic Biodegradation of Plastic Materials in the Presence of Municipal Sewage Sludge, American Society for Testing and Materials, PA, USA, 2007. [81] ISO 11734:1998(E), Water Quality − Evaluation of the ‘Ultimate’ Anaerobic Biodegradability of Organic Compounds in Digested Sludge − Method by Measurement of the Biogas Production, International Organization for Standardization, Genève, Switzerland, 1998. [82] ASTM D5511-11, Standard Test Method for Determining Anaerobic Biodegradation of Plastic Materials under High-Solids Anaerobic-Digestion Conditions, American Society for Testing and Materials, PA, USA, 2011. [83] ASTM D5526-94(2011)e1, Standard Test Method for Determining Anaerobic Biodegradation of Plastic Materials Under Accelerated Landfill Conditions, American Society for Testing and Materials, PA, USA, 2011. [84] M. Day, K. Shaw and J.D. Cooney, Journal of Environmental Polymer Degradation, 1994, 2, 121. [85] P. Puechner, W-R. Mueller and D. Bardtke, Journal of Environmental Polymer Degradation, 1995, 3, 133. [86] R. Bartha and A. Yabannavar in Proceedings of the 4th International Workshop on Biodegradable Plastics and Polymers and Fourth Annual Meeting of the Bio-environmentally Degradable Polymer Society, Durham, NH, USA, 1995, p.28. [87] A.L. Andrady, J.E. Pegram and Y. Tropsha, Journal of Environmental Polymer Degradation, 1993, 1, 171. [88] A. Yabannavar and R. Bartha, Soil Biology and Biochemistry, 1993, 25, 1469. [89] A.V. Yabannavar and R. Bartha, Applied Environmental Microbiology, 1994, 60, 3608. [90] S. Urstadt, J. Augusta, R-J. Müller and  W-D. Deckwer, Journal of Environmental Polymer Degradation, 1995, 3, 121. [91] M. Itävaara and M. Vikman, Chemosphere, 1995, 31, 4359. [92] B. Spitzer, C. Mende, M. Menner and T. Luck, Journal of Environmental Polymer Degradation, 1996, 4, 157. [93] A.L. Allen, J.M. Mayer, R. Stote and D.L. Kaplan, Journal of Environmental Polymer Degradation, 1994, 2, 237. [94] R. Courtes, A. Bahlaoui, A. Rambaud, F. Deschamps, E. Sunde and E. Dutriex, Ecotoxicology and Environmental Safety, 1995, 31, 142. [95] P. Barak, Y. Coquet, T.R. Halbach and J.A.E. Molina, Journal of Environmental Quality, 1991, 20, 173. [96] U. Pagga, D.B. Beimborn, J. Boelens and B. De Wilde, Chemosphere, 1995, 31, 4475. [97] U. Pagga, D.B. Beimborn and M. Yamamoto, Journal of Environmental Polymer Degradation, 1996, 4, 173. [98] M. Tosin, F. Degli-Innocenti and C. Bastioli, Journal of Environmental Polymer Degradation, 1998, 6, 79. [99] G. Bellia, M. Tosin, G. Floridi and F. Degli-Innocenti, Polymer Degradation and Stability, 1999, 66, 65. [100] ASTM D6340-98, Standard Test Methods for Determining Aerobic Biodegradation of Radiolabeled Plastic Materials in an Aqueous or Compost Environment, American Society for Testing and Materials, PA, USA, 2007. [101] ASTM D6692-01, Standard Test Method for Determining the Biodegradability of Radiolabeled Polymeric Plastic Materials in Seawater, American Society for Testing and Materials, PA, USA, 2001. {Withdrawn in 2010} [79]

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[102] ASTM D6776-02, Standard Test Method for Determining Anaerobic Biodegradability of Radiolabeled Plastic Materials in a Laboratory-Scale Simulated Landfill Environment, American Society for Testing and Materials, PA, USA, 2002. {Withdrawn in 2011} [103] A-C. Albertsson, C. Barenstedt and S. Karlsson, Journal of Environmental Polymer Degradation, 1993, 1, 241. [104] R.J. Komarek, R.M. Gardner, C.M. Buchanan and S. Gedon, Journal of Applied Polymer Science, 1993, 50, 1739. [105] C.M. Buchanan, D. Dorschel, R.M. Gardner, R.J. Komarek, A.J. Matosky, A.W. White and M.D. Wood, Journal of Environmental Polymer Degradation, 1996, 4, 179. [106] OECD 303A, Simulation Test − Aerobic Sewage Treatment: Coupled Units Test, Guidelines for Testing of Chemicals, Organisation for Economic Co-operation and Development, Paris, France, 2001. [107] L.R. Krupp and W.J. Jewell, Environmental Science and Technology, 1992, 26, 193. [108] D.L. Kaplan, J.M. Mayer, M. Greenberger, R. Gross and S. McCarthy, Polymer Degradation and Stability, 1994, 45, 165. [109] R. Dale and D.J. Squirrell, International Biodeterioration and Biodegradation, 1990, 26, 355. [110] K.J. Seal and M. Pantke, Material und Organismen, 1990, 25, 87. [111] R.M. Gardner, C.M. Buchanan, R. Komarek, D. Dorschel, C. Boggs and A.W. White, Journal of Applied Polymer Science, 1994, 52, 1477. [112] C.M. Buchanan, D.D. Dorschel, R.M. Gardner, R.J. Komarek and A.W. White, Journal of Macromolecular Science, Part A: Pure and Applied Chemistry, 1995, A32, 683. [113] R.A. Gross, J-D. Gu, D. Eberiel and S.P. McCarthy, Journal of Macromolecular Science, Part A: Pure and Applied Chemistry, 1995, A32, 613. [114] EN 14045:2003, Packaging − Evaluation of the Disintegration of Packaging Materials in Practical Oriented Tests under Defined Composting Conditions, European Committee for Standardization, Brussels, Belgium, 2003. [115] S. Maiti, D. Ray, D. Mitra, S. Sengupta and T. Kar, Journal of Applied Polymer Science, 2011, 122, 2503. [116] E. Rudnik and D. Briassoulis, Industrial Crops and Products, 2011, 33, 648. [117] ASTM D7475-11, Standard Test Method for Determining the Aerobic Degradation and Anaerobic Biodegradation of Plastic Materials under Accelerated Bioreactor Landfill Conditions, American Society for Testing and Materials, PA, USA, 2011. [118] ASTM D5526-94(2011)e1, Standard Test Method for Determining Anaerobic Biodegradation of Plastic Materials Under Accelerated Landfill Conditions, American Society for Testing and Materials, PA, USA, 2011. [119] G.P. Smith, B. Press, D. Eberiel, S.P. McCarthy, R.A. Gross and D.L. Kaplan, Polymeric Materials Science and Engineering, 1990, 63, 862. [120] V. Coma, Y. Couturier, B. Pascat, G. Bureau, S. Guilbert and J.L. Cuq, Packaging Technology and Science, 1994, 7, 27. [121] C.M. Buchanan, C.N. Boggs, D. Dorschel, R.M. Gardner, R.J. Komarek, T.L. Watterson and A.W. White, Journal of Environmental Polymer Degradation, 1995, 3, 1. [122] D. Goldberg, Journal of Environmental Polymer Degradation, 1995, 3, 61. [123] G. Iannotti, N. Fair, M. Tempesta, H. Neibling, F.H. Hsieh and M. Mueller in Degradable Materials − Perspectives, Issues and Opportunities, Eds., S.A. Barenberg, J.L. Brash, R. Narayan and A.E. Redpath, CRC Press, Boston, MA, 1990, p.425. [124] J. Mergaert, A. Webb, C. Anderson, A. Wouters and J. Swings, Applied Environmental Microbiology, 1993, 59, 3233. [125] L. Tilstra and D. Johnsonbaugh, Journal of Environmental Polymer Degradation, 1993, 1, 257. [126] D.S.G. Hu and H.J. Liu, Journal of Applied Polymer Science, 1994, 51, 473.

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H.B. Greizerstein, J.A. Syracuse and P.J. Kostyniak, Polymer Degradation and Stability, 1993, 39, 251. [128] L.V. Lopez-Llorca and M.F. Colom Valiente, Micron, 1993, 24, 457. [129] H. Nishida and Y. Tokiwa, Journal of Environmental Polymer Degradation, 1993, 1, 227. [130] C. Bastioli, A. Cerutti, I. Guanella, G.C. Romano and M. Tosin, Journal of Environmental Polymer Degradation, 1995, 3, 81. [131] M.J. Kay, R.W. McCabe and L.H.G. Morton, International Biodeterioration and Biodegradation, 1993, 31, 209. [132] N.S. Allen, M. Edge, M. Mohammadian and K. Jones, Polymer Degradation and Stability, 1994, 43, 229. [133] A. Löfgren and A-C. Albertsson, Journal of Applied Polymer Science, 1994, 52, 1327. [134] A-C. Albertsson and S. Karlsson, Macromolecular Symposia, 1995, 98, 797. [135] J. Schurz, P. Zipper and J. Lenz, Journal of Macromolecular Science, Part A: Pure and Applied Chemistry, 1993, A30, 603. [136] A-C. Albertsson, C. Barenstedt and S. Karlsson, Journal of Applied Polymer Science, 1994, 51, 1097. [137] J.P. Santerre, R.S. Labow, D.G. Duguay, D. Erfle and G.A. Adams, Journal of Biomedical Materials Research, 1994, 28, 1187. [138] A. Iwamoto and Y. Tokiwa, Polymer Degradation and Stability, 1994, 45, 205. [139] K.K. Leonas, M.A. Cole and  X-Y. Xiao, Journal of Environmental Polymer Degradation, 1994, 2, 253. [140] P. Halley, R. Rutgers, S. Coombs, J. Kettels, J. Gralton, G. Christie, M. Jenkins, H. Beh, K. Griffin, R. Jayasekara and G. Lonergan, Starch – Stärke, 2001, 53, 362. [127]

Rolf-Joachim Müller

2 Biodegradation behaviour of polymers in liquid environments 2.1 Introduction According to many definitions [1], the biodegradation of plastics is usually primarily induced by the action of various microorganisms, although often nonbiotic effects such as irradiation, thermal degradation or chemical hydrolysis contribute to the degradation process. The activity of microorganisms is closely connected to the presence of water. The supply of nutrients to microorganisms and the transportation of excreted enzymes and metabolic products occur via diffusion in the aqueous environment surrounding the cells. (Thus, it can be said that an aqueous environment is actually the natural one for a microbe.) However, in environments regarded as nonliquid such as soil, compost or surfaces of solids, microbes can also be active as long as a specific aqueous microenvironment allows the transportation processes necessary for biological activity. For example, in soil, microbial life takes place in the thin water-films located between the particles or in water-filled cavities in the soil components. A soil-humidity of around 50−60% is optimal for aerobic biological processes, where the humidity is given as a percentage of the maximum water-holding capacity, which also takes into account the structural elements of the soil (actually it reflects the filling of the cavities in the material). Although water is a basic component of the microbial world, many organisms need or prefer the contact to a solid matrix. For example, many fungi exhibit better growth on surfaces than in agitated liquids, which can, besides other effects, be attributed to the sensitivity of the fungal mycelium to mechanical forces. Differences in the optimal living conditions of different microorganisms results in the presence of very specialised microbial communities in various environments and thus, lead to specific degradation behaviours of substances (which act as energy and/or nutrient sources). Discussing the biodegradation of plastics in a liquid environment usually means natural degradation in freshwater (lakes, rivers), in a marine environment, or in aerobic and anaerobic sludges (wastewater treatment). However, many degradation studies of plastics in laboratories have been performed in defined synthetic or in complex liquid nutrient broths, which can also be regarded as degradation in a liquid environment. While studies using real natural aqueous systems enable information regarding the behaviour of biodegradable plastics in a distinct natural environment to be obtained, laboratory studies with special aqueous media are used for fundamental studies of biodegradation processes or to optimise the evaluation of the intrinsic biodegradability of a plastic, e.g., under typical conditions. Special liquid media provide defined and optimal living conditions for many organisms and thus, in many cases, https://doi.org/10.1515/9781501511967-002

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increase degradation rates leading to reduced test durations. Furthermore, analytical procedures to characterise the degradation process or to detect degradation intermediates are facilitated in a homogeneous and well- defined liquid medium. This chapter covers the biodegradation in real liquid environments as well as in especially designed laboratory test systems, and also reviews the role of aqueous test environments of national and international standards in evaluating the biodegradability of plastics.

2.2 Degradation in real liquid environments In most cases biodegradability is a property which is related to the behaviour of the plastic items after they become waste. The biodegradation of plastics in landfills was discussed at an early stage of the development of such materials to reduce the waste volume and thus, save deposit space. Nowadays, composting as an alternative waste treatment system to landfilling, incineration or recycling is of major interest. However, the biodegradability of plastics can also contribute to the property profile of a product during its application. In the agricultural field, mulching films made from biodegradable plastics are now being commercialised; in addition, controlled release formulations with fertilisers or agrochemicals are being developed. In this context, degradation testing of plastics in soil has been intensified over the last few years. Generally speaking, it can be stated that most of the investigations on biodegradable plastics in the past focused on solid environments such as landfills, compost or soil. However, the aspect of avoiding waste is not the only issue that needs to be addressed when considering the fate of biodegradable plastics in liquid environments; biodegradability as a novel property for special applications of plastics is also an important consideration. The prevention of marine environment pollution, for instance, is regulated by the MARPOL Treaty. This international convention prohibits the disposal of any plastics waste in the oceans, e.g., from ships or from offshore platforms. The international convention generated activities to check if biodegradable plastics, used as an alternative to conventional polymers, are suitable to be degraded in a marine environment [2]. A further problem exists from littering, where plastic items are washed away to the sea by rivers or blown by wind from the shores and can cause the death of numerous marine animals [3]. Notably in Japan, the Fisheries Agency is active in developing fishery equipment (e.g., fishery nets), which are biodegradable and do not cause permanent harm to sea life when lost during fishing [4]. Besides these aerobic environments, the biodegradability of plastics under anaerobic conditions should also be considered; especially with the collection and biological treatment of green waste from households (kitchens), anaerobic digestion

2 Biodegradation behaviour of polymers in liquid environments 

 25

(normally followed by an aerobic composting stabilisation) becomes more and more important, particularly in certain European countries. In addition to this waste management aspect, the introduction of biodegradable plastics to natural anaerobic environments (e.g., sediments in lakes, rivers or oceans), may occur and therefore the biodegradation behaviour of plastics in the absence of O2 is also of practical interest. Most of the investigations reported in the literature concerning biodegradation in natural liquid environments consider natural polyhydroxyalkanoates (PHA) such as polyhydroxybutyrate (PHB) or the copolyester containing valerate units poly (hydroxybutyrate-co-hydroxyvalerate) (PHBV). These biodegradable materials were of outstanding interest in the past; however, nowadays the commercial relevance of these materials is limited. For commercially important biodegradable plastics, mainly synthetic polyesters, not much data regarding degradation in natural liquid environments is available.

2.2.1 Degradation in freshwater and marine environment 2.2.1.1 Polyhydroxyalkanoates Doi and co-workers [5] exposed PHBV of different copolymer compositions to seawater (1.5m depth) at temperatures between 14−25 °C (depending on the season). There was no clear detectable influence of the degradation rate on the hydroxyvalerate (HV) content of the copolymer. Erosion rates (removal of polymer material from each surface of the film sample) were in the order of magnitude of 2.5 µm/week (at approximately 22 °C). A significant influence of temperature was found for the degradation of poly(3hydroxybutyrate-co-4-hydroxybutyrate) polymers. Increasing the temperature from approximately 14 to 24 °C nearly doubled the degradation rate of the polymers (erosion rate at 24 °C was approximately 3.8 µm/week). Imam and co-workers [2] tested the degradation of PHBV (12 mol% HV) and PHBV/starch- blends in tropical coastal waters (in baskets at 0.5 m depth and temperatures of 25−32 °C) and stated a significant weight loss for both materials (approximately 500 µm sheets). While pure PHBV degraded quite slowly (10−40% weight loss within 400 days), the starchblends were totally disintegrated in less than 150 days. From these data an erosion rate of about 0.4−1.7 µm/week can be estimated for PHB and >11 µm/week for PHBV. The degradation of a PHBV (Biopol) in freshwater at a depth of 20−85 m was investigated by Brandl and Püchner [6] at temperatures ranging only from 6−8 °C. Despite the low temperatures and the reduced O2 concentration in the deeper water layers, 17 µm films of PHBV (8 mol% HV) were totally disintegrated within 254 days. Erosion data on PHBV bottles demonstrated that the degradation rate significantly decreased with increasing water depth, although even at a distance of 85 m from the surface a clear biological degradation could be observed.

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2.2.1.2 Synthetic polyesters Besides the work on natural PHA-polyesters, degradation experiments in seawater with synthetic polymers such as poly(ε-caprolactone) (PCL) and modified polyethylene (PE) are also reported in the literature. Rutkowska and co-workers reported a complete defragmentation of PCL samples in seawater (Baltic Sea) at temperatures between 9−21 °C [7] within 8 weeks; temperature was stated to be a major influencing factor for degradation. For PCL, chemical hydrolysis and enzymatic surface erosion are responsible in parallel for the polymer degradation. The same research group, using polyester urethanes, found a significant weight loss in seawater (Baltic Sea) within 12 months, while a polyether urethane was not biologically attacked under the same experimental conditions [8]. Polyolefins such as PE and polypropylene (PP) are usually not accessible to direct microbial attack. For such polymers, biological degradability is achieved by the addition of starch, prooxidant additives or photosensitive components. Starch, a natural polymer, can be degraded by microorganisms which enhances the defragmentation of the polyolefins (if the starch is accessible to the microbes). The additives increase the initial reduction of polymer chain length by chemical processes to form short chain length oligomers which can finally be metabolised by microorganisms. However, no significant changes in material properties nor any reliable weight loss of differently modified PE and PP could be observed by Gonsalves and co-workers [9, 10] upon exposure to seawater (1−9 m depth, temperatures of 13−30 °C) for 5−12 weeks. The primary (chemical) degradation depends on the exposure temperature and the normal temperatures (maximum and minimum) found in seawater, the reaction rate is probably too slow to observe any changes in the materials within the duration employed. Photodegradable PE proved to be degraded slower under seawater and freshwater floating conditions compared with environmental exposure to air [11]. The temperatures (12−28 °C), a shielding from sunlight by the water and biofouling were stated as reasons for the slower loss of physical properties in water. However, the disintegration of some samples could be observed within a period of 30−66 days. Similar observations were made by Leonas and Gorden in a laboratory simulation test [12]. Recently, much interest has arisen regarding the behaviour of biodegradable and compostable carrier bags in the marine environment. While compostable bags are designed to be reused at home as bin liners to collect biowaste and hence be composted together, characterisation of the biodegradability under environmental conditions is necessary due to littering of this product, it is therefore of interest to characterise the potential residence time in sensitive environments such as the sea. Recent studies show that compostable carrier bags can undergo a strong physical degradation when exposed in the open sea [13]. Degradation is not just due to a mechanical effect, but it is the consequence of true biodegradation as shown by [14] in a study where the marine environment is subdivided into its different habitats and a specific test method is proposed or required for each habitat.

2 Biodegradation behaviour of polymers in liquid environments 

 27

The activated sludge stage of a wastewater treatment plant is a liquid environment exhibiting a high microbial activity. Gilmore and co-workers tested the behaviour of different polymers in this environment [15]. Sheets of PHBV (500 µm; 26.5 mol% HV) were disintegrated within 60 days (at 22 °C), corresponding to an approximate erosion rate of 30 µm/week; at lower temperatures (12−19 °C) an erosion rate of approximately 6 µm/week was observed. Starch-filled polyolefins (without prooxidants) and blends of polyolefins with the degradable polyester PCL exhibited no hint of any biological attack (weight loss or changes in mechanical properties), even in this active environment.

2.3 Degradation in laboratory tests simulating real aquatic environments Field tests in real environments have a number of limitations and problems. Parameters such as temperature or water quality can vary during the test period, and monitoring of the biodegradation process is usually limited to visual changes or at least to the determination of the weight loss of the samples. To overcome these deficiencies, controlled laboratory tests simulating natural (aquatic) environments are often used to investigate biodegradation processes.

2.3.1 Aerobic liquid environments Investigations of Tsuji and Suzuyoshi [16] on PHB, PCL and polylactic acid (PLA) (films of 50 mm thickness) in a laboratory test with seawater at 25 °C resulted in erosion rates of 0.6 µm/week for PHB and 0.2 µm/week for PCL. This data are comparable to findings in field tests. In contrast to both these polyesters, PLA did not show any significant weight loss in this experiment; this can obviously be attributed to the different types of degradation mechanisms. While PHB and PCL are primarily attacked by enzymes (PHB depolymerases, lipases) at the surface, PLA is known to be mainly degraded initially by a nonenzymatically catalysed hydrolysis mechanism, which is strongly temperature dependent. While, for instance, in compost (at temperatures up to 70 °C) PLA has been shown to be quite rapidly chemically depolymerised and then metabolised by microorganisms, this reaction mechanism is much slower at 25 °C, where PLA is in the glassy state below its glass transition temperature (Tg). Thus, it can be expected that PLA is, despite the presence of various polyester-degrading microorganisms, only degraded very slowly in liquid environments such as seawater or freshwater. A direct comparison of degradation rates in different liquid and non-liquid environments at different temperatures is given in a publication by Manna and Paul [17]

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using PHB as the degradable polyester (250 µm sheets). It is quite surprising that no significant general differences in the degradation rate between liquid environments (freshwater and sewage sludge) and solid environments (compost and soil) at the same temperature could be detected (Figure 2.1). A pronounced temperature dependence of degradation was observed, where in most cases the highest weight losses were obtained at 30 °C (except with freshwater where a maximum degradation was observed at 40 °C). Compared with freshwater, microbial attack is somewhat higher in sewage sludge, an environment of high microbial activity. Erosion rates estimated from the weight loss data in freshwater are comparable to those presented previously for field tests (0.7 µm/week at 20 °C; 1.3 µm/ week at 30 °C and 1.5 µm/week at 40 °C). 50

compost

soils

liquid environments

45

20 °C 30 °C

40

40 °C

Weight loss (%)

35 30 25 20 15 10 5

e

r

ud g

te Se

wa

ge

sl

wa sh Fr e

Ta r

in

e

So i

l

l So i dy

l e lin Sa

e rit te La

Sa n

So i

il so

il so ay Cl

Co

m

po

st

0

Figure 2.1: Weight loss of 250 µm sheets of PHB in different environments after 200 days of incubation. Reproduced with permission from A. Manna and A.K. Paul, Biodegradation, 2000, 11, 5, 323. ©2000, Springer.

A direct comparison of microbial degradation of PHBV (14 mol% HV; fibres of approximately 213 and 493 µm diameter) in fresh and seawater was performed by Ohura and co-workers [4], using natural water supplemented with some mineral salts to increase

2 Biodegradation behaviour of polymers in liquid environments 

 29

microbial activity. In this accelerated simulation test, a complete degradation (weight loss measurement and determination of the biological oxygen demand (BOD)) of the 213 µm fibres could be achieved within 2 weeks in freshwater (two sources) and within 4 weeks in seawater (two sources); furthermore, for the 493 µm fibres, the degradation in freshwater was almost twice as high as in seawater. Some years previously, the same research group had investigated the degradation of a number of PHA-copolymers and a number of synthetic polyesters under similar test conditions using water from a river [18] (100 µm films at 25 °C, weight loss and BOD). Again quite rapid degradation was achieved with the accelerated test system used. A 100 µm film of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) (14% HV) was almost totally degraded within one week. The erosion rates obtained were in a range of 30−50 µm/week and much higher than observed in field tests under natural conditions. The test system used allowed comparison of the biodegradation of different polymers in freshwater. The PHBV-copolymer degradation rate increased with the copolyester composition up to a HV content of 14 mol%, and then decreased to nearly zero for P(3HB-co-3HV) (80% HV). Copolyesters of 3-hydroxybutyrate with 4-hydroxybutyrate and a pure poly(4hydroxybutyrate) homopolyester were completely degraded within 4 weeks. For copolymers of 3HB with 3-hydroxypropionate (3HP) the copolyesters proved to be rapidly degradable, while pure 3HP was not attacked. Among a number of different synthetic aliphatic polyesters, PE succinate degraded at approximately the same rate as the PHBV copolymers. PE adipate, polybutylene adipate and polybutylene sebacate also exhibited a weight loss within 4 weeks of incubation, but degradation was slower than for the other materials. For PE sebacate, polybutylene succinate and polyhexylene succinate, no significant weight loss could be observed under the test conditions applied. Interestingly, there is no correlation between the degradation rate and the melting points of the substances, a finding which has been made via enzymatic degradation tests [19, 20]. For synthetic polyesters, the degradation behaviour in seawater compared with freshwater is different from that of natural PHA [21]. While PHB and PHBV copolymers are quite rapidly degradable in both liquid environments, seawater and freshwater, synthetic polyesters seem to be less degradable in seawater. PCL (100 µm films) degrades completely in freshwater within approximately 2 weeks. In seawater (from the ocean) a BOD of 50−60% could be observed after only 4 weeks. In seawater from a bay, degradation is surprisingly high, reaching the maximum BOD level after only one week. PE succinate is more rapidly degradable than PCL in freshwater, but exhibits no microbial degradation in seawater, from the ocean or from a bay. This behaviour can probably be attributed to the occurrence of different microorganisms able to degrade the natural and synthetic polyesters. PHA are natural polymers and nature developed many organisms to degrade and utilise this carbon and energy source. Thus, PHA degraders are present in many environments. PCL is a synthetic polyester, but its

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structure resembles cutin, a natural polyester from plants. Thus, it is likely that many organisms do exist which are able to degrade PCL. In contrast, other synthetic polyesters have been shown to be degraded, more or less by accident, by microorganisms producing lipase-like enzymes with a broad substrate spectrum; the enzymes, and corresponding organisms, are specific to a particular polyester. It is suggested that for many synthetic polyesters, the number of organisms able to attack these polymer structures is much smaller than for degraders of natural or natural-like polyesters. As a consequence, the probability of synthetic polyesters being degraded depends, in particular, on the absolute number of organisms present in a particular environment. As a general conclusion, it can be supposed that the degradation of synthetic, nonnatural polyesters is more dependent on the microbial population present in a distinct environment than is the case for natural materials such as PHB. From this viewpoint, the natural environments should be better identified, in terms of ecological habitats, and test approaches should in turn be based on the typical conditions experienced by the plastic products when entering each habitat, since the microbial population (and thus biodegradation activity) could be quite different. Tosin and co-workers [13] have identified 6 habitats where plastic waste can reside when littered in the marine environment: 1) pelagic domain (the plastic products float freely in estuaries and the open ocean water), 2) eulittoral zone (tides and storm waves bring great quantities of plastic waste to the shoreline, where plastic products get partly buried and kept wet by tidal inundation and waves), 3) supralittoral zone (the plastic products are washed onto the beach, exposed to a sandy soil with a low moisture level), 4) sublittoral zone (plastic products settle on marine sandy sediment where they are exposed to the seawater/sediment interface), 5) plastic products can otherwise sink to the bottom of the deep sea and 6) plastic products can be slowly buried within sediments on the sea-floor. A further specialised test system was used by Allen and co-workers [22], and Gonda and co-workers [23]. In these tests completely synthetic media, inoculated with individual microbial strains or defined microbial consortia, were used to investigate the biodegradation of polymers. Honda and Osawa [24] used a simulation test with synthetic wastewater inoculated with mud from a lake to investigate the behaviour of PCL for the denitrification of wastewater (at 25 °C); they found a remarkable degradation of the PCL plates used (erosion rates approximately 10−15 µm/week).

2.3.2 Anaerobic liquid environments Compared with investigations of polymer degradation under aerobic conditions, very little information is available in the literature regarding the degradation of plastics under anaerobic conditions. Again, most of the investigations published are focused on PHA.

2 Biodegradation behaviour of polymers in liquid environments 

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It can be expected that the degradation characteristics of polymers in an anaerobic environment are different from those observed in the presence of O2, since anaerobic microorganisms have a much more limited set of enzymes and thus, are more specialised with regards to substrates. Additionally, the energy benefit for the organisms is lower without having O2 as an electron acceptor, resulting mostly in slow growth of anaerobic microorganisms. In 1992, Budwill and co-workers published a paper on the degradation of PHB and PHBV copolyesters in an anaerobic mineral medium inoculated with sewage sludge [25]. It could be shown that PHB powder, as well as PHBV (13% HV and 20% HV) powder, degraded almost completely in the laboratory simulation test (35 °C, degradation monitored via CH4 production) over a period of less than 3 weeks. No clear difference in degradation behaviour was observed between the homopolyester PHB and the PHBV-copolyester. In a later study, the same authors extended the investigation to other conditions [26]. The degradation of PHB and PHBV with a microbial consortium from an anaerobic pond sediment at 15 °C was significantly slower than that with sewage sludge at 35 °C (6 week lag-phase; complete degradation after 14 weeks). Again PHB and PHBV exhibited nearly the same degradation behaviour. The anaerobic degradation of PHB and PHBV (8.4 mol% HV) in a mineral medium with sludge from a wastewater plant of the sugar industry at 35 °C was tested by Reischwitz and co-workers [27]. In this case, a significant degradation of the polyester powders (approximately 50 µm diameter) was also observed within 3 weeks, with no significant differences between PHB and PHBV. Urmeneta found an almost complete degradation of PHBV powder (7 mol% HV) in a liquid anaerobic slurry from a freshwater sediment within 6 weeks (at 15 °C) up to an amount of 0.5 mg PHB/cm3 sediment [28]. Shin and co-workers extended anaerobic biodegradation tests to other materials [29] (a synthetic mineral medium inoculated with anaerobic sludge from a municipal waste treatment plant at 35 °C). They found a rapid degradation of PHBV (8 wt% HV) and cellophane films (50−75 µm) within 3 weeks, whereas no degradation (via biogas formation) was observed for the synthetic polyesters PLA and polybutylene succinate. A corresponding finding was made by Gartiser and co-workers [30]. While a PHBV copolymer (60 µm film) was degraded under similar conditions to those used by Shin and co-workers in less than 3 weeks, no biogas formation could be observed for the synthetic polyester PCL over a period of 11 weeks. In this paper it was also demonstrated that cellulose acetate polymers (approximately 2.5 degrees of OH-substitution) are in principle degraded under anaerobic conditions, however, the rate of metabolisation is significantly slower than that of PHA. A detailed investigation on the anaerobic degradability of various starch- and cellulose-esters is given by Rivard and co-workers [31]. The dependence of the anaerobic degradation rate on the degree of substitution and the type of substituent is discussed. An extensive investigation on the anaerobic degradability of a number of natural and synthetic polyesters was performed by Abou-Zeid [32]. Along with PHA, PHB

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(44 mg)

100

(46 mg)

110

(46 mg)

(48 mg)

and PHBV (10 mol% HV), Abou-Zeid also tested PCL, the aliphatic homopolyester polybutylene adipate (SP4/6) and the copolyester polybutylene adipate-co- butylene terephthalate where about 40% of the diacid component consists of the aromatic terephthalic acid (BTA 40:60). Weight loss measurements of polymer films (40−74 µm thickness) in two different anaerobic sludges from wastewater treatment plants and an anaerobic river sediment demonstrated that both natural PHA were rapidly eroded (up to 100% within 14 weeks at 35 °C) compared with the synthetic polyesters. While PCL exhibited a low but significant weight loss, SP4/6 and the aliphatic/aromatic copolyester BTA 40:60 exhibited no clear indication of microbial attack under these test conditions (Figure 2.2).

methane-producing laboratory sludge wastewater sludge anaerobic river sediment

70

(48 mg)

60

PHB

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SP 4/6

(0.1 mg)

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(0.1 mg)

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90

BTA 40:60

Figure 2.2: Weight loss of various polyesters in different anaerobic environments after 14 weeks at 35 °C. (Polyester films: diameter = 25 mm; surface area: 39.3 cm2; initial film weights = 39−49 mg; 3 films per test.) Reproduced with permission from D.M. Abou-Zeid, Anaerobic Biodegradation of Natural and Synthetic Polyesters, Technical University Braunschweig, Braunschweig, Germany. ©2000, Technical University Braunschweig.

Similar results were obtained by Abou-Zeid when monitoring anaerobic biodegradation via biogas formation in a synthetic medium inoculated with anaerobic sludge (Figure 2.3). From these measurements it could be clearly demonstrated that PHB, in contrast to aerobic conditions, degraded faster than the copolyester PHBV. Again, the

2 Biodegradation behaviour of polymers in liquid environments 

 33

110 100 90 Degradation (%ThBiogas)

80

PHB PHBV PCL SP4/6 BTA 40:60

70 60 50 40 30 20 10 0 –10

0

7

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21 Time (d)

28

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42

Figure 2.3: Time-dependent mineralisation (percentage of the theoretical biogas volume) of various polyesters in anaerobic conditions and wastewater sludge at 37 °C over a period of 42 days. (Polyester films: Ø = 19 mm; surface area: 22.7 cm2; initial film weights: 35−40 mg; 2 films per test). Reproduced with permission from D.M. Abou-Zeid, Anaerobic Biodegradation of Natural and Synthetic Polyesters, Technical University Braunschweig, Braunschweig, Germany. ©2000, Technical University Braunschweig.

synthetic polyesters exhibited a significantly slower anaerobic degradation rate. Finally, Abou-Zeid showed that synthetic aliphatic polyesters are in principle degradable, but for aliphatic-aromatic copolyesters of technical relevance (approximately 40 mol% aromatic component in the acids) no clear indication of an anaerobic attack could be found. The different rates observed in the biodegradation of the analysed synthetic aliphatic polyesters calls for further studies. The reason why such differences in rate are recorded is still unknown, and further specific research is therefore needed in order to better understand the microbiology and the enzymology involved in the process. These observations may have some significance for the biological treatment of biodegradable plastics, as anaerobic digestion becomes more and more established alongside aerobic composting. It cannot be supposed that synthetic polyesters will be significantly degraded in an anaerobic process with typical residence times of about 3 weeks. However, in most cases, anaerobic digestion processes contain a final aerobic step for the stabilisation of the anaerobic compost. If the polyester materials disintegrate sufficiently in the anaerobic step and will not disturb the technical process, the final biodegradation can take place during the aerobic stabilisation.

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 Rolf-Joachim Müller

2.4 Degradation in laboratory tests with optimised and defined liquid media For many investigations on biodegradable plastics reported in the literature, degradation tests in defined synthetic media, inoculated with mixed microbial populations or with individual strains, have been used. These kinds of tests have significant advantages when investigating the basic biological degradation processes of polymers. Due to the usage of defined, in most cases synthetic media, and the possibility of controlling the environmental parameters (such as temperature, pH, salinity, nutrient supply and so on), these tests give better reproducible results than degradation tests under natural conditions. Compared with laboratory tests in a solid matrix, (e.g., soil burial and controlled composting tests), analytical procedures aimed at the analysis of intermediates or persistent residues are facilitated [33] in defined aqueous media. Monitoring of the biodegradation process in such tests can be performed with various methods. Weight loss measurements of films or formed items are the easiest way, but do not necessarily prove microbial metabolisation of the material. However, in combination with a detailed analysis of the intermediates and the residual polymer (also possible by quantitative chromatographic analysis when using fine polymer powders), useful information can be gained regarding the degradation mechanism and possible residual components, as demonstrated by Witt and co-workers, for the degradation of the aliphatic-aromatic copolyester Ecoflex with a thermophilic actinomycete (Thermobifidia fusca), previously isolated from compost [34]. The methods most frequently used to measure the biodegradation process in laboratory tests with liquid media determine the consumption of O2 (e.g., Sapromat test) [35, 36], or the release of CO2 (Sturm-test) caused by the metabolic activity of the microorganisms (respirometric tests) [37]. Due to the usually low amount of other carbon sources being present, in addition to the polymer itself, when using synthetic mineral media, only a fairly low background respiration has to be accounted for and the accuracy of the tests is usually good. These kinds of tests have already been used for a long time to evaluate the degradability of diverse substances and chemicals in water, (e.g., in the Organisation for Economic Co-operation and Development (OECD) guidelines (Table 2.1)) and have now been adapted to the application of nonwatersoluble polymeric materials; in particular, the type of analytical methods, especially for the determination of CO2, have been modified. The OECD guidelines describe the trapping of CO2 in barium hydroxide solution in combination with manual titration. More sophisticated methods use the detection of O2 and CO2 concentration in the air stream (used for aeration) with infrared detectors and paramagnetic O2 detectors. However, despite the advantage of an automated and continuous measurement, there are also some disadvantages with these methods. The exact air flow has to be measured, the signals of the detectors must be stable for quite a long period of time and,

2 Biodegradation behaviour of polymers in liquid environments 

 35

if slow degradation processes have to be determined, the CO2 concentration or the drop in the O2 concentration is only very low. This increases the possibility of systematic errors during such long-lasting experiments. Other concepts, e.g., trapping CO2 in a basic solution (approximately pH 11.5) with continuous titration or detection of the dissolved inorganic carbon [37] are useful alternatives. Other attempts to overcome the problems with CO2 detection are based on non-continuously aerated, closed systems. Here, either a sampling technique in combination with an infraredgas-analyser [38] or a titration system is applied [39]. Another closed system with a discontinuous titration method is described by Solaro and co-workers [40]. Tests using small closed bottles as degradation reactors, determining the CO2 in the headspace [41] or the decrease in dissolved O2 (closed bottle test) [42], are simple and quite insensitive to leakages and so on, but may cause problems due to the low amounts of material and inoculum used. A crucial point in applying laboratory tests with synthetic liquid media is the source of microorganisms and the procedure of inoculation preparation. An optimum has to be achieved between the minimal input of external carbon into the synthetic medium (reducing the background O2 consumption and CO2 evolution) and the overall microbial activity in terms of the number and diversity of microorganisms. Originally developed for evaluating the biodegradability of chemicals in wastewater treatment plants, laboratory tests described in the OECD guidelines use aerobic sewage sludge as the source of microorganisms. As an inoculum, the complete sludge or the particle-free supernatant solution of a sedimented sludge or a filtrate can be used, again there is a question of how much additional carbon source would be introduced into the system. It has been demonstrated that the type of sewage sludge pretreatment (e.g., homogenisation) has a significant influence on the degradation of the polymers in the test [43]. However, since the predominant environments where biodegradable plastics are supposed to be degraded are compost or soil, attempts have been made to use extracts from soil or compost to simulate the microbial population in these environments, as well as in the liquid-phase degradation tests. These sources of inoculum are also included in current standard tests such as the International Organization for Standardization (ISO) 14851 [44] or ISO 14852 [45]. However, there has been some critical discussion about the sense of transferring microorganisms, which are adapted to life in a solid matrix, into a liquid environment. Fungi, for instance, often involved in polymer degradation in soils, do not show optimal growth conditions in a liquid medium and thus, will be under-represented in the aqueous tests. van der Zee and co-workers discussed this in a paper on cellulose-acetate degradation [46] and found significant differences when comparing the degradation behaviour of cellulose-acetate in aquatic tests and a controlled composting test using mature compost as degradation matrix. However, beside differences in the microbial community other parameters such as the test temperature were also different (aquatic test at 20 °C; controlled composting test at 58 °C) in the tests compared by van der Zee and co-workers.

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 Rolf-Joachim Müller

Despite the limitations of aqueous degradation tests discussed previously, these tests, usually performed in a carbon free, synthetic medium, have one important advantage − the possibility of establishing a reliable carbon balance. The polymer, as an energy and carbon source for the microorganisms, is not completely transformed into CO2, but a part of the polymer carbon is used to build new biomass or natural metabolic products other than CO2 can be released into the medium; in addition, this part of the polymer can be regarded as biodegraded. In degradation tests using mature compost at about 60 °C (controlled composting test), very little biomass formation is observed and the carbon of the polymer is almost completely transformed into CO2. In aqueous media, the fraction of carbon going into new biomass can be in the range of 20−30% of the total carbon, and thus, taking CO2 solely as a measure for biodegradation, usually underestimates the degree of biodegradation which is reached. Determination of the entire fate of the carbon from the polymer, (i.e., establishing a carbon balance), has already been discussed for ready biodegradability testing of low molecular weight (MW) chemicals [47]. This has been extensively investigated by Urstadt and co-workers for the biodegradation of plastics [48]. In a system where a watersoluble substance is tested, the quantitative separation of biomass from the medium does not usually constitute a problem, but if residual, nonwater-soluble material is present, mechanical techniques for the separation of the two solid fractions are in many cases not applicable and thus, other methods have to be used [49, 50]. Carbon balances have been included in currently developed standard test methods for evaluating the biodegradability of plastics, (e.g., ISO 14851 [44] or ISO 14852 [45]), and are also applied in nonstandardised testing practice [51].

2.5 Standard tests for biodegradable polymers using liquid media The evaluation of the biodegradability of low MW chemicals has been an issue for many years, and a number of standard methods are available in this field [52] (Table 2.1). However, for polymers the point of view was totally different in the past, since plastics had been optimised for many years to be as stable as possible against various environmental influences, among them biological attack. Thus, standard test methods dealing with the interaction of microorganisms with plastics focused at that time on unwanted changes of the material properties (mainly optical or mechanical properties) caused by biological action. Such processes were called biocorrosion. Standard test methods for biocorrosion of plastics were not really suitable to evaluate the biodegradability of plastics (meaning a metabolic conversion of the plastic material by microorganisms) although often used at the very beginning of the development of biodegradable plastics [53].

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Table 2.1: Standard test methods for the biodegradability of chemicals. OECD Guidelines [54] 301

Ready biodegradability

301 A – 1992

DOC die-away test

301 B 1992

CO2 evolution test

301 C – 1992

Modified MITI test

301 D – 1992

Closed- bottle test

301 E – 1992

Modified OECD screening test

301 F – 1992

Manometric respirometry test

302

Inherent biodegradability

302 A – 1981

Modified SCAS test

302 B –1992

Zahn−Wellens test

302 C – 1981

Modified MITI test (II)

302 D – draft (2002)

Inherent biodegradability − Concawe test

303

Simulation test

303 A – 2001

Aerobic sewage treatment: activated sludge units

306 (2002)

Biodegradability in seawater, aerobic mineralisation in surface watersimulation biodegradation test

310 draft (2002)

Ready biodegradability CO2 in sealed vessels (headspace test)

311 draft (2002)

Ready anaerobic biodegradability: gas production from diluted anaerobic sewage sludge

ISO 7827 – 1994

Water quality − Evaluation in an aqueous medium of the ‘ultimate’ aerobic biodegradability of organic compounds − Method by analysis of DOC

ISO 9439 – 1999

Water quality − Evaluation of ‘ultimate’ aerobic biodegradability of organic compounds in an aqueous medium – CO2 evolution test

ISO 9408 – 1999

Water quality − Evaluation of ‘ultimate’ aerobic biodegradability of organic compounds in an aqueous medium by determination of oxygen

ISO 9887 – 1992

Water quality − Evaluation of the aerobic biodegradability of organic compounds in an aqueous medium − SCAS method

ISO 9888 – 1999

Water quality − Evaluation of the ultimate aerobic biodegradability of organic compounds in an aqueous medium − Static test (Zahn−Wellens Method)

ISO 10634 – 1995

Water quality − Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium

ISO 10707 – 1994

Water quality − Evaluation in an aqueous medium of the ‘ultimate’ aerobic biodegradability of organic compounds − Method by analysis of BOD (Closed- bottle test)

ISO 10708 – 1997

Water quality − Evaluation in an aqueous medium of the ultimate aerobic biodegradability of organic compounds − Determination of biochemical oxygen demand in a two-phase closed-bottle test

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 Rolf-Joachim Müller

Table 2.1 (continued) OECD Guidelines [54] ISO 11733 – 1995

Water quality Evaluation of the elimination and biodegradability of organic compounds in an aqueous medium − Activated sludge simulation test

ISO 11734 – 1995

Water quality − Evaluation of the ‘ultimate’ anaerobic biodegradability of organic compounds in digested sludge − Method by measurement of the biogas production

ISO 14592-1 – 2002

Water quality − Evaluation of the aerobic biodegradability of organic compounds at low concentrations − Part 1: Shake- flask batch test with surface water or surface water/sediment suspension

ISO 14592-2 – 2002

Water quality − Evaluation of the aerobic biodegradability of organic compounds at low concentrations − Part 2: Continuous flow river model with attached biomass

ISO 14593 – 1999

Water quality − Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium − Method by analysis of inorganic carbon in sealed vessels (CO2 headspace test)

ISO/TR 15462 – 1997

Water quality − Selection of tests for biodegradability

ISO 16221 – 2001

Water quality − Guidance for determination of biodegradability in the marine environment

EN ISO 7827 – 1995

Water quality − Evaluation in an aqueous medium of the ‘ultimate’ aerobic biodegradability of organic compounds − Method by analysis of DOC

EN ISO 9439 – 2000

Water quality − Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium − CO2 evolution test

EN ISO 9408 – 1999

Water quality − Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium by determination of oxygen demand in a closed respirometer

EN ISO 9887 – 1994

Water quality − Evaluation of the aerobic biodegradability of organic compounds in an aqueous medium − SCAS method

EN ISO 9888 – 1999

Water quality − Evaluation of ultimate aerobic biodegradability of organic compounds in aqueous medium − Static test (Zahn− Wellens Method)

EN ISO 10634 – 1995

Water quality − Guidance for the preparation and treatment of poorly water–soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium

EN ISO 10707 – 1997

Water quality − Evaluation in an aqueous medium of the ‘ultimate’ aerobic biodegradability of organic compounds − Method by analysis of BOD (Closed- bottle test)

EN ISO 11733 – 1998

Water quality − Evaluation of the elimination and biodegradability of organic compounds in an aqueous medium − Activated sludge simulation test

EN ISO 11734 – 1998

Water quality − Evaluation of the ‘ultimate’ anaerobic biodegradability of organic compounds in digested sludge − Method by measurement of the biogas production

2 Biodegradation behaviour of polymers in liquid environments 

 39

Table 2.1 (continued) OECD Guidelines [54] DIN 38412 – 26 – 1994 German standard methods for the examination of water, wastewater and sludge; bioassays (Group L); surfactant biodegradation and elimination test for simulation of municipal wastewater treatment plants (L26) EN: European Norms DIN: Deutsche Institüt für Norms DOC: Dissolved organic carbon MITI: Ministry of International Trade and Industry (Japan) SCAS: Semicontinuous activated sludge

At the beginning of the 1990s the first attempts were made to establish standard testing conditions to measure and evaluate the biodegradation of nonwater-soluble polymeric materials and the first standards, often modifications of existing standards to assess the biodegradability of low MW substances, were published by the American Society for Testing and Materials (ASTM). At the outset, environments where plastics were supposed to be degraded focused on the marine environment and landfills, however, with the upcoming discussion about composting as an alternative method of treating biodegradable plastic waste, standardisation then focused on this topic. Nowadays, the degradation of plastics in soil is of major interest and standardisation bodies are now starting to establish evaluation schemes for this environment. For biodegradation processes in liquid environments the standards established up to now can be structured as follows: – Standards for laboratory test methods determining the intrinsic biodegradability of plastics. – Standards evaluating the biodegradability of plastics in a marine environment. – Standards evaluating the biodegradability of plastics in a wastewater treatment (activated sludge). – Standards evaluating the biodegradability of plastics in anaerobic sludges. A list of currently published standards is given in Table 2.2. While most of these standards are predominantly focused on how to measure biodegradation in a specific environment, some standards represent evaluation schemes, especially for biodegradable plastics in composting processes (ASTM D6002-96 [54] and EN 13432 [55]) and also provide limit values and threshold levels for the evaluation of biodegradability. Generally, all test schemes reflect the problems in measuring biodegradation processes in complex environments such as in biowaste during a composting process and thus, they recommended to first measure the intrinsic biodegradability of a plastic in defined laboratory tests and then to evaluate the disintegration behaviour under real composting conditions. In all schemes, laboratory tests based on liquid media are acceptable to

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 Rolf-Joachim Müller

prove biodegradability. However, the requested threshold levels of 90% degradation (transformed carbon with respect to the carbon introduced), fixed in the evaluation schemes, requires the establishment of a carbon balance when using aqueous degradation tests, also including the biomass formed, into the calculation of the degree of polymer degradation, since in most cases, more than 10% of the carbon from the polymer will be used to form new biomass instead of being transformed into CO2. Table 2.2: National and international standards for biodegradable plastics. ASTM D5210-92 (2000) Standard test method for determining the anaerobic biodegradation of plastic materials in the presence of municipal sewage sludge ASTM D5271-02

Standard test method for determining the aerobic biodegradation of plastic materials in an activated-sludge-wastewater-treatment system

ASTM D5511-02

Standard test method for determining anaerobic biodegradation of plastic materials under high solids anaerobic-digestion conditions

ASTM D6340-98

Standard test methods for determining aerobic biodegradation of radiolabelled plastic materials in an aqueous or compost environment

ASTM D6691-01

Standard test method for determining aerobic biodegradation of plastic materials in the marine environment by a defined microbial consortium

ASTM D6692-01

Standard test method for determining the biodegradability of radiolabelled polymeric plastic materials in seawater

EN 13432 – 2000

Packaging − Requirements for packaging recoverable through composting and biodegradation − Test scheme and evaluation criteria for the final acceptance of packaging

DIN V 54900 – 1998

Testing of the Compostability of Plastics

ISO 14851 – 1999

Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous medium − Method by measuring the oxygen demand in a closed respirometer

ISO 14852 –1999

Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous medium − Method by analysis of evolved CO2

ISO/DIS 14853 1999

Determination of the ultimate anaerobic biodegradability of plastic materials in an aqueous system − Method by measurement of biogas production

ISO/DIS 15985 – 1999

Plastics − Determination of the ultimate anaerobic biodegradability and disintegration under high solids anaerobic-digestion conditions − Method by analysis of released biogas

ISO/DIS 17556 – 2001

Plastics − Determination of the ultimate aerobic biodegradability in soil by measuring the oxygen demand in a respirometer or the amount of CO2 evolved

JIS K6950 – 2000

Plastics − Testing method for aerobic biodegradability by activated sludge

JIS K6951 – 2000

Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous medium − Method by analysis of evolved CO2

DIS: Draft international standard JIS: Japanese Institute for Standards Organisation

2 Biodegradation behaviour of polymers in liquid environments 

 41

2.6 Summary Generally, any biological process is connected to the presence of water and thus, it could be stated that in principle all biological degradation takes place in a ‘liquid environment’. However, in a macroliquid environment such as in lakes, rivers, salt water or in special nutrient media in laboratory tests, the biodegradation of plastics differs significantly from that in soil or compost. This is connected on the one hand to differences in the type and concentration of the microbial population, but also diffusion characteristics of enzymes or intermediates will play a role. Compared with degradation in compost or soil, the current interest in investigations of (nonsoluble) plastics in aqueous environments is only limited. This is caused by the preferential application of biodegradable plastics as packaging materials (which are degraded in compost) or in agriculture (where degradation takes place in soil). However, in laboratory tests evaluating the intrinsic biodegradability of plastics, tests in liquid media play an important role, since such test systems are comparable, defined and reproducible due to the lack of a multiphase system.

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Y. Doi, K-I. Kasuya, H. Abe, N. Koyama, S-I. Ishiwatari, K. Takagi and Y. Yoshida, Polymer Degradation and Stability, 1996, 51, 3, 281. Y. Tokiwa, T. Ando, T. Suzuki and T. Takeda, Proceedings of the ACS Division of Polymeric Materials: Science and Engineering, 1990, 62, 988. E. Marten in Korrelationen Zwischen der Struktur und der Enzymatischen Hydrolyse von Polyestern, Technical University Braunschweig, Braunschweig, Germany, 2000. [PhD Thesis] {In German} http://opus.tu-bs.de/opus/volltexte/2000/136. K-I. Kasuya, K-I. Takagi, S-I. Ishiwatari, Y. Yoshida and Y. Doi, Polymer Degradation and Stability, 1998, 59, 1−3, 327. A.L. Allen, J. Mayer, R. Stote and D.L. Kaplan, Journal of Environmental Polymer Degradation, 1994, 2, 4, 237. K.E. Gonda, D. Jendrossek and H.P. Molitoris, Hydrobiologia, 2000, 426, 1, 173. Y. Honda and Z. Osawa, Polymer, 2002, 76, 2, 321. K. Budwill, P.M. Fedorak and W.J. Page, Applied and Environmental Microbiology, 1992, 58, 4, 1398. K. Budwill, P.M. Fedorak and W.J. Page, Journal of Environmental Polymer Degradation, 1996, 4, 2, 91. A. Reischwitz, E. Stoppok and K. Buchholz, Biodegradation, 1998, 8, 5, 313. J. Urmeneta, J. Mas-Castella and R. Guerrero, Applied and Environmental Microbiology, 1995, 61, 5, 2046. P.K. Shin, M.H. Kim and J.M. Kim, Journal of Environmental Polymer Degradation, 1997, 5, 1, 33. S. Gartiser, M. Wallrabenstein and G. Stiene, Journal of Environmental Polymer Degradation, 1998, 6, 3, 159. C. Rivard, L. Moens, K. Roberts, J. Brigham and S. Kelley, Enzyme and Microbial Technology, 1995, 17, 9, 848. D.M. Abou-Zeid in Anaerobic Biodegradation of Natural and Synthetic Polyesters, Technical University Braunschweig, Braunschweig, Germany, 2000. [PhD thesis] {In English} http://opus.tu-bs.de/opus/volltexte/2001/246 M. Itävaara and M. Vikman, Journal of Environmental Polymer Degradation, 1996, 4, 1, 29. U. Witt, T. Einig, M. Yamamoto, I. Kleeberg, W-D. Deckwer and R-J. Müller, Chemosphere, 2001, 44, 2, 289. P. Püchner, W-R. Müller and D. Bardtke, Journal of Environmental Polymer Degradation, 1995, 3, 3, 133. J. Hoffmann, I. Reznicekova, S. Vanökovä and J. Kupcec, International Biodeterioration and Biodegradation, 1997, 39, 4, 327. U. Pagga, A. Schäfer, R-J. Müller and M. Pantke, Chemosphere, 2001, 42, 3, 319. A. Calmon, L. Dusserre-Bresson, V. Bellon-Maurel, P. Feuilloley and F. Silvestre, Chemosphere, 2000, 41, 5, 645. W-R. Müller, LaborPraxis, 1999, September, 94. R. Solaro, A. Corti and E. Chiellini, Journal of Environmental Polymer Degradation, 1998, 6, 4, 203. M. Itävaara and M. Vikman, Chemosphere, 1995, 31, 11/12, 4359. K. Richterich, H. Berger and J. Steber, Chemosphere, 1998, 37, 2, 319. F. Degli-Innocenti in Labelling Biodegradable Products, Unpublished Data of EC- Project SMT4-CT97-2167. ISO 14851, Determination of the Ultimate Aerobic Biodegradability of Plastic Materials in an Aqueous Medium − Method by Measuring the Oxygen Demand in a Closed Respirometer, International Organization for Standardization, Genève, Switzerland, 1999.

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[56]

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ISO 14852, Determination of the Ultimate Aerobic Biodegradability of Plastic Materials in an Aqueous Medium − Method by Analysis of Evolved Carbon Dioxide, International Organization for Standardization, Genève, Switzerland, 1999. M. van der Zee, J.H. Stoutjesdijk, H. Feil and J. Feijen, Chemosphere, 1998, 36, 3, 461. P. Kuenemann, A. De Morsier and P. Vasseur, Chemosphere, 1992, 24, 1, 63. S. Urstadt, J. Augusta, R-J. Müller and W-D. Deckwer, Journal of Environmental Polymer Degradation, 1995, 3, 3, 121. A. Serandio and P. Püchner, Gas, Wasser Abwasser, 1993, 134, 8, 482. B. Spitzer, C. Mende, M. Menner and T. Luck, Journal of Environmental Polymer Degradation, 1996, 4, 3, 157. H.R. Stapert in Environmentally Degradable Polyesters, Poly(ester-amide)s and Poly(esterurethane)s, University of Twente, Enschede, The Netherlands, 1998. [PhD Thesis] U. Pagga, Chemosphere, 1997, 35, 12, 2953. J. Augusta, R-J. Müller and H. Widdecke, Chemie Ingenieur Technik, 1992, 64, 5, 410. ASTM D6002-96 (2002)e1, Standard Guide for Assessing the Compostability of Environmentally Degradable Plastics, American Society for Testing and Materials, PA, USA, 2001. EN 13432, Packaging − Requirements for Packaging Recoverable Through Composting and Biodegradation − Test Scheme and Evaluation Criteria for the Final Acceptance of Packaging, European Committee for Standardization, Brussels, Belgium, 2000. OECD Guidelines for Testing of Chemicals, Organisation for Economic Co-operation and Development, Paris, France, 1993.

Fernanda Farachi, Giulia Bettas Ardisson and Francesco Degli Innocenti

3 Environmental fate and ecotoxicity assessment of biodegradable polymers 3.1 Introduction Over the last 20 years, biodegradable polymers have been developed and designed for applications that gain an important advantage from biodegradation. Biodegradation is a naturally occurring phenomenon performed by saprophytic microorganisms that live by exploiting lifeless organic matter, which is converted into microbial energy and biological mass (biomass) with the release of inorganic compounds as by-products. The final effect of biodegradation is the removal of dead organic matter from the environment and the restoring of inorganic substances which are used by plants as nutrients. Biodegradation is exploited during waste management in order to convert biowaste (kitchen, food, garden waste) into compost for soil fertilisation by means of organic recycling. Another form of organic recycling is performed in anaerobic digesters leading to biogas and digestate which is then converted into compost. Biodegradable polymers are designed to be recycled together with biowaste in both composting plants and anaerobic digesters. The suitability of packaging to undergo organic recycling is regulated by European Norm (EN) 13432 [1], whereas the compostability of plastics is regulated by EN 14995 [2]. The compostability of plastics is also defined by the International Organization for Standardization (ISO) 17088 [3] and by the American Society for Testing and Materials (ASTM) D6400 standard [4], the latter being issued by ASTM International − formerly known as ASTM. Biodegradable polymers are also used in agricultural plastic items which are designed to be left and biodegraded in the field after their use. The biodegradation of plastics in soil is defined by Norme Française (NF) (French standard), U 52–001 [5], which regulates mulch films used in agriculture and horticulture, and by two Ente Nationale Italiano di Unificazione (Italian standards) UNI 11462 [6] and UNI 11495 [7]. Additionally, a certification scheme for biodegradation in soil has been developed by the Certification Institute Vinçotte (Brussels, Belgium) [8], in order to respond to the market need for a workable definition of plastics which are biodegradable in soil. Biowaste can also be home composted in smaller installations. Biodegradability requirements for plastics under home composting conditions are defined by Italian standards UNI 11183 [9] and UNI 11355 [10], which regulate the home composting of biodegradable plastic materials and articles. All of the above standards verify both the inherent biodegradability in a specific environment and the safe environmental impact of the plastic material. https://doi.org/10.1515/9781501511967-003

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 Fernanda Farachi, Giulia Bettas Ardisson and Francesco Degli Innocenti

Due to the growing volumes of biodegradable polymers and plastics, interest in their environmental safety is increasing. A recurring question concerns the environmental impact of substances released during polymer biodegradation and composting which could subsequently be spread into the environment during fertilisation with compost, or directly diffused during their biodegradation in soil. The current approaches to the standardisation of composting and soil biodegradation are considered satisfactory and suitable for the assessment of the environmental safety of biodegradable plastics. The reason lies in the very high mineralisation threshold levels (90%) required by these standards, which can be considered as an indicator of total biodegradation and no remaining residues. Additionally, the ecotoxicity tests applied to the compost or soil, which result after the biodegradation of the plastic, make sure that products of biodegradation do not affect any agricultural application. In this chapter we are going to explore whether a deeper investigation of the environmental fate and ecotoxicity of substances, released during the biodegradation of polymers and plastics, is possible by applying the principles of the European Regulation 1907/2006, called REACH: Registration, Evaluation, Authorisation and Restriction of Chemicals [11]. REACH does not focus on polymers; instead it prescribes a comprehensive collection of information and investigations on single substances, with the aim of characterising all the hazardous properties concerning human health and the environment, to outline the appropriate exposure scenarios, which include the usage and disposal conditions of the substances, in order to determine their safe management and disposal. Regarding environmental properties, REACH prescribes the gathering of information concerning the environmental partitioning and ecotoxicity of the substances. With this concept in mind, the idea explored herein is to adapt and apply the environmental and ecotoxicological approach of REACH to characterise the intermediate substances that are released during the biodegradation of polymers and plastics, in order to offer an additional assessment tool to the already developed testing schemes.

3.2 End of life scenarios of biodegradable polymers 3.2.1 Biodegradation end products In nature, chemical elements exist both as inorganic compounds (e.g., CO2, NH3 and PO43-) and as components of organic molecules (e.g., sugars, proteins, phospholipids).

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The chemical conversion between organic and inorganic status occurs in ‘exchange pools’ that belong to complex chemical cycles called ‘biogeochemical cycles’, which involve both biotic pools of exchange and abiotic reservoirs. Hereafter, organic and inorganic carbon exchanges are briefly described in order to give a quick overview of the principles that are involved and the role of biodegradation. Plants are able to synthesise organic molecules starting from inorganic substances. This is characteristic of autotrophic organisms which are able to uptake and assimilate inorganic compounds and convert inorganic elements into organic molecules. For example, Equation 3.1 shows the synthesis of glucose (C6H12O6), a core molecule in plant metabolism, starting from atmospheric CO2 and soil H2O: 6CO2 + 6H2O → C6H12O6 + 6O2

(3.1)

Heterotrophic aerobic organisms perform the reverse reaction, cellular respiration. More specifically, the conversion performed by microorganisms of lifeless organic matter into inorganic compounds is called biodegradation. For example, during aerobic biodegradation glucose molecules are oxidised back into those inorganic compounds originally used by plants to synthesise glucose. This process is also known as mineralisation because it ultimately results in the conversion of organic molecules into inorganic compounds and minerals. As shown in Equation 3.2 the final products of aerobic biodegradation are CO2 and H2O: C6H12O6 + 6O2 → 6CO2 + 6H2O

(3.2)

Similarly, the biodegradation process affects more complex organic molecules such as natural polymers (starch, cellulose and so on) and some man-made polymers. This is indeed the case of biodegradable polymers used for the production of plastic articles that have been designed with the aim of being biodegraded in the soil or in composting plants. Equation 3.3 represents the biodegradation of a complex polymeric molecule where part of the original carbon (Cpolymer) is mineralised (CO2), part is used by microorganisms for their own growth and reproduction (Cbiomass), while another part of the initial carbon can still be present as polymeric residue (Cresidue) [12]:    Cpolymer + O2 → CO2 + H2O + Cresidue + Cbiomass

(3.3)

Under anaerobic conditions, other types of microorganisms are involved in the biodegradation process, leading to the formation of different waste products: CH4 and CO2. Anaerobic biodegradation is described by Equation 3.4: CnH2nOn + CnH2nOn → nCH4 + nCO2

(3.4)

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3.2.2 Biodegradation during organic recycling Organic recycling is the biological treatment of biowaste which leads to the production of compost, a stabilised organic matter that is used in agriculture as a soil fertiliser. Organic recycling is an industrial and standardised biotechnology characterised by a multistep biodegradation process, which is carried out in aerobic composting plants or anaerobic digesters.

3.2.2.1 Industrial composting This technology is used to treat biowaste generated by household, industrial and agricultural activities, parks and gardens maintenance, and sewage treatment. Composting is a recycling technology for organic matter which is recycled into compost. Postuse biodegradable plastic articles (such as disposable cutlery and packaging) and biodegradable bags, liners and similar items used to collect biowaste have been designed to be a suitable feedstock for industrial composting. In industrial composting plants, biowaste is gathered into piles and various factors combine to create an ideal habitat for microorganisms to enhance the composting process: temperature, moisture and pH undergo fixed variations during the process. O2 provision is guaranteed during the process through aeration. The large mass of organic matter allows an intense biodegradation activity. Through this process, microorganisms obtain energy and the chemical elements necessary for their own life, growth and reproduction. Heat produced during microbial metabolism contributes to a higher pile temperature and as the temperature of the mass rises, faster reactions occur and the process of biodegradation is accelerated. A temperature increase influences microbial activity and promotes a switch from mesophilic to thermophilic microorganisms. Depending on the temperature, microbial populations and the mechanisms of biodegradation, four stages take place during industrial composting: mesophilic, thermophilic, cooling down and maturation. Completion of these four stages takes several months and leads to the formation of mature humic compost as described in the following 4 steps [11]: 1. Industrial composting starts at ambient temperature. The mesophilic stage is characterised by a 15−45 °C temperature range at which heterotrophic aerobic bacteria decompose most of the simplest organic substances into water and CO2 along with the release of heat. As the microorganisms rapidly multiply, the temperature increases and the pH becomes acidic. Under these conditions the microbial population switches from mesophilic bacteria to thermophilic microorganisms. 2. During the thermophilic stage, the temperature rises continuously and can reach a peak of 70 °C, while the pH progressively turns alkaline. This phase is

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characterised by a mixed population of heterotrophic and autotrophic microorganisms: bacteria and fungi. High temperatures promote water evaporation and the death of pathogens; hence, the composting mass becomes dry and sanitised. 3. Once the temperature peak is exhausted, the cooling down stage starts: when the temperature falls below 60 °C thermophilic fungi reinvade the mass. Ambient temperature is slowly achieved and the process enters the maturation stage. 4. The final stage is characterised by microorganism competition, caused by a lack of organic nutrients, where antibiotics are produced and lignin residues of the original waste react with proteins from dead microorganisms to form humic acids. Maturation lasts several months, during which the compost progressively stabilises, a necessity for its use in agriculture. Once compost is mature (i.e., stabilised) it can be used as a soil fertiliser.

3.2.2.2 Home composting Besides ‘industrial’ composting, ‘home’ composting is also practised by individuals on a voluntary basis. Home composting applies to a smaller amount of biowaste, generated during household activities or garden maintenance and is undertaken in a much more variable fashion compared with industrial composting. Therefore, home composting can lead to different results as several factors affect the process (e.g., ability of the practiser and efficiency of the composter, temperature, moisture, pH and microorganisms). In general, home composting is slower than industrial composting because the small dimensions of the composting mass and the installation, generally, do not enable the achievement of high temperature. Hence, mature compost can take more than one year to be produced.

3.2.2.3 Anaerobic digestion During anaerobic digestion, biowaste is degraded by a bacterial population in the absence of O2 with the production of biogas (CH4 and CO2) and digestate, without practically any release of exothermic heat. Most commercial anaerobic digestion systems perform a two-step process: the first step consists of anaerobic fermentation, followed by a second step of aerobic composting. This is due to the fact that often the anaerobic fermentation output (i.e., digestate) is not fully stabilised and needs to be aerobically composted in order to reduce the residual biological activity, and to obtain complete maturity and stabilisation of the compost end product. Furthermore, the agronomic value of digestate is greatly reduced in comparison with compost, a matter rich in humic substances and beneficial microorganisms.

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3.2.3 Biodegradation in soil Several applications based on biodegradable plastics that end up directly in soil after their useful life are under development and experiencing rapid market growth. Examples are mulch films, strings, hooks and clips, slow release pheromone dispensers and drip irrigation pipes. Furthermore, mature industrial compost, obtained from a feedstock that includes biodegradable polymers, also ends up in soil because of its application as a soil improver. Soil is a heterogeneous matrix influenced by the combination of several climatic and seasonal factors, which firmly control the formation and activity of the microbial population. The nature of soil together with the wide combination of climatic and seasonal factors combine to create several different habitats, in which only selected microorganisms can live and operate. For example, an alkaline-neutral humid soil is colonised mainly by bacteria, whereas fungi prefer to live and operate in an acid dry soil [14]. The main factors influencing the presence of microorganisms and their biodegradation activity in soil are briefly discussed below. 3.2.3.1 Soil texture and structure Soil can be made of different percentages of clay, sand or silt particles that combine to give soil its texture. Clay particles are smaller than sand particles which are smaller than silt particles. It is difficult to alter the composition of soil particles, whereas it is possible to operate on aggregates that soil particles form to create the soil structure. Soil texture and structure have a strong impact on microbial colony formation: a sandy, dry and well-aerated soil encourages fungal colonisation, whereas a poorly aerated clay compact soil hosts facultative aerobic or microaerophilic bacterial colonies. 3.2.3.2 Water content Soil texture, structure and organic matter content give soil a certain water-holding capacity which has a strong influence on the selection of microorganisms: humidity promotes the formation of bacterial colonies whereas dryness promotes fungal colonies. 3.2.3.3 Organic matter Organic matter represents the primary fuel source for biodegradation; hence, it is the favourite substrate for the formation and growth of microbial colonies. It also acts as a soil buffer (pH regulator) and contributes to soil aeration and humidity, and generally imparts a significantly positive contribution to microbial habitat preservation.

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3.2.3.4 pH Usually microorganisms can only adapt to a specific pH range and are strongly affected by pH fluctuations. Hence, the soil pH value is an important factor which limits microbial colonisation; usually bacteria prefer slightly alkaline-neutral pH, whereas fungi resist acid pH. 3.2.3.5 Temperature Temperature influences microbial presence and activity because it allows the formation of microorganism colonies that can only live at a certain temperature range. Warm temperatures promote the chemical reaction rate of microbial metabolism and enzymatic breakdown of polymers [15]. 3.2.3.6 Oxygen Soil O2 content discriminates between aerobic and anaerobic biodegradation, by promoting the growth of those microorganisms capable of living respectively with or without an O2 source. Usually, the O2 content is inversely proportional to the water and CO2 content [14]. 3.2.3.7 Sunlight Ultraviolet light prevents microbial growth on the soil surface. Hence, biodegradation occurs a few millimetres below the soil surface and is greatest in the first 10 cm where temperature, organic matter content, aeration, moisture and O2 reach optimum conditions for microbial growth and activity. The top 30 cm of the soil are those most affected by ploughing and milling before and after cultivation. Agricultural and horticultural biodegradable plastic applications, such as mulch films, are designed not to be removed after cropping. They remain on or in the soil and through milling and ploughing operations are cut into pieces and mixed with the most biologically active soil layer, thus promoting material biodegradation.

3.3 Investigation into polymer biodegradation Both biodegradable plastics and organic recycling are technologies that raise particular interest due to their contribution to solving the problems relating to plastic and organic waste recovery. By performing organic recycling of postuse biodegrad-

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able plastics together with biowaste, two important goals are achieved: the organic recycling of plastic waste with biowaste and the production of mature compost that can be used as a soil fertiliser. Recent studies carried out by Garaffa and co-workers showed that biodegradable plastic bags offer several advantages when used to collect the organic fraction of municipal waste by improving collection, quality and composting processes [16, 17]. Additionally, several nonbiodegradable plastic articles used in food contact applications, such as disposable tableware, cutlery and food packaging, are not suitable for material recycling after use because they are contaminated with food waste. Biodegradable plastics offer a simpler recycling procedure for such food contact plastic applications, since the item and food residues are both compatible for organic recycling. As mentioned in the introduction, biodegradability can be assessed by following standardised testing schemes: EN 13432 for packaging and EN 14995 for plastics are designed to verify that biodegradable articles effectively have the ability to safely biodegrade in organic recycling facilities; French standard NF U 52–001 and Italian standards UNI 11462 and UNI 11495 have been designed to verify that mulch materials used in agriculture and horticulture are able to safely biodegrade in soil once their functional life is exhausted; Italian standards UNI 11183 and UNI 11355 define requirements and test methods for plastic materials and articles deemed to be suitable for home composting at ambient temperature. All such standards have been based upon common evaluation schemes and principles hence, they have a common approach. Needless to say, a wide variety of composting plants and cycles, home composting procedures and environments exist. Hence, it has been necessary to establish and fix some parameters (temperature, substrate, pH, microbial population, moisture and so on), in order to obtain reproducible standard laboratory conditions which verify the ability and the potential of different materials to undergo safe biodegradation in the environment where they are allocated for treatment. As a representative example of a standard, the conditions and requirements of EN 13432 and EN 14995 are discussed in Section 3.3.1.

3.3.1 Standard on industrial composting ‘Compostability’ of a plastic article is verified by assessing four characteristics: inherent material biodegradability, disintegrability of the article, absence of negative effects on the composting process and the absence of ecotoxic effects of the compost obtained from a feedstock that includes the plastic. Regarding biodegradation, a 90% mineralisation level, measured as produced CO2 or consumed O2, within 6 months under controlled composting conditions (according to ISO 14855 [18]) is required. Industrial composting is simulated using

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mature compost as the substrate for biodegradation, rather than real biowaste, this is because the background respiration of fresh biowaste would be too high and not compatible with respirometric biodegradation tests. Moreover, fresh biowaste is extremely changeable and heterogeneous which, for standardisation purposes, makes it unsuitable for use as a substrate. Under laboratory conditions, the temperature is kept at a constant value of 58 °C which is representative of the thermophilic conditions found in most industrial composting facilities. Conversely, disintegration is verified in a pilot-scale composting installation using fresh biowaste. In order to quantify the total amount of specimens that did not fragment below 2 mm, a final sieving of whole compost is undertaken. This fraction is considered not disintegrated, while the fraction below 2 mm is considered disintegrated. 90% disintegration of the initial mass within 12 weeks is the pass-level required by both EN 13432 and EN 14995. It can be challenged that disintegration is tested for 3 months while biodegradation is monitored for 6 months. The difference in test duration lays in the following reasons. When assessing disintegration, standards pay more attention to the process, where the ‘time’ factor (the disintegration rate) is clearly very important for reasons of productivity: 90% disintegration in 3 months, guarantees that composting processes (most lasting a minimum of 3 months) are not impaired by nondisintegrated plastic items. On the contrary, when dealing with biodegradation, standards focus on ensuring the final result is reached: 90% mineralisation level within 6 months, as proof of the ability of the plastic to accomplish total biodegradation. Therefore, the biodegradation test focuses on the verification of the inherent and total material mineralisation, whilst considering the conditions and timing compatible with industrial composting. In practice, industrial composting undergoes several different procedures in each composting plant involving shorter or longer composting cycles. Composting cycles can be very short, while the maturation phase usually lasts several months, hence biodegradation could still be in progress when the compost enters the maturation phase. From this perspective, the 6 months observation time given to carry out biodegradation testing is a reasonable time span that, in a real context, would cover both active composting and the maturation phase. Moreover, it must be understood that the 90% mineralisation requirement is a very high biodegradation level which practically represents complete biodegradation. In fact, as shown in Equation 3.3, aerobic biodegradation is a metabolic conversion involving two parallel processes: one catabolic, used by living organisms to produce energy with the release of CO2 and water; the other, anabolic, involving complex chemical cycles that transform starting substances into biomass, which are necessary for the development and reproduction of the organisms. Therefore, the complete biodegradation and assimilation of a substance, does not necessarily imply complete mineralisation. Overall growth yields (grams dry weight biomass/grams substrate)

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have been found to span from 0.36 (Candida utilis growing on acetic acid) to 0.84 (bacterial species growing on n-pentane), as shown by Pirt [19]. This means that in the short-medium term, a significant fraction (50% as a rule of thumb) of the total carbon present in the initial organic matter is used to build new biomass and the remainder is converted into CO2. Laboratory respirometric test methods estimate biodegradation by measuring the release of CO2 and do not investigate biomass formation. Therefore, in the short-medium term, total biodegradation would be underestimated because the assimilated carbon would not be counted as biodegraded. After consumption of the test substance, a substantial starvation is imposed on the microbial population, and over a longer period, the newly formed biomass is also turned into CO2 hence, after 6 months testing most of the newly formed biomass will be catabolised into CO2. A final respirometric prolonged plateau phase is the indication that the microbial population has ‘burned’ any available substrate and no more mineralisation is expected in the short term. In conclusion and regardless of the nature and composition of the material, EN 13432 and EN 14995 allow for the quantifiable, reproducible, comparable, repeatable, and economically and practically feasible laboratory verification of the biodegradability and disintegrability of plastics or packaging. A successful conclusion of such an assessment can be considered as a general guarantee of compatibility with any industrial organic recycling technology. Regarding ecotoxicity, such standards pay attention to the initial ingredients of the material by prescribing some essential requirements on the starting composition, i.e., very low heavy metals content and a limited use of substances with unknown biodegradability. The latter can be used at a concentration in the material below 1% by weight per substance, on condition that the overall sum of such substances is below 5%. In addition, similarly and more severely, NF U 52–001 also requires the absence of specific classes of chemicals in the polymeric composition, i.e., polycyclic aromatic hydrocarbons and other persistent pollutants. The effects of any substance released during biodegradation are indirectly investigated by observing that biodegradable materials do not negatively influence the biodegradation process and by measuring the quality parameters of the resulting compost, in view of its application as a fertiliser. Additionally, ecotoxicity is assessed through bioassays performed on the compost (or soil) obtained after disintegration of the compostable article. Bioassays are a quick, reliable and economic instrument that allow the verification of the ecotoxicological effects of the overall degraded material, and a rapid comparison among different material formulations. Bioassays are a useful instrument for regulatory purposes and will be discussed in more detail in Chapter 4. Years of familiarisation with the aforementioned standards confirm that their purposes are met, and that they are a valid tool to assure the safe compostability of materials and articles, and the production of compost which does not have any toxic or side effects when used as a soil fertiliser.

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Though fulfilment of the above standards demonstrates the suitability of biodegradable polymers and plastics to undergo safe biodegradation and composting, further reassurance on their environmental safety is progressively requested due to the higher volumes that are expected to be marketed in the near future by the biodegradable plastic industry. With interest focused on the substances that could be released during biodegradation in soil, or might reach soil when compost is used as a fertiliser, for the remainder of this chapter such substances will be named ‘biodegradation intermediates’. Knowledge of the environmental fate and ecotoxicological profile of intermediates released during biodegradation would obviously enable a better characterisation of the environmental behaviour of the overall biodegradable polymer (or plastic), and would confirm and strengthen any conclusion on the absence of ecotoxicological effects, as already inferred from the bioassays prescribed by the aforementioned standards. To this purpose, a substance-by-substance approach that applies the REACH environmental and ecotoxicological scheme is hereinafter proposed as a deeper method of investigation to characterise the intermediates released during polymer biodegradation.

3.3.2 Identification of the intermediates of polymer biodegradation Biodegradable polymers are placed in soil or composting plants as solid materials. As such they are not available to microbial intracellular enzymes that mineralise organic molecules into inorganic compounds. The surface of the item is the interface between the plastic solid phase and the aqueous liquid phase, where microorganisms are present. In order to uptake large organic molecules from the outside, microorganisms need to reduce the polymer’s molecular length. Therefore, an earlier event takes place: microorganisms form colonies on the surface of the plastic article and produce extracellular enzymes that depolymerise the chain [15]. Released oligomers, dimers, monomers and derivatives can be assimilated by microorganisms: these substances subsequently undergo intracellular oxidation, before eventually being converted into inorganic compounds (i.e., CO2 and water). In the context of the current work, such products of extracellular depolymerisation are the biodegradation intermediates to be taken into account for further ecotoxicological investigation. In nature, extracellular enzymes are secreted to depolymerise natural polymers such as cellulose, and proteins, and complex substances such as triglycerides of plant oils. In the area of biodegradable polymers and plastics, the enzymes involved in this first step of biodegradation have been widely investigated and their mechanisms are well characterised [20].

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The success of extracellular polymer biodegradation primarily depends on the polymeric structure. Synthetic polymers can be grouped according to their backbone linkages; these are related to the functional groups of the starting monomers which react to form the polymeric chain. The groupings based on the polymer backbone bonds are: – Alkane bond is typical of polyolefins, a vast family of polymers that include polyethylene, polypropylene, polystyrene, polyvinyl chloride and many others. These polymers are derived from the reaction of starting alkene monomers and are not biodegradable. – Ester bond-based polymers, called polyesters, include several biodegradable and nonbiodegradable polymers such as polyethylene terephthalate (PET), polybutylene terephthalate, polybutylene succinate, polycaprolactone, polylactic acid, polyhydroxyalcanoate and aromatic-aliphatic polyesters. They are derived from starting monomers such as dialcohol, diacid and/or their esters (like methyl esters), and hydroxyl acids that are monomers with both an alcoholic and a carboxylic functional group. All such monomers react to form ester bonds with the release of water (or methanol if the starting monomers are the methylic esters of diacids). – Amide bond-based polymers, called polyamide (PA), have a peptide-like bond that is typical of proteins. For example, nylon is a popular family of PA and includes, among many others, Nylon 6,6 and Nylon 6,10. Another example of PA is polyglutamic acid, which is derived from the reaction of α-amino and γ-carboxyl terminations of glutamate amino acid monomers with the release of water. – Urethane bond-based polymers, called polyurethane (PU), include a wide family of polymers obtained from the reaction of starting monomers and/or prepolymers containing alcoholic and isocyanate functional groups: typically, polyesterspolyols (polyesters with alcoholic termination) and diisocyanates are reacted to obtain PU. Final products often consist of reticulated polymers rather than linear ones, depending on the final use and desired properties. – Ether bond-based polymers, called polyether, such as polyethylene glycol and polypropylene glycol, are produced by the reaction of starting monomers mainly consisting of cyclic ethers or glycols. Depending on the final polymeric structure and backbone linkages, extracellular biodegradation proceeds via different mechanisms [21]. Hydrolytic microbial enzymes recognise and hydrolyse mainly amide and ester bonds, releasing the starting monomers and oligomers of the polymer [21, 22]. Polyether and polyolefins need to be oxidised in order to be depolymerised. Polyether oxidation occurs on the ether bonds, whereas alkane oxidation occurs randomly in the polymer chain leading to the release of several intermediates [21]. The great majority of industrial biodegradable polymers and plastics developed in recent decades are polyesters. Not all polyesters are biodegradable: the

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biodegradation rate is influenced by the percentage and distribution of aromatic monomers in the polymer chain [23]. For this reason aliphatic-aromatic copolyesters, that combine biodegradability with enhanced mechanical properties, have been developed. In addition, natural polysaccharides, such as starch and cellulose, unmodified or partially modified, are widely enrolled in the development of biodegradable plastic materials. Biodegradable PU are also under development. However, as demonstrated by Degli Innocenti and co-workers [24], attention should be paid to the initial formulation because biodegradation can lead to the formation of toxic substances, such as diisocyanates and diamines: such substances do not necessarily correspond to starting monomers and their formation depends on the side of the urethane bond that is hydrolysed: amino or carboxyl. Polyolefins and aromatic polyesters, such as PET, have been developed with the aim of being resistant to any biotic or abiotic factor. Hence they are particularly recalcitrant to biodegradation, which nevertheless occurs at a negligible rate [21]. Recently, additives used in polyolefins and aromatic polyesters, such as PET, are reported to make them photo and thermodegradable, hence they are able to fragment and degrade. These additives are commercially known as ‘oxo-biodegradable’. The degradation mechanism is reported to be oxidation which gives rise to a vast class of intermediates. As mentioned above, the microbial extracellular depolymerising mechanism of polyester is hydrolytic and proceeds layer after layer from the surface to the core of the plastic article releasing the monomers, dimers and oligomers which can be predicted from the polyester backbone. Mueller and co-workers showed that intermediates released during the depolymerisation of polyesters can be identified by gas chromatography-mass spectroscopy (GC-MS) [22]; whereas the progressive release of acid monomers during polyester biodegradation can be followed by titration of the solution [15]. It should be noted that the extraction of biodegradation intermediates, for identification purposes, is complicated by their uptake and biodegradation by microorganisms; in fact the biodegradation rate of monomers is usually much faster than the depolymerisation rate, as shown by Siotto and co- workers [25, 26]. As shown by Mueller and co-workers, a microbial strain must be identified that is able to depolymerise the chain, but not able to biodegrade it; this is the only way that the intermediates of biodegradation can be isolated and identified by GC-MS [22]. As already explained, the intermediates of polyester biodegradation are the monomers and oligomers obtained by the cleavage of ester bonds. Therefore, these are the substances to be considered for environmental fate and ecotoxicity investigation. The initial depolymerisation step must be investigated through the extraction and identification of the released substances if the intermediates to be investigated are not so obvious (for example, if other polymeric classes are involved).

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Biodegradable plastic items often contain other constituents besides the biodegradable polymer such as fillers and additives which are used to obtain specific technological effects, such as plasticisation and stabilisation, or a change, for example, in optical, mechanical or processing properties. These additives can also be considered as ‘intermediates’ because they are expected to be released from the plastic item into the surrounding environment in a similar fashion to that of polymer intermediates released during the first extracellular step of biodegradation. It should be pointed out here that ISO recently modified the standard on the compostability of plastics (ISO 17088) to take this issue into consideration. The current ISO specification requires that constituents present in the plastic formulation in a concentration below 10% shall be tested individually for biodegradation. This is to make sure that minor constituents are also biodegradable, as these may not be detected by just measuring the overall biodegradation, which requires a final 90% level, and that could ‘hide’ some minor recalcitrant substances.

3.4 Environmental fate of biodegradation intermediates Once the extracellular polymer intermediates released during biodegradation have been identified, definition of their fate and behaviour in the environment becomes necessary for the evaluation of their ecotoxicological impact. As explained in the introduction, for the creation of an evaluation scheme suitable for biodegradation intermediates, the European Regulation REACH has been taken as a reference. REACH asks companies to collect data and/or perform tests, in order to prepare a dossier per substance that is produced in or imported into the European Union over 1 tonne/year. The chemical-physical properties, toxicological and ecotoxicological data have to be measured and/or collected. The extent of information to be provided is related to the substance tonnage-band that is produced or imported, and end-points and data must follow a defined sequence which, where appropriate, contemplates the application of scientific waiving rules. The regulatory obligations of REACH are managed by the European Chemicals Agency (ECHA) in Helsinki. With regard to the testing methods proposed for each end-point, most of them refer to the international Organisation for Economic Co-operation and Development (OECD) guidelines suggested by ECHA ‘End-point specific guidance’ [27–29]. However, other recognised international guidelines elaborated by geographical areas other than Europe can also be applied for the determination of single end-points (e.g., harmonised test guideline nr.850 for Ecological Effects developed by the Environmental Protection Agency Office of Chemical Safety and Pollution Prevention). Finally, it should be noted that REACH aims to minimise the efforts made by companies and priority is given to the prevention of animal suffering: through the use of

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published data, ‘read across’ deductions based on substance analogues, applications of validated algorithms and the ‘weight of evidence’ approach, which consists of scientific deductions based on the analysis of several data sources. Companies should not engage in new experiments on animals, rather they are invited to share existing data. Sharing available studies involving vertebrate testing is compulsory and new vertebrate testing may only be performed when no other information or strategies are available. The same approach is suggested to be adopted in the scheme outlined below.

3.4.1 Physico-chemical properties and behaviour of intermediates Biodegradation intermediates can distribute in the environment reaching different compartments (soil, water, air, biotic) depending on their physico-chemical properties. Therefore, the preliminary information that is required in order to apply the proposed scheme consists of the collection of some physical and chemical parameters, such as the physical status, dissociation constant (pKa) and n-octanol/water partition coefficient. These are the starting properties that allow one to understand the partitioning of a substance and its behaviour in the environment, which are critical for the settings and proper interpretation of experiments that might be required by the following scheme. Among the physico-chemical properties, the n-octanol/water partition coefficient (Kow) is key: it measures substance distribution between an n-octanol and water phase (OECD Methods 107 [30], 117 [31], 123 [32], 122 (draft, [33]) and OPPTS 830.7550 [34]). n-Octanol is representative of lipids in organisms and organic carbon in soil. Hence, Kow is used in numerous models and algorithms for the estimation of environmental partitioning, soil adsorption, bioavailability, bioconcentration/ bioaccumulation and also human and ecotoxicity [27]. In general, the higher the Kow is, the higher the likelihood is that the substance will preferably distribute into the biota. Therefore, the Kow may become critical in deciding the direction of the evaluation process. Once the physical and chemical parameters are collected, the evaluation scheme goes on with the estimation of the ‘ready biodegradability’, followed, if required, by the ‘adsorption to soil organic carbon’ and by the ‘Bioconcentration factor’ (BCF).

3.4.1.1 Ready biodegradability Ready biodegradability is a commonly tested property of chemicals because it definitively establishes the substance’s environmental fate. Ready biodegradability is the ability of a substance to undergo ultimate mineralisation into CO2 and H2O in a ‘ready biodegradability’ test carried out according to

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OECD 301 [35]. Said guidelines include 6 test methods (A−F) with specific pass levels to be satisfied within a 10-day window that starts when the substance reaches 10% mineralisation and ends within 28 days of the test period. Biodegradation pass levels are: – 70%, measured as dissolved organic carbon removal (TG 301 A and TG 301 E). – 60%, as theoretical CO2 (TG 301 B). – 60%, as theoretical oxygen demand (TG 301 C, TG 301 D and TG 301 F). The 10-day window does not apply to TG 301 C. With regard to these pass levels, it is important to note that such requirements practically represent complete and ultimate degradation of the test substance, since the remaining 30−40% is assumed to be assimilated as biomass [36]. Moreover, whenever a substance satisfies the ‘ready biodegradability’ test requirements, it can be assumed it will undergo rapid and ultimate biodegradation in any biologically active environment [36]. For such reasons, according to the REACH evaluation scheme once the ‘ready biodegradability’ of a substance is confirmed there is no need for further investigation. If any intermediate does not satisfy the ‘ready biodegradability’ test requirements, or its ‘ready biodegradability’ is not known, in order to prove ultimate biodegradation (i.e., mineralisation) of the substance in the final environment, a soil biodegradation test according to ISO 17556 [37] or other similar tests (such as the ASTM D5988 [36]) can be performed. This approach is used by the Vinçotte ‘OK Biodegradable SOIL’ certification scheme [8] which addresses polymer and plastic biodegradability in applications ending up in soil. According to the Vinçotte scheme, 90% mineralisation of the material (either absolute or relative to microcrystalline cellulose, tested in parallel) must be reached within 2 years. In the context of current work on the biodegradation of intermediates, which are nonpolymeric substances, we would suggest a 90% mineralisation level relative to glucose (tested in parallel as the positive reference substance) measured when the tested material reaches its plateau. In any case the test should not last more than 6 months. Glucose is the proper reference substance for testing monomers because it is the monomer of cellulose, the typical reference material used when testing polymers. In conclusion, the ‘ready biodegradability’ of the intermediates is also the relevant starting information in the proposed evaluation scheme: for biodegradation intermediates fulfilling the ‘ready biodegradability’ criteria, or alternatively the ‘soil biodegradability’ criteria; no further investigation of possible environmental effects is required and the assessment can stop at this point. As previously mentioned, polymers are considered ‘biodegradable’ according to EN 13432 and EN 14995 when they reach 90% mineralisation within 6 months. This mineralisation level can only be reached if the microbial uptake of polymer degradation intermediates followed by intracellular oxidation and mineralisation

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into inorganic end products, such as CO2 and H2O, is extensive and fast. This leads to the consistent conclusion that biodegradable plastics fulfil the requirements of the above standards only if most of the polymer’s intermediates are themselves biodegradable. Moreover, available literature shows that several typical monomers which are used for the production of biodegradable polymers, such as 1,4-butanediol, adipic acid and lactic acid, respond to the requirements of the ‘ready biodegradability’ test [39–41]. Hence, intermediates recalcitrant to ultimate biodegradation are not expected. However, these standards tolerate substances not yet investigated for biodegradability or not biodegradable in an overall concentration of 5%; it is primarily to these substances that attention should be paid. What if the intermediate under examination is neither ‘readily biodegradable’ nor ‘soil biodegradable’? According to the REACH guidelines [28], biodegradation pathways and metabolites of those substances that are not ultimately biodegraded can be predicted using expert systems such as CATABOL and META. These systems show the most likely pathways of biodegradation and possible metabolites, and predict if any metabolite that is recalcitrant to ultimate biodegradation is formed. If any recalcitrant substance (either the intermediate itself or any derived metabolites) is highlighted, the ecotoxicological assessment shall be applied (see Figure 3.1 ). Since only the substances that are neither readily biodegradable, nor ultimately biodegradable in soil are considered for further ecotoxicological assessment, it is suggested to adopt long-term ecotoxicity tests rather than short-term tests, because, once the substance is released in the environment, it (or its metabolites) will presumably remain there for a long period of time. Additionally, in order to choose an adequate testing strategy, the ultimate distribution of the substances, which are recalcitrant to ultimate biodegradation, in various environmental compartments must be investigated. Adsorption to soil organic carbon is measured as the organic carbon/water partition coefficient (Koc), calculated as the distribution among a soil and an aqueous phase, normalised to the organic carbon content of the soil [27]. At this stage, as noted, Kow may become very helpful. In fact, the adsorption to soil organic carbon is more often estimated rather than measured, starting from the Kow: since in Kow measurement n-octanol mimics soil organic carbon, a relation between Koc and Kow exists, and algorithms for the estimation of Koc as a function of Kow have been developed and largely applied. When using Kow for the estimation of Koc, it should be noted that the Kow for ionisable substances is usually measured for the nondissociated form. However, if the substance is normally dissociated at environmental pH, this has to be considered for the evaluation of both Kow and Koc. In such cases, physico-chemical properties, such as pKa, are therefore needed to correctly orient results interpretation and hence any further evaluation of the substance.

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IDENTIFICATION OF INTERMEDIATES

Substance-by-substance approach

Readily biodegradable?

YES

No further assessment

YES

No further assessment

NO

Soil biodegradable?

NO Intermediate of concern

Identification of possible further recalcitrant metabolites of the intermediate

EVALUATION OF ENVIRONMENTAL BEHAVIOUR OF THE RECALCITRANT INTERMEDIATE OR ITS METABOLITES Figure 3.1: Individuation of substances whose environmental behaviour has to be further investigated.

In fact, Koc is particularly valid for nonpolar organic substances, whereas ionic substances are more influenced by electrostatic interactions of the soil mineral matrix rather than the organic carbon content of the soil.

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For the adsorption/desorption test it should be noted that according to REACH: ‘The study does not need to be conducted if: – Based on the physico-chemical properties the substance can be expected to have a low potential for adsorption (e.g., the substance has a low octanol water partition coefficient), or – The substance and its relevant degradation products decompose rapidly.’ As already extensively explained, a rapid decomposition, i.e., ready biodegradation of a substance, is considered as one of the main criteria for low or no concern, thus allowing a simplification of the overall evaluation pathway. If this is not the case, the result of the measured or estimated Koc affects the subsequent evaluation steps. Substances estimated to leave soil (on the basis of the Koc value) and reach the water compartment are qualified for the estimation of the BCF.

3.4.1.2 Bioconcentration factor The BCF measures the potential of a chemical to accumulate in biota that lives in an aquatic environment. BCF does not consider uptake from diet, which is taken into account by another less commonly estimated or measured indicator, known as the ‘Bioaccumulation Factor’. BCF is measured under controlled laboratory conditions and it is the ratio between biota and water concentrations of a given chemical. BCF is important in order to establish if long-term exposures to external low concentrations of a substance can lead to a high internal concentration due to the accumulation of the substance within the organism. BCF can also be estimated and Kow is the starting indicator. A linear correlation between BCF and Kow has been established for nonionic, slowly metabolised substances with log Kow between 1 and 6. Substances with log Kow >6 have a lower BCF than would be predicted by a linear relationship because the low aqueous solubility reduces bioavailability; hence for substances with log Kow >6, BCF decreases and a parabolic/bilinear model can be applied [29]. According to REACH, a bioaccumulation study need not be conducted if: – ‘The substance has a low potential for bioaccumulation (for instance a log Kow ≤3) and/or a low potential to cross biological membranes (i.e., a low BCF, authors’ note), or – Direct and indirect exposure of the aquatic compartment is unlikely.’ It should be noted that the final compartment directly relevant to biodegradable plastics is soil, both for agricultural items and for those items which end up in composting plants. Hence BCF evaluation is only needed if the Koc of a substance indicates that the substance will preferably move to the water compartment.

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In such cases, only if insufficient information is gathered for the substance, or its analogues, from the literature or using predictive algorithms, new experiments may be performed on fish as the preferred animal and according to OECD 305 [42] (or equivalent OPPTS 850.1730 [43] or ASTM E1022-94 [44]).

3.4.2 Ecotoxicological assessment based on the environmental behaviour of the intermediates If the biodegradation intermediate is not ultimately biodegraded, further investigation on its fate is needed: the identification of its biodegradation metabolites and the environmental distribution of substances recalcitrant to ultimate biodegradation is the starting point for any further ecotoxicological evaluation. Environmental partitioning into soil or water on the basis of the Kow or Koc estimation must be predicted or measured in order to understand which compartment will be affected by the recalcitrant substances. If the highlighted compartment is water, and if the substance shows a high potential for bioaccumulation (based on its Kow and its capability to cross biological membranes), BCF estimation is needed in order to assess if the substance could accumulate in living organisms and eventually exert a toxic effect. In other words, the preliminary steps leading to the identification of the preferred partition of the substance into soil, water or biota, allow selection of the most appropriate scenario (terrestrial or aquatic) for the subsequent ecotoxicological assessment, as shown in Figure 3.2 .

3.5 Ecotoxicological assessment of biodegradation intermediates Based on environmental partitioning and the bioconcentration potential, the ecotoxicological impact of the substance is assessed on the relevant compartment (aquatic or terrestrial), for the most relevant period of time (short or long term), by observing acute and/or chronic effects. Acute toxicity tests last for a relatively short period of time and evaluate the ability of a substance to induce mortality or adverse effects in the tested organisms. On the contrary, chronic toxicity tests deliver exposures that are related to the organism’s life cycle and cover stages that are critical for the organism: survival, growth and/or reproduction are typically included and evaluated [28]. In the following sections aquatic and terrestrial, short- and long-term, acute and chronic toxicity tests will be discussed, paying particular attention to the most suitable test(s) for a particular substance profile.

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RECALCITRANT SUBSTANCE

Does the substance have a high Koc?

YES

TERRESTRIAL ECOTOXICITY ASSESSMENT

NO

Does the substance have a high BCF?

YES

NO NO FURTHER STUDY

AQUATIC ECOTOXICITY ASSESSMENT

Figure 3.2: Individuation of substance environmental distribution and most suitable ecotoxicity test.

Again it should be noted that in applying the evaluation scheme, every effort should be taken to minimise the need for testing by collecting available existing information on the substance itself or on similar substances (analogues), and by applying modelling, such as Quantitative Structure-Activity Relationship (QSAR), which are widely endorsed by REACH.

3.5.1 Aquatic toxicity The determination of aquatic toxicity has a double advantage: according to REACH: ‘in the absence of toxicity data for soil organisms, the equilibrium partitioning method (which converts aquatic toxicity values into terrestrial ones by using a partition coefficient, authors’ note) may be applied to assess the hazard to soil organisms.’ Hence, aquatic experiments can be used to evaluate effects both on aquatic and terrestrial organisms. For this reason aquatic toxicity experiments are generally preferred to other experiments and experimental data are already available in the literature for a large number of substances (especially critical ones). For the aquatic toxicity test it should be noted that according to REACH: ‘The study does not need to be conducted if there are mitigating factors indicating that aquatic toxicity is unlikely to occur, for instance, if the substance is highly insoluble in water or the substance is unlikely to cross biological membranes (i.e., it has a low BCF, authors’ note)’.

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The choice to perform a short- or long-term toxicity test mainly depends on the properties of the substance: if the substance is poorly soluble in water, a longterm toxicity test has to be preferred in order to detect effects due to slow substance diffusion [11]. Aquatic organisms used for ecotoxicity tests are: bacteria, algae, crustacea and fish.

3.5.1.1 Bacteria Biodegradability testing does not only provide information about the biodegradability of the substance but it also provides evidence that the substance is not toxic to microorganisms. In fact, if a substance is readily or inherently biodegradable, it can be assumed that it does not affect microbial biodegradation processes or metabolism, hence it is not toxic to biodegrading microorganisms (and microorganisms in general) [28]. If a substance is not readily biodegradable, this does not necessarily imply that the substance is toxic to microorganisms. Therefore, specific experiments on bacteria are carried out to obtain information on substance ecotoxicity. Examples are: the ‘respiratory inhibition test’ (e.g., OECD 209, [45]) and the ‘nitrification inhibition test’ (ISO 9509, [46]), which are performed on a mixed microbial population representative of a sewage treatment plant’s community, ‘cell multiplication inhibition test’ on Pseudomonas putida (ISO 10712, [47]) and ‘flash tests’ on luminescent bacteria Photobacterium phosphoreum [22] and Vibrio fischeri [24], which reduce their luminescence when exposed to toxic substances.

3.5.1.2 Algae Short-term toxicity tests on algae are carried out mainly according to OECD 201 [48] and OPPTS 850.5400 [49] to measure algal growth rate inhibition (chronic effect). The typical test duration for this study is 72 h (96 h is also often reported) and the preferred species are: Pseudokirchneriella subcapitata (previously named Selenastrum capricornutum) Scenedesmus subspicatus and Chlorella vulgaris.

3.5.1.3 Crustacea The crustacean most commonly used is Daphnia magna. It is used to assess both acute (OECD 202 [50] or OPPTS 850.1010 [51]) and chronic (OECD 211, [52]) aquatic toxicity. Acute toxicity tests usually last for 48 h (short term) and evaluate daphnia immobilisation. Chronic toxicity tests generally last 21 days (long term) during which

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daphnias reach maturity and reproduce: observations include time to first brood, number of offspring produced per female (reproduction), growth and survival (lethality).

3.5.1.4 Fish Fish tests include both short-term and long-term toxicity tests on various species. Short-term toxicity tests (OECD 203 [53] and OPPTS 850.1075 [54]) last for 96 h and measure fish mortality (acute effect). The REACH long-term toxicity test prescribes one of the following: ‘Fish, Early-Life Stage (FELS) Toxicity Test’ (OECD 210, [55]), ‘Shortterm Toxicity Test on Embryo and Sac-fry Stages’ (OECD 212, [56]) and ‘Fish, Juvenile Growth Test’ (OECD 215, [57]). Among them, FELS is the most sensitive because it covers the life cycle from egg fertilisation to early growth stages and it is also sensitive to effects caused by bioaccumulation. The other two tests are shorter, less expensive and less sensitive, however they offer an alternative for testing substances with log Kow   FRESHWATER   > MARINE WATER T + fungi + bacteria fungi + bacteria bacteria     dilute bacteria

(5.3)

As for aquatic biodegradation, the first standards for soil biodegradation were developed for simple and pure chemical substances, and published as OECD 304 and ISO 11266. For complex samples such as biodegradable polymers, modifications were needed and

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first introduced by ASTM D5988-96. In 2003, an ISO Norm was also published: ISO 17556 – Plastics − Determination of the ultimate aerobic biode- gradability in soil by measuring the oxygen demand in a respirometer or the amount of carbon dioxide evolved. – Principle: The test item is introduced into soil and incubated at ambient temperature under optimum O2 and moisture conditions. The soil acts at the same time as the carrier matrix, and the source of microorganisms and nutrients. Maximum duration is 6 months, while a typical minimum is 3−4 months. Either O2 consumption or CO2 production is monitored. To improve the reproducibility of the test it is advisable to use a blend of different soils as the texture of the soil is also important.

5.3.5 Aquatic, anaerobic biodegradation tests When O2 is available in a specific environment, this is called an aerobic environment; when no O2 is available, the conditions are anaerobic. Several anaerobic environments exist, especially in places where oxygen is consumed or depleted more rapidly than it is replaced by diffusion. Examples include the bottoms of rivers, canals and lakes with a lot of organic debris, landfills, the rumen of herbivores and so on. Besides these ‘natural’ examples anaerobic conditions also exist in several man-controlled environments such as septic tanks, anaerobic wastewater treatment plants, sludge digesters or solid waste biogasification plants. These anaerobic environments show a high biological activity that can be quite different from aerobic conditions. Through anaerobic biodegradation, organic carbon is converted into biogas, a mixture of CH4 and CO2 (Equation 5.2). As biodegradation can differ, the need was quickly recognised to develop separate anaerobic biodegradation tests. These tests can be further subdivided into two major categories according to moisture content: aquatic tests and high solids or dry tests. The aquatic, anaerobic biodegradation test was first published by the European Centre for Ecotoxicology and Toxicology of Chemicals Technical Report No.28 [10]. Later, more or less the same procedure was adopted as ASTM D5210 in 1992 and ISO 11734 in 1995. Within the field of bioplastics a new version with some minor modifications was published in 2005 as: ISO 14853 – Plastics − Determination of the ultimate anaerobic biodegradability in an aqueous system – Method by measurement of biogas production. – Principle: The test item is placed into an aqueous mineral medium, spiked with inoculum (anaerobic sludge) and incubated under batch conditions at a mesophilic temperature (35 °C). The duration of incubation is 60 days. Biodegradation is measured by following the biogas production (measured by volume displacement or pressure build-up) and the increase of dissolved inorganic carbon in the medium. Depending on the frequency of biogas production, determination of the kinetics of biodegradation can also be established.

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5.3.6 High solids, anaerobic biodegradation tests ISO 14853 is representative for anaerobic systems which are always operated under aquatic conditions (moisture content >95%) and at mesophilic temperatures. Yet, other commercial biogasification systems are being used which work under much drier conditions (moisture content as low as 60%) and also eventually at a higher thermophilic temperature (around 55 °C). These different conditions lead to different biodegradation characteristics and hence the need for a specific test procedure. For example, the lower moisture content results in a much higher concentration of microorganisms and therefore a much higher biodegradation rate. A new biodegradation test method for bioplastics was first developed at ASTM level as ASTM D5511-94. Later, in 2004, the same method was published as: ISO 15985 – Plastics – Evaluation of the ultimate anaerobic biodegradability and disintegration under high solids anaerobic digestion conditions – Method by analysis of released biogas. – Principle: A small amount of test item is added to a large amount of highly active inoculum that has been stabilised prior to the start of the biodegradation test. The inoculum consists of residue obtained directly from a high solids biogasification unit or obtained after the dewatering of anaerobic sludge. Optimal conditions are provided and the mixture is left to ferment batch wise. The volume of biogas produced is measured and used to calculate the percentage of biodegradation based on carbon conversion.

5.3.6.1 Landfill simulation tests Another category of dry, anaerobic biodegradation tests are landfill simulation tests. These tests have primarily been developed in the USA, where biologically active landfills represent a viable waste management option for the future. In Europe however, there is much less interest for biodegradation characteristics in landfills, especially after the adoption in 1999 of the EU landfill directive, which is phasing out the disposal of biodegradable materials in landfills. In landfill simulation tests, the biological activity is much slower compared with the high solids anaerobic digestion test due to the (much) lower concentration of microorganisms. Biodegradation is evaluated through loss of properties after exposure (ASTM D5525-94a) or measurement of biogas production (ASTM D5526-94).

5.3.7 Marine biodegradation tests Another category is marine biodegradation tests. Some promising applications of bioplastics are related to the marine environment (e.g., fishing lines, fishing nets,

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disposables on ships and so on). In particular, the US Navy has been spearheading the developments for many years. Again, the first standards were developed for simple and pure chemical substances, and published as OECD 308. For bioplastics, the first norm was published by ASTM in 1993: ASTM D5437-93 − Standard practice for weathering of plastics under marine floating exposure. – As the title clearly indicates, this norm only describes an exposure procedure in which the loss of properties is monitored, which is no proof of true biodegradation or mineralisation as represented in Equation 5.1. A standard for measurement of true biodegradation was published afterwards: ASTM D6691-09 − Standard test method for determining aerobic biodegradation of plastic materials in the marine environment by a defined microbial consortium or natural seawater inoculum. – This method is very similar to ISO 14852 (see above) in which the inoculum is replaced with seawater or selected marine microorganisms, and a different (more salty) mineral medium. Biodegradation is measured through CO2 production.

5.3.8 Other biodegradation tests Several other tests have also been used to demonstrate biodegradation. Some of these were originally developed to verify resistance against biodeterioration or biofouling. Examples are ASTM G21-90 (resistance to fungi), ASTM G22-76 (resistance to bacteria), AFNOR Norme Française (NF) X.41-601 and AFNOR NF X.41-514. Yet, these tests at most show a susceptibility to biological attack but are totally unsuited to demonstrate a far-reaching, let alone complete biodegradation or mineralisation. A similar judgement can be given on a wide variation of tests which basically consist of some sort of immersion or burial in a given environment (soil, compost, surface water) followed by physical or chemical analyses. The most used analysis is weight loss; yet, weight loss is dependent on disintegration and the possibility of retrieval and therefore, is no proof of a complete mineralisation as mentioned in Equations 5.1 and 5.2. Other analyses include tensile strength, elongation, MW and so on. For a better understanding of degradation mechanisms, tests have been reported in which specific microorganisms or enzymes have been used to evaluate the degradation of polymers or organic compounds. Some of these tests have even been normalised (e.g., ASTM D5247-92, aerobic biodegradability by specific microorganisms). Nonetheless, these tests are mostly used for internal evaluation purposes and only very rarely for outside communication, marketing or certification purposes. Moreover, the extrapolation of results obtained with single, specific species to multispecies, natural environments is very difficult.

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5.4 Norms on disintegration test methods 5.4.1 Introduction As explained already in Section 5.3.1, a distinction needs to be made between biodegradation and disintegration. Whereas the first is a degradation process on a micromolecular level and involves the conversion of organic carbon to CO2 (and CH4 if anaerobic), disintegration is the degradation on a macrophysical level and involves the degradation of a product into small, often invisible particles. Figure 5.6 further illustrates the difference between biodegradation and disintegration. As a consequence, biodegradation is determined by the chemical composition of the sample, while disintegration is also determined by physical dimensions such as thickness and density. Disintegration is especially important if degradation needs to take place in managed processes such as composting, biogasification or wastewater treatment, in which the physical degradation of a product needs to take place in a certain time frame. In natural environments, disintegration is often less critical.

5.4.2 Compost disintegration tests Disintegration has been evaluated in various tests, ranging from simple burial tests to labour-intensive full-scale tests. The first clear test procedure was published in 2002 by ISO: ISO 16929 – Plastics − Determination of the degree of disintegration of plastic materials under defined composting conditions in a pilot-scale test. CO2

DISINTEGRATION

BIODEGRADATION H 2O

Figure 5.6: The difference between disintegration and biodegradation.

The same procedure was also published by CEN in 2003 as: EN 14045 – Packaging Evaluation of the disintegration of packaging materials in practical oriented tests under defined composting conditions. – Principle: The test material is mixed with fresh biowaste in a precise concentration (1%) and introduced into a pilot-scale composting bin. Composting will start spontaneously via the presence of natural, ubiquitous microorganisms in the

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biowaste and will result in a temperature increase. The composting mass is regularly turned and mixed. Several parameters are followed and have to stay within certain limits to guarantee a proper and typical composting process, and good quality compost at the end, e.g., temperature, pH, moisture, gas composition, nitrogen balance and so on. After 12 weeks of composting, the test is terminated and disintegration of the test item is evaluated by sieving over 2 mm, retrieval of the remaining test item particles and calculation of a mass balance; in doing so, disintegration is determined in a quantitative way. The test can also be used to produce compost for subsequent ecotoxicity tests. In this case, the total concentration of sample should be 10% which is mostly achieved by adding an extra 9% in the form of a powder.

In 2004, another method was introduced by ISO which is easier to perform: ISO 20200 – Plastics − Determination of the degree of disintegration of plastic materials under simulated composting conditions in a laboratory-scale test. – The differences in ISO 16929 are: the use of smaller reactors (5−20 L), the use of synthetic waste instead of natural, a fixed temperature regime and the possibility for different options with regard to duration of the test, temperature regime and sample concentration. Disintegration is determined quantitatively in a similar way as for ISO 16929. Because of the different possible options, the test was originally mainly used for preliminary research. The test cannot be used for the production of compost for subsequent ecotoxicity tests. Other procedures for the evaluation of disintegration during composting in full-scale testing have been discussed in which the sample is introduced in net bags into a composting pile. However, national or international standards have not been published yet. In addition, for disintegration it is important to validate test procedures by the use of (natural) reference materials. In spite of being developed for the measurement of biodegradation, ISO 14855 has also been proposed and used for the evaluation of disintegration. Nonetheless, this method is less suitable as it has been demonstrated to give false positive and several false negative results for disintegration.

5.4.3 Disintegration in water For certain applications of biopolymers (e.g., feminine hygiene care), disintegration in water is also an important factor. This mainly has to be understood as dispersion or dissolution for, e.g., flushable products. Test procedures have been proposed in: EN 14987 − Plastics − Evaluation of disposability in wastewater treatment plants − Test scheme for final acceptance and specifications.

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In addition, ASTM D7081-05 includes a chapter on disintegration under marine conditions. After 3 months of exposure in an ASTM D6691 test, the disintegration is evaluated after sieving over 2 mm and calculating a mass balance.

5.4.4 Disintegration in other environments There are no norms to evaluate disintegration in other environments. Instead, several studies have been reported in which samples were buried in, immersed in or exposed to certain environments for certain time periods, and disintegration was mainly visually monitored and evaluated in a qualitative way.

5.5 Norms on specifications for degradability 5.5.1 Introduction At an early stage, it was widely viewed that the available test procedures were insufficient to normalise (bio)degradability or compostability. In addition, specifications and criteria with specific pass levels and requirements were needed. Obviously, this exercise is even more difficult as results in the failure or acceptance of specific materials. In addition, there was a common opinion to include not only degradability criteria but to immediately include criteria related to environmental safety; a biodegradable polymer cannot be accepted for degradability when at the same time it is toxic. The OECD Guidelines for Testing of Chemicals [2] is the first publication in which specifications for biodegradability were mentioned with requirements for ready and inherent biodegradability. For biodegradable polymers (industrial), composting was the first environmental process for which specifications were developed. In the nineties, two parallel developments took place in Europe, resulting in the publication of DIN V 54900 in 1998 and EN 13432 in 2000. In spite of some minor differences, both norms were largely similar. After a few years, the DIN norm was made redundant as several international norms (EN and ISO) dealt with the same issue. In addition, in North America a norm was published in 1999 on specifications for (industrial) compostability: ASTM D6400. On a global level, ISO 17088 in the field of plastics was published in 2008, while a similar norm for packaging is close to publication and now available under the form of a Draft International Standard (DIS), ISO DIS 18606. For aquatic environments, there has been much less development on standards for specifications for biodegradable polymers. At CEN level a standard was published in 2006, EN 14987, for biodegradation in freshwater, while ASTM produced a standard for the marine environment ASTM D7081-05.

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So far, no international standard had been published with specifications for soil biodegradation. Only France has a national norm, published by AFNOR for mulching film (NF U52-001). No specification standards exist for home composting, landfill or anaerobic digestion. A special case is ASTM D6954-04 for oxo-degradable plastics, which is a standard guide with mainly suggestions and advice, and is devoid of strict specifications except for the conversion of carbon to CO2, which must be 90% for heteropolymers (or 60% for homopolymers) within a maximum period of 2 years (see further).

5.5.2 (Industrial) Compostability An overview of the different standards on specifications for industrial compostability is given in Table 5.2. Overall, there is a large similarity between these various norms. Differences are relatively small and mostly limited to minor details. Table 5.2: Overview of (industrial) compostability standards related to material and geography.

Plastics

Worldwide

EU

USA

Australia

ISO 17088

EN 14995

ASTM D6400

AS 4736-2006

Packaging

ISO DIS 18606

EN 13432

−

−

Paper coating

−

−

ASTM D6868

−

The norms of compostability specifications can be considered as ‘umbrella’ norms in which various necessary characteristics are summarised, along with the test methods on how to check these and the necessary pass levels to be met. An overview of the main characteristics is given in Figure 5.7. Two main characteristics are related to environmental safety, which in the case of composting falls back to compost quality: chemical analyses and ecotoxicity. Two other characteristics are related to degradation; biodegradation and disintegration. 1) Chemical characteristics: Some analyses are required on the polymer or packaging itself; the content of organic matter and heavy metals. The organic matter, determined as volatile solids must be at least 50% on dry weight. A series of various metals, each with a specific limit, are given in the different norms. These limits are related to heavy metal limits for compost itself. Mostly, the polymer or basic packaging material itself poses little problem. Only in the case of colouring or printing inks, can heavy metals become a limiting factor and must carefully be checked. Heavy metal requirements are different between norms both with respect to type of metal and limit value. Yet, by analysing all metals mentioned and meeting the most stringent limit, it is easily possible to cover all standards.

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BIODEGRADATION (Chemical degradation)

ECOTOXICITY (Effect on plants)

DISINTEGRATION (Physical degradation)

 133

Figure 5.7: Overview of the main characteristics required for industrial compostability.

2) Biodegradation: As explained above, biodegradation is the complete breakdown to mineral end products (CO2, H2O) and biomass. EN 13432 specifies it is preferably determined by ISO 14855, while alternatively other suitable international standards such as ISO 14851 or 14852 may be used. The pass level is 90% biodegradation in absolute terms or in relative terms compared with the positive reference, cellulose, to be reached within a maximum period of 6 months. Constituents below 1% don’t have to be evaluated as long as the total of these ‘irrelevant’ constituents is below 5%. In addition, natural materials which are not chemically modified do not have to be evaluated for biodegradation. ISO 17088 is similar to EN 13432 with regard to biodegradation requirements. The new ISO DIS 18066 specifies that constituents between 1 and 10% must be separately tested for biodegradation. ASTM D6400 is different from EN 13432 in two respects. When radiolabelled samples are being tested and C14-CO2 is measured, the test duration may be 360 days instead of 180 days. Secondly, homopolymers need to reach (only) 60% biodegradation and not 90% as in heteropolymers or blends. The biodegradation percentage limit is only in relative terms, compared with the cellulose positive reference, and not in absolute terms. Although not really specified in ASTM D6400, the certification agency Biodegradable Products Institute (BPI) (see further) also requests separate biodegradation testing for constituents between 1 and 10%. 3) Disintegration: The test item must physically fall apart or degrade on a visual, physical level and disintegrate into invisible particles. EN 13432 requires it to be evaluated either in a pilot-scale composting test (ISO 16929) or in a full-scale test. The test material is added in a concentration of 1% (on wet weight basis). At the end of a 12-week composting cycle, a maximum of 10% of the original weight of the test material may be retrieved after sorting and careful manual selection in

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the >2 mm compost fraction. The physical form of the test item is critical and typically materials will be approved when a certain maximum thickness (plastics) or a certain maximum density or grammage (paper materials) is reached. ASTM D6400 allows the use of ASTM D5338 as well as ISO 16929 for determining disintegration, although this possibility is expected to be withdrawn in the near future at the next revision of ASTM D6400. ISO 17088 mentions ISO 14855 and ISO 20200 as alternative methods besides ISO 16929. The new ISO DIS 18066 only allows ISO 20200 as an alternative, although ISO 16929 is the preferred method. 4) Ecotoxicity: Compost quality may not be negatively influenced by the addition of a biodegradable material. This is assessed by comparing a blank compost (obtained from organic waste to which no test material was added) to test compost (obtained from the same organic waste to which 10% of the test material was added at the start of the preceding pilot-scale composting test). Note: the pilot- scale composting test for the measurement of disintegration can be combined with the pilot-scale test for compost preparation for ecotoxicity tests; in this case 1% of the test material is added in the final form and 9% in the form of a powder. The compost is analysed for typical physico-chemical parameters such as pH, salt content, density, nitrogen and so on. The ecotoxicity tests include two plant tests in which the germination and plant growth (biomass) are compared between the blank compost and the test compost. The test compost cannot show a significant negative difference with the blank compost. Requirements with regard to plant toxicity are identical for EN, ISO and ASTM norms. The only deviating standard is AS 4736-2006, which also requires an earthworm toxicity test as well as two plant toxicity tests. (It can be noted that in all other respects AS 4736 is similar to EN 13432). In 1996, the first document was published by the ASTM, ASTM D6002-96, which was a standard guide without specific requirements to be met by compostable plastics. Instead it gave an overview of the various tests which are available in the field of biodegradable plastics, ranging from simple screening tests to field and full-scale assessment, together with some very basic and general requirements. In 2011, this standard was withdrawn and the only remaining standards are the ones described above with clear requirements, specifications and pass levels.

5.5.3 (Home) Compostability In spite of some debate on the value of home composting – some saying it is a valuable and sustainable way of waste reduction and management, others saying it is an important source of greenhouse gases if improperly managed while hygienic aspects

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could also be a concern − it is an important waste management option in several countries. The major difference with industrial composting is the temperature profile: although heat generation is the same, heat losses are much greater, because of the smaller volume, and maximum temperatures reached are much lower. For some biodegradable polymers, which need a thermal trigger to start hydrolysing, this makes a big difference. So far, no international standards exist with regard to specifications for home compostability. On a national level, a norm was published in Australia: AS 5810-2010, which is largely inspired by the OK Compost Home programme (see further) which was published earlier. In essence, the requirements are largely the same as for industrial compostability; a major difference being the necessity to determine biodegradation at ambient temperature as well as disintegration. For the latter, a qualitative or visual evaluation of disintegration is sufficient and a quantitative determination, with calculation of a mass balance after sieving and retrieval, is not needed if such information is already available for industrial compostability. In Italy, two norms were also published relating to home compostability: Ente Nationale Italiano di Unificazione (UNI) 11183:2006 Plastic materials biodegradable at ambient temperature – requirements and test methods and UNI 11355:2010  – Plastic products biodegradable in home composting – requirements and test methods. Requirements again are very similar to OK Compost Home and the Australian norm.

5.5.4 Soil biodegradability In March 2012, no international standards existed which defined specifications for soil biodegradability. Over several years, CEN made a big effort to develop a standard but a consensus could not be reached. The main reason was a disagreement on the possibility for a weathering pretreatment step in which polymers would first be exposed to light, temperature and moisture. A standard was published in 2005, but only in France: AFNOR NF U 52-001. Biodegradable materials for use in agriculture and horticulture − Mulching products  – Requirements and test methods. Chemical analyses do not only include heavy metals or organic contaminants such as polychlorinated phenols, dioxins and furans; ecotoxicity tests include plants, algae and earthworms. Biodegradation must be checked using two different tests and disintegration is not included in the standard. It is unclear how widespread this French standard is employed. In general, for soil, it can be noted that disintegration is largely determined by the intended use. For some applications, the plastic product should preferably disintegrate after a few months, while for other applications the product should remain intact for much longer. Consequently, many experts share the opinion that disintegration requirements should not be included in a soil standard.

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5.5.5 Aquatic biodegradability Historically, the first standards with specifications on biodegradability were published by OECD [2]. Chemicals can claim ready biodegradability when 60/70% biodegradation (% determined by the parameter being followed) is reached within 28 days and a 10 to 60% increase is seen within a maximum period of 10 days. Preexposure of the inoculum is not allowed. Inherent biodegradability does not include the maximum test duration. Primary inherent biodegradability is defined as reaching more than 20% biodegradation. Ultimate inherent biodegradability is defined as reaching 70% biodegradation. Preexposure and preadaptation are allowed. At CEN level, EN 14987 was published in 2006: Plastics − Evaluation of disposability in wastewater treatment plants − Test scheme for final acceptance and specifications. The pass level for biodegradation is 90%, which must be reached within a maximum of 56 days. Aquatic biodegradation tests must be used, with sludge as the only inoculum source and the test temperature must be ambient. Specifications for water solubility and dispersability are also included. On the other hand, requirements for chemical analyses or ecotoxicity are not defined. A greater need for additional specifications on water degradability can be expected when more biodegradable flushable products are developed and put onto the market.

5.5.6 Marine biodegradability Although biodegradable polymers could have many applications under marine conditions, developments on standardisation have been limited to ASTM with the US Navy as the major driver. In 2005, ASTM D7081 was published: Standard specification for nonfloating biodegradable plastics in the marine environment. Besides full biodegradation in a composting test (ASTM D5338), 30% biodegradation in a marine test must be achieved within a period of 6 months, disintegration in a marine test must be at least 70%, smaller than 2 mm, within a period of 3 months and an aquatic toxicity test is also required.

5.5.7 Anaerobic digestion Criteria for acceptance as a starting material for anaerobic digestion are given as an option only in EN 13432 and are rather vaguely described. Biodegradation under anaerobic conditions must be at least 50% within 2 months while disintegration must be at least 90%, smaller than 2 mm, within a period of 5 weeks, combining anaerobic digestion and aerobic stabilisation.

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No norms exist with specifications for anaerobic digestion from other standards organisations. Nevertheless, several producers of biodegradable polymers felt this was a requirement and it can be expected that in the next few years (2014−2015) serious efforts will be undertaken to develop more detailed and valuable standards on specifications for anaerobic digestion.

5.5.8 Oxo-degradation A special case is the so-called oxo-degradable plastics which claim to degrade by a combination of oxidation and biodegradation. In 2004, ASTM published D6954 Standard guide for exposing and testing plastics that degrade in the environment by a combination of oxidation and biodegradation. This is not a standard in the strict sense as for ASTM D6400 with precise test procedures and specifications, but instead a guideline which can be used for comparison and ranking purposes. Three tiers are distinguished: Tier 1 consists of abiotic degradation under the influence of heat and/ or light, Tier 2 involves biodegradation and Tier 3 is ecotoxicity. Although precise pass levels and criteria are not given, it is mentioned that after Tier 1 and Tier 2 are combined, the carbon to CO2 conversion must be more than 90% (60% for homopolymers) within a maximum period of 2 years.

5.6 Certification 5.6.1 Introduction The intensive work on international standards related to the biodegradability of plastics is an important and vital element in market development and breakthrough of these materials. Still, this is only the first step in communication and build-up of credibility. The next step is an independent and reliable third party certification system linked to a logo. The certification body is needed to evaluate the often complex information and make a correct judgement on the overall characteristics of a given material. In a way, one could see it as standards being the theory and certification systems turning the theory into practice. Another role of certification is interpretation of the standard. Originally, standards were developed for basic materials which were fairly simple and straightforward. Since then however, products have become more complex with multiple combinations possible and minor differences (e.g., inks and adhesives). Full testing of each and every product is not feasible which led to the need for the development of a set of by-laws (often called a certification scheme) defining what tests are needed while still overall respecting the compostability standards. Imagine, for example, a plastic bag

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which is certified but for which a coloured (e.g., white or black) alternative is needed as well. The only difference is the colour. What testing is needed? The certification scheme should give the answer. Over the last 20 years, several certification systems for industrial compostability have been started. In Europe, the two major systems are the Seedling logo and the OK Compost logo while in the USA the BPI logo is best known. Attempts have been made to achieve mutual recognition but have not been successful so far. It must be noted that most certification systems are specifically aimed at the evaluation of industrial compostability, as this is the most frequently used end of life option. Some other systems are related to biodegradation in soil, water or home compostability.

5.6.2 (Industrial) Compostability certification systems 5.6.2.1 Seedling One of the best known and most used certification systems for compostability is the seedling logo. The property rights of the logo belong to European Bioplastics (an industry association of bioplastic producers) while management is contracted to DIN-Certco, a quality control organisation based in Berlin (Germany) and linked to DIN. On 1st April 2012, the Belgian quality organisation Vinçotte was subcontracted to manage the seedling logo. Products that are certified can carry a compostability logo (Figure 5.8).



ko

mp

osti e r ba

r

Figure 5.8: Seedling logo.

The system is based on EN 13432, although reference can also be made to ISO 17088 and ASTM D6400. In addition, a certification scheme has been developed which gives additional rules for interpretation and use of these standards.

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Testing must be performed in test laboratories which are approved by DIN-Certco. The approval is based on the ISO 17025 standard for quality assurance in test laboratories. The applicant for the compostability logo must submit a dossier to DIN-Certco. After review of information and test results, the material or product is (eventually) approved, receives a certificate and is allowed to carry the compostability logo (with a number). A distinction is made between materials, intermediates and additives, on the one hand, and products on the other. Whereas new materials have to go through the complete testing programme, other categories or products only have to be submitted to a reduced testing programme. If a packaging material is to be certified, the content also needs to be evaluated on its suitability for composting. For the initial approval, a sample of the material or product must be delivered for archiving and an infrared spectrum produced. The latter can be considered as a kind of fingerprint analysis for identification. After certain time intervals, samples are retrieved from the market for conformity checks. These new samples are submitted for IR analyses, which are used to check the similarity between the retrieved material and the originally certified material. More information on the seedling system can be found at: http://www.dincertco.de/en/products_made_of_compostable_materials.html. The seedling logo is well known on a business-2-business (B2B) level throughout Europe and beyond. On a business-2-consumer (B2C) level it is mainly known in Germany, Switzerland, Austria, The Netherlands, UK and Poland.

5.6.2.2 OK compost The second well-known (industrial) compostability certification system in Europe is OK Compost, managed by Vinçotte based in Brussels, Belgium. Historically, the system was launched in 1994, at the request of local governments who wanted to use compostable biowaste collection bags. OK Compost is based on EN 13432 and also uses an additional certification scheme for further interpretation and use of the norm. The procedure for application is similar to that of the seedling logo. Testing must be performed at recognised laboratories. Approved products can carry the OK Compost logo (Figure 5.9), with a number. More information can be found at: http://www.okcompost.be/en/recognising-ok-environment-logos/ok-compost-ampok-compost-home/. The OK Compost logo is well known on a B2B level throughout Europe and beyond. On a B2C level it is mainly known in Belgium, France, Italy and Spain.

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Figure 5.9: OK Compost logo.

5.6.2.3 Biodegradable Products Institute Logo In the USA, a compostability certification and logo (Figure 5.10) programme was started in 2000 by a joint effort of the Biodegradable Products Institute (BPI) and US Composting Council (USCC). BPI is an industry organisation of bioplastic producers and the USCC represents the interests of the composting industry. The certification programme is based on ASTM D6400-99 ‘Standard Specifications for Compostable Plastics’. Since January 2012, BPI has contracted the management of the logo to NSF International. Applicants have to submit a dossier, which is reviewed by NSF. Promotion of the logo and the actual licensing remains BPI’s responsibility. More information can be found at: http://www.bpiworld.org/.

Figure 5.10: BPI-USCC logo.

5.6.2.4 Cedar Grove logo In the USA, another compostability acceptance system is being operated by Cedar Grove, a composting company in Seattle (Washington) with several side activities including compostability testing and sales of compost, soil amendments as well as compostable products. On top of conformity with international standards, products need to pass full-scale testing in the Cedar Grove system.

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5.6.2.5 GreenPla certification system In Japan a certification system is being operated by the Japan BioPlastics Association (JBPA). Basic principles are comparable to that of the seedling logo and BPI. More information can be found at: http://www.jbpaweb.net/english/e-gp2.htm. A picture of the JBPA logo is given in Figure 5.11.

Figure 5.11: GreenPla logo.

5.6.2.6 The Australasian seedling logo and certification system The Australasian Bioplastics Association (ABA) represents members in Australia and New Zealand. ABA runs a certification scheme based on Australian standards. Consequently, industrial compostability is based on AS 4736-2006 including an earthworm toxicity test (Section 5.5.2). In addition, the seedling logo is used under license of European Bioplastics. More information can be found at: http://www.bioplastics.org.au/

5.6.2.7 Other certification and logo systems In some other, mainly European, countries initiatives have been taken to launch compostability logos; however, most of them can mainly be considered as local initiatives. One example is Italy with the compostability system managed by the Association of Compost Producers (Consorzio Italiano Compostatori (CIC)). More information can be found at: http://www.compostabile.com/prodotti_certificati.html. A picture of the CIC logo is given in Figure 5.12.

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ile

Co

ost mp ab

CIC Figure 5.12: CIC compostability logo.

Another system, only for compostable bags, is run in Catalonia by the Department of Environment and Housing (Departament de Medi Ambient i Habitatge). Bags which are ap-proved can be identified by the Environmental Quality Guarantee logo as shown in Figure 5.13.

Figure 5.13: Environmental Quality Guarantee logo of Catalonia for compostable bags.

5.6.3 (Home) Compostability certification systems 5.6.3.1 OK Compost home In 2003, the Belgian certification agency Vinçotte launched the OK Compost Home certification scheme for approval of home compostable products and packaging. Since no international standards on home composting exist, it is based on a scheme developed by Vinçotte itself. However, this scheme has been taken over by several

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other organisations as well (see further). Key elements are the need to demonstrate biodegradation and disintegration at ambient temperature. The general operation of this system is similar to that of OK Compost. More information can be found at: http://www.okcompost.be/en/recognising-ok-environment-logos/ok-compostamp-ok-compost-home/. The logo itself is represented in Figure 5.14.

Figure 5.14: OK Compost Home logo.

5.6.3.2 Other systems for home compostability In the UK, the OK Compost Home scheme was adopted by the Association for Organics Recycling (AfOR) in 2011. More information can be found at: http://www.organicsrecycling.org.uk/category.php?category=991&name=Certification. The associated logo for the AfOR can be seen in Figure 5.15.

Figure 5.15: AfOR Home Compostability logo.

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Furthermore, in the USA, the OK Compost Home scheme has been taken over by the Sustainable Biomaterials Collaborative for their specifications on home compostability. In 2011, OK Compost Home was referred to in Californian state legislation.

5.6.4 Other biodegradability certification systems Certification systems for other environments besides composting in which biodegradable polymers can play a role are much less developed. Only the Belgian certification agency Vinçotte has created some systems, all under the header of OK Biodegradable but with the environment additionally specified, e.g., OK Biodegradable Soil and OK Biodegradable Water (Figure 5.16).

Figure 5.16: OK Biodegradable logos.

OK Biodegradable Soil is based on a scheme developed by Vinçotte itself and includes requirements with regard to biodegradation, chemical characteristics and ecotoxicity. Disintegration is not included as for applications in soil it is supposed to be a product requirement instead of an environmental requirement. OK Biodegradable Water is mainly based on EN 14987. More information can be found at: http://www.okcompost.be/en/recognisingok-environment-logos/ok-biodegradable-soil-amp-ok-biodegradable-water/.

References [1]

[2] [3] [4] [5]

The Green Report – Findings and Preliminary Recommendations for Responsible Environmental Advertising, The Task Force (group of 10 US State Attorney Generals), St. Paul, MN, USA, 1990. OECD, Guidelines for the Testing of Chemicals, Organisation for Economic Co-operation and Development, Paris, France, 1981. R.N. Sturm, Journal of American Oil Chemists’ Society, 1973, 50, 159. W. Zimmermann, Journal of Biotechnology, 1990, 13, 119. T.K. Kirk in Degradation of Lignin in Microbial Degradation of Organic Compounds, Ed., D.T. Gibson, Marcel Dekker, New York, NY, USA and Basel, Switzerland, 1984, p.399.

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[6]

[7] [8] [9] [10]

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B. De Wilde, L. De Baere and R. Tillinger in Test Methods for Biodegradability and Compostability, Mededelingen Faculteit Landbouw, University of Gent, Gent, Belgium, 1993, 58/4a, p.1621. U. Pagga, D.B. Beimborn, J. Boelens and B. De Wilde, Chemosphere, 1995, 31, 11/12, 4475. G. Bellia, M. Tosin, G. Floridi and F. Degli Innocenti, Polymer Degradation and Stability, 1999, 66, 65. B. Spitzer and M. Menner, Dechema Monographs, 1996, 133, 681. Technical Report No. 28 − Evaluation of Anaerobic Biodegradation, ISSN-07773-8072-28, European Centre for Ecotoxicology and Toxicology of Chemicals Brussels, Belgium, 1988.

Catia Bastioli and Franco Bettarini

6 General characteristics, processability, industrial applications and market evolution of biodegradable polymers 6.1 General characteristics In the past, industrial and academic researchers traditionally focused on developing stable and durable polymeric materials that resisted exposure to natural forces such as heat, sunlight, oxygen (O2), water and microbial attack. The most widespread modern plastics such as polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET) and polyvinyl chloride (PVC) are inexpensive, easily processable, resistant and durable. In fact, most man-made polymers on the market cannot be biologically degraded because their carbon components are not broken down by microbial enzymes. The hydrophobic character of plastics, their low surface area and high molecular weights (MW) are all features which inhibit or decrease enzyme activity and enhance resistance to microbial attack [1].

The durability of conventional plastics is a serious environmental drawback when these materials are used in applications where recycling has a low prob-

ability of occurring, or is too expensive, or when plastics have a high probability of being pollutants for the natural environment or for organic waste. Over the past 30 years, increasing efforts have been dedicated to develop polymers designed to be biologically degraded in selected environmental conditions. In particular, industrial research has been focused on discovering and developing biodegradable polymers that are, at the same time, easily processable, exhibit good performance and the cost is competitive with conventional polymers [2]. When bioplastics are biodegradable according to the European Norm (EN) 13432 (the European reference for technical material manufacturers, public authorities, composters, certifiers and consumers), or its equivalents, the American Society for Testing and Materials (ASTM) 6400 and the International Organization for Standardization (ISO) 14855, besides other disposal options they can be organically recycled through composting. When composting infrastructures are available, such characteristics may therefore represent a significant advantage in sectors such as waste collection, catering, or food packaging with a high probability of being contaminated by food, or ending up in organic wastes or in the environment: in such cases organic recycling has to be preferred to mechanical recycling. The property of a plastic to biodegrade in household compost permits its use of the widespread composting infrastructures for its disposal, simultaneously optimising the quality of organic waste and maximising its diversion from landfill. The ability of a plastic to biodegrade via composting is also proof that its chemical structure is inherently biodegradable. Composting is the most aggressive

https://doi.org/10.1515/9781501511967-006

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environment for biodegradation; conditions are optimised, microbial metabolism is accelerated and the microbial load is very high. If a polymer is not biodegradable under these conditions it is very unlikely to be biodegradable under milder conditions such as the soil environment or other natural environments. Polymers purported to be biodegradable only under natural conditions but not via composting, probably rely on the chance dispersion in nature over a long period as a means of disappearing from sight. There is also significant literature to show that the most widespread compostable bioplastics currently on the market are also able to fully biodegrade in soil and even in the marine environment or through home composting. A range of standards are also available to certify the behaviour of these bioplastics in many different environments. These materials realise their full environmental potential when they are used in applications in which their unique performance brings advantages to the system, both during their use and at the end of their life. In this sense, rather than being considered a simple alternative for traditional plastics, this class of products must be seen as an opportunity to redesign the entire system, focusing attention on the efficient use and recovery of resources. An update is given in this chapter of the most successful biodegradable polymers commercialised up to now and of their main uses.

6.1.1 Polymer biodegradation mechanisms The biodegradation of polymers occurs through two basic steps [3]; the first one is chain cleavage (depolymerisation) in which long polymeric chains are broken down into oligomeric fragments. This step is very important because high MW macromolecules cannot pass through the outer membranes of living cells [4]. Hydrolysis and/ or oxidation are the main biochemical reactions involved. Extracellular enzymes may also participate in the depolymerisation of macromolecular chains, acting either in an endo (random cleavage on internal linkages of the polymer) or exo (sequential cleavage of the terminal monomer unit) manner. The second step is known as mineralisation and occurs inside the cell. Small-sized oligomeric fragments are converted into biomass, minerals, salts, water, and gases such as carbon dioxide (CO2) and methane (CH4). The extent of biodegradation is usually measured using respirometric methods, which detect CO2 evolution. Other parameters to assess are: the rate of MW loss, the loss of polymer physical properties (e.g., tensile strength per ASTM standard D3826-98 [5]), the rate of increase of the microbial culture colony size in contact with the material, O2 uptake (e.g., biochemical oxygen demand) and radioactive tracer techniques that use 14C labelling.

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Plastic substrates act as a carbon source for microbial metabolism and biodegradation, ultimately producing CO2 under aerobic conditions or CH4 under anaerobic conditions. Humic material, which is an important component because it enhances the productivity of agricultural soils, is also produced during the biodegradation process. Through the composting process, the carbon content of polymeric materials is thus recycled in the environment. Narayan defined composting as the ‘accelerated degradation of heterogeneous organic matter by a mixed microbial population in a moist, warm, aerobic environment under controlled conditions’ [1]. A typical compost system contains a mixed microbial population in a moist environment, in a temperature range of 40−70 °C and aerobic conditions [6]. The macromolecular structure, size and chemical composition, and environmental conditions such as darkness, high humidity, mineral and organic nutrients, temperature, pH, O2 requirements [3], and the microbial population and enzyme activity, are all important factors in order to obtain an efficient biodegradation of a polymeric material. Conventional plastics are resistant to biodegradation mainly because of their molecular size, chemical structure and composition. It has been reported that MW is the most important feature in the biodegradation process [7]. Considering high MW synthetic polymers, only aliphatic polyesters and some aliphatic/aromatic copolyesters were actually found to be biodegradable. It is deemed that PE oligomers become biodegradable at a MW below 500. Polyvinyl alcohol (PVA) is probably the only synthetic polymer formed by carbon chains to be biodegradable, although it has been reported that PE can be slowly biodegraded by pretreatment with surfactants or an oxidation process [8].

6.1.2 Polymer molecular size, structure and chemical composition The action of extracellular microbial enzymes breaks down a polymeric chain into products small enough to pass through the cellular membrane. Microorganisms preferentially attack the ends of large molecules. Since the number of ends is inversely proportional to the MW, to render plastics degradable it is necessary to cut them into very small particles with a large surface area. Such activities can be reduced or inhibited by chain branching and crystallinity. Thus, a lower degree of polymerisation of the plastic material provides a higher concentration of chain end groups to be attacked by microorganisms and suppresses the formation of crystalline domains, which are generally difficult to biodegrade [9]. Biodegradation of the carbon chain backbones of synthetic polymers may be enhanced by the presence of hydrolysable groups such as N-substituted amides, esters or, in particular conditions, ethers. On the other hand, the presence of certain additives, impurities or intermediate products, may adversely affect the biodegradation processes.

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6.1.3 Biodegradable polymer classes The most commercially important biodegradable polymers can be broadly divided into three families: 1. Natural biodegradable polymers (unmodified natural polymers). 2. Synthetic biodegradable polymers (mainly polyesters). 3. Modified, natural biodegradable polymers (natural biodegradable polymers modified with additives and fillers). Natural biodegradable polymers are produced in nature, thus are always renewable. Some synthetic biodegradable polymers are also renewable when produced from renewable feedstocks: one example is polylactic acid (PLA), derived from agricultural sources. Modified natural biodegradable polymers can be totally or partially renewable, depending on the modifying agent used.

6.1.4 Natural biodegradable polymers All living organisms produce natural polymers. Natural macromolecules containing hydrolysable linkages, such as protein, or polysaccharides such as cellulose and starch, are generally susceptible to biodegradation in aqueous media by the action of the hydrolytic enzymes of microorganisms. The hydrophilic/hydrophobic character of natural polymers greatly affects their biodegradability, as well as their performance and durability in humid conditions. Not considering natural fibres like wool and silk, polysaccharides such as starch and cellulose are the main natural biodegradable polymers in commercial use.

6.1.4.1 Starch Starch is composed of two polysaccharides: α-amylose and amylopectin. α-amylose is linear because of the exclusive α (1–4) linkages between the D-glucose monomers (Figure 6.1) and typically has a MW of 1.6 × 105 to 2.6 × 106 [9]. Amylopectin is branched because of the presence of α (1–6) linkages and α (1–4) linkages, as shown in Figure 6.2, and has a MW of 5 × 107 to 4 × 108 [10]. CH2OH O OH HO

O

OH Figure 6.1: α-Amylose.

CH2OH O OH OH

α O

CH2OH O OH OH

CH2OH O OH

O n

OH

O

6 General characteristics, processability, industrial applications and market evolution 

O

CH2OH O OH

O

OH

O

CH2OH O OH

CH2OH O OH OH

 151

a O

OH O

CH2 OH

O O OH

  Figure 6.2: Amylopectin.

The content of amylose and amylopectin varies according to the origin of the starch: normal corn starch is composed of 20−30% amylose and 70−80% of the water-soluble constituent amylopectin [11]. In plants, starch is stored in the form of quasi-crystalline granules [11] with sizes and shapes specific to the plant of origin. Since the melting point of starch granules is higher than their degradation point, in order to use starch in polymer applications it is necessary to change its original structure and allow it to be treated as a thermoplastic polymer. This process, known as destructurising, involves heating starch under pressure, in the presence of a limited amount of water, above the melting and glass transition temperatures (Tg) of its components so that they undergo endothermic transitions [12]. Even if thermoplastic starch alone could be processed as a conventional plastic, its sensitivity to moisture renders it unsuitable for many applications [13]. Thermoplastic starch can be used to obtain foams useful as effective alternatives to PS in loose-fill protective packaging, and possibly as thermal or acoustic insulating materials. Besides being readily biodegradable in the environment, starch-based foams offer superior antistatic properties. However, they are brittle and their densities are higher than expanded polystyrene (EPS) [14]. Bulk density is a very important property for packaging applications. The bulk density of starch-based foam loose fills is in the range of 5−13 kg/m3, corresponding to a specific density of 19−31 kg/m3 [15, 16]. The composition includes one or more thermoplastic polymers to give a high melt strength to the molten mass, and can also include a nucleating agents, lubricant, plasticisers, flame retardants and rodent repellents. The foam is prepared in two steps: a) starch, and other ingredients are mixed in an extruder in the presence of water to obtain a plasticised matrix (the total water content of the resulting pellets is 5−20 wt%) and b) the pellets are foamed using a single screw extruder. Currently, Starch-Tech in the USA, Storopack in Germany and GreenLight in the UK are among the main producers of starch-based loose fills, under the Novamont licence.

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It is worth mentioning the results of the Rebiofoam Project, financed by the European Union (EU) and has Novamont as the convenor, under the VII Framework Programme [17]. The deliverables of the project are: a new type of pellet, foamable under microwave irradiation, the technology to produce foamable pellets and a pilot plant for moulding shaped parts together with tailor-made moulds. The technology permits the generation of a foam structure able to overcome the natural brittleness of starch: the foam parts are as resilient as EPS, the specific density is in the range of 40 kg/m3 and the process is faster than the moulding process for EPS shaped parts. The original idea came from Blue Marble Polymers Limited, New Zealand [18].

6.1.4.2 Polyhydroxyalkanoates Another important class of natural biodegradable polymers is represented by the polyhydroxyalkanoates (PHA), a family of intracellular biopolymers synthesised by numerous bacteria as a reservoir of energy and carbon [19]. They are mainly produced via fermentation starting from renewable resources [20]. From a chemical point of view, PHA are aliphatic polyesters and represent some of the most easily biodegradable polymers found in nature. During sugar fermentation, many types of microorganisms produce and store the aliphatic polyester polyhydroxybutyrate (PHB). PHB can be extracted from microorganisms, dried, obtained as a powder or conventional resin, and finally moulded into a film or rigid forms. A range of copolymers based on hydroxybutyric and hydroxyvaleric acids, resulting in random copolymers called poly(hydroxybutyrate- cohydroxyvalerate) (PHBV), can be produced by certain bacteria by varying the carbon sources available to the microorganism. The structure of the PHBV copolymer is shown in Figure 6.3. CH3 H3C CH

CH2

O CH2

C

O x

CH

O CH2

C

O

y   Figure 6.3: Structure of PHBV.

Considering that microorganisms synthesise PHB and PHBV for use as carbon and energy reserve materials, it is inferred that many microorganisms are capable of degrading and metabolising such polymers. The degradation rate depends on many factors such as the environment, temperature, pH, O2 concentration, surface area, molecular mass and degree of crystallinity [21]. The physico-chemical properties of PHB and PHBV are quite different, as reported in Table 6.1: PHB is stiffer and more brittle, its solvent resistance is inferior, but it has

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Table 6.1: Properties of PHB and PHBV. Property

PHB

PHBV (10% HV)

Melting point, °C

180

140

130

40

25

20

Tensile strength, MPa Flexural modulus, GPa

3.5

Extension to break, %

8

1.2 20

PHBV (20% HV)

0.8 50

Adapted from J.M. Liddel in Chemical Industry: Friend to the Environment?, Ed., J.A.G. Drake, Special Publication No.103, Royal Society of Chemistry, Cambridge, UK, 1992, p.10 [21] and Anon, Modern Plastics, Breskin & Charelton Pub. Corp., New York, NY, USA 1981, 58, 7, 90 [22].

better natural resistance to ultraviolet (UV) weathering. PHBV properties depend on the valerate content: increasing the content of the hydroxyvalerate (HV) monomer in the copolymer reduces the crystallinity and melting point, resulting in a decreased stiffness but an increased toughness and impact resistance. The bacterial production of PHB was first reported by Lemoigne at the Pasteur Institute in Paris in 1925 and, since then, has been extensively studied [23]. Many organisations patented technologies related to the microbial production of PHA (in particular PHB, PHBV and related polyesters). WR Grace in the USA patented PHB, producing small quantities of the product for commercial evaluation in the late 1950s and early 1960s [24–26]. In the 1970s, Imperial Chemical Industries (ICI) in the UK continued the development of PHB and commercialised BIOPOL™ polymers in 1981. The BIOPOL™ business and related technology were sold to Monsanto in 1996 and subsequently acquired by Metabolix in 2001. A number of ICI’s patents in the 1980s disclosed the preparation of PHBV by cultivating Ralstonia eutropha in a two-stage fermentation process [27–29]. The first step is carried out as a conventional fermentation process with glucose as the carbon source and nutrient salts as the nitrogen source. In the second step, propionic acid is added as an additional carbon source, while the nitrogen nutrient is limited, in order to induce the microorganism to produce 3-HV units. PHBV polymers are accumulated as granules within the cell cytoplasm and each granule is thought to be surrounded by a lipid and protein membrane [21]. Recovery of the polymer from the cell may be accomplished by a number of extraction processes. Since the estimated cost of producing PHA via microbial fermentation was very high [30], research was focused on the reduction of PHA cost by trying to produce it in plants. Such an approach was theoretically possible because acetyl-CoA and acetoacetyl-CoA, the precursors to PHB synthesis in R. eutropha, are also involved in the synthesis of a variety of compounds in plants. PHB production in plants was demonstrated by using a weed belonging to the mustard family [31]. Procter and Gamble in the USA patented a process for recovering poly(3-hydroxyvalerate-co- hydroxyhexanoate) (Nodaxtm)

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from transgenic potatoes [32]. Researchers at the University of Warwick used the yeast protein GAL4 (a transcriptional activator) to produce PHB in oilseed rape plants [33]. Monsanto achieved PHA levels in plants of up to 5% [34]. Metabolix developed PHA production starting from genetically modified cultures and in 2010 announced the possibility of obtaining PHA in modified tobacco plants. Besides PHB and PHBV, a broad range of homopolymers and copolymers have been studied over time, mostly on a laboratory scale. Further examples of homopolymers are polyhydroxyhexanoate, polyhydroxycaproate, polyhydroxyoctanoate, polyhydroxynonanoate, polyhydroxydecanoate, polyhydroxyundecanoate, polyhydroxydodecanoate and polyhydroxyoctadecanoate. In 2007, Metabolix and the Archer Daniels Midland Company (ADM) started their joint venture, Telles, and built a 50,000 tonne plant at Clinton, Iowa, USA to produce Metabolix’s PHA commercialised under the Mirel trademark [35]. The plant started its production in 2008. However, due to the limited market, ADM announced the end of its commercial alliance with Metabolix on 8th February 2012. As a result of this decision, Telles dissolved and Mirel production on behalf of Telles stopped [36]. At present, the main companies involved in PHA production on a pilot or (semi)industrial scale include Metabolix (USA), Meredian (USA), Kaneka (Japan), Tianan (China), PHB Industrial/Copersucar (Brazil), Biomatera (Canada), Biomar (Germany), Bio-On (Italy), PolyFerm Canada (Canada), Tianjin and DSM (China), and Tianzhu (China). With regard to their potential applications, PHA materials are especially suitable for packaging to ensure the protection and safe transportation of goods. The existence of a range of cheaper biodegradable bioplastics superior to PHA in many properties such as transparency, toughness, thermal and environmental stability as well as processability in standard conversion machines, requires the identification of sectors able to give a value to the specific distinctive properties of PHA. One such sector could utilise the high barrier against oxygen permeation which would prevent the oxidative spoiling of food products [37]. Other possible applications include the use of PHA as components of biomedical devices, or as components of biodegradable blends for daily commodity items such as razors, nappies, hygiene products, cups and dishes, and mulch films in agriculture.

6.1.5 Synthetic biodegradable polymers In this chapter, the term synthetic biodegradable polymers defines polymeric materials produced by mankind either starting from renewable monomers of natural origin or from monomers of petrochemical origin. The reactions involved in the biodegradation of synthetic biodegradable polymers are the same as for natural polymers, i.e., enzyme-catalysed transformations occurring in aqueous media.

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Polyesters with easily hydrolysable bonds along the chain represent the major family of such polymeric products. Nonestereous PVA is also considered to be biodegradable.

6.1.5.1 Polylactic acid and polyglycolic acid The simplest aliphatic polyesters which are synthetically produced starting from natural monomers are PLA and polyglycolic acid (PGA). PLA was discovered by Carothers in the 1930s [38], but its tendency to hydrolytic degradation initially discouraged its development. In the 1970s, PGA was developed in medical applications, such as degradable surgical sutures [39], owing to the ability of the human body to degrade this kind of material. PGA is usually synthesised by polymerising diglycolide with a tin catalyst. Similarly, the ring-opening polymerisation of dilactide using stannous octanoate as the catalyst affords PLA. The structures of PGA and PLA are shown in Figure 6.4. O CH2

C

H3C O

x

CH

O C

O

y   Figure 6.4: Structures of a) PGA and b) PLA.

Owing to their high biocompatibility [40], biodegradable polyesters such as PLA, PGA and their copolymers, found a number of useful applications in the medical field, for the controlled release of drugs, biodegradable surgical sutures, and implants for the fixation of fractures such as screws, plates and pegs. After having effectively secured the orthopaedic prostheses to the bone(s), the devices are eventually biodegraded and absorbed by the body. The biodegradation process of such aliphatic polyesters initially proceeds through a random, nonenzymatic hydrolytic chain scission of the ester bonds [41]. When the chain scission has produced oligomers small enough to diffuse from the polymer bulk, a loss of mechanical strength occurs; the extent of degradation increases at higher degradation temperatures (between 40 and 60 °C) [5]. As the average MW of lactic acid oligomers approaches 10,000, microorganisms are able to digest them, producing CO2 and water [42]. The production process of PLA is usually carried out in two steps. Lactic acid is first oligomerised to a linear chain with a MW of less than 3,000 by removing water. The oligomer is then depolymerised to lactide, a six-membered cyclic dimer. After purification, lactide is subjected to a ring-opening polymerisation to produce a PLA of MW greater than 50,000. Patents related to this process were filed by Cargill [43, 44], Camelot Technologies [45] and Ecological Chemical Products [46]. Mitsui Chemical in Japan developed a process for making high MW PLA directly from lactic acid without the oligomerisation step [47]. Purac developed a process for the production of a stable

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form of L-lactide and cooperated with Sulzer to develop a polymerisation process for buyers of L-lactide. Synbra is the first company to install a 5,000 tonnes/ year capacity plant to produce PLA for moulded foams [48]. Initially, PLA was a high-cost material compared with conventional thermoplastics and its commercial exploitation was limited. Recent developments, particularly with regard to the sourcing of lactic acid, which is mostly obtained by the fermentation of plant-derived sugars (starch from corn and potatoes, sucrose from beets and sugar cane and so on), has allowed PLA to be on the market at a reasonable price (approximately 2 €/kg) [49]. Depending on the relative content of L and D enantiomers, the physical properties of PLA polymers may vary widely, from semicrystalline products with melting points of 130−180 °C to amorphous glassy polymers with a Tg of 60 °C [42]. Selection of the appropriate PLA stereochemistry has a major effect on the polymer’s physical properties, processability and biodegradability. Many of the basic properties of PLA lie between those of crystal PS and PET. Flexible PLA films can be produced by incorporating a plasticiser. One of the main applications today is in the fibre sector. In Table 6.2, the properties of PET and rayon fibres are compared with those of PLA fibre. Table 6.2: Fibre property comparison. Fibre property

PET

PLA

Rayon

Specific gravity

1.39

1.25

1.52

Melting temperature, °C

254−260

130−175

None

Tenacity, g/d

2.4–7.0

2.0–6.0

2.5

Elastic recovery at 5% strain

65

93

32

Moisture regain, %

0.2−0.4

0.4−0.6

11

Adapted from Technical Bulletin 180904, Fiber and Fabric Properties, NatureWorks LLC, Minnetonka, MN, USA [50].

They are similar to cotton in terms of hand and wickability/breathability, and are biodegradable. They have a good flammability resistance and UV resistance as well as excellent drapeability. They can be used in blends with cotton and polyester, or alone. End uses of nonwoven PLA fibres include: wipes and sanitary napkins, cosmetics, nappies, apparel and agri/industrial applications. PLA has been further developed (in particular by NatureWorks LLC with the trademark Ingeo®) for use in a wide range of applications beside fibres, such as packaging materials, thermoformed food containers, bottles, biomedical devices, automotive materials, electronic devices, or as a component of polymeric blends in a wide range of applications. The current production capacity of Natureworks is approximately 150,000 tonnes/year.

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6.1.5.2 Poly(ε-caprolactone) The first synthetic biodegradable aliphatic polyester commercially available was poly(ε-caprolactone) (PCL), produced under the trade name Tonetm by the Union Carbide Corporation in the USA. The product was the subject of a number of biodegradability studies [7] and was originally employed for medical sutures, and then as a component in biodegradable polyester/starch blends (e.g., Mater-Bi® Z-grade, Novamont, Novara, Italy) [13]. PCL is a semicrystalline flexible polymer produced by the ring-opening polymerisation of ε-caprolactone. Union Carbide patented a process which allows achieving a MW as high as 100,000 using stannous octanoate as a polymerisation initiator [51]. Due to its low melting point (approximately 60 °C) and its high price, the use of PCL is now reduced. The production of Tone was stopped and PCL is at present available from the Perstorp Group under the trade name CAPA®. The PCL structure is reported in Figure 6.5. Typical properties of PCL for 3 degrees of polymerisation are reported in Table 6.3. O (CH2)5

C

O

n  Figure 6.5: Structure of PCL.

Table 6.3: Typical properties of PCL. Property

CAPA®6400

Mean MW

CAPA®6500

CAPA®6800

37,000

50,000

80,000

59

58−60

58–60

140

360

580

660

800

900

Melting point, °C Tensile strength, kg/cm Elongation at break, %

2

Adapted from CAPA Product Data Sheets, Perstorp Group, Perstorp, Sweden, 1st August 2013 [52].

6.1.5.3 Diol-Diacid aliphatic polyesters Aliphatic polyesters derived from the polycondensation of diols with aliphatic dicarboxylic acids are another important class of synthetic biodegradable polymers. Since 1994, Showa Highpolymer in Japan has been producing a family of aliphatic polyesters known as Bionolle®, obtained from butanediol and succinic acid to give polybutylene succinate (PBS), and the copolymer polybutylene succinate adipate (PBSA) obtained from butanediol, succinic acid and adipic acid. Polyethylene succinate (PES) was also produced.

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 Catia Bastioli and Franco Bettarini

The chemical structures of PBS and PES are shown in Figure 6.6. O O

(CH2)4

O

C

O (CH2)2

C

O n

(CH2)2

O

O

C

O (CH2)2

C

n

Figure 6.6: Structures of PBS and PES.

At first, succinate polyesters with a MW of less than 5,000 were produced, and these polymers were weak and brittle. High MW aliphatic polyesters were subsequently synthesised by Takiyama and co-workers [53] as pressure sensitive or thermosetting adhesives. Further research efforts brought the development of succinate polyesters with an average MW in the range of 20,000−200,000. Such high MW polymers are prepared in two steps: condensation of a diol (e.g., 1,4-butanediol (1,4-BDO)) with succinic acid produces a hydroxy-terminated aliphatic polyester prepolymer which is then reacted with a diisocyanate chain extender to form a high MW succinate polyester [54]. In Table 6.4, the basic properties of commercial grade Bionolle® and conventional polyolefins are compared. Table 6.4: Basic properties of the Bionolle® pressed sheet. Property

PBSU #1000

MFR at 190 °C, g/10 min

1.5−26

PBSU Co. #2000 4.0

PBSU Co. #3000 28

LDPE 0.8

HDPE 11

Melting point, °C

114

104

96

110

129

Yield strength, kg/cm2

336−364

270

192

100

285

Elongation, %

560−323

710

807

700

300

Stiffness 103, kg/cm

2

Izod impact at 20 °C, kg-cm/cm

5.6−6.6 30−4.2

4.2 36

3.3 >40

1.8 >40

12 4

HDPE: High-density polyethylene LDPE: Low-density polyethylene MFR: Melt flow rate PBSU: Polybutylene succinate Reproduced with permission from T. Fujimaki, Polymer Degradation and Stability, 1998, 59, 209. ©1998, Elsevier Science [53].

PBS is a highly crystalline polymer (melting point around 114 °C) and its properties are suitable for thermally stable injection moulded and thermoformed products. Its main use today is in Japan, as a component of mulch films and compost bags with a market volume of approximately 2,000 tonnes/year. Several other companies produce aliphatic polyesters based on PBS homopolymers and copolymers: Mitsubishi Chemicals (GS-PLA) and Samsung Fine Chemi-

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 159

cals (Enpol G4000) in Japan, Hexing Chemical (Hexing-PBS), Xinfu Pharmaceuticals (PBS), Kingfa Science & Technology Co. Ltd. (PBSA) in China and Novamont among its range of Origo-Bi® polyesters.

6.1.5.4 Aliphatic/Aromatic copolyesters Since nonbiodegradable aromatic polyesters like PET provide excellent material properties [55], with respect to easily degradable aliphatic polyesters, a number of aliphatic/aromatic copolyesters were studied and developed in order to produce materials which combined good mechanical properties and biodegradability. Major polyester producers in Europe and the USA brought aliphatic/aromatic copolyesters for biodegradable applications to the market. The most studied aliphatic/aromatic copolyester is polybutylene adipate-co- terephtalate (PBTA), produced by the condensation of 1,4-BDO with terephtalic acid (TPA) and adipic acid. Eastman Chemical patented three different families of linear, random aliphatic/aromatic copolyesters [56]: 1) 1,4-BDO and glutaric acid/TPA, 2) 1,4-BDO and adipic acid/TPA and 3) 1,6-hexanediol and adipic acid/TPA, and launched Eastar Bio® aliphatic/aromatic PBTA copolyester. In 2004, Novamont acquired the patents portfolio of Eastman Chemical containing aliphatic/aromatic copolyesters. At present, PBTA is commercialised by BASF with the trade name Ecoflex® and produced by Novamont among the Origo-Bi® polyesters. A modified PET known as Biomax® was developed by DuPont. Aliphatic/aromatic copolyesters containing sulfo groups were also patented by DuPont [57–60]. Polyesters copolymerised with 5-sulfoisophthalic acid are readily hydrolysed and biodegraded, and can be processed at higher temperatures than other biodegradable materials. Other companies claiming to produce PBTA are Xinfu and Fuwin in China, and Ire Chemical in Korea. The structure of a typical aliphatic/aromatic copolyester is shown in Figure 6.7. Aliphatic/aromatic copolyesters can be prepared either as random copolymers or block copolymers. Random copolymers are more readily biodegraded than copolymers with long aromatic blocks. O

O

C

C

O O

(CH2)4

O

C

O (CH2)4

C

Figure 6.7: Aliphatic/aromatic copolyester.

High MW are needed when high melt viscosities are required, e.g., for blown-film production [61]. This aim can be achieved by incorporating diisocyanates into the polymer chain as a chain extender. The chain extension does not influence the bio-

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degradability of the polymer. Indeed, studies carried out at the Society for Biotechnological Research (Gesellschaft für Biotechnologische Forschung) concluded that, in a compost environment, biodegradation rates of chain-extended 1,4-BDO/adipic acid/ terephthalic acid copolyesters are the same as the nonextended copolyesters [61]. Biodegradable copolyesters patented by BASF [62, 63] are prepared by the initial condensation of 1,4-BDO and adipic acid to afford polytetramethylene adipate. This polymer is reacted with dimethyl terephthalate, 1,4-BDO, pyromellitic dianhydride and polyethylene glycol (MW of 600), in the presence of a titanium catalyst. A sulfonate compound is optionally incorporated into the polymer. Novamont patented a series of biodegradable aliphatic/aromatic copolyesters which are prepared by the copolymerisation of diol components with aromatic diacids and long chain aliphatic diacids of natural origin, such as azelaic, sebacic and brassylic acids [64–67]. An open issue concerning aliphatic/aromatic copolyesters regards the extent of their biodegradability, as aromatic polyesters such as PET are known to be resistant to microbial attack [4]. Researchers at the Gesellschaft für Biotechnologische Forschung in Germany, found that the biodegradability of such copolymers depends on the length of the aromatic sequence [4]. Block copolyesters with relatively long aromatic sequences are not rapidly degraded: polybutylene terephthalate oligomers having aromatic sequences ≥3 show very little degradation over a period of several months, whereas oligomers with aromatic sequences of 1 or 2 were degraded within 4 weeks [68]. In biodegradable PBTA aliphatic/aromatic copolyesters available on the market, the amount of aromatic acids in the polymer chain is maintained below 49 mol%, in view of the significant and sudden decrease of the biodegradation of polyesters above this threshold. This behaviour was attributed to the lower biodegradability of the butylene terephthalate sequences with length equal or higher than 3 which, above said threshold, represents more than 10% of the aromatic fraction of these products [69]. The new aliphatic/aromatic copolyesters developed recently by Novamont have the aliphatic dicarboxylic acid component predominantly based on long chain dicarboxylic acids of natural origin (sebacic acid, azelaic acid and brassylic acid). With respect to the aliphatic/aromatic polyesters comprising an aliphatic dicarboxylic component with shorter carbon chain length, such as PBTA polyesters, these copolyesters did not show the sudden decrease of biodegradation properties above 49 mol% of aromatic acids. This allowed the development of aliphatic/aromatic copolyesters with a higher molar fraction of aromatics with respect to PBTA polyesters, achieving an improved balance between mechanical and biodegradation properties. The mechanical properties of this family of copolyesters depend on the content of the terephthalic acid in the copolymer [61]. It was demonstrated that, in a range between 40 to approximately 50 mol% of terephthalic acid (referred to the acid components), materials combining sufficient biodegradability with promising technical properties can be obtained. Compared with an LDPE material, a PBTA- copolyester

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 161

with approximately 40 mol% terephthalic acid exhibited a comparable mechanical strength but a significantly higher flexibility (elongation at break). Aliphatic/aromatic copolyesters are very versatile biodegradable polymers with an interesting cost/performance ratio [30, 61]. They can be produced from widely available, low-cost monomers, such as adipic acid, butanediol and terephthalic acid, as well as from renewable long chain diacids such as azelaic, sebacic or brassylic acid. It is expected that they will gain a significant share in the biodegradable polymers market.

6.1.5.5 Polyvinyl alcohol PVA is a water-soluble polymer obtained by the hydrolysis of polyvinyl acetate (PVAc). It is easily biodegraded by microorganisms and enzymes [70]. The solubility and biodegradability, as well as other physical features, can be addressed by varying the MW and the degree of hydrolysis of the polymer [71]. It was reported that when PVAc is hydrolysed to less than 70%, it is nonbiodegradable under conditions similar to those that biodegrade the completely hydrolysed polymer [72]. Owing to the high degree of crystallinity, PVA cannot be processed as a thermoplastic. PVA thermally degrades at approximately 150 °C, well below its melting point (180−240 °C) [73], releasing water and forming conjugated double bonds. PVA films were thus produced by an expensive solution casting process. Several companies developed biodegradable PVA that can be processed as a thermoplastic: Environmental Polymers Group (EPG) in the UK, Idroplast in Italy, Millenium Polymers in the USA and PVAX Polymers in Ireland [74]. EPG patented an extrusion process together with PVA formulation technology to produce thermoplastic PVA pellets which can be converted into film and sheet products [75]. EPG PVA, which is typically 40−50% crystallinity, can be used to produce films with tensile and tear strengths superior to PE and PVC (Table 6.5). Table 6.5: Typical properties of EPG PVOH films. Property

EPG PVA

Cellophane

PVC

PE

Clarity (light transmitted), %

60−66

58−66

48−58

54−58

Water vapour transmission at 40 °C and 90% relative humidity

1,500−2,000

1300−2,000

120−180

35−180

Tear strength, Elmendorf Nm/m

147−834

2−4

39−78

29−98

Tensile strength, MN/m

44−64

55−131

20−76

17−19

Elongation at break, %

150−400

−

5−250

50−600

2

Reproduced with permission from N. Hodgkinson and M. Taylor, Materials World, 2000, 8, 4, 24. ©2000, Institute of Materials, Minerals and Mining [73].

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At present, the main PVA producers include Kuraray (Japan and Europe) and Sekisui Specialty Chemicals (USA), but a very large number of production facilities have been installed in China over the past decade, currently accounting for 45% of world capacity.

6.1.6 Modified, natural biodegradable polymers Carbohydrates, or saccharides, are naturally occurring organic compounds consisting of carbon, hydrogen and oxygen. They are extremely widespread in plants, accounting for as much as 80% of plant dry weight. Carbohydrates can be divided into four chemical groups: monosaccharides, disaccharides, oligosaccharides and polysaccharides. Polysaccharides are biodegradable polymers made up of simple monosaccharide units. Cellulose, a linear polysaccharide, is made of glucose units and is probably the most abundant organic compound on earth; it is the basic structural component of plant cells. The degradation of cellulose to glucose is catalysed by microbial enzymes (cellulases). Starch is the second most abundant polysaccharide and it is also made up of glucose units. In plants, starch is present in the form of small insoluble granules. Starch readily gelatinises in hot water to form a paste that can be cast into a film. Being sensitive to water, such films become brittle on drying. Less brittle films can be produced by combining starch with other materials such as plasticisers and synthetic polymers. Several approaches were followed to incorporate polysaccharides into synthetic polymers with the aim of enhancing their biodegradability [76–81]. The microstructures obtained play a fundamental role in determining the biodegradation rate of the final products. The proposed biodegradation sequence is the following: 1. Polysaccharide units are first consumed by microorganisms, a process which increases the surface area of the synthetic polymer and weakens the polymeric matrix. 2. The synthetic polymer chains are broken down, by various environmental mechanisms, into smaller fragments which are small enough to be assimilated by microorganisms. However, if the synthetic polymer is a recalcitrant product, such as PE, there is no reliable and reproducible demonstration that it could be completely mineralised, due to the presence of a polysaccharide or another additive [82]. The reference commercial starch-based biodegradable polymers are marketed by Novamont in Italy under the name Mater-Bi®. This starch-based technology permits going beyond conventional compounding. In the presence of different synthetic polymers, starch can undergo a thermoplastic transformation up to destruc-

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 163

turisation; the destructurised starch composites can reach starch contents higher than 50% [83]. In some polymers, the real complexation of a single helical amylose with the polymer backbone occurs, giving rise to supramolecular structures, whereas amylopectin remains in an amorphous state. Such complexes play an important role on the final properties of the starch-based polymer. Interesting products previously developed with the Mater-Bi® technology include: starch and ethylene-vinyl alcohol (EVOH) copolymers; starch and PVA; starch and aliphatic polyesters, in particular PCL. It is also possible to use aliphatic polyesters such as those formed by the reaction of glycols such as 1,4-BDO with succinic acid, sebacic acid, adipic acid, azelaic acid, dodecandioic acid or brassylic acid. The presence of compatibilisers between starch and aliphatic polyesters is preferred. Some examples are amylose/EVOH V-type complexes [84] and starch-grafted polyesters. Such materials are characterised by excellent compostability, excellent mechanical properties and reduced sensitivity to water. However, up to now the most important result in starch technology from Novamont is the attainment of nanostructured composites, where thermoplastic starch represents the dispersed phase and different types of aliphatic/aromatic copolyesters represent the continuous phase [85, 86]. In these products, nanostructured starch gives an important contribution to the mechanical performance in terms of toughness and stability at different humidity and temperature conditions. The development of new aliphatic and aliphatic/aromatic copolyesters, containing renewable monomers from vegetable oils, has further improved the performances of these kinds of products and their environmental compatibility. Modified starch-based polymers are mainly developed today as the most suitable solution to the current municipal solid-waste management problems, as well as for the production of compostable shopping bags and mulch films. With the Mater-Bi® trademark, Novamont commercialises a range of starch-based materials containing Origo-Bi® polyesters as the matrix. According to the level of renewability of the Origo-Bi® grades, linked to the upstream integration of the Novamont biorefinery, Mater-Bi® grades are currently divided into 4 generations. Eastar-Bio® is the polyester component of the first generation of Mater-Bi®, whereas the polyester of the fourth generation according to Novamont [87] contains azelaic acid and 1,4-BDO, both of which are from renewable resources. Azelaic acid will be produced by a flagship plant built by Matrica (a 50/50 joint venture (JV) between Novamont and Versalis), whereas renewable 1,4-BDO, currently at pilot scale, will be industrially produced by another Novamont flagship plant in 2015, using Genomatica’s direct fermentation process. As reported above, the combination of nanostructured starch and a tailor- made polyester matrix permits the production of a wide range of properties in terms of mechanical performance (Table 6.6), biodegradability in different environments and processability. These products are the most suitable candidates to replace PE when biodegrada-

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 Catia Bastioli and Franco Bettarini

bility is needed to avoid organic waste pollution, or high risks of dispersion in the environment or when biodegradability is a functional characteristic. The present capacity of Novamont for Mater-Bi® products is approximately 120,000 tonnes/year.

6.2 Processability With respect to processability, resins are classified as thermoplastic or thermoset, depending on their behaviour upon heating. By applying heat and pressure, thermoplastics soften and flow like liquids. Table 6.6: Typical Properties of Mater-Bi® films. Property

Mater-Bi® films

Method

MFR, g/10 min

3.5−7

ASTM D 1338

Young’s Modulus (MPa)

90−700

ASTM D 882

Stress at break (MPa)

22−36

ASTM D 882

Elongation at break (%)

250−600

ASTM D 882

Friction coefficient

0.10.6

DIN 53375 A

Haze (%)

26−90

ASTM D1003

Water vapour transmission rate (WVTR) (g × 30 µm/m2 × 24h)

200−900

ASTM E 96: 38 °C, 90% RH

O2 transmission rate (TR) (cc × 30µm)/ ( m2 × 24 h × atm)

500−2,000

ISO 15105-1: 23 °C, 50% RH

On cooling, they solidify; these phase changes can be repeated with little or no influence on the polymer’s physical properties. Thus, thermoplastics can be easily processed into finished products by a variety of thermoprocessing techniques, and can be easily recycled as well. Thermosetting resins are irreversibly transformed on heating by the formation of crosslinking bonds between polymeric chains. Once cooled, they cannot be reheated and moulded again [88]. Therefore, thermoforming and other thermoprocessing techniques have displaced thermosets in many applications [89]. The majority of commercially important biodegradable polymers are thermoplastics with the main exception of PVA, which can be processed as a thermoplastic only after modification or plastification. Thermoplastic polymers can be either amorphous or crystalline materials. Whereas crystalline thermoplastics have well-defined melting and freezing points, amorphous thermoplastics do not have melting points [88]. For an amorphous

6 General characteristics, processability, industrial applications and market evolution 

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polymer the transition from a liquid (or rubbery state) to a solid (or glassy state) is termed the Tg. In Table 6.7 the melting points of some conventional and biodegradable thermoplastics are reported. Table 6.7: Melting points for conventional and biodegradable thermoplastics. Conventional thermoplastics

Melting point, o C

Biodegradable thermoplastics

Melting point, oC

LDPE

110

PCL

60 [52]

HDPE

127

Succinate polyesters

96−114 [53]

PP

176

Copolyesters

79−137 [61]

Nylon 6

225

Meso PLA

130 [42]

Polyester 4GT

230

PHBV

130−140 [21]

Polyester 2GT

265

Polyesteramides

125−175

Nylon 6,6

265

100% L enantiomer-PLA

180 [42]

Reproduced with permission from P.N. Richardson in Encyclopedia of Polymer Science and Engineering, Ed., J.L. Kroschwitz, Wiley & Sons, New York, NY, USA, 1988, 11, 262.©1988, Wiley & Sons [88].

6.2.1 Extrusion Extrusion is the most widely used technique for processing thermoplastics at some stage during manufacturing. Many polymers are extruded for the first time during their manufacture and compounding operations, before the final extrusion step necessary to make the finished products [88]. Extruders can be single screw extruders or twin screw extruders. Single screw extruders are simple devices, less expensive and more widely used than twin screw extruders, which are mostly used for difficult compounding applications, devolatilisation and for extruding final products from viscous polymers which are not very stable upon heating (which include many biodegradable polymers) [90]. Starch-based polymers have been intensively studied for their extrusion characteristics, since extrusion processing plays a primary role in establishing the polymer’s properties. Starch can be made thermoplastic using technology very similar to extrusion cooking [13]. Granular beads of starch are approximately 15−100 µm in diameter and can be blended as a filler with other polymeric materials [91]. Under special heat and shear conditions during extrusion, starch can be transformed into an amorphous thermoplastic material by a process known as destructurisation. Starch can also be destructurised in the presence of polymers such as aliphatic polyesters, which are hydrophobic materials [92]. It is difficult to process aliphatic

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polyesters with low melting points by conventional techniques such as film blowing and blow moulding. For example, extruded PCL films are tacky, rigid and have a low melt strength over 130 °C. Moreover, the slow crystallisation rate may cause a change of the polymer’s properties. It has been found that blending starch with aliphatic polyesters improves their processability and biodegradability [93]. The addition of starch has a nucleating effect which increases the crystallisation rate of the polyester [92]. The rheology of the starch/PCL blends depends on the extent of starch granule destruction and the formation of thermoplastic starch during extrusion. Increasing shear and heat intensities can reduce the melt viscosity, but enhance the extrudate swelling properties of the composite [94]. Besides PCL and its copolymers, other examples of biodegradable polyesters which can be used in the presence of destructurised starch are those obtained by the polycondensation of glycols such as 1,4-BDO with succinic acid, sebacic acid, adipic acid, azelaic acid, dodecandioic acid, or brassylic acid and PBTA [85, 86]. Mixing thermoplastic starch with the polymer may take place in one or two steps. In the 2-step method, natural starch is first plasticised and then mixed in conventional extruders [95]. Temperature and pressure are usually regulated in such a way that the composition forms a thermoplastic melt.

6.2.2 Film blowing and casting Blowing and casting are the main processes used industrially for making films from thermoplastic polymers. Blown films are mostly used for food and rubbish bags. A blown film is extruded as a tube and the tube is filled with air to expand it to the desired size [88]. The film is then cooled, flattened and extruded again over an isolated bubble of air. Typical film thicknesses are 0.007−0.125 mm. High melt viscosity resin is required for processing a blown film so that the melt can be pulled upward from the die [88]. A cast film is prepared by drawing a molten web of polymer from the die onto a roll for controlled cooling. The cast film process is used to make a film with gloss and sparkle. In this process the melt temperature is higher than in the blown film process, since a higher melt temperature imparts better optical properties [88]. Many of the biodegradable materials described in this chapter are suitable for both film blowing and casting, although some process modifications are often necessary and productivities may not be as high as conventional thermoplastics. In general, starch-based Mater-Bi® films can be prepared with the same film blowing and casting equipment used for LDPE, with minor or no modifications. The productivity is reported to be 80−90% of LDPE [96]. Since lower welding temperatures are required with respect to traditional PE film production, small- to medium-sized production lines with good cooling capacity are preferred for processing starch-based films [97].

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PLA films with thicknesses of 8−510 µm have been obtained from commercial film casting equipment [98]. PLA can be difficult to process into a film due to instability at elevated processing temperatures. According to a Cargill patent, melt stable PLA, suitable for processing into films, can be made by controlling the polymer composition as well as adding stabilising or catalyst-deactivating agents [99]. The polymer MW plays a role in its processability; polymer morphology is also very important. Semicrystalline PLA is suitable for processing into films with desirable barrier properties. The desired range of compositions for semicrystalline PLA is less than 15 wt% meso-lactide and the remaining weight percent being L-lactide [98]. For efficient film processing, a thermoplastic must crystallise quickly, i.e., in a few seconds. Cargill patented four methods to increase the rate of PLA crystallisation [99]: – Adding a plasticising agent such as dioctyl adipate. – Adding a nucleating agent such as talc. – Orientation by drawing during film casting or blowing, or after it is cast or blown. – Heat setting, which involves holding a constrained oriented film at temperatures above the Tg. As mentioned in Section 6.1.5, EPG patented an extrusion process and a formulation technology to produce thermoplastic PVA pellets suitable to be converted into films and sheets [75]. Dual extrusion is also possible using this technology allowing films to be produced which combine layers of PVA film with different water solubility characteristics [100].

6.2.3 Moulding Thermoplastic polymers can be moulded into finished articles either by injection moulding or blow moulding. During injection moulding, by means of a high pressure, a molten thermoplastic is injected into a mould where it solidifies. During blow moulding a molten tube of resin is extruded, a mould is closed around the tube, and air is fed into the tube to expand it into the mould. It is the most common process for making hollow articles such as bottles [88]. Moulded articles can be produced using most of the biodegradable polymers discussed in this chapter. One typical example of injection moulding is the processing of PHBV into moulded articles. The degree of crystallinity of the finished product is a result of the processing history during moulding [101]. In the so-called fountain flow effect, hot melt flows into a cold mould and quickly forms a frozen layer on the surface of the mould, while material in the centre of the sample does not cool as quickly. This difference in orientation and cooling rate differentiates the crystallisation degree of the material close to the surface and the material close to the core. The degree of crystallinity of injection moulded PHBV affects both the physical properties

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and biodegradability of the article [101]. This is also true for many other biodegradable thermoplastics. Besides the aliphatic polyester PBS, the heteroaromatic polyester polyethylene furanoate (PEF), based on the renewable monomer furandicarboxylic acid, is particularly suitable for moulding processes. PEF is attracting increasing interest worldwide as a substitute for PET. Novamont has recently patented polymeric compositions comprising biodegradable polyalkylenefuranoates, aliphatic/aromatic polyesters, and starch in destructurised, gelatinised or filler form [102]. PLA is an example of a biodegradable polymer that may not be particularly suitable for injection moulding without nucleating agents, because its crystallisation rate is too slow to allow cycle times typical of common thermoplastics such as PS [42]. Stress induced PLA crystallisation is actually better exploited by processes such as fibre spinning or the biaxial orientation of film.

6.2.4 Fibre spinning Melt spinning, dry spinning and wet spinning are the most common commercial processes for making fibres. Melt spinning can only be applied to polymers stable enough, at temperatures above their melting point, to be extruded in the molten state without degradation. The process of orientation or drawing into fibre form can improve the properties of crystalline polymers. Increased strength, stiffness and dimensional stability of synthetic fibres can be attained using this processing. PLA fibres are commercially available from NatureWorks LLC (Ingeo®) in the USA and Kanebo Chemical Industries (Lactron) in Japan. PLA fibre properties compare well with both PET and rayon fibres, as reported in Section 6.1.5, Table 6.3. Fibre properties such as tensile strength and elongation depend on the conditions applied to the polymer during the spinning process [103]. Polymer degradation takes place during the melt spinning process even when using a dry polymer with less than 0.005% water content [104]. Fibres produced by dry spinning undergo very slight degradation. PLA can be spun both in a high-speed spinning process with a take-up velocity of up to 5,000 m/min and in a spin drawing process up to a draw ratio of 6 [105].

6.3 Industrial applications As already stressed in the Introduction, biodegradable polymers must not be a simple replacement for traditional plastics. They have to be used as an opportunity to redesign applications by focusing on the efficient use of resources and tending towards the elimination of waste, by transforming local issues into business opportunities

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and by developing a systemic vision to counterbalance the management culture that has contributed to the dissipative growth model we now are living in. The fundamental criterion needed to avoid any aggravation of the situation and to reverse the trend, is the efficient use of resources, in the knowledge that only a type of growth that restores the central focus on the territory and knowledge economy, the cascading model, and absence of waste and rejects, will lead to continuous harmonious growth. A similar approach requires the selection of standards which have to go, beyond the products, towards the systems. The objective should not be to maximise market volumes, but the local regeneration from an environmental, social and economic view point, promoting a cultural jump towards a system-based economy. For this purpose industry and institutions should select stringent quality rules to guarantee: a) compostability and biodegradability in different environments, b) nontoxicity of products, c) a low environmental impact for the whole product life cycle, and d) a continuous improvements programme in terms of the quality of raw materials, renewability level, production chain, in use efficiency and end of life options. A PRO-BIP Report of 2009 [106] identified the following main applications for compostable and biodegradable polymers: – Shopping and composting bags (36%) – Fruit and vegetable bags (21%) – Films for food packaging (9%) – Catering (cutlery plus laminated paper) (11%) – Extrusion coating of paper (15%) – Agricultural films (8%) The EU average market potential predicted for 2020 is estimated to be approximately 2 million tonnes/year, ranging between 1.5 and 4 million tonnes/year for the different scenarios listed above. Besides the applications identified above, several others have to be mentioned such as foam-cushioning materials (i.e., starch-based loose fills and starch- or PLAbased shape-moulded parts for electric and electronic applications), fishing nets, nonwoven fabrics for wipes and hygiene applications, auxiliary products for agriculture and greenhouses, bioresorbable prostheses in medical applications, slow release systems for active substances and so on. In the following paragraphs a few applications which are more representative of the systemic advantages activated by biodegradable polymers will be analysed.

6.3.1 Compost bags Compostable bags for organic waste have been one of the most natural applications for biodegradable polymers. The further development of this market is strictly related

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to the waste management policies adopted by public authorities in the various regions of the world and to the availability of efficient composting facilities. In Europe, the demand for compostable bags is driven by the European Council ban on landfilling or incinerating waste with more than 10% organic content. Germany bans anything from landfill with more than 5% organic content. As a result of these regulations, composting infrastructures have been highly developed in many EU countries [107]. Organic recycling is well established in Germany, Austria, Switzerland, Italy, The Netherlands, Belgium, Norway and, more recently, in the UK. Organic recycling is also established in California, in a few other areas of the USA, and in some areas of Canada. However, a big difference exists in the models which are adopted for the separate collection of organic wastes, and in their efficiency in diverting organic waste from the remaining waste. Whereas in Germany, organic waste is collected separately and is mainly constituted of green waste, the Italian model maximises the diversion of all food waste, preventing hygiene problems with aerated bins and biodegradable bags, as well as with higher collection frequency. Norway, Austria, Switzerland, the city of San Francisco, and more recently some UK areas, adopted a model similar to the Italian one. In Italy, DL152/2006 established that separate collection should constitute 65% of the total by 2012. It also defines ‘compost’ as the products coming from separate collection only. The organic waste has to be collected in containers to be emptied, or in compostable and biodegradable bags, certified according to the EN 13432 norm, or in paper bags. In 2013, the total volume of organic waste collected separately in Italy was 5 million tonnes, approximately twice the 2.6 million tonnes collected in 2006 and the quality of compost improved significantly, becoming a good product for farmers. The adoption of compostable bags and aerated bins by communities has contributed greatly to the improvement of organic waste quality and to its diversion from landfills. In Italy, the main compostability logo is given by the Italian Composters Consortium (Consorzio Italiano Compostatori) which certifies compliance with the EN 13432 norm and with the Italian composting plants at the same time. The development of composting has increased the consumption of compostable bags for collecting organic kitchen and garden waste. The first community to introduced compost bags in its waste system was Furstenfeldbruck, Bavaria in 1992, using a Mater-Bi® starch-based material produced by Novamont. Compost bags have been marketed in Germany since 1995. They are sold in supermarkets, but also by local authorities and composting plants. A compostability logo based on the German Institute for Standardisation (Deutsches Institüt für Normung) 54900 certification was introduced in Germany. In the UK, the lack of industrial composting infrastructures has promoted the compliance of bags with home composting standards such as ‘OK Compost Home’ developed by Vinçotte. Today, suppliers of compost bags are present in the EU and the USA; the widest range of producers are in Italy and Germany.

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Demand for biodegradable polymers in compost bag applications is also growing, at a lower rate, in the USA, although the most significant need for compost bags in the USA is to collect garden waste. Garden waste is collected by four different methods: in paper bags, in PE bags, in biodegradable bags or mechanically in bulk. The advantage of biodegradable bags in this application is that they can be simply disposed of, along with the waste, in a composting facility. Nonbiodegradable plastic bags have to be emptied and then disposed of separately. Paper bags tend to absorb moisture from the waste and then lose their strength as a result. In addition, paper bags tend to take up more shipping volume than plastic bags. In spite of some of these performance flaws, the majority of compost bags used in the USA are made from paper due to price advantages.

6.3.2 Carrier bags Thin single-use carrier bags are considered a clear case of over packaging all over the world. They are mostly used just once, which is a waste of resources and can lead to a litter problem. Carrier bags are airy, and tend to fly away and disperse into the environment, as shown by several studies. Bags are the highest ranking of the ‘top 10ʹ marine litter items, as reported in the United Nations Environment Programme’s Report ‘Marine Litter: A Global Challenge’ [108]. They are strongly resistant to biodegradation and tend to build up in the marine environment if not properly disposed of. In the long term, plastic bags are fragmented by the mechanical erosion caused by waves and marine currents, leading to the formation of microscopic fragments (the so-called ‘plastic soup’). The toxic chemicals present in the sea tend to be adsorbed onto the plastic fragments and concentrate on them. Because the microscopic plastic fragments are swallowed by fish and marine mammals as if they were plankton, there is a real risk of toxic chemicals entering the food chain, carried by the plastic fragments themselves. Plastic carrier bags are also a problem in the organic recycling of biowaste (kitchen and garden waste) in countries where waste streams are collected separately. The problem is that, whenever there are separate collections of biowaste in place (and this is an unwavering trend in Europe), the use of plastic carrier bags is critical, because they are not biodegradable. The organic recycling of biowaste requires plastic-free streams in order to assure high recycling rates. Plastic carrier bags are not ‘multipurpose’ waste bags, but contaminants of biowaste. Worldwide, all these factors have generated a series of initiatives intended to reduce the consumption of single-use carrier bags. Many retailers, committed to reducing their businesses’ environmental impacts, have tried to move towards more sustainable solutions. Specific legislation has also been introduced in some countries in order to speed up this shift in consumption habits, and various new laws have been announced.

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In Italy, a strategy was launched in 2011 aimed at eliminating the use of thin, nonbiodegradable single-use plastic bags, leaving only bags made from genuinely sustainable, thicker plastic and compostable materials, conforming to the harmonised European standard on compostable packaging (EN 13432), on the market. The Italian approach to single-use carrier bags can be considered an important case study whose initial results have been studied [109] and the implications of which should be fully assessed. The first lesson is that consumers are ready to change their habits quickly in order to adopt more sustainable behaviours, following a law promoting packaging prevention. Italians have been encouraged to adopt behaviours that have a positive impact on waste management. A study has shown that the use of single-use carrier bags dropped significantly (by 50%) after the strategy was enforced. Second point: the fewer single-use carrier bags in circulation, the lower the risk of littering. Therefore, the restriction of single-use carrier bags helps litter prevention. Fewer resources are consumed, less waste needs to be recovered and less pollution is produced. Third point: only biodegradable, compostable carrier bags can still be sold by Italian retailers as single-use bags, as a complementary tool, together with the reusable bags. The use of compostable carrier bags has very interesting consequences. There have been improvements in biowaste collection and recycling. After their first use, biodegradable compostable carrier bags can be reused as ‘multipurpose’ waste bags and are suitable for collecting residual waste (any waste that cannot be separated before collection), and biowaste (e.g., kitchen waste), This is usually well communicated to consumers, using catchphrases such as: ‘use and reuse for the separate collection of waste’ and similar slogans printed on the bags which become a vehicle of education. This approach is improving the quality and quantity of biowaste collection and recycling. Fewer nonbiodegradable plastics are contaminating compost. The risk of a nonbiodegradable bag being improperly used to collect biowaste is eliminated if the householder only receives thin biodegradable compostable bags and reusable thick bags. This in turn improves the quality of organic recycling and brings important environmental benefits. Plastic-free, high- quality compost maintains the fertility of the soil from which bioplastics originate, in a virtuous ‘cradle-to-cradle’ (or, strictly speaking, soil-to-soil) loop. This effect has been demonstrated in specific studies: impurities have decreased by 8% [110] and as a result compost is less contaminated by plastic waste. From a life cycle assessment viewpoint, this means substantial reductions of up to 30% in greenhouse gas emissions, mainly linked to the saving of the energy needed to recover and dispose of the plastic scraps. The biodegradable materials obtainable from starch modified with aliphatic or aliphatic/aromatic polyesters, discussed in Section 6.1.6, proved to be particularly suitable for the production of shopping bags and are today leading this sector.

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6.3.3 Mulch films The most important application of plastic in agriculture is represented by mulch films, which farmers use to cover their fields in various situations. Mulch films provide many advantages: increase the crop yield, eliminate the growth of weeds, cover soil fumigated with fumigants, save water consumption thanks to moisture retention, maintain the soil structure and avoid soil erosion (especially in windy areas). The dominant material used for agricultural films is LDPE. However, traditional mulch films present disadvantages such as their environmental impact on the agrosystem and the need for proper disposal at the end of their usage in the field. Removing plastic films represents a cost for the farmer, even in the case of mechanical removal, and recovered films are heavily contaminated with soil and biological waste. For these reasons, the substitution of mulch film made of traditional plastics with biodegradable mulches having an outdoor service life which matches the crop duration and which would later be incorporated by the agricultural system appears a very attractive solution [111]. After the end of the growing season, the mulch material would be ploughed into the soil where soil microorganisms should completely biodegrade the mulch into CO2 and water within a reasonable period of time. A number of commercially available biodegradable polymers and polymer blends can be employed to produce biodegradable agricultural mulches [112]. Studies carried out by Novamont on Mater-Bi®-based mulch films, during the 2002 EU Project Bioplastic, concluded that the material is biodegradable in a timeframe compatible with the agricultural cycle, and has no negative impact on the soil quality, on plants or on beneficial organisms living in the soil [113].

6.3.4 Other applications Biodegradable polymers can find high added value applications in the medical field. To be used as biomaterials, polymers must possess biocompatibility, bioabsorbability and mechanical resistance [114]. Current applications include surgical implants and plain membranes in vascular or orthopaedic surgery, as well as the controlled release of drugs. PLA is the most widely used material for medical applications. Other possible outlets are offered by the hygiene sector including, nappy back sheets, cotton swabs, disposable razors and so on. Provided that biodegradable polymers become widely accepted in composting systems and their prices decrease, a high potential demand exists in Europe for biodegradable polymers in applications such as food packaging, and disposable dishes and cutlery. In fast food restaurants, canteens, town festivals, sport events, feasts and so on, disposable tableware is usually distributed to the guests in place of traditional, durable tableware. Thus, plastic cutlery, plastic or laminated paper dishes, plastic or laminated paper cups, foam containers, paper and plastic bottles, when contaminated

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with food, are produced as waste which is difficult to be recycled and at the same time limits the quality and quantity of the organic recycling of food waste. When reusable cutlery cannot be used, it has been shown that by using biodegradable and compostable plastic cutlery, it is possible to collect the mixed waste as a whole homogeneous fraction and recover it by composting, or by anaerobic digestion followed by composting, thus avoiding residues being put in landfill or incinerated [115]. Besides mulch films, other potential agricultural applications for biodegradable polymers include devices for the controlled release of agrochemicals (such as fertilisers, soil applied active ingredients, insect pheromones and so on), disposable articles such as plant pots, composting containers and bags, envelopes of ensilage, seed trays and so on.

6.4 Market evolution In the 1990s, the USA was the dominant market for biodegradable polymers, accounting for approximately one-half of the world’s consumption, and this was primarily due to the demand for biodegradable loose fill in the USA; Western Europe reached approximately 40% and Japan accounted for less than 10%. In 1998, over 60% of the world’s production capacity was located in Europe which is, at present, the largest biodegradable polymer-consuming region, with approximately half of the global market. In the near future, Brazil, China and Thailand are expected to become some of the top producers of bioplastics in the world. According to a market study published by European Bioplastics on 16th December 2013, the Global Annual Production Capacity of biobased plastics has grown from approximately 1 million tonnes in 2010 to 1.4 million tonnes in 2012, and it is expected to grow to approximately 6.2 million tonnes in 2017 [116]. This is in fact still a minuscule fraction (0.5%) of the total world production of plastics, amounting to approximately 300 million metric tonnes in 2012 and absorbing 8−9% of the total crude oil production (5% directly and 4% for the processing energy). Table 6.8 shows the volumes for biobased plastics, divided into nonbiodegradable and biodegradable products, for the period 2010−2017. A 24% growth rate of biodegradable plastics is observed from 2011−2012 and it is expected that the market will continue to steadily grow up to 2017. A much higher growth rate is expected for biobased/nonbiodegradable products, due to the trend of shifting the sourcing of traditional monomers from petrochemical to renewable sources (e.g., ethylene from bioethanol). However, it is worth noting that just substituting the sourcing of traditional monomers to produce the same conventional polymers does not actually solve the environmental problems related to the

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Table 6.8: Global production capacities of bioplastics in the years 2010−2017. Year

Nonbiodegradable

Biodegradable

Total

2010

674,000

342,000

1,016,000

2011

675,000

486,000

1,161,000

2012

791,000

604,000

1,395,000

2017

5,185,000

1,000,000

6,185,000

Adapted from European Bioplastics Annual Report in Global Annual Production Capacity of Biobased Plastics, Institute for Bioplastics and Biocomposites, University of Hannover, Hannover, Germany, 16th December 2013 [116].

durability of such products. Biodegradable plastics, whether produced from renewable or nonrenewable sources, are in fact a real answer to specific needs like the ones illustrated in this chapter, when organic recycling is a preferred option and the risk of dispersion in the environment is high. In the case of shopping bags, for example, the adoption of renewable nonbiodegradable PE does not change the risks connected with littering and the probability of littering does not improve the recycling figures that, in this case, are very low (notwithstanding the EU Packaging Directive 64/92) and, moreover, does not negate the fact that shopping bags are one of the main pollutants of organic waste. The factors contributing to the steady growth of these materials are considered to be: – Continuous improvement of product properties and processing. – Growing interest of consumers in sustainable and ecological solutions (e.g., eco– friendly packaging) and in reduction of greenhouse gas emissions. – Increasing support of public authorities (at local and state level) to biodegradable plastics as a means to answer the compelling issue of solid waste disposal. – Existence and expansion of large-scale composting facilities. The share of by-products of the total bioplastic production capacity in 2011 is reported in Table 6.9. The market of starch-based biodegradable plastics has the second largest share, in terms of volume, with the main applications being represented by compost bags, shopping bags, loose-fill packaging and mulch films. Novamont recently started up its seventh line dedicated to the production of Mater-Bi® film grades in Terni, and claims a production capacity of 120,000 tonnes/ year. Established in 1990, the company started at the Fertec research facility set up by Montedison in 1989, acquired the Warner-Lambert’s bioplastic IP portfolio in 1997 (Warner-Lambert suspended the manufacture of starch-based materials in 1993) [118] and the Eastman’s Eastar Bio patents in 2004. Nowadays, Novamont holds a patent

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Table 6.9: Global production capacity of bioplastics by type (total capacity 1,161,200 tonnes in 2011). Bioplastic type

Volume share (%)

Biobased/nonbiodegradable

58.5

PET (bioPET)

39.0

PE (bioPE)

17.5

Polyacrylate (biopolyacrylate)

1.5

Others

0.5

Biodegradable

41.5

PLA

16.0

Starch blends

11.0

Biodegradable polyesters

10.0

Hydrate cellulose foils

2.5

PHA

1.5

Others

0.5

Adapted from European Bioplastics Annual Report in Global Annual Production Capacity of Biobased Plastics, Institute for Bioplastics and Biocomposites, University of Hannover, Hannover, Germany, 16th December 2013 [116].

portfolio which includes over 100 patent families and 800 internationally registered patents. Thanks to the upstream integration approach Novamont is also the producer of Origo-Bi® biodegradable polyesters with its proprietary technology and a capacity of approximately 70,000 tonnes/year. It has also developed technologies for two of the building blocks used in Origo-Bi. Azelaic acid and other chemicals will be produced by Matrica, the JV between Novamont and Versalis whose start up is forecasted for the second half of 2014. The first 30,000 tonnes/year bio 1,4-BDO plant, produced via the direct fermentation of sugars according to Genomatica technology, is under construction and will start production in mid2015. In 2005, the German company Biotec was jointly purchased by Sphere and Stanelco of the UK, changing the name to Biosphere; in 2013, Sphere acquired Stanelco’s shares taking control of Biosphere. Under the Bioplast trademark, the Sphere group produces various grades of bioplastic resins by blending starch with copolyesters and other materials. The group has a compounding facility, Biotec, in Germany and film blowing facilities in France and Spain; they manufacture biodegradable carrier bags, bin liners, catering objects and refuse bags. The claimed production capacity is 20,000 tonnes/year. The Australian research company Plantic Technologies Ltd offers products for thermoforming, obtained from nongenetically modified, hydroxypropylated corn starch with a high amylose content. Its production capacity is 7,500 tonnes/year [118].

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In 2008, Cardia Bioplastics Ltd (Australia) acquired the Biograde Limited’s plastic business; their Product Development and Manufacturing Centre is located in Nanjing, China. Their biomaterial is obtained by mixing starch and biodegradable polyesters in the presence of a transesterification catalyst [119]. In spite of intensive research efforts and the many potential applications, the world production of PHA is still low; according to European Bioplastics, the worldwide capacity production was estimated to be 88,000 tonnes/year in 2010. The leading company is Metabolix, which developed the production of PHA starting from genetically modified cultures. However, as recalled in Section 6.1.4, the 50,000 tonnes/ year production of Mirel stopped in 2012, following the end of the MDA-Metabolix JV (Telles). High production cost remains the primary obstacle to the full market exploitation for this class of natural biodegradable products. On the other hand, due to its reasonable price (approximately 2 €/kg) [46] and availability on the market, PLA is currently one of the biodegradable polymers exhibiting the highest potential for further development. PLA demand will benefit from the development of resins and compounds with enhanced performance for more durable applications such as fibres, and automotive and electronic parts. The product leader company is NatureWorks LLC (a Cargill/PTTGC joint venture), with 150,000 tonnes/ year of capacity in the USA, and a further 150,000 tonnes/year planned in Thailand [120]. Purac (The Netherlands) has approximately 75,000 tonnes/year in Thailand [120]. Other companies active in the PLA market are Mitsui Chemicals, Mitsubishi, Shimadzu and Teijin in Japan, Futerro (Total/Galactic) in Belgium and Zhejiang Hisun Biomaterials in China. Ecoflex® (BASF) and Origo-Bi® (Novamont), belonging to the family of biodegradable aliphatic/aromatic copolyesters, have also gained significant growth over the last 10 years, thanks to the possibility of being complexed with starch. These polymers offer improved properties at a good cost/performance ratio. The current capacity of Ecoflex® is approximately 60,000 tonnes/year. Similar compounds from totally aliphatic and aromatic monomers of vegetable origin are under development. Their success will depend to a large extent on the increasing acceptance of the sustainability concept and on the economics of renewable feedstock sourcing.

6.5 Conclusions After more than 30 years of research and development, it is now widely accepted that biodegradable polymers have achieved specific uses due to their unique performance and can adequately replace traditional plastic items in different application sectors where biodegradability is required or preferred: composting (bags and sacks), packaging (soluble foams for industrial packaging, film wrapping, laminated paper, food

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containers), goods transportation (shopping bags), agriculture (mulch films, nursery pots, envelopes of ensilage, seed trays, plant labels), fast-food tableware (cutleries, cups, plates, straws and so on), medical and pharmaceutical (surgical and orthopaedical devices, slow release of drugs), and hygiene (nappy back sheets, cotton swabs and so on). In particular, biodegradable bioplastics are able to significantly reduce the environmental impact in terms of energy consumption and greenhouse effect. Once they are composted through the action of living organisms, these materials offer ‘cradle-to-cradle’ solutions through the entire agroindustrial, nonfood chains. They can also offer possible alternatives to traditional plastics when recycling is unpractical or uneconomical, or when environmental impact has to be minimised.

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[18] J.G. Errington, A.J. Hornsey, T.J. Chapman, M.L. Wake and P.J. Quinn, inventors; Blue Marble Polymers Limited, assignee; WO 2005/051628, 2005. [19] K. Sudesh, H. Abe and Y. Doi, Progress in Polymer Science, 2000, 25, 1503. [20] E. Pollet and L. Averous in Biopolymers – New Materials for Sustainable Films and Coatings, Ed., D. Plackett, John Wiley and Sons, New York, NY, USA, 2011, p.65. [21] J.M. Liddel in Chemical Industry: Friend to the Environment?, Ed., J.A.G. Drake, Special Publication No.103, Royal Society of Chemistry, Cambridge, UK, 1992, p.10. [22] Anon, Modern Plastics, 1981, 58, 7, 90. [23] P.A. Holmes, Physics Technology, 1985, 16, 32. [24] J.N. Baptist, inventor; W.R. Grace, assignee; US3036959, 1962. [25] J.N. Baptist, inventor; W.R. Grace, assignee; US3044942, 1962. [26] J.N. Baptist and F.X. Werber, inventors; W.R. Grace, assignee; US3107172, 1963. [27] L. Hughes and K.R. Richardson, inventors; Imperial Chemical Industries, assignee; US4433053, 1984. [28] P.A. Holmes, S.H. Collins and L.F. Wright, inventors; Imperial Chemical Industries, assignee; US4477654, 1984. [29] K.R. Richardson, inventor; Imperial Chemical Industries, assignee; EP114086B1, 1988. [30] G.M. Bohlmann and R.G. Bray in Biodegradable Polymers, Process Economics Program Report No.115C, SRI Chemical and Health Business Services, Menlo Park, CA, USA, 1998. [31] Y. Poirier, D.E. Dennis, K. Klomparens and C. Somerville, Science, 1992, 256, 520. [32] I. Noda, inventor; The Procter & Gamble Co., assignee; EP0846184B1, 2002. [33] M. Roberts, Chemical Week, 1994, 154, 23, 23. [34] Monsanto Annual Report, Monsanto Company, St. Louis, MO, 1996, p.12. [35] ADM-Metabolix Press Release, Metabolix and ADM to Produce MirelTM, Cambridge, MA, USA, 23rd April 2007. http://ebook.future4.org/Mirel-s480. [36] Metabolix, Inc. Press Release, Metabolix Announces Termination of Telles Joint Venture, Cambridge, MA, USA, 12th January 2012 http://ir.metabolix.com/releases.cfm. [37] M. Koller, A. Salerno and G. Braunegg in Bio-Based Plastics: Materials and Applications, Ed., S. Kabasci, John Wiley & Sons, Chichester, UK, 2014, 7, p.137. [38] C.H. Holten in Lactic Acid Properties and Chemistry of Lactic Acid and Derivatives, Verlag Chemie, Vienna, Germany, 1971. [39] S.J. Huang in Encyclopedia of Polymer Science and Engineering, Eds., H.F. Mark, N.M. Bikales, C.G. Overberger, G. Menges and J.I. Kroschwitz, Wiley & Sons, New York, NY, USA, 1985, 2, 220. [40] X. Zhang, U.P. Wyss, D. Pichora and M.F.A. Goosen, Journal of Macromolecular Science: Pure and Applied Chemistry A, 1993, 30, 12, 933. [41] C.G. Pitt in Biodegradable Polymers and Plastics, Eds., M. Vert, J. Feijen, A. Albertsson, G. Scott and E. Chiellini, Royal Society of Chemistry, Cambridge, UK, 1992, p.7. [42] J. Lunt, Polymer Degradation and Stability, 1998, 59, 145. [43] P.R. Gruber and co-workers, inventors; Cargill, assignee; WO95/9879, 1995. [44] P.R. Gruber, E.S. Hall, J.J. Kolstad, M.L. Iwen, R.D. Benson and R.L. Borchardt, inventors; Cargill, assignee; US5357035, 1994. [45] I.D. Fridman, J. Kwok, R.J. Downey and S.P. Nemphos, inventors; Camelot Technologies, assignee; US5357034, 1994. [46] W.G. O’Brien, L.A. Cariello and T.F. Wells, inventors; Ecological Chemical Products, assignee; US5521278, 1996. [47] K. Enomoto, M. Ajioka and A. Yamaguchi, inventors; Mitsui Toatsu Chemicals, assignee; US5310865, 1994. [48] News-releases/purac-sulzer-chemtech-and-synbra, PR Newswire, London, UK. http://www.prnewswire.com.

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[49] L. Avérous and E. Pollet in Environmental Silicate Nano-Biocomposites, Ed., Averous and E. Pollet, Springer-Verlag, London, UK, 2012, p.13. [50] Technical Bulletin 180904, Fiber and Fabric Properties, NaureWorks LLC, Minnetonka, MN, USA. http://www.natureworksllc.com. [51] R.D. Lundberg and co-workers, inventors; Union Carbide, assignee; Canadian 900092, 1972. [52] CAPA Product Data Sheets, Perstop Group, Perstop, Sweden, 1st August 2013. https://www.perstorp.com/en/Products. [53] T. Fujimaki, Polymer Degradation and Stability, 1998, 59, 209. [54] E. Takiyama, T. Fujimaki, S. Seki, T. Hokari and Y. Hatano, inventors; Showa Highpolymer, assignee; US5310782, 1994. [55] R.J. Müller, I. Kleeberg and W-D. Deckwer, Journal of Biotechnology, 2001, 86, 87. [56] C.M. Buchanan, R.M. Gardner and A.W. White, inventors; Eastman Chemical, assignee; US5446079, 1995. [57] F.G. Gallagher, inventor; DuPont, assignee; WO95/14740, 1995. [58] F.G. Gallagher, C.J. Hamilton, S.M. Hansen, H. Shin and R.F. Tietz, inventor; DuPont, assignee; US5171309, 1992. [59] F.G. Gallagher, C.J. Hamilton, S.M. Hansen, H. Shin and R.F. Tietz, inventor; DuPont, assignee; US5171308, 1992. [60] R.F. Tietz, inventor; DuPont E.I. de Nemours, assignee; US5097005, 1992. [61] U. Witt, R.J. Müller and W-D. Deckwer, Journal of Environmental Polymer Degradation, 1997, 5, 2, 81. [62] V. Warzelhan, G. Pipper, U. Seeliger, P. Bauer, U. Pagga and M. Yamamoto, inventors; BASF AG, assignee; WO96/25446, 1996. [63] Warzelhan, G. Pipper, U. Seeliger, P. Bauer, D.B. Beimborn andYamamoto, BASF, assignee; WO96/25448, 1996. [64] C. Bastioli, T. Milizia, G. Floridi, A. Scaffidi Lallaro, G.D. Cella and M. Tosin, inventors; Novamont, assignee; WO2006/097353, 2006. [65] C. Bastioli, T. Milizia, G. Floridi, A. Scaffidi Lallaro, G.D. Cella and M. Tosin, inventors; Novamont, assignee; WO2006/097354, 2006. [66] C. Bastioli, T. Milizia, G. Floridi, A. Scaffidi Lallaro, G.D. Cella and M. Tosin, inventors; Novamont, assignee; WO2006/097355, 2006. [67] C. Bastioli, T. Milizia, G. Floridi, A. Scaffidi Lallaro, G.D. Cella and M. Tosin, inventors; Novamont, assignee; WO2006/097356, 2006. [68] U. Witt, R.J. Müller and W-D. Deckwer, Macromolecular Chemistry and Physics, 1996, 197, 1525. [69] U. Witt, M. Yamamoto, U. Seeliger, R-J. Muller and V. Warzelhan, Angewandte Chemie, International Edition, 1999, 38, 10, 1438. [70] Y. Watanabe, N. Hameda, M. Morita and Y. Tsujisaka, Archives of Biochemistry and Biophysics, 1976, 174, 575. [71] J.M. Mayer and D.L. Kaplan, Trends in Polymer Science, 1994, 2, 7, 227. [72] S. Matsumura, S. Maeda, J. Takahashi and Yoshikawa, Kobunshi Robunshu, 1988, 45, 4, 317. [73] N. Hodgkinson and M. Taylor, Materials World, 2000, 8, 4, 24. [74] Anon, British Plastics and Rubber, 1999, 11, 13. [75] R.L. Jack, inventor; British Technology Group, assignee; GB2291831B, 1997. [76] G.J.L. Griffin in Proceedings of the 166th Meeting of the American Chemical Society, Division of Organic Coatings and Plastics Chemistry, Chicago, IL, USA, 1973, p.159. [77] G.J.L. Griffin, Advances in Chemistry Series, 1974, 134, 159. [78] G.J.L. Griffin, Pure & Applied Chemistry, 1980, 52, 399. [79] C.L. Swanson, R.P. Westhoff, W.M. Doane and F.H. Otey, Polymer Preprints, 1987, 28, 2, 105. [80] G.F. Fanta and F.H. Otey, inventors; US Department of Agriculture, assignee; US4839450, 1989.

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[109] W. Ganapini in Bioplastics: A Case Study of Bioeconomy in Italy, E. Ambiente, Milan, Italy, February 2013, p.29. [110] M. Centemero in The Important Presence of Plastics in the Separate Collection of Organic Waste, Consorzio Italiano Compostatori’s Report on Organic Quality, Consorzio Italiano Compostatori, Rome, Italy, 12th January 2012. [111] L. Martin-Closas and A.M. Pelacho in Biopolymers-New Materials for Sustainable Films and Coatings, Ed., D. Plackett, John Wiley & Sons, New York, NY, USA, 2011, p.277. [112] D.G. Hayes, S. Dharmalingam, L.C. Wadsworth, K.K. Leonas, C. Miles and D.A. Inglis in Degradable Polymers and Materials: Principles and Practice, 2nd Edition, Ed, K. Khemani, ACS Symposium Series 1114, American Chemical Society, Washington, DC, USA, 2012, 13, 202. [113] S. Guerrini in Environmental Training Community Newsletter, Sino-Italian Sustainable Development Community, Venice, Italy, 2006, NL 04, 36, http://www.sdcommunity.org/ news-a-publications. [114] I. Vroman and L. Tighzert, Materials, 2009, 2, 307. [115] F. Razza, M. Fieschi, F. Degli Innocenti and C. Bastioli, Waste Management, 2009, 29, 1424. [116] European Bioplastics Annual Report in Global Annual Production Capacity of Biobased Plastics, Institute for Bioplastics and Biocomposites, University of Hannover, Hannover, Germany, 16th December 2013. [117] C. Tollefson, Chemical Marketing Reporter, 1993, 224, 20, 9. [118] L. Yu, G.B.Y. Christie and S. Coombs, inventors; Food and Packaging Centre Management Ltd, assignee; WO00/36006, 2000. [119] C. Changping, inventor; Biograde PTY LTD, assignee; WO2007/012142, 2007. [120] Morgan Stanley Research in Chemicals: ‘Green is Good’ – The Potential of Bioplastics, New York, NY, USA, 22nd August 2012.

Kumar Sudesh, Yoshiharu Doi, Paolo Magistrali and Sebastià Gest´ı Garcia

7 Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHA) is the term given to a family of polyesters produced by microorganisms. The most well-known among them is the thermoplastic poly(R-3hydroxybutyrate) (P(3HB)). In this chapter, an attempt has been made to summarise the present state of research and development of these interesting microbial polyesters. Several types of PHA homopolymers and copolymers with useful physical properties have been identified. It is now possible to tailor-make these PHA using suitable carbon sources in both wild type and/or recombinant microorganisms. The basic principles underlying the biosynthesis of various PHA will be emphasised. This will include the major biochemical pathways involved in the conversion of various carbon sources into suitable monomers that are polymerised by the key enzyme of PHA biosynthesis, PHA synthase. In addition, the development of various potential methods for the largescale production of PHA will be compared. The most attractive property of PHA is its biodegradability, which is a crucial factor in today’s polymer technology. Accordingly, this chapter will also include a brief overview of the biodegradation of PHA. Finally, the many potential applications of PHA, especially in the medical field, will be discussed.

7.1 Introduction PHA are a family of linear polyesters of 3, 4, 5 and 6-hydroxyacids, synthesised by a wide variety of bacteria through the fermentation of sugars, lipids, alkanes, alkenes and alkanoic acids. In the presence of an abundant carbon source when other essential nutrients such as oxygen (O2), phosphorous (P) or nitrogen (N2) are limited, many microorganisms usually assimilate and store PHA for future consumption [1]. They are found as discrete cytoplasmic inclusions in bacterial cells. These polymeric materials are able to be stored at high concentrations within the cell since they do not substantially alter its osmotic state [2]. Once extracted from the cells, PHA exhibit \ thermoplastic and elastomeric properties. PHA are recyclable, natural materials and can be easily degraded to carbon dioxide (CO2) and water. The assimilated carbon sources are biochemically processed into hydroxyalkanoate (HA) units, polymerised and stored in the form of water-insoluble inclusions in the cell cytoplasm. The ability to carry out this polymerisation process is dependent on the presence of a key enzyme known as PHA synthase. The product of this enzyme is a high molecular weight (MW) optically active crystalline polyester. The latter is https://doi.org/10.1515/9781501511967-007

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intriguingly maintained in an amorphous state in vivo [3]. Upon isolation however, this microbial polyester is a crystalline thermoplastic with properties comparable to that of polypropylene (PP) [4, 5]. PHA has a much better resistance to ultraviolet degradation than PP but is less solvent resistant. The research and development concerning PHA can be traced back to the beginning of the 20th century and a historical overview is available elsewhere [6]. Tremendous progress has been made over the past four decades, mainly motivated by the environmentally friendly properties of PHA. Unlike the present commodity plastics, PHA are produced from renewable resources. Petrochemical-based plastics currently in wide use are being regarded as a major threat of pollution. Plastics have found widespread application in our daily life because they are chemically inert and durable. Over the years however, these properties gave rise to the accumulation of plastic materials in our environment. Now, these nonbiodegradable polymers contribute to the pollution of the environment and therefore some attempts at recycling have been made. Nevertheless, a considerable amount ends up on beaches, in the oceans or clogging landfill sites. Attempts to dispose of them by other means, i.e., incineration, produces different kinds of equally unacceptable pollution. These problems teach us that it is essential for mankind to develop and use materials that are compatible with our natural ecosystem. This has been the primary motivating factor in the research and development of PHA as a potential substitute for petrochemical-based plastics. PHA are biocompatible as well as biodegradable, and its degradation product, 3-hydroxyalkanoate (3HA) is a normal mammalian metabolite [7]. Considerable work concerning PHA is in progress in many developed countries such as the USA, Germany and Japan, where waste disposal is becoming an increasingly serious problem. Cost factors will be critical in determining whether in the long term, PHA can enter into widespread use in fields presently dominated by conventional commodity plastics. Research on this microbial polyester has been, and still is, a great challenge to scientists in the fields of biotechnology and polymer chemistry. The final goal is to be able to produce various kinds of PHA from renewable carbon sources not only in a cost-effective manner, but also to overcome the intrinsic brittleness and low thermal stability.

7.2 Production of polyhydroxyalkanoates Microorganisms in nature are capable of synthesising various types of PHA depending on the types of carbon sources available and the biochemical pathways that operate in the cell. It is now possible to synthesise various PHA homopolymers and copolymers that have a certain monomer composition. Synthesis of the PHA polymer chain takes place within the cytoplasm of the bacterial cell, within inclusions known as granules.

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PHA production using pure cultures adopts a two-stage batch production process [8], with an inoculum of bacteria being introduced into a sterile solution of trace metal nutrients and a suitable carbon source and nutrients in the first (growth) stage. In the second stage, an essential nutrient (such as N2, P or O2) is deliberately limited and PHA accumulation takes place. The properties of the final polymer depend on the mix of carbon feedstocks fed during accumulation, the metabolic pathways that the bacteria use for the following conversion into precursors and the substrate specificities of the enzymes involved. PHA production based on open mixed cultures has been proposed as a means of lowering production costs [9]. No reactor sterilisation is necessary and the culture is able to adapt to various complex (cheap) waste feedstocks [10]. Organisms with elevated PHA storage capacity are favoured by taking the culture through a number of aerobic feast/famine cycles [11]. This method relies on the removal of a portion of the consortium during each cycle (the organisms that are able to store carbon during feast conditions) and then use that stored carbon to grow during famine conditions. A major advantage of mixed culture PHA production is the opportunity to use real fermented wastes as feedstock (such as agricultural or food industry by-product feedstocks) [12] as opposed to synthetic volatile fatty acids [13], such as acetate, propionate, butyrate and valerate, thus lowering substrate costs.

7.3 The various types of polyhydroxyalkanoates PHA can be divided into two main groups based on the number of constituent carbon atoms in their monomer units, these being the short-chain-length (SCL) PHA that contain monomer units with 3–5 carbon atoms, and the medium-chain-length (MCL) PHA that contain monomer units of 6–14 carbon atoms [2]. The most common PHASCL are P(3HB) and poly(3-hydroxybutyrate-co-3- hydroxyvalerate) P(3HB-co-3HV). These materials have mechanical properties that are comparable to those of PP and polyethylene (PE), although they have much lower elongation atbreak and are more brittle. PHAMCL are flexible, have low crystallinity, tensile strength and melting point. There have been reports of several bacteria that are able to synthesise PHA containing both SCL- and MCL-monomers [14].

7.3.1 Poly(R-3-hydroxybutyrate) P(3HB) (Figure 7.1) was the first type of PHA to be identified [15–17] and is the most common PHA found in nature. Based on the MW of the biosynthesised P(3HB), they can be divided into three distinct groups, i.e., low MW P(3HB) [18–20], high MW P(3HB),[1, 7, 16] and ultrahigh molecular weight (UHMW) P(3HB) [21].

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CH3 O

*CH

O CH2

R-3HB

C x

x = 120–200; low-MW P(3HB) x = 1,000–20,000; high-MW P(3HB) x ~ 100,00; UHMW P(3HB)

Figure 7.1: Chemical structure of P(3HB).

The low MW P(3HB) which is also known as complexed-P(3HB) (c-P(3HB)) is an ubiquitous cell constituent that exists in eubacteria, archaebacteria and eukaryotes [22, 23]. Studies have also revealed the presence of c-P(3HB) in humans [24]. This c-P(3HB) consists of about 120−200 3-hydroxybutyrate (3HB) units and has a MW of about 12,000 Da [25]. Depending on the strength of their association with macromolecules, chloroform-soluble and chloroform-insoluble c-P(3HB) have been identified [26]. The former forms a weakly bound (noncovalent) complex with polyphosphate salts while the latter usually forms a strongly bound (covalent) complex with proteins. These complexes are thought to function as ion (Ca2+) transport channels across cell membranes and may also facilitate the uptake of extracellular deoxyribonucleic acid (DNA) material [26, 27]. In contrast to the low MW c-P(3HB), high MW P(3HB) is synthesised and accumulated in the form of water-insoluble inclusion bodies in the microbial cell cytoplasm. They serve as carbon and energy storage compounds for the microorganisms. The MW of this storage P(3HB) are in the range of 200,000 to 3,000,000 Da and the precise value depends on the microorganism and its growth conditions [28]. In the 1960s and 1970s, much attention was directed on the high MW P(3HB) because of its thermoplastic property. The production of UHMW P(3HB) (MW >3,000,000) has been achieved using a recombinant Escherichia coli (E. coli) cultivated under specific fermentation conditions [21]. Unlike the high MW P(3HB) which is characterised by stiffness and brittleness, the UHMW P(3HB) seems to show improved characteristics. Mechanical properties of the stretched and annealed UHMW P(3HB) films remained unchanged for 6 months at room temperature [29]. In addition, it was also found that films prepared from this UHMW P(3HB) were completely degraded at 25 °C in a natural freshwater river within 3 weeks [30]. Strong fibres with high tensile strength were produced using UHMW P(3HB). By two-step drawing, the tensile strength increased from 38 MPa to 1.32 GPa, with an elongation at break of 65% [31]. High MW P(3HB) (MW = 200,000−3,000,000) was the first type of PHA to be identified, and because of its widespread occurrence, considerable work has been carried out to determine its physical properties and explore its potential applications. It is well established that P(3HB) samples obtained from various biological sources were all characterised by exceptional stereochemical regularity. They are linear polyesters and their chiral centres possess only the R absolute configuration [D(−) in

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traditional nomenclature]. The biosynthesised P(3HB) is therefore perfectly isotactic and upon extraction from the microorganisms, has a crystallinity of about 55−80% with a melting point at around 180 °C [8, 32, 33]. P(3HB) can crystallise into two different crystal structures. The most common is the a-form, which is produced under the typical conditions of melt, cold or solution crystallisation. The molecules in the crystalline regions have the conformational structure corresponding to a 21 (G2T2)2 lefthanded helix (two monomers being present in every one helical twist), and the unit cell is orthorhombic with a space group of P212121 (D24) and with dimensions a = 0.576 nm, b = 1.320 nm and c = 0.596 nm [32, 34, 36]. Under strain conditions a metastable structure (called β-form) is induced. The unit cell is orthorhombic with unit cell parameters of a = 0.576 nm, b = 1.320 nm and c = 0.598 nm, and space group P212121 [37]. The β-form has a near completely extended chain conformation, while in the α-form the ester group shows a gauche conformation [34]. Despite having similar physical properties to PP [38], the P(3HB) homopolymer produced by microorganisms is rather brittle and thermally unstable [39]. The brittleness is due to the formation of large crystalline domains in the form of spherulites. The formation of large spherulites is a special property of this biologically synthesised P(3HB) probably because of its exceptional purity. This makes the microbial P(3HB) an ideal system for the study of spherulites [40] but is definitely a major drawback to the commercial use of this homopolymer [8]. The brittleness can, however, be reduced to a certain extent by using suitable processing conditions (such as adding suitable plasticisers), enabling the production of ductile films [41]. A pure P(3HB) is commercialised under the trademark of Biocycle® 1000 by PHB Industrial S.A., a joint venture started in 1992 between a sugar producer (Pedra Agroindustrial S.A. formerly known as Irmaos Biagi) and an alcohol producer (Balbo group). The installation is located in Serrana (São Paolo, Brazil) and the nominal capacity of the plant is 50 tonnes/year. The implementation of a commercial production plant with capacity for 3,000 tonnes/year is expected soon [42]. Another commercial grade is available from Biomer Biopolyesters.

7.3.2 Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) One of the most direct solutions for improving the mechanical properties of P(3HB) is achieved by the incorporation of a second monomer into the 3HB sequence. Compared with P(3HB), P(3HB-co-3HV) has decreased stiffness and brittleness, higher elongation at break, and increased tensile strength and toughness. Depending on the amount of the 3-hydroxyvalerate (3HV) comonomer in the polymer chain, a significant decrease in the melting point can be achieved, increasing the difference between the melting point temperature and the onset of thermal degradation, thus improving the processing window. 3HV was first identified as a member of the PHA family in the 1970s [1]. This eventually led to the development, by Imperial Chemical Industries (ICI), of a biosynthesis

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process capable of producing a random copolymer of P(3HB-co-3HV) (Figure 7.2) containing various amounts (0−30 mol%) of 3HV units [5, 8]. The microorganism selected for this fermentation process is a nonpathogenic Ralstonia eutropha (R. eutropha) strain (formerly known as Alcaligenes eutrophus), which grows on glucose. Propionic acid was added to the culture medium as a precursor carbon source which gives rise to the incorporation of 3HV units [5]. This, for the first time enabled the production of PHA with properties that can be altered by controlling the content of the second monomer (i.e., both Young’s Modulus and tensile strength decrease with an increase in 3HV content) [5, 43, 44]. The development of P(3HB-co-3HV) copolymers also led to the discovery of a unique cocrystallisation behaviour known as isodimorphism [43, 45, 46], where two or more comonomer units can exist in the crystal unit structure of the other monomer unit in the copolymer. No other forms/types of microbial PHA show this behaviour. The easiest way to control the content of 3HV units in the P(3HB-co-3HV) copolymer is by changing the concentration of the carbon source which contributes to the formation of 3HV units. Doi and co-workers found that by using a combination of butyric and pentanoic acids, R. eutropha (NCIB 11599) can be made to produce P(3HBco-3HV) copolymers containing a wide range (0−85 mol%) of 3HV units (Table 7.1). CH3 CH3 O

*CH

CH2

O CH2

C

x

O

R-3HB

*CH

O C

CH2

R-3HV

y

Figure 7.2: Chemical structure of P(3HB-co-3HV).

Table 7.1: Production of P(3HB-co-3HV) copolymers using R. eutrophaa. Carbon source (g/l) Butyric acid

Cell dry weight (g/l)

PHA contentb (wt%)

Pentanoic acid

PHA compositionc (mol%) 3HB

3HV

20

0

7

48

100

0

15

5

8

55

85

15

8

12

6

37

70

30

6

14

6

48

55

45

2

18

6

43

40

60

0

20

7

46

15

85

References

[48, 49]

R. eutropha cells were grown in a nutrient-rich medium for 24 h and then transferred to a nitrogenfree medium containing the above carbon sources. b PHA content in cells incubated for 48 h in nitrogen-free medium. c Composition determined by 1H-nuclear magnetic resonance (NMR). a

7 Polyhydroxyalkanoates 

 189

In addition, the P(3HB-co-3HV) copolymers were shown to have a statistically random distribution of 3HB and 3HV units [49]. The homopolymer of poly(R- 3-hydroxyvalerate) (P(3HV)) has also been produced biologically using wild type microorganisms such as Rhodococcus sp., [50] and Chromobacterium violaceum [51], and also by using recombinant R. eutropha [52]. Single crystals of these biologically synthesised P(3HV) were found to be more perfect than those of synthetic P(3HV) although they both have a square shape as opposed to the characteristic lath shape of P(3HB) crystals [53]. P(3HV) crystallises is an orthorhombic unit cell with a space group of P212121 (D24) as P(3HB) does, with dimensions a = 0.592 nm, b = 1.008 nm and c = 0.556 nm [54]. P(3HB-co-3HV) is commercialised by Tianan Biopolymer (i.e., EnmatTM Y1000P) and Shenzhen Ecomann Biotechnology (i.e., EM5231A).

7.3.3 Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) Another type of PHA copolymer that shows useful physical properties is poly(R3- hydroxybutyrate-co-4-hydroxybutyrate) P(3HB-co-4HB) (Figure 7.3). Like 3HB, 4hydroxybutyrate (4HB) is a normal mammalian metabolite. 4HB has been found in extracts of brain tissue from rats, pigeons and man [55]. Synthetic 4HB in the form of a sodium salt was first made available in the early 1960s [56]. Approximately two decades later, Doi and co-workers reported the assimilation of this compound by R. eutropha to produce a random copolymer of P(3HB-co-4HB) [57, 58]. Subsequent studies resulted in the production of P(3HB-co-4HB) having a wide range of 4HB content (Table 7.2) [59, 63]. Other carbon sources such as, 4-chlorobutyric acid [57], γ-butyrolactone, 1,4-butanediol, 1,6-hexanediol, 1,8-octanediol, 1,10-decanediol and 1,12-dodecanediol also resulted in the incorporation of 4HB units [64]. By increasing the content of 4HB in P(3HB-co-4HB), the physical property of the copolymer (based on solvent-cast films) changes from one that is characterised by high crystallinity to one that is a strong elastomer [60, 62]. In addition, an increase in the 4HB content up to about 70 mol% was accompanied by an increased rate of enzymatic degradation [62, 65]. Further increase in the 4HB content, however, decreased the rate of enzymatic degradation of P(3HB-co-4HB). In general, there is an increase in the rate of enzymatic degradation of PHA films following a decrease in its crystallinity [66]. For P(3HB-co-4HB) films, crystallinity decreases with an increase in the 4HB content up to about 70 mol%. Further increase in the 4HB content contributed to an increase in the copolymer film crystallinity [63]. CH3 O

*CH

O CH2

R-3HB

C

O x

O

CH2

CH2 4HB

CH2

C

y

Figure 7.3: Chemical structure of P(3HB-co-4HB).

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Table 7.2: Production of P(3HB-co-4HB) copolymers containing various 4HB content. Microorganism

R. eutropha

C. acidovoransa

Carbon source (g/l)

Cell dry weight (g/l)

PHA contentb (wt%)

PHA compositionc 3HB

4HB

10

51

100

0 10

3HBA

4HBA

20

0

12

6.4

8

52

90

8

9.6

7

43

82

18

0

20

5

16

67

33

2

8

3

27

44

56

1.5

8.5

3

26

27

73

0.5

9.5

3

23

17

83

3

17

0

100

0

10

References

[47, 62]

[63, 64]

R. eutropha and C. acidovorans cells were grown in nutrient-rich medium for 24 h and then transferred to a nitrogen-free medium containing the above carbon sources. b PHA content in cells incubated for 48 h in nitrogen-free medium. c Composition determined by 1H-NMR. 3-HBA: 3-Hydroxybutyric acid. 4-HBA: 4-Hydroxybutyric acid. a

This shows that the accelerated enzymatic degradation of P(3HB-co-4HB) films may be due to a decrease in its crystallinity. P(3HB-co-4HB) is commercialised by Telles (i.e., Mirel®). Telles, a joint venture between the Archer Daniels Midland Company and Metabolix Inc., is in charge of the production of Mirel and its distribution. Similar properties are also shown by the incorporation of 3-hydroxypropionate (3HP) into the P(3HB) sequence [67]. Like 4HB, 3HP do not possess chirality. Since the discovery of 3HP as a member of the PHA family [68], considerable work has been carried out to produce poly(R-3HB-co-3HP) (P(3HB-co-3HP)) containing various amounts (0−88 mol%) of 3HP units [69, 70]. The investigation of the solid- state structure [71] and biodegradability [72] of these copolymers with various 3HP units showed a great deal of similarity to the copolymers of P(3HB-co-4HB) [72, 73]. However, in contrast to the poly(4HB) homopolymer, the homopolymer of poly(3HP) was hardly eroded in river water [72] but could be degraded by the P(3HB) depolymerise (EC 3.1.1.75) (Section 7.6) purified from Alcaligenes faecalis [74].

7.3.4 Polyhydroxyalkanoates containing medium-chain-length monomers Figure 7.4 shows the chemical structure of PHA copolymers composed of 3HB units and C6−C14 numbered R-3HA units of MCL R-3HA. In contrast to PHASCL made of

7 Polyhydroxyalkanoates 

 191

SCL HA (C4−C5), PHAMCL are thermoplastic elastomers with melting points of about 45−60 °C and glass transition temperatures near −40 °C [75]. The biosynthesis of PHA copolymers containing both the SCL and MCL units are relatively rare due to the substrate specificity of the polymerising enzyme, PHA synthase [76]. Most PHA synthases can efficiently polymerise only either SCL or MCL units. Since 1995, an increased number of PHA copolymers containing both the SCL and MCL units have been documented [52, 77, 81]. These copolymers, produced by PHA synthases, have a broad range of substrate specificity and show attractive physical properties (Table 7.3). Matsusaki and co-workers [80, 81] have shown that it is possible to produce poly(3HB-co-3HA) MCL copolymers containing a wide range of 3HAMCL units. CH3 CH3 *CH

O

CH2 n

O CH2

95 mol% 3HB

C

x

*CH

O

O CH2

C

y

5 mol% 3HAMCL(n=2, 4, 6, 8, 10)

Figure 7.4: Chemical structure of PHA copolymer containing an SCL monomer (3HB) as well as MCL monomers (3PHAMCL) (poly(3HB-co-3PHAMCL)). Table 7.3: Characteristics of PHA copolymers containing both SCL and MCL R-3HB in comparison to other polymers. Polymer samples

Tg (oC)

Tm (oC)

Tensile strength (MPa)

Elongation at break (%)

P(3HB)

4

180

43

5

P(3HB)

4

185

62

58

P(3HB-co-20% 3HV)

−1

145

20

50

P(3HB-co-16% 4HB)

−7

150

26

444

P(3HB-co-10% 3HHx)

−1

127

21

400

P(3HB-co-6% 3HD)

−8

130

17

680

0

176

38

400

−30

110

10

620

a

PP Low density PE

a UHMW P(3HB) [43]. P(3HB-co-3HD): Poly(3-hydroxybutyrate-co-3-hydroxydecanoate). P(3HB-co-3HHx): Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Tg Glass transition temperature. Tm Melting temperature.

It must be noted that some pseudomonads produce a blend of PHASCL and PHAMCL [82]. Such blends cannot be distinguished from a copolymer solely using gas chromatographic analysis of the dried cells. However, once the polymers have been

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 Kumar Sudesh, Yoshiharu Doi, Paolo Magistrali and Sebastià Gestı´ Garcia

extracted from the cells they can be separated using suitable solvents. Thermal analyses of the extracted polymers are also frequently used to distinguish between blends and copolymers. In the case of blends, it has been shown that PHASCL and PHAMCL are stored in separate inclusions in the bacterial cell cytoplasm [83]. When cells containing these blends are subjected to freeze-fracture electron microscopy, the PHASCL granules often deform plastically resulting in needle-type protruding structures [84, 86]. On the other hand, PHAMCL granules show distinct mushroomtype deformation structures [87]. Based on these morphological differences coexisting granules of PHASCL and PHAMCL can be distinguished quite accurately. However, the temperature at which the freeze-fracture process is carried out can greatly affect the deformation morphology; at -160 °C most P(3HB) granules show mushroom-type deformation structures although they show needle-type deformations at −110 °C [86]. Besides that, granules of P(4HB) show both mushroom-type and needle-type deformations when fractured at either −110 or −160 °C [86, 87]. Figure 7.5 shows the morphology of P(3HB-co-3HV) granules in Comamonas acidovorans cells. As mentioned earlier, both the needle-type and mushroom-type deformation structures can be observed. It has been shown that mushroom-like deformations do not always represent the PHAMCL granule. This type of long chain PHA is receiving great interest. A spin-out of UCD School of Biomolecular & Biomedical Science called Bioplastech Limited is developing PHAMCL from waste materials [88, 89]. An example of commercial PHAMCL is P(3HB-co-3HHx) produced by Kaneka Corporation.

Figure 7.5: Morphologies of bacterial cells containing PHA granules in the cytoplasm. A: phase contrast light microscopy picture of Comamonas acidovorans cells containing 38 wt% of the dry cell weight (DCW) P(3HB-co-71% 3HV). B: freeze-fracture electron micrograph of the same sample. The fracture process was carried out at −160 °C. N: needle-type, M: mushroom type and S: crater-like holes in the cell cytoplasm resulting from granules that have been completely scooped out.

An overview of the properties of commercial PHA is reported in Table 7.4.

−

−

2,200 (ASTM D790)

173 (ASTM D3418)

135 (ASTM D1525)

Tensile modulus (MPa)

Flexural strength (MPa)

Flexural modulus (MPa)

Tm ( C)

VICAT softening temperature (oC)

−

170−176

3,520−4,170

−

2,800−3,500

2

31−36

1.25

147 (ISO/A/120) 166

−

−

35

1,140−1,900

6−9

24−27

1.25

10 @ 180 oC 5 kg

Analytical standard is indicated between brackets when available. ASTM: American Society for Testing and Materials. ISO: International Organization for Standardization.

o

32 (ASTM D638)

4 (ASTM D638)

Elongation (%)

1.2 (ISO 1183)

Density (g/cm3)

Tensile strength (MPa)

6.5 @ 190 oC 2.16kg

Enmat Y100P Injection moulding blown films

Biocycle 1000 Injection Biomer P226 moulding Injection moulding TM

P(3HB-co-3HV) ®

P(3HB) ®

Melt flow rate (g/10 min)

Application grade

Table 7.4: Properties of commercial PHA.

124 (ASTM D1525)

160−165

2,000 (ASTM D790)

40 (ASTM D790)

3,000 (ASTM D638)

4 (ASTM D638)

25 (ASTM D638)

1.4 (ASTM D792)

−

P4001 Sheet extrusion

133 (ASTM D1525)

160−165

1,900 (ASTM D790)

−

−

5 (ASTM D638)

20 (ASTM D638)

1.4 (ASTM D792)

−

Mirel TM

P(3HB-co-4HB) Mirel P1003 Injection moulding TM

117 @ load 2.25 lbs

142

1,530 (ASTM D790)

−

1240 (ASTM D882)

26 (ASTM D882)

28 (ASTM D882)

1.2 (ASTM D792)

−

Kaneka

P(3HB-co-3HHx)

7 Polyhydroxyalkanoates   193

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 Kumar Sudesh, Yoshiharu Doi, Paolo Magistrali and Sebastià Gestı´ Garcia

7.3.5 Uncommon constituents of polyhydroxyalkanoates Besides the common PHA constituents mentioned earlier, various other monomer constituents have been identified. In total, about 150 different HA are known to be members of the ever-growing PHA family [90]. Many of the uncommon monomers are incorporated only when related precursor substrates are supplied as carbon sources to the microorganisms. An interesting addition to the PHA family is the identification of a new class of sulfur-containing PHA with thioester linkages [91]. Poly(3HB-co-3mercaptopropionate) was produced by R. eutropha when 3-mercaptopropionic acid or 3,3-thiodipropionic acid was provided as the carbon source, in addition to fructose or gluconic acid, under nitrogen-limited culture conditions. What is striking about this finding is the fact that the polymerising enzyme, PHA synthase, has a very versatile catalytic centre. The PHA synthases of the pseudomonads are probably the most versatile with broad substrate specificity. In Pseudomonas oleovorans (P. oleovorans), for example, it is possible to synthesise PHA consisting of saturated, unsaturated, halogenated, branched and aromatic 3HA with 6−14 carbon atoms [92, 95].

7.4 Mechanisms of polyhydroxyalkanoate biosynthesis Unlike other microbial storage materials such as glycogen or polyphosphate that have been studied in detail for their physiological importance, only the early studies focused on the physiology of PHA biosynthesis [1, 96, 97]. Considerable effort has been directed towards understanding the enzymes, metabolic pathways and conditions which generate substrates from simple and renewable carbon sources for the PHA synthase, while recombinant DNA technologies are increasingly used to further understand complex regulatory mechanisms that affect PHA biosynthesis [98].

7.4.1 Conditions that promote the biosynthesis and accumulation of polyhydroxyalkanoates in microorganisms Early studies revealed that the rate of PHA accumulation can be increased by increasing the ratio of the carbon to nitrogen source [99]. Eventually it became evident that PHA accumulation usually occurs when cell growth is impaired due to depletion of an essential nutrient such as sulfate, ammonium, phosphate, potassium, iron, magnesium or O2 [1, 100, 103]. Suzuki and co-workers [103] studied 51 methylotrophs for their ability to produce P(3HB) from methanol. Similar nutrient limitations were found to stimulate the formation of P(3HB). However, a kinetic study of the production

7 Polyhydroxyalkanoates 

 195

of P(3HB) by a fed-batch culture of Protomonas extorquens showed that a nitrogen source was necessary even in the P(3HB) production phase [104]. Feeding with a small quantity of ammonia resulted in a more rapid increase of intracellular P(3HB) than was the case without ammonia feeding. Excessive feeding of ammonia, however, caused not only degradation of accumulated P(3HB) but also reduction of microbial P(3HB) synthase activity. PHA accumulation can also take place during active cell growth, but this ability is limited to only a few microorganisms such as Alcaligenes latus (A. latus) which can accumulate P(3HB) up to 80% of the DCW without limitation of any nutrient [105, 106]. This characteristic may be due to a low activity of the β-ketothiolase, which catalyses the cleavage of acetoacetyl-CoA [107]. Besides A. latus, Paracoccus denitrificans also shows growth associated PHA accumulation depending on the type of carbon sources available to the bacterium. Kim and co-workers [108], tested linear primary C1−C9 alcohols and linear C2−C10 monocarboxylic acids and found that growth associated synthesis of PHA could only be obtained with the carbon sources containing an odd number of carbon, except for methanol. The advantage of using a bacterium that shows growth associated PHA accumulation for large-scale production is a shorter fermentation time. In addition, it also avoids the extra operations associated with the two-step fermentation process for PHA accumulation under nutrient-limited conditions. By using A. latus btF-96, Chemie Linz was able to produce more than 1,000 g of P(3HB) in a week using a 15 m3 fermenter [106]. ICI on the other hand chose R. eutropha as the production organism although this bacterium accumulates PHA under nongrowth conditions. R. eutropha was chosen over Azotobacter and Methylobacterium because of the higher polymer content, good molecular mass and also because of easier PHA recovery.

7.4.2 Carbon sources for the production of polyhydroxyalkanoates An attractive feature of microbial PHA is the ability to produce them using renewable carbon sources. The plastic materials widely in use today are synthesised from fossil fuels such as petroleum and natural gas. PHA on the other hand can be produced using renewable carbon sources such as sugars and plant oils, which is an indirect way of utilising the atmospheric CO2 as the carbon source. Various waste materials are also being considered as potential carbon sources for PHA production. Among them are whey [109, 110], molasses [111, 113] and starch [114, 115]. The carbon source available to a microorganism is one of the factors (others being the PHA synthase substrate specificity and the types of biochemical pathways available) which determines the type of PHA produced. For industrial-scale production, the carbon source significantly contributes to the final cost. This makes the carbon source one of the most important components in the production of PHA and is therefore a prime target for potential cost reduction.

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 Kumar Sudesh, Yoshiharu Doi, Paolo Magistrali and Sebastià Gestı´ Garcia

Figure 7.6 shows three general systems for the production of biodegradable polymers using CO2 as the starting material. The three-step process involves the utilisation of plant sugars derived from photosynthetically fixed CO2 as carbon sources in the fermentation of organic acids, alcohols and amino acids. These substances are then used as building blocks for the chemical synthesis of polymers. Examples of polymers produced using this three-step process include polylactic acid, polybutylene succinate and polyaspartic acid.

Three-Stage Production (Polylactides)

CO2

+

Plants Photosynthesis

Sugars

Bacteria Fermentation

Lactic acid

Catalyst Chemosynthesis

Polylactide

Two-Stage Production (PHAs)

CO2

+

Plants Photosynthesis

Sugars, Plant oils

Bacteria Fermentation

PHA

One-Stage Production (PHA)

CO2

+

Plants, Algae Photosynthesis

PHA

Figure 7.6: Systems for the production of biodegradable polymers from CO2 as the starting material.

On the other hand, the two-step process involves the direct conversion of plant sugars and plant oils into polymers by microorganisms. At present, the biosynthesis of PHA is largely carried out through this two-step process. Compared with the three- step process of polymer production, the two-step process can be more costeffective provided that excellent producers of PHA are identified and the fermentation process is highly optimised. It has been calculated that 2.5 kg of glucose must be used for each kilogram of polymer produced [116]. Studies in the author’s laboratory have shown that plant oils may be a better carbon source whereby a kilogram of oil can give rise to a kilogram of polymer. A recombinant strain of R. eutropha PHB-4 (a PHA-negative mutant), harbouring the PHA synthase gene from Aeromonas caviae (A. caviae), could produce a random copolymer of 3HB and 3-hydroxyhexanoate

7 Polyhydroxyalkanoates 

 197

(3HHx) from plant oils such as olive oil, palm oil and corn oil. The P(3HB-co-3HHx) content was approximately 80% of the DCW and the 3HHx mole fraction was 4−5 mol% regardless of the structure of the triglycerides fed [117]. The results demonstrate that inexpensive renewable plant oils are excellent carbon sources for the efficient production of PHA. Ideally, producing environmentally friendly polymers directly in plants would be the most energy efficient process (one-step process) (Figure 7.6), provided that suitable technologies are available for the extraction and downstream purification processes of the polymers from plant materials. At present however, plant derivatives such as sugars and oils are the most popular carbon sources for the production of PHA by microbial fermentation.

7.4.3 Biochemical pathways involved in the metabolism of polyhydroxyalkanoates In order to tap the full potential of microbial systems for PHA production, it is necessary to modify the existing metabolic pathways in a particular microorganism. This is to ensure that the major portions of the supplied carbon sources are channelled towards PHA biosynthesis. Recent knowledge of the complete genetic makeup of several microorganisms [118, 120] is facilitating the engineering of novel metabolic pathways. New pathways can be constructed by introducing relevant genes into suitable microorganisms. Likewise, unnecessary pathways can be shut down by inactivating the enzyme(s) involved in a certain reaction. Such manipulations have to be carried out judiciously to achieve maximum PHA production in the shortest possible time using cheap and readily available carbon sources, without compromising cell growth. Another important factor that is often overlooked in the experimental stage is the stability of the genetically modified strains over many generations. Recombinant strains that do not have this characteristic will not be attractive as an industrial strain for the large-scale product of PHA. Figure 7.7 shows the common metabolic pathways which are frequently encountered in the biosynthesis of PHA in various microorganisms. Along with the type of carbon source and specificity of the PHA synthase, the metabolic pathways play a crucial role in determining the type of PHA that can be produced by a particular microorganism. Most of the P(3HB)-producing microorganisms possess Pathway I through which acetyl-CoA is converted into (R)-3-hydroxybutyryl-CoA and subsequently polymerised by PHA synthase. It has been shown that a similar pathway also operates in the cyanobacterium Synechocystis sp., PCC6803 [121]. In some microorganisms, (S)-isomers of 3-hydroxybutyryl-CoA are generated instead of the (R)-isomers. Since the PHA synthase is active only towards the (R)- isomers, additional reaction steps catalysed by enoyl-CoA hydratases are present in microorganisms such as Rhodospirillum rubrum to convert the (S)-isomers into the (R)-isomers [122].

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 Kumar Sudesh, Yoshiharu Doi, Paolo Magistrali and Sebastià Gestı´ Garcia

Pathway I

Pathway II

Carbon soure (Sugars)

Fatty acid degradation (β-oxidation) Carbon source (Fatty acids)

TCA cycle

Acetyl-CoA PhaA

Acyl-CoA

Acetoacetyl-CoA PhaB

3-Ketoacyl-CoA

Enoyl-CoA

(R)-3-Hydroxybutyryl-CoA PhaC

FabG (?) PhaB (?)

PHA

PhaC

(S)-3-Hydroxyacyl-CoA

PhaJ

(R)-3-Hydroxyacyl-CoA PhaG FabD (?) (R )-3-Hydroxyacyl-ACP

PhaC 4-, 5-, 6-Hydroxyalkacyl-CoA

Other pathways

Related carbon sources

3-Ketoacyl-ACP

Enoyl-ACP

Acyl-ACP Malonyl-ACP Malonyl-CoA Pathway III Acetyl-CoA

Fatty acid biosynthesis

Carbon source (Sugars)

Figure 7.7: Common metabolic pathways that are involved in the biosynthesis of PHA in microorganisms. FabC: malonyl-CoA: acyl carrier protein (ACP) transcylase, FabD: malonyl-CoAACP transacylase, FabG: 3-ketoacyl-CoA reductase, PhaA: β-ketothiolase, PhaB: NAOH-dependent acetoacetyl-CoAreductase, PhaC: polyhydroxyalkanoates synthase, PhaG: 3-hydroxyacyl-ACP: CoA transferase, PhaJ: (R)-enoyl-CoA hydratase and TCA: tricarboxylic acid.

7 Polyhydroxyalkanoates 

 199

The fatty acid β-oxidation pathway (Pathway II) is known to generate substrates that can be polymerised by the PHA synthases of pseudomonads. PHAMCL from various alkanes, alkanols and alkanoates can be synthesised by pseudomonads that belong to the ribosomal nucleic acid-homology group I. The monomer composition of the PHAMCL produced is often related to the type of carbon source. Most of the pseudomonads belonging to this group, except P. oleovorans, can also derive (R)-3hydroxyacyl-CoA substrates for PHA biosynthesis from unrelated carbon sources such as carbohydrates. Huijberts and co-workers [123] presented evidence showing that the PHA synthases responsible for PHA synthesis from fatty acids are also involved in PHA synthesis from glucose. It was then presumed that there are at least two distinct substrate supply routes for PHA synthesis in Pseudomonas putida (P. putida), i.e., via the intermediates of fatty acid biosynthesis (Pathway III) [124] and via the intermediates of β-oxidation (Pathway II). Although it was known that the intermediates of the β-oxidation cycle are channelled towards PHA biosynthesis, only recently the precursor sources were identified. In A. caviae, the β-oxidation intermediate, trans-2-enoyl-CoA is converted to (R)-3hydroxyacyl-CoA via (R)-specific hydration catalysed by an (R)-specific enoyl-CoA hydratase [125, 126]. Subsequently, Tsuge and co-workers [127] reported the identification of similar enoyl-CoA hydratases in Pseudomonas aeruginosa. In the latter case, two different enoyl-CoA hydratases with different substrate specificities channelled both SCL and MCL enoyl-CoA towards PHA biosynthesis. In recombinant E. coli it was further shown that 3-ketoacyl-CoA intermediates in the β-oxidation cycle can also be channelled towards PHA biosynthesis by a nicotinamide adenine dinucleotide phosphate dependent (NADPH-dependent) 3-ketoacyl-ACP reductase [128]. A similar pathway was also identified in P. aeruginosa [129]. In addition, it was also reported that the acetoacetyl-CoA reductase (PhaB) of R. eutropha can also carry out the conversion of 3-ketoacyl- CoA intermediates in Pathway II to the corresponding (R)-3-hydroxyacylCoA in E. coli [130]. The results clearly indicate that several channelling pathways are available to supply substrates from the β-oxidation cycle to the PHA synthase. This explains why it was not possible to obtain mutants that completely lack PHA accumulation ability, unless the mutation occurred in the PHA synthase gene [131]. Among the various metabolic pathways that are involved in PHA biosynthesis, the fatty acid de novo biosynthesis pathway (Pathway III) is of particular interest because of its ability to supply various types of HA monomers from simple carbon sources such as gluconate, fructose, acetate, glycerol and lactate. It can be envisaged that the potential future production of PHAMCL using photosynthetic organisms will benefit through the exploitation of such pathways. This is because acetyl-CoA is the starting material (Figure 7.6, Pathway III) which is used to generate HA monomers for PHAMCL biosynthesis, and acetyl-CoA is a universal metabolite present in all living organisms. However, it must be noted that the intermediates of the fatty acid de novo biosynthesis pathway are in the form of (R)-3-hydroxyacyl-ACP, which is not recognised by the PHA synthase.

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Studies of PHAMCL biosynthesis in P. putida from glucose as the sole carbon source has identified an enzyme that is capable of converting 3-hydroxydecanoyl-ACP to 3-hydroxydecanoyl-CoA. The enzyme was referred to as a 3-hydroxyacyl-ACP: CoA transferase (PhaG) [132]. Since then, similar enzymes have been identified in several other pseudomonads [133–135]. P. oleovorans does not have the ability to synthesise PHAMCL from gluconate but shows this ability upon the introduction of the PhaG gene of P. putida. The genes for PhaG and PHA synthase from P. aeruginosa were expressed together in a nonPHA-producing pseudomonad, (Pseudomonas frugi). This resulted in the ability to produce PHAMCL by P. frugi from gluconate as the sole carbon source. Besides the PhaG protein, overexpression of transacylating enzymes such as FabD in E. coli, also seem to generate monomers for P(3HB) biosynthesis [136]. Besides the three main pathways mentioned above, there are several other metabolic pathways that can be manipulated to produce substrates for PHA biosynthesis. In recombinant E. coli, it has been shown that 4-hydroxybutyryl-CoA can be derived from the intermediates of the tricarboxylic acid (TCA) cycle [137]. By providing external precursor substrates such as 4-hydroxybutyric acid, 1,4-butanediol and γ-butyrolactone to certain wild type [59, 60, 62, 63] and recombinant microorganisms [138], 4HB monomers can be incorporated more efficiently.

7.4.4 The key enzyme of the biosynthesis of polyhydroxyalkanoates, polyhydroxyalkanoate synthase Without doubt, PHA synthase is the key enzyme in the biosynthesis of PHA. Unfortunately, the mechanism of this important enzyme is not yet fully understood. Based on genetic analysis, the primary structures of PHA synthases from a large number of microorganisms are available [76]. PHA synthases have been classified into three groups based on their primary structures and the types of PHA that they produce. The PHA synthases of R. eutropha and P. oleovorans represent groups I and II, respectively, while that of Chromatium vinosum represents group III. The latter differs from the two former groups by the fact that group III synthases consist of two different subunits (PhaC and PhaE), while the members of groups I and II only have one subunit (PhaC). As for the types of PHA produced, PHA synthases of groups I and III are efficient in the synthesis of PHASCL, while those of group II are superior in the synthesis of PHAMCL. A few exceptions to the above classification are the PHA synthases of A. caviae [125], Thiocapsa pfennigii [139] and Pseudomonas sp., 61–3 [140]. These PHA synthases are capable of producing PHA copolymers containing both the SCL- and MCL-PHA. These exceptional PHA synthases are of great interest because they can be used to biosynthesise PHA copolymers containing novel compositions which show promising physical properties [80, 81]. In order to elucidate the mechanism of PHA synthase, various site-specific [141, 142] and random mutagenesis [143] studies have been carried out. Results show that

7 Polyhydroxyalkanoates 

 201

many of the amino acid substitutions that affected PHA accumulation occurred in the conserved regions within an ‘α/β hydrolase fold’, while changes in the sequence of the first 100 amino acids at the N-terminal region did not show any significant effect. The α/β hydrolase fold is characteristic of a protein superfamily that includes lipases [144]. Initially it was thought that the PHA synthase catalytic mechanism might resemble that of fatty acid synthases [145, 146]. Present knowledge dictates that a lipasebased catalytic mechanism is perhaps more suitable as a model of PHA synthase [147].

7.5 Genetically modified systems and other methods for the production of polyhydroxyalkanoates E. coli offers a well-defined physiological environment for the production of recombinant proteins and other bioproducts because the physiology, biochemistry and genetics of this bacterium have been studied in great detail [148, 149]. Likewise, the production of PHA using photosynthetic organisms is attractive because atmospheric CO2 can be directly converted into plastic material [150]. Both these recombinant systems have been explored by various research groups with the hope of reducing the cost of PHA production.

7.5.1 Recombinant escherichia coli In the case of recombinant E. coli various strategies are available to achieve high cell-density cultures [151]. It is important to select the most suitable E. coli strain for a particular purpose. Detailed studies by Lee and co-workers [152] revealed that the recombinant E. coli strains, XL1-Blue (pSYL105) and B (pSYL105) were the best candidates for P(3HB) production. Although strain JM109 (pSYL105) produced the highest P(3HB) content, the cell mass was low. In any case, high gene dosage is necessary to produce high concentrations of P(3HB) in recombinant E. coli [153, 154]. The PHA biosynthetic genes of A. latus were cloned and used to produce P(3HB) in E. coli [155]; it was found that the genes of A. latus resulted in better P(3HB) production in E. coli compared with the PHA biosynthetic genes of R. eutropha. It must be noted that, recombinant E. coli can produce P(3HB) during active growth in nutrient-rich conditions [153, 154] just like A. latus [107]. Ever since the first successful expression of the R. eutropha PHA biosynthetic genes [156, 158], various other heterologous genes have been introduced into E. coli resulting in the ability to produce both PHA homopolymers and copolymers. Among the PHASCL, other than P(3HB), that have been produced in recombinant E. coli are, P(4HB) homopolymer and P(3HB-co-4HB) copolymer [137, 138, 159], and poly(4hydroxyvalerate) homopolymer and copolymers [159]. Besides that, the production

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of PHAMCL have also been demonstrated [160–164]. In addition, Fukui and co-workers [165] demonstrated the production of P(3HB-co-3HHx) copolymer in E. coli by the coexpression of the A. caviae PHA synthase gene and (R)-enoyl-CoA hydratase gene. P. aeruginosa (R)-enoyl-CoA hydratase (PhaJ1) (EC 4.2.1.17) [127] can also be used to supply 3HHx monomers for the production of the P(3HB-co-3HHx) copolymer in E. coli [166]. In recombinant microorganisms, plasmid stability is of crucial importance for continued PHA production. Some attention has been directed to this problem in recombinant E. coli [154,167]. Another advantage of using E. coli as the production host is the relative ease with which the accumulated PHA can be extracted from the cells [168].

7.5.2 Transgenic plants Ever since the first successful expression of PHA biosynthetic enzymes in plants [169], which resulted in the accumulation of small amounts (115 °C [19]. Besides Metabolix, other companies are active in the development and production of PHA. Tianan, a Chinese company, produces P(3HB) and P(3HB-co-3HV) (tradename Enmat) with a capacity of 2,000 tonnes/year, and has announced an increase in capacity to 10,000 tonnes/year. Enmat PHA are certificated as compostable. Applications suggested by the producer are injection moulding, extrusion, thermoforming, blown films, fibres and nonwovens [20]. Procter & Gamble developed a wide range of poly(hydroxybutyrate-cohydroxyalkanoates) [copolymers of P(3HB) and 3-hydroxyhexanoate, P(3HB) and 3-hydroxyoctanoate, P(3HB) and 3-hydroxyoctadecanoate] with the tradename Nodax. In 2007, Meredian Inc. purchased Procter & Gamble’s PHA technology. Meredian Inc. has announced the start-up of production at full industrial level (30 ktonnes/year) in its Bainbridge facility in the second half of 2014 [21]. Under the tradename Aonilex, Kaneka, a Japanese company, plans to start production of 1,000 tonnes/year of P(3HB) and 3-hydroxyhexanoate, of different ratios, in order to achieve a wide range of final properties. Kaneka claims it will increase Aonilex production up to 10,000 tonnes/year depending on the market feedback [22–23].

10.2.1.3 Polylactic acid As for PHA, a more detailed description of PLA (Figure 10.4) is given in Chapter 9 of this book so, in this chapter, only its main characteristics will be disclosed. PLA plays an important role among synthetic biodegradable polyesters, thanks to its low price. Moreover, it is made from 100% renewable resources (Section 10.4), which is an aspect becoming more and more important. PLA is synthesised by the ring-opening polymerisation of lactide [10, 11, 24], the cyclic dimer of lactic acid, which is produced on a large scale via fermentation. H *

CH3 C

O

C O

n  Figure 10.4: Chemical structure of PLA.

Properties of PLA are highly related to the ratio between the two optical isomers, D and L. With regards to PLA on the market, it is usually a copolymer of L-lactide containing 2−4% of D-lactide along its polymer chain. This causes a decrease in the melting

10 Biodegradable Polyesters 

 265

temperature (Tm) which can drop from nearly 180−190 °C for pure poly(L-lactic acid) to 140−150 °C for PLA containing almost 4% of D-lactide in the polymer chain [25, 26]. PLA is a rigid and stiff product with a Tg of 60−65 °C. As far as biodegradation is concerned, PLA is compostable in industrial composting facilities, while, due to the lower temperatures developed during the process, home composting does not allow complete degradation of the polymer. NatureWorks, a joint venture of Cargill and PTT Global Chemical, is the world leader in PLA production (tradename Ingeo). Its production capacity in the USA reaches 150,000 tonnes/year. According to the producer, different Ingeo grades have been specifically tailored to be used in a wide range of processes and applications, such as extrusion/thermoforming, injection moulding, films and sheet, fibres and foam injection stretch blow moulded bottles [27]. There are many other companies which produce PLA, amongst them: Mitsui (LACEA PLA), Futerro, which is a joint venture between Galactic and Total (Futerro PLA), Dainippon Ink & Chemicals Inc (CPLA PLA), Purac and Zhejiang Hisun. In 2011, the first plant to use a new polymerisation technology for PLA, developed by Sulzer Chemtech and Purac Biochem and built by Synbra Technology in the Netherlands, started industrial production of Synterra® PLA grades. The new plant has a capacity of 5,000 tonnes/year: it uses L- and D-lactides from Purac, produced from genetically modified organism (GMO)-free plant feedstock, which is 100% biobased.

10.2.1.4 Polyglycolic acid The family of polylactides (mainly represented by PLA – Section 10.2.1.3) are receiving great interest because they can be hydrolysed at a relatively high rate even at room temperature and neutral pH, without the help of hydrolytic enzymes if moisture is present. This property makes them interesting to the medical sector. Among them, polyglycolic acid (PGA) (Figure 10.5) can be hydrolysed in our body to the respective monomers and oligomers which are soluble in aqueous media [28]. PGA  and, in particular, the copolymer of lactic acid and glycolic acid referred to as poly(lactic-co-glycolic acid) (PLGA), exhibits excellent biocompatibility, biodegradability and mechanical strength. PLGA have been approved by the US Food & Drug Administration (FDA) for drug delivery. For example, PGA sutures are commercially available under the tradename Safil® and PLGA sutures under the tradename Vycril® or Novosyn®. CH2

* O

C O

n  Figure 10.5: Chemical structure of PGA.

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10.2.1.5 Long chain polyhydroxyacid Another new family of biodegradable polyesters containing a hydroxyacid repetitive unit is that of the long chain polyhydroxyacid or polyhydroxyfattyacid. These products, even though from a chemistry point of view can be considered of the PHA family (Section 10.2.1.2), due to their physico-chemical properties and polymer synthesis, have been classified separately.

*

O

CH2

xC O

* n  Figure 10.6: General chemical structure of poly(ω-hydroxyfattyacid) (x = 11−21).

Thanks to genetic science, engineered yeasts strain, such as Candida tropicalis, can lead to the production of ω-hydroxyfattyacid or α,ω-dicarboxylic acid containing 12−22 carbon atoms from renewable resources (i.e., natural oils) via fermentation processes. These fermentation processes avoid the poor yields and extensive purification required by chemical conversion [29]. Unlike PHA, where microbial activity gives rise to the final polymers, the fermentation process used for w-hydroxyfattyacid leads to monomers which can be copolymerised with a wide range of other monomers to manufacture polymers with different final properties. The polymer synthesis of long chain polyhydroxyacids can be performed either in bulk via condensation reactions or by the ring-opening polymerisation of lactones/ macrolactones obtained by w-hydroxyfattyacid cyclisation [30]. These sort of aliphatic polyesters are claimed to have melting points above 90 °C which give them suitable characteristics for commercial applications (i.e., films, injection moulded products) and the number average molecular weight (MW Mn) ranging from 45,000−65,000 D and MW ranging from 105,000−120,000 D. Poly(14-hydroxytetradecanoic acid), poly(16-hydroxyhexadecanoic acid) and poly(18-hydroxyoctadecanoic acid) have been synthesised in bulk from the respective w-hydroxyfattyacid. The resulting products have melting points of 91, 98 and 102 °C, respectively, with the average MW ranging from 170,000−230,000 D. Their average mechanical properties show Young’s Modulus ranging from 370−450 MPa, elongation at break ranging from 370−730% and tensile strength from 15−18 MPa [30].

10.2.2 Biodegradable aliphatic polyesters with a diol/ dicarboxylic acid repetitive unit A large number of biodegradable aliphatic and alicyclic polyesters obtained by the polycondesation of diols (mainly 1,2-ethanediol, 1,4-butanediol, 1,3-propanediol and

 267

10 Biodegradable Polyesters 

cylohexanedimethanol) and dicarboxylic acids (mainly succinic, adipic, sebacic, azelaic and dodecanedioic) have been studied. Unfortunately, apart from aliphatic polyesters of this group based on succinic acid, the majority have a very low melting point, which can range from nearly 40−70 °C depending on the diol/dicarboxylic acid coupling. This characteristic precludes their use at an industrial level. The most common industrial products are polyesters and copolyesters of succinic acid, as having a high melting point makes them suitable for applications requiring high temperature resistance. Polyethylenesuccinate is a highly crystalline polymer, with a high elongation at break, a Tg of nearly −9 °C and a Tm of nearly 96 °C [31] Polybutylene succinate (PBS) is a highly crystalline polymer, with a Tg of nearly −30 °C and a Tm of nearly 115 °C [32]. Poly(butylene succinate-co-adipate) is a random copolymer of PBS containing adipic acid (PBSA) usually at a molar ratio of 20%. Compared with the PBS homopolymer it shows both a lower Tg (nearly −40 °C) and Tm (nearly 93 °C) [33]. Moreover, it is less tough with a lower tensile strength and a higher elongation at break compared with PBS. The biodegradation is affected by the presence of an adipate unit, in fact, the biodegradation rate of PBSA film in activated sludge rises with the increase of butyleneadipate [34]. PBS (Bionolle 1000 series, Figure 10.7) and copolymer with butyleneadipate (Bionolle 3000 series, Figure 10.8) have been produced by Showa Highpolymer (Showa Denko) since 1994 [34–36]. O *

O

CH2

CH2

CH2

O

CH2

C

CH2

CH2

C

*

n

O Figure 10.7: Chemical structure of PBS. O *

O

CH2

CH2

CH2

CH2

O

C

CH2

CH2

O

O

C n

O

CH2

CH2

CH2

CH2

O

C O

CH2

CH2

CH2

CH2

C

* m

Figure 10.8: Chemical structure of PBSA.

According to the producer, Bionolle is suitable for the manufacture of films, sheets, fibres, laminates, moulded foam products and injection moulded products. In particular, Bionolle properties are considered to be suitable for film processing, and it is actually used for ‘mulching film’ and ‘composting bags’ in Japan. The biodegradability of the Bionolle family is certified according to the following standards: ‘GreenPla’, the label of the Japanese Bioplastic Association, European Norms (EN) 13432, DIN CERTCO (Compostable) and Vinçotte (OK Compost).

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Bionolle production capacity is around 6,000 tonnes/year, but Showa Denko is planning to expand the capacity to 10,000−20,000 tonnes/year shifting towards PBS based on bioderived succinic acid (Section 10.4) [37]. In addition to Showa Denko, many other companies produce polyesters based on PBS as the homo- or copolymer. GS-PLA, from Mitsubishi Chemicals, are a range of grades of PBS or PBSA suitable for compound or master applications, with a production capacity of about 3,000 tonnes/year. GS-PLA is claimed to be compostable and biodegradable in soil. Many grades are available with different mechanical and physico-chemical properties [38]. In 2011, Samsung Fine Chemicals acquired the technology and patents of IRE Chemicals for production of the Enpol PBS and PBSA families. The brand name of these resins is S-Enpol (G4000 series) and their properties are close to those of Bionolle products. The production capacity is 2,000 tonnes/year [39]. Moreover, in recent years China has drastically increased the production capacity for biodegradable PBS and its copolymers. [40]. In 2009, a facility capable of manufacturing 10,000 tonnes/year of PBS and its copolymers was constructed by Hexing Chemical (tradename Hexing PBS). The facility can manufacture resins for extrusion, injection, moulding, thermoforming and blowing grade [41]. Xinfu Pharmaceutical Co. produces different PBS grades (tradename Biocosafe 1800−1900 series) for injection moulding, extrusion and film applications. The claimed production capacity is 13,000 tonnes/year [42]. In 2012, Kingfa Science & Technology Co. Ltd. claimed the launch of 30,000 tonnes/ year of PBSA capacity, and China New Holding Material is planning the launch of 25,000 tonnes/year of PBS capacity.

10.2.3 Aliphatic polyesters biodegradation Aliphatic polyesters are degraded by microorganisms present in the environment. A list of microorganisms capable of degrading PCL [2], PHB [43], PLA [44] and PBS [45] can be found elsewhere. Enzymatic degradation is a complex process involving: water absorption from the polyesters, enzymatic attack on the polyester surface (lipases are only active after conformational changes induced by adsorptive binding at the substrate surface), ester cleavage of macromolecular chains, formation of oligomer fragments due to hydrolysis, surface erosion due to the solubilisation of oligomer fragments in the surrounding environment, diffusion of soluble oligomers by bacteria and finally, consumption of the water-soluble intermediates and formation of CO2, H2O and new biomass. For example, ε-hydroxy caproic acid has been detected as a degradation product of PCL [2], and acetate, propionate and butyrate after anaerobic fermentation of PHB [46].

10 Biodegradable Polyesters 

 269

The biodegradation of polyesters is also affected by the chemical structure, hydrophilic/hydrophobic balance within the main chain, MW and crystallinity, which acts as a hindrance to biodegradability. In fact, biodegradation first takes place in the amorphous region of the polymer, where the erosion rate is much higher than in the crystalline region [47]. Measurement of the weight loss in controlled soil burial conditions provides a realistic test of the biodegradability of polymeric materials, since mineralisation rates cannot be determined using enzyme assays. PCL shows a high rate of biodegradation at ambient temperature (30 °C) in different soil types (canal shore soil, garden soil, compost and peat moss) [48]. Biodegradation in soil of some aliphatic polyesters was studied by Teramoto and co-workers [49]. The soil used in the test was a 1:1 mixture of black soil and leaf mould for gardening, incubated at room temperature (23−30 °C). Water was supplied at intervals of 2 days to keep the soil wet. In this order, P(3HB-co-3HV), PBS and PLA showed less weight loss, under these conditions, compared with PCL. Rizzarelli and co-workers [50] studied a set of polyalkylene dicarboxylates derived from 1,4-butandiol and methyl esters of succinic (PBS), adipic and sebacic acids. The soil burial degradation test was performed at 30 ± 0.1 °C, under moisture controlled conditions, using a mixture of milled perlite and soil. The incorporation of butylene adipate units and butylene sebacate units in the polymer chain gave rise to an enhancement of biodegradation. Another type of biodegradation test commonly performed takes into account the degradation in natural waters. PHA show good biodegradation performances in water. Kasuya and co-workers [51] demonstrated that P(3HB), P(3HB-co-14% 3HV) and P(3HB-co-10% 4HB) degrade completely within 28 days in river, lake, bay and ocean waters from Japan. The biodegradation of aliphatic polyesters obtained from the polycondensation reaction of diol and dicarboxylic acid is dependent both on the chemical structure and the source of water. For example, no microorganisms able to degrade PBS were present in freshwater and seawater, while polybutyleneadipate was eroded in freshwater from a lake.

10.2.4 Properties of biodegradable aliphatic polyesters The main characteristics of biodegradable aliphatic polyesters are detailed in Table 10.1. Data has been taken from technical bulletins available from the producers. Table 10.1: The main characteristics of biodegradable aliphatic polyesters.

Tradename Density (g/cm ) 3

PHB

P(3HB-co-4HB)

PCL

Biocycle 1000*

Mirel P1004

CAPA 6800

1.2 (ISO 1183)

1.3 (ASTM D792)

−

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 Paolo Magistrali, Sebastià Gestí Garcia and Tiziana Milizia

Table 10.1 (continued) PHB

P(3HB-co-4HB)

PCL

Tm (°C)

170−175 (ASTM D3418)

160−165

60−62 (DSC)

MFI (190 °C/2.16 kg)

6.5 (ISO 1133)

−

7b (ASTM D1238)

Tensile strength (MPa)

32 (ISO 527)

24 (ASTM D638)

54 (ASTM D412-87)

Elongation at break (%)

3.5 (ISO 527)

7 (ASTM D638)

920 (ASTM D412-87)

Modulus (MPa)

2,250 (ISO 178)

1,600 (ASTM D638)

440 (ASTM D412-87)

Vicat VST (°C)

135 (ISO 306)

−

−

PBS

PBSA

PLA

Tradename

Bionolle 1001 series

Bionolle 3000 series

Ingeo 4043D

Density (g/cm3)

1.26

1.23

1.24 ( ASTM D1505)

Tm (°C)

114

94

145−160 (ASTM D3418)

MFI (190 °C/2.16 kg)

1−3b

1−3b

−

Tensile strength (MPa)

31 (ISO 527-3)

18 (ISO 527-3)

110 (ASTM D882)

Elongation at break (%)

660 (ISO 527-3)

780 (ISO 527-3)

160 (ASTM D882)

Modulus (MPa)

470 (ISO 527-3)

320 (ISO 527-3)

3,300 (ASTM D882)

Vicat VST (°C)

−

−

−

a

* No longer commercially available a  cm3/10 min b  g/10 min ASTM: American Society for Testing and Materials DSC: Differential scanning calorimetry ISO: International Organization for Standardization MFI: Melt flow index

10.3 Biodegradable aliphatic-aromatic copolyesters Since aromatic polyesters turned out to be quite resistant to hydrolytic degradation under physiological conditions, a number of attempts were made to implant structures open to biological attack in such polyesters. These attempts have predominately been effectuated by introducing aliphatic acid components into the aromatic polyester chains [52]. Table 10.2 gives an overview of the different aliphatic-aromatic copolyesters synthesised as degradable materials during the last few years. Part of the work reported in the literature dealt with hydrolytic degradation mechanisms which do not involve enzymatic catalysis (chemical hydrolysis). This kind of degradation is often present

10 Biodegradable Polyesters 

 271

Table 10.2: History of the development of biodegradable aliphatic-aromatic copolyesters. Aromatic polymer

Aliphatic component

Mode of degradation

References

PET (1979/1981)

Oxyethylene diols

Hydrolysis in buffer at 37 °C

PET PBT polyethylene isophthalate (1981)

ε-caprolactone

Hydrolysis with lipase from Rhizopus arrhizus in a buffer at 37 °C

PBT (1989)

Glycolic acid

Hydrolysis in water at 60 °C

[115]

PBT (1990)

Oxalic acid

Hydrolysis in water at 33 and 50 °C

[116]

PET (1992)

Adipic acid

Hydrolysis in water at 25−80 °C

[117]

PET (1993)

L-lactic acid and oxyethylene diols

Hydrolysis in a buffer at 60 °C

[118]

PET (1994)

ε-caprolactone

Hydrolysis with lipase from Pseudomonas sp., in a buffer at 37 °C, soil burial and composting

PET (1994)

Adipic acid

Hydrolysis in water at 25−90 °C

PPT (1994/95)

Adipic acid and sebacic acid

Degradation in a synthetic liquid medium by microorganisms

[52, 83]

PET PPT PBT (1995)

Adipic acid and sebacic acid

Degradation in a synthetic liquid medium by microorganisms, soil burial and composting

[84]

PET (1995)

Oxyethylene diols and oxybutylene diol

10% NaOH at 70 °C

[120]

PET (1996)

Succinic acid

No data on degradation given

[121]

PBT (1997)

Adipic acid

Composting and agar plate test with prescreened microorganisms

[85]

PET (1997)

Adipic acid, sebacic acid and ethylene glycol

Hydrolysis with lipase from Rhizopus arrhizus in a phosphate buffer at 37 °C

[122]

Polypropylene terephthalate (1998)

Fumaric acid

Hydrolysis with lipase from Chromobacterium viscosum in a potassium phosphate buffer at 40 °C

[123]

PET (1999)

Succinic acid, sebacic acid and 1,12 dodecane dicarboxylic acid

Hydrolysis with lipase from Rhizopus arrhizus in a phosphate buffer at 37 °C

[124]

PBT

Succinic acid

Composting

[125]

PBT (2001)

Succinic acid and 1,4-cyclohexane dimethanol

Hydrolysis in a buffer at pH 4, pH 7 and pH 10, and composting

[126]

[116] [4, 79, 82]

[80, 81]

[119]

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 Paolo Magistrali, Sebastià Gestí Garcia and Tiziana Milizia

Table 10.2 (continued) Aromatic polymer

Aliphatic component

Mode of degradation

References

PBT (2001)

Succinic acid

Hydrolysis with lipase from Rhizopus arrhizus at 37 °C

Polyhexamethylene terephthalate (2008)

ε-caprolactone

Hydrolysis with lipase

PBT (2008)

1,12-dodecanedioic

−

[130]

PBT (2009)

Suberic acid and sebacic acid

Hydrolysis with, Candida cylindracea lipase

[131]

[127] [128, 129]

PPT: Polypropylene terephthalate.

in medical applications of polyesters, e.g., as implants in living tissues. Enzymatic hydrolysis, in contrast, is usually connected to microbial degradation both in the environment and in composting facilities. Random copolyesters composed of 1,4-butanediol, terephthalic acid and adipic acid (BTA- copolyesters, Figure 10.9) turned out to be the first promising materials for technical applications, not only for their degradation behaviour and their material properties, but also for the availability of the monomers and the estimated price level. Their structure and morphology, including the crystal structure, were investigated by Kuwabara and co-workers, [53] and Herrera and co-workers [54]. O *

O

CH2

CH2

CH2

CH2

O

C O

(CH2)2

C (CH2)2

O

CH2

CH2

CH2

CH2

O

*

C O

O

n

Figure 10.9: Chemical structure of poly(butylene adipate-co-terephthalate) (BTA- copolyester).

Compared with a low-density polyethylene material, a BTA-copolyester with a molar ratio of terephthalic acid of nearly 40% showed not only a comparable mechanical strength but also a significantly higher flexibility (elongation at break). Moreover, in order to increase the polyester MW and mechanical properties, a chain extension of the polyester chains with diisocyanates up to a MW of 230,000 D was performed without affecting biodegradability. In the biodegradable BTA aliphatic-aromatic polyesters available on the market, the amount of aromatic acid in the polymer chain was always maintained below 49 mol% in view of the significant and sudden decrease of the biodegradation of polyesters above this threshold. This behaviour was attributed to the lower biodegradability of the butyleneterephthalate sequences with length equal or higher than 3 which, above the said threshold, represent more than 10 % of the aromatic fraction of these polyesters [55] (Section 10.3.6).

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In recent years, Novamont developed a family of aliphatic-aromatic copolyesters with its aliphatic dicarboxylic acid component predominantly based on long chain dicarboxylic acids of natural origin (sebacic acid, azelaic acid and brassylic acid) [56–58]. Compared with aliphatic-aromatic polyesters where the aliphatic dicarboxylic component is a shorter carbon chain length, such as BTA polyesters, these copolyesters do not show the sudden decrease of biodegradation properties above 49 mol% of aromatic acids. This allowed developing aliphatic-aromatic copolyesters with a higher molar ratio of aromatic acid compared with BTA polyesters, achieving an improved balance between mechanical and biodegradation properties. Being long chain dicarboxylic acids of natural origin, generally coming from vegetable oils, their use allows a reduction of CO2 emission into the atmosphere and replacing monomers of fossil origin, such as adipic acid. Novamont recently developed a new family of aliphatic-aromatic copolyesters where terephthalic acid is replaced by furan-dicarboxilic acid from a renewable origin. These copolymers can be 100% renewable and show interesting new performances [59–61]. As opposed to aliphatic polyesters, which can be easily classified on the different repetitive unit, aliphatic-aromatic copolyesters are, nowadays, constituted of terephthalic acid, adipic acid (or a long dicarboxylic acid) and 1,4-butanediol. For this reason, their classification is by brand name or producers.

10.3.1 Ecoflex 10.3.1.1 Producer/Patents: BASF AG, Germany Standard Ecoflex grade is based on a copolyester from terephthalic acid, adipic acid and 1,4-butanediol. BASF [62 –64] recently introduced a new Ecoflex grade onto the market where the adipic acid has been replaced with a long chain dicarboxylic acid following the Novamont Origo-Bi polyester method (Sections 10.3 and 10.3.2), in order to increase the amount of renewable monomers in the polymer chain. The content of terephthalic acid in the polymer is approximately 42−47 mol% (with regard to the whole dicarboxylic acid content). Modifications of the basic copolyester lead to a flexible material which is especially suitable for film applications. A down gauging to 10 µm films can be achieved [65] according to the producer. Ecoflex can be used for the manufacture of biowaste bags and several films applications (i.e., packaging, mulching, hygiene products and household applications). The biodegradation of Ecoflex was tested under composting conditions. After 100 days in a compost environment, more than 90% of the carbon in the polymer was converted to CO2 [55]. In a detailed investigation, digestion of more than 99% could be proved for Ecoflex using a thermophilic actinomycete strain [66]. From these tests it can also be concluded that aromatic oligomers are subject to biodegradation under

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conditions present in a composting process. Ecotoxicological tests with Photobacterium phosphorum and Daphnia magna revealed no toxic effects of the degradation intermediates. A risk assessment resulted in the statement that no toxic effects can be expected from copolyester composting (Section 10.3.6). Ecoflex meets the requirements of Deutsche Institüt für Norms (DIN) V 54900 [67] as a compostable material and is certified by DIN CERTCO (Compostable), EN 13432, ASTM D6400 and the Japanese standard GreenPla [68].

10.3.2 Origo-Bi 10.3.2.1 Producer/Patents: Novamont Origo-Bi is based on a range of aliphatic and aliphatic-aromatic copolyesters mainly composed of terephthalic acid, long chain dicarboxylic acids of renewable source and 1,4-butanediol. Depending on the dicarboxylic acid used, the ratio of terephthalic acid can be tuned, in order to fulfil biodegradability and specific performance criteria [56–58]. Origo-Bi is a key blend component for the production of Mater-Bi grades [69]. Novamont is also able to produce BTA copolyesters, since the acquisition of the EastarBio patents portfolio, technology and trademark from Eastman in 2004, and PBS for its Mater-Bi range of products. The industrial capacity of Origo-Bi is of 70,000 tonnes/year.

10.3.3 Biocosafe 2003F 10.3.3.1 Producer: Zhejiang Hangzhou Xinfu Pharmaceutical Co. Ltd The Biocosafe brand, from Zhejiang Hangzhou Xinfu Pharmaceutical Co. Ltd, is a family of biodegradable polyesters both aliphatic (based on succinic acid – Section 10.2.2) and aliphatic-aromatic. Biocosafe 2003F is based on a copolyester composed of terephthalic acid, adipic acid and 1,4-butanediol. The content of terephthalic acid in the polymer is approximately 42−45 mol% (with regard to the whole dicarboxylic acid content) [42].

10.3.4 S-EnPol 10.3.4.1 Producer: Samsung Fine Chemicals In 2011, Samsung Fine Chemicals acquired the technology and patents of IRE Chemicals for the production of EnPol, a biodegradable polyester resin [70, 71]. The IRE original resin EnPol G8000 series was based on a group of aliphatic copolyesters composed of adipic acid, succinic acid, 1,2-ethanediol and/or 1,4-butanediol [72]. The

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producer stated that EnPol polymers met the specifications of the FDA for food contact and the United States Pharmacopeia specifications for medical device applications. The biodegradation of EnPol polymers was tested in a controlled laboratory composting test (according to ISO 14855 [73]). Within 45 days a CO2 evolution of more than 90% of the carbon present in the copolyester was detected [74]. Samsung Fine Chemicals has slightly changed the brand name into S-EnPol which now comprises both aliphatic polyesters based on succinic acid (G4000 series – Section 10.2.2) and aliphatic-aromatic copolyesters based on a composition of terephthalic acid, adipic acid and 1,4-butanediol (PBG7000 series) which is changed with respect to the previous G8000 series. According to the producer, the S-EnPol PBG7000 series can be used in the following applications: mulch film, shopping bags, metre-rate trash bags, roll bags, disposable products (sanitary gloves or tablecloths) and paper cup coating [39].

10.3.5 Properties of biodegradable aliphatic-aromatic copolyesters The main characteristics of biodegradable aliphatic-aromatic copolyesters are detailed in Section 10.3.1 and Table 10.3. Data has been taken from the technical bulletins available from the producers. Table 10.3: Compilation of some characteristic material data of commercial biodegradable aliphaticaromatic copolyesters. Trade name

Ecoflex

Producer

Origo-Bi

Biocosafe

S-EnPol

BASF AG, Germany Novamont, Italy

Xinfu Pharm, China

Samsung Fine Chemicals, Korea

Chemical basis

Modified copolyesters from 1,4- butanediol, adipic acid and terephthalic acid

Modified copolyesters from 1,4-butanediol, terephthalic acid and aliphatic dicarboxylic acid

Copolyester from 1,4-butanediol, adipic acid and terephthalic acid

Copolyester from 1,4-butanediol, adipic acid and terephthalic acid

Density (g/cm3)

1.25−1.27 (ISO 1183)

1.18

1.18–1.28

1.25 (ASTM D792)

Tm (°C)

110−115 (DSC)

125−135 (DSC)

110−120 (DSC)

125 (ASTM D2117) 11357-3

MFI

3−6a (190 °C, 2.16 kg) (ISO 1133)

−

≤5b (150 °C, 2.16 kg)

3b (190 °C, 2.16 kg) (ASTM D1238)

Tensile strength (MPa)

34c (ISO 527)

35d (ASTM D882)

15

35 (ASTM D638)

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Table 10.3 (continued) Trade name

Ecoflex

Origo-Bi

Producer

BASF AG, Germany Novamont, Italy

Elongation at break (%)

560c (ISO 527)

Biocosafe

S-EnPol

Xinfu Pharm, China

Samsung Fine Chemicals, Korea

700d (ASTM D882) 650

675 (ASTM D638)

Shore D hardness 32 (ISO 868)

−

33

−

Vicat VST A/50 (°C)

80 (ISO 306)

−

−

−

Transparency (%)

82c (ASTM D1003)

−

−

−

Oxygen permeation cm3/ (m2 × d × bar)

1,600c(DIN 53380) −

−

−

Water vapour permeation g/ (m2 × d)

140c (DIN 53122)

−

−

480d (ASTM E96)

cm3/10 min g/10 min c 50 µm film; machine direction d 30 µm film; machine direction MFI: Melt flow index a

b

10.3.6 Biodegradation of aliphatic-aromatic copolyesters Polyesters which solely contain aromatic acid components, such as PET or PBT, are used for many technical applications and are commonly regarded as quite resistant to any hydrolytic degradation. Only by applying very drastic chemical treatments, (e.g., sulfuric acid at 150 °C), very far from any physiological conditions, can the hydrolysis of such polymers be achieved at reasonable rates and used for recycling purposes [75]. Furthermore, chemical hydrolysis has been applied in some cases for the analysis of aromatic polyesters [76]. Since there are no reliable indications available in the literature that microbes and enzymes can attack aromatic polyesters such as PET, PBT or polyethylene naphthalate [2, 77, 78], pure aromatic polyesters cannot be considered biodegradable or compostable according to EN 13432, ASTM D6400 and GreenPla standards. The first published papers on the biological degradation of aliphatic-aromatic copolyesters came to the conclusion that the copolymers showed a significant degradability only with a quite high fraction of aliphatic monomers [4, 79–82]. However, these authors

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only investigated quite short degradation times − degradation with lipases (EC 3.1.1.3) for a few days, composting for about 15 days − and thus, the relative slow degradation processes of copolyesters were not detectable under the nonoptimised test conditions. In 1994, Witt and co-workers [52] first reported the microbial degradation of a block- copolyester [poly(trimethylenedecanoate)-block-(trimethyleneterephthalate)] with a molar ratio of 50% terephthalic acid in the whole acid component. In a mineral medium inoculated with sewage sludge, Witt and co-workers observed a weight loss in polyester films of about 9% within 4 weeks. In 1995, the same authors published data regarding the degradation of random aliphatic-aromatic copolyesters from terephthalic acid, 1,3-propanediol and adipic acid or sebacic acid (with a molar ratio of 30% terephthalic acid in the whole acid component) in a soil burial experiment [83]. The melting points of these random copolyesters were above 100 °C, which could promise better performances in industrial applications compared with PCL. In general, a decreasing degradation rate occurred when the aromatic fraction component increased. In another paper, the biodegradation of statistical copolyesters composed of ethylene glycol/adipic acid/terephthalic acid and 1,3-propanediol/ adipic acid/terephthalic acid and 1,4-butanediol/adipic acid/terephthalic acid, was examined in composting simulation tests [84]. While the fraction of terephthalic acid in the copolymers predominantly determined the degradation behaviour, the type of dicarboxylic acid monomer did not exert much influence on material degradation. BTA-copolyesters (made of terephthalic acid/adipic acid/1,4-butanediol) were studied in more detail in 1997 both in terms of biodegradation behaviour and material properties [85]. It was demonstrated that, with a molar ratio of terephthalic acid (referring to the acid components) between 40 to about 50%, materials with sufficient biodegradability and promising technical properties could be obtained. In order to determine the range of copolyester composition suitable to obtain specific technical properties without affecting its biodegradability, a correlation of biodegradation rate (in terms of erosion rate) versus molar ratio of terephthalic acid was obtained in composting conditions. This correlation allows a rough estimation of the time needed for the complete deterioration of items of BTA-copolyesters of different composition. The correlation is shown in Figure 10.10.

10.3.6.1 Polymer-related parameters determining biodegradation While a number of aliphatic components which alter the biodegradation behaviour of aromatic polyesters have been tested, the aromatic component predominantly used was terephthalic acid. The materials commercially available on the market also contain this aromatic dicarboxylic acid. The degradation behaviour of aliphatic-aromatic copolyesters generally depends on the monomer composition, as well as on the structure of the polymer chains at a given composition.

 Paolo Magistrali, Sebastià Gestí Garcia and Tiziana Milizia

10.0

1.2

1.0

8.0

0.8 6.0 0.6 4.0 0.4

30

32

34

36

38

40

42

44

46

Rate of decrease in thickness(µm/week)

Specific degradation rate (mg/week * cm2)

278 

48

Figure 10.10: Dependence of the degradation rate of statistical polyesters of 1,4-butanediol, terephthalic acid and adipic acid on the content of aromatic dicarboxylic acid; degradation of polyester films on mineral agar inoculated with a mixed population from compost at 60 °C [85]. Degradation is given either as weight loss per film surface area (left y-axis; in mg/week cm2) or as rate of surface erosion (right y-axis; in µm/week), calculated from weight loss data and material density.

When introducing terephthalic acid units into an aliphatic polymer, at first an increase in degradability can be observed at low levels of the aromatic monomer. Increasing the fraction of terephthalic acid, the degradation rate decreases and above a level in the range of 60−70 mol% terephthalic acid (with regards to the dicarboxylic acid component) no significant biological attack is observed [83]. An example of this behaviour is shown in Figure 10.11. This phenomenon is attributed to the melting point of the material − a correlation already demonstrated by Tokiwa and co-workers for different aliphatic polyesters [79]. In aliphatic-aromatic copolyesters, the melting behaviour is mainly determined by the length of the aromatic sequences in the polymer chains, which depends both on the composition and structure [86, 87]. For many aliphatic polyesters, a correlation of the degradability with the melting point was observed [2]. Marten [86] interpreted it as a decrease in the mobility of the polyester chains at lower temperatures; in this situation the polymer chains are highly fixed in the polymer crystals and cannot adjust easily into the active sites of the extracellular enzymes. A random insertion of some aromatic monomers in aliphatic polymer chains disturbs the formation of crystals. The amount of crystals (crystallinity) is reduced and the melting point is lowered due to the less regular crystal structures; both effects cause an increase in biodegradability. In contrast, at a higher content of aromatic dicarboxylic acid, the formation of crystals rich in terephthalic acid leads to increasing melting points and decreasing

10 Biodegradable Polyesters  10 PTS copolyester

200

8

150

6

100

4

50

2 0 10

0 250 PTS copolyester 200

8

150

6

100

4

50

2

0

20

40

60

80

100

Weight loss (mg/cm2)

Melting point (°C)

Weight loss (mg/cm2)

Melting point (°C)

250

0

 279

0

Content of terephthalic acid with reference to the total amount of acid (mol%) Figure 10.11: Weight losses and melting points of statistical copolyesters of 1,3-propanediol, terephthalic acid, sebacic acid (PTS copolyester) and 1,3-propanediol, terephthalic acid, adipic acid (PTA colpolyester) as a function of the molar fraction of terephthalic acid in the copolyester. Degradation of 100 µm films at room temperature in an aerated mineral salt medium, inoculated with an eluate from soil for 8 weeks. Reproduced with permission from U. Witt, R-J. Müller and W-D. Deckwer, Journal of Macromolecular Science A, 1995, 32, 4, 851. ©1995, Taylor and Francis [83].

degradability. This is also related to the temperature difference between the melting point of the materials and the temperature at which degradation takes place. The smaller the temperature difference the higher the mobility of the polymer chains − which is of great importance, since the chains have to fit into the active sites of the enzymes to be cleaved. Similarly, the manner in which the aliphatic and aromatic sequences are lined along the polymer chain has a great role in enzymatic attack. In fact, Marten demonstrated that a diester of 1,4-butanediol with benzoic acid, which represents a sequence in a PBT homopolyester, was attacked by a lipase from a Pseudomonas sp. With a degradation experiment using a strictly alternating copolyester of terephthalic acid, adipic acid and 1,4-butanediol, it was shown that even a sequence of only one aliphatic acid is sufficient to enable the enzyme to cleave the ester bonds within the polymer chain, while a block-copolyester of the same overall composition was not enzymatically degraded − although it could be thought that the long aliphatic

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sequences would facilitate its degradation. In the alternating copolyester, which has a melting point of 85 °C, the polymer chains are less fixed in the crystals than in the block-copolymers exhibiting melting points between 200−224 °C (representing the aromatic component). Besides the fixation of the polymer chains in the crystalline domains, the flexibility of the chain itself also influences the degradation behaviour to some extent. Copolyesters with long aliphatic dicarboxylic acids exhibit a somewhat higher degradation rate than those with shorter ones [85]. However, this effect is usually masked by the much higher influence of the melting point. In the range of 30−60 mol% of terephthalic acid, which is of particular interest for industrial applications, the degradation rate drops linearly with an increase in the aromatic acid content [85, 88] (Figure 10.12).

CO2, H2O, CH4 other metabolic products excretion of extracellular enzymes enzymes attach to the surface and cleave polymer chains extracellular enzymes

micro

intermediates are assimilated into the cells

organ

short degradation intermediates are dissolved into the medium

ism

surface erosion O O

O O

water-soluble intermediates

Figure 10.12: General scheme of microbial polymer degradation.

While the amount of terephthalic acid strongly influences degradation behaviour, the type of aliphatic monomer has less influence on degradation. For copolyesters of terephthalic acid and adipic acid differing in the diol component (1,2-ethanediol, 1,3-propanediol and 1,4-butanediol), similar erosion rates were observed in soil and compost [84] (Figure 10.13); the effect of different kinds of aliphatic dicarboxylic acids in copolyesters with terephthalic acid were also reported [86]. While the reduced enzymatic susceptibility of the aromatic ester bonds is caused by the interaction of the long chains in the polymer, the chain length itself has no direct influence on the biodegradability above a minimum molar mass. Tests with copolyesters from terephthalic acid, adipic acid and 1,4-butanediol, which were chain extended with hexamethylene-diisocyanate, did not result in a decrease in the biological degradation rate (in a compost simulation test), although the molar mass of the prepolyesters of about 48,000 D was increased to 232,000 D by the chain extension [85].

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10.3.6.2 Degradation under composting conditions Composting, the biological treatment of biowaste under controlled technical conditions, has been discussed as the primary environment where biodegradable plastics − and thus biodegradable aliphatic-aromatic copolyesters − will be degraded, (e.g., as waste from biodegradable packaging or as biodegradable biowaste bags). Under composting conditions, in parallel with the enzymatic action − which takes place solely at the surface of the material − the nonbiological, purely chemical process of hydrolysis is involved due to the temperatures of the degradation process. Water penetrates into the polymer matrix, hydrolyses the ester bonds and thus, lowers the MW of the entire material. For this reason, a number of publications used test systems to evaluate the biodegradation of plastics, which reflect similar conditions to a composting process. In addition, some major standards concerning biodegradable materials are focused on the degradation of plastics under composting conditions [67, 89, 90]. Degradation time

soil at ambient temperature

4 weeks

8 weeks

12 weeks

44300

50100 50100 53100

37300 39200

44700

41800

31600

ETA 38:62 ETA 44:56 PTA 39:61 PTA 43:57 30000 87 °C 39000 135 °C 51000 96 °C 40000 119 °C

47700

0

38500

20

47500

40

37100 14800 33800

60

30400

80

30300

Weight loss (%) molecular weight of residuals (g/mol)

100

BTA 34:66 BTA 42:58 BTA 51:49 47000 88 °C 46000 117 °C 50000 142 °C

Polyester initial molecular weight (g/mol) melting point

Figure 10.13: Weight loss of films of different aliphatic-aromatic copolyesters in soil at ambient temperature and mature compost at 60 °C, film thickness 100 µm [13], components: E = 1,2-ethanediol, P = 1,3-propanediol, B: 1,4-butanediol, A: adipic acid, T: terepthalic acid; both numbers at the end of the identification reflect the ratio of aromatic/aliphatic acid component in mol%, (e.g., ETA38:62 copolyester from 1,2-ethanediol, adipic acid and terephthalic acid with 38 mol% terephthalic acid in the acid component). PTA39:61-copolyester from 1,3-propanediol, adipic acid and terephthalic acid with 39 mol% terephthalic acid in the acid component; BTA34:66 copolyester from 1,4-butanediol, adipic acid and terephthalic acid with 34 mol% terephthalic acid in the acid component.

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 Paolo Magistrali, Sebastià Gestí Garcia and Tiziana Milizia

Compost at 60 °C

4 weeks

Degradation time 8 weeks

12 weeks

34200

12400

80

0

ETA 38:62 ETA 44:56 PTA 39:61 PTA 43:57 30000 87 °C 39000 135 °C 51000 96 °C 40000 119 °C

20300 44000 35400

41000

45000 32600

37900

22400

20

16700

12800

40

22700

60 17600

Weight loss (%) molecular weight of residuals (g/mol)

100

BTA 34:66 BTA 42:58 BTA 51:49 47000 88 °C 46000 117 °C 50000 142 °C

Polyester initial molecular weight (g/mol) meltingpoint

Figure 10.13 (continued)

Jun and co-workers [81] studied the degradation of a copolyester of PET and PCL, and came to the conclusion that at a fraction of more than 50% (w/w) of the aromatic component, no degradation took place. However, the incubation time was only 15 days, which is too short to monitor slow degradation processes. However, Witt and co-workers [84] observed for a BTA-copolyester film (composed of 1,4-butanediol, adipic acid and terephthalic acid) with 51 mol% of terephthalic acid, a significant weight loss after a 3-month incubation in compost at 60 °C (Figure 10.4). With these experiments, the biodegradation of polyesters with a relatively high content of aromatic components could be demonstrated for the first time. In 1998, BASF AG (Ludwigshafen, Germany) presented a respirometric measurement of the copolyester Ecoflex (approximately 45 mol% of terephthalic acid in the acid component), showing that this material was more than 90% metabolised in compost within three months [91]. Kleeberg and co-workers succeeded in 1998 in isolating and identifying a number of thermophilic microorganisms from compost, able to depolymerise BTA-copolyesters [92]. Out of 61 isolates, 30 strains were able to attack a BTA 40:60 (40 mol% terephthalic acid, 60 mol% adipic acid) copolyester at a detectable rate (clear-zone method and weight loss of films on agar plates). It turned out that under thermophilic conditions, actinomycetes play a dominant role in copolyester degradation. Out of the 30 degrading isolates, 25 belonged to the group of actinomycetes and only 5 to bacte-

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 283

ria. Fungi were not found to be relevant in copolyester degradation in compost, since most fungi only grow at temperatures lower than 50 °C. Two of the most active strains were identified and belong both to the genus Thermobifida and are consistent with the Thermobifida fusca taxon. In a screening experiment with 1,328 actinomycete strains from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany), 34 strains were identified which attacked a BTA 40:60 copolyester [93]. The degrading strains were both mesophilic and thermophilic organisms; however, thermophilic actinomycetes exhibited the highest degradation rates. A copolyester depolymerising extracellular enzyme was isolated and characterised from the strain Thermobifida fusca DSM 43793. The enzyme exhibits a homology of 65% with a triacylglycerol-lipase from Streptomyces sp. (strain M11) [94], has a molar mass of approximately 27,000 D and an optimal temperature for hydrolysing BTA-copolyesters of about 60 °C. The identification of the enzyme having a lipase- like structure is in accordance with the observation that many lipases are able to attack polyesters [2, 86, 95] and are probably also predominantly responsible for the microbial induced depolymerisation of synthetic polyesters in nature. With this thermophilic actinomycete strain it was possible to investigate the degradation behaviour of BTA-copolyesters very accurately over a timescale of a few weeks [66], and using the enzyme, the hydrolysis of BTA-copolyesters could be measured within less than 24 h [93].

10.3.6.3 Degradation in soil Keeping in mind the potential applications of biodegradable plastics in agricultural applications (i.e., mulching films), the characterisation of the degradation behaviour of the copolyesters in soil is currently of great interest. Generally, compared with composting, degradation in soil is slower and less predictable, due to lower soil temperature and variability in environmental conditions (e.g., humidity, temperature) and soil composition. Witt and co-workers [84] showed that a number of copolyesters (acid components: adipic acid and terephthalic acid; alcohol components: 1,2-ethanediol, 1,3-propanediol and 1,4-butanediol) degrade both in compost and soil (Figure 10.13). The weight losses of copolyester films (100 µm thickness) in soil (gardening soil, 60% humidity, ambient temperature, incubation times 1, 2 or 3 months) were significantly lower than in compost at 60 °C. While for a BTA-copolyester with about 40 mol% terephthalic acid in the acid component, a weight loss of approximately 50% after 3 months was observed, the same material degraded completely in compost within 3 months. Increasing the aromatic monomer to 50 mol%, no further weight loss of films could be detected in soil within the 3-month period.

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During a 60 °C incubation in compost, the test specimen material exhibited a significant loss in MW, indicating the contribution of nonenzymatically catalysed hydrolysis (abiotic) which takes place not only at the surface, but throughout the entire material. This abiotic hydrolysis was not observed in soil burial experiments within the testing period. Using similar conditions [96], the weight loss of 55 µm films of a BTA-based copolyester containing approximately 55 mol% terephthalic acid was tested in the same kind of soil and was compared with the degradation of PCL (PCL Tone787, Union Carbide, films of 75 µm thickness) (Figure 10.14) in soil and compost at 60 °C. For the BTA-copolyester and PCL, the weight losses are much smaller in soil than in compost. However, for the copolyester with 55 mol% terephthalic acid, a degradation of the film of about 30% could be measured after an incubation of 10 weeks. 100

Weight loss (%)

80 PCL/soil BTA/soil PCL/compost BTA/compost

60 40 20 0

0

2

8 6 4 Degradation time (weeks)

10

Figure 10.14:  Degradation of a BTA-based copolyester (approximately 55 mol% terephthalic acid) and PCL in soil at ambient temperature and in compost at 60 °C. Weight loss of films (BTA: 55 µm thickness and PCL: 75 µm thickness) which grew a number of mycelium-forming microorganisms (fungi, actinomycetes), shown to be important for the degradation of copolyesters in compost.

As far as soil composition is concerned, in recent years a BTA-copolyester with about 47 mol% terephthalic acid in film form of about 800 µm thickness was biodegraded both in composting conditions and in three different soils (canal shore, garden, peat moss) at 30 °C and 60% moisture content. It turned out that under these conditions, canal shore and garden soils were able to biodegrade the BTA-copolymer as fast as composting conditions (95% of weight loss after 8 weeks), while in peat moss the biodegradation rate was much lower (69% of weight loss after 8 weeks). This behaviour was ascribed to the high organic carbon/nitrogen ratio of peat moss soil (nearly 21) compared with canal shore and garden soils (nearly 4), which is unfavourable for most microbial growth [48].

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10.3.6.4 Degradation in an aqueous environment Degradation results obtained in a compost environment at high temperatures or in soil differ significantly from those determined in a liquid system. In liquid media, the degradation rate is usually much slower for copolyesters. Besides the lower temperature, which is in most cases at ambient level, a different microbial population may be responsible for the variation. In degradation tests in a liquid environment, the inoculum to provide microbial activity is often taken from sewage sludge. Although in some cases eluates from soil or compost are used to inoculate the media, a liquid environment is not optimal for the biodegradation of aliphatic-aromatic copolyesters. Van der Zee [97] reported only a very slow degradation in a modified Sturm test (measurement of CO2 which is produced by the microorganisms during the degradation of plastic) for copolyesters from terephthalic acid, adipic acid and 1,4- butanediol with levels of aromatic dicarboxylic acid of more than 30 mol% (with regard to the acid component). In a study concerning the applicability of different test methods, the degradation of an aliphatic-aromatic copolyester was found to be strongly dependent on the specific conditions (especially the kind and pretreatment of the inoculum) of the aquatic test [98]. After 50 days of incubation, a conversion of the polymer to CO2 in a range from less than 10% up to more than 90% was observed depending on the inoculum. Recently, studies on BTA-copolyester/organoclay nanocomposites have been carried out in order to increase the biodegradation rate of BTA-copolyester in an aqueous medium containing activated sludges. The addition of sodium modified montmorillonite to BTA-copolyesters with about 47 mol% terephthalic acid at a ratio of 10% in weight, led to an increase in biodegradation (measured by biochemical oxygen demand) from 10 to 20% after 60 days at 25 °C in an aqueous medium containing activated sludge. This behaviour was attributed to the preferential paths that can be followed by enzymes, conferred by the hydrophilic character of the sodium modified montmorillonite compared with the BTA-copolyester [99].

10.3.6.5 Degradation under anaerobic conditions Whilst a large number of investigations have been published for the degradation of plastics in the presence of oxygen, there is very little data on anaerobic biodegradation. Some aliphatic polyesters such as PHA or PCL also turned out to be biodegradable under anaerobic conditions [16]. However, aliphatic-aromatic copolyesters of the BTA type seem to be quite stable in the absence of oxygen. For incubation of BTA- copolyesters with both 40 mol% and 45 mol% of terephthalic acid, in anaerobic sewage sludge (at 37 °C) and also in an anaerobic high solids sludge from an anaerobic biowaste treatment plant (at 50 °C), no significant biodegradation of BTA films could be observed within a test period of three months [100]. Small weight losses of less than 5% were obviously caused by abiotic effects, such as migration of low molecular compounds or abiotic hydrolysis.

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In the same work, a number of anaerobic individual microbial strains degrading PHA, PCL and a polyester from 1,3-propanediol and adipic acid (SP3:6) were isolated and identified. BTA-copolyesters were attacked by the individual strains only when the content of terephthalic acid did not exceed 20 mol%. Here it can be supposed that these organisms predominately attack the quite long aliphatic domains in these copolyesters. So far there is no proof of the existence of anaerobic microorganisms which can depolymerise BTA-copolyesters in the range of compositions of interest for technical applications. The results reported up to now indicate that a biological treatment of BTAwaste in anaerobic digestion plants would be difficult. However, as the residence time of biowaste in anaerobic reactors is only in the range of a few weeks, whatever the product, even if biodegradable in three months under anaerobic conditions it will not be biodegraded in the anaerobic section of biogas plants. In any case, it is worth mentioning that such plants have a final aerobic maturation step of the anaerobic sludge. This last step is essential to avoid the use of digestate which has not been stabilised in the soil and allows the biodegradation of compostable bioplastics, such as aliphatic-aromatic copolyesters, to such an extent that the final compost quality is not negatively affected. The ability of these polymers to biodegrade in soil and water, besides composting, is further assurance for the full reabsorption of these products in the natural environment.

10.3.6.6 Fate of aromatic sequences and risk assessment A major point of criticism for aliphatic-aromatic copolyesters is the final degradability of the aromatic sequences in the polymers. In such statistical copolyesters there are domains in the polymer chains, where several aromatic dicarboxylic acids are linked with the alcohol component, without being interrupted by an aliphatic dicarboxylic acid. The distribution of sequence lengths depends on the ratio of aliphatic and aromatic dicarboxylic acids, and can be calculated for an ideal random copolymerisation using (Equation 10.1): War(n) = {([Mar]/([Mar] + [Mar]))n−1} {([Mal]/([Mar] + [Mal])}

(10.1)

Where: War(n): fraction of the aromatic dicarboxylic acid (in mol%) located in sequences of the length n. [Mal]: fraction of the aliphatic dicarboxylic acid monomer in the polymer (in mol%). [Mar]: fraction of the aromatic dicarboxylic acid monomer in the polymer (in mol%). n: length of a sequence. As an example, the distribution of the sequence lengths for a BTA 45:55 copolyester (45% terephthalic acid in the acid component) are listed in Table 10.4. If biodegradation is monitored by weight loss or disintegration, or even when carbon conversion is determined in a respirometric test, it is difficult to assert whether the ester sequences of the pure aromatic acid are also subject to biological attack. The

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first attempts to evaluate the biodegradation behaviour of oligomeric aromatic intermediates from copolyester degradation, used a specially synthesised aromatic oligomers model [101–103] for degradation experiments (Figure 10.15). It turned out that in a liquid mineral salt medium (inoculated with a mixed microbial population), in soil and in mature compost at 60 °C, oligomers with one or two repeating units (1: BTB and 2: BTBTB) were selectively removed from the synthetic oligomer mixture (average molar masses in the range from MW: 680 D through MW: 2,600 D), while the amount of longer aromatic oligomers remained almost unchanged in these experiments. Table 10.4: Sequence length distribution of aromatic domains in a statistical aromatic copolyester with 55 mol% terephthalic acid in the acid monomers. Length of aromatic sequence (number of repeating units)

Fraction of terephthalic acid monomers in the sequences of length n (mol% of terephthalic acid monomers)

1

0.550

2

0.248

3

0.111

4

0.050

5

0.023

6

0.010

7

0.005

8

0.002

9

0.001

The big change in degradation behaviour is related to the water solubility of the oligomers. Only mono- and diesters of terephthalic acid and 1,4-butanediol (or 1,2-ethanediol or 1,3-propanediol) were water soluble to some extent (the oligomers were OH-terminated) and could therefore get into the microbial cells to be metabolised. The microbial transformation could be confirmed by following the degradation of the soluble oligomers in a liquid medium using CO2 measurements. However, despite the results with oligomeric model substances it was shown that under temperature conditions similar to a composting process, longer aromatic oligomers, which were generated as intermediates from the biodegradation of the copolyesters, can be totally degraded by microorganisms. In a degradation experiment with a BTA 40:60 copolyester in a liquid medium and on agar plates which were inoculated with a prescreened mixed microbial population from compost, the formation of various oligomeric intermediates could be proved using GPC measurements [103–105] (Figure 10.16). However, quantitative analysis revealed that the different oligomers, (e.g., with two or three repeating units), were present in lower concentrations than could be

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Liquid medium at RT 5 weeks

3

4

2

1

2a

Soil at RT 8 weeks

4

3

2 1

2a

Compost at 60 °C 8 weeks

4

3

2

1

2a

6

7

8 Elution volume (ml)

Before degradation

9

10 After degradation Blank test

Figure 10.15: Gel permeation chromatography (GPC) chromatograms of a synthetic oligomer mixture synthesised from 1,4-butanediol and terephthalic acid before and after incubation in different microbial active environments (synthetic mineral medium at room temperature inoculated with a compost extract); soil at room temperature and compost at 60 °C. Grey area: GPC profile of the oligomer mixture before incubation, dotted line: GPC profile of the oligomer mixture after incubation in sterile water under conditions comparable with the degradation experiment (blank test), and solid line: GPC profile of the oligomer mixture after incubation in the different microbial environments. RT: room temperature. Reproduced with permission from U. Witt, R-J. Müller and W-D. Deckwer, Journal of Environmental Polymer Degradation, 1996, 4, 1, 9. ©1996, Springer [102].

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Synthetic BT-oligomer mixture

BTB

Extract from agar

BTBTBTB

BTBTBTBTBTB BTBTBTBTB

BTBTB

Calibration with a synthetic BT-oligomer mixture

BTA degradation on an agar plate Elution profile of initial BTA polyester

* 10.2

Residual BTA polymer

Peak height expected from theory

* 2.2

Extract from agar

5

Residual oligomers

6

7

8

9

10

Time (min) Figure 10.16: GPC chromatogram of a BTA 39:61 copolyester after degradation for 11 weeks at 55 °C on a mineral medium agar plate, inoculated with a prescreened microbial population. The upper diagram represents the calibration with a synthetic oligomer mixture of known composition. The molar mass distribution of the degraded sample (solid line) changed to lower masses during degradation compared with the initial molar mass distribution (hatched area). The two aromatic oligomers formed during degradation could by identified (BTBTB and BTBTBTB). Their theoretically expected concentrations according to Equation 10.1 are marked with arrows and the factor of the theoretical concentration divided by the measured concentration is given above the arrows. Reproduced with permission from R-J. Müller, U. Witt and W-D. Deckwer, Fett/Lipid, 1997, 99, 2, 40. ©1997, Wiley [104].

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calculated from Equation 10.1. In Figure 10.16, aromatic oligomers formed during the degradation of the BTA polymer were identified by comparison with a synthetic BT-oligomer mixture. The concentrations of the BTBTB-oligomer (dimer) and the BTBTBTB-oligomer (trimer) were lower by a factor of 2.2 and 10.2, respectively, as would be expected from the aromatic sequence length distribution (Equation 10.1) without considering any degradation. The formation and the degradation of aromatic intermediates could be monitored by gas chromatography-mass spectrometry (GC-MS) and GPC analysis [66]. The commercial copolyester Ecoflex was degraded in a synthetic liquid medium using a thermophilic microbial strain which is able to depolymerise the polymer very effectively, but cannot metabolise the intermediates. Under these conditions, it was possible to generate high concentrations of degradation intermediates in the medium, allowing an accurate analysis. After three weeks of incubation, neither residual polymer nor nonsoluble intermediates could be detected. From the lower detection limit of the GPC method used, a 99.9% depolymerisation of the material was estimated. From Equation 10.1 it can be calculated that for Ecoflex, a material based on a BTA 45:55 copolyester, about 20 mol% of the terephthalic acid forms sequences of more than 2 aromatic repeating units, corresponding to about 10% (w/w) of the material consisting of aromatic sequences which were earlier shown to be water insoluble and hardly biodegradable; thus, it could also be concluded that these long aromatic intermediates were subject to degradation. In the medium of the degradation experiment, the monomers (1,4-butanediol, terephthalate and adipate) could be detected, along with some short aliphatic and aromatic mono-, di- and triesters. However, upon adding a mixed population to the monomeric and oligomeric intermediates, a complete metabolisation could be observed within 14 days. Depolymerisation is due to various reasons. The lack of biological accessibility seems to be determined predominantly by intramolecular interactions. The aromatic intermediates formed during polymer degradation are embedded in a different environment than aromatic oligomers in the model substances. Furthermore, the model esters were mostly OH-terminated, while during polyester hydrolysis COOH-endings of the oligomers will also probably occur. Both effects can cause the final hydrolysis and metabolisation of the aromatic copolyesters observed in the experiments by the individual microbial strain. In the same work, estimation of the environmental effect of a BTA-copolyester when treated in a composting plant and the resulting compost used for agriculture, was also assessed. Based on toxicological tests with Photobacterium phosphoreum and Daphnia magna a risk assessment was calculated. For both test organisms, no toxic effect of the intermediates produced during degradation were detected. Quite conservative assumptions were made, such as the compost is loaded with 1% (w/w) polymer, and that the entire polymer is depolymerised into oligomers and monomers, but these intermediates are not metabolised and remain in the compost material

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(actually it has been shown that the intermediates are rapidly metabolised by a mixed culture of microorganisms). After a 50% weight reduction of the biowaste during composting (and thus an increase in concentration of the intermediates by a factor of two), 30 tonnes of compost per hectare were applied to the fields over 3 years (the maximum value recommended by the German biowaste directive) and were ploughed 30 cm deep into the soil. The concentration of the degradation intermediates was then calculated to be 130 ppm. Toxic effects could be excluded up to a concentration of approximately 1,400 ppm in the toxicity tests and thus, it can be expected that no toxic effect will result from the application of compost from the copolyester treatment.

10.4 Renewable monomers for biodegradable polyester synthesis As mentioned before, there are a few biodegradable polyesters which were historically obtained from renewable sources as their production concerns fermentation or microbial processes from vegetable raw material (i.e., PLA, PHA and more recently polyhydroxyfattyacid). Moreover, in the last 5 years an increasing interest in renewable raw material for polymer synthesis has been developed, not only for biodegradable polyester production but also for nonbiodegradable polymers such as polyethylene or PET. This interest is due to the fact that reduction of the carbon footprint arising from human activity is becoming more and more important, both for decreasing the fossil resource consumption and trying to limit climatic changes due to the greenhouse effect (even though scientists partially disagree on this effect) [106]. With regards to biodegradable polyester production, the use of renewable monomers can be of particular interest as not only can the final products be biodegraded, hence avoiding plastic accumulation in landfills, but also the biodegradation products (usually CO2 and water) are used by plants to produce the renewable monomers. As can be deduced, this ‘cradle-to-grave’ cycle can significantly reduce the carbon footprint due to plastic production. In recent years, a breakthrough towards renewable monomer production has been possible with the help of genetic science, which can manipulate microorganisms to produce specific molecules from vegetable raw materials. The main monomers for the most common biodegradable polyester production, which can now be obtained from renewable resources are: succinic acid, 1,4-butanediol, 1,2-ethanediol, sebacic acid and azelaic acid. So far, wholly renewable biodegradable polyesters belong only to the aliphatic family, but efforts are being made to obtain aromatic dicarboxylic acids from renewable resources in order to fill this gap, as demonstrated by the research achievements involving aliphatic-aromatic copolyesters based on furandicarboxylic acid [59–61].

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The industrialisation of 1,4-butanediol and succinic acid production from biomass is in progress. Hence, aliphatic-biopolyesters containing a diol/dicarboxylic acid repetitive unit from 100% renewable resources will be available. Both Mitsubishi and Showa Denko are planning to expand their product portfolio with renewable-based PBS. Since January 2010, Bioamber, a joint venture between US-based Diversified Natural Products Inc. and Green Technology Agroindustrie Recherches et Développements has been producing biobased succinic acid in France, with an initial capacity of 2,000 metric tonnes, and is planning the building of a new facility in Canada of 30,000 tonnes/year capacity in partnership with Mitsui [107]. Reverdia, a joint venture between DSM and Roquette, is dedicated to the production, commercialisation and market development of sustainable succinic acid, Biosuccinium. Since 2010, Reverdia has been producing tonnes of Biosuccinium in a demonstration plant in France. In 2011, Reverdia completed the construction of a commercial-scale plant with 10,000 tonnes/year capacity of biobased succinic acid, using fermentation technology in Cassano Spinola, Italy; production started in December 2012 [108]. In 2009, BASF and Purac formed a partnership for the development of the industrial fermentation and downstream processing of biosuccinic acid, and are now establishing a joint venture for the production and sale of biobased succinic acid. The company has been named Succinity GmbH and started up officially in August 2013. The two companies have modified an existing fermentation facility of Purac in Montmelò, Spain, for the production of succinic acid. The annual capacity of the plant is 10,000 metric tonnes and production started in March 2014 [109]. Myriant Technologies, in the USA, has received a $50 million grant from the US Department of Energy to help build a commercial-scale biosuccinic acid facility in Louisiana of 15,000 tonnes/year capacity. Currently, the company is producing succinic acid via fermentation on a pilot scale [110]. 1,4-butanediol from renewable resources can be produced both by the hydrogenation of biosuccinic acid, or a more promising way developed by Genomatica, by the one- step fermentation of sugars. Mater Biotech, a subsidiary of Novamont, is building the world’s first 1,4 biobutanediol dedicated plant in Bottrighe, Italy, in a joint venture with Genomatica [112]. 1,2-ethanediol from renewable resources is going to be produced at a large scale of 500,000 tonnes/year by the joint venture between JBF Industries Ltd. and CocaCola, in Brazil [113]. In 2014 Matrica, the 50/50 joint venture between Novamont and Versalis, was the first plant worldwide able to produce azelaic acid from the oxidative cleavage of vegetable oil without ozonolysis. The plant, in Porto Torres, Sardinia, has an oil capacity of 30,000 tonnes/year [112].

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[90] EN 13432, Packaging − Requirements for Packaging Recoverable Through Composting and Biodegradation − Test Scheme and Evaluation Criteria for the Final Acceptance of Packaging, European Norms, Brussels, Belgium, 2000. [91] Ecoflex – A Biodegradable Plastic from BASF, Press release for K98, BASF AG, Düsseldorf, Germany, 17th March 1998. [92] I. Kleeberg, C. Hetz, R.M. Kroppenstedt, R-J. Müller and W-D. Deckwer, Applied Environmental Microbiology, 1998, 64, 5, 1731. [93] I. Kleeberg in Untersuchungen zum mikrobiellen Abbau von aliphatisch– aromatischen Copolyestern sowie Isolierung und Charakterisierung eines polyesterspaltenden Enzyms, Technical University Braunschweig, Germany, 1999. [Dissertation in German] http://opus. tu-bs.de/opus/volltexte/2000/90. [94] C. Perez, K. Juarez, E. Garcia-Castells, G. Soberon and L. Servin-Gonzales, Gene, 1993, 123, 1, 109. [95] T. Walter, J. Augusta, R-J. Müller, H. Widdecke and J. Klein, Enzyme and Microbial Technology, 1995, 17, 3, 218. [96] S. Basei in Influence of Starch on the Aerobic and Anaerobic Biodegradation of an Aliphatic and an Aliphatic-Aromatic Polyester, University of Venice, Italy, 1999. [Graduation thesis] [97] M. van de Zee in Structure-Biodegradability Relationships of Polymeric Materials, University of Twente, Twente, The Netherlands, 1997. [Dissertation] [98] U. Pagga, A. Schäfer, R-J. Müller and M. Pantke, Chemosphere, 2001, 42, 3, 319. [99] Y. Someya, N. Kondo and M. Shibata, Journal of Applied Polymer Science, 2007, 106, 730. [100] D.M. Abou-Zeid, R.J. Muller and W.D. Deckwer, Biomacromolecules, 2004, 5, 5, 1687. [101] E. Rantze, I. Kleeberg, U. Witt, R-J. Müller and W-D. Deckwer, Macromolekulare Symposia, 1998, 130, 319. [102] U. Witt, R-J. Müller and W-D. Deckwer, Journal of Environmental Polymer Degradation, 1996, 4, 1, 9. [103] U. Witt, Charakterisierung und Beurteilung der Biologischen Abbaubarkeit von anwendungsorientierten Biologische Abbaubaren Aliphatisch– Aromatischen Copolestern, Technical University Braunschweig, Germany, 1996. [Dissertation in German] [104] R-J. Müller, U. Witt and W-D. Deckwer, Fett/Lipid, 1997, 99, 2, 40. [105] E. Marten, R-J. Müller and W-D. Deckwer, Polymer Degradation and Stability, 2005, 88, 371. [106] United Nations Framework Convention on Climate Change, Bonn, Germany. http:/infccc.int/ kyoto_protocol/items/2830.php [107] BioAmber Inc., Plymouth, MN, USA. http://www.bio-amber.com/bioamber/en/company/ manufacturing_facilities [108] Reverdia, Geleen, The Netherlands. http://www.reverdia.com [109] BASF, Ludwigshafen, Germany. http://www.basf.com/group/pressrelease/P-14-0303-CI. [110] Myriant Corporation, Quincy, MA, USA. http://www.myriant.com/media/press-kit.cfm. [111] Genomatica Inc., San Diego, CA,USA/. http://www.genomatica.com/manufacturing/. [112] Novamont S.p.A, Novara, Italy. http://www.novamont.com/detail.asp?c=16&p=1&id=23319. [113] JBF Industries Ltd., Mumbai, India. http://www.jbfindia.com/project.htm. [114] D.K. Gilding and A.M. Reed, Polymer, 1979, 20, 12, 454. [115] P. Sharma and B. Gordon, Polymer Preprints, 1989, 30, 2, 197. [116] B. Gordon, P.P. Sharma and S.L. Hansen, Polymer Preprints, 1990, 31, 1, 507. [117] S. Heidary and B. Gordon, Polymer Materials Engineering and Science, 1992, 67, 190. [118] D. Kint and S. Munoz-Guerra, Polymer International, 1999, 48, 5, 346. [119] A. Niekraszewiecz, Polymeri (Warswa), 1993, 38, 8−9, 399. [120] S. Heidary and B. Gordon, Journal of Environmental Polymer Degradation, 1994, 2, 1, 19. [121] T. Kiyotsukuri, T. Masuda, N. Tsutsumi, W. Sakai and M. Nagata, Polymer, 1995, 36, 13, 2629. [122] D-K. Kim, Y.S. Shin, Y-T. Yoo and J-R. Huh, Polymer (Korea), 1996, 20, 3, 431.

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Stéphane Guilbert and Bernard Cuq

11 Material formed from proteins 11.1 Introduction Over the last two decades, there has been a renewal of interest in the development of recyclable, biodegradable and/or edible materials formed with raw materials of agricultural origin [1, 2]. These materials are often referred to as ‘agromaterials’ or ‘biopackagings’ (when used to make trays or packaging films). Natural biodegradable thermoplastic materials are commonly called ‘bioplastics’. The ‘agromaterial’ concept generally involves the use of renewable raw materials which can be recycled after utilisation [3–5]. The main uses of agromaterials and bioplastics are reviewed in Table 11.1. Proteins are natural polymers that have long been empirically used to produce edible packaging and materials (i.e., soybean lipoprotein sheets in Asia and collagen envelopes). At the beginning of the 20th century, proteins were considered interesting raw materials for making plastics to eventually replace cellulose. Formaldehyde crosslinking of milk casein (i.e., galalith) is a process that was invented as early as 1897 to make moulded objects such as buttons [6, 7]. The first patents were taken out in the 1920s on the use of zein to formulate different materials (coatings, resins, textile fibres). At that time, formaldehyde was widely used in blends with soybean proteins and slaughterhouse blood to make automotive parts, especially distributor caps [8]. In addition, gelatin was used to produce films for foods, drug capsules and photography; protein materials were subsequently used for many applications (Table 11.2). In the 1960s, synthetic plastics posed a serious threat to proteins for many of these applications. The abandonment of protein materials (except for gelatin) lasted for the next 30 years. Since the 1980s, the number of academic research programmes, and industrial research and development projects on protein-based bioplastics has increased exponentially, as a result of the present interest in using some field crops for renewable and biodegradable materials for nonfood applications, and also in order to explore the unique specific properties of proteins. The complexity of proteins and the diversity of their different fractions can be tapped to develop materials with original functional features which differ markedly from those of standard synthetic plastic materials. Apart from the previously mentioned proteins, many other proteins (wheat and maize gluten, cottonseed flour, whey proteins, myofibrillar proteins and so on), can be used as raw materials to produce films, moulded materials and various hollow items. Materials formed from proteins are biodegradable and even edible when foodgrade additives are used. In addition, they are generally biocompatible except for some traits associated with specific proteins (e.g., gliadins in wheat gluten are allergenic), their processing, and the presence of impurities or additives. https://doi.org/10.1515/9781501511967-011

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Table 11.1: Main applications of agromaterials and bioplastics. Plastics to be composted or recycled

– Food packaging (dried foods, short life cycle food, egg boxes, fresh or minimally processed fruits and vegetables, dairy products, organically grown products and so on) – Paper or cardboard (windows from paper envelopes or for cardboard packaging, coating for paper or cardboard) – Hygienic disposable (nappies, sanitary napkins, sticks for cotton swabs, razors, toothbrushes and so on) – Miscellaneous short life goods (pens, toys, gadgets, keys holders and so on) – Dishes and cutlery (trays, spoons, cups and so on) – Loose-fill packaging (shock absorbers); waste and carrier bag (compost bags) and blister packaging

Plastics used in – Biodegradable/soluble/controlled release materials for agriculture and natural environment fisheries (mulching plastic, films for banana culture, twine, flower pots, (no recovery) materials for controlled release fertilisers or agrochemical, high water retention materials for planting, soluble sachets, biodegradable containers for fertilisers or agrochemical, fishing lines and nets) – Civil engineering, car industry and construction materials (heat insulators, noise insulators, car interior door casings, retaining walls or bags for mountain areas or sea, protective sheets and nets for tree planting) – Disposable leisure goods (golf tees, goods for marine or mountain sports) Specialty plastics

– Edible films and coatings (barrier internal layers, surface coatings, ‘active’ superficial layers, soluble sachets for instant dry food and beverages or food additives) – Matrix for controlled release systems (slow release of fertilisers, agrochemical, pharmaceuticals and food additives) – O2 barrier, selective CO2/O2 barrier, aroma barrier (simple or multi-layer packaging) – Medical goods (bone fixation, suture threads, films, non-woven tissues and so on) – Super-absorbent materials (material for plant planting in desert, nappies and so on) – Adhesives (glue)

Adapted from S. Guilbert, Bulletin of the Research Institute of Food Science, Kyoto University, 1999, 56, 38 [4]. Table 11.2: Main applications of protein-materials until the 1960s. Applications Films, coatings, – Waterproofing of paper and cardboard bags for food packaging facings and – Paper glazing for magazine covers and sleeves for long-playing records adhesives – Edible coatings for pharmaceutical tablets – Edible coating for food products (protection against water absorption or lipid oxidation)

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Table 11.2 (continued) Applications – – – – – Textile fibres

Photographic supports (or papers) Pigment binder for printing inks Adhesives for pasting (wallpapers), sticking (labels on bottles) and wood veneers Adhesives for cork and chipboard, (e.g., ‘hardened wood’ based on egg white)

– ‘Vicara’ (corn zein-based fibres) – Casein textiles

Moulded plastic – Buttons, door handles, belt buckles and driving belts in car engines items – Jewellery (necklaces and earrings) Adapted from L. Di Gioia in Obtention et Etude de Biomatériaux à Base de Protéines de Maïs, ENSA Montpellier, France, 1998. [PhD Thesis] [9].

11.2 Structure of material proteins Until recently, research work on the structure, properties and applications of proteins were mainly considered within the scientific field of Food Science. To reach a better understanding of properties and to define the potential applications of material proteins, it is essential to compare their structural features with those of chemically synthesised organic polymers used to produce plastic materials. Novel research on nonfood uses of agricultural resources, and especially on ‘material proteins’, has led to the application of Polymer Science concepts and tools to investigate the structurefunction relationships of these macromolecular organisations. This involves: – Investigating the three-dimensional (3D) structure of proteins at different levels (atomic, molecular and supramolecular arrangements). – Studying structural variations according to temperature and the presence of functional additives. – Simulating the macroscopic properties of macromolecular arrangements (mechanical, optical, thermal and electrical properties). Proteins (except homopolymers or copolymers in which one or two monomers are repeated) are heteropolymers comprising more than 20 different amino acids, each with specific sequences and structures. The structure of the 20 natural amino acids, shown in Figure 11.1, highlights the high chemical variability conferred by the lateral groups. Amino acids are generally classified by groups that could interact via hydrogen bonds (nonionised polar amino acids), ionic interactions (ionised polar amino acids), nonpolar interactions (nonpolar amino acids) or covalent bonds (disulfide or dityrosine bonds). Amino acids are also classified on the basis of their relative hydrophobicity (Table 11.3).

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 Stéphane Guilbert and Bernard Cuq NH2 COOH

CH

R CH3

CH3 H

CH3

Glycine (Gly)

CH2

CH

Alanine (Ala)

CH2

S

Valine (Val)

CH3

CH2

C

CH2

CH

CH

CH2

C

C

CH

C

CH

CH

CH

Tryptophane (Trp)

Phenylalanine (Phe)

CH2

-A-

CH2

CH

COOH

CH2

CH2 CH2

CH2

Lysine (Lys)

CH2

Arginine (Arg)

CH2

CH2OH

NH

CH2OH Serine (Ser)

CH2

CO

NH2

Asparagine (Asn)

NH2

CH2

NH CH2

NH2

CO

Glutamine (Gln)

Threonine (Thr)

Aspartic acid (Asp)

CH2

CH2

CH3

COOH

Glutamic acid (Glu)

CH2

CH3

CH

NH

Proline (Pro)

CH

CH2

CH

Isoleucine (Ile)

CH

N CH

CH3

CH

Leucine (Leu)

CH2 CH2

Methionine (Met)

CH

CH2

CH3

CH3

C

NH2 -B-

C

Tyrosine (Tyr)

CH

CH

CH

CH

C

OH

C

CH

CH2

-C-

NH

N CH

-D-

CH2

SH

Cysteine (Cys)

Histidine (His)

Figure 11.1: Biochemical structure of amino acids and their different lateral chains. Classification: nonpolar amino acids (-A-), ionised polar amino acids (-B-),nonionised polar amino acids (-C-), amino acids able to form –SS– bonds (-D-).

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Table 11.3: Relative hydrophobicity (or polarity) of the different amino acids. Amino acid

Relative hydrophobocity

Polarity Aminoacid Relative hydrophobocity

Polarity

Arg

+176

Polar

Tyr

−2

–

Lys

+110

Polar

Cys

−4

Non-Polar

Asp

+72

Polar

Gly

−16

Non

Gln

+69

Polar

Ala

−25

Non

Asn

+64

Polar

Met

−26

–

Glu

+62

–

Trp

−37

Non

His

+40

–

Leu

−53

Non

Ser

+26

–

Val

−54

Non

Thr

+18

–

Phe

−61

Non

Pro

+7

–

Ile

−73

Non-Polar

Adapted from J.A. Rothfus, Journal of Agricultural and Food Chemistry, 1996, 44, 10, 3143 [10].

The molecular diversity means that proteins have considerable potential for the formation of various interactions and links that differ according to their position, nature and/or energy [11, 12]. This heterogeneous structure provides many reaction sites for potential crosslinking or chemical grafting − it even facilitates modification of the film-forming properties and end-product properties. The amino acid sequence formed by peptide bonds is called the primary structure. The secondary structure concerns the spatial pattern of the peptide chain. This involves λ-helix structures and λ structures, i.e., a zigzag structure which is more stretched than the λ-helix. These stretched chains bind to form folded structures. The lateral group structure of some amino acids upsets these ordered patterns, resulting in some of these proteins having a random coiled ‘less ordered’ structure. The tertiary structure corresponds to a 3D polypeptide chain organisation containing organised and unorganised secondary structure zones. In a polar solvent medium, hydrophilic amino acids are distributed over the surface of the molecule, while nonpolar amino acids tend to be located within the structure and give rise to hydrophobic interactions. The so-called quaternary structure is generally formed by noncovalent associations of 3D organised protein subunits that are sometimes identical. Several different types of interactions help to stabilise the secondary, tertiary and quaternary structures of material proteins. Concerning low-energy interactions, van der Waals interactions (especially London forces) have very little impact on protein arrangement structuring, as compared with synthetic polymers. As a comparison, Table 11.4 gives the molar interaction energy of material proteins and synthetic organic polymers. Material proteins are mainly stabilised by ionic or hydrogen

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interactions. Lower overall values for proteins compared with synthetic polymers could be explained by their highly heterogeneous structure, thus reducing the frequency of ordered zones that promote such interactions. Hydrophobic interactions, which only take place in polar solvent solutions, should also be mentioned in connection with proteins. The energy of such interactions depends on the hydrophobic or hydrophilic amino acids and the type of solvent involved [14]. Material proteins often resemble elastomers due to the presence of disulfide bonds between cysteine residues. These proteins are highly stabilised by hydrogen bonds, contrary to most elastomers that have very weak secondary bonds. Table 11.4: Comparison of molar interaction energy of material proteins and synthetic organic polymers. Interaction Type

Energy of interaction (kJ/mol) Proteins

Synthetic polymers

Van der Waals

0.1–0.3

2–17

Hydrogen bonds

8.4–42

≈40

Ionic interactions

21–84

160–560

Adapted from C. Oudet in Polymères – Structure et Propriétés, Masson, Paris, France, 1994 [13] and L.G. Phillips, D.M. Whitehead and J. Kinsella in Structure-Function Properties of Food Proteins, Eds., L.G. Phillips, D.A. Whitehead and J. Kinsella, Academic Press, Inc., San Diego, CA, USA, 1994 [14].

Material proteins could thus be defined as amorphous 3D arrangements, stabilised by low-energy interactions, which are eventually strengthened by covalent bonds (-SS- bonds). The thermomechanical behaviour of material proteins should be compared with the behaviour of thermoplastic compounds or thermoplastic elastomers. This means that they could be used to form materials by dissolution in a solvent filmforming solution, with subsequent spreading and drying (i.e., the ‘casting’ process), or via thermal processes (extrusion, thermomoulding, injection and so on). The functional properties of these material proteins depend on their structural heterogeneity, heat sensitivity and hydrophilic characteristics. In organic polymers, macromolecules can form regular ‘crystal network’ type arrangements. These arrangements have a marked effect on the properties of polymers, especially their mechanical strength. For proteins, α-helix or β-sheet secondary structures are highly stabilised by hydrogen bonds and can resemble crystalline zones (Figure 11.2). Rothfus [10] demonstrated that the presence of β-sheets determines a cereal’s potential use as an adhesive, coating or textile fibre. For example, X-ray studies revealed that the stretching of protein fibres can lead to the formation of crystalline structures, thus enhancing their mechanical resistance.

11 Material formed from proteins 

a)

O R

b)

-

O

C

C

O

N

O

N

C

C

O C N

C

O C C O N C

R C N

R

R

N

O

N

R

N

R

R

 305

N

C

R

O C

O

+

C N

Figure 11.2: Structural organisation (α-helix and β-sheet of proteins). a) structure of an α-helix (3.6 amino acids per turn, stabilised by H-bonds) and b) structure of a β-sheet (antiparallel linear chains, stabilised by H-bonds). Reproduced with permission from L.G. Phillips, D.M. Whitehead and J. Kinsella in Structure-Function Properties of Food Proteins, Eds., L.G. Phillips, D.A. Whitehead and J. Kinsella, Academic Press, Inc., San Diego, CA, USA, 1994. ©1994, Academic Press, Inc. [14].

Protein molecular weights (MW) have a substantial effect on the protein networkstructure. They also determine the presence of molecular entanglements, leading to the formation of physical nodes. As is the case for synthetic macromolecules, entanglements could occur beyond a critical molecular weight (Mc) of around 104 g/mol, and thus the material properties would be stable (Figure 11.3). Table 11.5 compares the mean MW of synthetic polymers and proteins commonly used in material production. A high mean MW also hampers polymer flow during material formation, which can lead to defects in the end product. Synthetic polymers are characterised by their MW heterogeneity (i.e., polydispersity), which could be explained by the fact that chain growth is stopped randomly by the absence of residual monomers (polycondensation) or by termination reactions (polyaddition). On the other hand, the structure and MW of proteins are determined during synthesis (by using the genetic code), which means that for a given subunit there is very little variability. However for each protein family, there are generally several subunits of different MW, hence polydispersity. As for synthetic polymers, protein materials with high ‘apparent’ polydispersity are generally easier to process but have inferior mechanical properties.

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Strength at break

Log (MW) Mc

4 Mc

Figure 11.3: Influence of MW on rupture strength for a polymeric material. Adapted from C. Oudet in Polymères − Structure et Propriétés, Masson, Paris, France, 1994 [13].

Table 11.5: MW and main sub-units of protein-materials used to form ‘bioplastics’ and comparison with some conventional synthetic plastics. Proteins

Nomenclature of main sub-units

Corn gluten

α, β, γ, δ Zein A, B, C, D, E, F Glutelin

Wheat gluten

α, γ, ω Gliadins Glutenins

MW (kDa)

References

10–28

[13, 14]

11–127 30–80

[15]

200–2,000

Soy proteins

Glycinin

363

[16]

Peanut proteins

Arachin

330

[16]

Cottonseed proteins

Albumins

10–25

[17]

Globulins

113–180

Gelatin

–

3–200

[18]

Caseins

αs1, αs2, β, κ, γ Caseins

19–25

[15]

Myofibrillar proteins

Myosin

16–200

[19]

Actin Polymethyl methacrylate

–

Polyethylene

High density Low density

42 100–200

[4]

20–60

[4]

200–400

Adapted from L. Di Gioia in Obtention et Etude de Biomatériaux à Base de Protéines de Maïs, ENSA Montpellier, France, 1998. [PhD Thesis] [9].

11.3 Protein-based materials Many plant and animal proteins have been considered as raw materials for producing films and coatings, which are generally characterised by functional properties of great interest [5, 12, 12–29].

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Corn zein has been the focus of considerable industrial interest, especially during the first half of the 20th century, in manufacturing films, lacquers, varnishes, adhesives, textile fibres and moulded plastic objects [30]. During World War II, this protein was considered as a strategically important substance, being used as a substitute for shellac, which was in short supply [31]. Zein is one of the four proteins (along with milk casein, soy glycinin and peanut arachin) that have been used for fibre manufacturing. In the 1950s, this provided a major outlet for zein, with 1,800 tonnes/year, sold to produce ‘Vicara’ fibres. A standard protein spinning process (developed especially for soy protein texturing) was used to produce zein fibres. The process involves an initial stage of protein solubilisation (in organic solvents or alkaline medium), spinning in an acidic coagulant bath, formaldehyde-induced hardening and drying. Recently, soy protein and zein blends were studied to produce textile fibres with the aim of improving fibre properties and decreasing their manufacturing cost [32]. Zein is not water-soluble because of its high nonpolar amino acid content. Zein solutions are thus obtained by solubilisation in a solvent (generally alcohol or a volatile organic solvent), or in an alkaline medium, sometimes with a soap supplement. Zein water dispersions (zein ‘latex’) are now commercially available (e.g., OptaGlaze from Opta Food Ingredients Inc., Bedford, MA, USA). Another technique involves direct hot press moulding of zein, after decreasing the glass transition temperature (Tg) via the addition of plasticisers [33]. Many studies and patents have focused on the production of moulded or calendered and stamped zein-based plastics [34–36]. The manufacturing process generally involves hot mixing (40−90 °C) of zein with nonvolatile plasticisers, water (up to 20% w/w) and other additives (crosslinking agents, pigments, extenders). The plasticised mass is hot press moulded (100−150 °C) for 1−2 min, or up to 15 min when formaldehyde is added (0.5−5% w/w) as the crosslinking agent. The mould is then cooled to around 90 °C and the piece is removed from the mould. When formaldehyde has been added, the piece can be left to harden for about 10 h, at atmospheric pressure and 60−90 °C. Zein has many amide functions that could form methylene bonds in the presence of formaldehyde and at temperatures above 40 °C [37]. Free formaldehyde is thus inserted in the protein before moulding. Hardening is then achieved by heat treatment (>40 °C) or acid catalysis [38]. Zein has been used to enhance the water resistance of starch-based plastics. It can be blended directly with starch (10−20% w/w) and the mixture plasticised and crosslinked by aldehydes or acid anhydrides [6]. A film-forming solution based on zein can also be spread on the surface of the starch objects [39]. In biodegradable packaging, it has also been shown that coating paper with zein gives heat-sealed products that are just as resistant as polyethylene (PE)-coated paper [40]. Beck and co-workers [41] recently conducted a study on the film coating of pharmaceutical drugs and demonstrated that zein-based coatings could be applied by a conventional extrusion coating technique. These coatings were also found to have mechanical and oxygen barrier properties comparable with or better than, standard cellulose derivatives. Hot press

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 Stéphane Guilbert and Bernard Cuq

moulding of plastics formed with corn gluten, (i.e., a corn starch industry co-product with around 60−70% protein content, 60% of which is zeins) was studied by Di Gioia and co-workers [42, 43], and Di Gioia and Guilbert [33]. The mechanical properties of corn gluten-based materials were found to be similar to those of polyvinyl chloride (PVC). Corn gluten materials are very inexpensive (approximately 0.5 €/kg). The film-forming properties of corn zein have also been investigated in detail [2, 44–55]. Zein films are water-insoluble, relatively shiny and greaseproof, but they sometimes have unsuitable organoleptic properties (off-odour, chewiness and so on). Zein-based films and coatings improve food shelf life, via their high barrier properties (to water vapour or especially to oxygen (O2)), or their retention or controlled release properties of active additives on the food surface. These films have been successfully used to protect dry fruits and various parts of frozen or intermediate moisture foods. Many studies have focused on the film-forming properties of wheat gluten [46, 47, 56–69]. Wheat gluten films are generally formed by spreading and drying of protein hydroalcohol solutions, in an acidic or basic medium, usually in the presence of disruptive agents such as sulfite. Wheat gluten-based films have also been produced by skimming off the skin formed on the surface of the heated protein solutions [70] and by wheat gluten extrusion, with or without the addition of disruptive agents [3, 71]. Wheat gluten-based films are water-resistant and their properties (thus their applications) are close to those of zein-based films. They have a more neutral taste and colour, but their use as an edible film or coating, or as a packaging material in contact with a food product, can be problematic for consumers with coeliac disease, (i.e., gluten intolerance). The film-forming properties of wheat gluten have been used particularly for encapsulating additives, enhancing the quality of cereal products, and maintaining antioxidant and antimicrobial agents on the surface of food products [72]. The remarkable gas (O2 and carbon dioxide (CO2)) barrier properties of these materials, because of their exceptional selectivity, can be utilised to improve the shelf life of fresh or slightly processed vegetables [61, 73] (Section 11.5). The viscoelastic and flow properties of plasticised malleable phases based on wheat gluten were investigated as a function of temperature, water content and time [71, 74, 75]. ‘Plasticised gluten’ resembles a structured viscoelastic solid with pseudoplastic behaviour. The pseudoplasticity index of plasticised gluten (m = 0.27−0.37) is comparable to that of plasticised starch (m = 0.32−0.37) and low-density polyethylene (LDPE) (m = 0.4). The consistency (k = 18−47 kPa-s) is higher than that of LDPE (k = 9.7 kPa-s) but comparable to that of plasticised starch (k = 11−40.3 kPa-s). A study of rheological functions (G´ and G´´ moduli, complex viscosity) revealed that the time/temperature superposition can be applied. For given mixing conditions, the complex viscosity of plasticised gluten can be characterised by a power law function with the temperature and plasticiser content as variables. Redl and co-workers [71, 74, 75] demonstrated that wheat gluten can form a homogeneous plasticised malleable phase under thermal (at temperatures above the Tg), mechanical (shear) and chemical (additives and degrada-

11 Material formed from proteins 

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tion) treatments. Redl and co-workers [71, 74] carried out studies on the extrusion of gluten-based materials in a twin corotating screw extruder, with simulation of flow properties and extrusion conditions. Due to the thermoplastic properties of wheat gluten and its high capability for chemical modifications, it is possible to adjust the extent of network crosslinking, the hydrophobicity of the network (e.g., using hydrophobic glutens obtained by lipophilisation treatment) and make it compatible with synthetic materials. This natural material could thus be developed for a wide range of nonfood uses [3], e.g., envelope windows, paper coatings, biodegradable plastic films for agricultural applications, soluble bags for fertilisers, detergents or additives, and moulded objects [3, 8, 76]. The film-forming properties of soy proteins are traditionally used in Asia to produce edible films. These traditional films, (i.e., ‘yuba’ in Japan) are obtained by skimming off the lipoprotein skin formed on the surface of heated soymilk [24, 77–80]. Proteins are the main components of these films, but significant quantities of polyosides (sucrose, raffinose and stachyose) and lipids (droplets trapped in the protein matrix) are also present. These films have good mechanical properties but are generally not very water-resistant. In addition, films have been formed from soy protein isolates dispersed in a hydroalcohol solvent system [81–83] or by spreading and drying a thin layer of solution [45, 84–86]. Soy protein films are often proposed to improve the shelf life of many foods and for making soluble sachets. Biodegradable plastics have also been produced from soy protein isolates and concentrates by hot press-moulding techniques [8]. However, these materials are highly water sensitive unless a chemical crosslinking agent, such as formaldehyde, is used. Peanut protein-based films and soluble sachets have been obtained by skimming off the skin on the surface of heated peanut milk, as described previously for soymilk [82, 83, 87]. Marquié and co-workers [88, 89] recently developed biodegradable and bioresorbable cottonseed protein-based films from a film-forming solution treated with different chemical crosslinking agents. A recent review on the formulation and properties of cottonseed protein films and coatings was proposed by Marquié and Guilbert [90]. Up until the 1960s, milk protein-based materials were used for making glossy record album covers, buttons and decorative items. Labels for some cheeses are still made with crosslinked casein. The film-forming properties of casein and whey proteins were investigated with the aim of developing edible films and coatings [91, 92]. Caseins dispersed in aqueous solutions can form transparent, flexible and neutral-flavoured films. Covalent bonds catalysed by peroxidases or transglutaminases have been proposed to improve the moisture resistance of casein materials and to immobilise active enzymes (e.g., β-galactosidase and α-mannosidase) [93, 94]. The film-forming properties of caseins have been utilised to improve the appearance of many foods, to make soluble sachets, quality labels for custom-cut cheeses, to maintain additives on the surface of intermediate moisture foods and to encapsulate polyunsaturated fats produced for livestock feed [45, 95–101]. The film-forming properties of whey proteins have been utilised to produce transparent, flexible, tasteless

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 Stéphane Guilbert and Bernard Cuq

and odourless films [5, 102]. Mahmoud and Savello [103, 104] formed films by the enzymic polymerisation of whey proteins using transglutaminases. Films have also been obtained by skimming off the skin formed on the surface of heated whey dispersions [82, 105]. Whey protein-based materials are stabilised by disulfide bonds and are therefore not water-soluble. The film-forming properties of collagen are traditionally used in the meat industry for the extrusion of edible casings [106-108]. Collagen-based materials are also used for medical applications [109–113]. Gelatin is conventionally used to produce transparent, flexible, and oxygen-resistant and oxygen-proof films [45, 114, 115]. These films are formed after cooling and drying an aqueous gelatin solution. Film- forming applications of gelatin are common in the pharmaceutical industry, i.e., for producing pills and capsules (dry or soft). Gelatin is also a raw material for photographic films and the microencapsulation of flavourings, vitamins and sweeteners [116]. In addition, studies were carried out to assess the use of gelatin films to protect frozen meats from oxidation [117], but the results showed a very limited protective effect unless an antioxidant was incorporated into the film. Anker and co-workers [56] developed insoluble keratin-based films obtained by spreading and drying a thin layer of alkaline dispersions. The high cysteine content in keratin prompts the formation of many disulfide bonds, which stabilise the protein network [83, 118]. However, consumer acceptance of keratin-based edible sachets for food products has been low [119]. The use of albumen proteins as a base for the encapsulation of hydrophobic organic compounds for cosmetic or food uses is the focus of several patents [120–122]. Application of albumen coatings can reduce raisin moisture loss in breakfast mixtures [123]. Albumen has also been used as an edible coating ingredient [124, 126]. Okamoto [83] reported the formation of films on the surface of heated albumen- based lipoprotein solutions, similar to the formation of soy films. The mechanical and water vapour properties of albumen-based films were studied by Gennadios and co-workers [125]. The materials are clearer and more transparent than wheat gluten-, soy protein- and corn zein-based materials. Albumen-based films could beused to produce soluble sachets to protect ingredients used in the pharmaceutical, food and chemical industries. Recent studies highlighted the film-forming properties of fish and meat myofibrillar proteins [127–134]. Films formed from an aqueous solution were found to be water- insoluble and completely transparent, with good mechanical and gas barrier properties [53]. Their mechanical strength is close to that of PE films. The thermoplastic features of myofibrillar proteins [135, 136] could also be tapped for industrial-scale production of these films using techniques commonly implemented to obtain synthetic thermoplastic polymers (e.g., extrusion or thermoforming). Table 11.6 gives a list of the main material proteins, along with the production techniques used for each of them. Other proteins have also been used for film-forming applications: rye, pea, barley, sorghum and rice proteins, silk fibroins, fish flesh proteins and serum albumin [26, 83, 137, 138].

11 Material formed from proteins 

 311

Table 11.6: Summary of the main proteins used as polymeric materials to form biopackagings. Proteins

Tested methods to obtain films Film forming solutiona

Corn zein, Corn gluten Wheat gluten Soy proteins Peanut proteins Cottonseed protein

+ + + +

Keratin Collagen Gelatin Caseins Whey proteins Egg albumin Myofibrillar proteins

+ + + + + +

Collect the‘skin’b

Enzymic treatmentc

+ + +

+ + +

Thermoplastic extrusion + +

+ +

+ +

Casting in thin layer and drying of a film-forming solution Collect ‘skin’ formed after boiling protein solutions c Enzymic polymerisation Reproduced with permission from B. Cuq, N. Gontard and S. Guilbert, Cereal Chemistry, 1998, 75, 1, 1. ©1998, AACC International [12]. a

b

11.4 Formation of protein-based materials Protein materials are obtained via the formation of a relatively organised, low hydration and continuous macromolecular network. Interactions between proteins therefore have to be quite numerous and uniformly distributed. The probability of interprotein links depends on the protein structure and denaturation conditions (solvent, pH and ionic strength, heat treatment and so on). High MW proteins (e.g., glutenins) and fibrous proteins (e.g., collagen, glutenins) have attractive film-forming features [9]. Conversely, globular or pseudoglobular proteins (e.g., gliadins, glycinin, caseins), have to be unfolded prior to network formation. Due to the level of current knowledge, it would be unrealistic to try to predict the functional properties of material proteins on the basis of their primary structure [139]. Nevertheless, a good understanding of the main physico-chemical characteristics of the raw materials is essential. The main protein raw material characteristics are summarised in Table 11.7. Several steps are required to form a protein network: – Rupture of low-energy intermolecular bonds stabilising systems in the native state, protein rearrangement.

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–

 Stéphane Guilbert and Bernard Cuq

The formation of a 3D network stabilised by new interactions or bonds, after removal of the intermolecular bond scission agent. Two different technological strategies can be used to make protein-based materials: the ‘wet process’ or ‘solvent process’ involving a protein solution or dispersion, and the ‘dry process’ or ‘thermoplastic process’ using the thermoplastic properties of the proteins under low-hydration conditions (Figure 11.4).

11.4.1 The solvent process The formation of materials by the coacervation of a protein solution or dispersion (i.e., the solvent process) has been widely studied [22, 23, 145]. This process (which is fully controlled on a laboratory scale) often involves spreading a thin layer of protein solution, which is why this is often called a ‘casting’ or ‘continuous flow’ process. The solubility of proteins, as defined by Osborne [144], seems to be highly variable (Table 11.7) and there are no specific solubilisation conditions for the casting of protein-based solutions. Therefore, it is generally useful to know the nature of the different intermolecular interactions before attempting to solubilise proteins [146]. For example, due to the presence of intermolecular disulfide bonds in keratin, disruptive agents have to be added to obtain homogeneous solutions [143]; the low water solubility of wheat gluten is attributed to the low content of ionised polar amino acids (14%), to the many hydrophobic interactions between nonpolar amino acids (39.6%) and to the presence of disulfide bonds [31]; and the water insolubility of zein is linked with the high nonpolar amino acid content (46.6%) [16, 147]. The properties and physico-chemical characteristics of proteins in an aqueous solvent system depend on the pH conditions. Many protein-based materials are sensitive to pH variations, which could be linked with the relatively high proportion of ionised polar amino acids in protein raw materials (Table 11.7). Zein and keratin materials can, for example, be produced within a broad pH range because these proteins (which have a low ionised polar amino acid content, 10% and 10.7%, respectively), are not very sensitive to pH variations [69]. Conversely, the high content of ionised polar amino acids in soy proteins (25.4%) limits film-forming applications to within a narrow pH range [148]. The coacervation of protein dispersions involves separation of the film-forming material in the solvent phase by precipitation or a phase change, through: – Modification of the solvent system (pH or polarity modification, addition of – electrolytes). – Heat treatments. – Solvent removal. Films can be formed by solvent removal as a result of an increase in the polymer concentration in the solution, leading to molecule aggregation and formation of a 3D network. Films that are obtained by skimming of the skin formed on the surface

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Table 11.7: Main physico-chemical characteristics of the proteins used as polymeric materials to form biopackagings. Proteins

References

Amino acid ratiosa

Main sub-units

A

B

C

Name

WR

MW (kD)

Sb

21–25

IV

Corn zein

[15]

36

10

47

λ-Zein

80

Wheat gluten

[15]

39

14

40

Gliadin

40

30–80

IV

Glutenin

46

200–2,000

III

Soy proteins

[16]

31

25

36

β-Conglycinin Glycinin

35 40

185 363

II II

Peanut protein

[16]

30

27

32

Arachin

75

330

II

Cottonseed proteins

[17]

41

23

32

Albumin Globulin

30 60

10–25 113–180

I II

Keratin

[115]

34

11

42

–

–

10

III

Collagen

[116]

13

13

40

Tropocollagen

–

300

III

Gelatin (A)

[18]

12

14

41

–

–

3–200

III

Caseins

[15]

31

20

44

αS1, αS2, β, κ, γ

–

19–25

c

[117, 118]

30

26

40

β-lactoglobulin

60

18

I

α-lactalbumin

20

14

I

Myosin Actin

50 20

16–200 42

Whey protein Myofibril: Sardine Beef meat

[19] [15]

27 27

31 27

35 39

IIII

50% Amidation rates of aspartic and glutamic acids are supposed for peanut proteins, keratin, collagen and gelatin a Amino acid ratios (mol/100 mol): A: non-ionised polar (Asn, Cys, Gln, His, Ser, Thr, Tyr), B: ionised polar (Arg, Asp, Glu, Lys) and C: non-polar (Ala, Ile, Leu, Met, Phe, Pro, Trp, Val) b S is the solubility of proteins according to Osborne [119]: I – in water, II – in diluted salt solutions, III – in diluted acidic or basic solutions, and IV – in ethanol (80%) solutions c Miscellaneous associations WR: is the rate of sub-unit weight (%) in raw materials Reproduced with permission from B. Cuq, N. Gontard and S. Guilbert, Cereal Chemistry, 1998, 75, 1, 1. ©1998, AACC International [12].

of heated protein milks, result from the polymerisation of heat-denatured proteins associated with solvent evaporation [24, 77]. Coacervation is said to be ‘simple’ when a single molecule is involved and is the main process applied for producing protein-based materials. For ‘complex’ coacervation, at least two macromolecules of opposite charge are combined to obtain a blend of insoluble molecules; associating proteins with chitosans could, for instance, be very appealing for material formation.

314 

 Stéphane Guilbert and Bernard Cuq SOLVENT PROCESS Structure

THERMOPLASTIC PROCESS -Proteins-

Dispersion condition Film-forming solution Spreading conditions Films or coatings

Structure Melt and tg

Powder, pellets Shaping conditions Films or biopackagings

FUNCTIONAL PROPERTIES OF BIOPACKAGINGS Figure 11.4: Schematic representation of the two technological processes used to form biopackagings based on proteins. Adapted from B. Cuq, N. Gontard and S. Guilbert, Cereal Chemistry, 1998, 75, 1, 1 [12].

Solvent systems used for preparing film-forming solutions or dispersions are generally water and/or ethanol based, sometimes even acetone based. The dispersion of molecules in a solvent medium sometimes requires the addition of disruptive agents (mercaptoethanol, sodium sulfite, cysteine, sodium borohydride and N-ethylmaleimide), pH adjustment by the addition of acids (lactic, hydrochloric, acetic acids and so on), or bases (ammonium, sodium, potassium, or triethylamine hydroxide and so on) or controlling the ionic strength by adding electrolytes. The functional properties of materials formed by the solvent process depend on the production conditions: molecule concentration in the solution, pH, choice of additives, polarity of the solvent system, solution drying temperature and rate [57, 149, 150]. The film-forming solution can be directly applied on to food with a brush, by spraying, by coating using a falling film system, by immersion and then draining, by spinning or fluidisation and so on. In some cases, the food product is subjected to a second processing phase with a crosslinking solution which stabilises the film [151]. When a draining step is required, products are heated (to decrease the coating viscosity) using a vibrating grate, centrifugal drainage or forced ventilation. The coating or film is then hardened by drying or cooling. Relatively quick film hardening is generally required for industrial reasons; however, it is still important to control the cooling temperature, or the drying conditions, so that the film does not harden too quickly, i.e., hardening, which is too rapid, can lead to an irregular coating that could tear or become wrinkled. Coating techniques generally require a high level of skill and experience. For direct coating, it is sometimes difficult to properly moisten the support, e.g., for the protection of food with a greasy surface (peanuts and so on). In such cases, a surfactant can be applied to the support or incorporated in the film-forming system. Another solution is to precoat the product with a suitable material that will stick to each component. Films can be preformed by the solvent process, without the food support, by spreading the solution on a smooth flat surface. This technique is applied by contin-

11 Material formed from proteins 

 315

uous feed on ‘carpets’ for the industrial production of soluble films, especially those containing a wheat gluten base. Protein films are easier to unstick from some surfaces, depending on the surface material (metal, PE, polycarbonate, Teflon and so on) and surface properties. Drying the film-forming solution on a drum dryer can also be used to produce films. In addition, it is essential to carefully prepare the support, (i.e., the food surface for coating and the mould for films). Mould-release agents can be required when moulding a film onto a support.

11.4.2 The thermoplastic process The thermoplasticity of material proteins has been utilised to produce materials using thermal or thermomechanical processes under low hydration conditions, as already employed for starch- or polyolefin-based materials [136, 152]. According to the thermoplastic behaviour of synthetic polymers, the Tg of the proteins involves sudden variations in their physical properties (thermal, mechanical, dielectric properties and so on). The molecular response associated with the transition from the glassy to the rubbery state involves an overall increase in the free volume and macromolecule mobility [153, 154]. As for synthetic polymers, the Tg of the proteins is affected by the MW, chain rigidity, size and polarity of the lateral groups, presence of intermolecular bonds or crystalline zones, and also by the plasticiser type and concentration [155, 156]. The Tg values of native proteins or materials developed from proteins are given in Table 11.8. Protein Tg values are obtained by differential scanning calorimetry (DSC) or dynamic mechanical thermal analysis (DMTA) [62]. They can be predicted [33] on the basis of the amino acid composition using the method described by Matveev [157]. The Tg of proteins is highly affected by moisture content (around 10 °C decrease per 1% added water) because of their hydrophilic nature, which varies between proteins. In practice, once proteins contain more than 15% water, (i.e., which generally occurs when they are in equilibrium with 85% relative humidity (RH) at ambient temperature), the Tg of the protein material is close to ambient temperature (Figure 11.5). This effect is even more obvious in the presence of plasticisers. The effect of adding water (or another plasticiser) on the Tg can be described by the equation developed by Couchman and Karasz [171], when the molar fractions of components in the blend and the Tg values and heat capacity change at the Tg of the ‘pure’ components are known. Other empirical equations such as the equations of Gordon and Taylor [172], or Kwei [173] can also be applied. For example, Figure 11.6 highlights the impact of RH on the Tg and on the complex viscosity of a wheat gluten-based film. The critical water content or RH (Figure 11.6) indicates a sharp change in the mechanical and barrier properties (Section 11.5) of the material, which can be estimated from this diagram.

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 Stéphane Guilbert and Bernard Cuq

Table 11.8: Tg temperature of protein materials in dry conditions. Proteins

Analytical methods

Tg (°C)

References

Corn gluten

DSC/DMTA

174–182

[41]

Gluteline

DSC/DMTA

198–209

[41]

Corn zein

DSC/DMTA

164–168

[41]

Purified zein

DSC

165

[133, 134]

Commercial zein

DSC

139

[133]

167a

[135]

190

[136]

Wheat gluten

DMTA

DSC

a

180

[137]

180

a

[138]

160 Glutenin Alkylated glutenin

a

[139]

DMTA

175

[140]

DSC

138

[141]

139

HMV glutenins

b

Gliadin

DMTA

121

[142]

DSC

125

[143]

α-Gliadin

DSC

144

[141]

γ-Gliadin

DSC

124

ω-Gliadin

DSC

145

Caseins

DMTA/DSC

Sodium caseinate

130

DSC

Collagen Extrapolated values at 0% moisture content High molecular weight glutenins DSC: Dynamic scanning calorimetry DMTA: Dynamic mechanical thermal analysis a

b

[144]

a

Myofibrillar proteins DMTA

Gelatin

140–150a

215–250

[111]

200a

[145]

180–210

a

11 Material formed from proteins 

 317

200 180 160

Temperature (°C)

‘Elastomeric state’

Tg

140 120

Tr

100 80 60 40 0 –20

‘Rubbery state’

‘Glassy state’

20

10

0

20

30

40

5

Water content (g/100 g dry matter) Figure 11.5: Effect of water content on the Tg and minimum thermosetting temperature (Tr) for wheat gluten proteins [163]. Hoseney and co-workers [163] (◊), Kalichevski and co-workers [164] (□), Nicholls and co-workers (○), Cherian and Chinachoti [154] ( ), and Pouplin and co-workers [187] (X); the thermosetting temperature (●) was determined for wheat glutenins [166]. Sorption isotherm: experimental data ( □ ) and GAB equation

200

Log(viscosity) (Pa.s)

15

10

Viscosity experimental data ( ∆ ) and WLF equation

Water content (%) 25 20

150

15

100

10 5

Tambient

50 0

Critical Mc

5

0

0.2

0.4

0.6

Water activity

0.8

1

0

Critical aw

Figure 11.6: Schematic representation of relationships between water activity, water content, Tg and viscosity for wheat gluten-based films. Calculated values were obtained using the (GAB) equation [176], Couchman and Karasz equation (CK) [171], and Williams Landel and Ferry equation (WLF) [153]. The critical water activity (aw) and Mc are indicated when Tg is equal to the ambient temperature.

318 

 Stéphane Guilbert and Bernard Cuq

In order to be able to describe and predict changes in the physical-chemical properties of proteins during dry processing according to temperature and RH, it is essential to construct the state diagram relative to the water (or plasticiser) content [3, 174]. Figure 11.7 shows the different steps involved in the formation of protein-based materials using the dry process [13, 136, 152, 175]: – Plasticiser addition. – Heating the plasticised material above its Tg. – Mechanical energy input to form a homogeneous blend and to shape the product. – Cooling to ambient temperature to retransform the rubbery product into a vitreous Temperature (°C)

160

Rubbery product

140

Shaping

120 100

-Cooling and drying-

80 60 40

Product (agromaterial)

20 0 –20

0

Soft and rubbery materials -Heating and PlasticisingRaw material (powder, pellets) 10 Plasticiser content (g/100 g dm)

Glass transition 25

Figure 11.7: Schematic representation of the thermoplastic process applied to shape agropackagings based on wheat gluten proteins in relation to the Tg. Adapted from B. Cuq, N. Gontard and S. Guilbert, Cereal Chemistry, 1998, 75, 1, 1 [12].

Thus, protein-based bioplastics can be obtained by extrusion, calendering, extrusion blow moulding, injection and thermoforming processes. These ‘thermoplastic processes’ are derived from synthetic material production processes. However, when compared to standard synthetic thermoplastic polymers, proteins have markedly different thermoplastic properties. The complex and specific molecular organisation of proteins could explain their specific behaviour during thermomechanical treatments in low hydration conditions. Polydispersity, heterogeneous intermolecular interactions, the common presence of physical nodes and entanglements in protein chains, and the formation of some intermolecular covalent bonds are generally considered. The specific behaviour of proteins is characterised by high elastic modulus values around the rubbery plateau, by the absence of a mass flow region, by the large Tg range and by the apparent reversibility of Tg [157, 166, 167, 176].

11 Material formed from proteins 

 319

The Tg of proteins is partly reversible depending on the density of covalent interactions (usually disulfide bonds) established as a result of heat treatments or variations in redox potential. Heat treatments associated with the ‘thermoplastic processing’ of film-forming materials facilitate the formation of covalent bonds [177, 178]. For wheat gluten, the crosslinking activation energy is weaker under intense shear conditions. Hence, for example, the thermal crosslinking activation energy of wheat gluten is 70 kJ/°C during film formation by extrusion, whereas the activation energy is just 30 kJ/°C in a static mixer with a high shear rate [179]. In some cases, for example, to produce wheat gluten materials, extrusion is easier when disruptive agents (such as cysteine or sulfur dioxide (SO2)) are added, which break the intermolecular disulfide bonds which stabilise native proteins. Covalent bonds will (re)form after extrusion and removal of the disruptive agents, or after the addition of crosslinking agents. In addition, Micard and co-workers [180], and Morel and co-workers [181] demonstrated that structural rearrangements could occur as protein materials age. The crosslinking rate of a wheat gluten network thus, increases with storage and levels off after 72 h; this crosslinking is due to the gradual oxidation of cysteine residues that are not yet involved in disulfide bonds. In order to optimise the process parameters (temperature, plasticiser content, residence time and so on), during the transformation of material proteins, the specific characteristics of each protein should be determined (thermal, mechanical and chemical sensitivities, and high viscosities of the rubbery phases above the Tg) for these new raw materials. However, the physico-chemical factors involved in these processes are unclear because very little is currently known about protein modifications that take place when processing at high temperature under low hydration conditions [182]. This has mainly been determined for wheat gluten-based materials [71, 74, 75]. Plasticisers are generally required for the formation of protein-based materials. These agents are small, relatively nonvolatile molecules which can modify the 3D structure of a polymer, and prompt a decrease in the attractive molecular bond energy, and an increase in the intermolecular space and chain mobility. Plasticisers modify the functional properties of protein-based materials, generally with a decrease in resistance, rigidity and barrier properties, and an increase in flexibility and maximal elongation of the materials [58, 105, 132, 183–186]. The main plasticisers used for protein materials and the observed effects are shown in Table 11.9. Adding water, polyhydroxyl compounds or amphipolar agents is called ‘external’ plasticisation. Plasticisers are generally used at concentrations ranging from 10 to 50% (weight base) and water is the most efficient plasticiser in weight-base terms. Polyols (e.g., glycerol, sorbitol, polyethylene glycol (PEG)), mono-, di- or oligosaccharides, di- and triethanolamine, and urea are the most common plasticisers for protein-based materials [33, 42]. For these polar compounds, the best plasticising effect on a molar base is often obtained for compounds with a high number of hydrophilic groups [42, 187]. Amphipolar plasticisers such as octanoic and palmitic acids, dibutyl tartrate and phthalate, and mono-, di- and triglyceride esters are also very efficient, at least for highly nonpolar proteins like zein and wheat gluten. In such cases, the plasticising

320 

 Stéphane Guilbert and Bernard Cuq

effect (characterised by a Tg drop) at a constant molar concentration seems to be proportional to the MW and inversely proportional to the percentage of hydrophilic groups in the plasticiser [40]. Generally, the effect of the Tg drop can be modelled on the basis of the number of potential hydrogen bonds between the plasticiser and the protein, or according to the respective hydrophilic/lipophilic ratios [33]. The plasticiser migration rate in the protein matrix during the formation process (mixing, extrusion and so on), is highly dependent on the physico-chemical characteristics of the plasticiser. Polar substances, therefore, quickly interact with readily accessible polar amino acids, while amphipolar plasticisers interact more slowly with nonpolar zones, which are often masked and not readily accessed. These kinetic aspects can be very important if nonpolar or amphipolar plasticisers are used when water-soluble compounds are not recommended, e.g., to limit plasticiser loss and thus changes in the properties (especially mechanical) of the protein material which could come in contact with water or an aqueous product. Chemical modifications are often aimed at enhancing water resistance and reducing the effects of RH on the properties of the protein material. However, no significant Tg modifications and especially no improvement in barrier and water-resistance properties have been noted for materials formed with chemically lipophilised glutens, at different lipophilisation levels [176]. Crosslinking agents are often used to improve water resistance, cohesion, rigidity, mechanical strength and barrier properties of materials, but in general to the detriment of the product appearance [45, 88, 151, 183, 194–196]. Thus, the functional properties of casein-based materials are substantially improved when calcium is added [151, 197]. The most common covalent crosslinking agents are glutaraldehyde, glyceraldehyde, formaldehyde, gossypol, tannic acid and lactic acid. Standard crosslinking agents (formol, glyoxal and glutaraldehyde) and specially designed crosslinking agents (bifunctional monosaccharides of variable carbon chain length (n = 2, 4, and 6), i.e., N,N´-suberoyl glucosamine, N,N´-hexamethylene glucuronamide or bis-1,1 [1,8-octyl] glucofuranosidurono-6,3-lactone types) have been used to crosslink wheat gluten materials. Enzymic crosslinking treatments involving transglutaminases or peroxydases were undertaken to stabilise protein materials [93]. Proteins crosslinked via heat treatments, crosslinking agents or radiation treatments (ultraviolet (UV), gamma and so on), form insoluble and infusible networks, characterised by elastomeric or thermosetting thermomechanical behaviour according to the covalent crosslinking density. Collagen-based materials obtained by extrusion can be chemically crosslinked and casein is often crosslinked by formaldehyde to form ‘galalith’. In practice, crosslinking treatments substantially modify the mechanical properties and solubility of protein materials but have very little effect on their water vapour barrier properties [180]. All treatments used with protein materials are shown in Table 11.10. The use of crosslinking agents, however, is unsuitable for edible films and coatings, and even those designed for contact with food products.

11 Material formed from proteins 

 321

Table 11.9: Main plasticisers used with proteins materials and the observed effects. Proteins

Plasticiser

Effect

References

Corn gluten

Glycerol, sorbitol, PEG 300, PEG 600, di- tri- ethanolamin, urea, octanoic acid, palmitic acid, dibutyl tartrate and phthalate and mono- di- tri- glycerids esters

C and D

Wheat gluten and gliadin

Water

D

[54]

Water, glycerol, sorbitol

D

[136]

Water, sucrose, glucose, fructose, caproic acid, hydrocaproic acid

D

[139, 163]

Di-, tri-, tetra-ethylene glycol, glycerol, 1,3-propane diol, 1,4-butane diol, 1,5-pentane diol

B and C

[164]

Soy proteins

Water, glycerol, glycerol mono-ricinoleate, triethanolamine, urea, triethylenen glycol, PEG

D

[165]

Lupin, colza protein

Water

D

[166]

Myofibrillar proteins

Water, glycerol, sorbitol and sucrose

A-D

[107, 110]

Caseins

Water, triethanolamine

A-C

[167]

Sodium caseinate

Water

D

[143]

Whey proteins

Water, glycerol

A and B

Elastins

Water, ethylene glycol, di-, tri- and tetra-ethylene glycol

D

[40, 41]

[83] [168]

A: Decrease in elastic modulus B: Decrease in strength at break C : Decrease in Tg D: Decrease in shaping temperature Adapted from L. Di Gioia in Obtention et Etude de Biomatériaux à Base de Protéines de Maïs, ENSA Montpellier, France, 1998. [PhD Thesis] [9].

Table 11.10: Main physico-, chemical and enzymic treatments applied to the protein materials and their effects on properties. Treatments

Main effects Physical treatments

– Fractionation (ultra-filtration and centrifugation) – Mechanical treatments (high pressures and shear) – Irradiation (UV and microwave) – Heating

– Changes in protein composition – Unfolding, changes in texturation properties, crosslinking, desulfuration and desamidation

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 Stéphane Guilbert and Bernard Cuq

Table 11.10 (continued) Treatments

Main effects Chemical treatments

– – – – – – – – – – – –

Chemical reactions: Grafting Acids Reducing agents Crosslinking agents Solvent Interactions with other components: Proteins Glucides Salts Pigments Plasticiser

– – – – – – – – – –

Hydrophobisation Hydrolysis Formation of S-sulfone derivatives Covalent bonds Variable effects as a function of solvent type Aggregation Maillard reaction Conformation changes Colour changes Decrease in density of low energy interactions

Enzymic treatments – Transglutaminase and peroxidase

– Specific modifications of primary structure (covalent bonds and chemical gels)

Adapted from L. Di Gioia in Obtention et Etude de Biomatériaux à Base de Protéines de Maïs, ENSA Montpellier, France, 1998 [PhD Thesis] [9]; B. Cuq, N. Gontard and S. Guilbert, Polymer, 1997, 38, 16, 4071 [136]; V. Micard, R. Belamri, M-H. Morel and S. Guilbert, Journal of Agricultural and Food Chemistry, 2000, 48, 7, 2948 [180]; and P. Kolster, J.M. Vereuken and L.A. De Graaf in Plant Proteins from European Crops: Food and Non-Food Applications, Eds., J. Guéguen and Y. Popineau, Springer-Verlag, Berlin, Germany, 1998, p.305 [198].

11.5 Properties of protein-based materials The macroscopic properties of the protein-based, 3D macromolecular networks partially depend on system stabilising interactions. The water solubility of protein materials depends on the nature and density of intermolecular interactions. Materials are soluble in water when the energy of the interprotein bonds is lower than the energy of the interactions that could be established between water and polar groups not involved in the network. The presence of ‘physical nodes’ (i.e., chain entanglements), covalent intermolecular bonds and/or a high interaction density is sufficient to produce films that are completely or partially insoluble in water [77]. For example, the presence of intermolecular covalent bonds in wheat gluten- or keratin-based materials makes them insoluble. The mechanical properties of protein-based materials can partly be related to the distribution and intensity of inter- and intramolecular interactions that take place in primary and spatial structures. The cohesion of protein materials mainly depends on

 323

11 Material formed from proteins 

the distribution and intensity of intra- and interprotein interactions, as well as interactions with other components. For example, in soy-based materials, hydrophobic interactions between soy proteins and lipids have a key role in network stability [199]. Cooperative phenomena are generally involved to achieve optimal thermodynamic stability within the system. Interaction effects depend on their occurrence probability and the energy involved. The mechanical properties of materials are relatively dependent on potential controlling interactions which stabilise the network. When covalent bonds stabilise the network or when the binding energy is high, materials are basically very resistant and relatively elastic (e.g., keratin films). Conversely, when low-energy interprotein interactions are mainly involved, the resulting materials are highly ductile. The mechanical properties of protein-based materials are substantially lower than those of standard synthetic materials, such as polyvinylidene chloride (PVDC) or polyester (Table 11.11). The mechanical properties of protein-based materials were measured and modelled as a function of film characteristics [74, 131, 132]. For ‘stronger’ materials (e.g., based on wheat gluten, corn gluten and myofibrillar proteins, critical deformation (DC) = 0.7 mm) and elastic modulus (K = 510 N/m) values are slightly lower than those of reference materials such as LDPE (DC = 2.3 mm, K = 135 N/m), cellulose (DC = 3.3 mm, K = 350 N/m) or even PVC films. The mechanical properties of corn gluten-based material are close to those of PVC. Table 11.11: Mechanical properties of various films based on proteins and comparison with synthetic films. Films Myofibrillar proteins

References Tensile Strength Elongation Film thickness Temperature RH (%) (MPa) (%) (µm) (oC) [102]

17.1

22.7

34

25

57

Whey protein isolate

[86]

13.9

30.8

–

23

50

Soy proteins

[125]

1.9

35.6

88

25

50

Wheat gluten proteins

[125]

0.9

260

88

25

50

[45]

0.4

–

81

26

50

Methylcellulose

[175]

56.1

18.5

–

25

50

Polyesters

[176]

178

85

–

–

–

PVDC

93.2

30

–

–

–

HDPE

25.9

300

–

–

–

LDPE

12.9

500

–

–

–

Corn zein proteins

HDPE: High-density polyethylene Adapted from B. Cuq, N. Gontard and S. Guilbert, Cereal Chemistry, 1998, 75, 1, 1 [12].

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 Stéphane Guilbert and Bernard Cuq

In Figure 11.8, the general mechanical properties of various wheat gluten-based films (obtained by casting and thermomoulding) are compared with the properties of conventional plastics, synthetic biodegradable plastics and biodegradable materials derived from agricultural products. This figure shows that the mechanical properties of plastic materials can be classified in the following order: conventional synthetic (PVC, PE) > biodegradable synthetic (BAK, Eastar) > biodegradable agricultural-based materials (wheat gluten-based materials). It is also interesting to note that proteinbased materials have either high strength at break or high elongation at break, but never both simultaneously. 50

-8-

-2-

-4-

Tensile stress at break MPa

40

-1-

-4-

-1-

-4-

-1- -4-

-8-

-4-

30

-7-6-7-

-1-1-

-3-

-4-

-7-

-5-2-

0

-1-5-

-1-5-

-3-6-

-8-

-2- -3- -1-3-3- -1-1-3-2-2-2- -1-3-

0

-2-

-3-

-3-

10

-1-

-3-

-7-

20

-1-1-

200

-1-

400

-1-

600

800

1,000

Elongation at break (%)

Figure 11.8: Mechanical properties of selected protein-based films compared with some biodegradable and nonbiodegradable materials. Adapted from S. Guilbert, N. Gontard, M.H. Morel, P. Chalier, V. Micard and A. Redl in Protein-based Films and Coatings, Ed., A. Gennadios, CRC Press, Boca Raton, FL, USA, 2002 [63]. All nonreferenced data are from Saechtling [6] and from commercial data sheets. Synthetic materials (○): -1- thermoplastic polyurethane elastomer (Dow Chemical), -2PVC, -3- PVC plasticised with di-2-ethylhexylphthalate, -4 polypropylene and -5- LDPE. Synthetic biodegradable materials (□): -1- BAK 1095: polyester amide (Bayer, G), -2- ECOFLEX: 1,4 butandiol adipinic-dicarbonic and terephthalate copolyester (BASF, G), -3- EASTAR 14766: poly(tetramethylene adipate-co-terephthalate) (Eastman, USA), -4- Bionolle 3000: polybutylene succinate/adipate (Showa, Japan). Biodegradable materials from agricultural origin (: -5- BIOTEC: starch/polyester (Biotec, G), -6Materbi: starch/polycaprolactone (Novamont, Italy), -7- Biopol: polyhydroxybutyrate (Monsanto, Italy), -8- Lacea: polylactic acid (Mitsui, Japan). Protein materials (■):-1- cast gluten films [184], -2Moulded gluten (unpublished results) and -3- moulded soy protein isolate materials [193, 205].

11 Material formed from proteins 

 325

The mechanical properties of protein-based films can be markedly improved by adding fibres (i.e., composite materials). Mechanical properties are always highly dependent on the temperature and RH of the protein material (Figure 11.9). This modification, (i.e., sharp increase in deformation at break and decrease in mechanical strength), occurs suddenly when the material crosses the Tg range [174].

Deformation at break (mm)

12

9

6

3

0

0

0.2

0.4 0.6 Water activity

0.8

1

Figure 11.9: Influence of temperature at 5 °C (Δ), 25 °C (●) and 50 °C (□), and equilibrium RH on the mechanical properties of myofibrillar protein-based films (from Cuq and co-workers [131]).

The barrier properties of protein materials depend on the nature and density of the macromolecular network, and more particularly on the proportion and distribution of nonpolar amino acids relative to polar amino acids [11, 27]. The protein composition and structural organisation of the network enables some chemical groups to remain free, which means that they are sites of potential interactions with permeating molecules. Generally for protein-based materials, most free hydrophilic groups are able to interact with water vapour and permit water transfer phenomena, to the detriment of hydrophobic gas transfers (e.g., nitrogen and O2). Protein-based materials generally have high water vapour permeability. Water vapour permeation through protein films is facilitated by the systematic presence of hydrophilic plasticisers, which promote water molecule adsorption. Protein-based materials have much higher water vapour permeability (around 5 × 10-12 mol/m/s/Pa) than synthetic materials (0.05 × 10-12 mol/m/s/Pa for LDPE). This feature could still be interesting for coatings on materials that need to ‘breathe’ (e.g., packaging of fresh products and films for agricultural or cosmetic applications). These properties can be significantly improved to resemble those of PE films by adding lipid compounds (e.g., beeswax and paraffin) to the film formulation [59, 60, 196]. As already noted for the mechanical properties, water barrier properties are highly dependent on the temperature and RH of the protein material, and decrease suddenly when materials cross the Tg range [174] (Figure 11.10).

326 

 Stéphane Guilbert and Bernard Cuq

Water vapour transmission rate (10–8 mol/m/s/Pa)

2

1.5

1

0.5

0

0

0.2

0.4

0.6

0.8

1

Water activity Figure 11.10: Influence of water activity and temperature on the water vapour barrier properties of wheat gluten-based films. At 5 °C (Δ), 20 °C (●) or 50 °C (□) (from Gontard and co-workers [58]).

The gas barrier properties (O2, CO2 and ethylene) of protein-based materials are highly attractive since they are minimal under low RH conditions. Oxygen permeability (around 1 amol/m/s/Pa) is comparable to ethylene vinyl alcohol (EVOH) properties (0.2 amol/m/s/Pa) and much lower than the properties of LDPE (1,000 amol/m/s/ Pa) [61] (Table 11.12). The O2 permeability of protein films is about 10-fold higher that EVOH-based films, mainly due to the high plasticiser content of protein-based films. While the barrier properties of synthetic materials remain quite stable at high RH, the gas barrier properties of material proteins (as for all properties of hydrocolloidbased materials) are highly RH- and temperature-dependent (Figure 11.11). The O2 and CO2 permeabilities are about 1,000-fold higher for moist films than for films stored at 0% RH. For proteins, this effect is much greater for ‘hydrophilic’ gases (CO2) than for ‘hydrophobic’ gases (O2). Changes in the RH of temperature modify the CO2/O2 selectivity coefficient, which rises from 3 to more than 50 when the RH rises from 0 to 100% and the temperature from 5 to 45 °C, as compared with constant values of around 3−5 for standard synthetic films (Figure 11.11). Gas permeability differences in protein materials are partly due to gas solubility differences in the film matrix, and could be mainly explained by the high affinity between CO2, the polypeptide chain and many lateral amino acid groups [188]. A film with good O2 barrier properties is interesting for the protection of oxidisable foods (rancidification, loss of oxidisable vitamins and so on). However, some extent of permeability to O2 and especially to CO2 is required to decrease the metabolic activity of many fresh fruits and vegetables. The development of protein films with selective gas permeability features could thus be highly promising, especially for controlling respiratory exchange and improving the shelf life of fresh or minimally processed fruits and vegetables [214]. Wheat gluten-based films were tested with the aim of creating atmospheric conditions suitable for preserving fresh vegetables.

11 Material formed from proteins 

 327

Table 11.12: O2 permeability (1018 mol/m/s/Pa) and CO2 permeability (1018 mol/m/s/Pa) of various films based on proteins and comparison with synthetic films and edible films. Film

References

P(O2)

P(CO2)

Temperature (°C)

aw

LDPE

[182]

1,003

4,220

23

0

HDPE

[182]

285

972

23

0

HDPE

[185]

224

–

23

1

Polyester

[182]

12

38

23

0

EVOH

[183]

0.2

–

23

0

[187]

6

–

23

0.95

[178]

522

29,900

30

0

Methyl cellulose Beeswax

[159]

480

–

25

0

Hydroxypropyl cellulose

[178]

470

28,900

30

0

Carnauba wax

[159]

81

–

25

0

[45]

35

216

38

0

[184]

2

–

23

0

[53]

1

7

25

0

[53]

1,290

36,700

25

0.95

[45]

3

–

38

0

Corn zein Soy protein Wheat gluten Wheat gluten protein Fish myofibrillar protein

[53]

1

9

25

0

[53]

873

11,100

25

0.93

[53]

0.6

–

25

0

[53]

472

8,010

25

0.93

Cellophane

[186]

130

–

23

0.95

Polyester

[185]

12

–

23

1

Pectin

[53]

1,340

21,300

25

0.96

Starch

[177]

1,085

–

25

1

Chitosan

Reproduced with permission from B. Cuq, N. Gontard and S. Guilbert, Cereal Chemistry, 1998, 75, 1, 1. ©1998, AACC International [12].

Measurements of changes in the gas composition of modified atmosphere packaged mushrooms under wheat gluten films confirmed the high selectivity of such materials, i.e., the CO2 and O2 composition ranged from 1−2% despite product respiration [73]. The aroma barrier properties of protein-based materials seem especially interesting for blocking nonpolar compound permeation. However, it is hard to determine the relationship between the physico-chemical properties of aroma compounds and their retention by protein films [215].

328 

 Stéphane Guilbert and Bernard Cuq

Solute retention properties (especially antimicrobial and antioxidant agents) were investigated and modelled for wheat gluten-based films [72], and the results indicated potential applications for the controlled release of functional agents. The antimicrobial efficacy of edible wheat gluten-based films containing antimicrobial agents has been very well documented [216–218]. The use of these films on high moisture model foods extended their shelf life by more than 15 days at 4 and 30 °C. Many patents and publications recommend adding antioxidant agents to protein films and coatings, as already occurs in some commercial edible films. Guilbert [45] measured α-tocopherol retention in gelatin films applied to the surface of margarine blocks. No migration was noted after 50 days storage when the film was pretreated with a CO2 Permeability (amol/m/s/Pa) 210,000 140,000 100

70,000 50

0

3 Temperature (°C)

45

RH (%)

0

Selectivity coefficient

40 100

20 0

50 3 Temperature (°C)

O2 Permeability (amol/m/s/Pa)

45

RH (%)

0

3,500 100 0

50 3 Temperature (°C)

45

0

RH (%)

Figure 11.11: Changes in gas barrier properties of wheat gluten-based films as a function of temperature and RH (from Barron and co-workers [73]).

11 Material formed from proteins 

 329

crosslinking agent (tannic acid), whereas α-tocopherol diffusivity was around 10 to 30 × 10−11 m2/s without the film. Few studies have focused on the biodegradability and environment-friendly aspects of protein-based products which degrade naturally or in compost. The construction of protein networks can induce marked changes in the conformation and resistance to enzymic hydrolysis and chemical attack of proteins [219]. However, Garcia- Rodenas and co-workers [220] showed that the susceptibility of casein and wheat gluten-based films to in vitro proteolysis did not significantly differ from that noted for native proteins.

11.6 Applications Proteins could be used as raw material for bioplastics with a wide range of agricultural, agrifood, pharmaceutical and medical industry applications. The functional properties (especially optical, barrier and mechanical) of these protein-based materials are often specific and unique. Plant proteins are generally inexpensive (0.5−1 €/kg for corn and wheat glutens, with 70−80% protein content, respectively), widely available and relatively easy to process. Animal proteins are more expensive (2−10 €/kg), but sometimes have no functional substitutes (e.g., gelatin). The casting process is generally adapted for coating seeds, drug pills and foods, for making cosmetic masks or varnishes, and pharmaceutical capsules. Heat casting of protein-based materials by techniques usually applied for synthetic thermoplastic polymers (extrusion, injection, moulding and so on) is more cost-effective. This process is often applied for making flexible films (e.g., films for agricultural applications, packaging films and cardboard coatings) or objects (e.g., biodegradable materials), that are sometimes reinforced with fibres (composite bioplastics for construction, automobile parts and so on). The complexity of proteins and the broad range of protein fractions could be used to produce materials with unique functional properties, which differ markedly from those of conventional plastic materials. Protein-based materials are biodegradable and even edible when food-grade additives are used. Moreover, they are often biocompatible, barring some protein-specific aspects (e.g., allergenic features of wheat gluten gliadins), processing aspects, and the presence of impurities or additives. Protein materials are generally homogeneous, transparent, resistant and water insoluble. Their high moisture permeability is especially attractive for cheese, fruit and vegetable packaging, and for agricultural material and cosmetic applications. Protein- based materials have slightly lower mechanical properties than reference materials such as LDPE or plasticised PVC, but the addition of fibres (composite materials) can considerably improve them. The thermoplastic properties of pro-

330 

 Stéphane Guilbert and Bernard Cuq

teins and their water resistance (for insoluble proteins) are especially interesting for natural resin uses to produce chipboard, and medium- and particleboard type materials. The gas barrier properties (O2, CO2 and ethylene) of protein-based materials can be utilised in designing selective or active materials for the modified atmosphere packaging of fresh products (such as fruits, vegetables and cheeses). Solute retention properties (especially antimicrobial and antioxidant agents) are attractive for designing controlled release systems for functional additives in the food (e.g., active coatings and encapsulation), agriculture (e.g., coated seed), pharmacy (drug delivery) and cosmetic industries. Multilayer ‘protein/paper’ and ‘protein/biodegradable polyester’ (polycaprolactone, polylactic acid (PLA) and so on) materials can be produced using some highly amphipolar proteins with a wide compatibility range. Composite agromaterials combining proteins with cottonseed, sisal, coconut and straw fibres were successfully tested (excellent compatibility) and have considerable application potential. Multilayer materials based on modified PE and proteins can be obtained using thermomoulding processes. Thermosetting protein/resin composite materials can also be produced. Materials could thus, be developed which combine the unique gasvapour- and solute-permeability properties of protein films with the mechanical performances of conventional synthetic materials. Material protein properties can generally be modified in a wide range of ways via raw material choices and combinations, the proper use of fractionating techniques and rheological modifying additives, and also by adjusting the product formation process variables.

References S. Guilbert, M.H. Morel, A. Redl, Andreas, M. Pommet and P. Menut in Proceedings of the 229th ACS National Meeting, San Diego, CA, USA, American Chemical Society, Washington, DC, USA, 13−17th March 2005. [2] S. Guilbert in Proceedings of the 227th ACS National Meeting, Anaheim, CA, USA, American Chemical Society, Washington, DC, USA, 28th March−1st April 2004. [3] S. Guilbert and N. Gontard in Foods and Packaging Materials: Chemical Interactions, Eds., P. Ackermann, M. Jägerstad and T. Ohlsson, The Royal Society of Chemistry, Cambridge, UK, 1995, p.159. [4] S. Guilbert, Bulletin of the Research Institute of Food Science, Kyoto University, 1999, 56, 38. [5] Protein-based Films and Coatings, Ed., A. Gennadios, CRC Press, Boca Raton, FL, USA, 2001. [6] W. Woebcken in Saechtling International Plastics Handbook for the Technologist, Engineer and User, 3rd Edition, Hanser Publishers, Munich, Germany, 1995. [7] I. Kruszewska in Clean Production Action Report of Greenpeace International Toxic Campaign, Greenpeace, Amsterdam, The Netherlands, 1997. [8] J.L. Jane, S.T. Lim, I. Paetau, K. Spence and S. Wang in Polymers from Agricultural Coproducts, Eds., M.L. Fishman, R.B. Friedman and S.J. Huang, ACS Symposium Series No. 575, American Chemical Society, Washington, DC, USA, 1994, p.92. [1]

11 Material formed from proteins 

 331

[9] L. Di Gioia in Obtention et Etude de Biomatériaux à Base de Protéines de Maïs, ENSA Montpellier, France, 1998. [PhD Thesis] [10] J.A. Rothfus, Journal of Agricultural and Food Chemistry, 1996, 44, 10, 3143. [11] S. Guilbert and J. Graille in Valorisations Non Alimentaires des Grande Productions Agricoles, Les Colloques No. 71, Ed., J. Gueguen, INRA Editions, Paris, France, 1994, p.195. [12] B. Cuq, N. Gontard and S. Guilbert, Cereal Chemistry, 1998, 75, 1, 1. [13] C. Oudet in Polymères − Structure et Propriétés, Masson, Paris, France, 1994. [14] L.G. Phillips, D.M. Whitehead and J. Kinsella in Structure-Function Properties of Food Proteins, Eds., L.G. Phillips, D.A. Whitehead and J. Kinsella, Academic Press, Inc., San Diego, CA, USA, 1994. [15] P.R. Shewry and A.S. Tatham, Biochemical Journal, 1990, 267, 1, 1. [16] J.S. Wall and J.W. Paulis in Advances in Cereal Science and Technology, Volume 2, Ed., Y. Pomeranz, VCH, New York, NY, USA, 1978, p.135. [17] F.W. Sosulski and G.I. Imafidon, Journal of Agricultural and Food Chemistry, 1990, 38, 6, 1351. [18] C.E. Boldwell and D.T. Hopkins in New Protein Foods, Volume 2, Eds., A.M. Altschul and H.L. Wilcke, Academic Press, Inc., Orlando, USA, 1985, p.152. [19] C. Defromont in Proceedings of Colloque CIDT, Abidjan, Ivory Coast, 1985, p.112. [20] P.I. Rose in Encyclopedia of Polymer Science and Engineering, Volume 7, Eds., H.F. Mark, N.M. Bikales, C.G. Overberger and G. Menges, John Wiley and Sons, Inc., New York, NY, USA, 1987, p.488. [21] E. Orban, G.B. Quaglia, I. Casini, I. Caproni and E. Moneta, Lebensmittel Wissenschaft und-Technology, 1992, 25, 371. [22] J.J. Kester and O. Fennema, Food Technology, 1986, 40, 47. [23] S. Guilbert and B. Biquet in L’Emballage des Denrées Alimentaires de Grand Consommation, Ed., G. Bureau and J-L. Multon, Technique et Documentation, Lavoisier, Paris, France, 1989, p.320. [24] A. Gennadios, T.H. McHugh, C.L. Weller and J.M. Krochta in Edible Coatings and Films to Improve Food Quality, Eds., J.M. Krochta, E.A. Baldwin and M.O. Nisperos-Carriedo, Technomic Publishing Company, Lancaster, PA, USA, 1994, p.201. [25] N. Gontard and S. Guilbert in Food Packaging and Preservation, Ed., M. Mathlouthi, Blackie Academic and Professional, Glasgow, UK, 1994,p.159. [26] J.A. Torres in Protein Functionality in Food Systems, Eds., N.S. Hettiarachchy and G.R. Ziegler, Marcel Dekker Inc., New York, NY, USA, 1994, p.467. [27] B. Cuq, N. Gontard and S. Guilbert in Active Food Packagings, Ed., M.L. Rooney, Blackie Academic and Professional, Glasgow, UK, 1995, p.111. [28] F. Debeaufort, J.A. Quezada-Gallo and A. Voilley, Critical Reviews in Food Science and Nutrition, 1998, 38, 4, 299. [29] S. Guilbert and B. Cuq in L’Emballage des Denrées Alimentaires de Grande Consommation, Eds., J-L. Multon and G. Bureau, Technique et Documentation, Lavoisier, Paris, France, 1998, p.471. [30] D.M. Rathmann in Mellon Institute Bibliographic Series, Bulletin No. 7, Mellon Institute, Pittsburgh, PA, USA, 1954, p.1891. [31] R.A. Reiners, J.S. Wall and G.E. Inglett in Proceedings of the 58th Annual Meeting of the AACC, Ed., Y. Pomeranz, St. Paul, MN, USA, American Association for Clinical Chemistry, Washington, DC, USA, 1973, p.285. [32] M. Zhang, C.A. Reitmeier, E.G. Hammond and D.J. Myers, Cereal Chemistry, 1997, 74, 5, 594. [33] L. Di Gioia and S. Guilbert, Journal of Agricultural and Food Chemistry, 1999, 47, 3, 1254. [34] D.N. Martin, inventor; Corn Products Co., assignee; US 3497369, 1970. [35] N. Hasegawa, K. Suzuki, T. Ishii, M. Hayashi and G. Danno, inventors; Sanei Touka KK, assignee; JP 6192577, 1994.

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[201] [202] [203]

David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

12 Enzyme catalysis in the synthesis of biodegradable polymers 12.1 Introduction

Nature is responsible for the synthesis of a diverse set of polymers. These polymers are involved in the storage and transfer of information (deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and proteins), energy storage (polyhydroxyalkanoates (PHA) and polysaccharides), architectural and mechanical systems (fibrous proteins, polysaccharides), and catalysis (proteins and RNA). These polymers provide all of the requirements for survival of the organisms that synthesise them. This includes protection from environmental variables and threats, such as changes in available food supplies, desiccation, mechanical integrity and other features. Furthermore, and perhaps most importantly for the present review, these polymers are ‘programmed’ in terms of structure and chemistry for finite lifetimes. Biological systems cannot ‘afford’ to generate polymers that are not recyclable, which can’t be put back into normal metabolic processes for reuse as the building blocks or elements in new structures and functions. Thus, biodegradability is an inherent feature of any biologically derived polymer. With this knowledge, it is logical to consider the key catalyst in these processes: enzymes, which are an important source for initiating reactions designed to generate biodegradable polymers that might have a wide range of potential uses, such as for information flow, energy storage and architectural functions. While biological systems are capable of generating a diverse set of polynucleotides, polysaccharides, polyesters, proteins and polyaromatics, isolating the catalysts responsible for these processes from the rest of the biological milieu should result in important control over the biosynthesis and the resulting structural and functional aspects of the polymers formed from these catalysts. The remarkable regioselective, chemoselective and enantioselective capabilities of enzymes, their ability to retain catalytic function in diverse and even nonnatural environments such as organic solvents and supercritical fluids, their robust nature under certain conditions, and their ability to be chemically and genetically manipulated to optimise or modify functions represents the extraordinary opportunity presented by this group of catalysts. This is the focus of the present chapter. We address the impressive progress in the last 10 to 15 years in terms of in vitro enzyme-based polymerisation reactions to generate new biodegradable polymers. Using our previous definition for biological catalysts, any polymer generated through enzyme catalysis should be biodegradable, presuming that the monomers are naturally occurring – thus, sufficient time has been available on the evoluhttps://doi.org/10.1515/9781501511967-012

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 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

tionary scale for a degradative enzyme to develop. This would also suggest that even in the case of nonnative building blocks, such as fluorinated amino acids or modified sugars, enzymes could evolve in time either in the laboratory, or naturally, to accommodate their biodegradation. Therefore, a key aspect to the field of enzyme-based polymer synthesis is that the products of such reactions should be biodegradable. This chapter will focus primarily on polyesters, polysaccharides and polyaromatics as products of in vitro enzymatic synthesis reactions. For other recent reviews see Kobayashi [1], Kadokawa and co-workers [2], and Kobayashi and co-workers [3]. We have neglected polyamides to maintain focus, however, recent reviews on this topic are available (e.g., Cheng [4], Poulhès and co-workers [5], and Stavila and co-workers [6]).

12.2 Polyester synthesis Microorganisms such as bacteria produce biodegradable PHA polyesters for use as intracellular energy and carbon storage materials from a variety of different substrates such as sugars, alcohols, n-alkanes, n-alkenes, alkanoic and alkenoic acids. Isolated enzymes, mostly lipases, have been used as catalysts for the construction of polyesters from various monomers, typically hydroxy acids or their esters, dicarboxylic acids or their activated derivatives with glycols, lactones, carbonates, oxirane with glycols, and anhydrides with glycols. The remarkable properties of lipases such as regio-, enantio- and chemoselectivity, and mild reaction conditions in comparison to chemical processes have been exploited to produce functional polyesters, most of which are difficult to synthesise using conventional methodologies. Polyesters are particularly attractive for a variety of commodity polymer applications as well as in speciality biomedical polymer uses, since their rates of degradation can be controlled through composition and processing.

12.2.1 Polycondensation of hydroxyacids and esters Many lipases, such as porcine pancreatic lipase (PPL), Candida cylindracea lipase (CCL), Chromobacterium viscosum lipase (CVL), Candida antarctica lipase (CAL), Candida antarctica lipase B (CALB, Novozyme-435), polyethylene glycol (PEG)- modified Pseudomonas fluorescens lipase (PFL), Pseudomonas cepacia lipase (PsCL), Candida rugosa lipase (CRL), Aspergillus niger lipase (ANL), Pseudomonas sp. lipase (PSL) and Mucor miehei lipase (MML), have been used for the construction of polyesters from the hydroxyacids and their esters (Table 12.1) in accordance with the following scheme [7]:

12 Enzyme catalysis in the synthesis of biodegradable polymers 

HORCO2X

Lipase

 341

O

ORC n –XOH X: H, alkyl, halogenated alkyl, vinyl, etc. Scheme 12.1: Reproduced with permission from H. Uyama and S. Kobayashi, Enzyme-Catalyzed Synthesis of Polymers, Eds., S. Kobayashi, H. Ritter and D. Kaplan, Springer, New York, NY, USA, 2006, 194, p.133 © 2006, Springer [7].

Table 12.1: Polymerisation of hydroxyacids and hydroxyesters with different enzymes. Monomer hydroxyacids

Enzyme

References

1

HO-CH2-COOH

PEG-modified PFL

2

HO-CH2-(CH2)8-COOH

PEG-modified PFL CAL

3

CH3-CHOH-COOH

PLA

[9]

4

HO-CH2-(CH2)14-COOH

CAL

[10]

5

HO-CH2-(CH2)10-COOH

CAL

[10]

5

HO-CH2-(CH2)8-COOH

CAL

[10]

6

HO-CH2-(CH2)4-COOH

CAL

[10]

7

HO-CH2-(CH2)10 -COOH HO-CH(C6H13)-(CH2)10-COOCH3

CALB

[11]

8

HO-CH(C6H13)-CH2-CH=CH-(CH2)7-COOH

PCL

[13]

9

HO-CH2-(CH2)7-COO-(CH2)7-COOH

CALB

[14]

10

HO-CH2-(CH2)5-CHOH-CHOH-(CH2)7-COOC3H7

CALB

[15]

11

CH3-CHOH-(CH2)4-COO-CH3

CALB + Ru(Shvo)

[16]

12

CH3-CHOH-(CH2)5-COO-CH3

CALB + Ru(Shvo)

[16]

13

CH3-CHOH-(CH2)11-COO-CH3

CALB + Ru(Shvo)

[16]

[7] [7, 8]

PLA: Polylactic acid.

Usually a low molecular weight (MW) polymer is produced. The first work reporting a lipase-catalysed condensation polymerisation of an oxyacid monomer, 10-hydroxydecanoic acid, in benzene was published in 1985, using a PEG-modified esterase (PEG-lipase) which was soluble in the medium [8]. The degree of polymerisation (DP) of the product was low (≥5). The PEG-lipase also induced the oligomerisation of glycolic acid, the smallest oxyacid (R = CH2, Scheme 12.1). O’Hagan and co-workers [9] reported that 10-hydroxydecanoic acid was close to optimum length for CAL-catalysed polymerisation, while monomers with shorter and longer

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 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

carbon chains (4-hydroxybutyric acid, DL-2-hydroxybutyric acid, glycolic acid and 16-hydroxyhexadecanoic acid) failed to polymerise. As described by Kiran and co-workers [10], the lipase-catalysed polymerisation of lactic acid gave a low MW PLA under various reaction conditions (R = CH(CH3), Scheme 12.1). Molecular sieves have been used to remove the water produced during the reactions to increase the product MW. Polyesters of higher MW were enzymatically produced from hydrophobic oxyacids. In the lipase CALB-catalysed polymerisation of 16-hydroxyhexadecanoic acid, 12-hydroxydodecanoic acid (HDDA) or 10-hydroxydecanoic acid, under vacuum at a higher temperature (90 °C) in bulk for 24 h, the DP value was beyond 100, whereas a polyester with lower MW was formed from 6-hydroxyhexanoic acid [11]. Copolymerisation of HDDA with methyl 12-hydroxystearate (both from seed oils) was obtained by a CALB catalyst in toluene in the presence of molecular sieves at 90 °C, to give a copolymer with a high MW ~1.0 × 105, and showing good elasticity and biodegradability [12]. Ohara and co-workers reported that the CALB-catalysed polycondensation of alkyl esters of lactic acid as the monomer produces oligoLAs (X = alkyl, n=2–7 in Scheme 12.1) [13]. The reaction is perfectly enantioselective: only the alkyl D-lactate monomer produced the oligomers. These results provide the first direct evidence that in the lipase-catalysed reaction mechanism ‘the enantioselection is governed by the deacylation step of lipase’. Ricinoleic acid was polymerised via dehydration with an immobilised poly(ɛcaprolactone) (PCL) catalyst to give a polymer with MW up to 8,500 Da [14]. Immobilised CALB was efficient as a catalyst for the dehydration polycondensation of cis-9,10 epoxy-18-hydroxyoctadecanoic acid (from the outer birch bark) in toluene, in the presence of molecular sieves at 75 °C, to give a polyester with the highest MW of 2.0 × 104 Da after 68 h [15]. As shown in Scheme 12.2 [16], the regioselective polycondensation of isopropyl aleuriteate was achieved by Veld and co-workers with a CALB catalyst, where the only primary alcohol was reacted at 90 °C. The polymer with a number-average MW (Mn) of 5,600 Da was obtained with a yield of 43%. The copolymerisation of isopropyl aleuriteate with ɛ-caprolactone (ɛ-CL) gave a random copolymer having a Mn up to 1.06 × 104 Da with a yield of ~70% [17]. O

OH

O lipase –iPrOH

OH

OH O

OH OH

O n

Scheme 12.2: Reproduced with permission from S. Kobayashi, Proceedings of the Japan Academy, Series B, 2010, 86, 4, 338. ©2010, The Japan Academy [16].

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As reported by Kanca and co-workers, the enantioselective transesterification polycondensation of racemic AB-type monomers containing a secondary hydroxy group and a methyl ester moiety led to chiral polyesters by iterative tandem catalysis. The concurrent actions of an enantioselective acylation catalyst (such as CALB) and a racemisation catalyst (Ru(Shvo)) resulted in the high conversion of the racemic monomers to enantioenriched polymers. AB-type monomers used were typically methyl 6-hydroxyheptanoate, methyl 8-hydroxynonanoate and methyl 13-hydroxytetradecanoate. The polycondensation at 70 °C in toluene gave a polyester of high yield with a Mn of around several thousand and an enantiomeric excess higher than 74% [18].

12.2.2 Polymerisation of dicarboxylic acids or their activated derivatives with glycols Various combinations of dicarboxylic acid and their activated derivatives with glycols have been reacted enzymatically to generate biodegradable polyesters under mild reaction conditions (Table 12.2), in accordance with the following general scheme of dehydration polycondensation mode (Scheme 12.3) [7].

XO2CRCO2X

+

HOR′OH

Lipase

O O

CRC OR′O n –XOH X: H, alkyl, halogenated alkyl, vinyl, etc.

Scheme 12.3: Reproduced with permission from H. Uyama and S. Kobayashi, Enzyme-Catalyzed Synthesis of Polymers, Eds., S. Kobayashi, H. Ritter and D. Kaplan, Springer, New York, NY, USA, 2006, 194, p.133 © 2006, Springer [7].

The first paper reporting a lipase A-catalysed dehydration polymerisation between a free dicarboxylic acid and a diol, which produced oligoesters, appeared in 1984. Okumura and co-workers [19] studied polyester formation from dicarboxylic acids (C6~C14) and diols; those from 1,13-tridecanedioic acid and 1,3-propanediol were studied extensively. A mixture of products were separated by gel permeation chromatography (GPC) and determined by infrared spectroscopy (IR) and mass spectrometry (MS). Trimer, pentamer and heptamer products were detected, while a small amount of dimer and no tetramer or hexamer were identified. Since the dimer was the key substrate used by the ANL for the construction of the polymer, it was assumed that the pentamer was produced from the dimer and trimer, and the heptamer from the pentamer and dimer. Uyama and co-workers [20, 21] performed polymerisations in solvent-free systems and reported that polymer yield and MW were strongly dependent on the methylene chain length of the monomers, i.e., the hydrophobicity of the

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monomers. The leaving groups (water or alcohol) were removed from the reaction mixture, leading to a shift of equilibrium towards polymerisation and a polymer with a Mn of more than 1 × 104 was obtained in reactions under reduced pressure. A high boiling point diphenyl ether was the preferred solvent for the polycondensation of adipic acid (p = 4) and 1,8-octanediol (q = 8) giving a product with a Mn of 28,500 Da (48 h, 70 °C). The reactions involving monomers containing longer alkylene chain lengths of diacids (sebacic and adipic acids) and diols (1,8-octanediol and 1,6-hexanediol) showed a higher reactivity than those involving monomers of shorter chain lengths [22]. Table 12.2: Polymerisation of dicarboxylic acid and their activated derivatives with glycols. Monomer

Enzyme

References

HOOC-(CH2)2-COOH with HO-(CH2)2-OH

CAL

[18]

HOOC-(CH2)2-COOH with HO-(CH2)4-OH

CAL

[18, 19]

HOOC-(CH2)4-COOH with HO-(CH2)2-OH

ANL, CAL

[17, 18]

HOOC-(CH2)4-COOH with HO-(CH2)3-OH

ANL, CAL

[17, 18]

HOOC-(CH2)4-COOH with HO-(CH2)4-OH

CAL, CALB

[18, 20]

HOOC-(CH2)4-COOH with HO-(CH2)6-OH

CALB

HOOC-(CH2)4-COOH with HO-(CH2)8-OH

CAL, PSL, CALB

HOOC-(CH2)5-COOH with HO-(CH2)2-OH

ANL

[17]

HOOC-(CH2)5-COOH with HO-(CH2)3-OH

ANL

[17]

HOOC-(CH2)6-COOH with HO-(CH2)2-OH

ANL

[17]

HOOC-(CH2)6-COOH with HO-(CH2)3-OH

ANL

[17]

HOOC-(CH2)6-COOH with HO-(CH2)4-OH

CAL

[17]

HOOC-(CH2)6-COOH with HO-(CH2)6-OH

PSL

[22]

HOOC-(CH2)6-COOH with HO-(CH2)8-OH

PSL

[22]

[20] [18, 20]

HOOC-(CH2)6-COOH with HO-(CH2)10-OH

PSL

[22]

HOOC-(CH2)6-COOH with HO-(CH2)12-OH

PSL

[22]

HOOC-(CH2)8-COOH with HO-(CH2)2-OH

ANL, CAL

[17–19]

HOOC-(CH2)8-COOH with HO-(CH2)3-OH

ANL, CAL

[17–18]

HOOC-(CH2)8-COOH with HO-(CH2)4-OH

ANL, CALB, MML, PCL, CAL

[18–21]

HOOC-(CH2)8-COOH with HO-(CH2)5-OH

CAL

HOOC-(CH2)8-COOH with HO-(CH2)6-OH

CAL, PCL, CALB

[18, 20, 22]

HOOC-(CH2)8-COOH with HO-(CH2)8-OH

CAL, PCL, CALB

[18–22]

[18]

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Table 12.2 (continued) Monomer

Enzyme

References

HOOC-(CH2)8-COOH with HO-(CH2)10-OH

CAL, PCL

[18, 22]

HOOC-(CH2)8-COOH with HO-(CH2)12-OH

CAL, PCL

[18, 19, 21,22]

HOOC–(CH2)6–COOH with

PCL

[22]

PCL

[22]

PCL

[22]

PCL

[22]

PCL

[22]

HOOC-(CH2)10-COOH with HO-(CH2)2-OH

ANL

[17]

HOOC-(CH2)10-COOH with HO-(CH2)3-OH

ANL

[17]

HOOC-(CH2)10-COOH with HO-(CH2)6-OH

PCL

[22]

HOOC-(CH2)10-COOH with HO-(CH2)8-OH

PCL

[22]

HOOC-(CH2)10-COOH with HO-(CH2)10-OH

PCL

[22]

HOOC-(CH2)10-COOH with HO-(CH2)12-OH

PCL

[22]

HOOC-(CH2)11-COOH with HO-(CH2)2-OH

ANL

[17]

HOOC-(CH2)11-COOH with HO-(CH2)3-OH

ANL

[17]

HOOC-(CH2)12-COOH with HO-(CH2)2-OH

ANL, CAL

HOOC-(CH2)12-COOH with HO-(CH2)3-OH

ANL

[17]

HOOC-(CH2)12-COOH with HO-(CH2)4-OH

CAL

[18, 19]

HOOC-(CH2)12-COOH with HO-(CH2)6-OH

PCL

[22]

HOOC-(CH2)12-COOH with HO-(CH2)8-OH

CAL, PCL

HOOC-(CH2)12-COOH with HO-(CH2)10-OH

PCL

[22]

HOOC-(CH2)12-COOH with HO-(CH2)12-OH

PCL

[22]

CH2OH CH2OH CH2–OH

CH2OH

HOOC-(CH2)8-COOH with

HOOC-(CH2)8-COOH with

HOOC-(CH2)8-COOH with

HOOC-(CH2)8-COOH with

CH2OH

HOH2C

HO

HOH2C

OH

CH2OH

[17, 18]

[18, 21, 22]

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 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

Table 12.2 (continued) Monomer

Enzyme COOH

HOOC

HOOC

[22]

PCL

[22]

PCL

[22]

PPL

[24–26]

with HO-(CH2)8-OH

COOH

with HO-(CH2)4-OH

O Cl3CH2COOCH2C-HC

PCL with HO-(CH2)8-OH

COOH COOH

References

CH-CH2COOCH2CCl3 with

HO-(CH2)4-OH CH3OOC-(CH2)2-COOCH3 with HO-(CH2)4-OH

CALB

[27]

HOH2C-(CHOH)4-CH2OH with HOOC-(CH2)4-COOH with HO-(CH2)8-OH

CAL

[28]

HOOC-(CH2)4-COOH with HO-(CH2)4-OH

HiC

[29]

HOOC-(CH2)4-COOH with HO-(CH2)6-OH

HiC

[29]

HOOC-(CH2)4-COOH with HO-(CH2)8-OH

HiC

[29]

HiC

[29]

HiC

[29]

HiC

[29]

HiC

[29]

CH3OOC-(CH2)4-COOCH3 with HO-(CH2)4-OH

CAL

[31, 32]

CH3OOC-(CH2)8-COOCH3 with HO-(CH2)4-OH

CAL

[31, 32]

HOOC-(CH2)2-COOH with

HOOC-(CH2)4-COOH with

HOOC-(CH2)6-COOH with

HOOC-(CH2)8-COOH with

HOCH2

CH2OH

HOCH2

CH2OH

HOCH2

CH2OH

HOCH2

CH2OH

Divinyl sebacate with the following diols HO-(CH2)2-OH

PCL, CAL

[34]

HO-(CH2)4-OH

PCL, CAL, MML, PFL, PPL PCL, CAL

[34]

HO-(CH2)6-OH

[34]

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 347

Table 12.2 (continued) Monomer

Enzyme

References

HO-(CH2)10-OH

PCL, CAL

HO-CH2-CH(OH)-CH2-OH

MML, CAL

[36, 38–40]

[34]

HO-CH2-CH(OH)-(CH2)2-OH

MML, CAL

[39]

HO-CH2-CH(OH)-(CH2)4-OH

MML, CAL

[39]

HO-CH2-CH=CH-CH2-OH

PCL

[34]

HO-CH2-C≡C-CH2-OH

PCL

[34]

HO ― CH2 ― CH ― CH ― CH ― OH ― CH2OH

CAL

[37]

HO-(CH2)2-OH

PCL

[33, 34]

HO-(CH2)4-OH

PCL

[34]

HO-(CH2)6-OH

PCL, PFL

[33, 34]

HO-(CH2)10-OH

PCL, PFL

[33, 34]

OH OH  OH OH

HO-CH2-CH=CH-CH2-OH

PCL

[34]

HO-CH2-C≡C-CH2-OH

PCL

[34]

Dehydration reactions are normally performed in nonaqueous media because water, which is a product of dehydration and is in equilibrium with starting materials, does not favour dehydration to proceed in an aqueous medium due to the ‘law of mass action’. Nevertheless, lipase catalysis enables the dehydration polycondensation of a dicarboxylic acid and a glycol in water at 45 °C, to afford a good yield of polyester. Some lipases such as CA lipase are active for the dehydration polymerisation of sebacic acid and 1,8-octanediol [23, 24]. In the polymerisation of an α,ω-dicarboxylic acid and a glycol, the polymerisation behaviour largely depended on the monomers comprising the methylene chain length. The polyester was obtained in good yields from 1,10-decanediol, whereas no polymer formation was observed from 1,6-hexanediol, suggesting that the hydrophobicity of monomers is a key factor for the production of polymers. Dehydration polycondensation in water is a new aspect in organic chemistry and has attracted considerable attention by organic chemists. Manabe and co-workers [25] realised dehydration reactions in water using a surfactant-type catalyst, dodecylbenzenesulfonic acid (DBSA). In these reactions, which include dehydrative esterification, etherification, thioetherification and dithioacetalisation, DBSA and substrates form emulsion droplets whose interior is hydrophobic enough to exclude the water molecules generated during the reactions. The principle of ‘dehydration in water’ may lead to environmentally benign systems. Condensation polymerisation via transesterification normally needs the activation of carboxylic acid groups. This activation is usually conducted by esterification of the acid group. Initially, alkyl or haloalkyl esters (Scheme 12.4, [16]) were employed and subsequently vinyl esters have often been used.

348 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

Wallace and co-workers [26] performed an enantioselective polymerisation of bis(2,2,2-trichloroethyl)trans-3,4-epoxyadipate with 1,4-butanediol (1,4-BDO) using the enzyme PPL as a catalyst at ambient temperature in anhydrous ethyl ether (Scheme 12.5). End group analysis of the polymer using nuclear magnetic resonance (NMR) gave a Mn of 5,300 Da, whereas GPC provided a MW of 7,900 Da. The same author used different diesters and diol combinations for the polymerisation employing PPL in ether, tetrahydrofuran (THF) or hexane. Polyester MW (Mn) were reported to be in the range of 3,200−8,200 Da using NMR and 4,900−11,800 Da using GPC for the polymers generated from the same set of monomers [27, 28]. O

O

XOC(CH2)mCOX + HO(CH2)4OH X: CH3—, CH3CH2—, ClCH2CH2—, CCl3CH2—, CF3CH2— O

lipase

O

C (CH2)mC

–XOH

O(CH2)4O

n

Scheme 12.4: Reproduced with permission from S. Kobayashi, Proceedings of the Japan Academy, Series B, 2010, 86, 4, 338. ©2010, The Japan Academy [16].

O Cl3CH2COOCH2C

HC

CH

CH2COOCH2CCl3 + HO

(CH2)4

OH

(±) Porcine pancreatic lipase, ether

O C

H2C H

O

H CH2CO2(CH2)4O

(–)–Polymer

H Cl3CH2CO2CH2C

O

CH2CO2CH2CCl3 H

(+)–Monomer

Scheme 12.5

Azim and co-workers [29] described the CALB-catalysed synthesis of polybutylene succinate (PBS) via condensation polymerisation using a monophasic reaction mixture of dimethyl succinate and 1,4-BDO in bulk and in solution.

12 Enzyme catalysis in the synthesis of biodegradable polymers 

 349

Diphenyl ether was a preferred solvent to give PBS with higher MW; at 60–90 °C after 24 h, the Mn values of PBS ranged from 2,000−8,000 Da. After 21 h, the reaction at 95 °C gave PBS with an Mn value of 38,000 Da. A regioselective direct dehydration polycondensation between a polyol sugar component, sorbitol, and an adipic acid was carried out with a CALB catalyst at 90 °C for 48 h. The product, polysorbityl adipate, was water-soluble; the Mn and MW values were 10,880 and 17,030 Da, respectively. Sorbitol was esterified at the primary alcohol group of 1-and 6-positions with high regioselectivity (85 ± 5%). To obtain a water-insoluble sorbitol polyester, sorbitol, adipic acid and 1,8-octanediol (molar ratio 15:50:35) were terpolymerised at 90 °C for 42 h. The methanol-insoluble portion (80%) had an MW of 1.17 × 105 [30]. As described by Hunsen and co-workers [31], cutinase HiC (cutin hydrolase from Humicola insolens) catalysed a dehydration polycondensation between a glycol and a diacid at 70 °C for 48 h under vacuum, containing 1% w/w enzyme. With the adipic acid component fixed, polyesters with Mn values up to 12,000 Da were obtained from diols. With the 1,4-cyclohexanedimethanol component fixed, polyesters obtained from succinic acid, adipic acid, suberic acid and sebacic acid had Mn values of 900, 4,000, 5,000 and 19,000 Da, respectively. For the synthesis of an optically active polyester from a racemic monomer, a new method of dynamic kinetic resolution was used. A mixture of stereoisomers of a secondary diol, α,α’-dimethyl-1,4-benzenedimethanol, were enzymatically polymerised with dimethyl adipate (Scheme 12.6, [1]) [32]. HO H3C

OH *

*

CH3

+

O CH3O

lipase CA Ru catalyst – CH3OH

O

CH2 4

OCH3

H3C O

CH3 (R)

(R)

CH2

O O

4

O

n

Scheme 12.6: Reproduced with permission from S. Kobayashi, Polymer Science: A Comprehensive Reference, 2012, 5, 10, 217. ©2012, Elsevier [1].

Due to the enantioselectivity of CALB, only hydroxyl groups at the (R) centre are preferentially reacted to form the ester bond with the liberation of methanol. The reactivity ratio was estimated as (R)/(S) = ~1 × 106. In situ racemisation from the (S) to the (R) configuration using Ru catalysis allowed the polymerisation to occur at a high conversion, which means the enzymatic polymerisation and the Ru-catalysed racemisation occurred concurrently. During the reaction, the MW increased to 3,000–4,000 Da and the optical rotation of the reaction mixture increased from − 0.6° to 128°.

350 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

As described by Uyama and co-workers [33], in an ionic liquid such as 1-butyl3-methyl-imidazolium tetrafluoroborate ([bmim][BF4]), a similar polycondensation between diethyl adipate or diethyl sebacate and 1,4-BDO, under reduced pressure at 60 °C after 72 h, gave the polyester with Mn ~1,500 Da and yield of 97%. Since the ionic liquid is nonvolatile, ethanol was removed under vacuum during the reaction. According to this work, the CALB-catalysed polymerisation of dimethyl adipate or dimethyl sebacate with 1,4-BDO was also performed in an ionic liquid such as [bmim][BF4], [bmim][PF6] and [bmim][(CF3SO2)2N] at 70 °C for 24 h to give a higher MW polyester with the Mn reaching several thousand. This, in combination with the tunable solvent hydrophilicity of ionic liquids, could represent an advantage in the polymerisation of highly polar monomers with low solubility in organic solvents [34]. Transesterifications are inherently reversible reactions. To shift the equilibrium towards the product polymer more effectively, the activation of esters was carried out using a halogenated alcohol, such as 2-chloroethanol, 2,2,2-trifluoroethanol and 2,2,2-trichloroethanol, instead of using methanol or ethanol for increasing the electrophilicity of the acyl carbonyl. This activation helps to avoid significant alcoholysis of the products by decreasing the nucleophilicity of the leaving alkoxy group. In 1994, an irreversible process was developed using a vinyl ester for lipase- catalysed acylation, where the product of vinyl alcohol tautomerises to acetaldehyde (Scheme 12.7, [16]) [35]. The reaction of an alcohol with a vinyl ester proceeds much faster than with an alkyl ester or a haloalkyl ester to form the desired product in higher yields; Kobayashi and co-workers [36–41] studied the polymerisation of the divinyl esters of dicarboxylic acids with diols, triols and sorbitol. The polymerisation behaviour was strongly dependent on the monomer structure, enzyme origin and reaction conditions. Under appropriate conditions, an aliphatic polyester with a MW higher than 2 × 104 Da was obtained. The polymerisation of divinyl adipate with 1,4-BDO by PFL, in isopropyl (a) Alkyl ester or haloalkyl ester O R1

C

OR2 + R3OH

lipase

O R1

C

OR3 + R3OH

(b) Vinyl ester O R4

C

OCH=CH2 + R5OH

lipase

O R4

C

OR5 +

CH2=CHOH CH3CHO

Scheme 12.7: Reproduced with permission from S. Kobayashi, Proceedings of the Japan Academy, Series B, 2010, 86, 4, 338. ©2010, The Japan Academy [16].

12 Enzyme catalysis in the synthesis of biodegradable polymers 

 351

ether at 45 °C for 48 h, produced a polyester with a MW of 6,700 Da and a yield of 50% (Scheme 12.8, [1]), whereas the use of CAL was also employed to produce crosslinkable polyesters [40]. Divinyl sebacate and glycerol were polymerised in the presence of the unsaturated fatty acids, oleic acid, linoleic acid and linoleinic acid. NMR analysis revealed that the reaction proceeded with regioselectivity during the condensation of divinyl ester and glycerol, and the pendent hydroxyl group of glycerol was acylated with fatty acids in the same reaction. O CH2

CHO

O

C(CH2)4C

OCH

CH2 + HO(CH2)4OH O

lipase

O

C(CH2)4C

— CH3CHO

O(CH2)4O

n

Scheme 12.8: Reproduced with permission from S. Kobayashi, Polymer Science: A Comprehensive Reference, 2012, 5, 10, 217. ©2012, Elsevier [1].

Unsaturation within the fatty acid chain did not disturb the process. In the polymerisation of divinyl sebacate and polyol (sorbitol), the regioselectivity was controlled to yield sugar-containing polyesters in which the 1- and 6-positions of sorbitol were regioselectively acylated. The terpolymerisation of divinyl esters, glycols and lactones produced ester terpolymers with Mn higher than 1 × 104 (Scheme 12.9, [19]). O C

O

(CH2)p

O + CH2

CHO

Lipase CH3CHO

O

C(CH2)qC

OCH

CH2 + HO(CH2)rOH

O

O

O

C(CH2)pO

C(CH2)qC

O(CH2)rO

n

Scheme 12.9: Reproduced with permission from S. Okumura, M. Iwai and Y. Tominaga, Agricultural Biology and Chemistry, 1984, 48, 2805. ©1984, The Japan Academy [19].

Lipases showed high catalytic activity for the terpolymerisation involving both condensation polymerisation and ring-opening polymerisation (ROP) simultaneously in one-pot to produce ester terpolymers, without involving homopolymer formation [42–43]. A similar terpolymerisation was performed using three kinds of monomers, ω-pentadecalactone (ω-PDL), diethyl succinate (DES) and 1,4-BDO, by a CALB catalyst optimally at 95 °C via a two-stage vacuum technique. The polymerisation produced a terpolyester reaching a MW of 77,000 Da with MW/Mn ~1.7–4.0 [44].

352 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

In addition to olefinic and epoxy reactive groups [45], a mercapto group can also be a crosslinkable group. The direct Candida antarctica lipase-catalysed condensation polymerisation of 1,6-hexanediol and dimethyl 2-mercaptosuccinate at 70 °C in bulk gave an aliphatic polyester containing free pendent mercapto groups with MW = 14,000 Da and good yield. The polyester was readily crosslinked by air oxidation via the disulfide linkage formation [46].

12.2.3 Ring-opening polymerisation of carbonates and other cyclic monomers The ROP of six-membered cyclic carbonate and 1,3-dioxan-2-one has been investigated using lipases derived from Candida antarctica, Candida cylindracea, porcine pancreas, Pseudomonas sp. and Mucor sp. (Scheme 12.10) [47–50]. In some of these reports, extraordinary differences in polymer MW achieved using the same enzyme under similar experimental conditions are reported (Table 12.3). Matsumura and co-workers [48] reported 1,3-dioxan-2-one polymerisation yielding a MW of 84,700 Da and DP of 3.9 with 0.5 wt% PPL, and a MW of 169,000 Da and DP of 3.5 with 0.25 wt% PPL, when reactions were carried out at 100 °C for 24 h. Polymerisation occurred with PPL, CCL and Pseudomonas cepacia (PC) lipases but not with CALB. On the other hand, the best result in trimethylene carbonate bulk (TMC) polymerisations was at 70 °C for 120 h using CALB, with almost quantitative monomer conversion (97%) and the highest MW (Mn = 15,000, PD = 2.2) of the seven lipases tested [49]. O O

C

O

Lipase

O O

C

OCH2CH2CH2

OH n

Scheme 12.10

PPL exhibited high monomer conversion (>80%) over the 120 h polymerisation period, but the MW of the polymer produced was low (Mn = 3,500). In contrast, Kobayashi and co-workers [47] reported the formation of low MW poly (TMC) (Mn = 800 Da, PD = 1.4) via a 50 wt% PPL-catalysed polymerisation at 75 °C. Furthermore, thermally treated CAL (heated in water at 100 °C for several hours) did not show catalysis at 75 °C, but the enzyme from Mucor miehei and PPL (thermally inactivated) showed monomer conversion (36% and 97%, respectively). These data show that lipase from Mucor miehei and PPL maintained catalytic ability even after thermal treatment or possible impurities in the enzyme (the enzyme contains basic and acidic groups in the side chain such as those found in lysine, glutamic acid and aspartic acid residues) acted as catalysts

12 Enzyme catalysis in the synthesis of biodegradable polymers 

 353

for the polymerisation. The authors claimed that polymerisation proceeded through enzymatic catalysis, as the unchanged monomer was recovered in the absence of the enzyme or using an inactivated enzyme. NMR spectroscopic results for polymer structural analysis have shown an absence of ether linkages and the presence of carbonate groups in the polymer chain, confirming that chain propagation proceeded without decarboxylation. It has been reported that partial decarboxylation takes place when the polymerisation of trimethylene carbonate was carried out in the absence of the enzyme via cationic chemical initiators. The lipase-catalysed polymerisation of the disubstituted trimethylene carbonate analogue 5-methyl-5-benzyloxycarbonyl1,3-dioxan-2-one (MBC) was also studied [51]. The bulk polymerisation, catalysed by lipase immobilised in Sol-Gel-AK from P. fluorescens for 72 h at 80 °C, yielded 97% monomer conversion and a product with a Mn of 6,100. The benzyl ester protecting groups of the polymer were removed by catalytic hydrogenation (palladium/charcoal (Pd/C)) in ethyl acetate to give the corresponding functional polycarbonate with pendant carboxylic acid groups in the main chain. The ROP of cyclic phosphate (ethylene isopropyl phosphate), was demonstrated at 100 °C for 24 h, with a Mn = 1,660 Da using 0.25 wt% PPL (Scheme 12.11) [52]; a higher polymerisation temperature and lipase concentration enhanced the polymerisation rate. A novel six-membered cyclic carbonate, 5-alloyloxy-1,3-dioxan-2-one, was synthesised from glycerol, and the corresponding polycarbonate poly(5-alloyloxyTable 12.3: ROP carbonates and other cyclic monomers. Monomer

Enzyme/reaction conditions and polymer analysis

References

1,3-Dioxan-2-one

Candida antarctica lipase: Mn = 2,500, DP = 3.4 using GPC against a polystyrene standard. Reaction time 72 h, conversion 100% at 75 °C

[45]

Mucor miehei lipase: Mn = 610, DP = 1.2 using GPC against a polystyrene standard. Reaction time 72 h, conversion 93% at 75 °C

[45]

Porcine pancreatic lipase: Mn = 800, DP = 1.4 using GPC against a polystyrene standard. Reaction time 72 h, conversion 80% at 75 °C

[45]

Porcine pancreatic lipase (0.25 wt%): MW = 169,000, DP = 3.5 using GPC against a polystyrene standard. Reaction time 24 h, conversion 96% at 100 °C

[46]

Pseudomonas sp. lipase (0.5 wt%): MW = 24,000, DP = 1.9 using GPC against a polystyrene standard. Reaction time 24 h, conversion 97% at 100 °C

[46]

Candida cylindracea lipase (1 wt%): MW = 1,000, DP = 1.2 using GPC against a polystyrene standard. Reaction time 24 h, conversion 5% at 100 °C

[46]

354 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

Table 12.3 (continued) Monomer

Enzyme/reaction conditions and polymer analysis

1,4-Dioxan-2-one

Candida antarctica lipase (5 wt%): MW = 28,000, DP = 9.5 using GPC against a polystyrene standard. Reaction time 48 h, conversion 69% at 60 °C

[60]

Porcine pancreatic lipase (5 wt%): MW = 3,890, DP = 2.2 using GPC against a polystyrene standard. Reaction time 48 h, conversion 66% at 100 °C

[54]

Bacillus thermoproteolyticus rokko: MW = 8,610, DP = 6.2 using GPC against a polystyrene standard. Reaction time 48 h, conversion 36% at 100 °C

[54]

Candida antarctica lipase (5 wt%): MW = 41,000, using GPC against a polystyrene standard. Reaction time 15 h, conversion 77% at 60 °C

[54]

Candida antarctica lipase (10 wt% ionic liquid coated): MW = 182,100, using SEM, FT-IR, GPC and MALDI-TOF. Reaction time 6 h

[55]

Pseudomonas fluorescens lipase: Mn = 6,100, DP = 1.6 using GPC against a polystyrene standard. Reaction time 72 h, conversion 97% at 80 °C

[49]

Porcine pancreatic lipase: Mn = 1,300, DP = 1.3 using GPC against a polystyrene standard. Reaction time 72 h, conversion 98% at 80 °C

[49]

Pseudomonas cepacia lipase: Mn = 1,450, DP = 1.0 using GPC against a polystyrene standard. Reaction time 24 h, conversion 50% at 80 °C

[49]

Candida antarctica lipase: Mn = 4,400, DP = 2.1 by GPC against a polystyrene standard. Reaction time 72 h, conversion 86% at 80 °C

[49]

Porcine pancreatic lipase (10 wt%): Mn = 14,300, DP = 1.07 using GPC against a polystyrene standard. Reaction time 72 h, conversion 92% at 120 °C

[58]

Pseudomonas sp. (4.7 wt%): Mn = 12,500, DP = 3.33 using GPC against a polystyrene standard. Reaction time 168 h, conversion 73.8% at 100 °C

[57]

Pseudomonas cepacia lipase (10 wt%): Mn = 4,500, DP = 1.84 using GPC against a polystyrene standard. Reaction time 72 h, conversion 20.8% at 100 °C

[57]

Porcine pancreatic lipase (10 wt%): Mn = 12,200, DP = 1.14 using GPC against a polystyrene standard. Reaction time 72 h, conversion 90% at 120 °C

[58]

5-Methyl-5-benzyl oxycarbonyl-1,3dioxan-2-one

3(S)-Isopropylmorpholine-2,5-dione

3(R)-Isopropyl-morpholine-2,5-dione

References

12 Enzyme catalysis in the synthesis of biodegradable polymers 

 355

Table 12.3 (continued) Monomer

Enzyme/reaction conditions and polymer analysis

3(R,S)-Isopropyl morpholine-2,5-dione

Porcine pancreatic lipase (10 wt%): Mn = 12,000, DP = 1.15 using GPC against a polystyrene standard. Reaction time 72 h, conversion 90% at 120 °C

References [58]

3(S, 6R, S)- Isopropyl-6- Porcine pancreatic lipase (10 wt%): Mn = 6,900, DP = 1.16 methyl-morpholine using GPC against a polystyrene standard. Reaction time -2,5-dione 72 h, conversion 9% at 120 °C

[62]

3(S)-Isobutyl morpholine- 2,5-dione

Porcine pancreatic lipase (10 wt%): Mn = 9,900, DP = 1.14 using GPC against a polystyrene standard. Reaction time 72 h, conversion 40% at 130 °C

[58]

3(S)-Sec-butylmorpholine-2,5-dione

Porcine pancreatic lipase (10 wt%): Mn = 11,500, DP = 1.09 using GPC against a polystyrene standard. Reaction time 144 h, conversion 90% at 110 °C

[58]

6(S)-Methylmorpholine- 2,5-dione

Porcine pancreatic lipase (10 wt%): Mn = 12,000, DP = 1.05 using GPC against a polystyrene standard. Reaction time 72 h, conversion 76% at 100 °C

[62]

6(R,S)-Methylmorpholine-2,5-dione

Porcine pancreatic lipase (10 wt%): Mn = 9,300, DP = 1.04 using GPC against a polystyrene standard. Reaction time 72 h, conversion 34% at 120 °C

[58]

Cyclobis(hexamethylene carbonate)

Candida antarctica lipase: Mn = 12,000, DP = 1.7 using SEC analysis against a polystyrene standard. Reaction time 72 h, yield 85% at 60 °C

[60]

Pseudomonas fluorescens lipase: Mn = 13,000, DP = 2.1 using SEC against a polystyrene standard. Reaction time 120 h, yield 29% at 60 °C

[60]

Candida antarctica lipase: Mn = 5,300, DP = 1.8 using SEC analysis against a polystyrene standard. Reaction time 72 h, yield 72% at 60 °C

[60]

Pseudomonas fluorescens lipase, Mn = 9,200, DP = 2.0 using SEC against a polystyrene standard. Reaction time 120 h, yield 57% at 60 °C

[60]

Candida antartica lipase (Novozyme-435), Mn = 54,000, yield 99%

[61]

Cyclobis(diethylene glycol carbonate)

Cyclobis(decamethylene carbonate)

DP: Degree of polymerisation FT-IR: Fourier transform- infrared MALDI-TOF: Matrix assisted laser desorption/ionisation-time of flight GPC: Gel permeation chromatography SEC: Size exclusion chromatography SEM: Scanning electron microscopy SEM: Scanning Electron Microscopy

356 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

1,3-dioxan-2-one) was polymerised by ROP in bulk at 120 °C, using porcine pancreas lipase (0.1 wt%) immobilised on silica particles as the catalyst. The highest MW was 48,700 g/mol [53]. The pendent allyl group was further oxidised to an epoxy group, which was then grafted to low MW polyethylenimine to construct novel, nonviral gene delivery carriers with low toxicity and high gene-transfection efficacy [54, 55]. Enzymatic ROP of other cyclic monomers, 1,4-dioxan-2-one, [56–58], 3(S)isopropylmorpholine-2,5-dione and its derivatives have been studied [59, 60]. Out of 12 enzymes (7 lipases, 2 esterases and 3 proteases) studied, immobilised lipase from C. antarctica gave the best results. In a recent study [58], the activity of CALB was improved by coating with an ionic liquid, 1-butyl-3-methylimidazolium hexafluorophoshphate, which protected the bound water layer surrounding the lipase. Poly(1,4-dioxan-2-one) (PPDO) with a MW of 182,100 g/mol was obtained via this method. The thermodynamics and kinetics of CALB [57] showed that the polymerisation of 1,4-dioxan-2-one reached equilibrium after 12 h at 60 °C in the presence of 5wt% lipase, and the ROP kinetics of PPDO was of the first order with regards to the monomer concentration.

O

O P

O

OiPr

Porcine pancreatic lipase

O H

O

P OiPr

O

CH2

CH2

OH n

Scheme 12.11

The enzymatic ROP of six-membered cyclic depsipeptides: 3(S)-isopropyl-morpholine2,5-dione, 3(R)-isopropyl-morpholine-2,5-dione, 3(R,S)-isopropyl-morpholine-2,5dione, (3S, 6R, S)-3-isopropyl-morpholine-2,5-dione, 3(S)-isobutyl-morpholine-2,5dione, 3(S)-sec-butyl-morpholine-2,5-dione, 6(S)-methyl-morpholine-2,5-dione and 6(R,S)-methyl-morpholine-2,5-dione has been reported [60]. Enantiomerically pure functional polycarbonate, having many potential biomedical uses, was synthesised from a novel seven-membered cyclic carbonate monomer derived from naturally occurring L-tartaric acid, using four commercially available lipases from different sources at 80 °C, in bulk. The highest number-average MW, Mn = 15,500 g/mol, polydispersity index (PDI) = 1.7, [α]D20 = +77.8 and melting temperature (Tm) = 58.8 °C, optically active polycarbonate was obtained with lipase CALB [61]. Cyclic dicarbonates, cyclobis(hexamethylene carbonate) and cyclobis(diethylene glycol carbonate) were polymerised by lipase from C. antarctica and P. fluorescens [62]. CALB was used for the ROP of cyclobis(decamethylene carbonate) (DMC2) giving a polymer with a MW of 5.4 × 104 and 99% yield, and an ultralow enzyme/substrate weight ratio of 1/200. Compared with six-membered trimethylene carbonate, a much lower reaction activity of large-sized DMC2 was observed, the opposite of the enzymatic polymerisation of lactones with different ring sizes [63].

12 Enzyme catalysis in the synthesis of biodegradable polymers 

 357

The same enzyme showed considerably higher catalytic efficiency and a high MW of up to 6 × 104 g mol−1 was obtained through the ROP of cyclobis(pentamethylene carbonate) [64]. Poly(lactide-co-trimethylene carbonate) was prepared by the lipase-catalysed ring- opening copolymerisation of different kinds of lactide (LA) (L,L-, D,D- and D,Llactides) and trimethylene carbonate (Table 12.4) [65]. PPL showed the best results for both the polymerisation rate and the MW attained (MW in the range of 20,000 Da) for the polylactide. The results indicated that poly(lactide-co-trimethylene carbonate) was a random copolymer and the glass transition temperature of the copolymer linearly decreased with increasing TMC content. Lipase-AK (from P. fluorescens) catalysed the ROP of TMC with MBC at 80 °C for 72 h [66]. Compared with MBC, the reactivity of TMC was higher and the polymers produced were not ordered structures but random polymers. The benzyl ester protecting groups of poly(TMC-co-BMC) were removed by hydrogenolysis using hydrogen (H2) over a Pd/C catalyst in ethylacetate to leave free pendent acid groups. CALB catalysed the ring-opening copolymerisation of trimethylene carbonate and ω-PDL in toluene at 70 °C giving random copolymers [67]. Changing the feed ratio of the comonomers resulted in regulation of the copolymer composition. Chemical catalysts such as stannous octanoate, methylaluminoxane and aluminium isopropoxide have been used for the copolymerisation of TMC and 15-pentadecanolide (PDL), and the results showed that TMC had much greater reactivity than PDL. In contrast, for the Novozyme-435-catalysed copolymerisation, PDL had a greater reactivity than TMC. Cyclic dicarbonates, cyclobis(hexamethylene carbonate) and cyclobis(diethylene glycol carbonate) have been copolymerised with e-CL and 12-dodecanolide (DDL) using CAL in toluene at 60 °C for 48 h (Scheme 12.12) [62]. Table 12.4: Ring opening copolymerisation of TMC with other cyclic monomers. Monomer + TMC

Enzyme, reaction conditions and polymer analysis

References

L,L-lactide [50:50]

Porcine pancreatic lipase: MW = 19,100, PD = 1.7 using SEC against a polystyrene standard. Reaction time 7 days, yield 34% at 100 °C

[63]

D,D-lactide [50:50]

Porcine pancreatic lipase: MW = 12,800, PD = 1.4 using SEC against a polystyrene standard. Reaction time 7 days, yield 38% at 100 °C

[63]

D,L-lactide [50:50]

Porcine pancreatic lipase: MW = 8,100, PD = 1.4 using SEC against a polystyrene standard. Reaction time 7 days, yield 25% at 100 °C

[63]

5-Methyl-5-benzyloxy Pseudomonas fluorescens lipase (4 wt%): Mn = 7,500, PD = 4.4 carbony-1,3-dioxan- using GPC against a polystyrene standard. Reaction time 72 h, yield 85% at 80 °C 2-one [50:50]

[64]

ω-Pentadecalactone

[65]

Novozyme-435 (10 wt%): Mn = 18,800, PD = 1.65 using GPC against a polystyrene standard. Reaction time 24 h, yield 90% at 70 °C

358 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

A mechanism was proposed for carbonate ROP (Scheme 12.13). The mechanism was based on the identification of propanediol, a dimer of trimethylene carbonate (DTMC) and a trimer of trimethylene carbonate (TTMC) in the reaction mixture, along with the presence of symmetrical hydroxyl end group structures in the low and high MW TMC polymerisation products and involves: – The reaction of TMC with lipase to form the lipase-TMC enzyme-activated monomer (EAM) complex. – Reaction of EAM with water followed by rapid decarboxylation to form 1,3-propanediol. – Propagation as defined by the presence of carbonate functionality involved in the formation of DTMC by the reaction of the EAM with 1,3-propanediol. – TTMC synthesis by the reaction of DTMC with EAM. – Subsequent reactions to form high MW chains. O O O

R

C

C O

O

R

O

O

R= —(CH2)6

O

+

R= —(CH2)2—O—(CH2)2—

m

Candida antarctica lipase

O

O O

R

O

C

O

(CH2)m

C n

m=2 m=8

Scheme 12.12

12.2.4 Ring-opening polymerisation and copolymerisation of lactones Four-membered ring lactones: β-propiolactone (β-PL) [68–70], β-butyrolactone (β-BL) [71–76], benzyl β-malolactonate (BBM) [77] and α-methyl-β-propiolactone (MPL) [78, 79] were polymerised using different lipases (Table 12.5). The lipase-catalysed ROP of the four-membered β-BL was first reported by Nobes and co-workers [74]. Poly(3-hydroxybutyrate), with a weight average MW ranging from 256 to 1,045 Da, were prepared after several weeks of polymerisation using approximately equal weights of β-BL and lipase. An enantioselective polymerisation of four-membered lactones was demonstrated. Racemic MPL was stereoselectively polymerised by PsCL to generate an optically active (S)-enriched polyester with an enantiomeric excess of 50%. Poly(3-hydroxybutyrate) (PHB)-depolymerase (EC 3.1.1.75) was also used to polymerise the BL and the rate of polymerisation was faster compared with PPL and CCL under the same reaction conditions at 80 °C in bulk. Benzyl β-malonate was polymerised by PPL and Novozyme-435 lipase at 60 °C to yield poly(benzyl β-maleate) with a MW greater than 7,000 Da. The benzyl group of

O

Initiation: E EAM

OH +

O

O

O

E

O

C

(CH2)3

O

OH

EAM

H2O

(CH2)3

HO

OH + CO2 +

E

OH

Propagation:

EAM + HO

O (CH2)3

Trimerisation HO

(CH2)3

(CH2)3

Scheme 12.13

(CH2)3

O O

C

O

C

Polymerisation HO

HO

OH

O

(CH2)3

EAM

OH

O

O

C

O

(CH2)3

O

C O n–1

OH +

E

OH

O HO

(CH2)3

O O

(CH2)3

(CH2)3

OH

O

EAM

C

O O

(CH2)3

O

C

O

(CH2)3

OH +

O HO

(CH2)3

O

C

E

OH

O O

(CH2)3

O

n

C

O

(CH2)3

OH

12 Enzyme catalysis in the synthesis of biodegradable polymers 

Dimerisation

 359

360 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

poly(benzyl β-maleate) was removed by catalytic hydrogenation using Pd/C to yield poly(β -D,L-malic acid). More recently, using a low weight ratio of the CALB enzyme to lactone (1:100) in an ionic liquid, polymers were obtained from MPL with a DP as high as 170, oligomers from β-BL with a DP of 5 and a copolymer of MPL and β-BL with a DP of 180 [80]. Table 12.5: Lipase-catalysed ROP of different sized lactones. Lactone

Enzyme

References

4-Membered ring β-PL

C. cylindracea, P.cepacia, porcine pancreatic, P. fluorescens, C. antarctica, P. aeruginosa and A. niger lipase

[66–68, 72, 81]

β-BL

Lipase ESL-001, C. cylindracea, P. fluorescens, porcine pancreatic, P. cepacia, PHB-depolymerase and Pseudomonas sp. lipase

[69–74, 79]

MPL

P. fluorescens, porcine pancreatic and C. cylindracea

[76, 77]

BBM

Novozyme-435

[75]

5-Membered ring γ-BL

P. cepacia, Pseudomonas sp. and porcine pancreatic lipase

γ-VL

Pseudomonas sp. lipase

[74]

γ-CL

Pseudomonas sp. lipase

[74]

[72, 79, 80]

6-Membered ring δ-VL

C. cylindracea, P. fluorescens, porcine pancreatic and R. japonicas lipase

MVL

C. antarctica lipase

δ-DL

Pseudomonas sp. lipase

[74, 79]

LA

C. antarctica lipase

[86–89]

[68, 81–83, 85] [84]

7-Membered ring ɛ-CL

C. antarctica, P. fluorescens, porcine pancreatic, P. cepacia, A. niger, C. cylindracea, P. delemer, R. japonicus, hog liver esterase and Pseudomonas sp. lipase

αMCL

C. antarctica lipase

[68, 72, 74, 79, 81–83, 92–98, 103, 106, 123] [84]

8-Membered ring 8-HL

C. antarctica lipase

[100]

9-Membered ring 8-OL

C. antarctica, C. cylindracea, P. cepacia and P. fluorescens lipase

[68, 81, 101]

12 Enzyme catalysis in the synthesis of biodegradable polymers 

 361

Table 12.5 (continued) Lactone

Enzyme

References

12-Membered ring UDL

P. fluorescens, C. cylindracea and C. antarctica lipase

[68, 81, 103–105]

13-Membered ring DDL

C. cylindracea, porcine pancreatic, Pseudomonas sp., P. fluorescens, C. antarctica and P. cepacia lipase

[68, 81, 103, 105−107]

16-Membered ring PDL

C. cylindracea, P. fluoroescens, Pseudomonas sp., Mucor sp., C. antarctica and M. meihei lipase

[68, 79, 81, 104, 105, 108–110, 113, 114]

17-Membered ring HDL

C. antarctica, C. cylindracea, P. cepacia, porcine pancreatic and P. fluorescens lipase

[68, 111]

8-HL: 8-Membered lactone 8-OL: 8-Octanolide HDL: 16-Hexadecanolide MVL: α-Methyl-δ-valerolactone UDL: 11-Undecanolide αMCL: α-Methyl-ε-caprolactone δ-DL: δ-Decalactone γ-BL: γ-Butyrolactone γ-CL: γ-Caprolactone γ-VL: γ-Valerolactone δ-VL: δ-Valerolactone

Five-membered, unsubstituted, lactone γ -butyrolactone (γ-BL) was polymerised by PPL or PCL [74, 81] into small oligomers with a DP of 8−11. More recently, γ-BL have yielded homopolymers of up to 50,000 [82]. Poly(γ-butyrolactone) is a very useful biomaterial, since its degradation gives γ-hydroxybutyric acid, a metabolite naturally occurring in the body. In the PSL-catalysed polymerisation of γ-valerolactone (γ-VL) and γ -caprolactone (γ-CL), less than 10% conversion was observed at 60 °C for 480 h [76]. Unsubstituted and substituted six-membered lactones δ-VL [70, 83–85] and α-methyl- δ-valerolactone (MVL) [86] were polymerised using Rhizopus japonicus lipase, and CCL, PFL, PPL and CAL enzymes. For unsubstituted δ-VL, the reactions were run for 5−10 days and the highest MW obtained were in the range of 2,000 Da. The CAL-catalysed polymerisation of MVL yielded polyester with a Mn of up to 11,400 at 60 °C in 24 h. Recently, the ROP of δ-VL was successfully catalysed by a thermophilic esterase from the archaeon Archaeoglobus fulgidus (AFEST) in organic solvents

362 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

[87]. Poly(δ-valerolactone) (PVL) was produced in a 97% monomer conversion, with a number-average MW of 2,225 g/mol, in toluene at 70 °C for 72 h. PVL is an important aliphatic polyester due to its good biodegradability, biocompatibility and permeability characteristics. LA is currently an important six-membered lactone used as a starting monomer for the production of PLA, a type of green plastic. LA polymerisation was first reported in 1997 [88] at temperatures between 80 and 130 °C to produce PLA with MW up to 1.26 × 105. The lipase-catalysed polymerisations of L-lactide were also carried out in four types of ionic liquids. [C4mim][BF4] was suitable to obtain higher MW polylactides and higher polymer yield at lower lipase content [89]. Using supercritical carbon dioxide (scCO2), the polymerisation of L-lactide by immobilised CALB was achieved at 65 °C in a biphasic media system, where the supercritical phase coexists with a liquid organic phase (melted monomer), and the growing poly(L-lactide) (PLLA) chains are soluble. A semicrystalline polymer with MW up to 12,900 g/mol was obtained, with a monomer conversion rate dependent on enzyme concentration and its initial water activity [90]. DD-lactide was converted, in a toluene solution and under mild reaction conditions (50−70 °C) using CALB, to form the corresponding polymer. By carefully selecting the reaction conditions, Hans and co-workers were able to obtain poly(D-lactide) of reasonable MW and in high yields using CALB catalysis [91]. During the syntheses of PLLA and poly(L-lactide-co-glycolide) (PLLGA) in the ionic liquid 1-hexyl-3-methylimidazolium hexafluoro-phosphate [HMIM][PF6] mediated by Novozyme-435, the highest PLLA yield (63%) was obtained at 90 °C with a MW (Mn) of 37,890 g/mol, while the yield for PLLGA was only up to 19% [92]. The lipase-catalysed oligomerisation of alkyl D- and L-lactate monomers was studied for the first time in 2010 [93]. In this study it was found that oligomerisation only occurs enantioselectively for D-lactates giving oligomers up to heptamers of LA in good to high yields, using primary C1 to C8 alkyl groups and the sec-butyl group for D-lactate monomers. No reaction occurred for any L-lactates in similar conditions. Among the seven-membered lactones, ɛ-CL is the most extensively studied. Several lipases (PPL, PFL, CAL, PCL and lipase from Yarrowia lipolytica (YLL)) have been used in the ROP of ɛ-CL (18, 28, 71, 75, 77, 81, 82, 84–90, 94). In recent years, a lot of work has been carried out to improve the monomer conversion rate and polymer MW. Using an immobilised form of CALB as a catalyst, PCL was obtained with 99% monomer conversion and a number-average MW (Mn) of 18,870 g/mol. In scaled up experiments, PCL was synthesised in an isolated yield of 78%, with a Mn of 41,540 g/mol. The PCL product showed a high degree of crystallinity and excellent thermal stability [95]. In a recent study [96], by changing the mixing and temperature, and using lipase CALB as a biocatalyst, PCL of MW up to 310,000 KDa were obtained, at a temperature of 70 °C for 3 h and with an impeller speed of 500 rpm. The effects of

12 Enzyme catalysis in the synthesis of biodegradable polymers 

 363

different reaction conditions on the ROP of e-CL using CALB were also examined by Ozsaguroglu and co-workers [97]. They were able to obtain higher molecular mass values and conversion rates in hydrophobic solvents: reactions in toluene were more stable at lower temperatures, but higher temperatures were needed to obtain an increase in the molecular mass and conversion rates in an n-hexane solvent. Short reaction time periods seemed enough to raise conversion rates at higher temperatures (97% conversion rate at 80 °C in n-hexane for 6 h), while an increasing molecular mass of PCL resulted in lower crystallinity. In other experiments [98], ultrasonic irradiation greatly improved, by 63%, the CALB mediated polymerisation of ɛ-CL to poly(6-hydroxyhexanoate) in the ionic liquid 1-ethyl-3-methylimidazolium tetraflorourborate. Even though CALB has been the most utilised catalyst, other lipases have been used for the ROP of e-CL. YLL, CRL and PPL were successfully employed in the presence of several ionic liquids. PCL with Mn in the range of 300–9,000 Da were obtained with a high degree of crystallinity in all the polyesters [99]. Ma and co-workers [100] reported the ROP of ɛ-CL catalysed by a thermophilic esterase of AFEST with almost 100% monomer conversion and an average MW of 1,400 in toluene at 80 °C for 72 h. Mathematical models have been developed to predict ɛ-CL conversion and to generate molecular mass distribution trends based on experimental data [101]. An 8-HL was reported to be polymerised by a CALB catalyst at 45 °C with the production of polyHL with a Mn of 23,600 Da [102]. The lipase-catalysed ROP of the nine-membered lactone, 8-OL, has been reported using various lipases in isooctane [70, 83, 103]. CAL and PFL showed high catalytic activity. In the polymerisation of 8-OL using PFL at 75 °C for 240 h, a polymer with a Mn of 1.6 × 104 Da was obtained. The ROP of lactones with various ring sizes (6- to 13- and the 16-membered ring) catalysed by CALB showed differences in their polymerisation rates. The KM was more or less independent of the ring size, suggesting similar affinities of the lipase for all lactones, while no obvious trend could be discerned for Vmax [102]. The ROP of 4-substituted ɛ-CL catalysed by the same lipase demonstrated dramatic differences in polymerisation rates and selectivity, depending on the size of the substituent. Quantification of the reaction rates showed that the polymerisation rate decreased by a factor of 2 upon the introduction of a methyl (Me) substituent at the 4-position. Moreover, 4-ethyl-ɛ-caprolactone (4-EtCL) polymerised 5 times slower than 4-methylɛ-caprolactone (4-MeCL) and 4-propyl-ɛ-caprolactone (4-PrCL) polymerised 70 times slower. The decrease in polymerisation rate is accompanied by a strong decrease in enantioselectivity [104]. Four macrolactones (12-, 13-, 16- and 17-membered rings respectively), undecanolide (UDL) [70, 83, 105−107], dodecanolide (DDL) [70, 83, 105, 107−109], pentadecanolide PDL [11, 70, 81, 83, 106, 110–112] and hexadecanolide (HDL) [70, 113] showed unusual activity towards enzymatic catalysis as compared with chemical polymeri-

364 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

sations. The PFL-catalysed polymerisation of macrolactones proceeded much faster than that of ɛ-CL. For the polymerisation of DDL, Candida cylindracea (CC), PC, PF and PP lipases showed high catalytic activity and the order of activity was as follows: PC lipase > PF lipase > CC lipase > PP lipase. The rate of UDL polymerisation using PFL was higher than that using CCL, whereas the polymerisation of UDL using CCL produced a polymer of higher MW (Mn = 9,400 Da) compared with that obtained using PFL (Mn = 8,400 Da) [106]. Lipase PS-30 immobilised on celite was used for bulk PDL polymerisation and poly(PDL) with a Mn of 62,000 Da and a DP of 1.9 was obtained [110]. Alternatively, instead of bulk polymerisation, the Novozyme-435 catalysed polymerisation of PDL was conducted in toluene (1:1 w/v) and poly(PDL) with the highest MW (86,000 Da) was obtained. The anionic polymerisation of small- and medium-sized lactones was reported to be fast (4-, 6- and 7-membered) when compared with macrolactones (12-, 13- and 16-membered) due to higher ring strain in the smaller lactones [114]. Comparing the anionic ROP using a metal catalyst with ROP by a lipase catalyst, the latter has shown a higher reactivity of larger ring-sized monomers which have a lower ring strain compared with medium ring-sized monomers. Poly(ω-pentadecalactone) (PPDL) is a lactone-based ring-opening polymer with good mechanical and biodegradation properties. Recently, the lipase-catalysed interfacial polymerisation of ω-PDL in an aqueous biphasic medium allowed an increase of the molar mass of oligo ω-PDL to 3,507 g/mol [115]. Furthermore, a novel technique has been reported for the formation of PPDL [116]. Using the synergistic effects of lipase and microwave technology, PPDL have been formed using lipase and MW irradiation at varying reaction time intervals (30−240 min). Synergistic effects allowed catalysing the polymerisation of PPDL giving a Mn of 24,997 g/mol and a PDI of 1.93 in 240 mins, compared with a Mn of 8,060 g/mol and PDI of 2.17 using lipase and traditional heating. A novel monomer, ambrettolide epoxide, a 17-membered functional macrolide, was polymerised by a CALB catalyst [117]. Among the 24-membered lactones, one derived from natural sophorolipid was polymerised via lipase-catalysed ROP to form a glycolipid-based polyester [118]. Namekawa and co-workers [119] studied the ROP of lactones (ɛ-CL, 8-OL, UDL, DDL and PDL) in water. Among the various lipases used, the best results (Mn = 1,200 and 1,300 at 60 °C) were obtained with PCL and PFL, respectively. Chemoselective ROP of the lactone, 2-methylene-4-oxa-DDL, was carried out using CAL, which yielded a polyester containing the reactive exomethylene group in the main chain [120]. According to the proposed mechanism for the enzyme-catalysed polymerisation of lactones, the hydroxyl group of the serine residue in the active site of the lipase opens the lactone ring to form an acyl-enzyme intermediate. A polymer chain initiated by the nucleophilic attack of water, which is probably contained in the enzyme on the acyl carbon of the intermediate to produce ω-hydroxycarboxylic acid, is the shortest propagating species. In the propagation

12 Enzyme catalysis in the synthesis of biodegradable polymers 

 365

stage, the enzyme-activated intermediate is nucleophilically attacked by the terminal hydroxyl group of a propagating polymer to produce an elongated polymer chain with one additional monomer unit (Scheme 12.14). Hence, the mechanism of lipase catalysis is generally accepted and considers the formation of an acylenzyme intermediate as a key step, followed by the initiation and propagation steps [114, 121]. O E

OH +

O

O

E

O

(CH2)5

C EAM

OH

O EAM + H2O

HO

C

(CH2)5

O E

O

C

OH +

E

OH

O (CH2)5

OH + HO

C

(CH2)5

O

n

H

O HO

C

(CH2)5

O

H n+1

E

OH

Scheme 12.14

Copolymerisation is an effective way to obtain materials with improved properties compared with those made by the respective homopolymers. Table 12.6 reports examples of lactones and lipases used in copolymerisation experiments. PCL catalysed the enzymatic copolymerisation of β-PL with e-CL in bulk at 60 °C. Low MW (Mn = 520) polyesters were produced. Ring-opening copolymerisation of another four-membered lactone, benzyl malolactanate (BML), was enhanced, based on yield and MW, through the addition of small amounts of β-PL. BML was polymerised in the presence of 17 mol% β-PL at 60 °C for 24 h. Poly(benzyl malolactnonate-co-propriolactone) (poly(BML-co-PL)) containing 91 mol% BML units was obtained with a MW of 32,100 Da[122]. β-BL has been copolymerised with ɛ-CL, DDL and HDDA [123, 124]. The PPL-mediated ROP of β-BL with HDDA at 45 °C produced a copolymer (yield 17%, Mn = 1,800 Da, PD = 1.11) containing hydroxy and carboxylic acid end groups. In the copolymerisation of (±)-β-BL with PDL, the (S)-isomer was preferentially reacted to give the (S)-enriched optically active copolymer with a 69% enantiomeric excess of β-BL units [124]. PSL (from P. fluorescens) catalysed the copolymerisation of γ-BL with e-CL and formed a copolymer with a low MW (Mn = 2,900 Da) at low conversion (56%) after 20 days [81]. The six-membered lactone, δ-VL has been polymerised with e-CL and PDL [125, 111]. In the copolymerisation of δ-VL with ɛ-CL in an equimolar ratio, using PFL at 60 °C for

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 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

10 days, a copolymer with a MW of 3,700 Da was obtained and found to be a random copolymer structure. The MW of the copolymer from PDL and δ-VL was lower (1,900 Da) when compared with the copolymer of δ-VL with e-CL obtained under the same reaction conditions. The lipase-catalysed copolymerisation of the nine-membered lactone, 8-OL with ɛ-CL and DDL produced random copolymers [103]. In the CAL-catalysed copolymTable 12.6: Lipase-catalysed ring opening copolymerisation of lactones.

References

Lactone 1

Lactone 2

Enzyme

1

β-PL

BML

C. cylindracea lipase

2

β-PL

ɛ-CL

P. cepacia lipase

3

β-BL

ɛ-CL

C. antarctica lipase

[121]

4

β-PL

DDL

C. antarctica lipase

[121]

5

β-PL

HDDA

Pancreatic lipase

[122]

6

γ-BL

ɛ-CL

Pseudomonas sp. lipase

7

δ-VL

ɛ-CL

P. fluorescens lipase

[123]

8

δ-VL

PDL

P. fluorescens lipase

[109]

9

ɛ-CL

δ-CL

C. antarctica lipase

[121]

10

ɛ-CL

8-OL

C. antarctica and P. cepacia lipase

[101]

11

ɛ-CL

PDL

C. antarctica lipase B, P. cepacia and P. fluorescens lipase

12

δ-CL

UDL

C. antarctica lipase

[121]

13

δ-CL

DDL

C. antarctica lipase

[121]

14

δ-CL

PDL

C. antarctica lipase

[121]

15

UDL

PDL

P. fluorescens lipase

[109]

16

DDL

8-OL

CAL

[101]

17

DDL

PDL

PFL

[109]

18

DO

PDL

C. antarctica lipase B

[134]

19

DES + 1,4-BDO

PDL

C. antarctica lipase B

[135]

20

DEC+ 1,4-BDO

PDL

C. antarctica lipase B

[136]

DO: p-Dioxanone DES: Diethyl succinate DEC: Diethyl carbonate 1,4-BDO: 1,4-Butanediol

[120] [66]

[79]

[109, 110]

12 Enzyme catalysis in the synthesis of biodegradable polymers 

 367

erisation, 8-OL showed less reactivity than ɛ-CL, whereas the opposite effect was observed when PCL was used. In the copolymerisation of 8-OL with ɛ-CL and DDL using a lipase CA catalyst, at 60 °C for 48 h with a 50:50% feed ratio, the Mn of the copolymers were 5.4 × 103 and 8.6 × 103 Da, respectively. Kobayashi and co-workers explored the polymerisation of PDL with UDL and DDL in bulk at 60 °C for 240 h using PFL or PCL [111]; copolymers with a Mn in the range of 2.0−2.1 × 103 Da were produced. In the Novozyme-435-catalysed copolymerisation of ɛ-CL and ω-PDL, at 70 °C for 45 min in toluene (toluene to PDL 2:1 v/w), a copolymer with a yield of 88% and Mn of 2,000 Da was formed. Studies on monomer (ɛ-CL and ω-PDL) reactivity showed that ω-PDL reacted 13 times faster than ɛ-CL and the copolymer produced had a random sequence of repeat units [97]. In the Pseudomonas sp. (PSL)-catalysed copolymerisation of ɛ-CL with hydroxyesters (ethyl lactate, ethyl 4-hydroxybutyrate and ethyl 15-hydroxypentadecanoate) low MW copolymers were produced [81]. In the CAL copolymerisation of racemic δ-CL with achiral ɛ-CL, UDL, DDL and PDL at 60 °C for 4 h, R-isomer enriched copolymers with Mn of 2,000, 5,900, 7,000 and 6,100 Da, respectively, were produced [123]. Uyama and co-workers [126, 127] utilised PFL in the single-step ROP of DDL and acylation of hydroxy termini with different vinyl esters to produce polymers containing polymerisable groups only at one terminus of the polymer chain. Kobayashi and co- workers [128] have reported that the lipase-catalysed (CAL and PCL) polymerisation of lactones (12-, 13- and 16-membered), divinyl esters of adipic and sebacic acid and α,ω-glycols using one-pot synthesis produced the corresponding ester copolymers in which two different types of polymerisation, ROP and polycondensation as well as transesterification, simultaneously occurred via the same enzyme intermediate to provide random copolymers. Polymerisation of macrolides (DDL and PDL) in the presence of preformed polyester (polycaprolactone) produced the corresponding copolyesters [129]. Cordova and co-workers [130] prepared macromonomers using CALB as the catalyst. The ROP of ɛ-CL was initiated by alcohols which included 9-decenol, cinnamyl alcohol, 2-(4-hydroxyphenyl)ethanol and 2-(3-hydroxyphenyl)ethanol. In another approach, acids and esters which included n-decanoic acid, octadecanoic acid, oleic acid, linoleic acid, 2-(3-hydroxyphenyl)acetic acid, 2-(4-hydroxyphenyl) acetic acid and 3-(4-hydroxyphenyl)propanoic acid were added to the prepolymerised ɛ-CL; consequently, acid-terminated PCL was formed. In the first approach, 9-decenol- initiated PCL was formed (24 h, 99% conversion of ɛ-CL) with an average MW of 1,980 Da. In the second approach, linoleic acid-terminated PCL was formed with an average MW of 2,400 Da (51 h, 99% conversion). In an effort to simultaneously control both the hydroxyl and carboxyl end groups of macromers and esters, e.g., 9-decenyl oleate, 2-(4-hydroxyphenyl)ethyl acrylate, 9-decenyl-2-(4-hydroxyphenyl)ethyl) acetate, methyl linoleate or a sequence of an alcohol and an acid, were added at various times

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 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

during the course of the CALB-catalysed CL polymerisation. Polyesters bearing hydrophilic sugar monomers at the polymer termini were synthesised by the CAL-catalysed polymerisation of ɛ-CL with sugar initiators [131, 132]. The enzyme selectively used the -OH group at the 6-position of the sugar to open the ring, thus no protection or deprotection for the other free hydroxyl groups was required. Similarly, the PPL and CLONEZYME ESL-001-catalysed graft polymerisation of ɛ-CL on hydroxyethylcellulose was used to obtain cellulose-graft-poly(ɛ-CL) with a degree of substitution from 0.10 to 0.32 [133]. Cordova and co-workers [134, 135] reported the selective synthesis of a poly(ɛ-CL) monosubstituted dendrimer using a hexahydroxy functional dendrimer. CALB was employed as the catalyst for the ring-opening copolymerisation of ω-PDL with p-dioxanone (DO) under mild reaction conditions (30,000 Da) and a wide range of comonomer content was synthesised using various PDL/DO feed ratios. The copolymers were typically isolated in 50−90 wt% yields as the monomer conversion was limited by the equilibrium between the monomers and copolymers [136]. The successful copolymerisation of dialkyl diester with diol and lactone to form aliphatic polyesters has been achieved using CALB lipase and a two-stage process: the first stage involves oligomerisation under low vacuum and is followed by a second polymerisation stage under high vacuum. Terpolymers of PDL, DES and 1,4-BDO with a MW of whole product (nonfractionated) up to 77,000 Da and MW/Mn between 1.7 and 4.0 were synthesised in high yields (95% isolated yield) at 95 °C [137]. More recently, two-stage polymerisation reactions were also applied to the copolymerisation of ω-PDL, diethyl carbonate (DEC) and 1,4-BDO. PDL-DEC-1,4-BDO copolymers were obtained with a MW of whole product (nonfractionated) up to 33,000 Da and MW/Mn between 1.2−2.3. The best reaction temperature for copolymerisation was found to be ≅80 °C. The synthesised PDL-DEC-1,4-BDO copolymers possessed near random structures with all possible combinations of PDL, carbonate and butylene units via either ester or carbonate linkages in the polymer chains [138]. Lipase-catalysed ROP is usually carried out in bulk or in organic solvents like heptane, toluene, diisopropyl ether and 1,4-dioxane. Due to the growing awareness for environmental protection, researchers are paying increasing attention to ‘green solvents’, working with water, scCO2 and ionic liquids. Good results were obtained with the PC lipase polymerisation of macrolides UDL, DDL and PDL in water [83, 139]. Also, polyPDL nanoparticles were obtained in miniemulsions of PDL monomer, water and exadecane [140]. scCO2 was used for the lipase CA-catalysed ROP of ɛ-CL to produce high yields of poly(ɛ-CL) with a MW of 1.1 × 104 Da, and for the ring-opening copolymerisation between ɛ-CL and DDL to produce poly(e-caprolactone-co-dodecanolide) (poly(ɛ-CL-co-DDL)) with a MW of 1.3 × 104 Da[141]. Further work demonstrated that the enzymatic route (CALB supported on macroporous beads) is viable in scCO2, yielding PCL of Mn 12,000−37,000 g/mol with MW

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 369

very similar to those obtained from the same enzymatic catalysts in organic solvents, but with lower polydispersities (typical PDI = 1.4−1.6) and higher polymer yields (typically 95−98%) [142]. On the contrary, in 2012, the structure of enzyme cp283∆7, a new variant of CALB, was simulated in both water and scCO2. Analysis of the root mean square deviation showed that structural variations of the enzyme in scCO2 are more than those occurring in the aqueous solution: therefore, dissolving the enzyme in scCO2 imposes undesirable instability on its structure [143]. Several ionic liquids (nonvolatile, thermally stable and highly polar) have been used for the ROP of ɛ-CL catalysed by different lipases (YLL, Candida rugosa (CR), PPL and CALB), 1-butyl-3-methyl-imidazolium salts, 1-ethyl-3-methyl-imidazolium salts and n-butyl pyridinium salts [33, 34, 144, 145].

12.3 Oxidative polymerisation of phenol and derivatives of phenol Phenol-formaldehyde polymers, including novolaks and resoles, have a number of applications in coatings, finishes, adhesives, composites, laminates and related areas. Concerns have been raised regarding the continued use of phenol-formaldehyde resins due to the various toxic effects of formaldehyde. Consequently, there has been active investigation for alternative sources of these types of oligomers and polymers with a consideration for environmental compatibility. Horseradish peroxidase (HRP) (EC 1.11.1.7) catalyses the covalent coupling of a number of phenols and aromatic amines using hydrogen peroxide (H2O2) as an oxidant; this process has been successfully used for the removal of toxic aromatic pollutants from industrial wastewaters [146–148]. Reactions were not feasible for the production of polyphenols because most phenols are insoluble in water, and the phenolic dimers and trimers formed are insoluble in water and immediately fall out of solution, thereby preventing further polymerisation to high MW polymers. Biswas and co-workers [149] investigated the feasibility of a two-step process for the removal of benzene from buffered synthetic wastewater. In order to remove benzene using an enzyme, a pretreatment via a modified Fenton reaction was employed, with the generation of the corresponding phenolic compounds without causing significant mineralisation. The pretreatment process was then followed by the oxidative polymerisation of the phenolic compounds, which was catalysed by a laccase from Trametes villosa. Under optimum Fenton reaction conditions, 80% conversion of the initial benzene concentration was achieved, giving a mixture containing an oxidative dimerisation product (biphenyl) and hydroxylation products. Ferreira and co-workers [150] analysed the potential use of white-rot fungi and hematin for phenol and aniline polymerisation, as a low-cost alternative to HRP. In

370 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

order to eliminate phenols by precipitation from aqueous solution, Pleurotus sajorcaju derived enzymes, in the absence of H2O2, produced insoluble polyphenol with similar efficiencies to those found with HRP or hematin in a one-step phenol treatment (near 40% phenol conversion). Fungal enzymes look promising for eliminating aniline/phenol from wastewaters, since they are able to polymerise and precipitate them from aqueous solutions. Polyphenols and their copolymers have been prepared from a series of phenol monomers [151–166] via several pathways by varying the polymerisation parameters, enzyme origin, buffer pH, mixed ratio of alcohol and buffer. The purity and amount of HRP, together with the concentration and addition rate of H2O2, strongly affect the MW and solubility of the polymers. The physical and chemical properties of these polymers such as Tm, solubility, elemental analysis, MW distribution, IR absorption, solid-state 13C-NMR, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were determined. The polymer structure was found to contain a mixture of phenylene and oxyphenylene units. The number of oxyphenylene units increased with increasing methanol content, varying in the range of 32−59%. The more oxyphenylene units present, the better the solubility of the polyphenols. Recently, the enzymatic polymerisation of phenol catalysed by HRP has been carried out in an aqueous micelle system using sodium dodecyl benzene sulfonate (SDBS) as a surfactant [167]: the addition of SDBS as a buffer greatly enhanced the polymer yield, and using SDBS over 0.4 g for 5 mmol of phenol monomer enabled the polymer to be obtained almost quantitatively. In an aqueous micelle system, the phenol polymerisation maintained high yields over a wide pH range (from 4 to 10), and the activity of the enzyme in the buffer was so high that the phenol polymerisation could be completed in only 2 h with a high yield. The resulting polymer was a kind of powdery material, which is partly soluble in N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and tetrahydrofuran (THF). IR analysis showed that the polymer structure contained a mixture of phenylene and oxyphenylene units. From TGA, the polymer was found to possess high thermal stability. Another enzymatic oxidative polymerisation of phenols was investigated by Kobayashi and co-workers [168] in the presence of the amphiphilic triblock copolymer Pluronic® in water. The PEG content of Pluronic greatly affected the polymerisation behaviour: the use of Pluronic with high PEG content improved the regioselectivity of the polymerisation of phenol, giving a polymer mainly consisting of a phenylene unit. Polymerisation in the presence of Pluronic F68 (EG76–PG29–EG76) produced a phenolic polymer with an ultrahigh MW (MW >106 Da). Soybean peroxidase (SBP) was used by Dordick and co-workers [169] to catalyse the polymerisation of phenols in room-temperature ionic liquids (RTIL). Phenolic polymers with an average MW number ranging from 1,200−4,100 Da were obtained depending on the composition of the reaction medium and on the nature of the phenol. Gel permeation chromatography (GPC) and matrix assisted laser desorption/ ionisation – time of flight (MALDI-TOF) mass spectrometry analysis indicated

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 371

that higher MW polymers can be synthesised in the presence of higher RTIL concentrations, with selective control over polymer size achieved by varying the RTIL concentration. The resulting polyphenols showed high thermostability and possessed thermosetting properties. A new approach to the enzymatic polymerisation of phenol was described by Zhu and co-workers [170] using a liquid-solid circulating fluidised bed (LSCFB) system, thus opening the possibilities for many bioprocesses where deactivation of the biocatalysts is a major problem and regeneration of the biocatalysts is required. About 8.5 kg of immobilised particles containing soybean seed hull peroxidase enzymes were applied in the LSCFB system. The continuous enzymatic polymerisation was carried out in the riser section by introducing phenol and H2O2 at the entrance of the riser. Under optimised hydrodynamic conditions and by keeping the molar concentration ratio of phenol to H2O2 at 1:2, 54% conversion of phenol was achieved. The downcomer pipe of the LSCFB was used for the regeneration of coated immobilised enzyme particles. The enzymatic polymerisation of phenol catalysed by HRP was efficiently performed in phosphate buffer (pH = 7.0) containing sodium dodecyl sulfate SDS, an environment- friendly system [171]. The obtained phenol polymer is partly soluble in common solvents, such as acetone, THF and DMF. IR analysis shows that the polymer is composed of phenylene and oxyphenylene units. The functionalisation of the phenol polymer was performed by reacting with epoxy chloropropane and triethylene- tetramine, the insoluble aminated phenol polymer was then obtained. The aminated phenol polymer was adopted as a carrier to prepare a novel supported palladium catalyst (PP-N-Pd) for the Heck reaction. A novel synthetic method for polyphenols was described by Inoue and co-workers [172]: m-cresol, 3-methylcatechol, 4-methylcatechol and 4-ethylcatechol were polymerised using a redox reaction between phenol derivatives and gold ions. The oxidised phenol derivatives produced by the reduction of gold ions formed water- insoluble polymers with MW of approximately 1,000 g/mol. Alternatively, Wei and co-workers [173] described the electrochemical polymerisation of phenol on 304 stainless steel anodes and the subsequent coating structure analysis. Anodic oxidation was carried out using 304 stainless steel anodes in a neutral 0.1 mol/l phenol solution with an electrolyte composed of 0.1 mol/l sodium sulfate. This oxidation generated a yellow brown polyphenol coating on the steel surface of the anode. In a monophasic solvent system containing 85% dioxane and 15% water, a polymer with a MW as high as 4 × 105 Da was produced by the polymerisation of p-phenylphenol. To carry out the reaction in reverse micelles (organised surfactant structures that form hollow spheres usually on the nanometre length scale in an organic solvent, the continuous phase, with an aqueous core within the sphere) the reaction conditions included the surfactant bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and HRP enzyme. The concentrations of reactants were adjusted to generate a reverse micellar

372 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

solution with a water to AOT molar ratio (Wo) of 15 and a final enzyme concentration of 12.5 µM. The monomer was then introduced and reactions initiated by the addition of 0.2 M of 30% H2O2. p-Ethylphenol polymerisation was extensively studied in reverse micelles to examine the feasibility of the reaction in terms of kinetics, monomer conversion and the morphology of the particles generated. In a comparative study of two systems (Table 12.7), 1) the monophasic organic solvent systems of dioxane plus water and 2) the reverse micellar system, the distinction in polymerisation lies in the oligomer-to-polymer ratio (soluble-to-insoluble product ratio). This difference could originate because the two systems have different solvating abilities and hence may sustain the growing chain in solution to differing extents. Oligomer products are more predominant in the dioxane/water system than in the reversed micellar system and the reactions are faster in the reverse micellar system compared with monophasic organic solvents. Polymers generated in reverse micellar systems had narrower MW distributions. In reverse micellar environments, the precipitated polymer particles acquired a spherical morphology (Figure 12.1a), while the polymer synthesised in bulk solvent systems of dioxane (85%) and water (15%) did not show this characteristic morphology (Figure 12.1b). Templating effects during polymerisation in reverse micellar environments could be the reason for the generation of spherical morphology particles. Table 12.7: Quantitative aspects of p-ethylphenol synthesised in various media. Reaction medium

Reverse micelles Wo = 15, (AOT) = 0.15 M

Monomer Polymer yield, Comments conversion polymer produced/ (after 2 hours) monomer converted ≈95%

≈95%

Monomer soluble, high enzyme dispersion (solution clear), minimal oligomer formation and polymer precipitates

Isooctane

< 5%

95−100%

Dioxane (85%) in water

95%

≈20%

Monomer soluble, fairly high enzyme dispersion (enzyme in suspension, cloudy solution), significant oligomer formation and polymer precipitates

Water

35%

≈55%

Poor monomer solubility, high enzyme dispersion (clearly soluble), some oligomer formation and polymer precipitates

Monomer soluble, poor enzyme dispersion (in insoluble aggregates), little oligomer formation and polymer at the air/isooctane interface

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 373

The morphology of the polymer is affected by the phase composition and a 3:1 surfactant (AOT) to monomer ratio has to be maintained for the generation of spherical morphology. Observations of interest from the micrographs are: comparison of Figure 12.1c and 12.1d indicates that increasing AOT at a constant p-ethylphenol concentration results in a small decrease in particle size. Secondly, a comparison of Figure 12.1d and 12.1a indicates an increase in average particle size when the monomer concentration was increased at a constant surfactant concentration. A further increase in monomer concentration results in a crossover from spherical to nonspherical morphology (Figure 12.1e and 12.1f). The spherical particles were soluble in benzene, THF, DMF and dimethylsulfoxide. MW measurements of polyethylphenol using GPC indicated a broad distribution typically centred at about 90 kDa. The efficiency of polymer synthesis decreased dramatically if the alkyl group on the monomer was longer than 3−4 units. Poly(p-butylphenol) synthesis was less efficient than poly(p-ethylphenol) synthesis, with only about 40% monomer conversion and negligible precipitation of the polymer. No detectable conversion was observed with p-octylphenol, p-nonylphenol and p-dodecylphenol in reverse micelles. In the polymerisation of alkylphenols in aqueous organic solvents [174−177], the position and chain length of the alkyl substituent, as well as the solvent type, significantly affected the polymerisation. In the polymerisation of unbranched p-alkylphenols, the yield of polymer increased with an increase of chain length of the alkyl group from 1 to 5 carbons, and the yield of polymer obtained from heptylphenol was almost the same as that from the pentyl derivative. The yield of the polymer from p-isopropylphenol was higher than that from the unbranched analogue at the p-position. No polymerisation was observed in the polymerisation of o- and m-isopropylphenols. In the case of the polymerisation of unbranched alkylphenols in aqueous 1,4-dioxane, polymer yield increased with an increase of the chain length of the alkyl group from 1−5 carbons, and the yield of the polymer from hexyl or heptylphenol was almost the same as that of the pentyl derivative. In the reverse micellar system, the highest yield was obtained from ethylphenol. The polymerisation of hexylphenol in the reverse micellar system produced no polymeric materials; on the other hand, the polymer was obtained in high yield in aqueous 1,4-dioxane. A possible explanation for the contrary results in reverse micelles is as follows; the enzyme is soluble in water and always present inside the reverse micelles. The phenol with the shorter alkyl chain length is relatively hydrophilic in nature and prefers to stay inside the micelle leading to an increased polymerisation rate. On the other hand, phenols with a longer alkyl chain are hydrophobic and prefer to be inside the nonpolar isooctane solution leading to a poor yield. The polymerisation behaviour of the m-substituted monomers depends greatly on the enzyme. HRP readily polymerised monomers with small substituents, whereas for monomers with large substituents, a high yield was achieved using SBP as the catalyst. The enzymatic oxidative polymerisation of p-alkylphenols by HRP gave a mixture of polyphenols containing phenylene and oxyphenylene units, as determined by NMR and IR as well as titration of the residual phenolic moiety of the polymer.

374 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

a

b

0061

c

15 kV

X5,000

1µm

HD15

d

0151

15 kV

X10,000

1µm

HD15

e

011

15 kV

X10,000

1µm

HD15

X5,000

1µm

HD15

f

0217

15 kV

X13,000

1µm

HD15

0217

15 kV

Figure 12.1: Scanning electron micrographs of polymer formed by enzymatic synthesis in different synthetic conditions. a) AOT 1.5 M, p-ethylphenol 0.3 M, b) Monophasic organic solvent system of 85% dioxane and 15% water (by volume), c) AOT 0.5 M, p-ethylphenol 0.15 M, d) AOT 1.5 M, p-ethylphenol 0.15 M, e) AOT 0.5 M, p-ethylphenol 0.3 M, and f) AOT 1.5 M, p-ethylphenol 1.5 M.

The enzymatic oxidative polymerisation of methoxyphenols by a fungal laccase was investigated to synthesise polymethoxyphenols, which would be environmentally benign materials since they are degraded by lignin-degrading fungi [178]. An industrially produced thermostable laccase from Trametes sp., Ha1, polymerised o-methoxyphenol and 2,6-dimethoxyphenol in McIlvaine buffer (pH 4.5) both with

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and without organic solvents, while the enzyme needed organic solvents to polymerise phenol, alkylphenols, and m- and p-methoxyphenols. The poly(o-methoxyphenol) synthesised was soluble in DMF while 80% of the poly(2,6-dimethoxyphenol) was insoluble in DMF. The weight-average MW of the poly(o-methoxyphenol) was 7,000−11,000 Da. o-methoxyphenol and 2,6-dimethoxyphenol would be suitable for the production of environmentally benign phenolic resins using enzymatic oxidative polymerisation, performed by laccase without formaldehyde, H2O2 or an organic solvent. Lignin-related polymers were synthesised. Coniferyl alcohol (4-hydroxy-3- methoxycinnamyl alcohol, CoA), a phenolic lignin monomer (monolignol) contained in plant cell walls, was polymerised in the presence of α-cyclodextrin (α-CD) in a HRP/ H2O2 system. The presence of α-CD led to the product polymer containing 8-O-4′-richer linkages, compared with the no-additive case. This is probably due to the inclusion complex formation between CoA and α-CD, which suppresses other linkages such as 8-5′ and 8-8′, due to the steric hindrance of the complex [179]. A lignin-based macromonomer, lignocatechol, was prepared from wood components and oxidatively polymerised by a laccase catalyst to produce crosslinked polymers with a good yield. The laccase-catalysed copolymerisation of lignophenol with urushiol was also performed to afford the corresponding copolymers with a high yield. The thermal properties were measured using DSC, TGA and thermomechanical (TMA) analyses, indicating that the polymers had high thermal stabilities because of the crosslinked structures [180]. Peroxidases (from horseradish and soybean) and laccases (derived from the fungi Pycnoporus coccineous and Myceliophthore) catalysed the polymerisation of 4-hydroxybenzoic acid derivatives (3,5-dimethoxy-4-hydroxy benzoic acid (syringic acid) and 3,5-dimethyl-4-hydroxy benzoic acid) in a mixture of a water- miscible organic solvent and acetate buffer at room temperature under air to give a good yield of poly(1,4-phenylene oxide) (PPO) (Scheme 12.15) [181–182]; both enzyme type and solvent composition greatly affected the polymerisation results. When laccases (EC 1.10.3.2) from P. coccineous and Myceliophthore were used as catalysts no polymerisation of syringic acid occurred when either pure acetone or the buffer were used. The highest yield (84%) was obtained in 50% acetone with acetate buffer (pH = 5), while the highest MW (7.7 × 103 Da) was obtained when 40% acetone was used. The enzymatic oxidative polymerisation of 4-hydroxybenzoic acid derivatives involves the elimination of CO2 and H2 from the monomer to form PPO. No polymerisation was observed when unsubstituted 4-hydroxybenzoic acid was used as a substrate for peroxidases and laccases under similar experimental conditions. R HOOC

OH

Peroxidase

R R=

O

R O R

CH3 ,

CH3

n Scheme 12.15

376 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

NMR, IR and MALDI-TOF results showed that the polymers were composed exclusively of 1,4-oxyphenylene units with a phenolic hydroxyl group at one chain terminus and a benzoic acid group at the other chain end. PPO was first prepared by the oxidative coupling polymerisation of 2,6-dimethylphenol using a copper amine catalyst [183]. PPO is synthesised commercially using this process, which involves side reactions resulting in the incorporation of a Mannich-base and 3,5,3′,5′-tetramethyl- 4,4′-diphenoquinone units into the polymer. The enzymatic polymerisation of 2,6-dimethylphenol using HRP, SBP and laccase derived from Pycnoporus coccineous, in an aqueous organic solvent at room temperature, produced polymers with MW in the range of several thousand Daltons. The polymerisation behaviour was dependent on the enzyme type as well as solvent composition. The resulting polymer was exclusively composed of dimethyl-1,4-oxyphenylene units according to NMR and MALDI-TOF determinations [184]. The enzymatic oxidative polymerisation of an azobenzene derivate 4-[(4phenylazo-phenyimino)-methyl]-phenol, P-(4-PPMP), using HRP as the catalyst in the presence of an oxidising agent, H2O2, was carried out in various solvents (acetone, methanol, ethanol, DMF and 1,4-dioxane) and phosphate buffers (pH 6, 6.8, 7 and 7.2) at room temperature [185]. Studies have shown that a black polymer having a Tm of 290 °C was successfully produced in good yields using aqueous 1,4-dioxane as the solvent at pH 6. P-(4-PPMP) showed good solubility in 1,4-dioxane, DMF and DMSO but it was only sparingly soluble in chloroform, THF, methanol and ethanol, whilst it was insoluble in diethyl ether. The characterisation of P-(4-PPMP) was carried out via ultraviolet-visible spectroscopy, FTIR, 1H-NMR, elemental analysis and SEC measurements. Mn, MW and DP of the polymer were determined to be 7,970.4 g/ mol, 8,146.2 g/ mol and 1.02, respectively. FTIR and 1H-NMR studies confirmed the presence of phenylene and oxyphenylene units within the polymer backbone. 4,4′-Biphenyldiol (HO-C6H4–C6H4-OH) [186], bisphenol-A (2,2-bis(4-hydroxyphenyl) propane) (HO-C6H4-C(CH3)2-C6H4-OH) [187] and 4,4 -̕ dihydroxydiphenyl ether (HO-C6H4O-C6H4-OH) [188] have been polymerised using HRP in aqueous organic solvents. Dordick and Wang used CAL to regioselectively acylate thymidine at the 5 -̕ hydroxyl position using a trifluoroethylester derivative of p-hydroxyphenylacetic acid in anhydrous acetonitrile [189]. This was followed by the polymerisation of the phenolic nucleoside derivative, catalysed by the peroxidase from soybean hulls in the presence of H2O2 and an aqueous buffer containing 60% (v/v) acetonitrile (Scheme 12.16). Bisphenol-A was polymerised by Coprinus cinereus peroxidase in an aqueous 2-propanol solution. Various polymerised products with different MW and hydroxyl values were synthesised depending on the reaction composition (the ratio of aqueous buffer to 2-propanol) [190]. Huang and co-workers [191] investigated the effects of a nonionic surfactant, Triton X-100, on the laccase-catalysed conversion of bisphenol A. It was found that the addition of Triton X-100 into the reaction system increased the conversion of bisphenol A (BPA), especially near the critical micelle concentration of Triton X-100. In addi-

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 377

tion, it was found that the stability of laccase was greatly improved in the presence of Triton X-100 and the binding of Triton X-100 to the laccase surface also mitigated the inactivation effect caused by the free radicals and polymerisation products. Under otherwise identical conditions, a lower dosage of laccase was needed for the higher conversion of BPA in the presence of Triton X-100. Redox-active polymers are useful in applications such as batteries, sensors, electrical conductors and antioxidants [192]. Dordick and co-workers synthesised polyhydroquinone using the enzymatic oxidative polymerisation of glucose-β-D- hydroquinone and subsequent acid hydrolysis [193]. In the first step, bovine liver β-glucuronidase (EC 3.2.1.31) was used to region specifically attach glycoside to one of the hydroxyl groups of hydroquinone in an aqueous solution to give glucose-β-D- hydroquinone (arbutin). In the second step, arbutin was polymerised by peroxidases from horseradish and soybean in an aqueous buffer to form water-soluble polymers (Scheme 12.17). Deglycosylation of the polyarbutin gave poly(1,4-dihydroxy-2,6-phenylene). This polymer was different from the electrochemically synthesised polyhydroquinone, which is poly(1,4-dihydroxy-2,5-phenylene). Kobayashi and co-workers [194] synthesised a new kind of polyhydroquinone derivative with a mixture of phenylene and oxyphenylene units using peroxidases (horseradish and soybean) to catalyse the polymerisation of 4-hydroxyphenyl benzoate and the subsequent hydrolysis of the resulting polymer (Scheme 12.18). Similarly, Tripathy and co-workers [195] synthesised a photoactive azopolymer, poly(4-phenylazophenol), via HRP- catalysed polymerisation in acetone and sodium phosphate buffer; bilirubin oxidase (EC 1.3.3.5) was shown to catalyse the regioselective polymerisation of 1,5-dihydroxynaphthalene to a polymer in a mixed solvent composed of dioxane, ethylacetate and acetate buffer [196]. Chalcones are intermediates in the biosynthesis of lignins in plants: oligomers were produced by the HRP-mediated polymerisation of aminochalcones in a mixture of 1,4-dioxane and phosphate buffer (Scheme 12.19) [197]. Some recyclable acyclic SO3H-functionalised ionic liquids have been used as catalysts for the synthesis of chalcones via Claisen–Schmidt condensation [198]. The chalcones could simply be separated from the catalyst by decantation. After removing water from the reaction mixture, the catalysts could be recycled and reused several times without a noticeable decrease in the catalytic activity. Recently, 2,4,5-trimetohoxy chalcones and analogues were synthesised from asaronaldehyde and their anticancer activity was tested [199]. Acetaminophen is a widely used analgesic and antipyretic drug. The HRPmediated polymerisation of acetaminophen was carried out in phosphate buffer at 25 °C [200− 201]. Using NMR spectroscopy it was shown that the reaction mixture was composed of two dimers, three trimers and one tetramer. Oligomer formation was due to the formation of covalent bonds between carbons ortho to the hydroxyl group and to a lesser extent, between the carbons ortho to the hydroxyl group and the amido group of another acetaminophen molecule.

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 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore O

CH2 — COOH CF3CH2OH

CH2 — COOCH2 — CF3 Thymidine

O

O

O

N

CAL/CH3CN

DCC/THF OH

OH

HN

O

OH H

O

H2O2 CH3CN/H2O Soybean peroxidase

HN

O O

OH

O

O

N

OH H

Scheme 12.16 OH

OH n

OH

Peroxidase

O

O HO OH

H2O2 OH

OH

HCl/H2O

O

O HO

OH

OH

n OH

OH

Scheme 12.17

Cardanol is an analogue of phenol with a 15-carbon unsaturated chain containing zero to three double bonds in the meta-position and is the main constituent obtained after the thermal treatment of cashew nut shell liquid. Cardanol was polymerised with SBP in a mixture of acetone:buffer (75:25), to form an oily polymer. 1H-NMR and FTIR analyses indicated that double bonds in the side chain were not affected by polymerisation conditions and polycardanol was a mixture of phenylene and oxyphenylene units (Scheme 12.20) [202]. Epoxide-containing polycardanol was enzymatically synthesised via two routes by two different enzymes, lipase and peroxidase [203]. One route was the synthesis of epoxide-containing cardanol from

12 Enzyme catalysis in the synthesis of biodegradable polymers 

OH

OH

O

O

O

Peroxidase O O

O

O OH

OH

O

OH

OH n

Scheme 12.18 HN

NH2 HRP, H2O2 Phosphate buffer

n R

O R=

R

O C 6 H 5,

C6H4

O

C2H5

Scheme 12.19 R

R

R

Peroxidase OH

OH R = = = = Scheme 12.20

O

n

n

 379

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 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

cardanol, H2O2 and an organic acid in the presence of lipase, followed by the polymerisation of the phenolic functional groups of cardanol using peroxidase. In the other route, polymerised cardanol was prepared from cardanol and H2O2 in the presence of peroxidase and, subsequently, the epoxide-containing polycardanol was synthesised from polycardanol, H2O2 and an organic acid in the presence of lipase. NMR and IR spectroscopy confirmed the polymer structure; the former route yielded epoxide-containing polycardanol in a higher yield of over 90%. Urushi is a Japanese traditional natural paint. The main component of urishi is urishiol (Scheme 12.21), whose structure is a catechol derivative with unsaturated hydrocarbon chains consisting of a mixture of monoenes, dienes and trienes at the meta or para position of catechol. Kobayashi and co-workers [204] have carried out laccase-catalysed crosslinking reactions with urishiolanalogues to prepare urishi. The HRP-catalysed polymerisation of m-ethynylphenol (HO-C6H4-C≡CH), containing more than one polymerisable group, showed that the phenol moiety was chemoselectively polymerised when an acetylene or methacryl group were present [205]. OH OH R R= (CH2)14CH3 or R= (CH2)7CH=CH(CH2)5CH3 or R= (CH2)7CH=CHCH2CH=CH(CH2)2 CH3 or R= (CH2)7CH=CHCH2CH=CHCH=CHCH3 or R= (CH2)7CH=CHCH2CH=CHCH2CH=CH2 and others Scheme 12.21

12.4 Enzymatic polymerisation of polysaccharides Cellulose is the most abundant compound produced photochemically by plants on Earth: it’s estimated that about 20 billion tonnes of cellulose is photosynthesised in nature every year. There are two types of high-ordered molecular structure in cellulose: cellulose I is the native form of cellulose, with parallel glucan chains and a thermodynamically metastable form, produced by living organisms, while cellulose II, has an antiparallel glucan chain and is the more stable form. Kobayashi and co-workers [206−219] synthesised cellulose, chitin, xylan and nonnatural derivatives of these polymers. Cellulose was produced in vitro by polycondensation reactions using β-cellobiose fluoride (β-CF) and cellulase (EC 3.2.1.4) (Scheme 12.22). The reaction proceeded with complete regio- and stereoselectivity, giving rise to cellulose containing β(1→4) linkages. The polymerisation reactions gen-

12 Enzyme catalysis in the synthesis of biodegradable polymers  OH HO HO

O

OH

HO O

OH

F

O

Cellulase CH3CN/ acetate buffer

OH OH O HO

 381

O OH

HO O

OH O OH

n

Scheme 12.22

erated cellulose with a DP of 22, and 54% yield, when carried out in an acetonitrile/ acetate mixed solvent buffer (5:1, v/v) at 30 °C with 5% w/w of cellulase. Kobayashi and co-workers [218] reported that cellulose I and cellulose II can be selectively synthesised in vitro using the enzymatic polymerisation of the β-cellobiosyl fluoride monomer, and this selectivity could be controlled by changing the enzyme purity and polymerisation conditions. Cellulase from Trichoderma viride was the most effective enzyme for the synthesis of cellulose when compared with cellulases from ANL or Polyporus tulpiferae. Novel mutant enzymes of endoglucanase II (EGII) from the fungus Trichoderma viride were prepared, and their hydrolysis and enzymatic polymerisation activities were investigated [220]. EGIIcore2 and EGIIcore2-His, which possess two sequential active sites of EGII with a His-tag probe at the N-terminal and with His-tag probes at the N and C terminals, respectively, catalysed the polymerisation of β-CF to form synthetic cellulose with a β(1→4) glycosidic linkage. They showed higher hydrolysis activities and polymerisation rates than EGIIcore with a single active site, even in comparison with the active-site concentration basis. The polymerisation products were identified as highly crystalline cellulose of type II. The enzymatic polymerisation on gold by immobilised genetically engineered EGII from Trichoderma viride was described by Kimura and co-workers [221]. They analysed the polymerisation behaviour and the produced cellulose in comparison with the results obtained by the use of free enzymes: the polymerisation catalysed by both immobilised EGIIcore2 and EGIIcore2-His EGIIcore2H produced highly crystalline cellulose in comparison with the free enzyme. Samejima and co-workers [222] achieved the in vitro enzymatic synthesis of cellulose II with high crystallinity from glucose and alpha-glucose 1-phosphate (alphaG1P) using cellodextrin phosphorylase (CDP). Although glucose had been considered not to act as a glucosyl acceptor of CDP, a significant amount of insoluble cellulose was precipitated when glucose was mixed with alphaG1P and CDP without an accumulation of the soluble cello oligosaccharides. 1H-NMR spectrometric analysis revealed that this insoluble cellulose had an average DP of 9.

382 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore

The direct and efficient enzymatic synthesis of long-chain cellulose was successfully achieved using a disaccharide monomer of cellobiose and a cellulase/surfactant complex as the catalyst [223]. The cellulase/surfactant complex consisted of a mixture of cellulase and a specific nonionic surfactant of dioleyl-N-D-glucono-L-glutamate. In the nonaqueous medium of dimethylacetamide (DMAc)/LiCl, a cellulose- solubilising solvent, polymerisation took place at 37 °C to form synthetic cellulose as a white powder. The DP value was high (over 100) and the product yield was low (up to 5%). The dehydration polycondensation reaction led to controlled regioselectivity and stereochemistry, involving an extensive transglycosylation of the product with a statistically significant even- and odd-numbered chain length. Another approach for the synthesis of cellulose was recently reported, extending the above method: the combination of a surfactant-enveloped enzyme (SEE) and a protic acid in an aprotic organic solvent, lithium chloride/N,N-DMAc system [224]. The SEE biocatalyst was prepared by protecting the surface of cellulase with the nonionic surfactant, dioleyl-N-D-glucona-L-glutamate, to maintain its enzymatic activity in a nonaqueous media. FT-IR and NMR analyses elucidated the successful synthesis of cellulose, β-1,4-linked D-glucopyranose polymer, through the reverse hydrolysis of cellobiose. Using protic acid cocatalysts, the synthesised cellulose reached a DP of more than 120, in a ~26% conversion, which was 5 times higher than that obtained in an acid-free SEE system. Usui and co-workers [225] achieved the enzymatic synthesis of a cellulose-like substance by transglycosylation, via a nonbiosynthetic pathway, in an aqueous system of the corresponding substrate, cellotriose, using the cellulolytic enzyme endoglucanase I from Hypocrea jecorina. A significant amount of water-insoluble product precipitated out from the reaction system: MALDI-TOF mass spectrometry analysis showed that the resulting precipitate had a DP up to 16: the solid-state 13 C-NMR spectrum revealed that all carbon resonance lines were assigned to two kinds of anhydroglucose residues in the corresponding structure of cellulose II, while X-ray diffraction measurement as well as 13C-NMR analysis showed that the crystal structure corresponded to cellulose II with a high degree of crystallinity. A new term ‘choroselectivity’ was therefore proposed, which is concerned with the intermolecular relationship in the packing of polymers having directionality in their chains. Various β-cellobiose derivatives (methyl β-cellobioside, allyl β-cellobioside, trifluoroethyl β-cellobioside, methyl β-thiocellobioside, phenyl β-cellobiosyl sulfoxide and 1-O-acetyl β-cellobiose) have been used in enzymatic polymerisations using cellulase as the catalyst. Among all the activated cellobiose substrates, β-cellobiosyl fluoride gave the best result in terms of DP. β-cellotriosyl fluoride (trimer) and β-cellotetreosyl fluoride (tetramer) were found to be rapidly hydrolysed in the enzymatic reactions. Nonnatural 6-O-methylated cellulose was produced with high regioand stereoselectivity by cellulase catalysis starting from 6-O-methyl-β-cellobiosyl fluoride.

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The enzymatic polymerisation of α-D-maltosyl fluoride using α-amylase (EC 3.2.1.1) as the catalyst in a mixed solvent methanol-phosphate buffer (pH 7) produced oligomeric products with α-(1→4)-glycosidic linkages [214]. Other substrates such as D-maltose, β-D-maltosyl fluoride and α-D-glucosyl fluoride gave no condensation products. These results indicated that α-D-maltosyl fluoride with an α-configuration was essential for an α-amylase-catalysed polymerisation. Malto-oligosaccharides are useful substrates as food additives, medicines and enzyme substrates for clinical research. Generally, they are produced by the degradation reaction of polymers such as amylose, amylopectin and glycogen. Xylan is a polysaccharide of xilose with a β(1→4)-glycosidic linkage and it is an important component of hemicellulose in plant cell walls. Xylan was synthesised by a transglycosylation reaction catalysed by cellulase with the use of β-xylobiosyl fluoride as the substrate [207]. Cellulose-xylan hybrid polymers were synthesised by the polycondensation of β-xylopyranosyl-glucipyranosyl fluoride catalysed by xylanase (EC 3.2.1.32) from Trichoderma viride, in a mixed solvent acetonitrile/ acetate buffer (Scheme 12.23) [208]. Chitin is widely found in invertebrates and is one of the most abundant and widespread natural structural polysaccharides normally found in animals, comparable to the predominance of cellulose in plants. Kobayashi and co-workers [210, 211] produced chitin through the chitinase-catalysed (EC 3.2.1.14) polymerisation of a chitobiose oxazoline derivative in a phosphate buffer (Scheme 12.24). The product structure, determined by cross-polarisation magic-angle-spinning and 13C-NMR spectroscopy, reported a (β1-4) linkage indicating region- and stereoselective linkages between the chitobiose units and the inversion of configuration at C1.

HO HO

HO O

O OH

OH F O OH

O HO

Xylanase CH3CN/ acetate buffer

O

HO O

OH

Cellulose-xylan hybrid polysaccharide

OH O OH

n

Scheme 12.23

A cellulose-chitin hybrid polysaccharide with alternating β(1→4)-linked D-glucose (Glc) and N-acetyl-D-glucosamine (GlcNAc) was synthesised via two modes of enzymatic polymerisation [226]. First, a sugar oxazoline monomer of Glcβ(1→4)

384 

 David Kaplan, Maria Dani, Antonio Verdoliva and Piero Bellofiore O CH3 N

OH O

HO HO H3C

NH

HO O

O O OH

C O

H3C

OH Chitinase Phosphate buffer, pH 10.6

HO O

O

O HO H3C

NH

C NH

O OH

n

C O

Scheme 12.24

GlcNAc was designed as a transition-state analogue substrate (TSAS) monomer for chitinase catalysis. The monomer was recognised by chitinase from Bacillus sp., giving rise to a cellulose-chitin hybrid polysaccharide via ring-opening polyaddition with perfect regioselectivity and stereochemistry, with a Mn value of 4,030 Da, which corresponds to 22 saccharide units. Second, a sugar fluoride monomer of GlcNAc β(1→4)Glc was synthesised for the catalysis of cellulase from Trichoderma viride. The enzyme-catalysed polycondensation resulted in a cellulose-chitin hybrid polysaccharide via a regio- and stereoselective manner, with a Mn value of 2,840 Da, which corresponds to 16 saccharide units. Synthesis of fluorinated chitin derivatives using chitinase from Bacillus sp. as a catalyst was carried out by Kobayashi and co-workers [227]. 6′-fluoro-, 6-fluoro- and 6,6′-difluoro-chitobiose oxazoline derivatives were newly prepared as TSAS monomers for chitinase. Ring-opening polyaddition of these monomers proceeded effectively at pH 8.0−9.0 and 30−40 °C, giving rise to alternating 6-fluorinated chitin derivatives and fully 6-fluorinated chitin derivatives, under total control of region selectivity and stereoselectivity. Finally, artificial polysaccharides produced via in vitro enzymatic synthesis [228] are new biomaterials with defined structures which either mimic natural polysaccharides or have nonnatural structures and functionalities. Polysaccharides are obtained by the enzymatic polymerisation of simple glycosyl donors via repetitive condensation. This approach not only provides a powerful methodology to produce polysaccharides with defined structures and morphologies as novel biomaterials, but also represents an available tool to analyse the mechanisms of polymerisation and packing in order to acquire high-order molecular assemblies.

12.5 Conclusions The challenges ahead are clear and do not involve the ability to make polymers in vitro using enzyme catalysis. As illustrated in the examples in this chapter, there

12 Enzyme catalysis in the synthesis of biodegradable polymers 

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are already many varied opportunities in this field. The challenges that remain are those of scale up, optimisation and economics in order to compete with high-volume commodity polymers already available, which all rely upon oil sources remaining reasonable in cost and supply. For biodegradable biomedical polymers, biological responses in terms of inflammation, rates of degradation in vivo and processing into suitable mechanically functional products are challenges that perhaps can be met in a shorter time frame than the needs in the commodity area. In either case, the place for enzymes in the world of polymer synthesis and polymer modification is already here. This role for enzymes should gradually expand as new insights are gained into the mechanisms involved as well as the opportunities for these polymerisation reactions.

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[217] E. Okamoto, T. Kiyosada, S-I. Shoda and S. Kobayashi, Cellulose, 1997, 4, 2, 161. [218] S. Kobayashi, E. Okamoto, X. Wen and S. Shoda, Journal of Macromolecular Science: Pure and Applied Chemistry, 1996, A33, 10, 1375. [219] S. Kobayashi, S. Shoda, M.J. Donnelly and S.P. Church in Carbohydrate Biotechnology Protocols, Methods in Biotechnology, Ed., C. Bucke, Humana Press, Totowa, NJ, USA, 1999, 10, 57. [220] I. Nakamura, Y. Horikawa, A. Makino, J. Sugiyama and S. Kimura, Biomacromolecules, 2011, 12, 3, 785. [221] I. Nakamura, A. Makino, J. Sugiyama, M. Ohmae and S. Kimura, International Journal of Biological Macromolecules, 2008, 43, 3, 226. [222] M. Hiraishi, K. Igarashi, S. Kimura, M. Wada, M. Kitaoka and M. Samejima, Carbohydrate Research, 2009, 344, 18, 2468. [223] S. Egusa, T. Kitaoka, M. Goto and H. Wariishi, Angewandte Chemie International Edition, 2007, 46, 12, 2063. [224] S. Egusa, M. Goto and T. Kitaoka, Biomacromolecules, 2012, 13, 9, 2716. [225] T. Hattori, M. Ogata, Y. Kameshima, K. Totani, M. Nikaido, T. Nakamura, H. Koshino and T. Usui, Carbohydrate Research, 2012, 353, 22. [226] S. Kobayashi, A. Makino, H. Matsumoto, S. Kunii, M. Ohmae, T. Kiyosada, K. Makiguchi, A. Matsumoto, M. Horie and S. Shoda, Biomacromolecules, 2006, 7, 5, 1644. [227] A. Makino, M. Ohmae and S. Kobayashi, Macromolecular Bioscience, 2006, 6, 10, 862. [228] M. Faijes and A. Planas, Carbohydrate Research, 2007, 342, 12−13, 1581.

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13 Environmental life cycle of biodegradable plastics 13.1 Introduction to life cycle thinking and assessment The environmental performance of bioplastics may be a competitive factor when they are compared with traditional, fossil-based plastics. Like all materials conceived for a wide range of industrial applications, including being major components in consumer products, bioplastics interact with the environment in several steps throughout their value chain. Due to this reason, the environmental performance of bioplastics is investigated with the life cycle thinking approach and its related quantitative method: life cycle assessment (LCA). Life cycle approaches emerged in the late 1960s and early 1970s from concerns about limited and nonrenewable natural resources, particularly oil. They came initially in the form of global modelling studies and energy audits, and were referred to as Resource and Environmental Profile Analysis and Net Energy Analysis. During the 1990s, the LCA methodology was further and quickly developed through a consensus-building process primarily driven by industry. This led to a standardisation of the methodology in the framework of the International Organization for Standardization (ISO). The ISO standards 14040 [1] and 14044 [2] provide comprehensive and detailed guidance on how to perform LCA studies, aiming to ensure the best attainable consistency and comparability of LCA results. LCA applies to all kinds of goods and services taking into consideration their cradle- to-grave environmental performance. The analysis usually embraces the whole product supply and value chain, from natural and energy resource extraction, transportation, processing, manufacturing, use and maintenance, to their final disposal or recycling (Figure 13.1). ISO 14040 and 14044 identify four phases in an LCA study: – The goal and scope definition. – The inventory analysis. – The impact assessment phase. – The interpretation phase. The goal definition specifies the reasons for carrying out the study and the intended application and audience. The scope relates to defining the unit of analysis, the system boundaries, the data quality and a number of other methodological choices.

https://doi.org/10.1515/9781501511967-013

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Material extraction

Material processing

Manufacturing

Use

Recycle

Remanufacture

Reuse

Waste management

Figure 13.1: Product life cycle.

The depth and breadth of LCA can differ considerably depending on the goal of the analysis. The life cycle inventory (LCI) analysis phase is a compilation of input/output data with regard to the system being studied. It involves the quantification of resources, material and energy carriers, and emissions into air, water and soil associated with the product life cycle stages (Figure 13.2). Inputs

Outputs Raw Materials Acquisition

Atmospheric Emissions

Manufacturing

Waterborne Wastes

Raw Materials

Energy

Use/Reuse/Maintenance

Solid Wastes Coproducts

Recycle/Waste Management

Other Releases

System Boundary Figure 13.2: Life cycle stages.

The life cycle impact assessment (LCIA) phase provides a comprehensive view of the potential environmental impact of the system by further modelling the inventory results to evaluate their effect on significant and global environmental problems (see  Table 13.1). The aggregation of inventory flows which contribute to the same impact category is made through characterisation factors reflecting the different effects caused by the individual substances. For example, contributions to climate change are usually quantified using global warming potential (GWP), expressed as carbon dioxide (CO2)-equivalents over a certain period of time (100 years as a rule).

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Table 13.1: LCA impact categories (selected list). Impact categories

Description

Climate change

CO2, CH4 and other GHG released into the environment allow sunlight to pass through the Earth’s atmosphere, but absorb the infrared rays that reflect off land and water. This inhibits their escape and therefore heats up the atmosphere

Ozone depletion

The release of substances, such as chlorofluorocarbons (CFC), hydrochlorofluorocarbons (HCFC), halons, methyl bromide, carbon tetrachloride and methyl chloroform, contribute to stratospheric ozone depletion and increased ultraviolet radiation to the Earth’s surface

Acidification

Emissions of chemicals such as sulfur dioxide, nitrogen oxides, ammonia and hydrochloric acid directly, or through conversion to other substances, lower the pH of soil and water bodies, affecting animal and plant life

Eutrophication

The release of nutrients, mainly nitrogen and phosphorus, from sewage outlets and fertilised farmland causes nutrient enrichment. This results in an altered species composition in nutrient-poor habitats and in algal blooms in water bodies, causing a lack of oxygen and fish death

Photochemical ozone creation

Ground-level ozone, which has impacts on animal and plant life, is produced by reactions of hydrocarbons and nitrogen oxides to light (‘summer smog’)

Human toxicity

Exposure to a chemical substance over a designated time period can cause adverse health effects to humans

Ecotoxicity

Emissions of substances (residues, leachate or volatile gases) that disrupt the natural biochemistry, physiology, behaviour and interactions of the living organisms which make up ecosystems. A distinction is made between different ecosystems, such as freshwater and terrestrial

Ionising radiation

Impacts as a result of radioactive substances in the environment and/or other sources of radiation

Land use

The use (occupation) and conversion (transformation) of land area by productrelated activities such as agriculture, roads, housing, mining and so on

Resource depletion

The consumption of nonrenewable resources such as water and crude oil, limiting their availability for future generations and affecting the areas they are taken from

Emissions of other greenhouse gases (GHG), such as methane (CH4) or nitrous oxide (N2O) are multiplied by their respective characterisation factor to convert their effect into an equal amount of CO2. Life cycle interpretation is the final phase of the LCA procedure, in which the results of an LCI or an LCIA, or both, are summarised and discussed as a basis for conclusions, recommendations and decision-making in accordance with the goal and scope definition. When LCA results are used to support decisions, they help consider potential trade-offs and prevent the shifting of burdens across different impact categories or stages in the product life cycle.

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In recent years, LCA has been increasingly adopted by a broad variety of sectors, ranging from business to public organisations. In the policy domain, life cycle approaches are useful in developing public strategies (e.g., for resources or waste management), understanding trends in product supply chains and where to best influence the environmental performance of the products. As an example, life cycle thinking and assessment stand at the heart of a growing number of policies and instruments in areas such as: Integrated Product Policy, the Sustainable Consumption and Production and Sustainable Industrial Policy Action Plan, Green Public Procurement, and the European Union (EU) Ecolabel and Ecodesign [3]. In business, a life cycle approach can help identify environmental hot-spots within the product value chain, i.e., understand which parts of a product’s life cycle have the greatest environmental impact as well as improvement potential. This in turn contributes to implement resource and energy efficiency, and to create an improved market position through more sustainable products. From the consumer’s point of view, a life cycle approach may play an important role in protecting the environment through the choices they make when buying products. Customers, but also more generally consumers and stakeholders, are showing a greater demand for product environmental performance information. To communicate such information on a fair and credible basis, ISO set up requirements for product environmental claims and labels, which are classified into three types: – Type I (ISO 14024) are environmental labelling programmes [4] which award their label to products meeting a set of predetermined requirements (e.g., the EU Ecolabel [5]). – Type II (ISO 14021) are self-declared environmental claims [6] about a significant environmental property of a product (e.g., recyclability). – Type III (ISO/TR 14025) is quantified environmental information [7] on a product life cycle, based on an independently verified LCA study and administrated by a programme operator (e.g., the International Environmental Product Declaration (EPD®) System [8]. LCA studies and applications present different levels of complexity, depending on several factors, the most important of which are the type of product under evaluation and the intended applications. Appropriate resources and expertise are often essential to perform LCA, such as knowledge of industrial processes and engineering approaches, technology assessment techniques and environmental impact assessment. A major issue concerns data availability and quality. Indeed, many current tools and databases, both commercial and free of cost have been developed over the years, ensuring a good coverage and representation of most business sectors. This led progressively to a significant improvement in the robustness of LCA results, which also contributed to enhancing public acceptance and confidence in this kind of assess-

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ment. However, full consistency and reproducibility of LCA studies is still to be achieved, hence the situation might arise where there are two ISO compliant studies on the same system providing different outcomes. To increase the consistency and quality assurance of LCA and to further facilitate its scientific development, a number of international initiatives are being carried out. In Europe, the International Reference Life Cycle Data System has been developed based on existing practice and through broad consultation between several partners. The project is coordinated by the Joint Research Centre of the European Commission [9] and consists of a series of methodological handbooks and a data network, which provide an authoritative basis for coherent LCA data, methods and applications. On the worldwide level, the United Nations Environment Programme-Society of Environmental Toxicology and Chemistry (UNEP-SETAC) initiative [10] has been launched to enhance the global consensus and relevance of LCA, as well as facilitate the diffusion of existing and emerging life cycle approaches, via the provision of methodological guidance and capacity building.

13.2 Bioplastics and life cycle assessment According to Comité Européen de Normalisation/ Comité Européen de Normalisation en Électronique et en Électrotechnique (CEN/CENELEC) Technical Specification TC 249 WI [11], bioplastics (and biopolymers as well) can be either biobased or biodegradable, or both. We refer to this definition when using the term bioplastics throughout the chapter. In recent years, bioplastics have been increasingly subject to LCA studies and applications, as documented in several published papers, reports as well as in unpublished grey literature [12– 15]. LCA is especially used to claim a positive environmental performance of bioplastics, due to the renewable origin of their raw materials and also their properties of biodegradability or compostability, which may be relevant in waste management operations. In general, bioplastics are expected to show environmental competitiveness with traditional plastics in terms of GWP, since their biological origin may in principle reduce GHG emissions. For this reason, LCA involving bioplastics are often comparative studies seeking to demonstrate their environmental superiority. In such respect, LCA appears one of the most suitable tools because it is able to depict the whole product value chain and to characterise a broad set of environmental indicators. In other cases, bioplastics producers make use of LCA to highlight the environmental performance of their products, not necessarily by comparison with other alternatives. This is, for instance, the case of ISO type III environmental declarations, such as EPD, which have been published for corn starch-based plastics [8]. Again, the EPD is a good instrument for the marketing and communication of products such as bio-

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plastics, which built their commercial success on environmental performance and being perceived as ‘more sustainable’ by clients and consumers. The purpose of this work is not to summarise the current literature on the LCA of bioplastics, which can be found using public information sources. This work, building on existing experience, aims rather to investigate and emphasise the peculiarities of bioplastics in relation to the LCA methodology, in particular whether LCA is the right tool to answer every question on bioplastics sustainability or for some aspects should it be replaced or complemented by other instruments. The basic issues are: – Which environmental properties of bioplastics are highlighted by LCA in a – scientifically robust manner? – Which environmental properties of bioplastics are not immediately displayed by – LCA and would need additional information by other methods? – Whether LCA is able, and under what circumstances, to provide clear and unambiguous guidance on the environmental performance of bioplastics versus competing materials. The following sections try to respond to such questions and provide some insight on the most typical peculiarities of bioplastics from a life cycle perspective.

13.2.1 Biodegradability and compostability Biodegradability and compostability are two different concepts, as laid down in the EU standards European Norms (EN) 13432 [16], EN 14995 [17] and in the US standard, American Society for Testing and Materials D6400 [18]: – Biodegradability is the capacity of an organic substance or material to be degraded – by biological agents. – Compostability is the capacity of an organic material to turn into ‘compost’, a humus-like stabilised substance. This occurs via an accelerated and controlled solid-state fermentation at high temperature. The inherent biodegradability of a plastic is inferred by studying an actual biodegradation process, under specific laboratory conditions, and the conclusion that the plastic is biodegradable (i.e., it can be biodegraded) can be drawn from the test results [19]. It must be noted that a fully biodegradable plastic can show a very limited biodegradation if environmental conditions are not suitable (e.g., bioavailability). The biodegradability of bioplastics is an interesting property which offers new solutions in waste management. While traditional plastics waste is generally treated through mechanical recycling or incineration with energy recovery, bioplastics after use can undergo an additional recycling option, i.e., composting or anaerobic digestion.

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The waste hierarchy established by the European Commission in 1989 [20] laid down a priority order in waste management options: 1. Prevention. 2. Recovery, with a preference for recycling and reuse. 3. Final disposal. Given that prevention of waste production is always the preferred option, recycling through biological treatment is a sound management practice for biowaste. The suitability of composting as a means to recycle biowaste has been observed through several life cycle studies [21–24]. Other authors [25] found that compost brings certain environmental benefits to agricultural soils. The spreading of compost increases the soil water-holding capacity and provides several nutrients, thereby reducing the need for irrigation and fertilisation. In addition, compost improves the soil structure by decreasing its density, which subsequently requires less energy during tillage operations, since the soil can be turned more easily. Lastly, compost is deemed to have a positive effect on crop yield and carbon stock, the latter through an increased carbon sequestration. In this scenario, compostable bioplastics can play a key role. Typical examples are: waste bags for the collection of household waste, disposable catering items used in fast-food restaurants and agricultural mulch films. Bioplastics do not need separation but can be collected and treated in a homogeneous way with the other organic waste, making the system more efficient. The environmental benefits induced by the use of bioplastics in biowaste management, notably when composting or anaerobic digestion are the preferred options, are readily measurable with LCA, provided that the following scope aspects are considered: – LCA should be performed from cradle-to-grave, i.e., accounting for the entire life cycle of bioplastics materials, including end of life management. – End of life bioplastics should not be separated from the associated biowaste management, which needs to be included in the system boundaries. This second aspect is essential in LCA applications to organic waste management scenarios, where bioplastics can compete with traditional nonbiodegradable materials. The use of bioplastics in the substitution of traditional nonbiodegradable plastics could in some cases improve the overall recyclability of the mixed waste fraction, whenever plastics and biowaste are mixed [26]. A typical situation where bioplastics can replace common plastics is found in town festivals where plates and cutlery are normally delivered in polystyrene. In such cases, separate collection of the different fractions can be a very difficult task, considering the usual crowding of town festivals. As a consequence, the collection of undifferentiated waste is the most common option. The waste management scenario involves the collection of a mixed heterogeneous waste composed of food waste and traditional plastics. The waste stream

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is then treated in local disposal facilities. The traditional plastics scenario can be replaced by an alternative scenario based on the use of bioplastics, enabling the collection of a mixed homogeneous waste composed of food waste and bioplastics. The final waste stream is then composted to produce high-quality compost. A reliable comparative LCA of the above two options would be based on an appropriate scenario where plates, cutlery and food waste are jointly modelled. In the case of traditional plastics materials, composting would not be an option within the waste management scenario. On the other hand, from a life cycle perspective, the possibility of shifting from heterogeneous waste (traditional plastics + food) to homogeneous waste (bioplastics+ food) makes the use of bioplastics particularly beneficial for the waste management ecoprofile. Whenever the organic ‘humid’ waste is contaminated with plastics and the possibility to avoid the plastic fraction is technically, economically or socially difficult, bioplastics products can represent a solution to produce uncontaminated waste which is recoverable via organic treatment. The plastic bags used to collect and transfer the organic waste to composting, and food packaging contaminated with food residues, represent two other typical examples of mixed food plastic waste. The same approach could be applied to these examples to further substantiate the environmental sustainability of using bioplastics products. As far as the use of compost is concerned, further environmental benefit is expected from a biowaste management system. The compost produced may be applied in agriculture as fertiliser or as a soil amender, having positive effects on, e.g., peat substitution, partial reduction of chemical fertilisers, carbon sequestration and the reduction of irrigation [23], (see Table 13.2). Table 13.2: Environmental benefits of using 1 kg of compost, obtained from organic waste and biodegradable and compostable (B&C) cutlery, in agriculture. Fertiliser savings (kg)

Water savings (m3)

Substituted peat (kg)

Carbon sequestration (kg CO2)

N

P

K

0.0053

1

0.4958

0.0037

0.0003

0.0012

−

−

−

Reproduced with permission from F. Razza, M. Fieschi, F. Degli Innocenti and C. Bastioli, Waste Management, 2009, 29, 1424. ©2009, Elsevier [26].

At present, LCA is able to emphasise the avoidable environmental burdens of materials which are potentially replaceable via composting, while benefits from carbon sequestration and the resulting variations in soil carbon stocks are still an open methodological issue, on which a full scientific consensus has not yet been achieved. Another issue which LCA cannot accurately address is the littering of fossil-based plastics. This can be important in comparative studies and represents a major point in favour of a higher use of bioplastics. The problem of plastic litter has been growing

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over the last few decades, becoming a permanent environmental issue. Plastic is the fastest growing component of the waste stream, and plastic pollution by single-use bags is amongst the most commonly found items during beach and coastal cleanups. According to different sources [27–29] about 60−80% of the overall marine debris and up to 90% of floating debris is caused by plastic. This massive amount has grave implications for marine wildlife, including filter feeders such as whales. More than 1 million seabirds, 100,000 marine mammals, and countless fish die annually in the North Pacific from ingesting or becoming entangled in marine debris. Furthermore, due to their chemical composition, plastic particles collect toxins on their surface which can harm the reproductive health of animals that consume them. All these environmental problems are not effectively covered by LCA, which considers broad environmental categories such as ‘marine or freshwater ecotoxicity’, but does not address in detail the complete cause and effect impact chain of marine debris. To solve this issue, further research on LCIA is needed, or complementary assessment methods should be used.

13.2.2 Renewable origin The renewable origin of bioplastics is seen as their major environmental asset in comparison with traditional plastics. This has an effect on GHG emissions and generally reduces GWP. However, this aspect is extremely complex and involves several methodological and modelling aspects in the product life cycle. Many findings from published comparative LCA show controversial data, depending on the system boundary definition and the assumptions made in modelling and the impact calculation [13]. Bioplastics [30] are partly or fully made from renewable feedstocks produced in agriculture and forestry. During their growth, plants absorb atmospheric CO2 and, powered by solar energy, convert it into carbon-rich plant matter. The valuable parts of the plants are extracted and processed industrially to produce clean and largely homogenous feedstocks such as starch, sugar or plant oil. Thus, the plant matter can start its ‘career’ as an input material for the production of bioplastics. The plant carbon is stored in a bioplastic product over the time of its useful existence. In this way, the CO2 is trapped in the material and released only at the end. When the product is burnt or composted, the stored carbon is released back into the atmosphere and turns into ‘fuel’ for new plant growth. In other words, bioplastics in their durable or easily recyclable form, remove carbon from the atmosphere for many years, thereby acting as a carbon sink. The above properties exert a great influence on LCA modelling principles and results, notably on the impact category of climate change which is expressed by the GWP. The characterisation factors of CO2 and CH4 from biogenic sources may in fact vary depending on whether fossil and biogenic emissions are reported separately or jointly:

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In the case of separate accounting, a characterisation factor of -1 should be assigned to CO2 biogenic removal and +1 to CO2 biogenic emission, while biogenic CH4 emission has a +25 emission factor. In the case of joint accounting, a characterisation factor of 0 is assigned to both CO2 biogenic emission and removal, and a factor of +22.25 is assigned to biogenic CH4 emission; the biogenic CH4 factor is determined by decreasing the fossil CH4 factor (25) by the ratio between the molecular weights of CO2 (44) and CH4 (16), i.e., 2.75.

The above numeric example (Table 13.3), which is purely illustrative, shows how GWP may be correctly evaluated in both cases, if the above rules are applied. Note: the amount of biogenic CH4 is stoichiometrically linked to the difference between CO2 removal and emission. Table 13.3: GWP evaluation: joint and separate accounting. Emission

Amount (kg)

GWP fossil (kg CO2 -equivalent)

GWP biogenic (kg CO2-equivalent)

GWP total (kg CO2-equivalent)

CO2 (fossil emitted)

2.00

2.00

−

−

CO2 (biogenic emitted)

0.50

−

0.50

−

Separate accounting

CO2 (biogenic removed)

1.00

−

-1.00

−

CH4 (fossil emitted)

0.10

2.50

−

−

CH4 (biogenic emitted)

0.18

−

4.50

−

−

4.50

4.00

−

CO2 (fossil emitted)

2.00

−

−

2.00

CO2 (biogenic emitted)

0.50

−

−

0.00

CO2 (biogenic removed)

1.00

−

−

0.00

CH4 (fossil emitted)

0.10

−

−

2.50

CH4 (biogenic emitted)

0.18

−

−

4.00

−

−

−

8.50

Total Joint accounting

Total

The current LCA practice gives strong preference to the separate accounting of GHG emissions from biogenic sources. Even the most internationally agreed standards and guidelines on LCA and carbon footprint, among which the Publicly Available Specification 2050:2011 [31], the GHG Protocol Product Life Cycle [32] and Corporate Value Chain [33] as well as the upcoming ISO 14067 [34] show an overall alignment on this methodological issue.

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Separate accounting is also acknowledged by European Bioplastics in its LCA position paper [35], where it is stated that omitting the biogenic carbon from the LCI is not supported for the following reasons: – The carbon neutral approach (i.e., joint accounting) does not convey the immediate carbon advantage that biobased materials provide. – The CO2 intake is just an input like any other input in the inventory analysis. In LCA methodology, there is no scientific basis for leaving out any specific input. Flows cannot be declared per se as neutral, rather they must be demonstrated by calculated inputs and outputs. – The carbon input is not automatically released at the end of the product’s life which, for example, happens in the case of incineration. Products can be durable or land-filled, where the carbon is sequestered; if the sequestration lasts more than 100 years, this result may be counted as a true contribution to GWP reduction. – Some bioplastics can be recycled and during this process the biogenic carbon is used for a second or even third time in a new product. If the carbon is left out of the LCA, errors in calculations and wrong conclusions can easily be made. If the biogenic carbon is ignored as an input or output, intermediate results will be biased. – Fossil and renewable fuels are used to drive all processes throughout the life cycle of bioplastics. The related carbon intake and emitted CO2 must also be taken into account. Regarding carbon sequestration (carbon storage) in bioplastics, it should be noted that a completely agreed approach for treatment of GHG delayed emissions is still lacking in the scientific community. Another important methodological aspect in the bioplastics life cycle relates to biogenic CO2 emission from land-use change. This is especially relevant when raw materials entering the bioplastics supply chain come from agricultural processes. Land-use change impacts include the following: – Biogenic CO2 emission and removal due to carbon stock change occurring as a result of land conversion within or between land-use categories. – Biogenic and nonbiogenic CO2, N2O and CH4 emissions resulting from the preparation of converted land, such as biomass burning or liming. Land-use changes can be direct or indirect. Direct land-use change (dLUC) occurs when the demand for a specific land use results in a change in carbon stocks on that land. A change in carbon stock can occur moving from one land-use category to another (e.g., converting forest to cropland) or within a land-use category (e.g., converting a natural forest to a managed forest or converting agricultural land from till to no-till). Indirect land-use change (iLUC) is defined as land-use change that occurs

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when the demand for a specific land use (e.g., an increased demand for crops as a bioenergy feedstock in the United States) induces a carbon stock change on another land (e.g., increased need for cropland in Brazil causing deforestation). This displacement is a result of market factors and calculated using data consistent with a consequential approach. The issue of iLUC is also linked to questions about sufficient farming land and/or land use for bioplastics production [36–38]. According to some authors, e.g., Stevens [39], if bioplastics were produced on a large scale, there could be competition between feedstock crops and food crops, possibly causing increases in food prices. However, other authors, e.g., Piotrowsky [40], argue that 92% of the cultivated land in the world is used for food and animal feed production, 6% for industrial materials and 2% for biofuels, while agricultural land used for bioplastics is less than 0.1%. It is now generally agreed in the LCA community that dLUC impacts should be included in the system boundaries, while iLUC may be excluded since a scientifically robust accounting methodology is not yet available. dLUC impacts are accounted as additional biogenic CO2 emissions which in the case of land- use variations (e.g., from a forest to a cropland) take into account the sacrificed sequestration potential of the previous, more natural land (the forest) compared with the resulting more antrophised land (the crop). These emissions should be accounted for in the system in the case where the last known land-use change occurred less than 20 years from the assessment period or for a single harvest period. For longer periods it is assumed that the variations in carbon stock are not attributable anymore to the system under assessment. The calculations are very complex since emission factors depend on several factors such as type of land and geography. A good guidance is provided in Appendix B of the GHG Protocol Product Life Cycle Accounting and Reporting Standard which makes reference to IPCC guidelines [41]. Despite these efforts, the evaluation of dLUC emissions still seems to be affected by significant value choices. The contribution of dLUC emissions in the life cycle of bioplastics is still difficult to assess in general terms. According to some authors, e.g., Tabone and co-workers [42], land-use change emissions can limit the attractiveness of bioplastics for the displacement of petroleum-based plastics, whenever the feedstock comes from lands diverted from their original use. Conversely, bioplastics from perennials grown on degraded cropland and from waste biomass would minimise habitat destruction, competition with food production and carbon debt. In this context, the commercial availability of technologies for producing bioplastics based on lignocellulosic feedstocks (such as for second generation biofuels) may induce changes in land use towards more lignocellulosic crop cultivation. If these new crops are established on croplands which have long been cultivated with conventional food/feed crops, both ground and soil carbon stock will likely increase.

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13.2.3 Optimisation potential Another nonnegligible aspect which may have a significant influence in the environmental assessment of bioplastics, notably in comparative studies with other materials, is linked to their optimisation potential. Bioplastics are still in their early stage of development [35]. They are produced on a small scale or in singular facilities, meaning that transport, conversion, product design and final disposal are not optimised. They are, however, quite often compared with mature materials whose life cycles have been optimised over several decades. This may lead to a biased comparison. The optimisation and improvement potential for bioplastics is huge and includes: – Switch to nonfood crops (switchgrass and wood) and agricultural waste streams. – Use of innovative and more efficient processes (new technology/increase production scale). – Further optimisation of conversion technology and product design. – Further development and introduction of innovative end of life options such as composting, anaerobic digestion and chemical recycling (cradle-to-cradle). – Installation of use cascades in which reuse and recycling is combined with thermal recovery. The above points are not immediately evident when performing an LCA of bioplastics. In general, the life cycle-based comparison between novel and mature products does not take this basic difference into account. As remarked by European Bioplastics, projections for improvement should be made and included in LCA studies. Besides, new materials and products, such as bioplastics, are often closely scrutinised while many existing products ‘on the shelf’ are far less thoroughly examined. Within their life cycle, bioplastics are often ‘put under the microscope’ while the impact of, e.g., oil or gas production is often modelled using fewer details, for instance, using data from generic databases.

13.3 Conclusions LCA is a reliable tool for measuring the environmental performance of bioplastics, and ensuring a fair and unbiased evaluation when bioplastics are compared with competing materials and products, built on the same functional unit. The main strength of LCA lies in its comprehensiveness, including the evaluation of all stages in the product supply chain, use and end of life, as well as a broad coverage of the environmental impact. This allows for the consideration of potential trade-offs and prevents unintended shifting of burdens when results are used in support of decision-making. LCA results are also widely communicated by the bioplastics industry to disclose verified and credible information on the environmental performance of products to

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clients, consumers and other interested parties. The ISO type III environmental declarations are seen as the most effective instrument. LCA does not provide universally valid answers, but can provide answers to specific and well-formulated questions. The environmental performance of bioplastic applications depend upon many different parameters. These include the type of bioplastics used, the raw materials which they originate from, the production and conversion technology, the transport means and distances, and the type of application, as well as the waste collection and disposal or recycling system. As remarked also by European Bioplastics, it is not possible to make generalisations such as ‘bioplastics are better or worse than other materials’. LCA is a complex tool which requires expertise and thorough knowledge. The methodology has been increasingly standardised over the last few years, but a certain degree of subjectivity still exists in the application and may lead to contradictory or misleading results. When LCA is applied to bioplastics, it is essential that the analysis is performed with a ‘cradle-to-grave’ approach, especially for materials where the greatest benefits arise from the end of life and disposal phase. Compostable bioplastics, for example, may contribute to increase recycling and recovery options in biowaste management and reduce the overall environmental footprint in the waste management scenario. In this respect, LCA still lacks adequate consideration of environmental benefits originating from compost application, which are generically measured in terms of substituted fertilisers or soil amenders. Other important benefits such as an increase in carbon stock or crop yield and reduction of water consumption are often neglected. LCA is also not able to account for environmental burdens due to the littering of traditional plastics after use. This aspect can be important in comparative studies and should be addressed by other complementary assessment methods. Regarding GHG emissions, LCA can provide a complete inventory for the life cycle of bioplastics. To ensure more transparent evaluations, emissions from biogenic sources should be made visible in the calculations and accounted separately from fossil emissions. This allows for the correct consideration of biogenic carbon storage processes which are an intrinsic property of bioplastics and may contribute to their environmental preferability under specific conditions. On the other hand, LCA applications in bioplastics cannot neglect additional GHG emissions resulting from dLUC, where attributable. This aspect is still not completely acknowledged in the common practice and is affected by methodological uncertainties. Further research and increased applications are envisaged in the near future. Lastly, it should be pointed out that bioplastics are novel products with a great potential for technological and environmental improvement, which are hardly assessable with a traditional LCA. Data used in the assessment are in fact generally based on current technologies or make use of secondary sources which date back some years. Efforts should be made in undertaking more prospective studies which can highlight this improvement potential.

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References [1] [2] [3] [4]

[5] [6] [7]

[8] [9] [10] [11] [12]

[13] [14] [15] [16]

[17] [18] [19] [20] [21] [22] [23]

ISO 14040, Environmental Management – Life Cycle Assessment – Principles and Framework, International Organization for Standardization, Geneva, Switzerland, 2006. ISO 14044, Environmental Management – Life Cycle Assessment– Requirements and Guidelines, International Organization for Standardization, Geneva, Switzerland, 2006. Making Sustainable Consumption and Production a Reality, European Commission, Publications Office of the European Union, Luxembourg, 2010. ISO 14024, Environmental Labels and Declarations – Type I Environmental Labeling – Principles and Procedures, International Organization for Standardization, Geneva, Switzerland, 1999. The EU Ecolabel, European Commission. http://ec.europa.eu/environment/ecolabel/ ISO 14021, Environmental Labels and Declarations – Self-declared Environmental Claims, International Organization for Standardization, Geneva, Switzerland, 1999. ISO 14025, Environmental Labels and Declarations – Type III Environmental Declarations – Principles and Procedures, International Organization for Standardization, Geneva, Switzerland, 2006. International EPD® system. http://www.environdec.com/ The European Platform on the Life Cycle Assessment, European Commission Joint Research Centre (JRC). http://lct.jrc.ec.europa.eu/ Life cycle Initiative, United Nations Environment Programme (UNEP) and Society for Enviornmental Toxicology and Chemistry (SETAC). http://lcinitiative.unep.fr/ Plastics – Environmental Declaration about Biopolymers and Bioplastics – Environmental Data Sheet, CEN/CENELEC Technical Specification TC 249 WI, Brussels, 2010. F. Degli Innocenti, F. Razza, M. Fieschi and C. Bastioli in Environmentally Degradable Materials based on Multicomponent Polymeric Systems, Eds., C. Vasile and G.E. Zaikov, CRC Press, Boca Raton, FL, USA, 2009. R. Narayan and M. Patel in Review and Analysis of Bio-based Product LCA’s, Chapter 27, Iowa State University, Ames, 2004. A. Steinbuchel in Biopolymers, Volume 10, General Aspects and Special Applications, Wiley-Backwell, Hoboken, NJ, USA, 2003. M. Patel, C. Bastioli, L. Marini and E. Würdinger, Life-cycle Assessment of Bio-based Polymers and Natural Fiber Composites, Biopolymers Online, 2005. EN 13432, Packaging: Requirement for Packaging Recoverable through Composting and Biodegradation − Test Scheme and Evaluation Criteria for the Final Acceptance of Packaging, European Committee for Standardization, Brussels, 2000. EN 14995, Plastics − Evaluation of Compostability − Test Scheme and Specifications, European Committee for Standardization, Brussels, 2004. ASTM D6400, Standard Specification for Compostable Plastics, American Society for Testing and Materials, Washington, DC, USA, 2012. A.A. Shah, F. Hasan, A. Hameed and S. Ahmed, Biotechnology Advances, 2008, 29, 246. Community Strategy for Waste Management of 18th September 1989, European Commission, Brussels, Belgium, 1989. M. De Bertoldi, M.P. Ferranti, P. L’Hermite and F. Zucconi in Compost: Production Quality and Use, Elsevier Applied Science, London, UK and New York, NY, USA, 1987. M. Odlare, M. Pell and K. Svensson, Waste Management, 2008, 28, 1246. G. Sharma and A. Campbell in Life Cycle Inventory and Life Cycle Assessment for Windrow Composting Systems, Recycled Organics Unit, NSW Department of Environment and Conservation and The University of New South Wales Sydney, Australia, 2006.

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[24] Plastic Shopping Bags – Analysis of Levies and Environmental Impacts − Final Report, Nolan-Itu Pty Ltd, Australia, 2002. [25] J. Barth, F. Amlinger, E. Favoino, S. Siebert, B. Kehres, R. Gottschall, M. Bieker, A. Löbig and W. Bidlingmaier, Final Report − Compost Production and use in the EU, European Commission, DG Joint Research Centre/ITPS, Belgium, Brussels, 2008. [26] F. Razza, M. Fieschi, F. Degli Innocenti and C. Bastioli, Waste Management, 2009, 29, 1424. [27] Californians Against Waste. http://www.cawrecycles.org [28] Ocean Conservancy. http://act.oceanconservancy.org/pdf/Marine_Debris_2011_Report_OC.pdf [29] D.W. Laist, Marine Pollution Bulletin, 1987, 18, 6, Supplement B, 319. [30] Renewable Resources for the Production of Bioplastics, European Bioplastics, Berlin, Germany, 2011. [31] Publicly Available Specification PAS 2050, Specification for the Assessment of the Life Cycle Greenhouse Gas Emissions of Goods and Services, BSI, 2011. [32] Product Life Cycle Accounting and Reporting Standard, WBCSD/ WRI Greenhouse Gas Protocol, World Business Council for Sustainable Development, Geneva and World Resources Institute, Washington, DC, USA, 2011. [33] Corporate Value Chain (Scope 3) Accounting and Reporting Standard, WBCSD/WRI Greenhouse Gas Protocol, World Business Council for Sustainable Development, Geneva and World Resources Institute, Washington, DC, USA, 2011. [34] ISO/DIS 14067, Carbon Footprints of Products, International Organization for Standardization, Geneva, Switzerland, 2012. [35] Life Cycle Assessment of Bioplastics, European Bioplastics, Berlin, Germany, 2008. [36] T. Searchinger, R. Heimlich, R.A. Houghton, F. Dong, A. Elobeid, J. Fabiosa, S. Tokgoz, D. Hayes and T.H. Yu, Science, 2008, 319, 1237. [37] R. Righelato and D.V. Spracklen, Science, 2007, 317, 902. [38] J. Fargione, J. Hill, D. Tilman, S. Polasky and P. Hawthorne, Science, 2008, 319, 1235. [39] E.S. Stevens in How Green are Green Plastics?, Biocycle, Emmaus, PA, USA, 2002. [40] S. Piotrowsky and A. Carus, Bioplastic Magazine, 2009, 4, 46. [41] H.S. Eggleston, L. Buendia, K. Miwa, T. Ngara and K. Tanabe, IPCC Guidelines for National Greenhouse Gas Inventories, Volume 4: Agriculture, Forestry and Other Land Use, Institute for Global Environmental Strategies (IGES), Hayama, Japan, 2006. [42] M.D. Tabone, J.J. Cregg, E.J., Beckman and A.E. Landis., Environmental Science and Technology, 2010, 44, 8264.

Enzo Favoino

14 The use of biodegradable polymers for the optimisation of models for the source separation and composting of organic waste 14.1 Introduction As source separation and recycling strategies evolve, we acquire more and more important information regarding waste streams on which the focus has been to reach higher recycling rates and an overall improvement of the environmental performance of waste management. Traditionally, source separation systems have only tackled dry recyclables and whose separate collection was simply added to the collection of mixed municipal solid waste (MSW). The collection of paper, glass and plastics by means of road containers did not require organisational changes to MSW collection. With such systems, separate collection rates are typically well below 30−40%, which falls short of important strategic drivers and targets, e.g., the material recovery target stipulated by Directive 98/2008/EC (European Union (EU) Waste Framework Directive (WFD)). In fact, the recently published (7th July 2014) EU package on Circular Economy includes proposals to increase the target to 70%; also, it stipulates a mandate on separate collection of biowaste by 2025. There is growing interest in source separation systems that also target ‘biowaste’ or ‘organic waste’, i.e., the compostable fractions as food waste and garden waste; not only does this require recycling/composting rates that are remarkably higher, but this also makes it possible to change the operational design of collection systems for residual waste (RW), with specific regard to the reduced collection frequency, which may lead to unexpected savings and economic optimisation of the system. In this respect, a central role is played by the source separation of food waste, i.e., the fermentable part of MSW. From a quantitative point of view, fermentable material (food waste) accounts for a major percentage of MSW; and this is particularly true in low-income economies (where organics may be as high as 70−80% of the total MSW) and, within the EU, in Southern Europe and former Eastern European countries. For instance, in Northern Italy the percentage of food waste ranges between 25−40% of the total MSW; whereas in southern regions it typically ranges between 35−50%, mainly due to the lower presence of packaging waste and the habit to have more meals at home instead of street food and convenience food (precooked and/or frozen products which produce less food waste). From a qualitative point of view, the more fermentable material which is sorted and recycled, the less production of biogas and leachate is to be expected in landfills, this is the reason why the Directive 99/31/EC on Landfills mandates phased diversion targets for biodegradable waste to be taken out of landfills. https://doi.org/10.1515/9781501511967-014

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14.1.1 The development of composting and schemes for the source separation of biowaste in Europe: A matter of quality Since the early 1980s, composting has been undergoing an impressive growth across Europe. Even prior to this, composting had been adopted as a management route for MSW, through the attempt to separate the organics of MSW mechanically; this strategy proved to be unsuccessful mainly due to the following reasons: – The increasing presence of contaminants inside MSW. – The lack of suitable refining technologies that could effectively clean-up the end product in order for it to be accepted by end users. – The consequent lack of confidence among farmers and other potential users. – The increasing awareness, among scientific bodies and institutions, of the importance to keep soils unpolluted, with specific reference to potentially toxic elements such as heavy metals. As a consequence, the recent and effective growth of composting programmes started in parallel with the growth of schemes for the source segregation of biowaste, which were increasingly adopted as the appropriate answer in order to obtain quality products suitable for a profitable use in farmlands and other cropping conditions (forestry, nursery, gardening, pot cultivation and so on). Programmes for the separate collection of organics were started as early as 1982 in Germany, followed shortly after by the Netherlands and Austria; such programmes then spread to South Europe starting in 1993 (Italy) and were then exported to Spain (Catalonia started the extensive roll-out of such schemes in the late 1990s). The system is also widespread in the UK and in many areas in Scandinavian countries, and is currently being diffused in central Eastern European countries, with many schemes already implemented, e.g., in Slovenia, while the first successful implementation in Greece took place in 2013. In a nutshell, the separate collection of organics is the driving force behind the evolution of strategies for the sustainable management of waste across Europe.

14.2 Main drivers for composting in the European Union Although implementation seems to show different speed in different areas of Europe, there is consensus on the fact that the separate collection of organics will continue to grow in the near future. As a matter of fact, many EU policies and thematic strategies are further propelling the interest in it.

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14.2.1 Directive 99/31/EC on landfills The Directive on Landfills basically requires landfilled biowaste to be substantially reduced over time. This is aimed at effectively reducing biogas production at landfill sites (one of the highest contributions to the global warming potential from waste management) and improving the conditions under which landfills are operated (e.g., lower chemical strength of leachates and a lower risk for the surrounding environment after the landfill gets shut down). Biowaste to be landfilled should be reduced by: – 25% (with reference to 1995) within 5 years. – 50% within 8 years. – 65% within 15 years. This target could also be achieved through thermal treatment; however, biological treatment and composting are likely to play a major role in this respect. In the end, composting is the most ‘natural’ way to manage biowaste, and its cost is typically lower than that of incineration, above all the latter has to comply with the provisions of the recent Directive on Incineration which mandates much tighter limits for emissions from incinerators. Also, one should consider that the proposed revision of the EU Waste Policy, included in the ‘Circular Economy ‘Package’’ (issued in early July 2014), includes a foreseen obligation for the separate collection of organics by 2025, which factually confirms the primacy of strategies aimed at separate collection and the subsequent application of end products onto farmlands.

14.2.2 The waste framework directive (Directive 2008/98/EC) The Waste Framework Directive (WFD) is the reference directive for waste management in the EU. Its main provisions relate to separate collection and biowaste management, and may be outlined as follows: – It establishes the ‘waste hierarchy’ (reduce, reuse, recycle, recover and dispose). – In order to supplement it, some ‘material recovery targets’ are stipulated (50% of MSW by 2020). – It stipulates (Article 22) that Member States should ‘encourage’ the separate collection of organics. See also previous note – provisions to make separate collection mandatory by 2025 have been included in the EU proposed package on Circular Economy.

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14.2.3 Other regulatory and political drivers The likely further diffusion of the separate collection of organics is enhanced by concurrent drivers such as: – The Thematic Strategy on Soils, which recalls the importance of replenishing organic matter in soils in order to ensure resilience and fertility, and fight erosion and desertification. – The EU Climate Change Programme, which has highlighted the importance of storing carbon in soils as a way to mitigate climate change (the more C in soils, the less CO2 in the atmosphere). – The Resource Scarcity Crisis, which ranks high on the geopolitical agenda, i.e., the need to recover as much resource from our food waste in order to secure the supply of raw materials to industry, in a world in which the growing economies (e.g., India, China, Brazil) are exerting an increasing pressure on the market of primary raw materials.

14.3 The source separation of organic waste: Schemes and results in the south of Europe As a consequence of a growing number of provisions in national or local legislation, and/or mandatory programmes, a growing number of districts, also in the Southern Member States, have lately adopted those strategies already well developed in central and northern Europe, aiming at the source segregation of the organic fraction from municipal waste. Over the last 20 years, the development has been particularly noticeable in Italy and Spain (Catalonia and the Basque Country). Italy has recently seen a huge growth of the source separation of food waste mainly due to provisions of the National Environmental Act (Decree 152/2006, then amended by Decree 205/2010). The decree, which enforces various EU Directives and Thematic Strategies, mandates a general midterm target of 65% for the separate collection from MSW, a goal which requires, no doubt, a widespread adoption of the separate collection of organic waste. As a matter of fact, since its early steps, the implementation of collection at the doorstep, including the separate collection of organics, has shown to be the only strategy able to meet the target. Table 14.1 reports on the best performing municipalities in 1999, and the contribution of food and garden waste to the overall quantity of recycled waste in such situations. Since the mid1990s, separate collection has been further diffused, with the two main directions of further development being:

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–

–

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Implementation in densely populated areas and large cities (with the outstanding case of Milan town, where the scheme covers more than 1 million (M) people, which has the largest scheme for the separate collection of food waste around the world). Remarkably, the scheme in Milan is showing very effective capture, in the region of approximately 90 kg/person a year, or a foreseen total of 130,000 tonnes/year of food waste [15]. Diffusion in southern regions, both in rural areas and large towns (e.g., Salerno, population 150,000, with 100% of the population covered; pilot neighbourhoods in Naples for a total covered population of approximately 200,000).

Table 14.1: Municipalities with the highest recycling rates (as of 1999) and the contribution of compostable fractions. Municipality

Inhabitants

% Recycling rate

Garden waste kg/ inh/year

Food waste kg/ inh/year

Masate

2,296

79.6

196

55

Villa di Serio

5,742

76.2

87

68

Presezzo

4,512

71.7

77

57

Mesero

3,430

70.8

106

66

Fara Gera d’Adda

6,533

70.1

41

43

Gambellara

3,166

69.0

−

42

Albairate

4,062

68.8

44

69

Cassago Brianza

3,936

67.7

65

41

16,495

67.3

43

66

Usmate Velate

8,252

67.3

70

62

Aicurzio

1,947

66.9

116

70

Fumane

3,736

66.1

37

52

3,923

66.0

45

55

11,425

66.0

55

82

Arcore

Bariano Trezzo sull’Adda Guido Visconti

1,307

65.5%

87

56

Azzano San Paolo

6,786

65.4%

37

58

In general, the intensive collection of dry recyclables alone (paper, glass, metal and plastic) does not allow municipalities to meet the 65% goal. Accordingly, most regions and provinces now plan to promote the source separation of food waste from households and major producers (restaurants, canteens, greengrocers and so on). Thanks to the wide diffusion of schemes, it is now possible to assess the effectiveness of these systems, in terms of: – Quantitative results: this is expressed as specific capture (in grams, per person, per day or kilograms per person, per year); the capture of food waste on its own

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is most often reported in Northern Italy at some 200 g/inh/day while schemes in southern regions often reach 250−300 g/inh/day as a consequence of the much more diffused habit of cooking and having meals at home, and of the higher percentage of vegetables and fish in the daily diet. On the contrary, we’ll see further on that schemes run using road containers deliver a much lower capture of food waste, while a high percentage of garden waste is deposited inside the containers. Purity of the fraction collected, as Table 14.2 clearly shows, and random analyses of food waste indicate the excellent quality of organic material collected. The level of purity allows adoption of relatively simplified screening/ refining equipment at compost sites (which decreases costs and reduces the total amount of rejected materials) and ensures production of a high-quality compost. Usually, where schemes with collection at the doorstep are being run, the purity (percentage of compostable materials inside the collected food waste) ranges between 95−99%. Table 14.2: Purity of collected food waste (assessed through compositional analysis of separately collected organics). Municipality/Area

Inhabitants

Milan province (17 municipalities)

493,673

Milan town

1.4 million

District ‘Padova 1ʼ (26 municipalities)

203,429

Compostable materials (wt%) 97.3 94.8−98.7 98.7

Adapted from Scuola Agraria del Parco di Monza, Analisi Merceologiche su FORSU Prodotta nel Comune di Milano − Campagna di Analisi 22−23 Gennaio 2013; Report to AMSA, Milan, Italy, 1999 [1]; Provincia di Milano in Il Quaderno: Gestione Rifiuti Solidi Urbani 1998, Indirizzi Programmatici e Azioni di Approfondimento, Milan, Italy 1998 [2]; and E. Favoino in Proceedings of the Biodegradable Plastics 99 Conference, Ed., A. Beevers, Frankfurt, Germany, European Plastic News, Croydon, UK, 1999 [3].

Composting is also under fast development in Spain. The start-up of pilot schemes for the source segregation of ‘basura orgánica’ (also worded as ‘form’ or ‘forsu’, the organic fraction of municipal waste) dates back to the mid1990s and has been developed in many Spanish districts, both rural and urban. Catalonia is undoubtedly the leading situation, more recently followed by a growing number of schemes in the Basque Country, based on the same operational model (originally borrowed from North Italy). The Catalan development took its implementation from a regional law (Law 6/93) that stipulated compulsory programmes for the source segregation of organic waste in all municipalities with a population over 5,000 inhabitants, which was then extended to all municipalities.

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As of November 2000, 72 municipalities in Catalunya were reported to source separate biowaste, for an overall population of some 640,000 inhabitants; in the Barcelona Metropolitan area, 21 out of the 33 areas covered 150,000 inhabitants. Catalan schemes were initially based on the collection of organic waste by means of road containers, as had been previously implemented in other Spanish districts. Later, on the spur of the effective outcomes reported in Northern Italy, doorstep schemes were introduced and developed in various municipalities (Tona, Tiana and Riudecanyes were the first) with a contrasting and improved outcome. Numbers are showing once again, as shown for a long time on a broader scale in Italy, the different and greatly improved outcomes that the doorstep collection of food waste can yield. Having stressed the higher contribution of food waste to the highest recycling targets met in doorstep schemes, we still have to consider the implications of its higher capture on the collection methods for residual waste, its simplified features and cost-optimisation. This can actually lead to optimised and cost-competitive schemes, and evidence will be provided further on.

14.4 ‘Biowaste’, ‘Vegetable, Garden and Fruit’, and ‘Food Waste’: Relevance of a definition on the performance of the waste management system In Germany and Austria, the fraction targeted by the source separation system is referred to as ‘Bioabfall’ (biowaste), which means a mixture of food scraps and garden waste; in the Netherlands, in Belgium (Flanders) and in many sites in Germany and Austria, the definition ‘groente-, fruit- and tuinavfal’ (GFT) or ‘vegetable, garden, fruit’ is used, addressing a mixture of garden waste and the food waste portion before cooking (not including, for instance, meat and fish scraps … ). This choice is due to the troublesome, highly fermentable nature of cooked food residues. On the other hand, we have to underline that the recycling of dry fractions and packaging materials (paper, glass, plastics and so on) determines – as an undesired side-effect – the concentration of the fermentable material inside ‘RW’, if food stuffs are not effectively sorted through high-capture systems; this is what actually occurs in those countries (Germany, Holland, Austria and so on) even though the source separation of biowaste has already evolved considerably in these locations. This means, in those countries, the separation of dry recyclables is likely to be more effective than that of food waste. For instance, in the Netherlands and Germany, the percentage of food waste inside ‘RW’ is often reported at 30−50% [5, 6]. When transferred to warmer climates, as in the Mediterranean area, this system would keep the need for the frequent collection of RW. Moreover, in central Europe, in the ‘biobin’ (the bin supplied to households to separate biowaste), a large proportion of garden waste can be found (up to 80−90%, wet weight basis, out of the total bin content) in addition to food waste. The delivery of garden waste is greatly encouraged as households, even in detached houses with

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gardens, are provided with large-volume bins that hold bulky materials found in garden waste. We would now like to focus on the possibility of adopting a different scheme for the collection of compostable organics, in which the collection of food waste and that of garden waste are kept separate. This means, one collection route has to target only ‘food waste’ as a whole (including cooked stuffs such as meat and fish), by means of small volume bins and buckets, and a different system targets garden waste only. This distinction between the two collection schemes takes into account: – The nature of food scraps (high putrescence and moisture). This asks for the adoption of specific tools, systems and collection frequencies in order to ensure the system is clean and ‘user-friendly’. When people feel comfortable, the overall participation is enhanced. This leads to better quality and higher quantity collections, and lowers the percentage of food stuffs inside the RW, making it possible to collect it less frequently. In effect, analytical measurements, where a door-todoor (DtD) collection is adopted, report the content of food stuffs inside RW at an average of 15% and even less [2], which is much lower than in previous source separation programmes across Europe (see numbers reported above). – The different biochemical and seasonal patterns of the food scraps compared with garden waste. In Italy, where a DtD collection for food waste is adopted and, in contrast with what is generally being done in central Europe, the collection of garden waste, which does not smell, doesn’t attract flies and rodents, and does not produce leachate, adopts different schemes and tools compared with that for food waste. This in turn makes an overall optimisation of the scheme possible, as ‘intensive’ features of the collection of food waste (high frequencies due to climatic conditions, watertight bags) do not apply to garden waste, which doesn’t need such intensive, expensive collection patterns. It is also possible to adapt the volume of the total bin/vehicle to fit the specific production of food waste, which does not show huge seasonal fluctuations as for garden trimmings; systems used for garden waste, on the contrary, can be seasonally adapted. – The different bulk density of garden and food waste. This compels the use of compacting vehicles (packer trucks) to collect garden waste, while in the case of food waste (which shows a much higher density) compacting vehicles can be replaced by small bulk lorries which are much cheaper at an equivalent working capacity. This is one of the most powerful means to optimise the operational features and costs related to systems for the source separation of compostable waste. A system that does not set any difference between food and garden waste is a system where a huge delivery of garden waste is to be expected. It is noteworthy that in central Europe, where a DtD bin collection for compostables is in place, an overall organic waste collection of some 200−250 kg/inh/year and more has often been recorded. This is due, above all, to the ease of delivering garden waste to the collection service (households are allowed to deliver it in the same bins adopted for food waste collection, even in detached houses with private gardens, which makes the percentage of

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garden waste, out of the total compostable waste collected, much higher). The general outcome is a high recycling rate, but the overall MSW production figure is also much higher. In such situations, it is common to record an overall MSW production of some 500−600 kg/inh/year. The same has been already reported in a few situations in Italy where similar collection systems have been adopted [7, 8]. Such a situation causes recycling rates to rise, but also increases the overall quantity of waste to be collected and treated. One should, for instance, mention the case of Forte dei Marmi (Tuscany) which after having implemented a curbside collection for garden waste, reached 462 kg/inh/year garden waste collected in 1998, though it led to an unfavourable 850 kg/ inh/year figure for the total waste generated [8]. Deliveries are much lower where the collection of garden waste is performed through transporting systems to Civic Amenity Sites, or by means of collection at the doorstep, but with much lower frequencies (once monthly). Such systems require, among householders, a certain attitude to participate in home composting programmes, as delivery is not made extremely easy, as it would be, on the contrary, with bins at the doorstep of detached houses with gardens.

14.5 The importance of biobags Due to its fermentable nature and high moisture content, maintaining the source separation of food waste, primarily by households, requires determining the best way to solve the specific troublesome features of such a material. In this respect, a comfortable feature of the service, where households are provided with tools to avoid nuisance, will result in an enhanced participation and will thus determine higher collection quantity/quality [3]. The answer to this problematic issue has been a ‘bespoke food waste collection’, which typically includes: – A relatively ‘intensive’ collection schedule (1–3 times a week; it has to be noted that in Southern Italy, as in Spain, Portugal and so on, collection is scheduled up to 6−7 times a week; in Northern Italy the collection for MSW is usually 3 times/ week). – The use of ‘DtD’ collection systems so they are more ‘user-friendly’ and enhance – participation. – The use of watertight, transparent though compostable tools to hold the waste (‘biobags’). In order to allow people to feel comfortable with the biowaste collection service, municipalities usually provide them with small watertight bags for food waste. The use of the bags: – Prevents insect proliferation and leachate production and keeps the bins clean; therefore, enabling the lower frequency for washing rounds. Actually, in many cases, bins are washed by householders themselves; in other instances the public

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 Enzo Favoino

cleansing service provides bin-washing at a much lower frequency than would be possible without using the bags. Avoids nuisances generally related to the delivery of ‘loose’ material inside the bin, makes it possible to collect even meat and fish scraps along with vegetables and fruit residues. Increases the capture of food stuffs, which in turn allows a significant reduction in the collection frequency for RW. The small bag size prevents bulky materials (e.g., bottles and cans) from being included in the collection, ensuring higher purity. The transparency of the bags allows an easy quality control of the material captured and defines the need for further information to be forwarded to households (e.g., in particular neighbourhoods).

The ‘biobag’ is then placed: – Directly on the roadside on the collection day, either as it is or, most frequently, inside the family small bin (6.5 litres); this system is often adopted in small towns and villages to reduce the pick-up time for each dwelling and to prevent households from delivering garden waste inside the bins. – In a bigger wheelie bin (cart) whose capacity usually ranges from 80−240 litres for 10−20 families depending on the collection frequency; this system is under adoption where dwellings are in high-rise buildings.

14.5.1 Features of ‘Biobags’: The importance of biodegradability and its cost-efficiency In general, it is possible to use polyethylene (PE) or biodegradable bags; as a matter of fact, both are used nowadays. Nevertheless, the biodegradable bag does not interfere with the composting process as it degrades during the composting cycle; whereas PE bags can be only used if the composting plant where the biowaste has to be delivered is provided with: – A pretreatment section (in general, a bag opener plus primary screen). – An aeraulic facility or equivalent in order to separate the nonbiodegradable plastic fragments. The separation itself of course is not 100% effective, and often compels plant managers to shrink the sieving size so as to get rid of the small PE fragments; very often screening holes are kept at 10 mm or less, whereas with biodegradable bags it is possible to screen at 12−15 mm and more (depending on the targeted use). But shrinking the screen holes leads, in turn, to a dramatic product loss, as many composted material particles are rejected and go to disposal. As for the rejects themselves, (which are on a weight basis and mainly comprised of wooden materials not yet degraded) recycling

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becomes much more difficult as the waste is contaminated with plastics that get more and more concentrated. This is why composting plants will only accept PE bags, if ever, with much higher tipping fees. The average additional cost related to the use of biodegradable bags (for the time being, in Italy, mainly corn starch-based materials with 6.5 or 10 litres unit volumes) is approximately €5 per tonne collected; this has to be compared with additional operational costs and fees (approximately €15−20 per tonne) incurred by composting plants when biowaste is delivered in PE bags. In both cases, a transparent bag allows an easy quality check. As a consequence, the waste hauler might reject the bag if it does not meet the quality criteria demanded by the composting plant. Furthermore, as we have already highlighted, it is possible to define the troublesome issues to be addressed in further information campaigns to improve collection quality. It has to be mentioned that the Italian law now stipulates that once bags are used for the collection of organic waste, such bags have to be biodegradable/compostable. In order to avoid any misuse of the term ‘biodegradable/compostable’ (and the subsequent introduction into the system of bags that are not truly and effectively compostable), an ancillary requirement should always stipulate that bags must comply with the EU standard, European Norms, EN 13432. The use of biobags is a very effective means to enhance participation and reduce collection costs. It also has to be mentioned that in some municipalities, watertight biodegradable liners have been adopted in bins to further prevent them from getting dirty; thus the goal to avoid expensive washing rounds is fully achieved. The liner is then collected with the biowaste itself as the collection truck empties the bin. In such situations, an average cost for the liner placed to line the bin is approximately €0.5 per bin for each collection round, including the manpower; but this makes it possible to save the much higher costs related to bin washing (approximately €1.5−2.5 per bin).

14.6 Cost assessment of optimised schemes One of the major waste management concerns across Europe is the lack of cost- competitiveness of source separation systems which aim to reach high recycling rates, as compared with the traditional mixed MSW collection. Operators, in general, think that sorting food waste leads to higher costs of the overall collection scheme. Cost analyses carried out to date across Europe have traditionally focused on costs per kilogram (or per tonne) for the collection of a single waste material. However, there is evidence that this biases the picture, because the more waste collected, the lower the costs of the collection service per kilogram. This distortion obscures some important outcomes of integrated source separation and waste management: – The reduction of total waste delivered as a consequence of effective waste reduction policies.

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The much lower delivery of industrial waste to the MSW collection route where large-volume road containers are substituted by curbside low-volume bins and bags. The contribution of home composting programmes to the overall reduction of collected organic waste.

Furthermore, evaluation of the cost for a single waste flow, does not allow one to compare its cost to the likely advantages of collection costs for other materials, flowing from ‘operational integration’. In effect, the collection of food waste, above all when it shows high capture, allows important changes in the collection scheme, by reducing, for instance, collection frequency for RW. It is therefore incorrect to express the cost of the service per kilogram collected, rather it should be expressed as cost per person. Once an overall cost of a certain scheme is given, the municipality could only be happy with lower deliveries which would, on the contrary, make the cost per kilogram higher! This is why we shall focus on cost per person. In order to allow a comparison among different collection systems, the Research Group on Composting and Integrated Waste Management at Scuola Agraria del Parco di Monza has conducted some surveys on the costs of different collection systems run in Italy [9], grouped by their main features and above all according to the way food waste is separated (or not). The three system groups can be described as follows: – Traditional source separation, based on the use of plastic bags or road containers (up to 3.3 m3) for mixed MSW and source separation through road containers for dry recyclables (paper, glass and plastics) only. The food waste is not sorted and it’s delivered along with the mixed waste; this holds pretty fermentable (actually, food waste gets concentrated in it due to the withdrawal of paper, board, glass, plastics and so on) and has to be collected frequently. – Intensive source separation, including that of food waste based on road containers (120−240 litres, up to 3.3 m3) both for food waste and dry recyclables; collection of the RW is achieved through road containers. This is usually referred to as the ‘double container’ collection (beside that for RW, households find the one designated for food waste). It’s pretty diffused in central Italy (Emilia, Tuscany) and has also been the most diffused, so far, in Spain. – Intensive source separation, including that of food waste, with DtD (also worded as ‘collection at the doorstep’) collection for food waste and RW. In general, some high-yield dry recyclables are also collected with a collection at the doorstep (usually paper and board, due to the much higher capture per person than with road containers). It’s the most diffused system in those municipalities and provinces where the highest recycling rates have been met (up to 70% in single municipalities). The system is well diffused in central Europe, as well, though the ‘Italian version’ adopts buckets in place of bins in the case of detached houses with gardens (to prevent deliveries of garden waste and reduce the unit collection times, as already mentioned).

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Surveys have led to some important outcomes: e.g., data from Region Lombardy [16] (Figure 14.1), show that schemes maximising separate collection rates, thanks to the intensive collection of organics, can be run with no substantial increase in cost for collection, relative to traditional collection (no segregation of food waste) or food waste segregation by means of road containers (in order to lower the number of pick-up points). The fact that optimised, intensive collection at the doorstep does not imply an increase in the cost of collection, leads then to benefits in terms of reduced costs of disposal, with a net decrease in the total cost of waste management.

Cost of collection (upper bars) and cost of treatment/disposal (lower bars) € 90

cTRAT/abeq

€ 80 cRACC/abeq

€ 70

Euro/person

€ 60 € 50 € 40 € 30 € 20 € 10 €0

70%

Figure 14.1: Cost comparison (€/inhab/year) for municipalities achieving different rates of separate collection (Region Lombardy, statistical basis: 1,547 municipalities grouped into different percentage classes). cTRAT: Cost of treatment per person equivalent; and cRACC/abeq: cost of collection per person equivalent [16].

Such a paradox requires, of course, a further detailed insight into the issue in order to understand which tools are best suited to optimise operational and cost features. Actually, we have consciously developed such tools since the very beginning in order to ensure the steady development of source separation as a cost-effective strategy across Italy; features of optimised schemes have also been adopted in national guidelines such as those reported in the National Handbook on Source Separation issued by the Italian National Environmental Protection Agency (Agenzia Nazionale per la Protezione dell’Ambiente) [10].

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14.6.1 Tools to optimise the schemes and their suitability in different situations To understand such unexpected outcomes, we must underline that if the source separation of food waste is added to that of commingled municipal waste, with no modification in the previous scheme for MSW collection, total costs are bound to rise; this actually happens with the segregation of food waste by means of road containers. But this does not happen when food collection is integrated into the overall collection scheme: namely, when schemes for collection at the doorstep are implemented. The trick is that intensive doorstep schemes for food waste, when made ‘comfortable’ for households, yields high capture. This cuts in turn the percentage of food waste in the RW, which can then be collected less frequently. Furthermore, food waste on its own needs no compaction, letting operators use cheaper collection vehicles; this holds true in those schemes where the delivery of garden waste with food waste (which is pretty high in areas with detached housing and private gardens) is prevented by means of low-volume buckets which allow households to deliver only their food waste.

14.6.1.1 Collection frequency for residual waste Obviously, collection frequencies for RW can only be cut when an effective separation of foodstuffs, yielding high capture, is run. Considering such a viewpoint, we have to mention (Table 14.3) that doorstep schemes enable a much better performance. A range of 170−250 g/person per day has been reported for food waste; outcomes tend to be higher in southern regions, thanks to a higher presence of food scraps in municipal waste (in addition, initial numbers reported in Catalunya, Spain, confirm the high capture). Large road containers yield much lower quantities; their capture is actually sometimes similar to doorstep collection, but a high percentage of garden waste contributes and the actual capture of food waste is low. We could therefore assume that ‘collection using road containers results in a lower participation rate’; which is quite obvious due to the higher average distance between households and the container. Cutting down collection frequencies for RW constitutes in itself one of the most important tools to optimise schemes for the source segregation of food waste. Its use is particularly effective in those areas where high collection frequencies are in place for traditional, mixed MSW collection (mostly Southern Europe). The following scheme (Table 14.4) shows the typical collection frequencies for mixed MSW and for ‘integrated’ collection systems where food waste is segregated. Frequencies applied in Southern Italy work perfectly in many Mediterranean situations, as well, where a mixed collection is traditionally run 6 times/week.

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14.6.1.2 Diversifying the fleet of collection vehicles Where doorstep schemes for food waste are in place, using small bags to be delivered in bins (for high-rise buildings) or small buckets (for single families in houses with gardens), a material with a high-bulk density (0.5−0.7 kg/litre) can be collected using bulk lorries instead of packer trucks. Table 14.3: Performances of different collection schemes for biowaste in Italy. System

Overall yield (typical) Garden waste %

Actual capture of food waste

DtD

170−250 g/ inhab/day

0% (where delivery is banned) to 10 % (maximum, due to low available volumes)

160−250 g/inhab/day

40−70% (seasonal)

60−120 g/inhab/day

Road 150−200 g/ containers inhab/day

Adapted from E. Favoino in the Proceedings of the Biodegradable Plastics 99 Conference, Ed., A. Beevers, Frankfurt, Germany, April 1999, European Plastic News, Croydon, UK, 1999 [3] and Provincia di Milano, Produzione, Smaltimento, Raccolte Differenziate Anni 1996/97, Milan, Italy 1998 [11]. Table 14.4: Frequencies for the collection of different types of household waste. Area

Mixed MSW (with Food waste no segregation of (both with DtD food waste) schemes and road containers)

RW in DtD schemes (frequencies cut down, thanks to the high capture of food waste)

RW in road container schemes (no difference from previous mixed collection)

Northern Italy

3 times weekly

2 times weekly (sometimes once weekly during wintertime)

1−2 times weekly

3 times weekly

Southern 6 times weekly Italy

3−4 times weekly

2−3 times weekly

6 times weekly

These are only suitable when schemes effectively prevent the delivery of garden waste with food waste. So it is advisable to limit the size of containers supplied to households where gardens are available (6−10 litres, up to 30 litres); bins (80−240 litres) should only be supplied to high-rise buildings to serve many families with a single pick-up. The use of buckets in detached houses also requires much less time per pick-up point (20 to 40 seconds on average, while bins require 2 to 3 min) as bins have to be hung up on the loading device, then unloaded and put back in their place: too time-consuming, for a single household, as compared with the simple, quick action of picking up and emptying a bucket manually. Assessment of course leads to a different

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outcome if we consider high-rise buildings, where a single bin can serve up to 10−20 families, thus making the single pick-up much more time-effective. Households can manage garden waste through: – Home composting, promoted effectively by the municipality. – Delivery to local recycling centres (‘Déchetteries’ in France, ‘Civic Amenity Sites’ in the UK, ‘Recyclinghöfe’ in German-speaking countries, usually worded as ‘Piattaforme Ecologiche’, or ‘Ecocentri’ in Italy). – The specific collection of garden waste at the doorstep with low frequency (e.g., once monthly, only in the growing season, in general April−October). We could therefore say that collection rounds for food waste will have reduced costs through the use of low-tech vehicles and time-saving containers. In our surveys, we calculated and determined that a two-shift scheme for food waste collection using bulk lorries tends to equal the cost of a single-shift collection for RW with packer trucks (Table 14.5). This is partly due to the higher cost of a packer truck itself and partly due to the much longer time spent on each pick-up point. Table 14.5: Costs of collection routes (€/inhab/year) for food waste and RW in DtD schemes. Municipality (Province) Calcio (Bergamo)

Population Cost for collection of RW (once Cost for collection of food waste weekly, with packer trucks) (twice weekly, with bulk lorries) 4,765

5.14

4.21

Caravaggio (Bergamo)

14,181

5.46

6.01

Sommacampagna, Sona (Verona)

26,036

7.28

8.88

14.7 Conclusions According to the numbers shown, it is clear that the main mistake made when planning sorting schemes, is the ‘added’ nature of the scheme; which means, a new collection scheme is run in addition to the previous mixed MSW collection, and cannot therefore yield savings to fund a new scheme. It is vital that the new separate collection is integrated into the established waste management system, e.g., changing frequencies and volumes of RW collections, providing the collection of food waste yields high capture through a comfortable scheme. Furthermore, ‘integration’ has to take into account the features of the area where the scheme is to be put in place; above all considering the need to find specifically suited systems for food and garden waste, where a large amount of garden waste is expected (areas with many gardens). We have to remember that collection frequencies of RW can be cut only where a high capture of food waste reduces the fermentability of RW. From such a standpoint, the use of

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comfortable tools such as watertight, biodegradable bags has proven to be very effective. This is why an ‘intensive’ collection, run through DtD schemes, notwithstanding a much higher number of pick-up points, has be shown to be suitable for cost-optimisation, thanks to the integration of the system and much lower collection costs for RW. Collection of food waste at the doorstep allows municipalities to achieve much higher recycling rates (even topping 60−70% and more in small municipalities, 50% in Monza, with a population of approximately 120,000) and a much better quality of collected food waste [12–14]. A further tool to optimise the scheme is the use of suitable vehicles to collect food waste, due to its high bulk density when garden waste is kept out of the collection scheme for food waste.

References  [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]

Scuola Agraria del Parco di Monza, Analisi Merceologiche su FORSU Prodotta nel Comune di Milano − Campagna di Analisi 22−23 Gennaio 2013, Report to AMSA, Milan, Italy, 1999. [In Italian] Provincia di Milano in Il quaderno: Gestione Rifiuti Solidi Urbani 1998, Indirizzi Programmatici e Azioni di Approfondimento, Milan, Italy 1998. [In Italian] E. Favoino in Proceeding of the Biodegradable Plastics 99 Conference, Frankfurt a/M, Germany, Ed., A. Beevers, European Plastic News, Croydon, UK, April 1999. F. Girò in Proceedings of the 2nd National Conference on Composting: RICICLA 2000, Rimini, Italy, 2000. K. Wiemer and M. Kern in Abfall-Wirtschaft: Neues aus Forschung und Praxis, Witzenhausen, Germany, 1995. Baden Baden Amt für umweltschutz in Versuchsergebnisse Restmüllaufbereitung, Provate Communication, 1996. [In German] Legambiente in Comuni Ricicloni 1997: Standings of the National Award to Highest Municipal Recycling Rates, Rome, Italy, 1998. [In Italian] Legambiente in Comuni Ricicloni 1998: Standings of the National Award to Highest Municipal Recycling Rates, Rome, Italy, 1999. [In Italian] Scuola Agraria del Parco di Monza in Proceedings of the Comuni Ricicloni 1999: National Award to Best Performing Municipalities in Source Separation, Rome, Italy, July 1999. [In Italian] Agenzia Nazionale per la Protezione dell’Ambiente in La Raccolta Differenziata – Aspetti Progettuali e Gestionali, ANPA − Osservatorio Nazionale Rifiuti, Rome, Italy, 1999. [In Italian] Provincia di Milano, Produzione, Smaltimento, Raccolte Differenziate Anni 1996/97, Milan, Italy, 1998. [In Italian] Consorzio Provinciale della Brianza Milanese, Rapporto Sulla Gestione dei Rifiuti Urbanied Assimilati: Anno 1997, Seregno, Italy, 1997. [In Italian] E. Favoino in Proceedings of the Jornadas Sobre Compostaje, La Rioja, October 2000 Provincia di Lecco, Rapporto Sulla Produzione di Rifiuti Solidi Urbani e Sull’andamentodella Raccolta Differenziata, Lecco, Italy, 1997. [In Italian] M. Giavini, C. Garaffa, S. Cantoni and E. Favoino in Proceedings of the ISWA Annual Conference, Vienna, Austria, 2013 Regione Lombardia in Valutazione Statistico – Economica dei Modelli di Gestione dei Rifiuti Urbani in Lombardia, Milan, Italy, 2010. [In Italian]

Christian Garaffa and Francesco Degli Innocenti

15 Collection of biowaste with biodegradable and compostable plastic bags and treatment in anaerobic digestion facilities: Advantages and options for optimisation 15.1 Introduction The main goal of this chapter is to analyse situations where items made of bioplastics (mainly biodegradable and compostable waste bags) interact with different state-ofthe-art anaerobic digestion (AD) technologies treating municipal source separated biowaste. This chapter will demonstrate that, despite the heterogeneity of AD technologies and processing conditions, an efficient and optimised treatment of municipal biowaste collected with compostable plastic bags is generally possible, hence preserving the advantages given by the bags in the collection phase and at the same time securing the most efficient treatment of the collected feedstocks to obtain the highest inputs and minimum production of residues. The first part of the chapter presents the background on the increasing importance of biowaste in the context of European waste and renewable energy strategies and policies, followed by an overview of the role of compostable plastic bags in municipal source separation schemes and their contribution to creating a higher acceptance of the collection service by householders, higher capture (especially of food waste) and lower contamination rates of the collected feedstocks. The third part addresses the interactions between compostable plastic bags and AD technologies with a discussion about organic recycling standards and the general performance of compostable bags in AD. The fourth and last part of the chapter will briefly describe the range of current state-of-the-art AD technologies used for the treatment of municipal biowaste in Europe today, followed by the description of some case studies of industrial AD facilities processing biowaste collected in compostable bags.

https://doi.org/10.1515/9781501511967-015

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15.2 Current European policies regarding biowaste, renewable energy, emission reduction and resource management Biowaste as a raw material and as a renewable energy source is gaining higher status in European waste strategies and policies. European Union (EU) legislation has been promoting the landfill diversion of biowaste since the end of the 1990s [1] and recently has started openly encouraging its source separation for recycling. In 2008, the European Commission (EC) published a Green Paper on the Management of Biowaste in the EU [2] and a subsequent Impact Assessment [3] identified significant environmental and economic benefits from improved management of biowaste in the EU, concluding that the market for quality compost could be increased by a factor of 2.6 to reach about 28 megatonnes (Mt). This in turn could help to improve the quality of depleted agricultural soils in Europe by 3 to 7%. Maximising composting could also substitute 10% of phosphate fertilisers, 9% of potassium fertilisers and 8% of lime fertilisers. Biogas from biowaste as a vehicle fuel could contribute to one-third of the 2020 EU target for renewable energy in the transport sector, set by the directive on the promotion of the use of energy from renewable sources [4]. If all the biowaste produced in the EU in 2020 were treated by AD and the gas used by public vehicle fleets, the potential gain would be around 13 Mt carbon dioxide equivalents (CO2 e) [5]. The Impact Assessment was followed in May 2010 by the ‘Communication on future steps in biowaste management in the European Union’ [6]. In this document, the EC states that a better alignment of the management of biowaste with the waste hierarchy and other provisions of the revised Waste Framework Directive could result in environmental and financial benefits of €1.5 billion (moderate increases of recycling) to €7 billion (ambitious recycling and prevention policies). Furthermore, ‘Composting and AD offer the most promising environmental and economic results for unpreventable biowaste. An important precondition is a good quality of the input to these processes. This would, in the majority of cases, be best achieved by separate collection. Member States should make strong efforts to introduce separate collection in order to meet high-quality recycling and AD’. Biowaste has now been fully recognised as a resource for valuable raw materials and renewable energy, and correctly put into the broader context of resource management by the ‘Roadmap to a resource efficient Europe’ [7]. In this document, the EC openly declares that the era of plentiful and cheap resources is over and that a transformation process needs a policy framework rewarding innovation and resource efficiency, creating economic opportunities and improved security of supply. This can be achieved by redesigning products and the sustainable management of environmental resources, promoting the reuse and recycling of products and materials, hence saving resources. In this context, waste has to become a resource to be fed back into the economy as a raw material with a specific need to give reuse and recycling

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a much higher priority. A combination of policies would help create a full recycling economy, such as product design integrating a life cycle approach, better cooperation between all market actors along the value chain, better collection processes, appropriate regulatory framework, incentives for waste prevention and recycling, as well as public investments in modern facilities for waste treatment and high-quality recycling. The EC will ensure that public funding from the EU budget gives priority to activities higher up the waste hierarchy as defined in the Waste Framework Directive (i.e., priority to reuse and recycle over energy recovery, and lowest priority to waste disposal in landfills) and facilitate the exchange of best practices on the collection and treatment of waste among Member States. The biowaste management guidelines issued by the German federal state of Baden-Württemberg [9] identify four areas of improvement and optimisation for the best exploitation of municipal biowaste and green waste from agricultural activities (Figure 15.1). Area 4 Gas pipeline Biowaste

Combined heat and power Biogas production

Area 2

High Yields High Emission Standards

Green waste

High Yields High Emission Standards

Wood chips

Area 3

Compost production

Mature Compost High Emission Standards

Area 1

Figure 15.1: The green waste and biowaste recovery system: four areas for optimisation. Reproduced with permission from F. Knappe, R. Vogt, T. Turk, Hüttner, G. Dehoust and T. Schneider in Optimierung des Systems der Bio- und Grünabfallverwertung, Ein Leitfaden, Ministerium Für Umwelt, Klima und Energiewirtschaft Baden-Württemberg, Stuttgart, Germany, 2012. ©2012 [9].

To minimise the environmental impact, a short delivery distance of the produced compost to a close market and several potential customers needs to be guaranteed

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(Area 1). For optimal energy use, heat needs to be efficiently exploited all year round along with electric power, by placing the combined heat and power (CHP) unit as close as possible to the final heat user (Area 2). Mitigation of greenhouse gas (GHG) emissions (methane (CH4), nitrous oxide (N2O)) and ammonia must be assured through high emission standards and proper digestate treatment with the production of fully aerobically stabilised compost (Area 3). Finally, an organic recovery system able to guarantee the best efficiency in the three above-mentioned areas must be supported by a collection system able to guarantee the highest participation and capture rates (Area 4). Appropriate waste fee structures, logistics, collection and communication tools are all key factors in determining the success of a waste collection scheme. Among the collection tools, compostable bags, which are certified according to recognised international standards as the European EN 13432 or the American ASTM D6400, play an essential role.

15.3 The role of compostable plastic bags in biowaste source separation schemes To reach the challenging targets described above, a key issue is the ability of municipal biowaste collection schemes to maximise the collection of potential feedstocks by securing the highest participation and capture rates. Another essential factor is the ability to secure the best possible quality of the collected biowaste by keeping contamination low, because the presence of noncompostable materials in the feedstock, e.g., conventional plastic bags, generates higher treatment and disposal costs and has a related environmental impact. Today, among those communities supporting the use of certified compostable plastic bags, the value of these tools for collecting biowaste, especially food scraps, is well recognised. Compostable plastic bags make it possible to keep high participation, high capture rates and lower contamination levels, both in areas of single-family houses and areas with a denser population and multifamily buildings. The advantages of compostable plastic bags are summarised below [10]: – Compostable plastic bags merge the strengths of conventional plastics (watertight, flexible and mechanically strong) with those of paper (compostable). – Make the source segregation of food scraps cleaner in the household (North Americans would say they reduce the ‘yuck factor’). – Favour higher participation rates, especially where collection programmes are voluntary, the use of liners can be a critical factor for the active participation of property managers and householders. – Favour higher capture rates: user friendly systems help in diverting more food waste from residual waste. – Without liners the householders tend not to put wetter food wastes into the kitchen containers.

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–

– – – –

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In multifamily buildings, liners make it possible to drop the food scraps on the way out (e.g., going to work) without the need to return the kitchen container. Less frequent washing of kitchen containers and outdoor bins is required. Liners can also extend the life of kitchen containers and outdoor bins. Compostable bags made with specific bioplastics containing native starch have high water vapour transmission rates and are therefore breathable. In combination with vented kitchen containers they allow water evaporation and the drying of food waste, hence reducing fermentation processes and the production of unpleasant odours in the household. By losing up to 15% water and weight during storage in the vented kitchen container, breathable liners act as a waste reduction tool, minimising the organic waste to be transported. Liners are preferred by collection crews as food scraps don’t stick to the bins and all food waste is tipped easily into the collection vehicle. Collection vehicles are kept cleaner and spills are reduced. Transport and intermediate storage of residential food waste are easier to manage. Compostable plastics can act as a communication tool during outreach and education campaigns positively influencing the sorting behaviour of householders and consumers.

Studies have been carried out showing the importance of using compostable plastic bags in the separate collection of biowaste. This has been proven in different countries such as the UK [11, 12], Spain [13], Norway [14], Austria and in the region of Bayern in Germany [15]. In Switzerland, compostable plastic bags are actively promoted by the main national energy and AD company (Axpo Kompogas AG) [16, 17]. Worldwide, Italy is the country with the most extensive use of compostable plastic bags. In 2010, 257 industrial composting facilities and 23 AD facilities treated about 4 Mt of biowaste; 2.5 Mt was food waste of which 1.9 Mt was composted and 0.6 Mt digested. In 2011, over 24 million people were connected to a food waste collection scheme [18]. The most successful biowaste collection schemes are able to capture around 90% of available food scraps, leaving less than 10% of putrescible waste in the residual fraction. In addition to appropriate collection frequencies, the highest performing systems rely on pay-as-you-throw pricing structures, clear communication and household tools to make participation easy such as kitchen bins and certified compostable bags.

15.4 Compostable plastics in anaerobic digestion: Standards and performance The European standard, European Norms (EN) 13432 [19] defines the characteristics which packaging must demonstrate in order to be considered suitable for organic

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recycling; for nonpackaging plastic items the exact same characteristics are defined in the European Standard EN 14995 [20] (EN 14995 Plastics, Evaluation of Compostability: Test Scheme and Specifications). Other European packaging standards similarly define the suitability of a packaging material for recycling (EN 13430) [21] and energy recovery (EN 13431) [22]. The standards EN 13432 and EN 14995 determine the compostability and anaerobic treatability of packaging and plastics, respectively. Plastic and packaging items that meet the requirements of the European standards can be recovered via organic recycling, i.e., composting and AD. To comply with either standard, products must show enough biodegradability, disintegrability and absence of ecotoxic effects. Compostable plastic bags for the collection of biowaste are today one of the most successful applications of certified compostable materials. Organic recycling is achieved by converting original waste into compost, a product that can be used in agriculture or in horticulture to improve soil quality. Compost is produced directly through a sole composting stage or through a previous fermentation step (AD) followed by the aerobic stabilisation of the resulting digestate. In the second case, transit through the anaerobic reactor can give rise to anaerobic biodegradation with biogas production. Disintegration and biodegradation are completed during the two stages providing mature compost and biogas. The behaviour of compostable plastics in anaerobic environments varies depending on the temperature (i.e., thermophilic/mesophilic), solids retention time, composition of the material, and shape and thickness of the compostable items being tested. EN 13432 provides guidance on how to measure anaerobic degradation as a further characterisation of the tested material if the specific potential contribution of biogas production from the bags is of interest. In a study published in 2007 by the California State University Chico Research Foundation [23], several materials were evaluated under anaerobic conditions for 43 days and characterised with methods established for the digestion of food waste. The biogas production changed considerably from material to material, ranging from 0.8 litres/g of volatile solids (in the case of bags made from polyhydroxyalkanoate, a polymer extracted from bacteria) to about 0.2 L/gVS (in the case of Kraft paper or starch-based bioplastic bags). This implies that most polyhydroxyalkanoates are biodegraded and converted into biogas during the first stage (with scarce contribution to the formation of compost, but with a high biogas recovery), while biogas production from paper and starch-based materials is limited, which benefits compost production. As previously mentioned, packaging (either made with bioplastics or paper) and bioplastics that comply with EN 13432/14995 are expected to complete organic recycling in a full cycle that includes the anaerobic stage and the final aerobic stage (stabilisation of the digestate via postcomposting).

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15.5 Anaerobic digestion facilities treating biowaste: Technologies, pretreatment options and management of compostable plastic bags 15.5.1 Combined anaerobic and aerobic versus anaerobic only processes: Pros and cons As previously described, the recycling of biowaste by means of AD is usually carried out in two steps. Biowaste is first subjected to an anaerobic process with the production of biogas and digestate. Biogas is used as a biofuel to produce electricity and heat. The digestate is then aerobically stabilised into compost in order to obtain a high-quality product. This aerobic step ensures a complete breakdown of the organic components as well as fixing the mineral nitrogen into a humus-like fraction, which reduces nitrogen loss. Digestate, used as an additive to the composting process, provides a good source for speeding up the process. At the same time, it enriches the compost in phosphorus and micronutrients such as magnesium (Mg) and iron (Fe). In some plants, especially those which were originally designed to treat animal sludges from breeding farms and are located in rural areas, the aerobic phase is missing and fresh digestate is first stored in tanks and then spread directly onto the surrounding fields. In most European countries plants built to treat biowaste are generally designed to produce both biogas and compost because the direct spreading of digestate onto fields has some drawbacks and is strictly regulated or even not allowed in some countries. The main drawbacks are listed below: – Whole digestate is an active matrix and prone to release residual biogas (i.e., CH4) into the atmosphere, contributing to the GHG effect. – Whole digestate is a pumpable matrix and its storage requires large tanks placed directly within the treatment plant. Compost storage is more flexible with lesser environmental impact, since it is a stable, low emission product. – The aerobic phase guarantees a permanence of the digestate for a longer time at high temperatures (>60 °C) assuring further sanitisation, making the products suitable to satisfy the standards foreseen by the European Animal By-products Regulation [24]. – The digestate is more difficult to transport and distribute to users via a commercial network. Besides the process, and regulatory and logistic reasons, the biological and agronomical properties make the production and commercial use of compost preferable over the direct spreading of digestate on the fields around the AD plants.

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Compost is rich in humic substances [25] and beneficial microorganisms [26]. The addition of compost to soil affects disease incidence by means of antagonistic mechanisms [27]. Biological control, based on competition, antibiosis, hyperparasitism and induced systemic resistance in the host plant, have been described for compostamended substrates [28]. Compost improves the number and functions of nitrogen fixers and vesicular-arbuscular mycorrhizae [29], which have positive effects on the physical properties of soil [30] and improves the organic matter content, contrasting soil desertification [31]. On the other hand, digestate can be considered a mixture of water, partially degraded organic matter (solid polymers and short chain molecules, such as intermediate degradation products and microorganisms) and inorganic compounds. From a microbiological viewpoint, digestate is composed of anaerobic bacteria that die as soon as they come into contact with air; the other microorganisms are facultative anaerobes, such as coliforms, and do not exert any relevant role in soil microbiology; therefore, the digestate does not provide structural benefits to the soil, humic substances or beneficial microorganisms. On the other hand, it is rich in ammonia and phosphorus, and can be used as a fertiliser. However, compost has a higher slow release nitrogen content and its availability is different in comparison with digestate; a relevant point when considering the nitrate directive.

15.5.2 Dry and wet technologies The choice of the most appropriate technology and process set-up is mainly dependent upon the substrates to be treated. Municipal biowaste includes two distinct fractions: green waste from gardens and parks, and food waste from residential and commercial sources. These fractions have a different composition and structure. Green waste has a high structure and total solids (TS) content with a high lignocellulosic and fibrous component. Food waste, on the other hand, usually has a poor structure and TS 10 mm and joins the oversized fraction of the first 80 mm screening step for composting. They are mixed with the solid digestate and with the oversized lignocellulosic fraction originating from a parallel green waste composting process. The prepared mix is put into a tunnel in a static aerated pile, where a 16 day active composting phase takes place. The compostable bag fragments, separated during the screening and mash separation steps, start their degradation here. Part of the process

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BAGGED FOOD WASTE

GREEN WASTE

BAG SPLITTING

SHREDDING

SCREENING (80 mm)

ACTIVE COMPOSTING (16 DAYS)

Oversize fraction

Finer fraction

Oversize fraction

SCREENING (40 mm) MASH SEPARATION (10 mm)

Solid fraction Finer fraction

Liquid fraction ANAEROBIC DIGESTION

CURING IN TUNNEL COMPOSTING (45 DAYS)

BIOGAS

Whole digestate DEWATERING

REFINING: METAL SEPARATION, AIR CLASSIFIER, SIEVING (12 mm)

Solid digestate

Process water WATER TREATMENT

CLEAN WATER

Solid sludge

Non compostable residues

DISPOSAL

QUALITY COMPOST

Figure 15.7: Material flow at the AD and composting facility of S.E.S.A., Este (Italy).

water from the AD is added to keep the moisture constant and to evaporate part of the excess water from AD. After the active phase, the feedstock is screened again at 40 mm and the oversized fraction is sent for further active composting. The finer fraction is moved into a second tunnel for a 45 day maturation phase. After maturation, the compost is screened for

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refining. According to the S.E.S.A. facility manager, during the total composting cycle of 61 days, the compostable plastic bags are completely degraded. The case study of the S.E.S.A. facility in Este shows a situation of wet AD combined with composting where the compostable bags skip the digestion phase (Route 1 in Figure 15.3) and go to a complete material recovery (Route 2 in Figure 15.3). S.E.S.A. is a public municipal company also managing the collection of biowaste from the surrounding towns. In order to guarantee low noncompostable plastic contamination levels in the treated biowaste, the company buys compostable plastic bags and distributes them to the households.

15.6.3 Case study 3: Dry plug flow digestion: Compostable bags going partly to digestion (Route 1) and partly to material recovery (Route 2) Dry technologies started gaining a significant market share in the 1990s with plug flow systems [36]. Dry fermenters operate with shovelable or solid substrates with TS content ranging between 20 and 40%, depending on their specific composition. Dry fermenters are less sensitive to sedimentation issues and handle substrates with larger particle size compared with wet systems (between 40−80 mm). They can treat garden waste or mixtures of garden and food waste. The different structure and higher TS content of the feedstocks require a different approach to pretreatment compared with wet systems [37]. The feedstock is subjected to particle size reduction and screening. It has a high viscosity and moves via a plug flow inside the digester. With input substrates having TS >60%, part of the liquid digestate or process water is recirculated and added to the incoming material in order to bring the TS into a range between 20 and 40%. Dry plug flow technologies include, e.g., Kompogas, Dranco (OWS), Valorga and Strabag (former Linde). The dry plug flow AD and composting facility in Mondercange (Luxembourg) managed by the waste management public authority ‘Syndicat Minett-Kompost’ is here described as a case study. Syndicat Minett-Kompost also promote and sell compostable plastic bags for food waste collection to 185,000 residents of 21 municipalities in the southern part of Luxembourg [38]. In 2011, Minett-Kompost added a new AD facility, based on technology supplied by the company Strabag Umweltanlagen GmbH, to an already existing 20,000 tonnes/ year composting facility based in the town of Mondercange. The plant treats mixed green and food waste with a capacity of 30,000 tonnes/year including the pretreatment and postcomposting stages. The capacity of the sole digester is 25,000 tonnes/ year. In the winter months, when municipal green waste production is low, up to 4,000 tonnes/year of energy crops (silage) can also be used. The ‘Syndicat Minett-Kompost’ also promotes the collection of biowaste in the 21 municipalities in the southern part of Luxembourg. In order to keep a high par-

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ticipation in the scheme and a good quality of the collected material, in 1999, the Syndicat introduced specifically labelled compostable plastic bags for the collection of residential food waste. Food waste bags and liners of different capacity (10, 20, 120 and 240 litres) along with 60 litre garden waste bags are distributed in different local supermarkets. Also, certified compostable food service ware items, listed on the website of the public waste management company, are accepted in the biowaste bin [39]. The Syndicat Minett-Kompost also organises a yearly promotional event using compostable food service ware and promoting the use of compostable plastic bags. Figure 15.8 shows the material flow in the Minett-Kompost facility. The collected biowaste is delivered in the waste reception area and sent to pretreatment through a walking floor conveyor system. After size reduction, the substrate is sieved at 80 mm by a drum screen and the oversized fraction (predominantly lignocellulosic) is sent directly to the postcomposting stage and used as structural material by mixing it with the digestate. Some of the shredded compostable plastic bags follow this material recovery route (Route 2 in Figure 15.3). The finer fraction is delivered to boxes where a short aerobic degradation and hydrolysis step of 2−3 days takes place. Preaeration buffers and homogenises the substrate, thus avoiding the build-up of lower organic acids that usually inhibit the start-up of the fermentation process. It also promotes the breakdown of the lignocellulosic materials, making them more readily available for microorganisms in the subsequent fermentation phase. In addition, the smaller fragments of compostable plastic bags start breaking down during the preaeration step. After hydrolysis, a second size reduction to 106 years

Fossil Resources (oil, coal and natural gas) -- OLD CARBON Rate and timescales of CO2 utilisation is in balance using biobased/plant feedstocks (1–10 years) as opposed to using fossil feedstocks - zero material carbon footprint short (in balance) sustainable carbon cycle using biobased carbon feedstock Figure 16.4: Illustration of the zero material carbon footprint using plant/ biomass resources. Reproduced with permission from R. Narayan, Biobased & Biodegradable Polymer Materials: Rationale, Drivers, and Technology Exemplars, American Chemical Society Symposium Series 1114, 2012, p.13 and American Chemical Society Symposium Series 939, 2006, p.282. ©2006, American Chemical Society [1].

rate and timescale of carbon sequestration to fossil resources (oil, coal, natural gas) is millions of years, whereas the use and ultimate disposal is in the 1−10 year time frame. This makes the use of fossil carbon resources out of balance and unsustainable. This represents the fundamental, intrinsic value proposition for using biobased carbon from plant/biomass, agricultural crops/residues and algae as opposed to fossil carbon resources. The zero material carbon footprint is attributable solely to the carbon’s origins (petro/ fossil versus plant/biomass) and does not address the carbon and environmental footprint arising from the conversion of the feedstock/resource into the product, its use and disposal, and ultimate release of carbon emissions back to the environment  −  the process carbon environmental footprint. Life cycle assessment (LCA) methodology and standards [16] (ISO 14040 standards) are the accepted tools to calculate the process carbon and environmental footprint. Unfortunately, LCA focuses almost exclusively on the process (carbon and environmental) footprint. The impact of the origins of the carbon present in the product, the material carbon footprint, is neglected or treated as feedstock energy or embodied carbon energy for potential use in the next product cycle. This is true for many LCA’s where the system boundaries are cradle-to-factory gate and therefore does not address the material carbon footprint arising from the carbon feedstock/resource selection. For example, compare the case of polylactic acid (PLA), a biobased product obtained from corn, with fossil-based polyolefin products (PE and PP) and PET. Figure 16.5 shows the process and material carbon footprint of the products.

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700 600

 467

Kg of CO2 emissions per 100 kg of polymer resin Material carbon footprint

Process carbon footprint

500 400 300 200 100 0

Zero PLA

PET

PE

Figure 16.5: Process and material carbon footprints for PLA, PET and PP. Reproduced with permission from R. Narayan, MRS Bulletin, 2011, 36, 9, 716. ©2011, Cambridge University Press [2].

As can be seen from Figure 16.5, the process carbon footprint for PE/PP is 205 kg CO2 emitted per 100 kg plastic manufactured [17], whereas that for PLA is around 385 kg CO2 emitted per 100 kg plastic manufactured [18], a much higher carbon footprint for the biobased product. However, if one includes the material carbon footprint, then PLA has a lower total carbon footprint − 385 kg CO2 emission per 100 kg plastic manufactured versus 519 kg CO2 emission per 100 kg plastic manufactured. This is because the material carbon footprint for PLA plastic is zero, whereas PE/PP has a material carbon footprint of 314 kg CO2 emission per 100 kg plastic manufactured. In the case of PLA, the total (net) CO2 released to the environment, taking into account the intrinsic carbon footprint as discussed earlier, is lower and will continue to get even lower as process efficiencies are incorporated and renewable energy is substituted for fossil energy [19].

16.8.1 Illustrating zero material carbon footprint using basic stoichiometric calculations Based on the earlier biological carbon cycle discussion and using basic stoichiometry, we calculate that for every 100 kg of polyolefin (PE and PP) manufactured, a net 314 kg CO2 is released into the environment at its EOL (100 kg of PE contains 85.7% kg carbon and upon combustion will yield 314 kg of CO2 (44/12) × 85.7). Similarly, PET contains 62.5% carbon and would result in 229 kg of CO2 released into the environment at EOL. However, if the carbon in PET or PE/PP comes from a plant/ biomass feedstock, the net release of CO2 into the environment is zero, because the CO2 released is fixed in a

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short time period by the next crop or plant/biomass plantation (Figure 16.6). This is the intrinsic zero material carbon footprint value proposition for using a bio/renewable feedstock. Thus, the fundamental driver to biobased products is the material carbon footprint reduction arising from the ‘short- term’ biogenic carbon cycle − the rate and timescale of CO2 sequestration is in balance with the use and release, resulting in a carbon neutral footprint. This is in contrast to the ‘long-term’ carbon cycle for petro/fossil feedstock; the rate and timescale of CO2 sequestration is millions of years, and the rate and timescales of use and release is in a 1−10 year time frame.

16.8.2 Measuring biobased carbon content A key requirement to document this material carbon footprint reduction is a transparent and accurate test method to unequivocally determine the amount of biocarbon present in a product. The biocarbon present in a material as a percentage can be readily calculated from the C-14 signature of the product, as shown in Figure 16.7 [1, 2]. The CO2 in the atmosphere has 12CO2 in equilibrium with radioactive 14CO2. Radioactive carbon is formed in the upper atmosphere through the bombardment of cosmic ray neutrons on 14N. It is rapidly oxidised to radioactive 14CO2 and enters the Earth’s plant and animal life through photosynthesis and the food chain. Plants and animals that use carbon in biological food chains take up 14C during

350

H2C

CH2 PE

300

n

Material carbon footprint kg of CO2 per 100 kg resin

314 kg of CO2 emissions reduction for every 100 kg of PE resin in which the petro carbon is replaced with bio carbon

250

Experimentally determined using ASTM D6866 based on the principle of C-14 analysis

200 150 100

Zero carbon footprint

50 0

PE/PP

PET

Bio-PE/PET/PLA

Figure 16.6: Illustrating zero material carbon footprint by substituting petro/fossil carbon with biobased carbon. Reproduced with permission from R. Narayan, Biobased & Biodegradable Polymer Materials: Rationale, Drivers, and Technology Exemplars, American Chemical Society Symposium Series 1114, 2012, p.13 and American Chemical Society Symposium Series 939, 2006, p.282. ©2006, American Chemical Society [1].

16 Principles, drivers, and analysis of biodegradable and biobased plastics 

14CO — 2

Solar radiation

C-14 signature forms the basis of Standard test method to quantify biobased content (ASTM D6866)

14N

Biomass (12CH2O)x

12CO 2

Cosmic radiation 14CO 14 C 2 12CO 2

 469

(14CH2O)x

NEW CARBON >106 years Fossil Resources (petroleum, natural gas and coal) (12CH2)n OLD CARBON

Figure 16.7: Measuring the biobased carbon content using radiocarbon analysis. Reproduced with permission from R. Narayan, Biobased & Biodegradable Polymer Materials: Rationale, Drivers, and Technology Exemplars, American Chemical Society Symposium Series 1114, 2012, p.13 and American Chemical Society Symposium Series 939, 2006, p.282. ©2006, American Chemical Society [1].

their lifetimes. They exist in equilibrium with the 14C concentration in the atmosphere; that is, the numbers of C-14 atoms and nonradioactive carbon atoms stay approximately the same over time. As soon as a plant or animal dies, the metabolic function of carbon uptake ceases; there is no replenishment of radioactive carbon, only decay. Since the half-life of carbon is around 5,730 years, the petrochemical feedstock formed over millions of years will have no C-14 signature. The quantity of biobased carbon can be readily determined by combusting the test material and analysing the CO2 gas evolved to provide a measure of its 14C/12C content relative to the modern carbon-based oxalic acid radiocarbon standard reference material 4990c (referred to as HOxII). This methodology, to determine the biobased carbon content, has an accuracy of +/−3% and has been codified into an ASTM standard [20] D6866 titled ‘Standard Test Methods for Determining the Biobased Content of Solid, Liquid and Gaseous Samples using Radiocarbon Analysis.’

16.8.3 Calculating and reporting biobased carbon contents Percent biobased carbon content = mass of biobased (organic) carbon/total mass of (organic) carbon × 100. Inorganic carbon like calcium carbonate is excluded from the calculations and in the ASTM D6866 method for measuring biobased carbon content, any carbonate present is removed before measuring the biobased carbon content. The percent biobased carbon content, using ASTM D6866, gives the ratio of the mass of biobased (organic) carbons to total mass of (organic) carbons present in the

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product, for example, if a product A has 60% biobased carbon content, it means that for every 100 kg of carbon present in product A there is 60 kg of biobased carbon. It is not that for every 100 kg of Product A, there is 60 kg of biobased carbon! This is because product A includes elements other than carbon, like hydrogen, oxygen and other elements. This does not pose a problem, because there is a straightforward and wellestablished method in organic chemistry to experimentally determine the elemental analysis − which gives the percent of carbon present in the product. In the above example, let us assume that elemental analysis of Product A gives us 50% organic carbon, 5% hydrogen and 45% oxygen − in other words 100 kg of Product A contains 50 kg of carbon. If the biobased carbon content, determined experimentally (using ASTM D6866), is 60% then 100 kg of Product A will contain 30 kg of biobased carbon [60/100 × 50]. This can be extended to calculating the biobased carbon content of a complex product comprising ‘n’ components as shown in the equation below. However, the biobased carbon content (using ASTM D6866), organic carbon content and mass of each of the ‘n’ components should be known. Alternatively, the complex product can be directly tested for biobased carbon content using ASTM D6866. BCC (product) = Σwn × BCCn × OCCn / Σwn × OCCn

(16.1)

Where: wn = mass of the nth component. BCCn = biobased carbon content of nth component. OCCn = organic carbon content of the nth component.

16.9 Example of bio polyethylene terephthalate Several major brand owners led by The Coca-Cola Company introduced PET bottle packaging. PET is prepared by the condensation polymerisation of terephthalic acid (TA) and ethylene glycol (EG). Only the EG component comes from plant/ biomass (sugar cane to ethanol to ethylene to EG). The TA component still comes from petro/ fossil resources (see Figure 16.8). Two of the ten carbons in PET are biobased and so the biobased carbon content is 20%. Experimental determination of the biobased carbon content using ASTM D6866 also gives 20%. However, on a total mass basis there is 31.25% of plant biomass present in the product. So, if one is reporting the plant biomass content of a product, this can be validated from the molecular formula and the experimentally determined biobased carbon content (using ASTM D6866).

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ROOC

COOR

+

OH

CH2

Diol

Diacid

C

C

O

O

O

CH2

CH2

CH2

 471

OH

O n

PET Terephthalic Acid = 8C; Ethylene glycol = 2C; biocontent is 20% Acid component = 68.75%; glycol component = 31.25% on total mass basis Figure 16.8: Biobased PET in which glycol comes from plant resources.

16.10 Summary Biodegradability is an EOL option for single-use disposable, packaging and consumer plastics that harnesses microbes to completely utilise the carbon substrate and remove it from the environmental compartment through the microbial food chain. However, biodegradability must be defined by: – The disposal system − composting, anaerobic digester, soil and marine. – Time required for complete microbial utilisation in the selected disposal environment − short defined time frame, and in the case of composting this is defined as 180 days or less. – Complete utilisation of the substrate carbon by microorganisms as measured by the evolved CO2 (aerobic) and CO2 + CH4 (anaerobic), leaving no residues. – Degradability, partial biodegradability or will eventually biodegrade is not an option! Serious health and environmental consequences can occur as documented in the literature and discussed in this chapter. – Specification standards with specific pass/fail criteria exist only for biodegradability in composting conditions − compostable plastics. There are a number of standard test methods for conducting, measuring and reporting biodegradability; however, they do not have pass/fail criteria associated with it. Therefore, an unqualified claim of biodegradability using a standard test method is misleading unless the biodegradability claim is qualified by the rate and extent of biodegradation in the test environment, and validated by an independent third-party laboratory using internationally adopted standard test methods. Biobased plastics, in which the fossil carbon is replaced by biobased carbon from plant-biomass resources, offer the intrinsic value proposition of a sustainable, zero

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material carbon footprint which is in balance with the rates and timescale of the biological carbon cycle. The process carbon and environmental footprint using LCA methodology is important and needs to be conducted as well. However, it does not capture nor convey the true, intrinsic value proposition of the zero material carbon footprint arising from the selection of the plant-biomass carbon resources. Identification and quantification of biobased content is based on the radioactive C–14 signature associated with (new) biobased carbon. Not all biobased plastics are biodegradable and not all biodegradable polymers are biobased.

References [1]

[2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]

R. Narayan, Biobased & Biodegradable Polymer Materials: Rationale, Drivers, and Technology Exemplars, American Chemical Society Symposium Series 1114, 2012, p.13 and American Chemical Society Symposium Series 939, 2006, p.282. R. Narayan, MRS Bulletin, 2011, 36, 9, 716. R. Narayan in Renewable Resources and Renewable Energy, Eds., M. Graziani and P. Fornasiero, CRC Press, Taylor & Francis Group, Boca Raton, FL, USA, 2006 and 2011, Chapter 1. J.H. Song, R.J. Murphy, R. Narayan and G.B.H. Davies, Philosophical Transactions of the Royal Society B, 2009, 364, 2141. P. Galgali, A.J. Varma, U.S. Puntambekar and D.V. Gokhale, Chemical Communications, 2002, 23, 2884. G. Scott in Degradable Polymers: Principles and Applications, Kluwer, New York, NY, USA, 2002, Chapter 3. T.F.J. Ojeda, E. Dalmolin, M.M.C. Forte, R.J.S. Jacques, F.M. Bento and F.A.O. Camargo, Polymer Degradation and Stability, 2009, 94, 965. R.C. Thompson, Y. Olsen, R.P. Mitchell, A. Davis, S.J. Rowland, A.W.G. John, D. McGonigle and A.E. Russell, Science, 2004, 304, 838. Algalita Marine Research Foundation, Long Beach, CA, USA. http://www.algalita.org/pelagic_ plastic.html. Y. Mato, T. Isobe, H. Takada, H. Kahnehiro, C. Ohtake and T. Kaminuma, Environmental Science & Technology, 2001, 35, 318. E.L. Teuten, J.M. Saquing, D.R.U. Knappe, M.A. Barlaz, S. Jonsson, Björn, S.J. Rowland, R.C. Thompson, T.S. Galloway and R. Yamashita, Philosophical Transactions of the Royal Society B, 2009, 364, 2027. R.C. Thompson, C.J. Moore, F.S. Vom Saal and S.H. Swan, Philosophical Transactions of the Royal Society B, 2009, 364, 1526. Federal Register Rules and Regulations; Federal Trade Commission 16 CFR Part 260: Guides for the Use of Environmental Marketing Claims, 2012, Volume 77, No.197. US Federal Trade Commission, Washington, DC, USA. http://www.ftc.gov/os/2012/10/ greenguides.pdf US Federal Trade Commission, Washington, DC, USA. http://www.ftc.gov/os/fedreg/2012/10/ greenguidesstatement.pdf ISO 14040, Environmental management – Life Cycle Assessment: Principles & Framework and ISO 14044, Environmental Management – Life Cycle Assessment: Requirements & Guidelines, International Organization for Standardization, Geneva, Switzerland. http://www.iso.ch.

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[18] Association of Plastics Manufacturers in Europe, Brussels, Belgium. http://www.plasticseurope.org/. [19] E.T.H. Vink, Polymer Degradation and Stability, 2003, 80, 403. [20] E.T.H. Vink, D.A. Glassner, J.J. Kolstad, R.J. Wooley and R.P. O’Connor, Journal of Industrial Microbiology and Biotechnology, 2007, 3, 1, 58. [21] ASTM International Annual Book of Standards, Standards ASTM D6866; D6400, D6868 and D7021, ASTM International, Philadelphia, PA, USA, 2010, Volume 8.03. http://www.astm.org.

Thomas Hirth and Rainer Busch

17 Biorefineries for renewable monomers 17.1 Introduction The use of renewable resources as raw materials for technical applications is not at all new. Mankind has already used natural materials during early civilisations in order to meet their basic needs and the first industrial activities were also largely based on the use of renewable resources [1, 2]. This continued until the beginning of the industrial revolution. Biobased polymers or bioplastics, as they are often called, are chemical products made from monomers from plant-based resources. They either have petrochemical equivalents with the same chemical structure and properties against which they have to compete in the market, or can show different structures and performances versus traditional plastics. On a large scale, such biobased monomers are produced in socalled biorefineries. The significant advances that have been made in the implementation of the biorefinery concept with the production of biofuels, such as bioethanol and biodiesel from sugar cane, corn starch and plant oils, have set a solid foundation for the expansion of the concept into other applications such as chemicals and will help to reach the goal of integrated biorefineries.

17.2 Biorefinery concepts The term biorefinery is nowadays widely used and several definitions have been developed over the last few years. Nevertheless, the core component of all definitions is the conversion of biomass into several products (food, feed, materials, chemicals and energy), and the integration of various technologies and processes in the most sustainable way. The International Energy Agency developed the following definition for biorefineries in its ‘Bioenergy Task 42’ which has been widely accepted due to its general and broad character [3]: ‘Biorefining is the sustainable processing of biomass into a spectrum of marketable products and energy’.

In biorefineries, biomass is used for the production of high added-value chemicals, materials and intermediates together with the production of energy carriers, preferably in a liquid phase due to its higher energy content and easier transportation. In that respect, biorefineries can be compared with fully integrated petrochemical refineries, except that they are supplied with renewable, plant-based materials instead of nonrenewable hydrocarbon-based feedstock. A related approach is the concept of https://doi.org/10.1515/9781501511967-017

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the ‘BioCascade’, which uses dedicated biomass in such a way that all constituents of a plant (oils, proteins, fibres, cellulose and lignin) result in a total product mix that offers the highest economic value [4]. Bozell [5] defines three primary basic operations, which comprise a biorefinery (reproduced with permission from J. Bozell ‘Products and Platforms for the Biorefinery − An Overview of Biobased Products from Carbohydrates’): 1. Supply: a biorefinery must be supplied with a dependable source of raw material inputs harvested from agricultural and silvicultural (forestry) sources. After harvest, these feedstocks may be processed using size reduction or densification technology at the point of collection or as the initial operation at the biorefinery. 2. Separation: upon delivery to the biorefinery, raw materials are separated into their constituent parts. In its simplest form, the biorefinery generates and uses three primary building blocks: a) Carbohydrates, in the form of cellulose, starch, hemicellulose and monomeric sugars. b) Aromatics, in the form of lignin. c) Hydrocarbons, in the form of plant triglycerides. 3. Separation greatly reduces the complexity of the starting biomass by converting an extensive range of inputs to a much smaller number of monomers and polymers. Depending on the feedstock, carbohydrates can make up 75% or more of the starting biomass and are therefore of particular interest as renewable raw materials. 4. Conversion: after separation, biorefinery process streams are subjected to chemical, thermal or biochemical conversions. The output of this operation is a portfolio of biobased fuels and chemicals. Of the three primary operations, conversion is the least well developed for the biorefinery. While the petrochemical industry can describe many high yield, selective conversions of their primary building blocks (ethylene, propylene, benzene and so on) only a scant number of biorefinery conversions, comparable in efficiency and breadth to the existing chemical industry, are available. The three basic operational steps are well illustrated in Figure 17.1. Although the concept of deriving chemicals from biomass is well known, the chemical industry has developed and grown during the first half of the 20th century using relatively cheap crude petroleum as a feedstock. The technology used in the petrochemical industry is well developed and defined, in contrast to the complex biorefinery concept, where a single technology will not provide a solution. Using biomass as a sustainable renewable resource is the only way to replace carbon from fossil sources for the production of carbon-based products such as chemicals, materials and liquid fuels. In order to be competitive with crude-oil-based products, an integrated biorefinery strategy has been developed to optimise the added value from biomass. This strategy is mainly based on the transfer of petroleum refineries thinking to biomass (raw material fractionation, integration of mass and energy fluxes, integration of processes) in order to be able to produce a spectrum of products

17 Biorefineries for renewable monomers  Supply (Input)

Conversion (Output)

Corn Wood Switchgrass Wood Ag residues Sorghum Soybean Stover Apple pomace Beet molasses Sugar cane Potatoes

Butadiene Polylactic acid Pentanes, pentene Succinic acid Phenolics Ethanol Butanol Organic acids Furfural Polyols Levulinic acid Sorbitol Xylitol Others

Separation Starch Cellulose Lignin Other carbohydrates Oils

 477

Figure 17.1: Generic block flow diagram of a biorefinery. Reproduced with permission from J. Bozell, Products and Platforms for the Biorefinery − An Overview of Biobased Products from Carbohydrates, BioWeb, SunGrant. ©BioWeb, SunGrant [5].

and therefore maximising the added value. The approach requires the valorisation of the whole biomass, based on a zero waste concept. Biorefineries are large industrial factory complexes in which agricultural feedstocks are processed and fractionated into intermediate basic products, which are then partially converted into final products [4]. Most conventional biomass processing plants (such as oil crushing, starch extraction and cellulose pulp extraction) carry out the primary biomass refining and some perform a first conversion step. Integrated biorefineries go one step further in their transformation capabilities, with the integration of further conversion steps, based on the industrial logic of sustainable optimisation (maximising profit and minimising waste). A range of different technologies can be used to industrially convert the available biomass into renewable materials or energy carriers. Industrial activities using renewable materials are very often linked to the food sector. Many of the renewable raw materials that can be considered for technical industrial applications can be made in food processing plants. For example, sucrose or starch, or natural oils for human food use are also important raw materials for industrial processes. The following industrial sectors supply the most important renewable raw materials: – The sugar and starch sector produces carbohydrates such as sucrose, glucose, starch and molasses from plants materials like sugar beet, sugar cane, potatoes, wheat, corn and so on. – The oil and fat processing sector produces numerous oleochemical intermediates such as triglycerides, fatty acids, fatty alcohols and glycerol from oil plants like rapeseed, soybeans, palm oil and coconut.

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The wood processing sector, in particular the cellulose and paper industries, produce mainly cellulose, cellulose derivatives and lignin from wood [4].

The majority of industrial biorefineries will be built upon these sectorial platforms.

17.2.1 Starch and sugar biorefineries This biorefinery processes starch crops (see Figure 17.2), such as wheat, maize and potatoes, or sugar crops (see Figure 17.3), such as sugar beet or sugar cane. Its most important output is glucose, which can be further used for the production of ethanol and organic acids.

Wheat Saccharification Fractionation and separation Starch Hydrolysis Fermentation

Glucose Biochemical Transformation

Feed (sugar beet pulp)

Food (sugar)

Chemicals

Modification

Surfactants

Ethanol

Figure 17.2: Schematic diagram of a starch biorefinery [6]. Reproduced with permission from Joint European Biorefinery Vision for 2030, Strategic Targets for 2020 – Collaboration Initiative on Biorefineries, Star-COLIBRI Project. © Star- COLIBRI [6].

17.2.2 Oilseed biorefineries Current oilseed biorefineries (see Figure 17.4) mainly produce food and feed ingredients, and biodiesel and oleochemical products from oilseeds, such as rape, sunflower and soybean.

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Sugar Beets Fractionation and separation

Saccharose

Feed (sugar beet pulp)

Food (sugar)

Fermentation

Esterification

Fermentation

Chemicals

Surfactants

Ethanol

Figure 17.3: Schematic diagram of a sugar biorefinery. Reproduced with permission from Joint European Biorefinery Vision for 2030, Strategic Targets for 2020 – Collaboration Initiative on Biorefineries, Star-COLIBRI Project. © Star-COLIBRI [6].

17.2.3 Green biorefinery Green biorefinery processes (see Figure 17.5) include wet biomass, such as grass, clover and lucerne. The wet biomass is pressed to obtain pressed juice and pressed cake. The pressed juice contains proteins, free amino acids, organic acids and enzymes. Lactic acid and its derivatives as well as ethanol, proteins and amino acids are the most favourable end products from pressed juice.

17.2.4 Lignocellulose biorefinery Lignocellulose biorefinery (see Figure 17.6) processes include lignocellulosic biomass from different sources: agricultural residues and wood. A lignocellulose biorefinery can be run either as a thermochemical or as a biochemical unit. The biochemical approach (see Figure 17.6a) is based on the fractionation of the lignocellulosic raw material into three separate product precursors: cellulose, hemicellulose and lignin. This is achieved during a primary refining step. These chemical fractions are then treated separately and converted into value-added products during a secondary refining step. Cellulose can be hydrolysed into sugars, which are then used as the fermentation substrate to produce ethanol and other chemicals such as organic acids and solvents. The second fraction, hemicellulose, can be converted to xylose, gelling agents, barriers, furfural and other furan derivatives. Finally, lignin can be applied as a binder and adhesive or can be used for the production of fuels and carbon fibres for materials or syngas production, which can be used for energetic or

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Rapeseed Extraction and separation Rapeseed oil Hydrolysis Transesterification Long-chain fatty acids

Long-chain fatty acids

Feed

Modification

Fermentation

Chemical conversion

Lubricants

1,3Propanediol

Triacetin

Biodiesel

Figure 17.4: Schematic diagram of an oilseed (rape) biorefinery. Reproduced with permission from Joint European Biorefinery Vision for 2030, Strategic Targets for 2020 – Collaboration Initiative on Biorefineries, Star-COLIBRI Project. © Star- COLIBRI [6].

Green biomass Fractionation and pressing

Green fibres Modification

Green juice

Anaerobic digestion

Extraction

Biogas Upgrading Materials

Biomethane

Chemicals and building blocks

Feed

Figure 17.5: Schematic diagram of a green biorefinery. Reproduced with permission from Joint European Biorefinery Vision for 2030, Strategic Targets for 2020 – Collaboration Initiative on Biorefineries, Star-COLIBRI Project. © Star-COLIBRI [6].

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industrial purposes. In addition, the use of lignin as a source for aromatic compounds is currently under investigation in many research projects [7]. The thermochemical (see Figure 17.6b) approach is based on gasification of lignocellulosic residues and further processing of the syngas for production of fuels and chemicals. Many different biomass types can be utilised as raw materials for this type of biorefinery: dry agricultural residues (e.g., straw, peelings and husks), wood, woody biomass and biogenic residues (e.g., recovered paper and residues from recovered paper pulping and lignin). These are relatively dry biomass feedstocks and well suited for new thermochemical conversion processes such as gasification. Liquid biooils are also suitable feedstocks for the gasification process [6].

C5 Sugars

Lignocelulosic crops/ wood

Lignocelulosic residues

Pretreatment

Pretreatment

Hydrolysis

Gasification

Lignin

C6 Sugars

Syngas

Fermentation

Chemicals

Bioethanol a)

Biomaterial fibre products

Alcohol synthesis

FT synthesis

Chemicals (products)

Synthetic biofuels (FT) b)

Figure 17.6: Schematic diagram of a lignocellulose biorefinery (biochemical approach (a) and thermochemical approach (b)). FT: Fischer−Tropsch. Reproduced with permission from Joint European Biorefinery Vision for 2030, Strategic Targets for 2020 – Collaboration Initiative on Biorefineries, Star-COLIBRI Project. © Star-COLIBRI [6].

17.2.5 Aquatic biorefinery Aquatic biomass such as microalgae and seaweed is a high-volume feedstock and characterised by a remarkably high productivity per area of land. It contains relatively high quantities of oils, proteins, polysaccharides and other valuable compounds. Microalgae and seaweed are therefore highly suited for biorefining (see Figure 17.7) with end products ranging from fuels and bulk chemicals to speciality chemicals and food and feed ingredients.

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Extraction of fatty acids and purification

Oil fraction Sunlight Nutrients CO2 Aquatic biomass cultivation

Chemicals Value-added product (omega fatty acids) Biodiesel

Transesterification

Cell disruption, product extraction and separation

Minerals Fertiliser/ nutrients

Oleochemistry

Carbohydrate fraction

Protein fraction

Value-added products Chemicals (amino-acids, N-chemicals,...) Feed Biogas

Fermentation

Fuels and chemicals (ethanol, butanol, lactic acid,...) Value-added products (iodine)

Figure 17.7: Schematic diagram of an aquatic biorefinery. Reproduced with permission from Joint European Biorefinery Vision for 2030, Strategic Targets for 2020 – Collaboration Initiative on Biorefineries, Star-COLIBRI Project. © Star-COLIBRI [6].

17.3 Monomers based on renewable raw materials The demand for chemicals derived from renewable resources is growing due to a volatile and generally rising crude oil price, concerns over the long-term supply of petrochemical commodity chemicals and consumer demand for ‘greener’ alternatives. Governments, notably the European Union are also seeking to encourage the use of bioresources and the production of sustainable bioproducts including chemicals [8]. A report by the US Department of Energy in 2004 listed a large number of chemicals and materials that can be derived from biomass, of which 12 value-added chemicals were identified as potential building blocks [9]. This list was recently revised by Bozell and Petersen [10], and now comprises the following monomers which can be considered as promising monomers for a whole series of industrially interesting chemicals: – Ethanol – Furans – Glycerine and derivatives – Lactic acid – Succinic acid – Hydroxypropionic acid/aldehyde – Levulinic acid – Sorbitol – Xylitol – Biohydrocarbons

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Ethanol is currently produced to a large extent (>90%) by the fermentation of sugars derived from agricultural or forestry biomass, either directly (sugar cane and sugar beets) or indirectly (starch, cellulose or hemicellulose hydrolysis products). The stateof-the-art production of biotechnical ethanol involves the large-scale fermentation of sugars or starch by yeasts, and the purification of the resulting ethanol by distillation. This form of ethanol production has been gradually improved over decades and can now be considered as technically mature. Ethanol from sugars or starch is used in large quantities either as a fuel or fuel additive, or increasingly as a raw material for the production of (green) ethylene and polyethylene (PE). The principle of the dehydration of ethanol to ethylene can also be applied to other biobased alcohols, thus giving access to bioolefins and biohydrocarbons [11]. Furfural is the starting material for the industrial production of almost all furan compounds and is industrially produced from a pentosan-rich biomass like corn cobs, oat hulls, almond husks, cottonseed hull bran, birch wood, bagasse and sunflower husks in large quantities (>200,000 mt/a). Several process improvements have been developed at pilot scale in recent years which lead to higher yields (up to 80%) due to reduced side reactions and improved product recovery [12, 13]. Important furan derivatives (see Figure 17.8) are furfural, 5-hydroxymethyl furan (HMF) and 2,5-furane dicarboxylic acid (FDCA), of which only furfural has industrial significance.

CHO

O

OHC

O

CH2OH

HOOC

O

COOH

Figure 17.8: Furfural and derivatives.

Lactic acid (2-hydroxypropionic acid) is both an alcohol and an acid, and one of the most recognised biobased chemical products (see Figure 17.9); it has been commercially produced for many years via glucose fermentation using organisms such as Lactobacillus delbrueckii along with a wide range of other organisms and biomass sources [14]. OH COOH Figure 17.9: Lactic acid.

Lactic acid is commercially used in food, beverages and industrial applications, as well as in pharmaceuticals and personal care products. It is also the monomeric precursor for polylactic acid (PLA). Market growth in the industrial applications segment is expected to result primarily from lactic acid-based biodegradable polymers for food and nonfood packaging, bottles and fibre applications, and lactate esters [16].

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Glycerine is chemically a triol (see Figure 17.10) and is present in all natural fats and oils in the form of fatty acid glycerides from which it can be obtained by the catalysed splitting of fats and oils. This technology is mature. HO OH

OH

Figure 17.10: Glycerine.

Due to its trifunctionality, glycerine is a versatile molecule, which can easily undergo oxidation, reduction and dehydration processes. It can also serve as a substrate in fermentation reactions. It is therefore industrially used for the synthesis of: – Esters, mono-, di- and triesters of inorganic and organic acids. Acetins are the most important esters of glycerol from short-chain carboxylic acids. The most widely used type of acetin is triacetin. – Acrolein and acrylic acid by the catalytic dehydration of glycerine. – 1,3-Propanediol. Conversion to 1,3-propanediol, either chemically or fermentatively and further derivatisation. 1,3-propandiol is today produced in a Tate & Lyle plant via the fermentation of glucose [17]. – 1,2-propanediol. Research is underway to convert glycerol to propylene glycol by hydrogenolysis. – Epichlorohydrin. – Polymers. 3-hydroxypropionate (3HP) is a difunctional C-3 intermediate (see Figure 17.11) and as such may give access to 1,3-difunctional molecules such as 1,3-propanediol, acrolein and acrylic acid, acrylamide or malonic acid. Currently, there is no commercial production process for 3HP from fossil feedstocks, but a lot of research work is going on to elaborate and scale up fermentation routes starting from glucose or glycerine, respectively [15]. All routes go through 3-hydroxypropionic aldehyde as an intermediate. Other projects aim to synthesis polyesters using 3HP as the primary building block. The projects comprise strategies for the preparation of biodegradable polyesters from 3HP, the preparation and characterisation of novel copolyesters that contain 3HP and other building blocks from renewable resource materials (e.g., lactic acid, glycolic acid), and the establishment of structure/property relationships in new 3HPbased materials [16]. O HO

OH

Figure 17.11: 3-Hydroxypropionate.

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Succinic acid is an aliphatic C-4 dicarboxylic acid (see Figure 17.12), which is currently produced at a low volume (22,000 tonnes/year) via the oxidation of 1,4-butanediol (1,4-BDO) or catalytic hydrogenation of maleic acid or fumaric acid. HOOC

COOH

Figure 17.12: Succinic acid.

Succinic acid can also be produced by fermentation from carbohydrates. A whole series of industrial processes have been developed in the last decade by companies such as BASF [18], Roquette and DSM [19], and BioAmber [20] to improve the yield of the fermentation and to bring it to industrial maturity. Succinic acid is a wonderful precursor for many industrially valuable chemicals, intermediates and processes and is used: – In the synthesis of 1,4-BDO, and tetrahydrofuran and their derivatives. – As the diacid building block for polyesters like polybutylene succinate. – In the synthesis of esters as fuel additives or ‘green’ solvents. – As the diacid building block for polyamides. According to a new technology, developed by Genomatica, 1,4-BDO can be also obtained by the direct fermentation of sugars [21]. The first plant of this type should be operative in 2015 and is a joint venture between Novamont and Genomatica [22]. Levulinic acid as the simplest γ-oxocarboxylic acid (see Figure 17.13) is present in hemicellulose hydrolysates. O COOH Figure 17.13: Levulinic acid.

It is industrially produced by the acid-catalysed hydrolysis of carbohydrates such as cellulose or starch. The global production of levulinic aid is very small (500,000 mt/a). Although a fermentative process was suggested 10 years ago, in which Zymomonas mobilis converts fructose and glucose to sorbitol and gluconic acid [23], this process will most probably not replace the technically mature hydrogenation; however, the process could be further improved by converting it from batch to continuous operation.

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 Thomas Hirth and Rainer Busch OH OH

HO

OH

OH OH

Figure 17.14: Sorbitol.

Sorbitol is used in the synthesis of the following significant industrial nonfood chemicals: – Ascorbic acid (vitamin C). – 1,4-Sorbitan. – Polyetherpolyols for the synthesis of polyurethanes (PU). – Isosorbide. – Glycols such as 1,2-propylene glycol (produced from renewable instead of fossil feedstocks). – Light alkanes by the hydrogenolysis of sorbitol and stepwise OH-removal [24]. Xylitol, another sugar alcohol (see Figure 17.15), is the hydrogenation product of xylose, which forms, together with arabinose, the main constituents of pentosans in hemicellulose. The process is very similar to the hydrogenation of glucose to sorbitol.

HO

OH OH

OH

OH Figure 17.15: Xylitol.

A biochemical route starting from crude hemicellulose hydrolysates rather than from isolated and purified xylose would be very advantageous for future applications of xylitol. Biohydrocarbons means hydrocarbons in which the carbon stems from renewable raw materials, mostly biomass. Such biohydrocarbons will provide a direct drop-in interface between a biorefinery and the existing petrochemical industry. In general, biomass can be converted by standard thermochemical processes like gasification to carbon monoxide and hydrogen, with the subsequent polymerisation to hydrocarbons in a Fischer−Tropsch (FT) reaction. Biotechnological processes, which lead directly to hydrocarbons, are very new and not well understood, although significant research work is going on in this field. One example is isoprene (see Figure 17.16), an unsaturated 5-carbon terpenoid which is produced in nature by plants and for which fermentative pathways, starting from sugars, have been recently detected [5, 24].

Figure 17.16: Isoprene.

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The commercial significance of isoprene is enormous, as it is industrially used as a raw material in rubber production for tyres. As no processes exist for the direct biochemical production of biohydrocarbons, most routes from biomass to hydrocarbons have to go through the respective alcohols as the precursor, which have to be dehydrated to the corresponding olefins (ethanol to ethylene, propanol to propylene, butanol to butylene and so on). The 10 chemicals, which were briefly presented in this overview, have a high potential as platform chemicals in a future bioeconomy, which will be based on biorefineries instead of fossil oil refineries. The following, Table 17.1, shows that all 10 chemicals can be produced in biorefineries and it also shows which biorefinery type produces which chemical. Table 17.1: Biorefineries and monomers produced in them. Type of biorefinery Feedstock

Intermediates

Platform

Monomers

Starch and sugar

Starch crops, sugar Saccharose and Glucose and crops and potatoes starch fructose

Ethanol, lactic acid, 1,3-propanediol, succinic acid, levulinic acid, furans, 3-HP and isoprene

Oilseed

Seeds of sunflower, Sunflower oil, Glycerine, rapeseed and rapeseed oil and long chain soybean soy bean oil fatty acids

Fatty alcohols, fatty acids, dicarboxylic acids, 1,3-propanediol and 3HP

Green

Wet biomass (grass, clover and lucerne)

Lactic acid, ethanol, lysine and 3HP

Lignocellulose

Wood and woody Cellulose biomass, and straw hemicellulose and lignin

Glucose, Ethanol, xylitol, sorbitol, pentoses and phenol, levulinic acid, phenolics succinic acid, furans, 3HP and isoprene

Aquatic

Aquatic biomass (seaweeds and microalgae)

Glycerine, fatty acids and glucose

Proteins, fatty − acids and amino acids

Oils and carbohydrates

Fatty alcohols, fatty acids, dicarboxylic acids, 1,3-propanediol, 3HP and isoprene

As already mentioned in Chapter 2, going from an intermediate to a monomer through a platform usually requires an additional chemical or biochemical conversion step. The latter is the domain of industrial or white biotechnology. While industrial biotechnology has already been applied to the production of pharmaceuticals, fine chemicals and speciality chemicals, there is uncertainty about when, how and to what extent biotechnology will also play a role in the production of bulk chemicals.

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It can be seen from Table 17.1, that glucose and glycerine are key components in biochemical conversion processes. Almost each of the monomers in the rightmost column in Table 17.1 can be produced from either glucose or glycerine, depending on the applied microorganism. Figures 17.17 and 17.18 illustrate this versatility very well. They show – in a simplified manner − how a number of monomers and polymers are derived from glucose and glycerine. Glucose C6H12O6

Ethanol C2H6O

Lactic acid C3H6O3

Succinic acid C4H6O4

5-HMF C6H6O3

Sorbitol C6H14O6

Ethylene

Lactic acid/ Acrylic acid

Succinic acid Butanediol

Furandicarboxylic acid

Sorbitol

PE

PLA Polyacrylic acid

Polyester Polyamide

Polyhydroxyfuranoate

PU

Figure 17.17: Monomers and polymers derived from glucose.

Triglycerides

Glycerine

Glycerine

Glycerine

Carboxylic acid

1,3-Propanediol

Epichlorohydrin

Acrylic acid

Dicarboxylic acid

Polyol

Polypropyleneterephthalate

Epoxy resins

Polyacrylic acid

Polyester Polyamide

PU

Figure 17.18: Monomers and polymers derived from triglycerides.

Carboxylic acid

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17.4 Summary and outlook Unlike energy, chemicals and materials can only be produced from carbon-containing resources such as crude oil or biomass. As the price of crude oil increases, traditional mass chemicals such as ethylene, propylene and benzene will become more expensive and less widely available. Therefore, chemicals and materials derived from biomass are the only alternatives to oil-derived chemical products, which means that we have to redesign those central chemical processes and molecules which form the basis of the current chemical industry. New monomers and processes based on new technologies, such as biorefinery and industrial biotechnology, will replace them and help the chemical industry to stay competitive. The list of monomers described in this chapter is only a 2014 snapshot of technological developments, which, if successful, may lead to commercial opportunities. A similar list of the top 10 biobased monomers in few years will probably look completely different. Chemicals and materials based on monomers from renewable resources are already providing opportunities today, and will definitely continue to do so in future. This will not merely involve replacing conventional plastics in individual application areas but also means that completely new ones will be developed. It is very uncertain how rapidly this can be realised and depends on multiple factors, e.g., the legislative framework conditions, price developments of raw materials and the general economic climate. In the future, biorefineries will process a variety of biomass-based feedstocks in order to produce new base chemicals for the production of many new and already existing bulk chemicals and polymers. White biotechnology will play an important role in this development, as it does already in the production of pharmaceuticals, fine chemicals and speciality chemicals. At the end of the day, however, production economy and market acceptance will be crucial for the commercial success of new monomers.

References R. Busch in Renewable Raw Materials New Feedstocks for the Chemical Industry, Eds., R. Ulber, D. Sell and T. Hirth, Wiley-VCH Verlag GmbH, Weinheim, Germany, 2011. [2] J. Clark and F. Deswarte in Introduction to Chemicals from Biomass, Wiley-VCH, Weinheim, Germany, 2008. [3] International Energy Agency, Bioenergy, Task 42 ‘Biorefinery’. http://www.biorefinery.nl/ fileadmin/biorefinery/docs/Brochure_Totaal_ definitief_HR_opt.pdf. [4] J. Thoen and R. Busch in Biorefineries: Industrial Processes and Products, Eds., B. Kamm, P. Gruber and M. Kamm, Wiley-VCH Verlag GmbH, Weinheim, Germany, 2006. [1]

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J. Bozell, Products and Platforms for the Biorefinery − An Overview of Biobased Products from Carbohydrates, BioWeb, SunGrant. http://bioweb.sungrant.org/Technical/Bioproducts/ Bioproducts+from+ Carbohydrates/Biorefinery+Overview/Biorefinery+Overview.htm. Joint European Biorefinery Vision for 2030, Strategic Targets for 2020 – Collaboration Initiative on Biorefineries, Star-COLIBRI Project. B. Kamm, M. Kamm, Th. Hirth and M. Schulze in Biorefineries – Industrial Processes and Products, Eds., B. Kamm, P. Gruber and M. Kamm, Wiley-VCH, Weinheim, Germany, 2006, Volume 2. A. Kazmi and J. Clark in Renewable Raw Materials New Feedstocks for the Chemical Industry, Eds., R. Ulber, D. Sell and Th. Hirth, Wiley-VCH Verlag GmbH, Weinheim, Germany, 2011. T. Werpy and G. Petersen in Top Value Added Chemicals from Biomass: Volume I − Results of Screening for Potential Candidates from Sugars and Synthesis Gas, Pacific Northwest National Laboratory, Richland, WA, USA; National Renewable Energy Laboratory, Washington, DC, USA; and Office of Biomass Program, Office of Energy Efficiency & Renewable Energy, Washington, DC, USA, 2004. J.J. Bozell and G.R. Petersen, Green Chemistry, 2010, 12, 539. J. Rass-Hansen, H. Falsig, B. Jorgensen and C.H. Christensen, Journal of Chemical Technology and Biotechnology, 2007, 82, 329. T. Tuercke, S. Panic and S. Loebbecke, Chemical Engineering and Technology, 2009, 32, 11, 1815. A.S. Dias, M. Pillinger and A.A. Valente, Journal of Catalysis, 2005, 229, 414. R. Datta and M. Henry, Journal of Chemical Technology and Biotechnology, 2006, 81, 1119. T. Banner, A. Fosmer, H. Jessen, E. Marasco, B. Rush, J. Veldhouse and M. de Souza in Biocatalysis for Green Chemistry and Chemical Process Development, Eds., J. Tao and R.J. Kazlauskas, John Wiley & Sons, Hoboken, NJ, USA, 2011. M. Patel, M. Crank, V. Dornburg, B. Hermann, L. Roes, B. Hüsing, L. Overbeek, F. Terragni and E. Recchia, Medium and Long-term Opportunities and Risks of the Biotechnological Production of Bulk Chemicals from Renewable Resources − The Potential of White Biotechnology: The BREW Project, University of Utrecht, Utrecht, The Netherlands, 2006. DuPont Tate & Lyle Bio Products Company, LLC., USA. http://www.duponttateandlyle.com/ E. Scholten, T. Renz and J. Thomas, Biotechnology Letters, 2009, 31, 12. Roquette S.A. Press Release 12th May 2011. http://www.roquette.com/2011-275/dsm-androquette-to-open-commercial- scale-bio-based-succinic-acid-plant-in-2012/. BioAmber, Press Release 8th November 2011. http://www.bioamber.com/bioamber/en/news/ article?id=490. H. Yim, R. Haselbeck, W. Niu, C. Pujol-Baxley, A. Burgard, J. Boldt, J. Khandurina, J.D. Trawick, R.E. Osterhout, R. Stephen, J. Estadilla, S. Teisan, H.B. Schreyer, S. Andrae, T. Hoon Yang, S.Y. Lee, M.J Burk and S. Van Dien, Nature Chemical Biology, 22nd May 2011, DOI:10.1038/ NChemBio.580. C. Bastioli in Proceedings at Ecomondo 2013 Conference ‘Le Bioraffinerie nel quadro della Strategia Europea sulla Bioeconomia’, Rimini, Italy, 7th November 2013. http://newweb. riminifiera.it/upload_ist/AllegatiProgrammaEventi/Bastioli_876095.pdf M. Silveira and R. Jonas, Applied Microbiology and Biotechnology, 2002, 59, 400. G.W. Huber and J.A. Dumesic, Catalysis Today, 2006, 111, 119. Amyris Inc., Press Release 27th September 2011.

Alessandra Perrazzelli

18 Research and development funding with the focus on biodegradable products 18.1 Introduction The increasing demand for a sustainable supply of food, raw materials, products and fuels, together with recent scientific progress, has been the major economic driving force behind the growth and interest in the bioeconomy of Europe over the last few decades [1]. The concept of a knowledge-based bioeconomy (KBBE) was developed and introduced into European jargon in 2004 by the European Commission (EC). The concept addresses the growing need for safer, healthier, higher quality food, as well as the use and production of renewable bioresources based on a sustainable and secure crop production system. The bioeconomy, i.e., the sustainable production and conversion of biomass for a range of food, health, fibre and industrial products, and energy – shall take advantage, according to the EC, of the advances in life sciences and biotechnologies, in convergence with other technologies, such as chemistry, nanotechnologies or information technologies (constituting the knowledge base). In the words of the EC, the KBBE can be defined as the transformation of ‘life sciences knowledge into new, sustainable, ecoefficient and competitive products’ [2]. The development and subsequent market entry of new, innovative materials and products from renewable resources, such as bioplastics and other biodegradable polymer-based products, as well as the current research in this field, is of the utmost importance for the future of Europe. That is why since its introduction in 2004, the concept of KBBE has become a major sector of European Union (EU)-funded research, and currently represents a great share in EU research programmes and EU policy initiatives. The overall objective of this chapter is to show how the interest in biopolymers and other biobased products and their application on the market has grown progressively in Europe over the years, and is recognised today as a major driver for the economy and a tool for reaching a more sustainable and smarter society. This chapter will analyse the main policy initiatives recently undertaken at EU level to foster the innovation capacity in Europe, reaching the goal of building a Knowledge Biobased Economy, and to favour the market entry of new and innovative biobased products. Many of the initiatives and plans adopted by the EU in the bioeconomy sector have tried to involve the main stakeholders, i.e., the industry sector (with a strong focus on small and medium-sized enterprises (SME)), European associations, European technology platforms and researchers. The chapter will then provide a brief overview of the main funding instruments of the EU for research and innovation in the bioeconomy field, such as the Competitiveness https://doi.org/10.1515/9781501511967-018

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and Innovation Framework Programme (CIP), and the Framework Programmes (FP) for research and technological development (RTD), and finally, focus on projects financed under the FP research activities on biopolymers and biochemistry for highly added-value nonfood products and processes. This aims to show how EU research priorities have developed and changed during the succession of FP, and mostly, how specific topics of research (e.g., the biorefinery concept) have been introduced in the FP and how the importance of these topics has grown both in terms of industrial interest and EU funding.

18.2 Policy initiatives and plans in the field of biopolymers and their applications The paragraphs that follow will provide a brief overview on the policy initiatives the EU has undertaken in order to build the bioeconomy, and anticipation of how research activities are influenced by policies, and vice versa. Support at legislative, political and, of course, financial level is required to boost the areas of research the EU deems necessary for its future; at the same time, if research had not reached the present level and results, many of the current policies would not have been undertaken.

18.2.1 The Lead Market Initiative One of the main challenges Europe faces is to close the gap between research and the market entry of innovative products. This is the aim of the Lead Market Initiative (LMI) for Europe. The EU needs a favourable environment for effective capitalisation of research results in products [3]. It also needs to promote demand which requires a more targeted approach: the LMI is the European policy for six sectors that are supported by specific actions to lower barriers in order to bring new products or services onto the market [4]. The LMI was launched by the EC following the EU’s 2006 broad based innovation strategy, the so-called Aho Report [5]. While implementing a new research programme that ran until 2013 (the FP7), the EC has requested an independent panel of experts to explore how the effectiveness of the EU’s research spending could be improved in order to strengthen Europe’s competitiveness [6]. This report presents a strategy to create an innovative Europe through the combination of a market for innovative goods and services, focused resources, new financial structures and mobility of people, money and organisations. The aim of the LMI is, therefore, to promote and stimulate innovation by strengthening the demand base, which in turn should enable enterprises to gain a better return on their innovation efforts. The added value of the LMI lies in the development of a

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prospective, concerted and tailored approach to regulatory and other policy instruments. LMI in fact includes demand-side measures on the one hand, and supply- side measures on the other. On the demand side, the policy tools used are: regulation, public procurement, standardisation, labelling and certification, and complementary instruments. On the supply side, measures mainly include: research and development (R&D) funding, financial support and fiscal incentives [7]. One of the areas selected by the EC is the area of biobased products. The Biobased Products LMI covers a broad range of intermediate products, product components and ready-made products, e.g., bioplastics, biolubricants, biofibres for textiles, composite materials for construction and automotive, chemical building blocks, enzymes and amino acids (see Table 18.1). In accordance with the action plan, and thanks also to FP7 funds, a mapping of existing biorefineries at pilot plant or demonstration scale in Europe has been carried out, and the results per country have been published on the website http://www. bio-economy.net (last accessed 10th April, 2011). We will see later that a prior mapping of existing industrial biorefineries in the EU (not solely pilot plants) was conducted within the projects Biorefinery Euroview and Biopol, both funded under FP6. Moreover, in 2008, an FP7 specific joint call on biorefineries was published with a financial allocation of €57 million. It aimed to develop inter alia biobased chemicals and demonstrate the performance, sustainability and viability of the proposed biorefinery concepts. Although the Ad-hoc Advisory Group has developed a series of recommendations to stimulate market uptake and development, these measures still have to be implemented. At the European level, in fact, a policy framework to support biobased materials is still missing. As a result, up to now biobased products have not benefitted, for instance, from tax incentives or other supports.

18.2.2 Key Enabling Technologies At the end of 2009, the EC published an action plan on ‘Key Enabling Technologies’ (KET) [9] as part of the preparation for the new European plan for innovation [10]. KET are of systemic relevance as they enable the restructuring of industrial processes needed to modernise EU industry and to secure the research, development and innovation base in Europe [11]. Industrial biotechnology is one of the five technologies selected by the EC under this initiative. The purpose is to develop an action plan with measures to remove obstacles hindering further development and to fully exploit the results of research. The most strategically relevant KET have been chosen because of their economic potential, contribution to solving societal challenges and knowledge intensity, and the use of biopolymers will address both the waste management and the progressive replacement of nonrenewable materials.

Ensure the coherent, comprehensive and coordinated development of policies and regulations that impact the development of biobased product markets

Legislation

Standardisation, labelling, certification

Timetable

Establish a network between public purchasers of biobased products to apply the EC guide on public procurement for innovation, to identify good practices in the field of biobased products and promote their application across the EU

EC and stakeholders

Actors involved

EC, CEN and stakeholders

2008 −2010 EC, Member States and industry

Establish a high-level advisory group, including Member 2008 States and industry, to assist the thematic interservice task force on biobased products in the follow-up of the present action plan and including in the analysis the impact of legislative proposals in relevant policy domains on the development of markets for biobased products

Action

Aggregate demand for biobased products Establish standards/labels for specific biobased 2008−2011 through a coordinated approach for standard products involving all relevant actors by: analysing setting and labelling the potential of biobased products standards/ labels, launching a mandate to CEN, in cooperation with EC services developing standards/labels, including cost-effective assessment criteria and procedures, building upon the current work of the European platform on LCA proposing a first set of standards

Public procurement Encourage Green Public Procurement for biobased products

Objectives

Policy instruments

Table 18.1: Lead Market Initiative (LMI) scheme [8].

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Conduct an information campaign via different media with the focus on SME Promote the establishment of strategically important biorefinery pilot plants and demonstrators involving all actors and investments at EU, national and regional level (PPP)

Communication of policies regarding biobased products as well as the benefits of biobased products.

Support access to finance for Research & Development & Innovation

CEN: European Committee for Standardization LCA: Life cycle assessment PPP: Public-private partnerships

Complementary Actions

Table 18.1 (continued)

2008−2010

2008−2012

EC, Member States and stakeholders

EC

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In fact, the communication outlines policy areas which need to be addressed, including focusing innovation policy more on KET, promoting more EU-wide technology transfer, as well as more joint strategic programming and demonstration projects [11]. Advanced materials are also suggested as a strategically relevant KET which could be used in various fields to facilitate recycling and lowering the carbon footprint. The EC also suggests that KET should be closely linked to climate change policies and be placed at the top of the EU’s international trade agenda. The EC has set up a High-Level Expert Group tasked with proposing specific policy recommendations for a more effective industrial deployment of KET in the EU [12].

18.2.3 The Innovation Union On 6th October 2010, the EC launched the Europe 2020 flagship initiative ‘Innovation Union’, aiming to set out a coherent and strategic approach to innovation policy in Europe, and containing over 30 concrete action points. The European Innovation Partnerships are one of these proposed action points [13]. To face societal changes and needs, Europe should combine research efforts and achieve a critical mass. From this point of view, the European Innovation Partnerships are meant to combine EU, national and regional efforts in R&D and innovation to achieve the targets quicker and more efficiently. In 2011, the EC launched the first pilot European Innovation Partnerships on Active and Healthy Ageing [14]; today the five European Innovation Partnerships are: Active and Healthy Ageing, Agricultural Productivity and Sustainability, Smart Cities and Communities, Water and Raw Materials. The European Innovation Partnership − Agricultural Productivity and Sustainability works to foster competitive and sustainable farming and forestry that ‘achieves more and better from less’. It contributes to ensuring a steady supply of food, feed and biomaterials, developing its work in harmony with the essential natural resources upon which farming depends [15].

18.2.4 The Bioeconomy Strategy The EC has recently raised the bar and launched a new strategy on the Bioeconomy entitled ‘Innovating for Sustainable Growth: a Bioeconomy for Europe’ [16]. The proposal will help to drive the transition from a fossil-based economy to a sustainable bioeconomy in Europe, with research and innovation at its core. According to the document, published on 13th February 2013, the EU bioeconomy includes agriculture, forestry, fisheries and food production, as well as parts of the chemical, biotechnological and energy industries, and it encompasses the sustainable production of renewable biological resources and their conversion, as well as that of waste streams, into biobased products, biofuels and bioenergy. The stronger innovation drive and reinforced policy interaction

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prescribed by the Bioeconomy Strategy, combined with the increased research funding for the bioeconomy under Horizon 2020 (see below), is estimated to generate an added value of about €45 billion and create new jobs in bioeconomy sectors by 2025.

18.3 European Union-funded research on biopolymers and their applications The paragraphs that follow will present the main features of FP for RTD. An analysis will be conducted on the succession and development of FP, focusing on specific programmes in the fields of biochemistry, biopolymers and biotechnology for nonfood products and processes.

18.3.1 Why the need for European Union-funded research? The EU research policy finds its legal basis in the EU Treaty. The EU, as we know it today, is the product of more than 50 years of development and integration. It is the merger of three communities: the European Coal and Steel Community (ECSC), founded in 1951 in Paris, the European Economic Community (EEC) and the European Atomic Energy Community (EURATOM), both founded by the Treaties of Rome in 1957. Although the three communities focused on different objectives, the EURATOM and ECSC Treaties made some reference to research activities in their fields of competence. In the third treaty, setting up the EEC, some provisions were made for research intended to boost agricultural productivity (Article 41), but did not originally include any reference to research in other sectors. However, one of its general articles (Article 235) was the legal basis that allowed the implementation, during the 1960s and 1970s, of a certain number of research programmes in areas such as energy, environment, biotechnology and so on [17]. A newly introduced Title (Title VI, Articles 130f−130q) in the Single European Act, signed in 1986, conferred upon the Community a specific competence in the field of RTD and described the objectives of the Community RTD policy. Article 130i, acknowledging the practise [18], clearly stipulates that the Community RTD policy should be based on multiannual FP, in order to complement the research activities carried out by Member States and to foster their cooperation in the field. Today, after several amendments of the Treaties, and reforms of the institutional structure of the EU, the legal basis for the European RTD policy is to be found in Title XIX, Articles 179− 190 of the Treaty on the Functioning of the EU (the so-called Lisbon Treaty), that came into force on 1st December 2009 [18]. The main stages of the EU integration and FP are listed in Table 18.2.

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Table 18.2: Main stages of EU integration and FP. 1951: ECSC is founded by Belgium, France, Germany, Italy, Luxembourg and the Netherlands (Treaty of Paris) 1957: The Treaties establishing the EEC and EURATOM are signed in Rome 1973: First enlargement: Denmark, Ireland and the UK join the European Communities 1979: The first elections to the European Parliament are held 1981: Greece joins the Communities 1984: First FP (1984−1987) 1986: Spain and Portugal join the Communities 1987: Second FP (1987−1991) 1991: Third FP (1990−1994) 1993: Maastricht Treaty enters into force. The European Community becomes the EU 1994: Fourth FP (1994−1998) 1995: Austria, Finland and Sweden join the EU 1998: Fifth FP (1998−2002) 2002: Euro starts circulating in 12 EU Member States; Sixth FP (2002−2006) 2004: Czech Republic, Cyprus, Estonia, Latvia, Lithuania, Hungary, Malta, Poland, Slovenia and Slovakia join the EU 2007: Bulgaria and Romania join the EU, which totals 27 Member States; Seventh FP (2007−2013) 2009: The Treaty of Lisbon comes into force 2014: Horizon 2020 − the FP for Research and Innovation (2014−2020)

18.3.2 The Framework Programmes FP were firstly introduced in the 1980s in an attempt to rationalise already existing Community research activities and include them in a more comprehensive plan, both in terms of identifying scientific and technological priorities and planning Community financial involvement. The Council of Ministers, in its resolution of 25th July 1983 [19], approved the principle of FP covering a multiyear period (4 years in the case of the first FP, 5 years from the second to the sixth ones, and 7 years for the current FP7 and for the next FP) and defined the scientific and technical objectives and selection criteria for the period 1984−1987. FP adopted by the Council and the European Parliament set the main objectives and priorities, the overall framework of Community activity in the area of RTD and its breakdown into main areas, together with total financial allocation. FP form the basis for decisions on specific implementation programmes, i.e., the programmes implement the FP.

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From the inception, the purpose of the FP has been to encourage transnational collaborative research projects, and this explains why one of the essential requirements for all EU-funded projects is that the participants in research teams have to come from more than one of the EU Member States. A common characteristic for all FP is that most of the funding (with the exception mainly of mobility grants for individual researchers) supports research activities, defined in a ‘top down’ approach: research projects selected for funding should fit into politically and scientifically determinate thematic topics, fulfilling the objectives and criteria set out in the FP and in specific programmes [20]. FP have evolved in several ways: a steady increase of the budget, from several hundred million euro up to €7 billion per annum in FP7 (reaching €10.5 billion in 2013); an extension of EU activities in new scientific and technological fields; a shift from hard scientific and technological research towards a multidisciplinary research focused on social demands and challenges; a decreased relative importance of areas such as energy and, to some extent, information and communication technologies (ICT), in favour of areas such as life sciences and the environment; and the diversification and introduction of new mechanisms of financial support and intervention methods, resulting in the present-day portfolio which ranges from projects to transnational networks for collaboration in research, and includes individual grants to researchers, specific measures for SME, support schemes for cooperation and coordination at various levels, as well as studies, publications and conferences [20] (see Figure 18.1).

18.3.3 Specific programmes with focus on biopolymers and their applications As previously mentioned, FP define the general objectives of EU RTD policy for a given period of time and are implemented through specific programmes that cover various fields of research. The quantity and content of specific programmes have developed over the years with relation to the evolution and progress of research and industrial application, and to the interest shown by stakeholders and policymakers (and the public) in a particular subject. Since the beginning, biotechnology and biochemistry were taken into account and appeared among the research priorities, usually covered by a dedicated specific programme, but specific research themes on biochemistry for nonfood products and processes were introduced in FP2 (see Table 18.3). FP1, in fact, made some general provisions for nonfood activities, but these were carried out under the general heading of research on energy from biomass. The interest in biobased nonfood products grew, even if not linearly, in the following FP: an explanation of the interest shown by the EC, apart from moving to a more sustainable economy, can be found in the increasing stock surplus of agricul-

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 Alessandra Perrazzelli Total budget of the EU FP

€ Million

50,521

19,235

3,750

5,396

FPI (1984– 1987)

FP2 (1987– 1991)

6,600

FP3 (1990– 1994)

13,100

14,960

FP4 (1994– 1998)

FP5 (1998– 2002)

FP6 (2002– 2006)

FP7 (2007– 2013)

FP annual budget growth

€ Million

7,217.3

3,847 2,620

937.5

FPI (1984– 1987)

1,079.2

FP2 (1987– 1991)

2,992

1,320

FP3 (1990– 1994)

FP4 (1994– 1998)

FP5 (1998– 2002)

FP6 (2002– 2006)

FP7 (2007– 2013)

Figure 18.1: Total budget and annual budget growth of EU FP [21].

tural production as a consequence of the Common Agricultural Policy (CAP). A critical point, strictly connected with European research in the field of chemistry and biotechnology, was in fact the reform of CAP in order to avoid the increasing surplus of agricultural production. In recent years, though, the situation somehow reversed, and the exploitation of biological resources for products (including energy products) from biomass has become a concern, and it is even actively opposed by some sectors of civil society (environmental nongovernmental organisations mainly). In fact, the use of land to produce biomass is perceived as a competitive use of land for the production of food. The paragraphs that follow will give a brief overview of the specific programmes or subprogrammes taking into account the research into biopolymers and nonfood products and processes. Focus will be then on research activities funded by FP in

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the field of biopolymers, with particular attention to fields such as biorefineries, biochemistry for nonfood products, bioplastics and food packaging. Analysis of several projects will be conducted in relation to the specific programme under which they have been funded. As mentioned previously, in FP1, some general provisions for nonfood activities were inserted under the general heading of research on energy from biomass. In FP2, which covered the years 1987 to 1991 [22], the specific programme, ECLAIR was established. Under Subactivity 4.2 ‘Agroindustrial technologies’ of FP2, the ECLAIR programme was established [23] with the aim of improving the interface of agriculture and industry, by taking advantage of Europe’s strength in biotechnology and life sciences. Thus, the objective was to encourage the transfer of technology in those areas where it would have the most practical and economic benefits. ECLAIR was the first programme to be directed specifically towards development of the use of agricultural raw materials as feedstock in the industrial sector. Table 18.3: Nonfood subprogrammes in FP. FP1 1984−1987

–

FP2 1987−1991

ECLAIR

FP3 1990−1994

AIR

FP4 1994−1998

FAIR

FP5 1998−2002

QoL – KA5

FP6 2002−2006

NMP

FP7 2007−2013

KBBE, NMP, health and the environment

AIR: Specific research and technological development and demonstration programme in the field of agriculture and agroindustry, including fisheries ECLAIR: European Collaborative Linkage of Agriculture and Industry through Research FAIR: Specific RTD programme in the field of fisheries, agriculture and agro-industrial research KA: Key Action NMP: Nanotechnologies and nanosciences, knowledge-based multifunctional materials, and new production processes and devices QoL: Quality-of-life

The financial allocation for ECLAIR was 80 million European Currency Unit (ECU) [24], of which 65 million ECU was devoted to shared cost projects and the remaining part to training, mobility grants, workshops and management of the programme [25]. The main objectives of ECLAIR were: 1) research, adaptation and development of agricultural products intended for industrial use, as well as the research and promotion of new industrial techniques for processing and transforming agricultural raw materials with the view to obtaining, under economically viable conditions, indus-

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trial products which meet the need of the market, 2) research and development of industrial inputs in agriculture, such as pesticides and fertilisers, and eradication and disease control systems less harmful or better adapted to the environment, and 3) the reduction and elimination of processing by-products through recovering resources and thus reducing waste [26]. ECLAIR funded 67 projects in various research areas: the application of molecular biology to crop plants, nonfood uses of crops, processing of crops, animal health and monitoring, and biological pest control [17]. The first project on biorefinery funded under the ECLAIR programme was the Whole Crop Biorefinery Project, as shown in Table 18.4. Table 18.4: Whole Crop Biorefinery Project [27]. The first biorefinery project was funded by the European Commission in 1991 under the ECLAIR programme (FP2). The Whole Crop Biorefinery Project was coordinated by a Danish research organisation and involved 11 partners from 5 Member States. It developed and tested technologies for the treatment and fractionation of agricultural crops, in order to allow technical and economic assessment of new areas of utilisation of whole crops, especially within the nonfood area. The project covered the whole integrated production chain from primary producers to end user, but concentrated mainly on the following activities: selection of the best-suited plant species based on botanical, physical, chemical and cultivation-related characteristics in relation to the utilisation of the entire plant; methods for the pretreatment and storage of unprocessed or wetseparated crops to achieve maximum capacity utilisation of the biorefinery; mechanical, physical and biochemical techniques for the separation and refining of oil crops, protein crops, starch crops, fibre crops and various by-products. In association with the technical tasks, an economic systems analysis model was developed in order to enable economic assessments of various types of biorefineries in various regions of the European Union

The successor of ECLAIR, the AIR programme was established under FP3 (1990−1994) [28] Subactivity II.4 ‘Life Sciences and Technologies’; however, drawing comparisons on the succession of ECLAIR-AIR is not straightforward, since they had a different scope; it must be highlighted that the budget allocation significantly increased. In fact, the AIR Programme (specific RTD and demonstration programme in the field of agriculture and agroindustry, including fisheries) had a budget of 377 million ECU [29]. The AIR Programme funded a total of 426 precompetitive RTD projects in four areas of research, involving more than 3,000 participants: 1) primary production in agriculture, horticulture, forestry, fisheries and aquaculture, 2) inputs to agriculture, forestry, aquaculture and fisheries, 3) processing of biological raw materials from agriculture, forestry, aquaculture and fisheries, and 4) end use and products (including nonfood demonstration projects) [30]. In particular, in the bioeconomy area, research was focused on the identification of new, more environmentally friendly types of products, such as: biodegradable

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materials, new composite products, biobased chemicals (detergents, lubricants) and pharmaceuticals, biocompatible polymers for medical applications, e.g., slow-release agents for medicines, seed, fertilisers, pesticides, herbicides and so on. An example of a project funded under the AIR programme in the field of bioeconomy is shown in Table 18.5. Table 18.5: The Biodegradability of Bioplastics Project [31]. Under FP3, a prenormative research project was carried out by a team led by a Belgian University on the biodegradability of bioplastics. The Biodegradability of Bioplastics: Prenormative Research, Biorecycling and Ecological Impacts project had as main objective the development of methods for the assessment of biodegradability and of standard systems, as well as the evaluation of methods for biorecycling and of the ecological impacts of bioplastics. The research team focused on rapid in vitro tests using cultures of microorganisms to determine the biodegradability of plastics. The project lasted 36 months, brought together three universities, two research centres and a company, and was granted €1,793,723 of EU funding

In the life sciences and technologies theme of FP4 [32], FAIR (a specific programme of research, technological development and demonstration in the fields of agriculture and fisheries, including agroindustry, food technologies, forestry, aquaculture and rural development) and BIOTECH 2 (a specific programme of research and technological development, including demonstration, in the field of biotechnology) programmes were established for the years 1994 to 1998. As a continuation of AIR, the FAIR programme [33] had a financial allocation of 739.5 million ECU, with the main objective of promoting the harmonising research in the food and nonfood production sectors of agriculture, horticulture, forestry, fisheries and aquaculture. It was implemented through demonstration activities (DEMO) and specific measures in support of SME (CRAFT), shared cost projects, concerted action and thematic networks, and research training grants. Six areas of research were covered by the FAIR programme: 1) integrated production and processing chains, 2) scaling up and processing methodologies, 3) generic sciences and advanced technologies for nutritious foods, 4) agriculture, forestry and rural development (this area was prioritised, accounting for 22% of the programme budget), 5) fisheries and aquaculture, and 6) ethical, legal and social aspects [34]. Area 1, Integrated Production and Processing Chains, was devoted to the nonfood agroindustrial and forestry sectors, from the production and processing of plant raw materials to the development of optimised, ‘cleaner’ processing methodologies and products with higher added value. The programme aimed to establish closer links between the input and processing industries, the end-user and the consumer, and matching the production of biological raw materials to the needs and requirements of the processing industry, end-users and consumers. In this area, Activity 1.2 dealt

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with ‘Green chemical and polymer chain’: under this theme 18 projects were funded, mostly covering precompetitive research for the development of new highly functional biobased materials. An example of a project funded under FP4 in the field of bioeconomy and biomaterials is shown in Table 18.6. Table 18.6: The Labelling Biodegradable Products Project [35]. The Labelling Biodegradable Products project was funded under FP4. Led by a French research centre, the project aimed to develop guidelines, testing scheme and predictive models for labelling the biodegradability of (agro) industrial polymers in relation to their environmental fate in agriculture and waste treatment. The project was funded under the specific research and technological development programme in the field of standards, measurements and testing (SMT programme) which aimed to provide research and technical support for the continued institution of harmonised systems of measurements, reference materials and written standards, essential to ensure the efficient operation of the Single Market and other EU policies and the competitiveness of European industry

Under FP5 (1998−2002) [36], research activities on biological material for nonfood use are to be found in the specific programme QoL, under KA5. Key Actions (KA), as a newly introduced concept, consisted of a cluster of small and large, applied, generic and, as appropriate, basic research projects directed towards a common European challenge or problem. The idea behind it was to organise research around multidisciplinary areas, rather than into compartmentalised disciplines. In the KA that were identified, European research had to be able to make a contribution, through innovative products, processes or services, to the resolution of the given societal problem. KA were targeted at socio- economic needs and the community policy objectives, for example, in the fields of agriculture and fisheries, industry and consumers, and health and the environment. Additional RTD activities of a generic nature and support to research infrastructures aimed to build up knowledge in specific areas over the longer term. Therefore, previous specific programmes were reorganised and life sciences and biotechnology research themes were merged into the QoL Thematic Programme [37]. The QoL Programme had a budget of €2.413 million and consisted of the 6 following KA: 1) food, nutrition and health, 2) control of infectious diseases, 3) the ‘cell factory’, 4) the environment and health, 5) sustainable agriculture, fisheries and forestry, and the integrated development of rural areas, including mountain areas, and 6) the ageing population and disabilities. Under KA5, Sustainable agriculture, fisheries and forestry, with a budget of €520 million (representing 22% of the QoL Programme budget), 593 research contracts were signed (involving almost 3,000 European scientists) and 305 out of 593 were cooperative RTD actions [38].

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A special provision in KA5 was made for the integrated production and exploitation of biological materials for nonfood uses. Subtheme 5.2 (nonfood products) sought to encourage the production and exploitation of biological materials for nonfood use by offering a broad range of possible applications: biopolymers for packaging and construction, fine chemicals for use in pharmaceuticals, bulk chemicals for lubricants, solvents and detergents, and biofuels for energy and transport. Specifically focused on nonfood uses of agricultural material, the work programme had a strong industrial focus based on a ‘chain’ concept, i.e., starting with products and working back to primary production. Thus, three aspects were taken into account: the market for products such as bioplastics, composites, bulk and fine chemicals, including market and LCA studies with results applied in the promotion of standards, codes and guidelines; the processing of biological raw materials for volume or higher added-value chemicals, polymers, composites and biofuels including scale-up processes and recovery of materials from wastes; matching agricultural and forestry production to industrial requirements, with an emphasis on molecular farming and systems that aid recovery of valuable components. Funded projects had to demonstrate the potential use of nonfood materials, including hydrogen from biomass, biopolymers from vegetable oil, biomedical textiles from chitin and algae for the production of bioplastics [39]. However, the overall contribution of the subtopic to the nonfood sector was limited to some extent by the size of the budget, which restricted the number of funded projects (21 out of 593 projects in KA5) as well as the topics that were actually selected for funding. The low level of funding gave a clear indication of the declining interest in research in the nonfood sector in the late 1990s, which was to culminate in the total absence of a nonfood-related programme in FP6 [40]. An overall lack of innovation was noted for the projects funded under QoL KA5, accompanied by a lack of industrial lead partners, although industrial participation in these projects was high, representing, on average, 33% of the partnerships. Many projects were based, according to the ex post report on FP5, on existing technologies and results were not taken to the marketplace in the short term. This is perhaps an indication of the lack of a strong commercial ‘pull’ for these technologies during those years, as biobased materials were still generally more expensive than their fossil-based equivalent, thus deterring companies from investing in new technologies [38]. Nonfood research at the genomic and cellular level was also funded under the biotechnology and cell biology section of the Cell Factory in KA3, while other nonfood aspects were covered by the renewable energy in KA4 and the materials and processing in KA3. Some examples of research projects funded under QoL KA5 are described in Table 18.7. Although there was diffusion of nonfood research activities throughout many thematic areas of FP5, demonstrating the strong interest and potential of agricultural and forest raw material feedstocks for industrial use, the situation evolved in a nonlinear manner. In fact, FP6 did not contain specific nonfood activities or programmes,

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Table 18.7: Some research projects funded under QoL KA5. The BIOPLASTICS (biodegradable plastics for environmentally friendly mulching and low tunnel cultivation) project was funded with EU contribution of €1,396,820. Bringing together 8 partners (3 industries, 3 universities and 2 research centres), the BIOPLASTICS project aimed to investigate the agronomic, engineering and environmental aspects involved in the use of bioplastic films in agriculture, and integrate them into well-justified techniques for enhancing sustainable, environmental friendly, economically optimised low-tunnel and mulching cultivation in Europe, and to improve or develop new bioplastic films and determine their overall performance, including eco-toxicological impact, in field experiments at a European level [41]. In 2001, the project Development of a 100% biodegradable and photoselective mulch film for sustainable agriculture was granted €22,500 by the European Commission to develop new and innovative 100% biodegradable and photoselective mulch film vegetable crops, at a competitive cost. The project focused on finding an environmentally friendly and economically viable solution for the management of plastic agriculture waste (both postharvest and production), through the complete biodegradation of the material [42]. The BIO-PACK project (biodegradable extruded starch-based plastics for packaging material) involved two partners and had a funding of €22,500. The project addressed the problem of packaging waste by developing new types of proactive biobased packaging introducing microscale blends of starches and synthetic polymers using compatabiliser techniques [43]

but some research topics on biobased materials for nonfood use were to be found in the nanotechnologies and nanosciences, knowledge-based multifunctional materials and new production processes and devices (NMP) thematic area and in the specific programme for SME [44]. This can be explained as a reflection of the concerns that were raised by public opinion in the early 2000s relating to plant genetic engineering and genetically modified organism (GMO) products in foods: the EC decided to focus research activities on the areas of biotechnology and genomics for health and food safety and health risks, which were perceived to be of higher priority [45]. Some examples of research projects funded under FP6 in the field of biorefineries are shown in Table 18.8. Table 18.8: The BIOPOL and BIOREFINERY EUROVIEW projects. Under FP6, the BIOPOL (assessment of biorefinery concepts and the implications for agricultural and forestry policy) and BIOREFINERY EUROVIEW (current situation and potential of the biorefinery concept in the EU: strategic framework and guidelines for its development) projects were funded. Bringing together 4 universities and 4 research institutes, the BIOPOL project assessed the status (from the technical, social, environmental, political, and implementation point of view) of innovative biorefinery concepts and their implications for European agricultural and forestry policy. The project was granted €551,000 for the duration of 24 months [46]. BIOREFINERY EUROVIEW consortium (made up of 8 partners) studied the existing or planned European biorefineries taking into account the different aspects of their economic, social and environmental impact, in order to develop a theoretical scenario for biorefinery development in Europe at the 2020 horizon. The project has been funded under the heading ‘Policies’ and has been granted €436,078 of EU funds [47]

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18.4 The seventh Framework Programme With a budget of over €50 billion, FP7 ran from 2007 to 2013, in line with the financial perspectives of the EU. Thus, FP7 was the largest publicly funded research programme in the world both in terms of budget and time covered [48]. The decision to align FP7 to financial perspectives resulted in extending the time frame to 7 years, and this gave the EC the possibility of gaining more experience of the ongoing FP and to have time for the evaluation and proper assessment of results, before starting the preparation of the next FP. FP7 was based on four major specific programmes: cooperation (€32.413 million), people (€4.750 million), ideas (€7.510 million) and capacities (€4.097 million). A fifth programme, EURATOM, with a budget of €2.751 million, deals with nuclear research. The Cooperation Programme, which aims to foster collaborative and transnational research on policy defined themes, according to a top-down approach, was clearly prioritised in terms of financing: it absorbed nearly 65% of the overall budget. The Cooperation Programme was subdivided into 10 thematic areas: health, food, agriculture, fisheries and biotechnology (KBBE), ICT, NMP, energy, environment (including climate change), transport (including aeronautics), socio-economic sciences and humanities, security and space (see Figure 18.2). Socio-economic sciences and humanities 623 2%

Security 1,400 4%

Transport (including aeronautics) 4,160 13%

Health 6,100 19% Food, agriculture and biotechnology 1,935 6%

Environment (including climate change) 1,890 6% Energy 2,350 7%

Space 1,430 4%

Nanosciences, nanotechnologies, materials and new production technologies 3,475 11%

Information and communication technologies 9,050 28%

Figure 18.2: Cooperative Programme breakdown (€ million and percentage).

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Within the Cooperation Programme, €1.9 billion was allocated to the food, agriculture, fisheries and biotechnology thematic area. Its main objective was to build the European KBBE by bringing together research organisations, industry and other stakeholders to exploit new and emerging research opportunities which systematically address the main societal, environmental and economic challenges [49]. €3.5 billion was allocated to NMP, the core objective of which is to improve the competitiveness of European industry and to generate the knowledge needed to transform it from a resource-intensive to a knowledge-intensive industry by financing, e.g., projects on the development of suitable biopolymers for packaging. Health was a major theme of the Cooperation Programme and the EU earmarked a total of €6.1 billion for funding it over the duration of FP7, in order to support new medical technologies using, among others, new materials and biopolymers. The main objective of research for the environment under FP7 was, instead, to promote the sustainable management of both the man-made and natural environment, and its resources. To this end, increased knowledge on the interaction between the climate, biosphere, ecosystems and human activities was sought, and new environmentally friendly technologies, tools and services were developed and supported by €1.9 billion for 7 years. Therefore, research on nonfood products resumed in FP7 and many activities were entirely devoted to life sciences, biotechnology and biochemistry for nonfood products and processes in a systematic approach, the production of improved renewable raw materials, the processing of renewable raw materials and environmental biotechnology. In particular, the KBBE thematic area had a financial allocation of €1.935 million, which was based on the rationale that innovations and advancement of knowledge in the sustainable management, production and use of biological resources would provide the basis for new, sustainable, safer, ecoefficient and competitive products for agriculture, fisheries, feed, food, health, forest-based and related industries. In a systematic approach, the KBBE thematic area intended to take stock of research results to increase the competitiveness of European companies working in the agricultural, food and biotechnology sectors. Areas such as industrial biotechnology, and novel high added-value bioproducts and bioprocesses, addressed the development and application of industrial biotechnology for the production of high-value products. These activities enable industries to deliver novel products which cannot be produced by conventional industrial methods; in addition, it enables replacing chemical processes with more resourceefficient biotechnological methods which have a reduced environmental impact. Biorefineries addressed the development and application of industrial biotechnologies for the conversion of renewable raw materials into sustainable and cost-efficient bulk bioproducts (e.g., chemicals such as lactic acid, biopolymers), and/or bioenergy. As we have seen, setting up biorefinery demonstration projects via public-private partnerships (PPP) had been one of the measures undertaken in the LMI for biobased products.

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The biopolymers issue in FP7 also regarded biofuels; here the focus was on the development of second generation biofuels, with improved energy and environmental balance, and avoiding the potential food/fuel conflict. Aimed at achieving an integrated and whole crop use of biomass, biorefineries can use a broad range of biomass feedstocks, ranging from dedicated agricultural, aquatic and forest biomass chains to residues/waste and by-products of biomass-based industrial sectors. Emphasis was put on: the discovery, characterisation and development of novel enzymes and strains, with optimised biocatalyst and microbial function for improved production of energy and bioproducts; characterisation of the structure and composition of the feedstock for optimised pretreatment and fractionation of the biomass into its components; development of improved bioprocesses with increased yield, quality and purity through bioprocess design, process optimisation and integration, as well as optimising downstream processing, fermentation science and engineering. Table 18.9 describes some examples of projects funded under the FP7 joint call on biorefineries. Table 18.9: Projects funded under the joint call on biorefineries. Under a joint call on biorefineries, the EC funded a specific support action, the Star-COLIBRI project (Strategic Research Targets for 2020 – Collaboration Initiative on Biorefineries). This project involved 5 European Technology Platforms and 5 major European research organisations. Star-COLIBRI aimed to overcome fragmentation and promote cross-fertilisation in the area of biorefineries research. The project supported innovation by speeding up and facilitating the industrial exploitation of research results in the biorefinery sector, as well as promoting coordination in the field of future R&D funding and facilitating the creation of PPP [50]. Started in 2008, the SUSTOIL project (developing advanced biorefinery schemes for integration into existing oil production/transesterification plants) aimed to develop advanced biorefinery schemes to convert whole European oil-rich crops into energy, food and bioproducts, making optimal use of the side streams generated during farming/harvesting, primary processing and secondary processing [51]. The overall aim of BIOREF-INTEG was the development of advanced biorefinery schemes to be integrated into existing industrial fuel-producing complexes. The main project objectives were: to make the production of biofuels more competitive, to identify and develop the optimal integrated biorefinery schemes for the production of best-suited ‘building blocks’ in terms of processes and bioproducts, and to identify opportunities in various biomass-based sectors to produce fuels while increasing their market competitiveness by coproducing high added-value products [52]

18.5 Funded projects: Biopolymers and their applications Success stories of EU-funded projects on biopolymers and their application have been analysed under the successive FP. Data have been collected, through a search in the Community Research and Development Information Service database (CORDIS) [53],

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to give some examples on how the EU has funded the biopolymers issue in their different applications throughout the development of the research programmes. Starting from FP4, several projects were financed, differing largely in budget allocation, consortium composition and the subprogramme under which they were funded. Moving from the older FP through to FP7, it can be noted that the latter projects have been funded under a higher number of subprogrammes, demonstrating the growing interest and awareness of the importance of the research and application of biopolymers and their application, in areas such as health. Recently, a new study, using the electronic research system in CORDIS, has been carried out by the University of Graz focusing on polyhydroxyalkanoates (PHA), polylactic acid (PLA) and starch-based plastics throughout the FP. The task was extremely complex due to the fact that information in the CORDIS system is lacking certain data and information on the project objectives is often very weak, but the study also reached the conclusion that the importance of bioplastics and bioderived plastics has not grown linearly. Much less money was spent in FP2−FP4 than in FP5−FP7, whilst funding reached its maximum in FP6 and FP7 [54]. The importance and growing interest in bioplastics also stems from the analysis conducted by the University of Graz, using a different methodology and keywords. The study proved an equal distribution of funding among the 64 projects found in the fields of starch-based plastics (20 projects), PHA (20 projects) and PLA (24 projects) from FP2 to FP7 in the CORDIS database. Comparing the different FP, it can be stated that European funding increased particularly in the latest programmes. A total of €88.068 million has been spent for financing sustainable plastics in the three fields mentioned above from FP5 to FP7 [54]. Regarding consortia composition, we note that the highest success rate in the projects belongs to academia, whereas in terms of industry participants [55], it can be noted that industry, which actually participates extensively, is deterred to a greater degree than other researchers which may be due, in part, to the weight of bureaucratic burdens and, on occasion, by a perception of insufficient flexibility in work programmes. For these reasons, the EU is currently devoting more effort to achieving greater impact regarding innovation, stimulating the participation of industry and SME, and focusing on the whole innovation process; as stated in the Interim Evaluation of FP7, the role of industry as the bridge between research and ‘commercialisation’ has to be stressed [56].

18.6 The Eco-innovation initiative One of the main actions the EU has already promoted to support innovation and the ‘commercialisation’ side is the Eco-innovation Initiative.

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 511

As part of the Entrepreneurship and Innovation Programme of the CIP [57], the Eco-innovation First Application and Market Replication Projects Initiative [58] aimed at filling the gap between research and the market uptake of innovative products. The programme aimed, in fact, to support projects which contribute to removing obstacles which hinder the uptake of eco-innovation into the market. Eco-innovation is defined as ‘any form of innovation aiming at significant and demonstrable progress towards the goal of sustainable development, through reducing impacts on the environment or achieving a more efficient and responsible use of natural resources, including energy’ [59]. The programme showed a special focus on biodegradable polymers as an important resource for reducing environmental contamination and waste. With a budget of €320 million for the years 2007−2013, the Eco-innovation supported, through the means of cofinancing, projects concerning the first application or market replication of ecoinnovative techniques, products, processes or services (beyond the prototype phase), which had already been technically demonstrated, but due to remaining risks, needed further incentives to significantly penetrate the market. Projects aimed to reduce environmental impacts, increase resource efficiency or improve the environmental performance of enterprises. In this sense, environmental impacts and optimisation of resources were considered in a nonisolated way throughout the life cycle of the related activities: from extraction of raw materials to production, distribution, use and final disposal or recycling using a full life cycle approach [60]. Over the last few years, the programme funded projects involving several priorities due to their importance for environmental protection and eco-innovation markets, and because of the expected added value of projects in these areas, which also took into consideration other EU actions in connected fields. These priorities covered, among others, recycling materials, clean and innovative products, processes and services aimed at a reduction of waste, water efficient processes, products and technologies, and funding to help SME reduce their environmental impact. Table 18.10 shows an example of a project funded by the Eco-innovation Initiative. Table 18.10: Example of a project funded by the Eco-innovation Initiative. Started in 2010, the project ‘A Green Optical Storage Medium for a Greener Europe (Pla Optical Disc)’ was funded under the priority area “Greening Businesses” and aimed to support the changeover from petroleum-based polymers to biopolymers made out of renewable resources. Under this project, two partners produced an optical disc replacing petroleum-based standard polycarbonate for optical media, with the environmentally compatible alternative polylactide acid (PLA), produced from the surplus European production of sugar beets, and then introduced onto the market with a certification of biodegradability [61]

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18.7 Horizon 2020 Horizon 2020 is the financial instrument implementing the Innovation Union, a Europe 2020 Flagship Initiative aimed at securing Europe’s global competitiveness. Running from 2014−2020 with a budget of just over €80 billion, the EU’s new programme for research and innovation is part of the drive to create new growth and jobs in Europe. One of the specific objectives of Horizon 2020 is to secure sufficient supplies of safe, high-quality food, and other biobased products, by developing productive and resource-efficient primary production systems in order to accelerate the transition to a sustainable European bioeconomy [62]. The EC’s proposal for Horizon 2020 also mentions the use of PPP in the form of a Joint Technology Initiative (JTI). The PPP is an instrument to support industrial research and innovation, to overcome the innovation ‘valley of death’, the path from research to the marketplace. It encourages partnership with the private sector to fund and bring together the resources needed to address the challenges involved in commercialising new technologies which have a major impact on society. On 10 July 2013, the EC launched its Innovation and Investment Package containing 5 JTI, including a newcomer on Biobased Industries; these industries are organised into a Biobased Industries Consortium. The consortium currently brings together approximately 70 full members (European large and small companies, clusters and organisations) and more than 100 associated members (universities, research and technology organisations, associations, European trade organisations, European technology platforms) across technology, industry, agriculture and forestry. They have all committed to invest in collaborative research, development and demonstration of biobased technologies within the PPP [63].

18.8 Conclusions There is still a long way to go in order to successfully build the KBBE in Europe, but the considerable effort that has been undertaken over the last few years is now showing results. In the field of research, the steady growth in FP budget shows the importance of research for the future of Europe. In this context, the importance of research activities related to biobased products and their application will probably continue to grow over the coming years under the new FP for research and innovation (Horizon 2020). Important and significant references to bioeconomy and bioindustries in the EC proposal highlights how the EU has, in a sense, understood the importance of speeding up a transition to a sustainable economy at the European level. The trend shows that several goals have been achieved, but evidence also suggests that extensive effort is still needed and areas of improvement such as legislation on market development and product-specific legislation are necessary. An interven-

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tion at the legislative level, such as the Italian ban of plastic shopping bags in favour of biodegradable bags, can powerfully boost the market development and uptake of biobased products. Moreover, encouraging the contracting authorities in all EU Member States to give preference to biobased products in tender specifications (the so-called ‘Green Public Procurement’) could also be fostered. Above all, there must be consistency and coherence across policies, funding programmes and legislative interventions, together with a strong political impetus to ensure that the goal of achieving the KBBE in Europe is treated as a priority [64].

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europa.eu/search/index.cfm?fuseaction=proj.document&PJ_LANG=EN&PJ_ RCN=9643255&pid=0&q=28141E1312D4D7C8FF58DB3755FD984C&type=adv. Decision 1982/2006/EC of the European Parliament and of the Council of 18 December 2006 concerning the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007−2013), Official Journal of the European Union, 30th December 2006, L412, p.1 Council Decision 2006/971/EC of 19th December 2006 concerning the Specific Programme Cooperation, Official Journal of the European Union, 30th December 2006, L400, p.86. Project Reference: 241535, STAR-COLIBRI – Strategic Targets for 2020 – Collaboration Initiative on Biorefineries Running from 2009- 11-01 to 2011-10-31. Project Reference: 213637, SUSTOIL – Developing Advanced Biorefinery Schemes for Integration into Existing Oil Production/Transesterification plants Running from 2008-06-01 to 2010-05-31. http://cordis.europa.eu/search/index.cfm?fuseaction=proj.document&PJ_LANG=EN&PJ_ RCN=10193633&pid=0&q=6DE30F5A5EF18B1E5EB6388AFFABD366&type=adv. Project Reference: 212831, BIOREF-INTEG – Development of Advanced Biorefinery Schemes to be Integrated into Existing Industrial Fuel Producing Complexes Running from 2008- 06-01to 2010-05-31. http://cordis.europa.eu/search/index.cfm?fuseaction=proj.document&PJ_ LANG=EN&PJ_RCN=10233259&pid=0&q=F4A4520ACB61EFC23BE700011B059CE8&type=adv CORDIS, European Commission. http://cordis.europa.eu/ G Braunegg in PLASTiCE Conference: Europe for Sustainable Plastics, Bologna, Italy, 2011. http://www.plastice.org/fileadmin/files/Lecture_Report_on_Biodegradable_polymers_and_ plastics.pdf As indicated on the CORDIS database, CORDIS, European Commission. http://cordis.europa.eu/ projects/home_en.html. Interim Evaluation of the Seventh Framework Programme, Report of the Expert Group, 2010. The Eco-innovation initiative was itself part of the Competitiveness and Innovation Framework Programme (CIP) for the years 2007–2013. With SMEs as its main target, the CIP Framework Programme supported innovation activities (including the eco-innovation initiative), provided for better access to finance and delivered business support services, such as the Enterprise Europe Network. Decision 1639/2006/EC of the European Parliament and of the Council of 24th October 2006 establishing a Competitiveness and Innovation Framework Programme (2007 to 2013), Official Journal of the European Union L 310/15, 09.11.2006. Eco-innovation, European Commission. http://ec.europa.eu/environment/eco-innovation Recital 25 of Decision 1639/2006/EC. Call for Proposals 2011 – Cip Eco-Innovation First Application and Market Replication Projects. http://ec.europa.eu/environment/eco-innovation/files/docs/getting-funds/text-of-the-2011call-for-proposals.pdf Eco-innovation, European Commission. http://ec.europa.eu/environment/eco-innovation/ projects/en/projects/pla-optical-disc Proposal COM(2011) 809/3 for a regulation of the European Parliament and of the Council Establishing Horizon 2020, The Framework Programme for Research and Innovation (2014−2020). Bio-based Industries Consortium. http://biconsortium.eu/ EuropaBio Policy Guide, Building a Bio-based Economy for Europe in 2020, 2010. http://www. europabio.org/positions/white/EB_bio-based_brochure.pdf

Abbreviations 1,4-BDO 3D 3HA 3HB 3HBA 3HHx 3HP 3HV 4HB 4HBA 8-HL 8-OL ABA ACP AD ADM AFEST AFNOR AfOR AIR alphaG1P ANL AOT APE AS ASTM ATP aw BBM BCF BML BOD BPA BPI BS BTA CAL CALB CAP CCL CDP CEN CHP CIC CIP

1,4-Butanediol Three-dimensional 3-Hydroxyalkanoate 3-Hydroxybutyrate 3-Hydroxybutyric acid 3-Hydroxyhexanoate 3-Hydroxypropionate 3-Hydroxyvalerate 4-Hydroxybutyrate 4-Hydroxybutyric acid 8-Membered lactone 8-Octanolide Australasian Bioplastics Association Acyl carrier protein Anaerobic digestion Archer Daniels Midland Company Archaeoglobus fulgidus Association Française de Normalisation Association for Organics Recycling Specific research and technological development and demonstration programme in the field of agriculture and agroindustry, including fisheries Alpha-glucose 1-phosphate Aspergillus niger lipase Bis(2-ethylhexyl)sulfosuccinate sodium salt Aliphatic polyester Australia standards American Society for Testing and Materials Adenosine triphosphate Critical water activity Benzyl β-malolactonate Bio Concentration Factor Benzyl malolactanate Biological oxygen demand Bisphenol A Biodegradable Products Institute British standard 1,4-Butanediol, terephthalic acid and adipic acid Candida antarctica lipase Candida antarctica lipase B Common Agricultural Policy Candida cylindracea lipase Cellodextrin phosphorylase Comité Européen de Normalisation (European Committee for Standardization) Combined heat and power Consorzio Italiano Compostatori Competitiveness and Innovation Framework Programme

https://doi.org/10.1515/9781501511967-019

518 

CK COD CORDIS c-P(3HB) CRL CS CVL DBSA DC DCW DDL DDT DEC DES DEV DGGE DIN DIS dLUC DMAc DMC2 DMF DMSO DMTA DNA DO DOC DP DSC DtD DTMC EAA EAM EC ECHA ECLAIR ECSC EEC EG EGII EN EOL EPD EPG EPS ETA EU EURATOM EVOH

 Abbreviations

Couchman and Karasz equation Chemical oxygen demand Community Research and Development Information Service database Complexed poly(R-3-hydroxybutyrate) Candida rugosa lipase China standards Chromobacterium viscosum lipase Dodecylbenzenesulfonic acid Critical deformation Dry cell weight 12-Dodecanolide Dichlorodiphenyltrichloroethane Diethyl carbonate Diethyl succinate German Standard Procedures for Investigation of Water, Wastewater and Sludge Denaturing gradient gel electrophoresis Deutsches Institut für Normung (German Institute for Standardisation) Draft international standard Direct land-use change Dimethylacetamide Cyclobis(decamethylene carbonate) N,N-Dimethylformamide Dimethyl sulfoxide Dynamic mechanical thermal analysis Deoxyribonucleic acid p-Dioxanone Dissolved organic carbon Degree of polymerisation Differential scanning calorimetry Door-to-door Dimer of trimethylene carbonate Ethylene-acrylic acid Enzyme-activated monomer European Commission European Chemicals Agency European Collaborative Linkage of Agriculture and Industry through Research European Coal and Steel Community European Economic Community Ethylene glycol Endoglucanase II European Norms End-of-life Environmental Product Declaration Environmental Polymers Group Expanded polystyrene 1,2-Ethanediol, adipic acid and terephthalic acid European Union European Atomic Energy Community Ethylene-vinyl alcohol

Abbreviations 

FabC FabD FabG FAIR

 519

Malonyl-CoA: acyl carrier protein Malonyl-CoA-ACP transacylase 3-Ketoacyl-CoA reductase A specific programme of research, technological development and demonstration in the fields of agriculture and fisheries, including agroindustry, food technologies, forestry, aquaculture and rural development FDA US Food & Drug Administration FDCA 2,5-Furane dicarboxylic acid FELS Fish, early-life stage FP Framework Programme FT Fischer−Tropsch FTC US Federal Trade Commission FTIR Fourier-Transform infrared GC-MS Gas chromatography-mass spectroscopy GHG Greenhouse gas Glc β(1→4)-linked D-glucose GlcNAc N-acetyl-d-glucosamine GPC Gel permeation chromatography GWP Global warming potential(s) HDDA 12-Hydroxydodecanoic acid HDL 16-Hexadecanolide HDPE High-density polyethlene HiC Cutin hydrolase from Humicola insolens HMF 5-Hydroxymethyl furan HRP Horseradish peroxidase HV Hydroxyvalerate ICI Imperial Chemical Industries ICT Information and communication technologies iLUC Indirect land-use change IR Infrared ISO International Organization for Standardization JBPA Japan BioPlastics Association JIS Japanese Institute for Standards Organisation JTI Joint Technology Initiative KA Key Action(s) KBBE Knowledge-based bio-economy KET Key Enabling Technologies LA Lactide LCA Life cycle assessment LCI Life cycle inventory LCIA Life cycle impact assessment LDPE Low-density polyethylene LMI Lead Market Initiative LOEC Lowest effect concentration level LSCFB Liquid-solid circulating fluidised bed MALDI-TOF Matrix assisted laser desorption/ionisation - time of flight MBC 5-Methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one MC Critical molecular weight MCL Medium-chain-length

520 

MFI MFR MITI MML Mn MPL MSW MVL MW NF NMP

 Abbreviations

Melt flow index Melt flow rate Ministry of International Trade and Industry (Japan) Mucor miehei lipase Polymer with a number-average molecular weight α-Methyl-β-propiolactone Municipal solid waste α-Methyl-δ-valerolactone Molecular weight Norme Française (French Standard) Nanotechnologies and nanosciences, knowledge-based multifunctional materials, and new production processes and devices NMR Nuclear magnetic resonance NOEC Maximum effect concentration level OECD Organisation for Economic Co-operation and Development P(3HB) Poly(R-3-hydroxybutyrate) P(3HB-co-3HD) Poly(3-hydroxybutyrate-co-3-hydroxydecanoate) P(3HB-co-3HHx) Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) P(3HB-co-3HP) Poly(R-3-hydroxybutyrate-co-3-hydroxypropionate) P(3HB-co-3HV) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-4HB) Poly(R-3-hydroxybutyrate-co-4-hydroxybutyrate) P(3HV) Poly(R-3-hydroxyvalerate) P(4HB) Poly(4-hydroxybutyrate) P-(4-PPMP) 4-[(4-Phenylazo-phenyimino)-methyl]-phenol PA Polyamide(s) PBS Polybutylene succinate PBSA Polybutylene succinate adipate PBSU Polybutylene succinate PBT Polybutylene terephthalate PBTA Polybutylene adipate-co-terephtalate PC Pseudomonas cepacia PCB Polychlorinated biphenyl(s) PCL Poly(ε-caprolactone) Pd/C Palladium/charcoal PDI Polydispersity index PDL 15-Pentadecanolide PE Polyethylene PEF Polyethylene furanoate PEG Polyethylene glycol PES Polyethylene succinate PET Polyethylene terephthalate PF Pseudomonas fluorescens PFL Pseudomonas fluorescens lipase PGA Polyglycolic acid PHA Polyhydroxyalkanoates PhaA β-Ketothiolase PhaB NAOH-dependent acetoacetyl-CoA reductase PhaC Polyhydroxyalkanoates synthase PhaG 3-Hydroxyacyl-ACP: CoA transferase

Abbreviations 

PhaJ PHB PHBV PLA PLGA PLLA PLLGA PP PPDL PPDO PPG PPL PPO PPP PPT PS PsCL PSL PTA PTS PU PVA PVAc PVC PVDC PVL QoL QSAR R&D REACH RH RNA ROP RRM RT RTD RTIL RW SBP SCAS scCO2 SCL SDBS SEC SEE S.E.S.A. SME TA TCA

(R)-Enoyl-CoA hydratase Polyhydroxybutyrate Poly(hydroxybutyrate-co-hydroxyvalerate) Polylactic acid Poly(lactide-co-glycolides) or poly(lactic-co-glycolic acid) Poly(L-lactic acid) Poly(L-lactide-co-glycolide) Polypropylene Poly(ω-pentadecalactone) Poly(1,4-dioxan-2-one) Polypropylene glycol Porcine pancreatic lipase Poly(1,4-phenylene oxide) Public-private partnerships Polypropylene terephthalate Polystyrene Pseudomonas cepacia lipase Pseudomonas sp. lipase 1,3-Propanediol, terephthalic acid and adipic acid 1,3-Propanediol, terephthalic acid and sebacic acid Polyurethane Polyvinyl alcohol Polyvinyl acetate Polyvinyl chloride Polyvinylidene chloride Poly(δ-valerolactone) Quality-of-life Quantitative Structure-Activity Relationship Research and Development Registration, Evaluation, Authorization and Restriction of Chemicals Relative humidity Ribonucleic acid Ring-opening polymerisation Renewable raw materials Room temperature Research and technological development Room-temperature ionic liquids Residual waste Soybean peroxidase Semicontinuous activated sludge Supercritical carbon dioxide Short-chain-length Sodium dodecyl benzene sulfonate Size exclusion chromatography Surfactant-enveloped enzyme Società Estense Servizi Ambientali SpA Small and medium-sized enterprises Terephthalic acid Tricarboxylic acid

 521

522

Abbreviations

Tg TGA THF Tm TMS TOD TPA TPPS TPS Tr TS TSAS TTMC UDL UHMW UNI USCC USDA UV WAXD WFD WLF Wo YLL ZAW-SR β-CF β-BL β-PL α-CD ε-CL γ-BL γ-CL γ-VL δ-CL δ-VL ω-PDL

Glass transition temperature Thermogravimetric analysis Tetrahydrofuran Melting temperature Trimethylene carbonate bulk Theoretical oxygen demand Terephthalic acid Thermoplastically processable starch Thermoplastic starch Minimum thermosetting temperature Total solid(s) Transition-state analogue substrate Trimer of trimethylene carbonate 11-Undecanolide Ultrahigh molecular weight Ente Nationale Italiano di Unificazione (Italian standards) US Composting Council US Department of Agriculture Ultraviolet X-ray diffraction Waste Framework Directive Williams Landel and Ferry equation Water to AOT molar ratio Yarrowia lipolytica Zweckverband Abfallwirtschaft Straubing Stadt und Land β-Cellobiose fluoride β-Butyrolactone β-Propiolactone α-Cyclodextrin ε-Caprolactone γ-Butyrolactone γ-Caprolactone γ-Valerolactone δ-Caprolactone δ-Valerolactone ω-Pentadecalactone

Index (R)-Enoyl-CoA hydratase 198, 202 1,2-Ethanediol, adipic acid and terephthalic acid 281 1,3-Dioxan-2-one 352, 353, 356 1,3-Propanediol, terephthalic acid and adipic acid 279 1,3-Propanediol, terephthalic acid and sebacic acid 279 1,4-Butanediol 61, 158, 189, 200, 231, 266, 272, 273, 274, 275, 277, 278, 279, 280, 281, 282, 283, 285, 287, 288, 290, 291, 292, 348, 366, 485 – terephthalic acid and adipic acid 272, 278, 280 1,4-Dioxan-2-one 354, 356 11-Undecanolide 361 12-Dodecanolide 357 12-Hydroxydodecanoic acid 342 15-Pentadecanolide 357 16-Hexadecanolide 361 2,2,2-Trifluoroethanol 350 2,5-Furane dicarboxylic acid 483 2GT 165 3(R)-isopropyl-morpholine-2,5-dione 356 3(S)-sec-butyl-morpholine-2,5-dione 355, 356 3-Hydroxyacyl-ACP: CoA transferase 198, 200 3-Hydroxyalkanoate 184 3-Hydroxybutyrate 29, 186, 263, 358 3-Hydroxybutyric acid 190 3-Hydroxyhexanoate 196, 264 3-Hydroxypropionate 29, 190, 484 3-Hydroxyvalerate 187 3-Ketoacyl-CoA reductase 198 4-[(4-Phenylazo-phenyimino)-methyl]-phenol 376 4GT 165 4-Hydroxybutyrate 29, 189, 263 4-Hydroxybutyric acid 190, 200, 342 5-Hydroxymethyl furan 483 5-Methyl-5-benzyloxycarbonyl-1, 3-dioxan-2-one 353 6(R,S)-methyl-morpholine-2,5-dione 355, 356 6(S)-methyl-morpholine-2,5-dione 355, 356 8-Membered lactone 361 8-Octanolide 361 α-Amylase 8, 383 α-Chymotrypsin 7 https://doi.org/10.1515/9781501511967-020

α-Cyclodextrin 375 α-Methyl-β-propiolactone 358, 360 α-Methyl-δ-valerolactone 361 α-Trypsin 7 β(1→4)-linked D-glucose 383–384 β-Amylase 8 β-Butyrolactone 358, 360, 365 β-Cellobiose fluoride 380, 381 β-Ketothiolase 195 β-Propiolactone 358, 365 γ-Butyrolactone 189, 200, 361, 365 γ-Caprolactone 361 γ-Valerolactone 361 δ-Caprolactone 367 δ-Valerolactone 361, 365, 366 ε-Caprolactone 154, 262, 342, 357, 362–369 ρ-Dioxanone 368 ω-Pentadecalactone 351, 357, 364, 367, 368 Absorb 95, 171, 395, 401, 460 – Absorbance 94 Absorption 93, 124, 125, 222, 226, 234, 252, 268, 300, 370 Acceptor 31, 381 Accumulation 2, 3, 7, 63, 81, 82, 86, 104, 184, 185, 194, 195, 199, 201, 202, 204, 291, 381 Accuracy 10, 34, 103, 469 Acid 4, 5, 27, 32, 33, 49, 50, 51, 54, 56, 57, 61, 82, 84, 90, 99, 100, 150, 152, 153, 155, 156, 157, 158, 159, 160, 161, 163, 166, 168, 176, 184–186, 188–190, 194–196, 198–201, 206, 228–231, 234, 245–257, 261, 262, 264–292, 301–305, 307, 312–315, 319–326, 329, 330, 339–344, 347, 349–353, 356, 357, 360, 361, 364, 365, 367, 371, 375–377, 380, 382, 395, 446, 466, 469, 470, 471, 477–480, 482–488, 493, 508, 510, 511 – Acidic 48, 206, 246, 253–256, 307, 308, 313, 352 Acquisition 237, 274, 394 Acrylic 229, 233, 484, 488 – acid 229, 484, 488 Acrylonitrile 230 Actin 306, 313 Activation 319, 347, 350

524 

 Index

– energy 319 Activator 154 Acyclic 377 Acyl carrier protein 198 Acylation 343, 350, 367 Additives 26, 57, 58, 69, 81, 139, 149, 150, 204, 221, 228, 230, 233, 252–254, 257, 299, 300, 301, 307, 308, 309, 314, 329, 330, 383, 455, 485 Adenosine triphosphate 5 Adhesive 137, 158, 262, 300, 301, 304, 307, 369, 479 Adipic acid 61, 157, 159, 160, 161, 163, 166, 231, 267, 271–275, 277–283, 285, 286, 344, 349 Administration 250, 265 Adsorption 59, 61, 63, 230, 325 Aerobic biodegradation 3, 9, 10, 40, 47, 53, 119–122, 125, 128 Aeromonas caviae 196 Age 319 – Ageing 5, 230, 254, 496, 504 Agent 4, 10, 14, 96, 150, 151, 167, 168, 221, 229, 233, 234, 235, 249, 307, 308, 309, 312, 314, 315, 319, 320, 322, 328, 329, 330, 376, 398, 479, 503 Aggregation 87, 312, 322, 394 Agreement 2, 118, 257 Agriculture 2, 41, 45, 48, 49, 52, 77, 78, 80, 105, 109, 135, 154, 169, 173, 178, 238, 290, 300, 330, 395, 400, 401, 432, 439, 455, 465, 496, 501–508, 512 Agromaterial 299, 300, 318, 330 Alanine 302 Albumin 306, 310, 311, 313 Alcaligenes eutrophus 188 Alcaligenes faecalis 190 Alcaligenes latus 195 Algae 66, 88, 92, 93, 95, 96, 98, 102, 110, 135, 196, 466, 505 Alicyclic 266 Aliphatic 7, 29, 32, 33, 101, 104, 149, 152, 155, 157–161, 163, 165, 166, 168, 172, 177, 231, 232, 250, 252, 253, 261, 262, 263, 266–281, 285, 286, 290, 291, 350, 352, 362, 368, 485 – -aromatic polyester 232, 261, 272, 273 – Polyester 7, 29, 33, 56, 149, 152, 155, 157–159, 163, 165, 166, 168, 231, 252, 253, 261–269, 273, 275, 278, 285, 350, 352, 362, 368 Alkali 11, 249

– Alkaline 48, 307, 310 Alkyl group 362, 373 Allyl group 356 Alpha-glucose 1-phosphate 381 Aluminium 207, 249, 357 – chloride 249 American Society for Testing and Materials 3, 39, 45, 76, 116, 117, 147, 193, 257, 270, 398, 456 Amino acid 56, 196, 201, 301–305, 307, 312, 313, 315, 320, 325, 326, 340, 479, 482, 487, 493 Amorphous 156, 163–165, 184, 219, 224, 228–230, 234, 250, 251, 269, 304 Amylopectin 150, 151, 163, 218–221, 224, 226, 228, 229, 233–236, 383 Amylose 150, 151, 163, 176, 218–221, 223, 224, 226–229, 231, 233–237, 383 Anaerobic 3, 6, 10, 11, 13, 15, 23–25, 30–33, 37–40, 45, 47–49, 51, 77, 80, 82, 89, 99, 105, 119, 120, 126, 127, 129, 132, 136, 137 – biodegradation 3, 10, 31–33, 40, 47, 51, 119, 126, 127, 285, 432 – test 31, 126, 127 – condition 10, 15, 24, 30, 31, 33, 47, 82, 89, 119, 126, 136, 149, 203, 263, 285, 286, 432, 457 – digestion 3, 6, 10, 15, 24, 33, 40, 49, 77, 80, 105, 127, 132, 136, 137, 174, 286, 398, 399, 405, 427, 431, 433, 435–437, 444, 447, 450, 457, 458, 460, 480 – liquid 30 Analogue 59, 64, 65, 353, 373, 377, 378, 380, 384 Analysis 11, 34, 37, 38, 40, 59, 69, 76, 78, 81, 84–86, 90, 91, 93, 97, 98, 101, 103, 105, 108–110, 121, 122, 124, 127, 128, 139, 191, 200, 203, 221, 229, 234, 235, 276, 287, 290, 315, 316, 348, 351, 353, 354, 355, 357, 369–371, 376, 381, 382, 393, 394, 403, 406, 414, 439, 440, 441, 442, 450, 455, 459, 468–470, 494, 497, 501, 502, 510 Anhydrous 348, 376 Animal 24, 58, 59, 64, 82, 87, 91, 95, 100, 105, 205, 306, 329, 383, 395, 401, 404, 433, 437, 438, 462, 468, 469, 502 Anion 204, 249, 250, 364 – Anionic 249, 250, 364 – polymerisation 249, 364 Antimicrobial 308, 328, 330

Index 

Antioxidant(s) 308, 310, 328, 330, 377, 461 Antistatic properties 151 Aquatic environment 27, 63, 76, 81, 98, 101, 105, 115, 123, 131 Aquatic test 35, 87, 109, 123, 126, 285 Aqueous 23, 24, 34–38, 40, 41, 55, 61, 63, 87, 91, 92, 94, 95, 97, 121, 125, 126, 150, 154, 222, 245, 250, 252, 253, 265, 285, 309, 310, 312, 320, 347, 364, 369, 370, 371, 373, 376, 377, 382, 459 – phase 61 – solution 309, 310, 369, 370, 377 Arabidopsis thaliana 202 Arachin 306, 307, 313 Archaeoglobus fulgidus 361 Archer Daniels Midland Company 154, 190 Arginine 302 Aromatic 7, 32, 33, 54, 56, 57, 80, 82, 101, 104, 107, 109, 149, 159–161, 163, 168, 172, 177, 194, 232, 261, 270–283, 285–287, 289–291, 369, 476, 481 – polyester 57, 159, 160, 168, 172, 232, 261, 270, 272, 273, 276, 277 – sequence 160, 278, 279, 286, 287, 290 Ascorbic acid 486 Asparagine 302 Aspergillus niger lipase 340 Assessment 6–8, 11, 13, 45, 46, 54, 60–62, 64, 65, 69–71, 83, 86, 134, 172, 204, 274, 286, 290, 393, 394, 396, 397, 401, 404–406, 419, 423, 428, 451, 466, 494, 495, 502, 503, 506, 507 Association for Organics Recycling 143 Association Française de Normalisation 118 Asymmetric 245 Atactic 247 Atmosphere 82, 273, 327, 330, 395, 401, 412, 433, 468, 469 – Atmospheric pressure 307, 368 Australasian Bioplastics Association 141 Australian Standard 117, 141 Autocatalytic 253 Backbone 4, 56, 57, 163, 228, 376 Bacteria 5, 8, 48–51, 66, 68, 87, 88, 94, 95, 100, 107–110, 120, 122, 125, 128, 152, 183, 185, 196, 203, 207, 257, 263, 268, 340, 32, 434

 525

Barrier 154, 167, 207, 300, 307, 308, 310, 315, 319, 320, 325–330, 479, 492 – properties 167, 307, 308, 310, 315, 319, 320, 325–328, 330 Beads 165, 221, 368 Benzyl β-malolactonate 358 Binder 301, 479 – Binding 204, 268, 323, 377 Bio concentration factor 59, 63 Bioassay 39, 54, 55, 79, 80, 81, 85, 86, 87, 88, 90–100, 102, 103, 105, 107–110 Biobased 91, 98, 99, 100, 174–176, 232, 265, 292, 397, 403, 455, 456, 458, 465–472 475–467, 483, 489, 491, 493–496, 499, 503–506, 508, 512, 513 Biocompatible 184, 299, 329, 503 Biodegradable 1–4, 6, 9, 13, 15, 23–26, 33–36, 39–41, 45–48, 50–52, 54–58, 60–63, 66, 68–71, 75–83, 86, 87, 89, 91, 92, 95, 97–101, 103, 104, 106–110, 115–118, 121, 122, 125, 127, 131, 133–137, 140, 143, 144, 147–152, 154–157, 159–162, 164–178, 184, 196, 203–205, 207, 217, 222, 232, 237, 238, 257, 261–264, 266, 268, 269, 270, 271, 272, 274, 275, 276, 281, 283, 285, 286, 290, 291, 299, 300, 307, 309, 324, 329, 330, 339, 340, 343, 385, 393, 397, 398, 400, 409, 414, 418, 419, 421, 423, 425, 427, 441, 442, 451, 455–463, 465, 466, 468, 469, 472, 483, 484, 491, 502, 504, 506, 511, 513 – Biodegradability 1–15, 23, 24, 26, 35–41, 45, 52, 54, 57, 59, 60, 61, 66, 69, 71, 77, 86, 104, 107, 109, 115, 116, 118, 121, 122, 124, 126, 127, 128, 131, 135, 136, 137, 143, 150, 156, 157, 159–164, 166, 168, 169, 177, 183, 190, 204, 206, 222, 230, 231, 233, 238, 261, 262, 265, 267, 269, 272, 274, 277, 278, 280, 329, 339, 342, 362, 397, 398, 418, 432, 451, 455–464, 471, 503, 504, 511 – Biodegradation 1–15, 23–41, 45–48, 50–61, 63, 64, 66, 67, 69, 70, 71, 75–110, 115, 116, 118, 119–138, 143, 144, 148–150, 154, 155, 160, 162, 171, 183, 203, 230, 231, 232, 234, 236, 246, 265–269, 272, 273, 275–277, 281, 282, 284, 285–287, 291, 340, 364, 398, 432, 438, 441, 451, 456–459, 461, 464, 471, 506 – behaviour 14, 23, 25, 125, 277

526 

 Index

– in the environment 52, 76 – intermediate 55, 57–60, 64, 67, 70, 87, 99 – polymer 1, 2, 36, 45–71, 75–78, 81, 87, 91, 92, 95, 97–100, 103–109, 117, 121, 122, 125, 131, 135–137, 143, 147–177, 184, 196, 205, 207, 237, 257, 261, 263, 339, 409, 458, 466, 468, 469, 472, 483, 491, 511 Biodegradable Products Institute 133, 140 Biodeterioration 128 Biological 4–6, 9, 23–27, 29, 33, 34, 36, 41, 45, 48, 49, 63, 64, 65, 75, 79, 84, 97, 106–108, 115, 119, 121, 126, 127, 128, 173, 186, 204, 206, 261, 262, 270, 276, 278, 280, 281, 286, 290, 339, 385, 397, 398, 399, 411, 433, 434, 451, 455, 456, 460, 466, 467, 468, 472, 496, 500, 502, 503, 504, 505, 508 – activity 4, 23, 49, 97, 106–108, 119, 126, 127 – oxygen demand 9, 29, 121 Biomass 1, 3–5, 7, 11, 15, 36, 38, 40, 45, 47, 53, 54, 60, 68, 75, 79, 83, 89, 95, 97, 106, 119, 121, 123–125, 133, 134, 148, 217, 268, 292, 403, 404, 455, 464–472, 475–477, 479–483, 486, 487, 489, 491, 499–501, 505, 509 Biomaterial 143, 173, 177, 237, 256, 361, 384, 496, 504 Biomax® 159 Biomedical Applications 1, 205, 257 BIOPOLTM 153 Biotest 79–81, 85–87, 89, 91–93, 95–98, 103, 107 Bis(2-ethylhexyl)sulfosuccinate sodium salt 371 Bisphenol A 376 Blend 126, 191, 232, 274, 313, 315, 318 – Blending 166, 176, 231 Blind 96, 98, 99, 109 Block 8, 159, 160, 277, 280, 484, 485 Boiling 228, 233, 234, 311, 344 – point 228, 344 Bond 4, 56, 57, 249, 250, 252–255, 312, 319, 349 – Bonding 102 Bone 1, 155, 205, 245, 256 BPI logo 138 Branch 218 – Branched 150, 194, 218, 247 – Branching 149

Breaking 207, 446 British Standard 118 Brittle 151, 152, 158, 162, 185, 187, 263, 461 – Brittleness 152, 184, 186, 187 Buffer 51, 271, 370, 371, 373, 375–378, 381, 383, 438 Building 98, 176, 196, 292, 339, 340, 393, 397, 398, 476, 482, 484, 485, 491, 493 – block 8, 159, 160, 277, 279, 280, 484, 485 Bulk 151, 155, 171, 228, 250, 251, 253, 266, 342, 348, 352, 353, 356, 358, 364, 365, 367, 368, 372, 416, 423–425, 481, 487, 489, 505, 508 – density 151, 416, 423, 425 Butadiene 230, 477 Butane diol 321 Butanol 234, 235, 477, 482, 487 By-product 5, 45, 82, 104, 175, 185, 207, 217, 246, 247, 254, 255, 257, 433, 502, 509 Calcium carbonate 469 Calorimetry 14, 221, 315, 370 Candida antarctica lipase 340, 352, 353–355 – B 340 Candida cylindracea lipase 272, 340, 353 Candida rugosa lipase 340 Capacity 3, 50, 82, 90, 96, 124, 156, 162, 164, 166, 174–177, 185, 187, 237, 238, 253, 264, 265, 268, 274, 292, 315, 397–399, 416, 418, 438, 445, 446, 491 Carbon dioxide 1–3, 5, 9–12, 15, 34–36, 40, 46–49, 51–55, 59–61, 75, 77, 93, 119–126, 128, 129, 132, 133, 137, 148, 149, 155, 173, 183, 195, 196, 201, 202, 205, 263, 268, 273, 275, 285, 287, 291, 308, 326, 327, 330, 362, 375, 394, 395, 401–403, 412, 428, 457, 458, 460–462, 464, 467–469, 471 – evolution test 37, 38 Carbon source 8, 9, 12, 34, 35, 36, 119, 123, 149, 152, 153, 183–185, 188–190, 194–200, 204, 207, 458 Carbonate 340, 352, 353, 356–358, 368, 469 Carrier 122, 124, 126, 356, 371, 394, 457, 475, 477 Carrier bag 26, 126, 171, 172, 176, 222, 237, 440 Casein 299, 307, 309, 320, 329 Casting 161, 166, 167, 304, 312, 324, 329 Catalunya, Spain 415, 422

Index 

Catalysis 4, 5, 252, 254, 270, 307, 339–385 – Catalyst 155, 160, 167, 177, 203, 237, 250, 252, 258, 339, 342, 343, 347–349, 351, 356, 357, 362–364, 367, 368, 371, 373, 375–377, 382–384 – Catalytic activity 351, 363, 364, 377 – Catalytic hydrogenation 353, 360, 485 Cationic 249, 250, 353 Cell 4, 5, 66, 83, 84, 93, 96, 102, 104, 148, 153, 183, 184, 186–190, 192, 194, 195, 197, 201, 205, 206, 220, 283, 375, 383, 457, 464, 504, 505 – growth 83, 194, 195, 197, 205 – wall 4 Cellodextrin phosphorylase 381 Cellophane 31, 161, 327 Cellular 47, 149, 458, 505 Cellulases 162, 381 Cellulose 8, 12, 31, 35, 47, 55, 57, 60, 82, 98–101, 103, 104, 123, 133, 150, 162, 204, 233, 261, 299, 307, 323, 368, 380–384, 476–479, 483, 485 – acetate 31, 35, 233 Centrifugation 91, 321 – Centrifuge 218 Certification 45, 60, 115–144, 170, 465, 493, 495 Chain 5, 7, 13, 26, 55, 56, 57, 82, 84, 99, 104, 107, 148, 149, 155, 158–162, 164, 169, 171, 178, 184, 185, 187, 190, 192, 205, 224, 231, 232, 234, 238, 245, 249–257, 264–266, 268–270, 272–274, 277–280, 286, 303, 305, 315, 318–320, 322, 326, 342–344, 347, 351–355, 358, 362, 367, 372, 373, 376, 378, 380, 382, 393, 396, 397, 401–403, 405, 429, 434, 456, 457, 460, 468, 471, 484, 504–505, 509 – end 149, 251, 252, 254, 255, 376 – extender 158, 159 – extension 159, 272, 280 – length 26, 160, 185, 190, 273, 280, 320, 343, 347, 373, 382 Characterisation 12, 26, 55, 93, 283, 376, 394, 396, 401, 402, 432, 484, 509 Chemical 3–6, 8, 9, 13–15, 23, 26, 46–48, 51, 53, 58, 59, 63, 71, 77–83, 85–87, 90, 95–97, 100, 102–104, 106–110, 119, 121, 122, 125, 128, 129, 132, 134–136, 144, 147, 149, 152, 153, 155, 158, 159, 162, 168, 186–191, 196, 206, 221, 234, 237, 246, 247,

 527

253, 255, 262–270, 272, 275, 276, 281, 301, 303, 308–313, 318–322, 324, 327, 329, 340, 353, 357, 363, 370, 400, 401, 405, 411, 461, 462, 466, 468, 469, 475, 476, 479, 483, 485, 487, 489, 493, 496, 504, 508 – attack 329 – bond 4 – composition 15, 77, 79, 86, 95, 97, 104, 129, 149, 253, 401 – environment 6 – industry 153, 476, 489 – modification 221 – oxygen demand 9, 121 – properties 59, 61, 63, 152, 221, 266, 268, 318, 327, 370 – reaction 5, 51, 85 – structure 102, 104, 107, 147, 149, 186, 188–191, 262–267, 269, 272, 475 Chemistry 3, 6, 102, 115, 184, 204, 208, 252, 266, 339, 347, 351, 397, 470, 482, 491, 500 China Standards 117 Chiral 186, 245, 247, 250, 251, 253, 254, 343 Chitosan 327 Chlorella sp 92 Chromatium vinosum 200 Chromatographic analysis 11, 34, 191 Chromatography 57, 253, 290, 343, 370 Chromobacterium violaceum 189 Chromobacterium viscosum lipase 340 Classification 6, 80, 200, 262, 273 Clay 50, 124, 125 Clean 85, 401, 410, 416, 417, 449, 511 Clear 9, 14, 25, 31–33, 69, 86, 91, 115, 118, 125, 129, 133, 171, 221, 282, 384, 398, 424, 431, 451, 505 Cleavage 5, 14, 57, 69, 148, 195, 246, 249, 251, 252, 254, 255, 268, 292 Closed bottle test 35, 37, 38 Cluster 504 Coacervation 312, 313 Coated 80, 307, 330, 354, 371 – Coating 87, 132, 169, 275, 300, 304, 307, 308, 310, 314, 315, 329, 356, 371 Coefficient 59, 61, 63, 65, 164, 253, 326, 328 Collagen 299, 310, 311, 313, 316, 320 Collected food waste 414, 425 Collection frequency 170, 409, 418, 420, 422

528 

 Index

Collection route 416, 420, 424 Collection vehicle 422, 423, 431 Colloid Colour 85, 87, 93–95, 132, 138, 308, 322 Combined heat and power 429, 430 Combustion 467 Comité Européen de Normalisation (European Committee for Standardization) 3, 78, 116–118, 129, 131, 135, 136, 257, 397 Common Agricultural Policy 500 Community Research and Development Information Service database 509 Comonomer 187, 188, 368 Compaction 422 Compatibiliser 232 – Compatibility 54, 116, 163, 330, 369 – Compatible 15, 52, 53, 110, 173, 184, 207, 221, 309 Competitiveness and Innovation Framework Programme 492, 511 Complex 5, 10, 12, 23, 39, 47, 53, 55, 75, 80, 81, 85, 87, 96, 101–103, 105, 110, 124, 125, 137, 185, 186, 194, 218, 233, 234, 252, 261, 268, 308, 313, 318, 358, 375, 382, 401, 404, 406, 460, 470, 476, 510 Complexation 163, 228, 233, 236, 249 Complexed poly(R-3-hydroxybutyrate) 186 Compliance 170, 222, 233, 464 Component 2, 3, 14, 15, 23, 26, 32–34, 46, 77, 80, 96, 97, 99, 101, 105, 108, 109, 115, 147, 149, 151, 154, 156–158, 160, 162, 163, 195, 218, 221, 226, 227, 229, 230, 234, 246, 262, 270–274, 276–278, 280, 282, 285, 286, 309, 314, 315, 323, 349, 375, 380, 383, 393, 401, 433, 434, 438, 448, 470, 475, 488, 493, 505, 509 Composite 166, 325, 329, 330, 493, 503 Composition 4, 6, 9, 10, 15, 29, 50, 54, 77, 79, 84–87, 91, 92, 94–98, 100, 102, 104, 129, 130, 149, 151, 166, 167, 184, 199, 203, 204, 206, 230, 231, 235, 236, 245, 251, 253, 258, 275, 277–279, 283, 284, 289, 315, 325, 327, 340, 357, 370, 373, 375, 376, 401, 432, 434, 440, 442, 445, 509, 510 Compost 12, 23, 24, 27, 28, 33–36, 41, 45, 46, 48–50, 52–55, 67, 78–82, 87, 89, 91–96, 99, 101, 102, 105, 107, 109, 110, 116, 120–125, 128–130, 132, 134, 135, 138–143,

147, 149, 158, 160, 169, 170–172, 175, 203, 232, 237, 267, 269, 273, 280, 282, 283–291, 329, 398–400, 406, 428–430, 432–436, 444, 446, 450, 456, 458, 460–462, 464, 465 – bag 171 Composting 3, 6, 10, 12, 13, 15, 24, 25, 33–36, 39, 45–49, 52, 53, 55, 63, 67, 71–82, 99, 107, 115–118, 120, 122–125, 129–136, 139, 140, 143, 147–149, 169, 170, 171, 173–175, 177, 217, 237, 238, 246, 257, 265, 267, 272–275, 277, 281, 283, 284, 286, 287, 290, 291, 398–400, 405, 409–411, 414, 417–420, 424, 428, 431–434, 436, 437, 439, 442–447, 449–451, 456–461, 464, 471 – test 10, 35, 36, 107, 122–124, 133, 134, 136, 275 Compostability certification 138–140, 143 Compound 9, 119, 160, 162, 189, 236, 238, 250, 251, 254, 255, 268, 327, 380 – Compounding 162, 165, 176 Contaminated 52, 80, 147, 172, 173, 400 Concentration 10, 25, 34, 35, 41, 54, 58, 61, 63, 71, 81, 84, 86, 92, 93, 98, 99, 100, 102, 119, 120, 125, 127, 129, 130, 133, 149, 152, 188, 203, 224, 225, 291, 312, 314, 315, 320, 353, 356, 362, 369–373, 376, 381, 451, 469 Condensation 158–160, 266, 341, 347, 348, 351, 352, 377, 383, 384, 470 – polymerisation 341, 347, 348, 351, 352, 470 Conditioning 438, 464 Conformational 5, 187, 268 Consensus 135, 393, 397, 400, 410, 463, 465 Consorzio Italiano Compostatori 141, 142, 170 Construction 176, 292, 300, 329, 340, 343, 492, 505 Consumer 6, 115, 139, 237, 310, 393, 455, 461, 471, 482, 503 Consumption 10, 34, 35, 54, 90, 98, 121, 123, 125, 126, 170, 171, 173, 174, 178, 183, 218, 221, 236, 238, 268, 291, 396, 406 Container 420, 422, 431, 438 Contamination 78, 427, 430, 434, 436, 437, 440, 443, 445, 451, 511 Controlled composting test 10, 35, 36, 122, 123 Conversion 11, 36, 47, 53, 91, 120, 122, 127, 129, 132, 137, 154, 183, 185, 196, 199, 266, 285, 286, 343, 349, 352, 353, 361–363, 365,

Index 

367–373, 376, 377, 382, 403, 405, 406, 458, 460, 466, 475–477, 481, 484, 487, 488, 491, 496, 508 – Converting 200, 232, 403, 432, 455, 476, 485 Cooling 48, 49, 164, 166, 167, 224, 225, 231, 310, 314, 318 – rate 167 Copolyester 25, 29, 31, 32, 34, 159, 160, 250, 263, 272–275, 277, 279–291 Copolymerisation 160, 246, 247, 255, 286, 342, 357, 358, 365–368, 375 Copper 102, 376 Core 47, 57, 167, 234, 371, 475, 508 Corn 151, 156, 176, 197, 217–219, 221, 224, 226, 237, 247, 307, 308, 310, 323, 329, 397, 419, 466, 475, 477, 483 – gluten 306, 308, 316, 321, 323 – starch 151, 176, 218, 221, 224, 226, 235, 236, 237, 308, 397, 419, 475 – zein 307, 308, 310 Correlation 29, 63, 277, 278 Cosmetics 156 Cost 81, 110, 125, 147, 153, 156, 161, 173, 177, 184, 195, 196, 201–203, 205, 217, 247, 261, 307, 329, 369, 385, 396, 411, 415, 418–421, 424, 425, 501, 503, 508 – assessment 419 – -efficiency 418 Cotton 156, 173, 178, 204, 238, 300 Cottonseed 299, 309, 330, 483 Couchman and Karasz equation 317 Coupling 221, 247, 249, 255, 267, 369, 376 Covalent bond 249 Criteria 2, 3, 60, 63, 77–79, 116, 131, 136, 137, 247, 419, 460, 471, 498, 499 Critical deformation 323 Critical micelle concentration 376 Critical molecular weight 305 Critical water activity 317, 327 Crosslinkable 351, 352 – Crosslinked 307, 309, 320, 352, 375 – Crosslinking 164, 221, 229, 303, 307, 309, 314, 319, 320, 329, 380 – agent 307, 309, 329 Crude oil 174, 395, 476, 482, 489 Crystal 156, 187, 188, 220, 272, 278, 304, 382 – Crystalline 149, 151, 158, 164, 168, 183, 184, 187, 206, 219, 220, 227, 234, 251, 253, 256, 267, 269, 280, 304, 315, 381

 529

– polymers 158, 220, 267 – structure 227 – Crystallinity 149, 152, 153, 161, 167, 185, 187, 189, 190, 204, 223, 224–228, 233, 251–253, 269, 278, 362, 363, 381, 382 – Crystallisation 166–168, 187, 224, 231 Culture 97, 104, 148, 169, 185, 188, 194, 195, 291 Cutin 30, 349 – hydrolase from Humicola insolens 346, 349 Cyclic 56, 155, 245–247, 255, 264, 352, 353, 356, 357 Cyclobis(decamethylene carbonate) 356 Cyclobis(diethylene glycol carbonate) 356, 357 Cyclobis(hexamethylene carbonate) 356, 357 Cyclodextrin 375 Cysteine 302, 304, 310, 314, 319 D,D-lactide 357 D,L-lactide 247, 357 Damage 110 Daphnia 66, 95, 109, 110 – Daphnia magna 66, 88, 95, 274, 290 Deactivation 371 Decarboxylation 353, 358 Decay 469 Decomposition 12, 63, 222, 464 Definition 2, 3, 45, 58, 339, 393, 395, 397, 401, 415, 475 Deform 192 – Deformation 192, 323, 325 Degradability 7, 11, 26, 31, 34, 77, 80, 118, 131, 136, 160, 261, 276, 278, 279, 286, 463, 471 Degradation 1–5, 7–15, 23–36, 39–41, 57, 60, 63, 67, 69, 75–80, 82, 86–91, 96–101, 103–110, 119, 125, 128, 129, 132, 133, 137, 149, 151, 152, 155, 158, 160, 162, 168, 184, 187, 189, 190, 195, 203, 204, 206, 221, 230, 231, 245–247, 251–257, 261, 265, 268–272, 274, 276–291, 340, 361, 383, 385, 432, 434, 443, 446, 450, 456, 457, 464 – behaviour 29, 31, 35, 272, 277, 280, 283, 287 – mechanism 34, 57, 252–254 – temperature 220 Degree of crystallinity 152, 161, 167, 362, 363, 382 Degree of polymerisation 104, 149, 341, 342, 352–355, 360, 361, 364, 376, 381, 382 Dehydration 89, 342, 343, 347, 349, 382, 483, 484

530 

 Index

Denaturing gradient gel electrophoresis 101 Dense 76, 218 Density 2, 129, 134, 151, 152, 193, 201, 230, 231, 252, 269, 270, 272, 275, 308, 319, 320, 322, 325, 399, 416, 423, 425, 461 Deoxyribonucleic acid 84, 186, 339 Depletion 7, 194, 395 Depolymerisation 5, 7, 55, 57, 69, 148, 206, 283, 290 – step 57 Derivatisation 484 Derivative 233–236, 373, 376, 377, 380, 383 Design 76, 79, 107, 208, 405, 409, 429, 437, 509 Destructurised 163, 165, 166, 168, 218, 226–229, 231, 233, 235, 236 – starch 163, 166, 226, 228, 229, 231, 233, 235, 236 Detection 11, 34, 35, 78, 81, 84, 105–107, 206, 290 Deterioration 2, 277 Deutsches Institüt für Normung 39, 76, 86, 87, 91, 92, 95, 116, 117, 131, 138, 139, 170, 267, 274 Dicarboxylic acid 160, 262, 266, 267, 269, 273, 274, 277, 278, 285, 286, 292, 343, 347, 483, 485 Dichlorodiphenyltrichloroethane 462 Die(s) 89, 90, 166, 226, 401, 434, 469 Dielectric 315 Dienes 380 Diethyl carbonate 366, 368 Diethyl succinate 351, 366, 368 Diethylene glycol 356 Differential scanning calorimetry 14, 221, 223, 224, 315, 370, 375 Differentiation 110, 253 Diffraction 219, 234, 382 – Diffractometry 14 Diffusion 23, 41, 66, 89, 126, 246, 253, 268, 397, 412, 413, 505 – Diffusivity 329 Difunctional 484 Dilute 11, 125, 220 – Dilution 86, 89, 91, 225 Dimensional stability 168 Dimer 155, 264, 290, 343, 358 – of trimethylene carbonate 358 – Dimerisation 369

Dimethyl sulfoxide 370, 376 Dimethylacetamide 382 Dimethylformamide 370 Direct land-use change 403, 404, 406 Directive 127, 175, 291, 409, 411, 428, 429, 434 Disease 82, 206, 308, 434, 502 Disintegration test 78, 129 Dispersion 86, 130, 148, 164, 175, 217, 225, 226, 312, 314, 372 Displacement 126, 404 Dissolution 130, 253, 304 Dissolved organic carbon 7, 11, 13, 39, 60, 122 Dissolving 1, 369 Distillation 483 Distortion 264, 419 Distribution 13, 57, 59, 61, 64, 97, 104, 189, 190, 231, 245, 247, 250, 251, 253, 254, 286, 290, 322, 323, 325, 370, 373, 510, 511 Dodecylbenzenesulfonic acid 347 Domain 30, 204, 396, 487 Door-to-door 416, 417, 420, 423–425 Dosage 201, 377 – Dose 83, 84, 86 – -response 83, 84, 86 – -response relationship 83 Draft International Standard 40, 131–134 Dried 152, 191, 218, 221, 300 – Drier 127 Droplet 230, 233, 234, 236 – -like structure 234, 236 Drug 1, 205, 206, 245, 258, 265, 299, 329, 330, 377 Dry cell weight 192, 195, 197, 202 – Drying 162, 218, 304, 307–311, 314, 315, 431, 441, 442 Ductile 187, 323 DuPont 159 Durability 147, 150, 175 – Durable 147, 173, 177, 184, 401, 403 Dye 258 Dynamic 315, 349 – mechanical thermal analysis 315, 316 Earthworm 88, 91, 96, 103, 105, 107, 108, 134, 141 Eastar Bio® 159, 163 Ecoflex® 159, 177

Index 

Ecosystem 76, 78, 80, 82–84, 87, 92, 93, 100, 103, 105, 110, 184, 207, 462 Ecotoxic 52, 71, 81, 103–107, 110, 432 – Ecotoxicity testing 67, 70, 75, 88, 96, 103 – Ecotoxicological 46, 54, 55, 58, 61, 64, 67, 68, 70, 75–109, 274 Edible 299, 308–310, 320, 328, 329, 442 Efficiency 13, 49, 169, 170, 202, 218, 357, 373, 396, 418, 428, 430, 437, 476, 511 Eisenia foetida 88, 91, 105 Elastic 156, 318, 323 – modulus 318, 323 – Elasticity 236, 342 Elastomer 189, 324 Electric 169, 430 – Electrical 301, 377 – Electricity 433 Electrolyte 371 Electron 31, 192, 203, 234 Electronic 156, 169, 177, 510 Electrostatic 62, 254 Elemental analysis 370, 376, 470 Elongated 85, 365 – Elongation 128, 157, 158, 161, 164, 168, 185–187, 229, 266, 267, 272, 319, 323, 324 – break 99, 161, 186, 187, 229, 252, 266, 267, 272, 319, 324, 325 Elution 92, 288, 289 Emission 70, 273, 402–404, 428, 430, 433, 451, 467 Enantiomer 165, 247 – Enantiomeric 343, 358, 365 Encapsulating 308 – Encapsulation 87, 310, 330 End group 252, 348, 358 End-of-life 120, 455–457, 471 Endoglucanase II 381 Endothermic 151, 223, 226 End-point 58, 85 Energetic 438, 479 Energy 5, 10, 23, 29, 31, 36, 45, 48, 53, 75, 83, 93, 152, 172, 174, 178, 186, 197, 204, 218, 238, 292, 303, 304, 311, 318, 319, 322, 323, 339, 340, 393, 394, 396, 398, 399, 401, 427–432, 436, 437, 445, 451, 455–457, 465, 466, 467, 475–477, 482, 489, 491, 496, 497, 499–501, 505, 507, 509, 511 – consumption 178, 238 – source 10, 29, 428, 456, 465

 531

Engineering 197, 205, 207, 233, 256, 396, 506, 509 Enhancement 87, 93, 269 Enpol 159, 268, 274–276 Ente Nationale Italiano di Unificazione (Italian Standards) 45, 52, 69, 135 Enthalpy 75, 220 Environment 1, 2, 4–8, 12–15, 23–31, 35, 39, 41, 45, 46, 52, 58–61, 63, 69, 71, 75–79, 87, 93, 98, 103–105, 108–110, 115, 116, 118, 123–129, 131, 136, 139, 142, 143, 147–149, 151, 152, 160, 164, 171, 175, 184, 201, 203, 204, 207, 217, 221, 222, 252, 256, 264, 268, 272, 273, 281, 285, 286, 290, 300, 329, 371, 393, 395–397, 411, 451, 455, 456–467, 471, 492, 499, 502, 504, 507, 508, 511 Environmental 1, 3, 4, 6, 7, 14, 15, 26, 34, 36, 45, 55, 57–61, 64, 68–71, 75, 76, 78, 79, 84, 86, 102, 104, 109, 110, 115, 116, 119, 120, 122, 131, 132, 142, 144, 147, 149, 154, 161–163, 169, 171–174, 178, 184, 197, 203, 204, 207, 217, 232, 235, 238, 246, 257, 283, 290, 339, 347, 368, 369, 374, 375, 393, 394, 396–401, 405, 406, 409, 412, 421, 428–430, 433, 451, 455–457, 460, 462, 463, 466, 471, 500, 502, 508, 509 – niches 119–120 – Polymers Group 161, 167 – Product Declaration 396, 397 – protection 58, 78, 110, 154, 300, 314, 326, 339, 368, 421, 460, 511 Enzyme 4, 5, 7, 8, 12, 23, 27, 30, 31, 41, 55, 56, 83, 84, 104, 128, 147–150, 154, 161, 162, 183, 185, 191, 194, 197, 200, 202, 203, 204, 206, 224, 230, 246, 250, 252, 263, 265, 269, 276, 278–280, 283, 285, 309, 339–341, 343, 344–350, 352–360, 361, 362, 364–373, 375, 376, 378, 381–385, 479, 493, 509 – -activated monomer 358, 359, 365 – catalysis 4, 5, 252, 254, 270, 307, 339, 343, 347, 349, 352, 353, 362, 363, 365, 382, 384 – Enzymic hydrolysis 329 – Enzymic polymerisation 311 Epoxy group 356 Equilibrium 65, 254, 315, 325, 344, 347, 350, 356, 368, 468, 469 Equipment 24, 166, 167, 222, 414 Escherichia coli 186, 201

532 

 Index

Ester 8, 56, 57, 69, 81, 149, 155, 187, 233, 246, 250, 252–255, 261, 268, 269, 279–281, 286, 290, 319, 321 Esterase 341, 356, 360, 361, 363 Esterification 221, 233, 254, 347, 479 Esterified 349 Ethanol 217, 218, 255, 313, 314, 350, 367, 376, 470, 477–479, 482, 483, 487, 488 Etherification 221, 347 Ethylene 163, 174, 229–232, 271, 277, 321, 326, 330, 353, 461, 470, 471, 476, 483, 487–489 – -acrylic acid 229, 230, 484, 488 – copolymer 229 – glycol 227, 321, 470, 471 – -vinyl alcohol 163, 230, 231, 233, 234, 235, 237, 326, 350 European Atomic Energy Community 497, 498, 507 European Chemicals Agency 58 European Coal and Steel Community 497, 498 European Collaborative Linkage of Agriculture and Industry through Research 501, 502 European Commission 190, 202, 204, 277, 358, 369, 375, 377, 380, 383, 397, 399, 409, 411, 428, 429, 491–496, 499, 502, 506, 507, 509, 512 European Committee for Standardization  3, 257, 495 European Community 462, 498 European Economic Community 497, 498 European Norms 15, 38–40, 45, 52–54, 60, 67, 69, 76, 77, 79, 116, 121, 123, 129–134, 136, 138, 139, 143, 144, 147, 170, 172, 267, 274, 276, 398, 419, 430–432, 440, 451, 456–460, 463, 464 European Union 58, 117, 127, 132, 152, 169, 170, 173, 175, 217, 236, 396, 398, 409–412, 419, 428, 429, 482, 491–500, 502–504, 506–508, 510–513 Evaluation 3, 23, 36–40, 46, 52, 58–65, 67–71, 80, 81, 83, 84, 86, 96, 103, 107, 122, 127–130, 135, 136, 138, 153, 396, 402, 404–406, 420, 432, 462, 503, 507, 510 Evaporation 49, 313, 431 Evolution test 10, 11, 37, 38 Exothermic 49 Expanded 124, 151, 230, 231, 237, 246 Expanded polystyrene 151, 152 Expansion 175, 475

Exposure 6, 7, 12–15, 26, 46, 63, 64, 68, 70, 85, 88, 105, 125, 127, 128, 131, 147, 206, 221, 222, 395, 462 – period 12, 85 Expression 84–86, 201, 202 Extension 83, 153, 159, 272, 280, 499 Extraction 57, 101, 124, 153, 187, 197, 202, 205, 393, 394, 441–443, 446, 448, 461, 477, 480, 482, 511 Extrapolation 103, 128, 223, 457, 459, 460 Extrude – Extruded 165–168, 226, 231, 506 Extrusion 161, 165–167, 169, 193, 225, 226, 229, 264, 265, 268, 304, 307–311, 318–320, 329 – coating 169, 307 Fabric 156, 169, 246, 262 Factor 1, 26, 48–51, 53, 57, 59, 63, 65, 93, 104, 119, 120, 130, 132, 149, 152, 171, 175, 183, 184, 195, 197, 203, 204, 224, 228, 237, 247, 252, 253–255, 289–291, 319, 347, 363, 393–396, 402, 404, 428, 430, 438, 489 Farming 404, 496, 505, 509 Feed 87, 91, 217, 309, 315, 357, 367, 368, 404, 434, 475, 478, 479–482, 496, 508 – Feeding 105, 121, 122, 195, 437, 438 – Feedstock 48, 50, 52, 76, 177, 185, 207, 265, 404, 430, 434, 436, 442–445, 449, 451, 455, 466–469, 475, 476, 481, 487, 501, 509 Fertiliser 48, 49, 52, 54, 55, 82, 98, 400, 434, 482 Fibre 7, 28, 29, 150, 156, 168, 177, 186, 218, 238, 264, 265, 267, 269, 301, 304, 307, 325, 329, 330, 438, 441, 442, 462, 476, 479–481, 483, 491, 502 – Fibrous 311, 339, 434, 435 Filled 23, 27, 116, 221, 222, 403, 438 – Filler 58, 150, 165, 168, 221 – Filling 23, 511 Film(s) 11–13, 23–25, 27, 29, 31–34, 45, 50, 51, 125, 132, 152, 154, 156, 158, 159, 161–164, 166–169, 173–178, 186, 187, 189, 190, 193, 203, 205, 207, 221, 222, 229, 230–232, 234, 236–238, 253, 256, 257, 262–268, 273, 275, 276–279, 281–285, 299, 300, 303, 304, 306, 307–312, 314, 315, 317, 319, 320, 322–330, 399, 455, 460, 461, 506 – blowing 166, 167, 176, 222, 230, 231, 236 – casting 167

Index 

– forming 303, 304, 307–312, 314, 315, 319 – production 159, 166, 229, 263 – properties 229 – thickness 166, 281, 323 Filter 401, 443 – Filtrate 35 – Filtration 91, 321 Fischer-Tropsch 481, 486 Fish 66, 67, 88, 171, 310, 327, 395, 401, 414–416, 418 – early-life stage 67 Flammability 156 Flexibility 161, 272, 280, 319, 510 – Flexible 156, 157, 218, 262, 273, 309, 310, 329, 430, 433 Flexural modulus 153, 193 Flexural 153, 193 Flow 34, 38, 88, 158, 164, 167, 193, 223, 225, 270, 276, 305, 308, 309, 312, 318, 339, 394, 403, 420, 435, 437–439, 444–450, 477 – rate 158, 193 Fluorescence 113 Foam 151, 152, 156, 169, 173, 177, 226, 230, 231, 238, 265, 267 – Foamed 151, 226 Food 11, 15, 45, 52, 78, 82, 84, 119, 147, 154, 156, 166, 169–171, 173, 174, 177, 178, 185, 202, 207, 217, 232, 238, 247, 250, 265, 275, 300, 301, 303–305, 307, 308, 310, 314, 315, 320, 322, 329, 330, 339, 383, 399, 400, 404, 409, 412–425, 429, 430–432, 434, 435, 437–440, 443–447, 449, 450, 456, 457, 460, 468, 471, 475, 477–479, 481, 483, 491, 496, 500, 501, 503, 504, 506–509, 512 – additive 250, 300, 383 – industry 185 – packaging 11, 15, 52, 147, 169, 173, 207, 300, 400, 483, 501 – waste 52, 170, 174, 399, 400, 409, 412–417, 419–425, 427, 430–432, 434, 435, 437–440, 443–447, 449, 450 Force 102, 207, 410, 463, 491, 494, 497, 498 Forestry 401, 410, 466, 476, 483, 496, 502–506, 512 Formation 7, 11, 31, 32, 36, 47, 48, 50, 51, 54, 57, 81, 82, 84, 90, 91, 101, 102, 104, 149, 164, 166, 171, 187, 188, 194, 221, 224, 226,

 533

229, 234, 235, 245–247, 253, 256, 257, 268, 278, 287, 290, 303–305, 310–313, 318–320, 322, 330, 343, 347, 351, 352, 358, 364, 365, 372, 375, 377, 432, 446, 449, 461 Forming 5, 102, 161, 224, 225, 284, 303, 304, 307, 308–312, 314, 315, 319 Formulation 24, 54, 57, 58, 78, 161, 167, 207, 229, 230, 236, 309, 325 Foundation 432, 462, 475 Fourier-transform 234, 441 – infrared 234, 343, 355, 395, 441–443 Fraction 2, 11, 36, 52–54, 134, 160, 174, 197, 220, 222, 223, 224, 226, 229, 272, 276–279, 282, 286, 287, 289, 400, 412, 414, 415, 431, 433, 438–444, 446, 447, 450, 451, 479, 482 Fractionation 321, 476, 478–480, 502, 509 Fracture 115, 192, 245, 256 Fragment 3, 53, 57, 222 – Fragmentation 2, 119, 509 Frame 2, 129, 385, 456, 464–466, 468, 471, 507 Framework 152, 394, 409, 411, 428, 429, 489, 492, 493, 498, 506, 507–509 – Programme 152, 492, 498–501, 507, 512 Free-flow packaging 178 Free radical 5, 377 Free volume 315 Frequency 126, 170, 304, 409, 417, 418, 420, 422, 424 Fruit 169, 329, 415, 418 Fuel 50, 195, 217, 218, 236, 401, 403, 428, 466, 476, 479, 481–483, 485, 491, 509 Fumaric acid 271, 485 Functional group 56, 205, 208, 263, 380 Functionalisation 371 Functionality 358, 455, 484 Fungi 5, 8, 23, 35, 49–51, 120, 122, 125, 128, 203, 263, 283, 284, 369, 374, 375 Fusion 220 Gas(es) 1, 5, 10–12, 37, 57, 99, 130, 134, 147, 172, 175, 191, 195, 207, 220, 231, 237, 290, 308, 310, 325–328, 330, 395, 405, 428–430, 448, 449, 458, 466, 469 – barrier properties 310, 326, 328, 330 – chromatography-mass spectrometry 290 – chromatography-mass spectroscopy 57 Gaseous 3, 11, 469

534 

 Index

Gel(s) 9, 101, 288, 322, 343, 353, 355, 370 – permeation chromatography 288, 343, 355, 370 – Gelling 479 Gelatin 299, 306, 310, 311, 313, 316, 328, 329 Gelation 222, 224 Gene 84, 196, 199–202, 356 – Genetic 4, 154, 176, 177, 197, 200–202, 207, 250, 265, 266, 291, 305, 339, 381, 506 – Genetically modified 154, 177, 197, 201, 202, 265, 506 German Normalisation Institute 116 German Standard Procedures for Investigation of Water, Wastewater and Sludge 88 Glass 27, 89, 96, 151, 191, 224, 252, 254, 257, 262, 307, 357, 410, 413, 415, 420 – transition temperature 27, 151, 191, 224, 252, 254, 257, 262, 307, 357 – Glassy 27, 156, 165, 315, 317 Gliadin 299, 306, 311, 313, 316, 321, 329 Global warming 394, 411, 456 – potential 394, 411 Glutamic acid 302, 313, 352 Glycinin 306, 307, 311, 313 Glycol 56, 160, 271, 277, 319, 321, 340, 347, 349, 355–357, 470, 471, 484, 486 – Glycolic acid 265, 271, 341, 342, 484 Gold 371, 381 Graft 368 – Grafted 163, 231, 356 – Grafting 303, 322 Green chemistry 204 Greenhouse gas 172, 175, 237, 395, 397, 401–404, 406, 430, 433, 451 GreenPla 141, 267, 274, 276 Grow 9, 89, 93, 98, 174, 185, 188, 238, 249, 283, 410, 512 – Growth 8, 9, 11, 15, 23, 31, 35, 47, 48, 50, 51, 53, 64, 66–68, 75, 79, 81–83, 85, 89–98, 104–108, 110, 134, 169, 173–175, 177, 185, 186, 194, 195, 197, 201, 205, 233, 237, 238, 247, 250, 254, 284, 305, 401, 410, 412, 455, 460, 465, 483, 491, 496, 500, 512 Handling 12, 103, 451 Hardening 307, 314 – Hardness 276 Health 15, 46, 395, 401, 457, 460, 462, 463, 471, 491, 501, 502, 504, 506–508, 510

Heat 48, 49, 135, 137, 147, 164–167, 223, 225, 226, 249, 264, 300, 304, 307, 311–313, 315, 319, 320, 329, 429, 430, 433, 448 – flow 223, 225 – generation 135 – Heating 151, 164, 165, 223, 225, 226, 318, 321, 364, 448, 449 Hemicellulose 383, 476, 479, 483, 485–487 Heterogeneous 50, 53, 67, 149, 253, 254, 256, 303, 304, 318, 399, 400, 451 High conversion 343, 349 High-density 158, 323 – polyethylene 158, 323 High molecular weight 183, 316 High pressure 167 High-speed 168 High temperature 49, 250, 267, 319, 398 High viscosity 445 Higher plant 78, 80, 87, 88, 95, 108 Homogeneity 449 – Homogeneous 24, 87, 174, 252, 308, 312, 318, 329, 399, 400, 449 Homopolymer 187, 189, 190, 201, 202, 267, 351 Horseradish peroxidase 369–371, 373, 375–377, 379, 380 Hot-melt 262 Housing 142, 395, 422 Humidity 23, 50, 149, 161, 163, 218, 228, 230, 235, 283, 315, 462 Hybrid 383, 384 Hydration 199, 311, 312, 315, 318, 319 Hydrocarbon 380, 475 Hydrochloric acid 206, 249, 395 Hydrogen peroxide 205, 369 Hydrogenation 292, 353, 360, 485, 486 Hydrolysable 149, 150, 155 Hydrolysis 1, 4, 7, 14, 23, 26, 27, 81, 82, 125, 148, 161, 203, 230, 245, 246, 253–255, 263, 268, 270–272, 276, 281, 283–285, 290, 322, 329, 377, 381, 382, 446, 447, 478, 480, 481, 483, 485 Hydrophilic 150, 221, 233, 250, 269, 285, 303, 304, 315, 319, 320, 325, 326, 368, 373 – Hydrophilicity 4, 350 Hydrophobic 4, 91, 147, 150, 165, 221, 231, 255, 269, 303, 304, 309, 310, 312, 323, 325, 326, 342, 347, 363, 373, 462 Hydroxyacid 262, 266

Index 

Hydroxy group 343 Hydroxyl group 351, 364, 365, 376, 377 Hydroxylation 369 Hydroxyvalerate 25, 29, 152, 153, 185, 187, 189, 201, 203 Identification 1, 55, 57, 62, 64, 69, 101, 139, 154, 194, 199, 205, 255, 281, 283, 358, 472, 502 Immersion 128, 314 Immobilisation 66 – Immobilised 92, 102, 250, 342, 353, 356, 362, 364, 371, 381 Impact 2, 3, 45, 46, 50, 58, 64, 76, 79, 80, 82, 86, 89, 98, 103, 120, 153, 158, 169, 171–173, 178, 207, 217, 222, 232, 238, 252, 303, 315, 393, 394–396, 401, 403–405, 428–430, 433, 451, 455, 460, 466, 494, 503, 506, 508, 510–512 – assessment 393, 394, 396, 428 – resistance 153 – strength 252 Imperial Chemical Industries 153, 187, 195, 263 Impurities 80, 149, 172, 299, 329, 352 In situ 349 In vitro 203, 207, 208, 246, 252, 254, 329, 339, 340, 380, 381, 384, 503 In vivo 1, 184, 206, 246, 254–256, 385 – degradation 1, 246, 255 Incubation 7–10, 28, 29, 125, 126, 282–285, 288, 290 Indicator 46, 63, 102, 110, 397 Indirect land-use change 403 Industrial 48–50, 52–54, 56, 82, 97, 131–133, 135, 138, 139, 141, 147, 154, 156, 168–174, 170, 177, 187, 195, 197, 206, 207, 217, 222, 225, 230, 232, 236, 238, 257, 264, 265, 267, 274, 277, 280, 292, 299, 307, 310, 314, 315, 369, 393, 396, 404, 420, 427, 431, 434, 460, 475, 477, 478, 481, 483, 485–487, 489, 491–493, 496, 499, 501, 502, 504, 505, 508, 509, 512 – application 236, 499 – Industry 31, 39, 55, 117, 138, 140, 153, 169, 185, 225, 238, 300, 308, 310, 329, 393, 405, 412, 438, 443, 476, 486, 489, 491, 493, 494, 501, 503, 504, 508, 510, 512 Inert 69, 76, 89, 107, 124, 184, 261 Inflammation 256, 385

 535

Influence 25, 35, 36, 48, 51, 54, 75, 78–83, 85, 87, 90, 92, 93, 95, 97, 98, 102, 104, 105, 107, 109, 120, 137, 159, 164, 203, 226, 229, 250, 252, 255, 277, 280, 306, 325, 326, 396, 401, 405 Information and communication technologies 499, 507 Infrared 11, 14, 34, 139, 234, 343, 355, 395, 441 – spectroscopy 343, 380 Inherent biodegradability 37, 45, 104, 131, 136, 398 Inhibition 8, 66, 67, 84, 86, 89–97, 100, 105–108 Initiation 250, 254, 255, 359, 365 Initiator 157, 246, 250, 251, 255, 257, 353, 368 Injection moulding 167, 168, 193, 207, 222, 230, 233, 238, 263–265, 268, 329 Injection 159, 167, 168, 193, 207, 222, 230, 233, 238, 263–268, 304, 318, 329 Ink(s) 94, 132, 137, 265, 301 Innovation 428, 491–496, 498, 505, 508, 509–512 Inorganic 5, 35, 38, 45–47, 55, 61, 92, 102, 108, 126, 434, 469, 484 Insect 174, 417 Insecticide 207 Insoluble 5, 11, 12, 65, 69, 162, 183, 186, 204, 218, 253, 290, 308, 310, 313, 320, 322, 329, 330, 349, 369–372, 375, 376, 381, 382, 441 Instability 167, 369 Institute 40, 45, 76, 116, 117, 133, 140, 153, 161, 170, 175, 176, 208, 300, 506 Instrument 54, 396–398, 406, 491, 493, 494, 512 Integration 163, 176, 232, 420, 424, 425, 475–477, 497, 498, 509 Intensity 83, 93, 94, 322, 323, 493 Interaction 5, 36, 76, 82, 97, 108, 220, 227, 229, 254, 280, 303, 304, 322, 323, 451, 496, 508 Interface 30, 55, 372, 486, 501 Intermediate 3, 11, 12, 24, 34, 41, 46, 55–62, 64, 67, 69–71, 76, 81, 87, 90, 97, 99, 108, 119, 139, 149, 199, 200, 225, 247, 268, 274, 280, 287, 290, 291, 308, 309, 364, 365, 367, 377, 403, 431, 434, 461, 475, 477, 484, 485, 487, 493

536 

 Index

International Organization for Standardization 3, 8, 15, 35, 36–38, 40, 45, 52, 58, 60, 66, 86, 88, 94, 116–118, 121–134, 138, 139, 147, 164, 193, 269, 270, 275, 276, 393, 396, 397, 402, 406, 456–460, 463, 464, 466 Intrinsic 23, 39, 41, 184, 217, 406, 466–468, 471, 472 Inventory analysis 393, 403 Investigation 24, 25, 27, 30, 31, 34, 41, 46, 51–58, 60, 64, 67, 78, 80, 81, 84, 86–88, 93, 95, 97, 103–105, 107, 108–110, 190, 203, 255, 261, 273, 285, 369, 442, 481 Ion 82, 89, 92, 102, 186 – exchange 82, 92, 102 – Ionic 62, 203, 204, 249, 301, 303, 304, 311, 314, 350, 354, 360, 362, 363, 368–370, 377 – liquid 350, 354, 356, 360, 362, 363 – Ionisation 355, 370 – Ionised 301, 302, 312, 313 Irradiation 23, 152, 321, 363, 364 Irreversible 223, 350 Isolate 93, 282, 309, 324 – Isolation 93, 184 Isomer 197, 206, 264, 365, 367 Isoprene 486–487 Isotactic 187, 247–248, 250 Italy 135, 139, 141, 154, 157, 161–162, 170, 172, 262, 292, 324, 409–410, 412, 414–424, 431, 438–440, 443 Japan BioPlastics Association 141 Japanese Institute for Standards Organisation 117 Joint 1, 140, 154, 163, 176–177, 187, 190, 237, 265, 292, 397, 400–403, 438, 478–482, 485, 493, 496, 509, 512 Joint Technology Initiative 512 Keratin 310–313, 322–323 Key Action(s) 501, 504 Key Enabling Technologies 493–496 Kinetic 7, 13, 126, 194, 320, 349, 356, 372 Knowledge-based bio-economy 491 L,L-lactide 357 Laboratory 10, 13–14, 23–24, 26–33, 34–36, 39, 41, 52–54, 63, 85–86, 97, 105–108,

110, 124, 130, 154, 196, 208, 275, 312, 340, 398, 441, 460, 464, 471 – test 14, 24, 27–33, 34–36, 39, 41, 86, 110 Lactide 155–156, 167, 245–250, 255–256, 264–265, 357, 362 Lactone 320, 360–366, 368 Large scale 100, 110, 175, 195, 197, 237, 264, 292, 404, 475, 483 Laser 355, 375 Lateral 301–303, 315, 326 Latex 207, 307 Lattice 220, 224, 228, 235 Layer 25, 51, 57, 89, 97, 167, 230, 234, 300, 309–312, 330, 356, 446 Leaching 223 Lead Market Initiative 492–493 Legislation 117, 143, 171, 412, 428, 512 Life cycle assessment 172, 204, 393, 397–405, 451, 466 – methodology 393, 466 – studies 393, 397, 451 – Life cycle impact assessment 394 – Life cycle inventory 394 Light 51, 76, 93–95, 102, 135, 137, 161, 192, 220, 226–228, 233–234, 438, 441–442, 455, 463, 486 Lignin 49, 122, 374–375, 476, 478–479, 481, 487 Limitation 10, 27, 36, 105, 115, 194–195, 230 Linear 56, 63, 150, 155, 159, 162, 183, 186, 195, 218, 245, 262, 280, 305, 357, 450, 460, 499, 505, 510 Linkage 5, 56 Lipase 9, 30, 204, 206, 279, 283, 340–343, 345, 347–358, 362–380 Lipid 153, 300, 305 Lipophilic 92, 233, 320 Liquid 350, 356, 360, 362, 363, 371, 378, 438, 443, 445, 446, 469, 475, 476, 481 – environment 23, 27, 35, 41, 285 – media 23, 34–36, 41, 285 – phase 35, 55, 475 – -solid circulating fluidised bed 371 Littering 6, 24, 26, 172, 175, 400, 406 Load 148, 440 – Loading 13, 423, 449 Long chain 160, 161, 192, 266, 273, 274, 382, 480, 487

Index 

Long-term 13, 61, 63, 64, 66–68, 80, 81, 83, 91, 95, 122, 205, 468, 482 Loose-fill packaging 175, 237, 300 Loss 2, 7, 8, 13–15, 25–29, 32, 34, 79, 110, 125, 127, 128, 148, 155, 221, 223, 253, 269, 277, 278, 282–284, 286, 310, 320, 326, 418, 426, 461 Low-density 2, 230, 272, 308, 461 – polyethylene 230, 272, 308, 461 Low molecular weight 36, 115, 121, 262, 341 Low temperature 250 Low toxicity 356 Lowest effect concentration level 81, 86 Luminescent 66, 94, 109, 110 – bacteria 66, 94, 109, 110 Lysine 302, 352, 487 Machine 450 Macromolecular 4, 13, 14, 148, 149, 235, 247, 268, 279, 301, 311, 322, 325 – Macromolecule 257, 315 Macroscopic 15, 247, 255, 301, 322 Magnetic 14, 234, 251, 348 Main chain 5, 251, 269, 353, 364 Maintenance 48, 49, 393, 394 Malonyl-CoA-ACP transacylase 198, 200 Malonyl-CoA: acyl carrier protein 198 Manufacture 165, 175, 176, 237, 266–268, 273 – Manufacturing 165, 177, 228, 237, 261, 268, 307, 393, 394 Marine environment 23–26, 30, 39, 125, 127, 128, 131, 136, 148, 171, 222, 264, 457, 463 Market 45, 50, 78, 81, 110, 116, 136, 137, 139, 147–177, 228, 233, 237, 238, 256, 261, 263, 264, 272, 273, 277, 292, 396, 412, 429, 443, 445, 448, 475, 483, 489, 491–494, 502, 504, 505, 509, 511–513 – share 238, 445 MARPOL Treaty 24 Mass spectroscopy 57 Mater-Bi® 157, 162–164, 166, 170, 173, 175, 233, 262 Material(s) 2–4, 6, 8, 13–15, 23, 25, 26, 34–36, 45, 51–54, 60, 75, 79, 81, 82, 86, 87, 98–100, 104, 105, 108–110, 119, 120, 124, 129, 132–134, 136, 137, 139, 147–149, 155, 156, 159, 160, 165, 167, 170, 173, 186, 196, 199, 201, 203–207, 217, 221, 222, 230, 231, 237, 245, 247, 251, 252, 261, 268,

 537

272–275, 277, 278, 281–284, 290, 291, 299–301, 303, 305–330, 370, 394, 398, 401, 409, 411, 414, 415, 417–419, 423, 428, 432, 436–439, 442–451, 455, 458, 460, 464–469, 472, 476, 479, 483, 487, 504–506 Matrix 12, 23, 34, 35, 50, 67, 82, 86, 87, 89, 90, 92, 96, 98, 101, 102, 105, 107, 108, 122, 124, 126, 144, 151, 162, 163, 221, 253, 254, 256, 281, 300, 309, 320, 326, 370, 433, 458 – assisted laser desorption/ionisation - time of flight 370, 376, 382 Maximum effect concentration level 84, 86 Measure 8, 10, 11, 14, 34, 36, 39, 66, 67, 81, 85, 432, 457, 458, 461, 469 – Measured 11–13, 15, 34, 52, 58, 60, 61, 63, 64, 67, 85, 90, 94, 95, 101, 120, 121, 123, 125–128, 133, 148, 283–285, 323, 328, 375, 406, 458, 460, 461, 462, 464, 471 – Measurement 9, 10, 29, 34, 61, 79, 83, 86, 104, 106, 123–128, 130, 134, 269, 282, 285, 382, 458 – Measuring 1, 6, 10, 11, 39, 54, 58, 121, 122, 126, 405, 457, 461, 464, 465, 468, 469, 471 Mechanical properties 2, 9, 13, 14, 27, 36, 57, 159, 160, 163, 185–187, 230, 231, 266, 272, 305, 308, 309, 320, 322–325, 329 Mechanical resistance 173, 304 Mechanical strength 155, 161, 265, 272, 304, 310, 320, 325 Mechanism 5, 13, 27, 34, 57, 200, 201, 208, 220, 230, 249, 252–255, 342, 358, 364, 365 Mediterranean 415, 422 Medium-chain-length 185, 190, 191, 199, 200 Melt 151, 158, 159, 166–168, 187, 228, 231, 236, 250, 262, 314 – flow index 270, 275, 276 – flow rate 158, 164 – temperature 166 – viscosity 166, 228 Melting 29, 151, 153, 156, 157, 158, 161, 164–166, 168, 185, 187, 191, 220–222, 224, 226, 231, 246, 261, 262, 264, 266, 267, 277–281, 356 – point 151, 153, 157, 158, 161, 168, 185, 187, 220, 231, 261, 262, 267, 278–281 – temperature 156, 220, 246, 252, 356

538 

 Index

Membrane 69, 102, 149, 153 Metabolism 4, 12, 47, 48, 51, 66, 69, 85, 101, 148, 149, 197, 206 Metabolite 61, 70, 105, 184, 189, 199, 361 Metastable 187, 380 Methodology 71, 384, 393, 398, 403, 404, 406, 466, 469, 472, 510 Methylcellulose 323 Micellar 4, 371–373 Micelle 370, 373, 376 Microbe 23, 458 Microbial 1, 5, 6, 8, 9, 11, 13, 14, 23, 26–32, 34, 35, 40, 41, 45, 48, 50, 51, 52, 54–57, 60, 66, 69, 75–77, 79, 82, 83, 85, 90, 91, 98–101, 104, 119, 124, 147–149, 153, 160, 162, 183, 184, 186–188, 194, 195, 197, 203–205, 207, 208, 221, 236, 246, 262, 263, 266, 272, 277, 280, 283–291, 456–458, 460–462, 471, 509 Microbiological 5, 84, 434 Microcrystalline cellulose 60 Microencapsulation 310 Microorganism 49, 51, 152, 153, 186, 188, 190, 195, 197, 280, 488 Microscope 405 – Microscopy 192, 203, 220, 226–228, 234 Microsphere 233 Microstructure 204 Microwave 152, 364 – irradiation 152 Migration 184, 230, 284, 320, 328 Mineral bed composting test 123 Mineral 8, 9, 12, 28, 31, 34, 62, 75, 80, 82, 92, 93, 94, 96, 97, 105, 107, 121, 123, 124, 126, 128, 133, 149, 277–279, 287–289, 433 – Mineralisation 1, 3–5, 7, 10, 11, 14, 15, 46, 47, 52–54, 59, 60, 69, 77, 125, 128, 148, 269, 369 Minimum thermosetting temperature 317 Ministry of International Trade and Industry 37, 39, 117, 121 Miscibility 229 MITI test 121 Mix 185, 443, 449, 450, 476 – Mixed 8, 34, 49, 51, 66, 86, 87, 89, 122, 129, 149, 151, 166, 174, 185, 221, 278, 287, 290, 291, 370, 377, 381, 383, 399, 400, 409, 419, 420, 422–424, 443, 445, 446, 449 – Mixing 101, 166, 177, 225, 237, 307, 308, 320, 362, 435, 446, 448

– Mixture 11, 85, 91, 92, 96, 122, 126, 127, 245, 269, 287, 288, 289, 290, 307, 343, 344, 348, 349, 358, 369, 370, 373, 375, 377, 378, 380, 382, 415, 434, 462 Mobility 278, 279, 315, 319, 492, 499, 501 Model 38, 63, 169, 170, 201, 235, 236, 287, 290, 328, 414, 460, 502 – Modelling 61, 393, 394, 401 Modification 1, 124, 164, 221, 231, 254, 303, 312, 325, 385, 422, 478, 480 – Modified 8, 10, 11, 26, 34, 57, 58, 109, 121, 133, 150, 154, 159, 162, 163, 172, 176, 177, 197, 201, 202, 221, 222, 228, 231, 237, 265, 275, 285, 292, 327, 330, 340, 369, 506 – Modify 70, 197, 205, 319, 320, 326, 339 Moduli 308 – Modulus 188, 266, 270, 318, 323 Moiety 343, 373, 380 Moisture 13, 30, 48, 49, 51, 52, 76, 119, 122, 126, 127, 130, 135, 151, 156, 171, 173, 207, 219, 226, 235, 265, 269, 284, 308–310, 315, 328, 329, 416, 417, 444, 446 Molar mass 245–247, 250, 251, 253, 254, 257, 258, 280, 283, 289, 364 Molar ratio 267, 272, 273, 277, 349, 372 Molecular 1, 11, 36, 55, 69, 84, 115, 119, 121, 147, 149, 152, 183, 185, 195, 218, 223, 224, 226, 229, 235, 247, 250, 253, 262, 266, 281, 282, 285, 301, 303, 305, 315, 318, 319, 341–343, 380, 384, 402, 461, 470, 502, 505 – mass 152, 195, 250, 363 – size 11, 149 – structure 219, 226, 380 – weight 1, 36, 69, 115, 121, 183, 185, 218, 253, 262, 266, 281, 282, 305, 341, 461 – Molecule 234, 235, 245, 250, 303, 312–314, 325, 377, 484 Molten 151, 166–168 Monitor 11, 84, 122, 282 – Monitoring 1, 7, 27, 32, 34, 79, 84, 502 Monomer 5, 7, 55–57, 60, 61, 69, 81, 104, 150, 153–155, 161, 163, 168, 174, 177, 183–185, 187, 188, 190, 191, 194, 199, 200, 202–207, 217, 232, 245, 246, 249, 251, 255, 258, 261, 265, 266, 272, 273, 276–278, 280, 283, 286, 290, 291, 301, 305, 339–344, 347–358, 362–365, 367, 368, 370, 372, 373, 375, 381–384, 475–489

Index 

Montmorillonite 285 Morphology 85, 167, 192, 234, 251, 255, 272, 372, 373 Mould 167, 269, 307, 315 – Moulded 152, 156, 158, 164, 167, 222, 265–267, 299, 307, 309 – Moulding 152, 166–168, 207, 222, 230, 233, 238, 263–265, 268, 307–309, 315, 318, 329 Mucor miehei lipase 340, 352 Mulching 24, 125, 132, 256, 257, 273, 283 Multifunctional 255, 506 Municipal solid waste 6, 163, 409–412, 417, 419, 420, 422, 424 Myofibrillar 299, 310, 323 – protein 299, 310, 323 N,N-Dimethylformamide 370, 371, 373, 375, 376 N-acetyl-d-glucosamine 383–384 Needle 192 Network 224, 305, 309, 310, 311, 312, 319, 320, 322, 323, 325, 329, 397, 433, 499, 503 Neutral 50, 51, 265, 308, 309, 371, 403, 468 Neutralisation 229, 253 Nitrogen 5, 68, 93, 98, 99, 101, 109, 130, 134, 153, 183, 194, 195, 284, 433, 434 Nitrous oxide 395, 430 Nonionic 63, 376, 382 Non-polar 303, 313 Norme Française (French Standards) 45, 52, 54, 67, 69, 128 Nuclear magnetic resonance 14, 234, 251, 348, 351, 353, 370, 373, 376–378, 380–383 – spectroscopy 377, 383 – spectrum 382 Number average molecular weight 266, 342–344, 348–357, 361–365, 367, 368, 376, 384 Nutrient 23, 34, 41, 80, 83, 98, 119, 153, 185, 194, 195, 201 Nylon 56, 165 OK Compost 116, 135, 138–140, 143, 170, 267 Olefins 487 Oligomer 7, 155, 268, 287, 290, 372, 377 – Oligomeric 5, 9, 148, 287, 290, 383 One-pot 351, 367 One-step 197, 292, 370 Optical 36, 58, 166, 220, 252, 264, 301, 329, 349 – microscopy 220, 227 – properties 166

 539

Optimisation 231, 385, 405, 409–425, 427, 429, 437, 451, 477, 509, 511 – Optimise 23, 319, 339, 416, 421, 422, 425, 476 – Optimised 34, 36, 76, 89, 148, 196, 236, 371, 405, 415, 419, 421, 427, 442, 451, 503, 509 Organic 5–7, 45–52, 54, 55, 59–62, 69, 75–82, 92, 96, 99–102, 105, 108, 109, 118, 119, 122, 126, 128, 129, 132, 134, 135, 147, 149, 162, 164, 169–172, 174, 175, 196, 203, 232, 250, 252, 255, 284, 301, 303, 304, 307, 310, 339, 347, 350, 361, 362, 368, 369, 371–376, 380, 382, 398–400, 409, 412, 414–416, 419, 420, 427, 430–436, 438–443, 446, 451, 455, 460, 469, 470, 477–479, 484 – phase 362 – recovery 76, 80, 81, 105, 109, 118, 430, 451 – solvent 250, 307–372, 375, 376, 382 – waste 6, 51, 76, 78, 134, 147, 164, 169, 170, 175, 232, 399, 400, 409, 412, 414–416, 419, 420, 431, 443 Organisation for Economic Co-operation and Development 34, 35, 37–39, 58–60, 64, 66–69, 87–89, 91–93, 95, 97, 115, 117, 121, 125, 128, 131, 136, 460 Orientation 167, 168 – Oriented 1, 129, 167, 218 Output 49, 394, 403, 443, 449, 458, 476–478 Oven 441 Oxalic acid 271, 469 Oxidant 369 Oxidation 9, 55–57, 60, 69, 82, 99, 121, 137, 148, 149, 199, 221, 310, 319, 352, 371, 484, 485 – Oxidative 154, 292, 369, 370, 373–377, 456 – polymerisation 369, 370, 373–377 – Oxidisation 205 Oxybutylene diol 271 Oxygen 3, 6, 9–11, 13, 29, 51, 60, 77, 119, 121, 126, 147, 148, 154, 162, 183, 285, 307, 308, 310, 326, 470 Ozone 395 Packaged 327, 438 – Packaging 6, 8, 11, 13, 15, 40, 41, 45, 48, 52, 54, 69, 71, 76, 80, 110, 118, 129, 131, 132, 139, 143, 147, 151, 154, 156, 169, 171–173, 175, 177, 205, 207, 211, 230, 237, 238, 246,

540 

 Index

256, 257, 273, 281, 307, 308, 325, 329, 330, 400, 409, 415, 431, 432, 451, 455, 460, 470, 471, 483, 501, 505, 508 – Packing 382, 384 Palladium/charcoal 353, 357, 360 Paper 31, 35, 80, 106, 134, 169, 170, 171, 173, 177, 207, 225, 238, 275, 277, 307, 309, 330, 343, 403, 409, 413, 415, 420, 428, 430, 432, 438, 461, 462, 478, 481 – industry 225, 438 – sizing 225 Paracoccus denitrificans 195 Particle(s) 23, 35, 50, 129, 130, 133, 149, 221, 233, 356, 371–373, 401, 418 – size 96, 97, 373, 438, 443, 445 Pathway 3, 61, 70, 75–77, 91, 99, 104, 105, 183–185, 194, 197–200, 370, 382, 486 Peanut protein 309, 313 Performance 147, 148, 150, 161, 163, 171, 177, 203, 205, 207, 218, 232, 238, 269, 273, 274, 277, 330, 393, 396, 397, 398, 405, 406, 409, 415–417, 422, 423, 427, 431–432, 455, 475, 493, 511 Permeability 102, 236, 325–330, 362 Permeation 154, 288, 325, 327, 343, 370 Permit 10, 84, 124, 147, 152, 162, 163, 217, 232, 238, 325 Peroxide 205, 369 Petrochemical 154, 174, 184, 204, 205, 217, 469, 475, 482 – industry 476, 486 pH 4, 7, 13, 34, 48, 51, 61, 79, 82, 85, 91, 93, 96, 119, 130, 134, 149, 152, 204, 224, 253, 265, 311, 312, 314, 370, 376, 384 Phaeodactylum tricornutum 92 Pharmacology 246 Phase separation 224 Phenol 135, 369–380 – -formaldehyde 369 Phenylalanine 302 Phenylene oxide 375 Phosphate 99, 194, 199, 353, 371, 376, 377, 381, 383, 428 Photobacterium sp 94 Physical properties 15, 26, 58, 80, 96, 97, 103, 148, 156, 164, 167, 183, 186, 187, 189, 191, 200, 202, 206, 263, 315, 434 Physics Pigments 104, 307, 322

Pipe(s) 50, 371 Pipeline 429 Plant 27, 30, 31, 32, 35, 45, 47, 48, 52, 53, 55, 63, 66, 67, 68, 75, 79, 80–83, 86, 87, 89–91, 95–100, 104, 105, 108–110, 120, 122, 126, 130, 134–136, 151–154, 156, 162, 163, 170, 173, 174, 176, 187, 195–197, 202, 207, 208, 217, 232, 237, 238, 256, 265, 285, 286, 290, 291, 292, 306, 322, 329, 375, 377, 380, 383, 401, 418, 419, 433, 434, 436–439, 443, 445, 449, 451, 455, 460, 465–472, 475–477, 484–486, 493, 502, 503, 506 – toxicity 104, 134 Plasma 205 Plastic 1, 8, 9, 11–13, 15, 23–25, 30, 33–36, 39–41, 45–48, 50–58, 60, 61, 63, 67–69, 71, 76, 89, 110, 115, 116, 118, 121–132, 134–137, 140, 147–149, 151, 153, 168, 171–175, 177, 178, 184, 195, 201, 202–205, 207, 218, 221–222, 228, 230, 232, 237, 238, 261–263, 281, 283, 285, 291, 299, 301, 307, 308, 309, 324, 329, 362, 393–406, 409, 418–420, 427–451, 455–472, 475, 489, 510, 513 – Plasticisation 58, 319 – Plasticised 151, 166, 307, 308, 318, 324, 329 – Plasticiser 9, 151, 156, 162, 204, 226, 228, 230, 307, 308, 315, 318–321, 325, 326 – Plasticising 167, 318, 319 Plate 30, 155, 287, 289, 399, 400 – test 8–10, 271 Platform 24, 202, 263, 476–478, 485, 487, 491 Polar 101, 302, 312, 319, 320, 322, 325, 350 – solvent 303, 304 – Polarisation 234, 383 – Polarity 314, 315 Poly(1,4-dioxan-2-one) 356 Poly(1,4-phenylene oxide) 375 Poly(3-hydroxybutyrate-co-3hydroxydecanoate) 191 Poly(3-hydroxybutyrate-co-3hydroxyhexanoate) 191 Poly(3-hydroxybutyrate-co-3hydroxyvalerate) 29, 185, 187–189 Poly(4-hydroxybutyrate) 29 Poly(hydroxybutyrate-co-hydroxyvalerate) 152 Poly(lactic-co-glycolic acid) 265 Poly(lactide-co-glycolides) 256

Index 

Poly(L-lactic acid) 245, 265 Poly(L-lactide-co-glycolides) 265 Poly(R-3-hydroxybutyrate) 183, 185–187, 263 – -co-3-hydroxypropionate) 190 – -co-4-hydroxybutyrate) 189 Poly(R-3-hydroxyvalerate) 189 Poly(δ-valerolactone) 362 Poly(ε-caprolactone) 14, 26, 157, 222, 262 Poly(ω-pentadecalactone) 364 Polyacetals 4 Polyamide(s) 4, 56, 233, 340, 485 Polybutylene adipate 29 – -co-terephthalate 32, 159 Polybutylene sebacate 29 Polybutylene succinate 29, 31, 56, 157, 196, 267, 324, 348, 485 – adipate 157, 324 Polybutylene terephthalate 56, 160, 261 Polycaprolactone 7, 56, 324 Polycarbonate 4, 353, 356 Polychlorinated biphenyl(s) 462 Polycondensation 157, 166, 246–250, 257, 269, 340–344, 347, 349, 350, 367, 380, 382–384 Polydispersity index 356 Polyester 4, 9, 25, 26–33, 56, 57, 69, 149, 152, 155–160, 163, 165, 166, 168, 172, 176, 177, 183, 184, 186, 208, 222, 231–233, 237, 245, 252, 253, 261–292, 323, 324, 330, 339, 340, 342, 343, 347–352, 358, 361–365, 367, 368, 441, 442, 484, 485 – resin 274 – synthesis 291–292, 340–369 Polyethylene 1, 26, 56, 147, 185, 218, 230, 257, 272, 291, 307, 308, 418, 455, 461, 483 – furanoate 168 – glycol 56, 160, 246, 319, 340 – naphthalate 276 – oxide 255 – succinate 157, 267 – terephthalate 56, 147, 257, 261, 455, 470–471 Polyglycolic acid 155–156, 245, 265 Polyhexylene succinate 29 Polyhydroxyalkanoate(s) 25, 152–154, 183–208, 263–264, 432, 510 – synthase 198, 200–201 Polyhydroxybutyrate 25, 101, 152, 202, 263, 324 Polylactic acid 27, 56, 150, 155–156, 196, 245, 261, 264–265, 324, 330, 466, 483, 510

 541

Polylactide 265, 357, 362 Polymer 1–15, 23–41, 45–71, 75–110, 115, 117, 119, 121, 122, 125, 128, 131, 132, 135–137, 143, 147–178, 183–185, 187, 191, 192, 195–197, 203–208, 217, 218, 220–222, 224, 226–233, 235, 237, 238, 245–258, 261, 262–267, 269, 272–281, 285, 286, 288, 290, 291, 299, 301, 303–306, 310, 312, 315, 318, 319, 329, 339–385, 409–425, 432, 458, 460, 466, 468, 469, 472, 475, 476, 483, 484, 488, 489, 491, 503, 504 – backbone 56, 163, 228, 376 – morphology 167 – -related parameters 277–280 – resin 467 – Polymeric 1, 3–6, 8, 10, 11, 14, 15, 34, 39, 47, 54, 56, 104, 105, 147–149, 154–156, 162, 164, 165, 168, 183, 217, 218, 250, 253, 269, 306, 373, 461 – Polymerisation 104, 149, 155–157, 183, 245–251, 255, 262, 264–266, 310, 313, 339, 341, 342, 343–385, 470, 486 – conditions 378, 381 – mechanism 255 – rate 353, 357, 363, 373, 381 Polymethyl methacrylate 257, 306 Polyolefin(s) 2, 14, 26, 27, 56, 57, 158, 221, 232, 233, 315, 461, 462, 466, 467 Polypropylene 26, 56, 147, 184, 324, 455 – glycol 56 – terephthalate 271, 272 Polysaccharide 57, 150, 162, 204, 219, 339, 340, 380–384, 481 Polystyrene 56, 147, 151, 218, 257, 399, 455 Polyurethane 4, 56, 105, 324, 486 Polyvinyl acetate 161 Polyvinyl alcohol 7, 149, 161–162, 229, 231 Polyvinyl chloride 56, 147, 222, 308 Polyvinylidene chloride 323 Porcine pancreatic lipase 340, 348, 352, 353, 356–358, 361–363, 365, 368, 369 Porosity 221, 253, 449 Porous 254, 256 Powder 12, 31, 123, 130, 134, 152, 253, 382, 460 Power 308, 429, 430, 456 Precipitate 370, 382 – Precipitated 372, 381, 382 Precursor 153, 185, 188, 194, 199, 200, 479, 483, 485, 487

542 

 Index

Preparation 35, 91–92, 102, 134, 153, 403, 438, 484, 493, 507 Prepolymer 56, 158 Prerequisites 97–102, 104 Press 307, 309, 437, 438, 441–446 – Pressed sheet property 158 – Pressure 10, 11, 151, 158, 164, 166, 167, 203, 221, 225, 226, 307, 344, 350, 412 Pretreatment 35, 135, 149, 369, 418, 433–440, 443, 445, 446, 448, 450, 509 Price 78, 156, 157, 171, 173, 177, 217, 261, 262, 264, 272, 404, 482, 489 Procedure 11, 24, 34, 35, 52, 53, 78, 86–96, 105, 115–144, 395, 459, 461, 463 Process 2, 3, 5, 6, 13, 14, 23, 24, 27, 33, 34, 39, 41, 47–49, 52–54, 59, 75–110, 118, 119, 129–131, 149, 151–153, 155–157, 161–163, 165–168, 183, 185, 188, 192, 195–197, 202–204, 207, 218, 220, 221, 223, 224, 228, 231, 250, 253, 254–266, 268, 274, 281, 287, 299, 305, 307, 312–322, 329, 330, 350, 351, 368, 369, 376, 393, 398, 403, 418, 428, 433, 434, 437–439, 442, 443, 445, 446, 448, 449, 451, 455–457, 466–467, 472, 476, 481, 483–486, 489, 509, 510 – Processability 69, 147–178, 229, 231 – Processed 151, 159, 161, 164, 183, 217, 218, 222, 228, 229, 261, 308, 326, 401, 441, 476, 477 – Processing 58, 78, 165–168, 174, 175, 187, 222, 226, 230, 233, 236, 237, 253, 261, 263, 267, 292, 299, 314, 318, 319, 329, 340, 385, 393, 427, 437, 475, 477, 478, 481, 501–503, 505, 508, 509 – conditions 451 Producer 77, 110, 115, 137, 140, 141, 151, 159, 162, 170, 174, 176, 187, 196, 202, 217, 264, 265, 267, 269, 273–275, 397, 413 Product 3, 5–7, 11–13, 23, 24, 26, 30, 31, 36, 45–47, 49, 55, 56, 61, 63, 67, 71, 76–80, 82, 87, 102–105, 109, 110, 115, 119, 125, 129, 130, 133, 135–140, 143, 144, 148, 149, 153, 154–158, 160–165, 167, 169, 170, 174–177, 183, 184, 185, 197, 204, 205–207, 217, 218, 221, 222, 224–226, 228, 230, 232–234, 237, 238, 253, 261, 263–268, 273–275, 286, 291, 292, 305, 307, 308, 310, 314, 318, 320, 324, 325, 327, 329, 330, 340–344,

347, 349, 350, 353, 358, 362, 368, 369, 372, 375–377, 381–383, 385, 393–397, 400–406, 409–411, 418, 428, 429, 432–434, 451, 455, 456, 460–468, 470, 475, 476–479, 481, 483, 486, 489, 491–513 – Production 47–49, 52, 54, 61, 75, 77, 78, 83, 89, 97, 121, 123, 125, 126–128, 130, 153, 154–157, 159, 162, 163, 166, 169, 172, 174–177, 183–190, 194–197, 199, 201–203, 205, 207, 208, 217, 218, 229, 231, 232, 237, 238, 246, 261, 263–266, 268, 274, 291, 292, 305, 307, 310, 314, 315, 318, 347, 362, 363, 369, 375, 396, 399, 401, 404–406, 409, 411, 414, 416, 417, 427, 430–433, 435, 436, 438, 445, 446, 449, 451, 475, 476, 478, 479, 481–485, 487, 489, 491, 496, 500, 501, 503, 505, 506, 508, 509, 511, 512 – cost 177 Profile analysis 86, 393 Profile 24, 55, 54, 67, 68, 86, 103, 105, 135, 288, 393 Propagation 230, 249, 353, 358, 364, 365 Properties 2, 7, 9, 13–15, 26, 36, 46, 56–59, 61, 63, 66, 69, 76, 80, 85, 86, 91, 92, 96, 97, 102, 103, 106, 127, 128, 148, 151–154, 156–161, 163–168, 175, 177, 183–192, 200, 202, 204–206, 218, 221, 226, 228–231, 233, 234, 237, 245, 247, 250–252, 258, 261–264, 266–270, 272, 273, 275–277, 299, 301, 303–312, 314, 315, 318–320, 322–330, 340, 364, 365, 370, 371, 375, 397, 398, 401, 433, 434, 455, 475 Propylene 26, 56, 147, 184, 324, 455, 476, 484, 486–489 Protection 58, 110, 154, 314, 326, 339, 368, 421, 460, 511 Protein 11, 121, 150, 153, 154, 198, 200–202, 218, 299, 303–322, 325–330 – composition 325 – material 299, 305, 311, 315, 319, 320, 322, 324–326, 329 – structure 311 Protocol 402, 404, 464 Protomonas extorquens 195 Pseudomonas aeruginosa 199 Pseudomonas cepacia lipase 340, 354 Pseudomonas cepacia 352 Pseudomonas fluorescens 364 – lipase 340, 354, 355, 357

Index 

Pseudomonas frugi 200 Pseudomonas putida 66, 88, 199 Pseudomonas sp. lipase 340, 353, 360, 366 Pseudoplastic 236, 308 Public-private partnerships 508, 512 Purification 155, 197, 205, 245, 255, 266, 483 – Purified 7, 8, 190, 203, 204, 249, 250, 251, 253, 256, 257, 486 – Purity 187, 205, 208, 370, 381, 414, 418, 442, 509 Quality 27, 52, 54, 77–82, 106, 107, 109, 119, 130, 132, 134, 138, 139, 142, 147, 169, 170, 172–174, 203, 207, 219, 286, 308, 309, 393, 396, 397, 410, 414, 416–419, 425, 428, 430, 432, 436, 440, 442, 446, 451, 463, 491, 509 – assurance 139, 397 – control 80, 81, 109, 138, 418 – -of-life 504, 505 Quantity 10, 11, 172, 174, 195, 412, 416, 417, 440, 451, 463, 469, 499 – Quantitative 7, 34, 36, 65, 130, 135, 287, 352, 393, 409, 413, 451 Quantitative Structure-Activity Relationship 65, 68 Quantitative 7, 36, 65, 130, 135, 287, 352, 393, 409, 413, 451 Quaternary 303 Quenching 225, 253 Radiation 206, 320 Radical 5 Radioactive 12, 148, 245, 468, 469, 472 Ralstonia eutropha 153, 188 Rate of polymerisation 358 Ratio 7, 63, 161, 168, 177, 194, 219, 226, 228, 233, 234, 236, 264, 267, 272–274, 277, 281, 284–286, 349, 356, 357, 360, 365, 367, 370, 371–373, 376, 402, 469 Raw material 99, 291, 310, 311, 329, 330, 428, 476, 479, 483, 487, 505 Rayon 156, 168 Reaction 5, 7, 26, 27, 47, 51, 56, 83, 85, 102, 163, 197, 231, 249, 251, 252, 269, 303, 340–344, 348–351, 356, 358, 362–364, 366, 368–372, 376–380, 382, 383, 486 – conditions 340, 342, 343, 350, 358, 362, 363, 366, 368, 369, 371 – mechanism 27, 342 – mixture 344, 348, 349, 358, 377

 543

– rate 26, 51 – temperature 368 – time 363, 364 Reactivity 250, 255, 344, 349, 357, 364, 367 Reactor 185, 432, 446, 448 Recombinant 183, 186, 189, 194, 196, 197, 199, 200–202, 207 Recovery 51, 76, 78, 80, 81, 105, 109, 118, 148, 153, 156, 195, 398, 399, 405, 406, 409, 411, 429, 430, 432, 436, 437, 439, 442–451, 483, 505 Recycle 203, 399, 411, 429 – Recycled 45, 48, 147, 149, 164, 174, 299, 377, 403, 409, 412, 465 Reductase 198, 199 Reduction 26, 79, 110, 134, 153, 175, 195, 236, 237, 273, 291, 371, 400, 403, 406, 418–420, 428–431, 440, 445, 446, 451, 468, 476, 484, 502, 511 Reflect 39, 281 – Reflection 506 Regeneration 169, 256, 371 Registration, Evaluation, Authorization and Restriction of Chemicals 46, 48, 51, 55, 58, 60, 61, 63, 65, 67–69, 71, 83, 95, 99, 133, 163, 228, 245, 301, 409, 414, 419, 428, 430, 475 Regulation 46, 58, 69, 78, 357, 433, 493 Relative 60, 85, 86, 92, 98, 99, 103, 133, 156, 202, 236, 261, 277, 301, 315, 318, 325, 421, 440, 469, 499 – humidity 315, 318, 320, 325, 326 – hydrophobicity 301 Renewable raw materials 299, 476, 477, 482–488, 508 Renewable resource 476, 484 Replacement 125, 168, 203, 205, 218, 493 Replication 511 Reproducibility 13, 126, 397 Reproduction 47, 48, 53, 64, 68, 92, 93, 96 Research 1, 26, 29, 33, 75, 82, 87, 90–92, 95, 103–107, 109, 110, 130, 147, 153, 158, 160, 175–177, 183, 184, 201, 203, 207, 208, 237, 238, 256, 291, 299, 301, 383, 397, 401, 406, 420, 432, 462, 463, 481, 484, 486, 491–513 – and Development 177, 183, 184, 208, 238, 299, 491–513 – and technological development 492, 497–499, 502–504

544 

 Index

Residual waste 76, 172, 409, 415, 416, 418, 420, 422, 424, 430 Residue 3, 47, 77, 85, 87, 92, 127, 226, 251, 364, 434, 443 Resilience 231, 412 Resin 152, 166, 167, 237, 274, 330, 462 Resolution 349, 498, 504 Resonance 14, 234, 251, 348, 382 Retention 13, 173, 308, 327, 328, 330, 432, 446 Reuse 172, 232, 339, 399, 405, 428 Review 118, 139, 203, 205, 206, 221, 309, 339, 437, 456, 463 Rheology 166 Rhodococcus sp 189 Rhodospirillum rubrum 197 Ribonucleic acid 339 Rigid 152, 166, 218, 231, 265 – Rigidity 224, 315, 319, 320 Ring 234, 356, 358, 363, 364, 368 – -opening – -opening polymerisation 155, 157, 245, 247, 249, 250, 262, 264, 266, 351, 352–369, 384 Risk 80, 103, 164, 171, 172, 175, 217, 218, 250, 411, 449, 462, 506, 511 – assessment 70, 71, 83, 86, 274, 286–291 Roadmap 428 Roll 166, 275, 410 Room temperature 186, 265, 269, 279, 288, 370, 375, 376 – ionic liquids 370, 371 Rubber 236, 287 – Rubbery 315, 318, 319 Safety 15, 46, 55, 58, 67, 69–71, 79, 110, 116, 131, 132, 451, 460, 506 Salinity 34, 95, 96 Sample 10, 12, 26, 27, 75, 85–87, 89–100, 102–105, 108, 110, 119, 125, 129–131, 133, 139, 167, 186, 227, 228, 440–442, 461, 462 – Sampling 35 Sanitisation 433 Saturated 96, 194, 462 – Saturation 90 Scale 13, 110, 119, 154, 163, 254, 256, 263, 264, 292, 312, 340, 371, 385, 404, 405, 415, 483, 486, 493 Scanning electron microscopy 354 Scenedesmus subspicatus 66, 92

Screening 8, 10, 85, 101, 134, 283, 414, 418, 443, 445, 449 Seal 9 – Sealed 221, 307 Sebacic acid 160, 163, 166, 231, 269, 273, 277, 291, 347, 349, 367 Sediment 30–32, 35, 81–83, 85, 95–97, 99, 103, 109, 446 – Sedimentation 97, 438, 445 Selectivity 308, 326, 327, 363, 381, 384 Selenastrum capricornutum 66, 92 Semicontinuous activated sludge 122, 230 Semicrystalline 156, 157, 167, 250, 262, 362 Sensitivity 14, 23, 92, 95, 103, 151, 163, 228, 231, 245, 304 Separation 36, 224, 312, 399, 409–425, 427, 428, 430, 437, 441, 443–445, 450, 451, 476 Serine 204, 364 Services 393, 492, 504, 508, 511 Setting 59, 167, 467, 508 Shape 87, 151, 152, 189, 246, 253, 254, 318, 432, 460 Shear 165, 166, 221, 225, 226, 236, 319, 438 Sheet 27, 161, 167, 173, 178, 238, 265, 267, 299 Shore 24, 284 Short-chain-length 185, 191, 199 Side chain 205, 352, 378 Simulation 13, 26, 29, 30, 31, 122, 123, 127, 277, 309 Size 5, 8, 11, 96, 109, 148, 149, 151, 166, 219, 227, 228, 231, 234, 246, 253, 254, 315, 356, 363, 371, 373, 418, 423, 438, 445, 446, 448, 505 – exclusion chromatography 253, 376 – Sizing 225 Skeletonema costatum 92 Small and medium-sized enterprises 491, 499, 503, 506, 510, 511 Società Estense Servizi Ambientali SpA 443, 445, 448 Sodium dodecyl benzene sulfonate 370 Sodium hydroxide 229 – -dependent acetoacetyl-CoA reductase 198–199 Soft 310 – Softening 193 Soil 11–13, 15, 23, 24, 28, 30, 35, 39, 41, 45–52, 54, 55, 59–65, 67, 68, 70, 75, 78–83, 87, 89–93, 96–104, 106–110, 116, 120, 121,

Index 

125, 126, 132, 135, 138, 140, 144, 148, 149, 172–174, 207, 217, 221, 232, 264, 268, 269, 277, 280, 283–287, 291, 394, 399, 400, 404, 406, 410, 412, 428, 432, 434, 456, 462, 471 – biodegradation 50–51, 81–83 – environment 148, 221, 456 – organic matter 48, 50, 80, 82, 412, 434 Solar 401 Sole 9, 10, 200, 432, 445, 450, 458 Solid 4, 6, 11, 23, 24, 28, 34–36, 55, 69, 87, 91, 92, 96, 97, 119, 126, 127, 132, 165, 175, 237, 246, 250, 285, 308, 432, 438, 443, 445, 463, 475 – waste 6, 126, 163, 175, 237, 409, 463 Solubilisation 226, 268, 307, 312 – Solubility 63, 87, 92, 93, 136, 161, 167, 226, 253, 255, 287, 312, 320, 322, 326, 350, 370, 376 – Soluble 11, 66, 91, 95, 96, 218, 221, 234, 253, 256, 257, 265, 268, 287, 309, 310, 315, 322, 341, 362, 370, 371, 373, 375, 376, 381, 441 Solution 34, 35, 57, 91, 92, 94, 96, 161, 163, 171, 173, 175, 178, 185, 187, 237, 238, 247, 250, 255, 304, 307–310, 312, 314, 315, 348, 362, 369–373, 376, 377, 398, 400, 448, 476 Solvent 192, 203, 224, 250, 258, 303, 304, 307, 311, 339, 344, 349, 350, 361, 363, 368, 369, 371–373, 375–377, 381–383, 461, 479, 485, 505 – extraction 461 – process 312–315 – resistance 152 Sorption 11 Source separation 409–425, 427, 428, 430–431, 450, 451 Soy protein 307, 309, 310, 312, 323 Soybean peroxidase 370, 373, 376, 378 Spain 139, 176, 237, 292, 410, 412, 414, 417, 420, 422, 431 Special prerequisites 97–102 Species 54, 67, 80, 83, 84–87, 89, 90, 92, 93, 95, 96, 99–101, 103, 105, 106, 108–110, 119, 128, 249, 364 Specific gravity 156 Specification 15, 58, 116, 117, 119, 131–137, 143, 233, 275, 460, 461, 464, 471, 513 Specificity 5, 191, 194, 195, 197, 206

 545

Spectra 234, 236 Spectrometry 290, 343, 370, 382 Spectroscopy 14, 57, 376, 377, 380, 383, 441 Speed 171, 224, 226, 231, 362, 410, 433, 512 Sphere 176, 237, 371 Spherical 372, 373 Spinning 168, 234, 307, 314 Spreading 304, 308–310, 312, 314, 399, 433 Stabilisation 33, 49, 58, 136, 432, 435, 437, 451 – Stabilise 8, 48, 49, 127, 235, 286, 303, 304, 310, 312, 314, 319, 320, 323, 398, 430, 433, 446 – Stability 154, 163, 168, 184, 197, 202, 232, 253, 323, 362, 370, 377 Standard deviation 440 Standard procedure 86–96 Standard test 3, 11, 35, 36–40, 460, 461, 471 Standardisation 2, 3, 8, 15, 39, 46, 53, 76, 78, 115, 116–118, 136, 393, 493 Starch 2, 9, 10, 15, 28, 32–33, 39, 58, 68, 98, 107, 117–119, 123, 125–126, 137, 178–180, 185–186, 193–194, 196–199, 201–202, 204, 207–209, 233, 265–293, 295–299, 314, 321, 323, 364, 378–379, 387, 398, 401, 494, 498 – -based foam 151 – -based polymer 163, 165, 218 – -based technology 162, 217–238 Static 11, 319, 443, 448 Steady state 83 Step growth 247 Stereochemistry 156, 382, 384 Steric hindrance 253, 375 Sterilisation 185 Stoichiometry 9, 467 Storage 185, 186, 194, 224, 235, 319, 328, 339, 340, 406, 431, 433, 448, 449 Strain 30, 34, 57, 187, 188, 196, 197, 201, 266, 273, 282, 283, 286, 290, 364, 509 Strategy 61, 172, 410, 412, 421, 451, 476, 492, 496, 497 Strength 2, 125, 128, 151, 155, 161, 166, 168, 171, 185, 186, 187, 188, 204, 229, 231, 249, 265, 266, 267, 272, 304, 310, 311, 314, 320, 324, 405, 411, 430, 501 Stress(es) 83, 84, 168, 235, 236, 415, 510 Stretch 265 – Stretched 186, 303 – Stretching 304

546 

 Index

Structural 23, 162, 221, 222–236, 247–249, 261, 301, 304, 319, 325, 349, 353, 369, 383, 434, 446 – modification 221 – organisation 325 – Structure 4, 5, 30, 39, 50, 56, 75, 82, 102, 104, 107, 124, 147, 149, 151, 152, 155, 157–159, 163, 173, 187, 188, 190, 192, 197, 200, 204, 217, 219–221, 224, 226–228, 230, 233–236, 245, 247, 249, 252, 253, 255–257, 261–263, 269, 270, 272, 277, 278, 283, 301–306, 311, 319, 322, 339, 350, 357, 358, 366, 368–371, 375, 380, 382–384, 399, 430, 431, 434, 445, 449, 475, 484, 492, 497, 509 – -activity relationship 65 Sturm test 10, 34, 97, 109, 120, 121, 230, 285 Substance 10, 15, 23, 29, 34, 36, 39, 45–49, 53–55, 57–71, 76, 79–83, 87, 90–93, 95, 101–106, 108–110, 119, 122, 125, 128, 169, 196, 205, 287, 290, 307, 320, 382, 394, 398, 434, 462 Substituted 361, 363, 406, 420, 467 – Substitution 8, 31, 173, 201, 232, 368, 399, 400 Substrate 3, 5, 7, 9–11, 14, 30, 31, 50, 53, 54, 67, 75, 89, 97, 99, 101, 119, 149, 185, 191, 194, 195, 199, 200, 203, 340, 343, 347, 356, 375, 382, 383, 434, 438, 443, 445, 446, 448, 449, 455–458, 461, 471, 479, 484 Succinate polyester 158 Succinic acid 157, 158, 163, 166, 231, 267, 268, 274, 275, 291, 292, 349, 482, 485 Sugar 31, 101, 152, 156, 176, 183, 187, 195–197, 217, 224, 247, 257, 292, 340, 349, 368, 383, 384, 401, 476–479, 483, 485, 486 Sulfonate 160, 370 Sulfur 98, 101, 109 Sunlight 26, 51, 82, 147, 202, 465 Supercritical carbon dioxide 362, 368–369 Supercritical fluids 203, 205, 339 Supply 13, 23, 34, 199, 202, 307, 385, 393, 396, 403, 405, 412, 428, 476, 477, 482, 491, 493, 496 Surface 4, 5, 8, 9, 14, 23, 25–27, 51, 55, 57, 95, 102, 128, 147, 149, 152, 162, 167, 203, 204, 207, 222, 228, 230, 253, 268, 281, 284, 303, 307–310, 312, 314, 315, 328, 371, 377, 382, 401, 438, 462

Surfactant 149, 314, 370, 371, 373, 376, 382 – -enveloped enzyme 382 Suspension 9, 222, 438, 441, 442 Sustainable 134, 143, 171, 172, 175, 204, 237, 292, 396, 398, 410, 428, 455, 465, 471, 475, 476, 477, 482, 491, 496, 499, 504, 508, 510, 511, 512 Swell 223, 224, 225 Symposium 458, 466, 468, 469 Syndiotactic 247, 250 Synergistic 107, 364 Synthesis 1, 12, 47, 152, 153, 155, 158, 183–187, 189, 194–196, 199, 200, 203, 205, 208, 246, 247, 249–251, 255, 261, 262, 264, 266, 270, 287, 291–292, 301, 305, 339–385, 484–486 Synthetic 12, 13, 23, 25, 26, 29–36, 80, 89, 96, 97, 104, 130, 149, 150, 154–162, 168, 185, 189, 221, 230, 233, 262, 264, 283, 287, 290, 299, 303, 305, 309, 310, 318, 323–326, 329, 330, 369, 371, 381, 382 – polymers 5, 12, 26, 56, 100, 149, 162, 221, 226–233, 235, 303, 304, 305, 315 Talc 124, 167 Tank 126, 433, 438, 448, 449 Target 12, 87, 195, 202, 236, 409, 411, 412, 415, 416, 418, 428, 430, 451, 457, 458, 464, 492, 496, 504 Technical 33, 78, 105, 118, 147, 160, 232, 238, 269, 272, 275, 276, 277, 281, 286, 400, 475, 477, 485, 498, 511 Temperature 4, 6, 8, 10, 13, 25, 26, 27, 28, 34, 35, 48, 49, 51, 52, 53, 82, 119, 120, 122, 125, 126, 127, 130, 135, 136, 143, 149, 151, 152, 155, 159, 163, 166, 167, 168, 186, 187, 191, 192, 203, 204, 220, 221, 222, 224, 225, 226, 228, 231, 232, 235, 246, 250, 253, 257, 262, 264, 265, 267, 269, 278, 279, 281, 283, 285, 287, 301, 307, 308, 314, 315, 318, 319, 325, 326, 342, 348, 353, 356, 357, 362, 363, 368, 375, 376, 398, 432, 433, 448, 449, 458 – range 48, 51, 149 Tensile properties 13, 14, 229 Tensile strength 125, 128, 168, 185, 186, 187, 188, 229, 266, 267 Tensile stress 324

Index 

Terephthalic acid 32, 160, 161, 272–275, 277–287, 290, 470 Termination 56, 305 Tertiary 250, 303 Test 3, 6–15, 24, 26–41, 46, 52–54, 58–61, 63–68, 77–81, 83–87, 89–100, 103–107, 109, 110, 115, 116, 119–137, 139, 141, 230, 231, 269, 273–275, 277, 280, 281, 284–286, 290, 291, 398, 432, 457, 458, 460, 461, 463, 468, 469, 471 – material 8, 10, 98, 124, 129, 133, 134, 458, 460, 469 – method 7, 9–11, 26, 36, 39, 52, 54, 80, 87, 94, 96, 116, 119–131, 132, 285, 458, 460, 461, 468, 471 Tetrahydrofuran 348, 370, 485 Texture 50, 126, 226 Theoretical oxygen demand 9, 60, 121 Thermal analysis 192 Thermal degradation 23, 187 Thermal history 251 Thermal properties 205, 375 Thermal stability 184, 362, 370 Thermal treatment 352, 378, 411 Thermobifidia fusca 34 Thermodynamic 323, 356 Thermogravimetric analysis 370, 375 Thermoplastic(s) 156, 161, 162, 164, 165, 166, 167, 168, 183, 184, 186, 191, 204, 207, 221, 228, 231, 262, 299, 304, 309, 310, 315, 329 – polymers 151, 164, 166, 167, 221, 310, 318, 329 – process 312, 315–322 – starch 151, 163, 166, 221, 222, 226, 228–234, 236 – Thermoplastically processable starch 228 Thermoset 164 – Thermosetting 158, 164, 320, 330, 371 Thickness 129, 134, 166, 167, 236, 284, 432, 460 Thiocapsa pfennigii 200 Three-dimensional 5, 301, 303, 304, 312, 319, 322 Threonine 202 Threshold 39, 40, 46, 160, 272, 460 Time 2, 3, 13, 14, 26, 33, 34, 36, 53, 61, 64, 67, 77, 80, 85, 86, 89, 90, 91, 95, 96, 99, 100, 103, 105, 106, 108, 109, 122, 126, 129, 131, 139, 147, 154, 165, 168, 170, 173, 174, 188,

 547

195, 197, 205, 207, 217, 222, 226, 231, 247, 251, 252, 256, 258, 277, 282, 286, 299, 308, 319, 339, 340, 362–364, 367, 369, 377, 382, 385, 394, 401, 403, 411, 415, 417–420, 423, 424, 427, 432, 433, 446, 448, 449, 455, 457, 458, 460–462, 464, 468, 469, 471, 492, 499, 507 Tissue 205, 256 – engineering 244, 263, 313 Titration 34, 35, 57, 373 TONETM 157 Tool 8, 10, 46, 54, 69, 71, 172, 204, 207, 301, 384, 396, 397, 398, 405, 406, 416, 417, 421, 422, 425, 430, 431, 451, 466, 491, 493, 508 Total solid(s) 434, 438, 445, 446, 449 Tough 267 – Toughness 153, 154, 163, 187, 232 Toxic 15, 54, 57, 64, 66, 77, 79, 80, 82, 83, 87, 91, 92, 93, 96, 98, 100, 104, 105, 107, 109, 291, 369, 410, 462 – Toxicity test 64–68, 85, 86, 89, 97, 134, 136, 141, 291 Trade 157, 159, 205, 463–465, 496, 512 Training 501, 503 Transfer 4, 89, 325, 339, 400, 476, 496, 501 Transformation 15, 68, 101, 154, 162, 221, 228, 238, 287, 319, 428, 477, 491 Transition 27, 151, 165, 191, 221, 224, 226, 235, 257, 262, 307, 315, 357, 496, 512 – -state analogue substrate 384 Transmission 234, 431 – electron microscopy 234 Transparency 154, 230, 418 – Transparent 309, 310, 329, 406, 417, 419, 468 Transportation 23, 154, 178, 393, 475 Trend 118, 169, 171, 174, 363, 396, 512 Triblock copolymer 370 Tricarboxylic acid 200 Trimer 343, 358, 369, 377, 382 – of trimethylene carbonate 358 Trimethylene carbonate bulk 352 Tube 166, 167 Turning 137, 446, 450 Tyre(s) 236, 487 Ultrahigh molecular weight 1, 185–186 Ultraviolet 14, 51, 153, 156, 320

548 

 Index

Unstable 187, 462 Untreated 97 US Composting Council 140 US Department of Agriculture 465 US Food & Drug Administration 265, 275 Vacuum 342, 349, 350, 351, 368 Vapour 236, 308, 310, 320, 325, 431 Velocity 168, 225 Vessel 89, 91, 93, 96, 205 Vibrio fischeri 66, 94 Viscosity 166, 224, 226, 228, 308, 315, 445 – Viscous 165 Volatile 132, 185, 254, 432, 482 Volume 10, 11, 24, 46, 55, 92, 126, 127, 135, 158, 169–171, 174, 175, 220, 222, 223, 224, 226, 228, 237, 238, 315, 416, 424, 485, 505 – fraction 220, 222–224, 226 Washing 417, 419, 431 Waste disposal 175, 184, 237, 429 Waste Framework Directive 409, 411, 428, 429 Water 1, 3–5, 10, 15, 23, 25–28, 34, 41, 48–51, 53, 56, 59, 61, 63, 64, 66, 75, 78, 83, 87, 89–92, 95–97, 101, 102, 115, 116, 119, 120, 122, 125, 130, 136, 138, 143, 144, 147, 148, 151, 155, 161–163, 167, 168, 173, 183, 190, 202, 203, 217, 218, 220–226, 228–231, 233, 236, 247, 250–253, 255, 257, 263, 268, 269, 281, 286, 287, 290, 307–310, 312, 314, 315, 318–320, 322, 325, 329, 330, 342, 347, 356, 358, 362, 364, 368–373, 377, 394, 406, 431, 434, 437, 438, 443–445, 464, 496, 511

– activity 317, 326, 362 – content 50, 89, 90, 101, 151, 168, 203, 221, 224, 229, 308, 315, 317 – to bis(2-ethylhexyl)sulfosuccinate sodium salt molar ratio 371–372 – uptake 4, 257 – vapour 236, 308, 310, 320, 325, 431 – Waterborne 394 Weight 12, 54, 69, 86, 90, 91, 124, 125, 132, 133, 162, 167, 229, 285, 291, 319, 358, 402, 415, 418, 431, 440, 441, 455, 510 – loss 7, 13–15, 25–29, 32, 34, 128, 253, 269, 277, 282–286, 461 – ratio 356, 360 Wheat gluten 299, 308–310, 312, 315, 319, 320, 322–324, 326–329 Williams Landel and Ferry equation 317 Wood 82, 97, 106, 204, 375, 405, 418, 438, 478, 479, 481, 483 X-ray diffraction 219, 234, 235, 382 Yarrowia lipolytica 362, 363, 369 Yield 53, 81, 82, 119, 173, 202, 217, 218, 219, 236, 250, 266, 342, 343, 347, 350–353, 356, 358, 360, 362, 365, 367–370, 373, 375, 376, 380–382, 399, 406, 415, 422, 424, 449, 463, 467, 476, 483, 485, 509 – Yielding 352, 368, 422 Young’s Modulus 188, 266 Zein 299, 307, 308, 312, 319 Zweckverband Abfallwirtschaft Straubing Stadt und Land 449