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Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

AIR, WATER AND SOIL POLLUTION SCIENCE AND TECHNOLOGY SERIES

SLUDGE

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

TYPES, TREATMENT PROCESSES AND DISPOSAL

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AIR, WATER AND SOIL POLLUTION SCIENCE AND TECHNOLOGY SERIES Trends in Air Pollution Research James, V. Livingston (Editor) 2005. ISBN: 1-59454-326-7 Agriculture and Soil Pollution: New Research James, V. Livingston (Editor) 2005. ISBN: 1-59454-310-0 Water Pollution: New Research A.R. Burk (Editor) 2008. ISBN: 1-59454-393-3 Air Pollution: New Research James, V. Livingston (Editor) 2007. ISBN: 1-59454-569-3

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Air Pollution Research Advances Corin G. Bodine (Editor) 2007. ISBN: 1-60021-806-7 Marine Pollution: New Research Tobias N. Hofer (Editor) 2008. ISBN: 978-1-60456-242-2 Complementary Approaches for Using Ecotoxicity Data in Soil Pollution Evaluation M. D. Fernandez and J. V. Tarazona 2008. ISBN: 978-1-60692-105-0 Complementary Approaches for Using Ecotoxicity Data in Soil Pollution Evaluation (Online Book) M. D. Fernandez and J. V. Tarazona 2008. ISBN: 978-1-60876-411-2 Lake Pollution Research Progress Franko R. Miranda and Luc M. Bernard 2008. ISBN: 978-1-60692-106-7

Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Lake Pollution Research Progress (Online Book) Franko R. Miranda and Luc M. Bernard (Editors) 2008. ISBN: 978-1-60741-905-1 River Pollution Research Progress Mattia N. Gallo and Marco H. Ferrari (Editors) 2009. ISBN: 978-1-60456-643-7 Heavy Metal Pollution Samuel E. Brown and William C. Welton (Editors) 2008. ISBN: 978-1-60456-899-8 Cruise Ship Pollution Oliver G. Krenshaw (Editor) 2009. ISBN: 978-1-60692-655-0 Industrial Pollution including Oil Spills Harry Newbury and William De Lorne (Editors) 2009. ISBN: 978-1-60456-917-9

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Environmental and Regional Air Pollution Dean Gallo and Richard Mancini 2009. ISBN: 978-1-60692-893-6 Environmental and Regional Air Pollution (Online Book) Dean Gallo and Richard Mancini 2009. ISBN: 978-1-60876-553-9 Traffic Related Air Pollution and Internal Combustion Engines Sergey Demidov and Jacques Bonnet (Editors) 2009. ISBN: 978-1-60741-145-1 Sludge: Types, Treatment Processes and Disposal Richard E. Baily (Editor) 2009 ISBN: 978-1-60741-842-9

Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

AIR, WATER AND SOIL POLLUTION SCIENCE AND TECHNOLOGY SERIES

SLUDGE

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TYPES, TREATMENT PROCESSES AND DISPOSAL

RICHARD E. BAILY EDITOR

Nova Science Publishers, Inc. New York Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Baily, Richard E. Sludge types, treatment processes and disposal / Richard E. Baily. p. cm. Includes index. ISBN H%RRN 1. Sewage sludge. 2. Sewage--Purification. 3. Sewage disposal. I. Title. TD767.B35 2009 628.3'64--dc22 2009024631

Published by Nova Science Publishers, Inc. † New York

Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

CONTENTS Preface Chapter 1

Chapter 2

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Chapter 3

ix Synthetic Sludge as a Physical and Chemical Analogue of Real Sludge in the Activated Sludge Process Nick Hankins, Nidal Hilal and Tan Phong Nguyen Processes to Recovery Profitable Products from Water Degumming Sludge of Vegetable Oils Liliana N. Ceci and Diana T. Constenla A Survey of Methods for Characterization of Sulfate-Reducing Microorganisms Bidyut R. Mohapatra, W. Douglas Gould, Orlando Dinardo and David W. Koren

Chapter 4

Nitrogen and Excess Sludge Management Lei Zhang, Deokjin Jahng, Rong Cui, Sunkeun Se and Jiyeon Hwang

Chapter 5

Sewage Sludge Treatment in the European Union Lucie Houdková, Jaroslav Boráň and Thomas Elsäßer

Chapter 6

Evaluation of in-situ Sludge Reduction Technologies for Wastewater Treatment Plants J.L. Campos, M. Figueroa, J.R. Vázquez-Padín, A. MosqueraCorral, E. Roca and R. Méndez

Chapter 7

Feasibility of Using a Mixture of Sewage Sludge and Incinerated Sewage Sludge as a Soil Amendment Silvana Irene Torri

Chapter 8

Potential of Sludge Treatment Xiaoyi Yang

Chapter 9

A Culture-Independent Novel Approach to Monitoring the Activity and Stability of Activated Sludge in Wastewater Treatment Hikaru Suenaga ,Takahiro Kanagawa and Kentaro Miyazaki

Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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229

Contents Chapter 10

Chapter 11

Chapter 12

Organic Contaminants in Sewage Sludge: Determination and Occurrence E. Pocurull, A. Nieto and R.M. Marcé Using ANFIS to Predict the Softening, Melting and Pouring Points of Sewage Sludge Ash During Formation of Slag Tzu-Yi Pai, Horng-Ming Chen, Tien-Ching Chang, Yao-Sheng Tsai, Hsiao-Hsing Chu and Chaio-Fuei Ouyang Sewage Sludge Treatment and Recycling Systems in Japan: Trends, Challenges and Future Perspectives Keishiro Hara

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Index

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273

289 297

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PREFACE Wastewater treatment plants usually generate millions of tons of sewage sludge every year. Sewage sludge results from the accumulation of solids from chemical coagulation, flocculation and sedimentation during wastewater treatment. Worldwide, sludge production is steadily increasing, driven by the increasing percentage of households connected to central treatment plants, the increasing tightening of pollution limits on the effluent discharged, as well as the availability of technologies capable of achieving higher efficiency of wastewater treatment. Sewage sludge contains undesirable hazardous substances such as trace elements, pesticides and endocrine disruptors, pathogens and other microbiological pollutants. Therefore, sludge has to be properly treated and disposed of to prevent environmental contamination and health risk. Sludge processing is intended to improve dewatering characteristics, eliminate disease-causing bacteria, reduce smell and decrease the quantity of organic solids. In this way, the end product can be treated further or disposed of with less handling problems and environmental consequences. This new important book gathers the latest research from around the globe on this issue. Chapter 1 - The most widespread process in use today for the biological treatment of municipal and industrial wastewaters is the activated sludge process (ASP). The ASP is an aerobic bioreactor process, in which micro-organisms are grown for the digestion and removal of soluble organic matter to low levels. It is flexible and reliable, capable of producing a high quality effluent which is low in suspended solids due to the tendency of the biomass to flocculate. The ASP consists of two liquid-stream unit operations: the biological reactor for pollutant digestion; and a secondary clarifier, in which solid flocs are separated (usually by gravity) from the effluent. The formation of stable biological flocs is essential for the successful operation of the process. Poorly flocculated sludge can also have an adverse effect on sludge dewatering. The thickened sludge that is produced as waste from the process is often dewatered to reduce its handling costs; a well-flocculating sludge translates into one that will dewater easily. Activated sludge flocculation is a very complex physical, chemical and biological process, in which many factors interact and have an influence. Since many of these influences are poorly understood, the characteristics of floc-formation remain difficult to predict and control. Yet an improved knowledge of the flocculation step is an essential requirement for optimal biological wastewater treatment. The living consortium of micro-organisms in activated sludge is dynamic, complex and unstable, making it extremely difficult to carry out controlled experiments during biological sludge studies. Fortunately, Sanin and Vesilind

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Richard E. Baily

(1996, 1999) developed a novel physical and chemical surrogate for activated sludge, which they termed synthetic sludge, to study sludge de-watering, settling and conditioning characteristics. It is made out of non-living components that resemble activated sludge components physically and chemically, if not biologically. The components of synthetic sludge include: polystyrene latex particles of bacterial size, to simulate individual bacteria; alginate, to simulate extracellular polymeric substances (EPS) such as polysaccharides; fibrous cellulose, to simulate the filamentous micro-organisms found in activated sludge; and calcium ions, as bridging cations for the EPS. All components are present at representative quantities. The early work of Sanin and Vesilind showed that it is possible to create a chemical sludge in this fashion, having close resemblance to biological sludge. Such a stable, physically and chemically well-defined system was used here as a surrogate for sludge during tests. The current article aims to present the results of an extensive and ongoing study to link the properties of synthetic and activated sludge, including floc formation, floc structure and stability, settling, dewatering and conditioning. Cations, biopolymer, filamentous organisms and shear all play important roles in the flocculation of both synthetic and activated sludge; calcium (and aluminium) plays a role in forming bridges between alginate or biopolymer adsorbed on the particles or bacteria. Calcium also has a major impact on settling and dewatering, while polymer conditioning has a major impact on the latter. Our work has shown that the stable and well-defined nature of synthetic sludge makes it very useful as a simple, non-biological and non-complex surrogate for studying the physical and chemical properties of biological activated sludge, including polymer conditioning. Nonetheless, some quantitative discrepancies are seen to arise in settling and sludge volume index, supernatant turbidity, sludge dewatering, and floc strength. For such studies, the inclusion of a filamentous material in synthetic sludge, such as cellulose, is recommended. Chapter 2 - Oilseed world production in 2007/2008 was 391.2 Mt with a contribution of 56.4 % for soybean and 7.0 % for sunflower, according to United States Department of Agriculture (USDA) data. During the same period, 128.0 Mt of oils from seeds were produced along the world, including 29.3 % of soybean oil and 7.8 % of sunflower oil. During processing, some millions of tons of sludge or gums are generated in water degumming step, to remove impurities and obtain oils without turbidity and stable with respect flavor and odor. Degumming sludge is a complex mixture comprising high water content, phospholipids (PLs), oil, and minor amounts of other constituents like phytoglycolipids, phytosterols, tocopherols, and fatty acids. PL fraction in degumming sludge principally includes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidic acid (PA). In this chapter, some traditional industrial processes to produce, purify, and fractionate lecithin, from degumming sludge are presented. These processes include drying and deoiling with acetone, a solvent in which PLs, glycolipids and related compounds are almost insoluble. Lecithin is used as emulsifiers, dispersing and release agent in food, pharmaceutical and cosmetic industries. Methods still not applied at industrial scale for extraction and partition of PLs using supercritical fluids, and processes for chemical and enzymatic modifications of lecithin are also reviewed in this chapter, with their advantages and disadvantages. Enriched lecithin in any component such us PC or PI, with distinctive surface-active properties can be obtained by lecithin fractionation with solvents. Special attention is dedicated in this review to novel structured PLs that can be obtained by enzymatic

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reactions to exchange fatty acids in natural PLs. These PLs are high-priced fine chemicals for membrane and lipoprotein investigation, cosmetic industries, and liposome technology to release food and medicament components. The extensive availability of different lipases and phospholipases, fatty acids and derivatives, including polyunsaturated fatty acids, to modify natural PLs and generate products with new physical and chemical properties, open a large investigation field at immediate future. Results of recent own investigation about recovery occluded oil in soybean degumming sludge, by water elimination and acetone extraction, and study on quality and stability indexes in recovered oils, in view to their re-insertion in productive process, are also shown in this review. Chapter 3 - Sulfate-reducing microorganisms (SRM) comprising of anaerobic bacteria and anaerobic archaea are an integral part of the global sulfur and carbon cycle for dissimilatory reduction of sulfate using sulfate as electron acceptor for the degradation of organic compounds with concomitant production of hydrogen sulfide. SRM are known to be ubiquitous in natural and engineering environments, including in the methanogenic and sulfidogenic sludge generated by municipal and industrial wastewater treatment facilities. Additionally, SRM are responsible for ca. 50% of the organic matter mineralization in wastewater treatment systems. SRM are detrimental to the safety, reliability and integrity of wastewater treatment facilities, and to the public and environmental health because hydrogen sulfide is highly corrosive, neurotoxic and malodorous. However, SRM have attracted significant industrial interest as potential biocatalysts for environmentally friendly remediation of acid mine and rock drainage, removal and reuse of sulfur compounds from waste effluents and off gases, recovery of heavy metals from wastewater and sludge, and biotransformation of petroleum- and hydrocarbon-containing sludge. Considerable efforts have been devoted for development of robust techniques to provide an early detection of SRM occurrence, to identify novel strains for bioremediation, and/or to evaluate their ecophysiological roles in the natural and engineering environments. This chapter provides an overview on the distribution and phylogenetic diversity of microorganisms associated with dissimilatory sulfate reduction, and the recent development of techniques used for the characterization of SRM in natural and industrial environments, including in the process of biological remediation of toxic wastewater and sludge. Chapter 4 - A large amount of excess sludge is generated from the activated sludge process as a result of biological removal of organic pollutants in the wastewater. Since the management of the excess sludge accounts for 25-65% of the total operational cost, the minimization of excess sludge production is now considered as one of the urgent issues that should be addressed. In order to reduce excess sludge production, growth yield could be lowered by introducing metabolic uncouplers into the aeration tank. One of the best studied metabolic uncouplers, 3,3',4',5-tetrachlorosalicylanilide (TCS), was found to reduce growth yield of activated sludge by over 60% at the concentration of 0.4 mg/L. The reduction of sludge production was confirmed in a laboratory-scale anoxic/oxic (A/O) process operated for 6 months. However, the TCS-fed continuous system failed to remove ammonia in the influent because of the inhibitory effect of TCS on nitrification. Net production of excess sludge can also be reduced by recirculating solubilized sludge into the main treatment stream. Solubilization of excess sludge was achieved by ozonation, sonication, and high-pressure homogenization. No matter which methods were used, net production of excess sludge was successfully reduced. In addition, it was shown that the recycle of solubilized sludge to the main process provided carbon source for denitrification but nitrogen was also introduced into

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Richard E. Baily

the system in the form of cellular proteins. Nitrogen mass balance analysis indicated that the carbon source contained in ozonated excess sludge was not sufficient to completely reduce the extra nitrogen released from sludge solubilization. Since absolutely no excess sludge process is practically impossible no matter which method is adopted, it was attempted to purify the cellular protein from the excess sludge. To release cellular proteins, excess sludge was disintegrated through ultra-sonication at alkaline pH. Subsequently proteins were precipitated at pH 3.3. At optimized conditions, the efficiency of protein recovery reached 80.5%. Nutrient compositions of recovered protein were comparable to the commercial protein feeds. Heavy metals were found to be removed during the protein recovery process, and aflatoxin B1, ochratoxin A and Salmonella D groups were not detected. It was also found that the crude protein recovered from excess sludge did not show any acute toxicity against rates. Since microbial cells were broken and intracellular proteins were removed, mass and dewaterbility of the residual sludge were reduced and improved, respectively. Anaerobic digestion of this deproteinized excess sludge showed a higher methane production rate and better biogas quality than that of untreated excess sludge. From these results, it was concluded that the combination of different processes can generate novel and efficient total solutions for excess sludge management. Chapter 5 - In first part of the chapter sludge treatment and management in the European Union is described. The European Council prepares legislation that is obligatory for all members of the Union. Council Directive 86/278/EEC of 12 June 1986 on the protection of the environment, and in particular of the soil, when sewage sludge is used in agriculture is the main regulation regarding sewage sludge treatment. Except for sludge application in agriculture, sludge is landfilled or incinerated. Council Directive 1999/31/EC of 26 April 1999 on the landfill of waste and Directive 2000/76/EC of 4 December 2000 on the incineration of waste also have to be observed. In the EU there are big differences in sludge treatment and management between individual countries. It is obvious from statistically recorded data that some countries prefer that sludge is used in agriculture; other countries keep landfilling major parts of produced sludge. Second part of the chapter is focused on a case study of several possibilities of sludge management for one big waste water treatment plant (WWTP). Designed capacity is 1.5 mil PE, wastewater flow is 5 m3/s on average. Reconstruction of the WWTP is planned for 2010. The reconstruction of mechanical and biological stage of wastewater treatment will cause increase of sludge production. Suitable technology for a new sludge management is being opted currently. In this chapter the mass and heat balances of few alternatives are shown and the results are described. The compared alternatives are as follows: anaerobic digestion of sludge, incineration of digested sludge and incineration of mixed raw sludge. Limiting conditions of the specific area, legislation and owner requirements are considered. The economical evaluation is based on the mass and heat balances. Chapter 6 - Excess sludge treatment and disposal accounts for between 50 and 60% of the operating costs of wastewater treatment plants (WWTPs). Nowadays the main alternative methods for sludge disposal in the EU are land application (agriculture and silviculture) (49%), landfill (40%) and incineration (11%). All these methods represent several disadvantages with regard to economical factors (need for dewatering, transport, or drying) and environmental factors (risk of heavy metal or pathogen discharges). Therefore, an alternative way to solve sludge-associated problems is to reduce sludge production at the WWTP rather than to change post-treatment of all the sludge produced.

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A state-of-the-art review of in-situ technologies for reducing sludge generated from both primary and biological treatments is provided in this work. The application of disintegration techniques seems to be very interesting due to the high reduction of excess sludge achieved and the possibility of energy recovery as biogas. However full-scale investigations have shown that disintegration technology is only efficient if the total sludge disposal costs are high, the limiting factor being the efficiency of disintegration units. Future research should lead to improved energy recovery by increasing the efficiency of the disintegration units and to recover phosphorus as a usable product. Chapter 7 - The accumulation of sewage sludge poses nowadays a growing environmental problem. Incineration is a feasible means of reducing sewage sludge‘s volume. Public acceptance of this technology is, however, hampered by concerns about potential adverse environmental impact, mainly due to non-volatile hazardous constituents that are concentrated in the ash. Application of incinerated sewage sludge ash to agricultural soils presents the opportunity of recovering nutrients considered essential for plant growth, reducing the need for commercial fertilizers. However, this practice can contribute to the pollution of agricultural soils by heavy metals. Combined use of sewage sludge and its incinerated ash may prove to be a beneficial means of disposal, improving soil quality and crop production. Very little attention has been dedicated to asses the potential of the application of this mixed waste. This Chapter evaluates the effects of a mixture of sewage sludge and its own incinerated ash on soil properties when used as a soil amendment. Three typical soils of the Pampas Region were used in order to predict the feasibility of using similar mixtures in large-scale degraded-land application. The application of the mixture of sewage sludge and its incinerated ash significantly increased soil organic carbon, pH and EC in the three amended soils compared to control. An increase in Lolium perenne L aerial biomass in amended soils compared with plants grown in unamended soils was observed. Cadmium and Pb concentrations were in all cases below detection limits in aerial part of L. perenne L. On the contrary, Cu and Zn concentration in the above ground tissue was significantly higher in the amended soils than control, indicating a high Cu and Zn availability. Nevertheless, no significant differences between Cu or Zn concentration in aerial biomass was observed between soils amended with the mixture of sewage sludge and its incinerated ash compared to soils amended with sewage sludge. Overall, this assay showed that the use of this mixed waste as a soil amendment may not pose a significant risk of soil, water or plants contamination. Therefore, the mixture of sewage sludge and incinerated sewage sludge may play a significant role as a soil amendment in land reclamation, especially if nonfood chain crops are grown. Chapter 8 - As the lack of fossil fuel is increasing, sludge management is not only to satisfy the disposal criteria but also to obtain energy and resource. For a better sewage sludge disposal and more efficient energy reclamation, a series of analysis and experiments have been performed to study the potential of anaerobic digestion. Anaerobic digestion techniques have traditionally been employed to reduce the volume and the weight of sludge and produce corresponding amounts of biogas. As most of the organics present in sewage sludge are enveloped by poor biodegradable cell walls and extracellular biopolymers, the rate-limiting step in sludge digestion is generally believed to be the hydrolysis of particulate organic matter to the soluble substance. Pretreatments including thermal, chemical, ultrasonic or mechanical disintegration have been confirmed to accelerate the solubilization (hydrolysis) of sludge and reduce the particle size. In addition, the majority

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of organic substance that enwrapped by microbial cell wall oxidizes into small molecule and shift into aqueous phase and thus enhances the biogas production by pretreatment. Wet air oxidation (WAO) and thermal treatment become potential alternatives in the pretreatment of sewage sludge. Therefore, it is necessary to investigate the mechanisms of WAO in order to achieve a better understanding of the reaction process and get a better reactor design. For this purpose kinetics studies have been performed. The two-stage first-order reaction kinetic model and the generalized kinetic model are applied to study the kinetics of the wet air oxidation process of industrial waste activated sludge. The MLVSS/MLSS rate is chosen as the model parameter instead of COD or TOC to shorten the error that results from sampling. The results showed that the generalized kinetic model is relatively available to predict the WAO process of waste sludge. The values of point-selectivity show that there is a strong presence of acetic acid and short chain organic substance in the WAO treatment process, which are experimentally confirmed by the chromatogram-mass spectrograph. Chapter 9 - Activated sludge systems are used worldwide for wastewater treatment. Their treatment capacity is dependent on the action of complex microbial communities, which include bacteria that are useful as well as harmful for the systems. The monitoring of the presence and activity of such bacteria is important as a means of directing a plant operation toward higher elimination rates and overall stability. Traditional methods using microscopes and agar plates suffer from severe limitations for the identification of the key microorganisms. During the past decade, various molecular approaches have been developed and used in a culture-independent manner. The most common molecular methods use 16S ribosomal RNA (16S rRNA) or its gene (16S rDNA) as a target molecule to analyze the specific groups of bacteria. Recently, author developed a novel method for the quantitative detection of 16S rRNA of a filamentous bacterium, Sphaerotilus natans, using catalytic DNA. This approach may be useful for early detection of bulking caused by filamentous bacteria in activated sludge systems. Other alternative targets for monitoring are functional genes involved in the removal of pollutants. We applied a metagenome approach to retrieve functional genes, extradiol dioxygenases, from activated sludge for the treatment of cokeplant wastewater. This gene may be used as a good indicator of plant performance. In this article, author focus on these two novel approaches, catalytic DNA and metagenome, which may be effective for assessing the activity and stability of the microbial community. Their fundamental principles, application results, and future prospects are described. Chapter 10 – This chapter focuses on the determination and occurrence of the main organic contaminants in sewage sludge. Analytical methods are usually based on chromatographic techniques, such as gas chromatography or liquid chromatography, in combination with an extraction technique, which usually is the main difference between all those methods. First, the sludge pre-treatment and the main analytical techniques used for the analysis are briefly discussed. Then, an extended overview of the fundamental features of the main extraction techniques, their main experimental parameters and applications to the extraction of organic contaminants from sewage sludge is included in this chapter. Finally, the occurrence of organic contaminants in sludge in recent years is reported. Chapter 11 - In this study, the compositions of sewage sludge ash (SSA) and the softening, melting and pouring points during formation of slag were determined. Then these measured data were used as input layer and output layer to train adaptive network-based fuzzy inference system (ANFIS). The results indicated that the softening points fell within the range of 1126.2 – 1239.2 centigrade. The melting points lay between 1155.2 and 1295.4

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Preface

xv

centigrade. The pouring points fell within the range of 1160.7 – 1310.2 centigrade. The values of mean absolute percentage error (MAPE) were 0.0 % when training ANFIS. When predicting, the MAPEs of softening point were 4.3 % - 16.3 %. Those of melting point fell in the range between 2.6 % and 7.3 %. Those of pouring point lay between 3.3 % and 7.6 %. According to calculation, ANFIS could predict the softening, melting and pouring points of SSA during formation of slag precisely. Chapter 12 - Given the increasing volume of sewage sludge and limited capacity of the final disposal sites, building a sustainable sludge management system with proper treatment and recycling options has been and will be crucial for municipalities in Japan. This chapter aims to overview sewage sludge treatment and recycling practices in Japan with a particular focus on recent trends for the sewage sludge management. Besides material productions using the treated sludge, sludge utilization as an alternative energy source has been encouraged lately as a promising option amid the increasing attention to carbon neutral energy. Some innovative approaches addressing such options, which have currently been developed and promoted at the municipality level, are introduced. The chapter then briefly describes a unique sludge treatment and recycling system adopted in Tokyo where a shortage of final disposal sites for sludge has been serious and, thus, sludge incineration and recycling have been essential for reducing the volume of treated sludge conveyed to the disposal sites. The recycling options in Tokyo since the late 90s had included the productions of brick, slag, aggregate and RDF (Refuse Derived Fuel). An environmental assessment of these recycling options in Tokyo indicated that the processes of materials production were costly as well as energy consuming. Recently, different practices, such as sludge utilization for cement production and sludge carbonization, draw attention as promising options in Tokyo and they are found to be desirable from the viewpoints of cost, energy consumption and associated environmental loads. These technologies and practices about sludge treatment, recycling, and disposal in Japan shall provide useful lessons for other Asian countries where proper sludge treatment, disposal and recycling strategies are becoming very important amid the rapid urbanization and increasing sewage sludge generation as a consequence.

Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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In: Sludge: Types, Treatment Processes and Disposal Editor: Richard E. Baily

ISBN: 978-1-60741-842-9 © 2009 Nova Science Publishers, Inc.

Chapter 1

SYNTHETIC SLUDGE AS A PHYSICAL AND CHEMICAL ANALOGUE OF REAL SLUDGE IN THE ACTIVATED SLUDGE PROCESS Nick Hankins1*, Nidal Hilal2 and Tan Phong Nguyen3 1

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Research Director, The Oxford Centre for Sustainable Water Engineering, Department of Engineering Science, The University of Oxford, Parks Road, Oxford OX1 3PJ, UK. email: [email protected] 2 Director of the Centre for Clean Water Technologies, School of Chemical and Environmental Engineering, The University of Nottingham, University Park, Nottingham NG7 2RD, UK. email: [email protected] 3 Faculty of Environment, University of Technology, 268 Ly Thuong Kiet Street, District 10, Ho Chi Minh City, Vietnam. email: [email protected]

ABSTRACT The most widespread process in use today for the biological treatment of municipal and industrial wastewaters is the activated sludge process (ASP). The ASP is an aerobic bioreactor process, in which micro-organisms are grown for the digestion and removal of soluble organic matter to low levels. It is flexible and reliable, capable of producing a high quality effluent which is low in suspended solids due to the tendency of the biomass to flocculate. The ASP consists of two liquid-stream unit operations: the biological reactor for pollutant digestion; and a secondary clarifier, in which solid flocs are separated (usually by gravity) from the effluent. The formation of stable biological flocs is essential for the successful operation of the process. Poorly flocculated sludge can also have an adverse effect on sludge dewatering. The thickened sludge that is produced as waste from the process is often dewatered to reduce its handling costs; a well-flocculating sludge translates into one that will dewater easily.

*

Corresponding author: Tel: +44(0)1865 273 027

Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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Nick Hankins, Nidal Hilal and Tan Phong Nguyen

Activated sludge flocculation is a very complex physical, chemical and biological process, in which many factors interact and have an influence. Since many of these influences are poorly understood, the characteristics of floc-formation remain difficult to predict and control. Yet an improved knowledge of the flocculation step is an essential requirement for optimal biological wastewater treatment. The living consortium of micro-organisms in activated sludge is dynamic, complex and unstable, making it extremely difficult to carry out controlled experiments during biological sludge studies. Fortunately, Sanin and Vesilind (1996, 1999) developed a novel physical and chemical surrogate for activated sludge, which they termed synthetic sludge, to study sludge de-watering, settling and conditioning characteristics. It is made out of non-living components that resemble activated sludge components physically and chemically, if not biologically. The components of synthetic sludge include: polystyrene latex particles of bacterial size, to simulate individual bacteria; alginate, to simulate extracellular polymeric substances (EPS) such as polysaccharides; fibrous cellulose, to simulate the filamentous micro-organisms found in activated sludge; and calcium ions, as bridging cations for the EPS. All components are present at representative quantities. The early work of Sanin and Vesilind showed that it is possible to create a chemical sludge in this fashion, having close resemblance to biological sludge. Such a stable, physically and chemically well-defined system was used here as a surrogate for sludge during tests. The current article aims to present the results of an extensive and ongoing study to link the properties of synthetic and activated sludge, including floc formation, floc structure and stability, settling, dewatering and conditioning. Cations, biopolymer, filamentous organisms and shear all play important roles in the flocculation of both synthetic and activated sludge; calcium (and aluminium) plays a role in forming bridges between alginate or biopolymer adsorbed on the particles or bacteria. Calcium also has a major impact on settling and dewatering, while polymer conditioning has a major impact on the latter. Our work has shown that the stable and well-defined nature of synthetic sludge makes it very useful as a simple, non-biological and non-complex surrogate for studying the physical and chemical properties of biological activated sludge, including polymer conditioning. Nonetheless, some quantitative discrepancies are seen to arise in settling and sludge volume index, supernatant turbidity, sludge dewatering, and floc strength. For such studies, the inclusion of a filamentous material in synthetic sludge, such as cellulose, is recommended.

1. INTRODUCTION Modern society generates vast quantities of waste-water by both domestic and industrial activity. The generation of this wastewater and its accumulation in the environment both pose hazards to human health and the environment. Wastewater contains numerous pathogenic microorganisms that can dwell in the human intestinal tract and lead to serious diseases, and it may contain toxic compounds which are potentially mutagenic or carcinogenic. It also contains nutrients which can stimulate the undesirable growth of aquatic plants and eutrophication. The decomposition of the organic matter it contains will lead to the production of malodorous gases. For all these reasons, the immediate collection of wastewater from its source of generation, followed by its effective treatment prior to reuse or re-dispersal, is necessary to protect public health and the environment (Figure 1).

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Figure 1. Schematic Diagram of a Wastewater Management Infrastructure.

With proper analysis and control of treatment conditions, the biodegradable components of wastewater can be treated biologically. But it is essential that the water resources engineer understands the characteristics of each process, to ensure that the correct conditions are created and controlled effectively. The most widespread process in use today for the biological treatment of municipal and industrial wastewater is the activated sludge process (ASP). The ASP is an aerobic, suspended growth process, in which micro-organisms are grown in a variety of bioreactor configurations for the digestion and removal of soluble organic matter to low levels. It is flexible and reliable, and capable of producing a high quality effluent which is low in suspended solids, due to the tendency of the biomass to flocculate. Activated sludge is a heterogeneous mixture of particles, micro-organisms, colloids, organic polymers and cations, whose composition depends on the origin of the sample and the date of sampling (Li and Ganczarczyk, 1990).

Bioflocculation The flocculated microbial aggregates, known as flocs, are the essential components of the system. Flocs typically vary in size from 10 to 1000 μm (Andreadakis, 1993). In the activated sludge process, the flocs remove both colloidal matter and soluble BOD (biochemical oxygen demand) by absorption and biodegradation, and their settling characteristics must also be such that the discharge standards of the final effluent are met to a high degree of consistency (Steiner et al, 1974). In general, the concept of the structure of activated sludge comprises three levels: bacteria, micro-colonies and flocs (Jorand et al, 1995; Snidaro et al, 1997). The first level of structure is made up of bacteria tightly bound together by a polymeric matrix to form the second level of structure called micro-colonies. The extracellular polymers arise from two

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origins; either from metabolism and cell lysis of microorganisms or from incoming wastewater (Urbain et al, 1993). These extracellular biopolymers or extracellular polymeric substances (EPS) are typically made up of proteins, polysaccharides, humic compounds, nucleic acids and lipids and contribute 15-20% by weight of mixed liquor suspended solids, MLSS (Urbain et al, 1993). Polymers and cations further link these micro-colonies to produce the third and final level of structure, activated sludge flocs. The flocculation of activated sludge is an active process, and depends on physical, chemical and biological factors. The basis of activated sludge floc formation lies in the ability of microorganisms to stick to each other and to non-biological particles. Microbial adhesion mechanisms have been studied widely, but are still not understood. It appears that the EPS form the bridges between microorganisms. At the approximately neutral pH values typical of activated sludge, these polymers carry net negative charges. It is thought that divalent cations such as Ca2+ and Mg2+ interact with negatively charged polymers to form bridges that allow the cells to adhere to each other. When built up by biopolymer bridging of relatively spherical microorganisms, the flocs themselves will be roughly spherical in shape. To form the irregularly shaped flocs often seen in activated sludge, other ingredients—filamentous organisms—are required (Sezgin et al, 1978). The filamentous organisms provide networks or ―backbones‖ for the flocs. The networks direct floc growth into shapes other than spherical, and allow the flocs to grow larger (Sezgin et al, 1978). The schematic structure of activated sludge is shown in Figure 2. Since floc strength depends on the integrity of the biopolymer bridging, it is possible for strong and weak flocs to exist both with and without filamentous organisms. Due to the complex nature of the flocs, they display a wide variation in physical, chemical and biological properties (Jin et al, 2003). Many major operating problems in the ASP, such as those which occur in solid-liquid separation, can also be attributed to the properties of the flocs. An understanding of the flocs and their formation is therefore critical to the optimum operation of the activated sludge process.

Figure 2. Schematic Structure of Activated Sludge (not to scale). Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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Review of Sludge Floc Formation DLVO theory DLVO theory, named after its developers Derjaguin, Landau, Verwey, and Overbeek, is a classical theory of colloidal stability, in which the surface charge of the particles and the counter-ion charge in the adjacent solution form an electrical double layer surrounding the particle. The first layer of surface charge, including adsorbed ions, is often referred to as the Stern layer (Adamson, 1980), and the second layer as the diffuse layer. The concentration of counter-ions in the counter-ion layer decreases with distance from the particle surface, until the concentration of ions equals that of the bulk solution. This results in an electrical potential which develops around the particle. The double layer results in osmotic repulsion of adjacent particles, and inhibits aggregation. As the ionic strength increases, the size of the double layer decreases, which in turn decreases the repulsion between the particles and allows short-range Van der Waals attractive forces to promote aggregation. However, Sobeck and Higgins (2002) state that results from monovalent cation addition in their reactor study conflict with the DLVO theory. According to DLVO, the addition of any ions such as Na+ and Cl- will increase the ionic strength and compress the double layer, thereby improving bioflocculation and the settling and dewatering properties. On the contrary, as results from their trial indicated, increasing the concentration of sodium in the system results in a substantial deterioration in settling and dewatering properties, as well as floc strength.

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Divalent cation bridging (DCB) theory The role of divalent cations has been demonstrated in experiments that have examined floc formation during the growth of monocultures, finding that calcium and magnesium were important to the bioflocculation process (Tezuka, 1969). A depiction of the divalent cation bridging (DCB) model is shown in Figure 3. According to the DCB theory, divalent cations bridge negatively charged functional groups within the EPS, and this bridging helps to aggregate and stabilize the matrix of biopolymer and microbes and therefore promote bioflocculation. The DCB theory can be used to explain the results of sodium trials. Previous research has shown that high concentrations of sodium ions were able to displace divalent cations from within the floc, by ion-exchange type reactions (Tezuka, 1969; Higgins and Novak, 1997). The replacement of divalent cations from within the floc by monovalent cations results in a deterioration in floc properties, due to the lack of bridging by the now monovalent cations.

The role of cations Ions such as calcium and magnesium are commonly found in natural water systems, while iron and aluminium may be prevalent if these are used as conditioners. Such metal ions, common to water and wastewater systems, are believed to be predominantly complexed by the extracellular polymeric materials. Even though calcium and magnesium ions have similar properties, findings indicate that their interactions with polymers are not similar. The addition of magnesium ions to sludge had been found to have no effect on the bound water content,

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whereas the addition of calcium ions was observed to reduce the bound water content remarkably (Forster and Lewin, 1972). A column of precipitated polymer was packed with sand, and solutions containing calcium and magnesium ions were passed through it. The results showed that there was a remarkable removal of calcium ions by the column; however, magnesium ions were not removed (Forster and Lewin, 1972). Results like this clearly indicate that calcium ions are more preferentially bound to sludge polymers than magnesium ions. The findings of a more recent study have also demonstrated the deflocculation of activated sludge when the calcium ions are extracted from sludge flocs by several different methods (Bruus et al, 1992). This causes the sludge turbidity to increase, and filterability to decrease. The same research work proposed that sludge polymers could be alginate, or another polymer with properties very close to those of alginates. In later work, synthetic sludge flocs were formed from stable particles by adding calcium ions and alginate (Sanin and Vesiland, 1996), further suggesting that one of the mechanisms of bioflocculation could be the interaction of alginate and calcium ions. Cations have been shown to have a significant effect on the bulk properties of activated sludge. Novak and co-workers (Higgins and Novak, 1997b,c; Novak et al, 1998) have shown that, for both lab-scale and full-scale wastewater treatment systems, sludge settling and dewatering properties could be improved by the addition of cations to the influent wastewater. In each case, settling properties were improved with the addition of calcium or magnesium. Batch addition of cations to activated sludge also showed improvement in the sludge settling characteristics (Higgins and Novak, 1997b).

Figure 3. Depiction of Divalent Cation Bridging within Floc Matrix (not to scale).

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Activated Sludge Process The ASP consists of two liquid-stream unit operations (Figure 4): a biological reactor for pollutant digestion; and a secondary clarifier, in which solid flocs are separated (usually by gravity, and possibly flocculant aids) from the effluent. In many cases, the efficiency of the clarifier is the limiting factor in producing a high quality effluent, and it is often regarded as the bottle-neck of the process. The settling properties of the sludge are determined primarily by the conditions prevalent in the aeration basin. If this settling process is not efficient, the effluent becomes turbid, and a large amount of the suspended solids are discharged into the receiving waters, leading to an unwanted increase in chemical oxygen demand (COD) and other nutrients (phosphorus and nitrogen).

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Figure 4. Schematic Flow Diagram of Complete-Mix Activated Sludge Reactor.

Therefore, to ensure that existing standards on effluent quality are met in full, an efficient flocculation process must prevail. Changes in the composition of activated sludge will lead to changes in the nature of the flocs, which can result in the poor formation of biological flocs. In short, the formation of stable biological flocs is essential for the successful operation of the process. Poorly flocculated sludge can also have an adverse effect on sludge flow and dewatering. The thickened sludge that is produced as waste from the process is often dewatered to reduce its handling costs; a well-flocculating sludge translates into one that will dewater easily.

Synthetic Sludge Activated sludge flocculation is a very complex physical, chemical and biological process, in which many factors interact and have an influence. Since many of these influences are poorly understood, the characteristics of floc-formation remain difficult to predict and control. Yet an improved knowledge of the flocculation step is an essential requirement for optimal biological wastewater treatment. The living consortium of micro-organisms in activated sludge is dynamic, complex and unstable, making it extremely difficult to carry out controlled experiments during biological sludge studies. Fortunately, Sanin and Vesilind (1996, 1999) developed a novel physical and chemical surrogate for activated sludge, which they termed synthetic sludge, to study sludge dewatering, settling and conditioning

Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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Nick Hankins, Nidal Hilal and Tan Phong Nguyen

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characteristics. It is made out of non-living components that resemble activated sludge components physically and chemically, if not biologically. The components of synthetic sludge include: polystyrene latex particles of bacterial size, to simulate individual bacteria; alginate, to simulate extracellular polymeric substances (EPS) such as polysaccharides; fibrous cellulose, to simulate the filamentous micro-organisms found in activated sludge; and calcium ions, as bridging cations for the EPS. All components are present at representative quantities. The early work of Sanin and Vesilind (1996) showed that it is possible to create a chemical sludge in this fashion, having close resemblance to biological sludge, by using bacteria-like particles, polysaccharides, and cations common to activated sludge at quantities typical of those in activated sludge. Such a stable, physically and chemically well-defined system was used as a surrogate for sludge during tests. This article presents the results of an ongoing study to link the properties of synthetic and activated sludge (Figure 5), including floc formation, floc structure and stability, settling, dewatering and conditioning. Our work has shown that the physical and chemical properties of synthetic sludge match those of a biological sludge reasonably well. For comparison with live activated sludge, a five-litre, bench-scale, continuous-flow reactor was used as an ASP (Figure 6). The reactor consisted of a complete mixing zone and a settling zone, separated by a slanted baffle. An aeration stone provided air and mixing to the system. Wastewater and activated sludge for mixed-liquor feed were obtained from Stoke Bardolph sewage wastewater treatment plant in Nottingham, UK (Figures 7 and 8). It is the largest such works in the East Midlands region, serving half a million people and 200,000 ‗industry equivalents‘; on average, it handles 170 million litres of sewage per day. It takes 16 hours to completely treat the liquid phase, before returning it as a high quality final effluent to the River Trent.

(a)

(b)

Figure 5. Samples of (a) Synthetic Sludge (b) Activated Sludge.

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Figure 6. Laboratory Scale Activated Sludge Reactor.

Figure 7. Stoke Bardolph Wastewater Treatment Plant in Nottingham, UK.

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Figure 8. Post-treatment Discharge to the River Trent.

2. MATERIALS AND METHODS

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Polystyrene Latex Particles The concentrated dispersion of sulphate polystyrene latex particles was 5% solid by weight, with 1 μm mean particle diameter to simulate individual bacteria. The coefficient of variation of particle diameters was usually less than 5%. The procedure for preparing the sulphate latex particles followed the guidelines reported elsewhere (Goodwin et al, 1973). About 20% of the surface area of particles were covered with sulphate groups to give them the necessary stability and negative surface charge. The zeta potential was measured as −14mV. The stock solution was diluted to 0.05% by weight with distilled, de-ionized water, forming a colloidal dispersion which matches the design particle concentration with that of bacteria in activated sludge.

Polysaccharide Alginate (low viscosity, sodium form) from brown algae was supplied by Sigma Chemical Company. Polysaccharide in the supernatant was measured using the method of Dubois et al (1956).

Fibrous Cellulose Fibrous cellulose was supplied by Sigma Chemical Company to simulate filamentous microorganisms in activated sludge. A medium fibrous cellulose having a mean size of 50– 350 μm was chosen for the experiments.

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Preparation of Synthetic Sludge Samples with a 0.05% particle concentration and an added alginate concentration of 100 mg/L were rotated horizontally at 12 rpm in an incubator at 25 ºC for 12 hours. During the incubation period, alginate was adsorbed onto the particles to simulate EPS. Since calcium ions are known to act as sludge conditioning agents, Ca(II) was added to samples in varying concentrations when the incubation period was completed, in order to monitor flocculation dynamics. In a similar way, calcium ions were also added to real sludge samples Numerous experiments were conducted to determine the minimum, or slight excess, concentrations of alginate, calcium, and cellulose required for floc formation in synthetic sludge. Alginate concentration was varied between 0 and 125 mg/L. The concentration of alginate required for floc formation was observed to be 100 mg/L. Calcium concentration was varied from 0 to 25mM Ca(II). Tiny flocs were formed after reaching 15mM Ca(II) concentration in synthetic sludge; larger flocs were observed at 20mM Ca(II). There are many chemical constituents which affect the flocculation process. To overcome the complexity caused by these effects, the elimination method has been applied in this research by fixing some parameters, such as polystyrene latex particle concentration, sodium chloride concentration and shear rate. A standard sludge was therefore defined as 20mM Ca(II), 100 mg/L alginate, and 0.05% latex particles. The standard sludge was used as a reference point to compare the changes that occur in the flocculation dynamics and final properties of synthetic sludge upon changing the chemical composition in terms of calcium, alginate, cellulose concentration, and pH in these experiments. The target pH was adjusted by adding predetermined amounts of NaOH or HCl to the suspension.

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Preparation of Activated Sludge - Laboratory System Setup and Operation A semi-continuous flow bench-scale reactor of five litre capacity was used to simulate the activated sludge process. The reactor configuration is shown in Figure 9. The reactor consisted of a complete mixing zone and a settling zone, separated by a slanted baffle. An aeration stone provided air and mixing to the system.

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Preparation of Activated Sludge - Steady-state Determination Wastewater and activated sludge were obtained from Stoke Bardolph municipal wastewater treatment plant in Nottingham, UK. After collection, the samples were returned to the laboratory (within 1 hour) and stored at 40C. All the samples were kept for a maximum of five days. Bactopeptone, a microbiological enzymatic digest of protein for use in culture media, was chosen as the food source for seeding to the reactor at a concentration of 300 mg/L. The concentration of several cations and nutrients in the bactopeptone seed are given in Table 1 (Nguyen et al, 2007b). The influent pH was consistently near 7 for all seed conditions. The hydraulic retention time (HRT) was 0.5 days, and sludge age was maintained at 10 days. Table 1. Cation and Nutrient Concentration in Bactopeptone Seed Used in Laboratory Activated Sludge Reactor.

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Constituent Na+ NH4+ K+ Mg2+ Ca2+ PO43NO3TKN COD

mg/L 20.9 0.5 2.1 3.8 13.7 2.3 15.9 46.5 300

mM/L 0.45 0.0015 0.0025 0.16 0.34 0.024 0.26 9.38

The activated sludge in the reactor was first fed with mixed liquor from Stoke Bardolph. The activated sludge reactor was operated until steady state was achieved, typically after 20 days of operation. Measured treatment efficiency parameters were plotted as a function of time to determine the steady state period. The reactor was considered to be at steady-state when the variability in treatment efficiency was less than 20 % between sampling periods. The steady-state values of each parameter were calculated as the average of values during the steady-state period. The sampling results of activated sludge from the reactor at steady-state are given in Table 2 (Nguyen et al, 2007b). Table 2. Sampling Results from Activated Sludge Reactor at Steady State. Determinant COD BOD5 Nitrate Phosphate MLSS Ca2+ Mg2+ Na+ K+

mg/L 220 154 0.1 9.0 1148 20.8 16.32 81.88 5.46

mM/L 3.44 0.0016 0.095 0.52 0.68 3.56 0.14

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180

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Examples of steady state determination, as a function of time for the reactor, are plotted in Figures 10 and 11 (Nguyen et al, 2007b).

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35 30 25 20 15 10 5 0 1

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Figure 10. COD and BOD of the Effluent from the Activated Sludge Reactor Seeded with Bactopeptone.

SS Turbidity

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Figure 11: Suspended Solids and Turbidity of the Effluent from the Activated Sludge Reactor Seeded with Bactopeptone

Settleability and Turbidity Settling was measured in 100mL graduated cylinders. The height of the interface was recorded after 60 minutes of settling. The small size of the cylinder was thought to produce

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an unwanted wall effect, but the cost of producing larger quantities of various types of synthetic sludge would have been prohibitive. The low solids concentration would also minimize the effect of the small cylinder diameter. The turbidity of the supernatants was measured using a Hach 2100AN turbidimeter after 60 min of settling.

Settling and Dewatering Properties Total suspended solids (TSS) were analyzed using method 2540D in Standard Methods (APHA, 1998). The settling properties of biological suspensions were characterized by sludge volume index (SVI), as described by method 2710D in Standard Methods (APHA, 1998).

Polymer Conditioning Three cationic polymers, polydiallyldimethyl ammonium chloride (PDADMAC), Clarifloc and Stockhausen, were used for the conditioning of thickened sludges from the reactor. Polymer of 20 wt % in water stock solution was diluted to the final design concentration, by mixing the concentrated polymer with distilled water. During conditioning and dewatering, the polymer was added to a 100 mL sludge sample and mixed for 30s in a beaker. The mixing speed was 250 rpm (200 s-1). After mixing, the capillary suction time (CST) was measured, and the optimum dosage was considered as the dose which resulted in the minimum CST.

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Floc Size, Strength and Structure A Malvern Mastersizer-S instrument was used to measure the floc size and size distribution. This instrument is a light-scattering instrument that operates on the principle of Fraunhofer diffraction. Scattered light from the diluted sample is detected on the custom designed detector. The size distribution is based on the volume, and the average size is quoted as the mass mean, based on volume equivalent diameter. Floc strength was analyzed by measuring CSTs following every 30s interval of stirring, using a Triton-WRC (Essex, England) stirrer/timer. The floc structure of sludge was examined through a Scanning Electron Microscope (SEM).

Filamentous Organism Quantification Microscopic observations of the biological suspensions were performed periodically to assess the culture characteristics, and also to quantify the filamentous organism content. Filamentous organism content was quantified using the method of Jenkins et al. (2004), in which the number of filamentous organisms was rated on a scale of 0-6, where 0 corresponds to no filamentous organisms present and 6 corresponds to excessive growth of filamentous organisms. All data reported in this study represent studies with a filamentous count less than or equal to 2.

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Calcium Analysis Calcium used in this investigation was added as chloride salt. Analysis of the cation Ca2+ was carried out by a Flame Atomic Absorption Spectrometer, using method 3111 in Standard Methods (APHA, 1998).

Monitoring the Dynamics of Flocculation

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A simple but sensitive optical technique, the Photometric Dispersion Analyser (PDA 2000, Rank Brothers, Cambridge) is a useful tool in this work for monitoring the state of aggregation of colloidal suspensions rapidly and non-invasively. Light scattering in a flowthrough detector is used to monitor the dynamics of coagulation - either aggregation (flocculation) or disaggregation (particle break-up and dispersion) (Gregory and Nelson, 1984). It is applicable over a wide range of suspension concentrations and particle sizes (Figure 12). Valuable information is provided about the dynamics of sludge flocculation.

Figure 12. Experimental Set Up for Monitoring the Dynamics of Flocculation by PDA 2000.

The output value of the PDA 2000 can accurately reflect the state of formation of flocs. A 1 litre sludge latex particle suspension was used in the flocculation dynamic experiments. When a particular chemical was added to the suspension, rapid mixing was performed with a stirrer in a batch reactor at a speed of 250 rpm (200 s−1) for 30 seconds, in order to provide blending of the chemical with synthetic sludge, and was followed by slow mixing at a speed of 100 rpm (50 s−1) for 100 seconds to promote flocculation. The flowing suspension is illuminated by a narrow beam of light perpendicular to the direction of flow. The light source is a high intensity light-emitting diode and transmitted light is continuously monitored by a sensitive photodiode. The output from the photodiode is

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converted to a voltage, which consists of a large dc component, together with a small, fluctuating ac component. The dc component is simply a measure of the average transmitted light intensity and is dependent on the turbidity of the suspension. The ac component arises from random variations in the number of particles in the sample. Because the suspension flows through the cell, the actual sample in the light beam is continually being renewed and local variations in particle number concentration give fluctuations in the transmitted light intensity. These fluctuations cease when the flow is stopped. The root mean square (RMS) value of the fluctuating (ac) signal is related to the average number concentration and size of the suspended particle. For uniform suspensions, estimates of particle size and number concentration can be made, but the main use of the PDA 2000 is in the monitoring of flocculation and dispersion processes. The RMS value of the fluctuating signal increases when aggregation of particles occurs. Measurable changes in the RMS value occur long before any visible signs of aggregation are apparent. Conversely, when aggregates are disrupted, the RMS value decreases, reaching a minimum when disaggregation (or dispersion) is complete. The term ―flocculation index‖ (FI) present in this paper refers directly to the state of flocculation.

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3. EFFECT OF CALCIUM ON FLOCCULATION DYNAMICS During experiments, an appropriate concentration of calcium ions were added to 500 ml batch suspensions of both synthetic and activated sludges, followed by rapid mixing with a stirrer at a speed of 250 rpm (200 s-1) for 30 seconds. This was then followed by slow mixing at a speed of 100 rpm (50 s-1) for 100 seconds; the gentle shear promotes flocculation by bringing separate particles into contact. There was no significant change in the pH of the sample after calcium addition. The results for both systems are shown in Figures 13 and 14 as flocculation index (related to aggregation state) versus time for different calcium ion concentrations (Nguyen et al, 2007b). For synthetic sludge (Figure 13), the results show that no significant floc formation occurs without calcium ion addition, and begins when Ca2+ concentration reaches 15 mM, dramatically increasing for 20 mM Ca2+ and above. For activated sludge (Figure 14), 1 litre samples were withdrawn from the bench-scale reactor at the steady state. The concentration of total suspended solids (TSS) was 1g/L, similar to that in synthetic sludge, and remained constant for all experiments. The results show that some floc formation started to occur without the addition of calcium ions. This phenomenon results from the natural characteristics of activated sludge collected from a wastewater treatment plant; at the beginning, a certain amount of calcium ions already exists in the sample. The floc formation was readily apparent for 10 mM Ca, and above (Figure 14). The flocculation index (FI) dramatically increased at 20 mM Ca ions. For each sludge type, larger flocs are observed visually above 20 mM Ca and start to settle to the bottom of the reactor during slow mixing. It is also evident from Figures 13 and 14 that aggregation increases rapidly until the flocs reach a steady state. Increasing calcium concentration causes the rate of aggregation to increase towards a limiting value. The flocculation dynamic behaviour of synthetic sludge is qualitatively similar to that of activated sludge, supporting the idea that it is useful as a less-complex analogue.

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8

Flocculation Index

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Figure 13. Synthetic Sludge Flocculation Dynamics via PDA with Varying Concentrations of Calcium.

8

Flocculation Index

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7 6

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Tim e(s)

Figure 14. Activated Sludge Flocculation Dynamics via PDA with Varying Concentrations of Calcium.

4. EFFECT OF CALCIUM ION ON FLOC SIZE DISTRIBUTION Sludge flocs are fragile, heterogeneous and have very broad size distributions, so that absolute size measurements are a challenging task. A Malvern Mastersizer-S instrument was used to measure the volume-based floc size distribution by light scattering from a diluted sample.

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In both cases (Figures 15 and 16), the final floc size increases with increase in calcium concentration, and the size distribution shifts to the right (Nguyen et al, 2007a). These and the earlier results are in agreement with the work of Biggs et al (2001), who also found that the rate of floc growth increases with increases in calcium concentration until approaching a constant value at the higher concentrations. At such concentrations, it would seem that saturation of the floc surface has occurred, and the rate of change of floc size is independent of calcium concentration. This supports the postulate that cations are directly involved in flocculation, most likely through cationic bridging. Figure 17 suggests further that the mean floc size of synthetic sludge increases linearly with calcium concentration.

100

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40

Ca 20mM

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Ca 25mM

20 10 0

10

1000 Log Size (m icrons)

100 90 Cumulative Volume (%)

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Figure 15. Change in Floc Size of Synthetic Sludge with Increasing Concentration of Calcium-Log Size.

80 70

Ca 5mM

60

Ca 10mM

50

Ca 15mM

40

Ca 20mM

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Ca 25mM

20 10 0 10

1000 Log Size (m icrons)

Figure 16. Change in Floc Size of Activated Sludge with Increasing Concentration of Calcium – Log Size Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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Figure 17. Change in Floc Size of Synthetic Sludge with Increasing Concentration of Calcium – Mean Floc Size.

Table 3. Calcium Ion Concentration Change in Solution During Experiments.

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Calcium Concentration Added (mM/L) 0 5 10 15 20 25

Ca2+ in Samples after Flocculation (mM/L) 0 4.89 9.85 14.58 19.40 24.03

Change in Ca2+ Concentration (mM/L) 0.0 -0.11 ± 0.02 -0.15 ± 0.06 -0.42 ± 0.05 -0.60 ± 0.12 -0.97 ± 0.10

The uptake of calcium was measured as the difference in calcium concentration before and after flocculation during batch tests with synthetic sludge, and is presented in Table 3 (Nguyen et al, 2007a). The concentration of calcium ions in the solution after flocculation was generally less than the initial concentration for each experiment. This indicates directly that an uptake of calcium ions was occurring during the flocculation process, again supporting the hypothesis that calcium ions play a role in floc formation.

5. EFFECT OF ALGINATE AND FIBROUS CELLULOSE ON FLOCCULATION DYNAMICS Studies on the effect of the addition of alginate on the flocculation dynamics of synthetic sludge demonstrated that the slope of the flocculation index curve and the rate of aggregation increased dramatically with alginate concentration (Nguyen et al, 2007a) in the presence of calcium ions. Again, this is consistent with a cationic bridging process: the calcium plays a role in the attachment of alginates to surfaces, to alginate bridging, and to flocculation of the system.

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It has been observed that the presence of filamentous organisms has an effect on the structure of the flocs (Sezgin et al, 1978). It is probable that the network of filamentous organisms provides a ―backbone‖ for the build up of the floc, which is subsequently formed with the additional assistance of various polymer bridges between primary particles and smaller flocs. The slope of the FI curve was observed to increase dramatically upon addition of cellulose, and the flocs became larger (Nguyen et al, 2007a).

6. RELATIONSHIP BETWEEN CATIONS AND POLYSACCHARIDE ON FLOCCULATION BEHAVIOUR Synthetic Sludge Polysaccharide (alginate in the case of synthetic sludge) and cations in the form of calcium, iron as Fe3+ and aluminium as Al3+ were added to the samples in each experiment. The calcium concentration of the samples was varied between 0 and 20 mM Ca2+ with fast feeding for 10 minutes. Again, no floc formation occured without calcium addition. The mass of polysaccharide in the final supernatant (following settling) is plotted as a function of feed calcium concentration in Figure 18 (Nguyen et al, 2008 a,b). At lower concentrations of added calcium, the concentration of polysaccharide in the final supernatant was higher, and the latter decreased at higher calcium concentrations.

Polysaccharide(mg/L) in the supernatant

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35.00 30.00 25.00 20.00 15.00 10.00 5.00 0.00 0

1.25(F)

2.5(F)

3.75(F)

5(F)

10(F)

15(F)

20(F)

Ca(m M)

Figure 18. The Effect of Calcium Ions on Polysaccharide Concentration in the Final Supernatant of Synthetic Sludge.

The concentration of calcium ions in the solution after flocculation was generally less than the initial feed concentration, confirming that an uptake of calcium ions occurs during the flocculation process, as suggested before in Table 3. These results are in agreement with the work of Higgins and Novak (1997a). They found that there was a relationship between

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exocellular biopolymer concentration and feed cation concentration in laboratory scale activated sludge reactors, with bactopeptone as a feed. An increase in feed divalent cation concentration was associated with a decrease in exocellular biopolymer concentration in the supernatant. Tests in the presence of low added concentrations of iron (1.2 mg/L) and aluminium ions (0.25 mg/L) indicate that trivalent ions can also play a role in the flocculation.

Activated Sludge Two separate sets of six bench reactor experiments were conducted to investigate the effect of cation ions on the flocculation behaviour of activated sludge. The first four reactors were slug fed (S) slowly for 5 hours in semi-continuous mode, and the last 2 reactors were fast fed (F) for 10 minutes in batch mode. Ca2+ and Al3+ ions in a bactopeptone feed were added to the reactors, which were operated until steady-state conditions were obtained. The concentration of calcium ions was increased in both slug and fast feeding. In the first set, Al3+ was fed at 0.25 mg/L, and in the second set at 0.5 mg/L. Polysaccharide concentration in the final supernatant is plotted versus calcium ion concentration in the feed, as shown in Figure 19 (Nguyen et al, 2008 a,b).

Polysaccharide (mg/L) in the supernatant

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12 10 8 6 4 2 0 0.25(S)

1(S)

2.5(S)

5(S)

.25(F)

2.5(F)

Ca (m M)

Figure 19. The Effect of Ca2+ and Al3+ ions on Polysaccharide Concentration in the Supernatant of Activated Sludge (Al3+ = 0.25 mg/l).

In Figure 19, it can again be seen that at lower added calcium concentrations the polysaccharide concentration in the supernatant is higher, while the latter decreases at higher added calcium concentrations. Again, this suggests that an increase in calcium ions results in an increase in bound polysaccharide. Note also that the polysaccharide concentration in the supernatant was higher with slow, slug feeding than with fast, batch feeding at the same concentrations of calcium feeding (0.25mM and 2.5mM). Higgins and Novak (1997a) suggested that when cations are slow fed, they are better able to incorporate within the microbe-biopolymer network into flocs,

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resulting in a denser floc that is more resistant to shear. In contrast, during fast feeding, the cations may not become completely enmeshed and bound. Indeed, floc density changes little during fast feed tests, while it increases over time in the slow feed tests. During fast feed, cations are thus freed up to bridge and precipitate free polysaccharide, leading to a lower concentration in the supernatant when compared to slow feed. The interactions among microorganisms, polysaccharide, and cations are thus very important for flocculation, and will depend on the feeding regimes. Tests at the higher Al3+ concentration show polysaccharide in the supernatant decreases as Al3+ concentration increases, and Al3+ has a positive effect on the flocculating capability of activated sludge. When sludge is deficient in Al3+, many organic components would remain unflocculated and washed out of the system (Keiding and Nielsen, 1997). However, it is not yet clear how Al3+ coagulates biopolymer in activated sludge flocs, and this area is worthy of further study.

Results for the settling of synthetic and activated sludge are illustrated in Figure 20 (Nguyen et al, 2007b). In each case, the flocs remain dispersed in a turbid environment below 10mM Ca2+, while settling improves dramatically above this concentration, in agreement with Figures 13 and 14. Note that the settleability of activated sludge is then somewhat greater; this is thought to be due to the lack of a network of filamentous micro-organisms in the case of synthetic sludge, which provides a backbone for floc building with help from polymer bridging. The improved settling when filamentous cellulose is added confirms this. Earlier in this paper, it was observed that cellulose improves the flocculation dynamics of synthetic sludge. Such a finding agrees with the view of Örmeci and Vesilind (2000), in that the addition of cellulose fibres to simulate the filamentous micro-organisms found in activated sludge also ensured good settling and compaction of synthetic sludge.

100 Final interface height (% of original)

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7. SETTLEABILITY AND TURBIDITY

90 80 70

Synthetic sludge

60 Synthetic sludge w ith cellulose

50 40

Activated sludge

30 20 10 0 0

5

10

15

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Figure 20. Settleability of Synthetic and Activated Sludge. Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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800 700 600

SVI (mL/g)

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Synthetic sludge w ith cellulose

300

Activated sludge

200 100 0 5

10

15

20

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Figure 21. Synthetic and Activated Sludge Settling Increases with Increasing Concentration of Calcium.

In Figure 21, a similar relationship may be seen between sludge volume index (SVI, related to settleability) and added calcium concentration (Nguyen et al, 2008c). The settleability of both types of sludge improved with added calcium, though in this case the improvement is much more marked in the case of synthetic sludge. A marked decrease in supernatant turbidity above 15mM calcium is also seen for synthetic sludge, while the activated sludge shows a less marked decrease. As before, agreement in turbidity change is improved by cellulose addition to the synthetic sludge samples. The work of Jenkins et al (2004) concluded that when a filamentous, bulking activated sludge settles, it produces a very clear, low turbidity supernatant, since the filamentous organism network filters out the small particles that cause turbidity. Sanin and Vesilind (2000) demonstrated that the removal of calcium ions from the sludge floc matrix causes the sludge flocs to disintegrate, as indicated by a decrease in filterability and particle size. These results once again support the divalent cation bridging theory, improving floc formation, sludge settleability, and decreasing turbidity of the supernatant. In real activated sludge, it is apparent that filamentous material plays a significant role, and this should be borne in mind during the monitoring and prediction of activated sludge settling properties.

8. SLUDGE DEWATERING The effect of calcium ions on the dewatering of synthetic and activated sludge is shown in Figure 22 (Nguyen et al, 2007b; Nguyen et al, 2008c). In the absence of calcium, no floc formation occurred in synthetic sludge, and all the particles remained dispersed, while a nonfilamentous bulking occured for the activated sludge. The dewatering tendency was measured by capillary suction time (CST), and followed a trend similar to that of the sludge volume index (SVI) seen earlier. A relatively poor dewatering occurred at the lowest calcium

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Nick Hankins, Nidal Hilal and Tan Phong Nguyen

concentrations, and improved as the calcium concentration increased. Most of the improvement in SVI and CST occurred after the first incremental addition of Ca(II) ion, with a modest improvement beyond this level.

60

CST(s)

50 40

Synthetic sludge

30

Synthetic sludge w ith cellulose Activated sludge

20 10 0 5

10

15

20

25

Ca (m M)

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Figure 22. Sludge Dewatering of Synthetic and Activated Sludge.

Higgins and Novak (1997a) demonstrated that the cation content of a wastewater has a major impact on the settling and dewatering characteristics of an activated sludge. In our work, these characteristics show a qualitatively similar response to calcium addition in synthetic and activated sludge. Once again, the existence of filamentous organisms in activated sludge explains the latter‘s superior settling and dewatering ability. In Figure 22, filamentous cellulose causes an improvement in the settling and dewatering of synthetic sludge. Filamentous organisms also cause an improvement in the compaction and settling of synthetic sludge, depending on the organism involved (Jenkins et al, 2004).

9. SLUDGE CONDITIONING Dewatering can be improved with the addition of a sludge conditioning agent. Thickened sludge samples with 1g/L TSS were conditioned with varying amounts of the cationic polymer polydiallyldimethyl ammonium chloride (PolyDADMAC), and CST plotted versus calcium addition in Figures 23 and 24 (Nguyen et al, 2007b). Clearly, both added polymer and calcium have a similarly strong beneficial effect on the conditioning of both synthetic and activated sludge. The CST of the samples decreased over time with added calcium, but the samples with added polymer showed a bigger improvement in sludge conditioning. Although the solid content was similar in both types of sludges, variability associated with real sludge, such as viscosity and surplus polyelectrolyte, could affect the CST values.

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55

CST(s)

50 45

No polymer add

40

Polymer 3 mg/g TSS

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Polymer 6 mg/g TSS Polymer 9 mg/g TSS

30

Polymer 12 mg/g TSS

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Polymer 15 mg/g TSS

20 15 10 5

10

15

20

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Ca (m M)

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CST(s)

Figure 23. Synthetic Sludge Conditioning with PolyDADMAC Polymer.

24 23 22 21 20 19 18 17 16 15 14 13 12 11 10

No polymer add Polymer 3 mg/g TSS Polymer 6 mg/g TSS Polymer 9 mg/g TSS Polymer 12 mg/g TSS Polymer 15 mg/g TSS

5

10

15

20

25

Ca (m M)

Figure 24. Activated Sludge Conditioning with PolyDADMAC Polymer.

Further results are presented in Figures 25 and 26 for the commercially available cationic polyelectrolytes Clarifloc and Stockhausen (Nguyen et al, 2008a; Nguyen et al, 2008c). The CST is plotted versus concentration of conditioner for both types of sludge and two different calcium concentrations. Note that, in the cases of these two polymers, the overdosing of conditioner generally leads to increases in CST at high polymer dosages. Overdosing in dilute suspensions is attributed to a reduced ability to aggregate the colloidal solids, associated with saturation of the colloidal surfaces by polymer and subsequent re-dispersion. This process is normally accompanied by a reversal of the surface charge. The optimal polymer dosage is commonly associated with only partial coverage of the colloidal surface, and with a minimum in surface charge (Christensen et al, 1993; Lee and Liu, 2000).

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60.0 50.0 Ca 2.5mM synthetic sludge

CST (s)

40.0

Ca 5mM synthetic sludge 30.0

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20.0

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10.0 0.0 0

2

4

6

8

10

12

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16

Clarifoc (m g/g TSS)

Figure 25. Effect of Clarifloc Polymer on Sludge Conditioning of Synthetic and Activated Sludge.

60.0 50.0 Ca 2.5mM synthetic sludge

CST (s)

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40.0

Ca 5mM synthetic sludge 30.0

Ca 2.5mM activated sludge

20.0

Ca 5mM activated sludge

10.0 0.0 0

2

4

6

8

10

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Stockhausen (m g/g TSS)

Figure 26. Effect of Stockhausen Polymer on Sludge Conditioning of Synthetic and Activated Sludge.

The results for conditioning agents support the work of Eriksson et al (1993), who state that polyelectrolyte is mainly consumed in the neutralization of biopolymers and the flocculation of colloids, and to a lesser extent in rebuilding or improving floc fragments at high degrees of stirring. The addition of calcium can also decrease polymer demand for the conditioning. Therefore, the soluble calcium content in synthetic activated sludge should be included in evaluations where flocculation, settling, and dewatering issues are important.

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10. FLOC STRENGTH AND FLOC STRUCTURE CST measurements are also a relative indicator of floc strength. Weak flocs break up under shear, creating a more porous structure and causing an increase in CST; stable flocs don‘t. Figures 27 and 28 (Nguyen et al, 2007b) show the response of flocs in mixed liquor samples to constant shear during mixing at 250 rpm (200 s-1). They show the same qualitative behaviour: the strongest flocs occur at the highest calcium concentration. They also show activated sludge flocs are stronger than synthetic sludge flocs, due again to the presence of filamentous organisms. It has been seen already that synthetic sludge flocs have similar physical and chemical properties to activated sludge flocs. The photomicrographs in Figure 29 of dried samples show that they also look rather similar (Nguyen et al, 2007b).

100 90

CST (s)

80

Ca 5mM

70

Ca 10 mM

60

Ca 15 mM

50

Ca 20mM Ca 25mM

40 30 20 1

2

3

4

5

6

7

8

9

10

Figure 27. Floc Strength of Synthetic Sludge.

50 45 40 Ca 5mM 35

CST (s)

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Tim e (m in)

Ca 10mM

30

Ca 15mM Ca 20mM

25

Ca 25mM 20 15 10 1

2

3

4

5

6

7

8

9

10

Tim e (m in)

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(a)

(b) Figure 29. Scanning Electron Microscope (SEM) Comparison of (a) Synthetic Sludge and (b) Activated Sludge (200 X Magnification).

SEM was used to examine the typical floc size of synthetic sludge, without and with calcium addition (Nguyen et al, 2007b). In Figure 30, when 20 mM Ca2+ is added to the sample, the floc sizes of synthetic sludge are in the range 10-60µm. A two-stage floc formation process is suggested. Initially, latex-particle flocculation forms 2-3 micron flocs (a) and, subsequently, calcium ions further bridge the flocs to form larger 10-60 micron flocs (b).

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(a)

(b) Figure 30. Structure of Synthetic Sludge Flocs (a) without Calcium; (b) with a Calcium Concentration of 20 mM (40 000X magnification).

11. CONCLUSION Cations, biopolymers, filamentous organisms and shear all play important roles in the flocculation of both synthetic and activated sludge; calcium (and aluminium) form bridges between alginate or biopolymer adsorbed on the particles or bacteria. Calcium also has a major impact on settling and dewatering, while polymer conditioning has a major impact on the latter. The stable and well-defined nature of synthetic sludge makes it very useful as a non-biological and non-complex surrogate for studying the physical and chemical properties of activated sludge, including polymer conditioning. However, quantitative discrepancies arise in settling and SVI, supernatant turbidity, sludge dewatering, and floc strength. For such

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studies, the inclusion of a filamentous material in synthetic sludge, such as cellulose, is recommended. The PDA 2000 has proven itself a useful tool for flocculation monitoring, establishing a link between the flocculation dynamics and final sludge properties, and establishing optimum conditions for flocculation.

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REFERENCES Adamson A.W. Physical Chemistry of Surfaces. Wiley, New York NY, 1980. Andreadakis A.D. Physical and Chemical Properties of Activated Sludge. Water Research 1993, 27 (12), 1707–1714. APHA (American Public Health Association). Standard Methods for the Examination of Water and Wastewater. Managing Editor: M.H.Franson. American Public Health Association, Washington, 1998. Biggs, C. A.; Ford A.M.; Lant P.A.. Activated Sludge Flocculation: Direct Determination of the Effect of Calcium Ions. Water Science and Technology. 2001, 43(11),75-80. Bruus, J.H.; Nielsen, P.H.; Keiding K. On the Stability of Activated Sludge Flocs with Implications to Dewatering. Water Research. 1992, 26(12), 1597-1604. Christensen, J.R., Sorensen P.B.(1993). Mechanisms for Overdosing in Sludge Conditioning. Journal of Environmental Engineering. 119(1): 159-171. Dubois M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F. Colorimetric Method for Determination of Sugars and Related Substances. Anal. Chem. 1956, 28, 350-356. Eriksson, L;Alm B. Characterization of Activated Sludge and Conditioning with Cationic Polyelectrolytes. Water Science and Technology. 1993, 28(1): 203-212. Forster C.F.; Lewin D.C. Polymer Interactions at Activated Surfaces. Effluent and Water Treatment J. 1972, 12, 520-525. Goodwin, J.W.; Hearn, J.; Ho C.C.; Ottewill, R.H.. The Preparation and Characterisation of Polymer Latices Formed in the Absence of Surface Active Agents. British Polym. J. 1973, 5, 347-362. Gregory, J. ; Nelson, D.W. A New Optical Method for Flocculation Monitoring, in: Solid– Liquid Separation ; J. Gregory (Ed.); Ellis Horwood: Chichester, UK 1984, pp. 172–182. Higgins, M. J.; Novak J. T. Characterization of Exocellular Protein and its Role in Bioflocculation. J. Environ. Eng. 1997a, 123: 479-485. Higgins M.J.; Novak J. T. The Effect of Cations on the Settling and Dewatering of Activated Sludges: Laboratory Results. Water Environment Research. 1997b, 69, 215-224. Higgins M.J.; Novak J.T. Dewatering and Settling of Activated Sludges: the Case for Using Cation Analysis. Water Environment Research. 1997c, 69, 225-232. Jenkins, D.; Richard M.G.; Daigger G.T. Manual on the Causes and Control of Activated Sludge Bulking, Foaming and Other Solids Separation Problems. 3rd Edition (illustrated), IWA Publishing, 2004. Jin B.; Wilen, B.M.; Paul, L. A Comprehensive Insight into Floc Characteristics and Their Impact on Compressibility and Settleability of Activated Sludge. Chem. Eng. Sci. 2003, 95,221–234.

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Jorand F., Zartarian F., Thomas, F., Block, J.C. ; Bottero, J.Y.; Villemin, G.; Urbain V.;Manem J. Chemical and Structural (2d) Linkage Between Bacteria Within Activated Sludge Flocs. Water Research. 1995, 29 (7), 1639– 1647. Keiding, K. ;Nielsen, P.H. Desorption of Organic Macromolecules from Activated Sludge: Effect of Ionic Composition. Water Research. 1997, 31(7), 1665-1672. Lee, C.H.; Liu, J.C. Enhanced Sludge Dewatering by Dual Polyelectrolytes Conditioning. Water Research. 2000, 34(18): 4430-4436. Li, D.H.;Ganczarczyk, J.J. Structure of Activated Sludge Flocs. Biotechnol. Bioeng. 1990, 35,57–65. Nguyen, T.P; Hankins N.P.;Hilal N. Effect of Chemical Composition on the Flocculation Dynamics of Latex-Based Synthetic Activated Sludge. Journal of Hazardous Materials. 2007a, Vol B139, 265-274. Nguyen, T.P; Hankins N.P.;Hilal N. A Comparative Study of the Flocculation Behaviour and Final Properties of Synthetic and Activated Sludge in Wastewater Treatment. Desalination. 2007b, 204, 277-295. Nguyen, T.P.; Hilal, N; Hankins, N.P; Novak, J.J. Determination of the Effect of Cations and Cationic Polyelectrolytes on the Characteristics and Final Properties of Synthetic and Activated Sludge. Desalination. 2008a, 222, 307-317. Nguyen, T.P.; Hilal, N; Hankins, N.P; Novak, J.J. The Relationship Between Cation ions and Polysaccharide on the Floc Formation of Synthetic and Activated Sludge. Desalination. 2008b, 227, 94-102. Nguyen, T.P.; Hilal, N; Hankins, N.P; Novak, J.J. Characterization of Synthetic and Activated Sludge and Conditioning with Cationic Polyelectrolytes. Desalination. 2008c, 227, 103-110. Novak J.T.; Love N. G.; Smith M.L.; Wheeler E.R. The Effect of Cationic Salt Addition on the Settling and Dewatering Properties of an Industrial Activated Sludge. Water Environment Research. 1998, 70, 984-996. Örmeci, B.; Vesilind, P.A. Response to Comments on ―Development of an Improved Synthetic Sludge: a Possible Surrogate for Studying Activated Sludge Dewatering Characteristics‖. Water Research. 2001, 35(5): 1365-1366. Sanin, F. D.; Vesilind, P.A. Synthetic Sludge: a Physical/Chemical Model in Understanding Bioflocculation. Water Environment Research. 1996, 68, 927-933 . Sanin, F. D.; Vesilind P.A. A Comparison of Physical Properties of Synthetic Sludge with Activated Sludge. Water Environment Research. 1999, 71, 191-196 . Sanin, F.D.; Vesilind, P. A.. Bioflocculation of Activated Sludge: the Role of Calcium Ions and Extracellular Polymers. Environmental Technology. 2000, 21, 1405-1412. Sezgin M.; Jenkins D.; Parker D.S. A Unified Theory of Filamentous Activated Sludge Bulking. J. Water Pollut. Control Fed. 1978, 50, 362– 381. Snidaro D.; Zartarian, F.; Jorand, F.; Bottero, J.Y; Block J.C.; Manem, J. Characterization of Activated Sludge Flocs Structure. Water Sci. Technol. 1997, 36 (4) 313–320. Sobeck, D.C.; Higgins, M. J. Examination of Three Theories for Mechanisms of Cationinduced Bioflocculation. Water Research. 2002, 36, 527-538. Steiner, A.E.; McLaren, D.A. ; Forster, C.F. The Nature of Activated Sludge Flocs. Water Research. 1974, 10, 25–30. Tezuka, Y; Cation-dependent Flocculation in a Flavobacterium Species Predominant in Activated Sludge. Appl. Microbiology. 1969, 17, 222-226.

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Urbain V.; Block, J.C.; Manem, J. Bioflocculation in Activated Sludge: an Analytic Approach. Water Research. 1993, 27 (5) 829–838.

ACKNOWLEDGEMENTS The authors thank the Ministry of Education and Training (MOET) of Vietnam for financial support of TPN. They also thank Stoke Bardolph sewage wastewater treatment plant; and the Environmental Engineering Laboratory, Virginia Tech, USA.

CONTRIBUTORS Nick Hankins ([email protected]) is a University Lecturer in Chemical Engineering and Research Director of the Centre for Sustainable Water Engineering, within the Department of Engineering Science at the University of Oxford. His research interests involve the application of colloids and interfaces in separation processes. Nidal Hilal is Professor of Chemical and Process Engineering and Director of the,Centre for Clean Water Technologies, within the School of Chemical and Environmental Engineering at The University of Nottingham. His research interests include water treatment, membrane technology and atomic force microscopy.

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Tan Phong Nguyen is a Senior Lecturer within the Faculty of Environment at The University of Technology, Ho Chi Minh City, Vietnam. His research interests include waste water treatment and the activated sludge process.

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In: Sludge: Types, Treatment Processes and Disposal Editor: Richard E. Baily

ISBN: 978-1-60741-842-9 © 2009 Nova Science Publishers, Inc.

Chapter 2

PROCESSES TO RECOVERY PROFITABLE PRODUCTS FROM WATER DEGUMMING SLUDGE OF VEGETABLE OILS Liliana N. Ceci* and Diana T. Constenla Planta Piloto de Ingeniería Química (PLAPIQUI), UNS-CONICET, Camino Carrindanga Km 7, 8000-Bahía Blanca, Argentina.

ABSTRACT

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Oilseed world production in 2007/2008 was 391.2 Mt with a contribution of 56.4 % for soybean and 7.0 % for sunflower, according to United States Department of Agriculture (USDA) data. During the same period, 128.0 Mt of oils from seeds were produced along the world, including 29.3 % of soybean oil and 7.8 % of sunflower oil. During processing, some millions of tons of sludge or gums are generated in water degumming step, to remove impurities and obtain oils without turbidity and stable with respect flavor and odor. Degumming sludge is a complex mixture comprising high water content, phospholipids (PLs), oil, and minor amounts of other constituents like phytoglycolipids, phytosterols, tocopherols, and fatty acids. PL fraction in degumming sludge principally includes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidic acid (PA). In this chapter, some traditional industrial processes to produce, purify, and fractionate lecithin, from degumming sludge are presented. These processes include drying and deoiling with acetone, a solvent in which PLs, glycolipids and related compounds are almost insoluble. Lecithin is used as emulsifiers, dispersing and release agent in food, pharmaceutical and cosmetic industries. Methods still not applied at industrial scale for extraction and partition of PLs using supercritical fluids, and processes for chemical and enzymatic modifications of lecithin are also reviewed in this chapter, with their advantages and disadvantages. Enriched lecithin in any component such us PC or PI, with distinctive surface-active properties can be obtained by lecithin fractionation with solvents. Special attention is dedicated in this review to novel structured PLs that can be obtained by enzymatic reactions to exchange fatty acids in *

e-mail: [email protected].

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Liliana N. Ceci and Diana T. Constenla natural PLs. These PLs are high-priced fine chemicals for membrane and lipoprotein investigation, cosmetic industries, and liposome technology to release food and medicament components. The extensive availability of different lipases and phospholipases, fatty acids and derivatives, including polyunsaturated fatty acids, to modify natural PLs and generate products with new physical and chemical properties, open a large investigation field at immediate future. Results of recent own investigation about recovery occluded oil in soybean degumming sludge, by water elimination and acetone extraction, and study on quality and stability indexes in recovered oils, in view to their re-insertion in productive process, are also shown in this review.

Keywords: gums, sludge, lecithin, phospholipids, oil recovery

1. INTRODUCTION

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The oilseed world production in 2007/2008 was 391.2 Mt according to United States Department of Agriculture (USDA) data being oil vegetable world production about 128 Mt in the same period (Figure 1). Soybean oil represents 29.3 % of total production of vegetable oils. In last years, a slight increase in oil production was observed (Figure 1). The crude vegetable oils, obtained by pressing or solvent extraction, have up to 3 % of PLs which may exert a negative influence on the taste, odor, appearance and stability. Water degumming is the classical process to reduce the hydratable PL content and other compounds such as glycolipids, metals, and free sugars (Table 1). Gums or sludge are generated during this process as by-product (Figure 2). In this way, some millions of tons of gums were produced in water degumming process during 2007/2008.

Figure 1. The world production of oilseeds and vegetable oils. Data from Foreign Agricultural Service, USDA, October 2008.

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Some degumming processes are utilized principally during refining of vegetable oils and can reduce non-hydratable PLs and offer oils with phosphorous content lower than 10 mg/kg. In the literature, are available a lot of papers and reviews about acid, acid/basic, membrane and enzymatic degumming as soon as process with chelating agents (Soft Degumming) to reduce non hydratable PL content (1-9). Table 1. Reduced compounds in crude soybean oil by water degumming [1]

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Compound (g/100g) Phospholipids Glycolipids Free Sugars Free Fatty Acids

Crude Oil 2.00-3.00 0.15-0.30 0.10-0.15 0.50-1.50

Water Degummed Oil 0.3-0.8 0.02-0.03 0.02-0.03 0.30-1.20

Figure 2. Flow sheet for classical water degumming process.

In this chapter is presented and discussed the treatment of water degumming sludge for obtain profitable products with possible applications in food, pharmacy, cosmetics, and other industries. A quickly glance of classical methods, for produce lecithin from water degumming sludge, is included and special attention is dedicated to modern methods to process and modify lecithin and recover oils. The methods described in this chapter contribute to reduce the volume of wastes to dispose and the risk of environmental contamination.

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2. WATER DEGUMMING SLUDGE Water degumming sludge is a complex mixture with high water content (Table 2) from that, quickly degrades and spoils at room temperature, and it is crucial a good conservation before processing. In the frozen state (-20 °C), no changes in general composition, and acid, iodine and peroxide values were observed during 24 months [10]. Table 2. Wet Gum Composition of Soybean and Rapeseed

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Compound Rapeseed [10] Soybean [11] g/100 g Wet Gum Moisture 43.0a 46.2 ± 4.8 Acetone Insoluble Material 35.0 29.3 ± 6.2 Occluded Oil 22.0b 24.1 ± 1.5c PC 17.1 12.0 ± 3.1 PE 9.1 6.8 ± 1.5 PI 12.5 4.3 ± 1.6 PA -2.8 ± 0.5 a Water and volatile substances. b Neutral lipids. c Estimated by difference 100 - % Moisture - % Acetone Insoluble - % Hexane Insoluble.

Two important components in water degumming sludge are PLs and occluded oil (Table 2). PLs are insoluble in acetone and a measurement of its content is the determination of acetone insoluble material including phosphatides, glycolipids and carbohydrates all together. Lecithin is meant in commercial sense refers to the natural mixture of neutral and polar lipids from vegetable or animal sources. Neutral lipids are mainly triglycerides, whereas polar lipids consist of glycolipids and PLs, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidic acid (PA). In chemical nomenclature the term ―lecithin‖ is used to refer specifically to PC. PLs are amphiphilic molecules, with a backbone of glycerol esterified by two fatty acids in 1 and 2 positions as large non-polar tails, and a polar phosphate group in third carbon linked to a nitrogenous base or a sugar functioning as polar head (Figure 3). PLs act as emulsifiers in the interface oil/water due to their amphiphilic structures. Wet gums also contain metals in traces such as potassium, magnesium, calcium, iron with contents of 326, 48, 20 and 1 mg/kg, respectively for soybean wet gums [3]. In oils, PC is always hydratable as zwitterion, PI is complexed with potassium and fully hydratable, PE and PA are hydratable when combined with potassium and non-hydratable when complexed divalent metals. During water degumming process is convenient eliminate some metals related to oxidation in oils such as iron. The composition of wet gums is variable and the source of this variability may be genetic (plant cultivars), seed quality (maturity, harvest-induced damage, and handling/storage conditions), and oil processing variables [12].

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Figure 3. Molecular Structure of Phospholipids from wet gums.

3. PROFITABLE PRODUCTS FROM WATER DEGUMMING SLUDGE 3.1. Animal Feeds By far the largest portion of the soybean and other oilseed meals and cakes are used as a protein source in animal feed including ruminants, swine and poultry. Soybean meals have relatively high concentration of protein (up to 44-50 %) and excellent profile of highly digestible amino acids. One practice commonly used in processing plants is the addition of gums back to the meal but may affect nutrient content and quality of the resultant meal reducing its nutritive value [13].

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When a biologically active substance is orally administered to ruminants, a substantial part of the substance is decomposed by rumen microflora, making it difficult or impossible for the animal to effectively utilize all of the administered proteins and amino acids contained in feed. When inadequate amounts of essential amino acids escape the rumen, productions of milk and meat, as well as reproduction, are all negatively affected. Feedstuffs with soybean meal coated with wet gums tend to pass through the rumen without being degraded resulting in a rumen by-pass value for the protein of 73 % and an increase of protein escaping degradation of 21 % due to addition of wet gums [14]. At least three reasons are important when rumen-bypass is increased: PC contribution, methioninesparing effect, and larger bypass of essential amino acids. PC is necessary for mobilization of fats out of the liver and an inadequate contribution with diet causes reductions in feed consumption and milk yield, health problems, and reduced breeding performance. If dietary PC is deficient, dietary methionine, which is essential for milk production, can be used to synthesize the needed PC. Figure 4 shows flow sheet of a friendly environmentally process for produce coated animal feeds. This process can be applied to seeds and/or grains such as soybean and can be carried out oil extraction by pressing without solvents or other contaminant additives. The degummed oil has high quality and the gums are used to coat the pressing cake and obtain a coated animal feed without generate residual gums. The whole seeds and/or grains are fractured into pieces by means cracking rolls, hulls can be eliminated, and then the pieces are heated in a cooker/dryer to reduce the solubility of the proteins in the rumen. Crude oil is extracted by screw press (expeller) and then is degummed by water and temperature and separate from wet gums by centrifugation. Finally, the fresh gums wet and warm and the cake still warm and fresh are entering in auger mixer with the addition of warm water. There is no loose water and all of the gums obtained are placed onto all the cake being also possible the application of additional gums from another degumming operation. After leaving the mixer, the cake or the meal coated with gums may be broken into smaller particles of a desired size or formed into desired shapes. The coated cake is cooled at room temperature in a cooler for its stabilization. A feedback control for the amount of water entering to mixer allows produce coated cake 10.0-12.5 % of moisture by weight and a content of protein at least of 42.0 %. Additional additives, such as vitamins, non essential and essential amino acids, additives and supplements may also be added to animal feed, included the cake and the coating.

3.2 Crude Lecithin About ninety years ago, was obtained the first commercial product from wet gums, crude soybean lecithin as alternative to egg lecithin, which was expensive and only available in small quantities. Due to historical reasons, lecithin from soybean, sunflower and rapeseed play a predominant commercial place today. Crude lecithin is produced from wet sludge of water degumming by drying in vacuum batch dryers (60-70 °C, 20-60 mmHg, 3-5 h) or film evaporators (80-105 °C, 25-300 mmHg, 1-2 min) [15, 16]. Dried lecithin has 1-2 % of moisture. The drying operation serves not only to remove moisture, but to lower the peroxide value as well. The success of the drying operation depends on using the lowest possible temperature and exposing the lecithin to temperature for the least possible time and quick cooling after process.

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Figure 4. Flow sheet of a process to produce animal feed coated with wet gums from grains and seeds [14].

The color of lecithin can be attributed to natural pigments from seeds, such as carotenes and porphyrins, in addition to the Maillard and Amadori reaction products generated by non enzymatic browning during processing. Lecithin can be bleaching by hydrogen peroxide (H2O2) and/or benzoyl peroxide [15]. The addition of 0.3-1.5 % of 30% H2O2, directly to the gums, or the addition of H2O2 to water for degumming of oil allows obtaining bleached lecithin. Benzoyl peroxide is not permitted for food use in some countries because this lecithin can only be used for technical applications. Crude lecithin can be standardized (62-64 % acetone insoluble) and fluidized with fatty acids and oil to reduce its viscosity [15].

3.3. Oil Occluded oil in water degumming sludge from soybean oil has been recovered by direct extraction with cold acetone (Method I) and extraction after water elimination under vacuum (Method II) and by solvent partition with hexane/ethanol (Method III) (Table 3). Higher oil yields (up to 58.8 % of occluded oil) were obtained when water was eliminated before extraction (Methods II and III). Recovered oils had stability and quality indexes compatible with their reinsertion in the productive process [11]. All recovered oils had total PLs contents practically in the range accepted for degummed products (Table 3). A water degumming step during refining process could reduce the PLs in the recovered oils without drawbacks, because PC, the principal PL in these oils, is easily hydratable.

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Liliana N. Ceci and Diana T. Constenla Table 3. Oil Recovery from Water Degumming Sludge of Crude Soybean Oil and Phospholipid Composition for Recovered Oils.

Recovered Oil Yield Method I Method II Method III g/100 g wet gums 10.4 ± 1.8 13.5 ± 2.6 14.2 ± 1.4 g/100 g occluded oil 42.9 ± 7.1a 55.6 ± 9.6b 58.8 ± 6.0 b Phospholipid (% rel.) PE 17 ± 5a 11 ± 6a 12 ± 5a a,b a PA 7±5 10 ± 4 5 ± 3b PI 14 ± 4a 8 ± 2b 6 ± 6b a a PC 62 ± 11 71 ± 10 77 ± 12a Total (g/kg) 1.56 ± 0.64a 9.56 ± 3.43b 5.10 ± 4.16a,b Means within a row followed by the same letter are not significantly different ( = 0.05).

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3.4. Deoiled Lecithin The triglycerides from oils are soluble in acetone, in contrast to the other more polar components like PLs of standard lecithin. Deoiled lecithin can be produced mixing and agitating the lecithin containing oil with acetone [15, 17, 18]. PLs and adherent carbohydrates precipitate as sediments, which are centrifuged and/or filtered. Acetone extraction can be carried out in a continuous process. A careful drying process is required to eliminate the residual acetone, preventing the formation of undesired off-flavors. Flushing with nitrogen is often applied for preventing oxidation. Commercial deoiled lecithin has only 2-5 % remaining oil and acetone insoluble matter of at least 95 %. The reduction of occluded oil enhances the emulsification function, moderates the taste and has the dosing more convenient. These products are used as emulsifying in food processing, nutritional health foods, choline supplementation and pharmaceutical ingredients. An alternative process without solvents is extraction with supercritical fluids, which was developed for the recovery of high value products, such as flavors and oleoresins and for decaffeination of coffee beans. Some of the advantages of this process are: i) free of oxygen (no oxidation of the lecithin) ii) no solvent residues either in the deoiled lecithin or in the oil iii) no risk of inflammability iv) no environmental problems v) no drying of the product is required. The first developed method for deoiling lecithin used supercritical carbon dioxide (SCCO2) at pressures up to 70 MPa and temperatures near ambient temperature (40-60 °C) which dissolves neutral lipids (oils) leaving the polar substances (principally PLs) [19, 20]. CO2 and oil are easy to separate and CO2 can be recovered and used again in the process. CO2 is inert, non toxic, and generally recognized as safe (GRAS) for use in pharmacy and foods. Figure 5 shows a simplified flow sheet for oil extraction with SC-CO2 from crude lecithin.

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Figure 5. Simplified flow sheet for deoling of crude lecithin with supercritical carbon dioxide.

The use of pure SC-CO2 as solvent has some disadvantages such as low dissolving capacity of CO2 requiring high excessively pressures, high solvent to feed ratio, and low yields. The recovery of PLs can be achieved by addition of a polar entrainer or co-solvent to SC-CO2. Presence of a co-solvent enhances the solubility in the supercritical fluid at the same temperature and pressure, making it possible to conduct the extraction at lower pressures. Cosolvent must not only have convenient thermodynamic properties but also be GRAS especially for food applications. Propane was studied as co-solvent (80 % propane and 20 % CO2) for deoling crude lecithin in a countercurrent column [21]. Also, SC-CO2 with 5 wt % ethanol or 10 wt % acetone, were assayed increasing the solubility of oil without coextraction of PLs [22]. Co-solvents in SC-CO2 can co-extract some PLs depending of the amount of co-solvent added resulting an obstacle if the objective is to retain all PLs in deoiled lecithin. In last years, a new process, known as Gas Anti-Solvent (GAS), was successful assayed in laboratory scale for deoiling lecithin [23]. This process requires much less pressure for high recovery and purity of lecithin, as compared to the jet extraction technique. Nowadays, GAS process is industrially used for crystallization of phytochemicals and bioactive compounds and for obtaining very small solid particles with controlled size distribution [24]. In GAS process the product (lecithin for example) is firstly dissolved in an organic solvent (hexane for lecithin) and the precipitation of particles is performing by reducing the solubility through adding a dense gas (CO2) as anti-solvent, which dissolves partly into the solvent. The gas and the solution are separated from the particles by filtration. The gas can be recovered spontaneously, but the organic solvent needs and additional separation step.

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3.5. Lecithin Fractionation PC, PE, and PI have different head groups, and this gives them different polarities and different emulsification properties. Therefore lecithin fractionation is desirable for certain applications. PC-enriched fractions may be a better oil-in-water (o/w) emulsifier than the unfractionated lecithin, whereas the PI-enriched fraction can be used as water-in-oil (w/o) emulsifier in the confectionery industries. Today, the production of plant lecithin fractions largely makes use of ethanol or ethanolwater mixtures with up to 10 % of water. This fractionation process takes advantage of the differences in solubility of the various PLs in ethanol [18]. PC in particular is readily soluble in ethanol, whereas PI and PA are virtually insoluble. PE, like the neutral lipids, is found in booth fractions. Other solvents, such as methanol and isopropanol, have been assayed, but for toxicological and economic reasons nowadays ethanol is used. The process can be applied to crude, deoiled or modified lecithin. Lecithin is mixed with the solvent and then soluble phase (PC-enriched) is separated from insoluble phase (PI-enriched). The solvent can be recovered from soluble phase. The higher the water content in water-alcohol mixture, promote lower oil content in the soluble phase. Soybean, rapeseed, and sunflower lecithin behave in the same way [18]. PLs have different adsorption characteristics and, therefore, different retention times by chromatography on columns with silica gel or other adsorbents. With this process fractions with PC content greater of 80 % can be obtained [17]. For food applications this fraction is mixed afterwards with carriers, such as special oils, cocoa butter and maltodextrin for convenient handling, dispersion and dosing. Figure 6 shows block diagram for large-scale fractionation of crude soybean lecithin using aqueous ethanol (95 % ethanol) at room temperature and fractionation by aqueous ethanol at high temperature (40 °C) and cooling at low temperature (0 °C), high-low temperature fractionation [25]. The oil solubility in ethanol may change more with temperature than the solubility of PLs, thus the treatment at low temperature may increase the purity of the PC fraction. Fractionating twice with ethanol increases the purity of the PC fraction and high-low temperature fractionation increases the purity and PC percentage in the PC fraction. The ethanol-soluble PC fraction (twice extracted from oil-containing lecithin) contained 36.9 % PC, 6.5 % PE, and undetectable quantities of PI; the ethanol-insoluble PI fraction (after deoiling) contained 27.1 % PI, 14.6 % PE, and 3.3 % PC. Practically pure PC can be produced using alcohol and solutions of some inorganic salts with divalent ions, such us CdCl2, which form insoluble complexes with PC [18]. Other precipitation methods use earth-alkaline salt solutions, which have to be added to ethanolic solutions of deoiled lecithin [18]. Acid PLs react with magnesium sulfate or calcium hydroxide to form insoluble precipitates. In the supernatant PC concentrations up to 90 % can be obtained. A new degumming protocol has been recently developed which employs electrolyte solutions (NaCl and KCl) to remove non-hydratable gums (mostly PA and PE) from soybean [26].

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Figure 6- Block diagram for fractionation of crude soybean oil. Yields are calculated in percentage from crude lecithin. EF: Aqueous ethanol fractionation (95 % ethanol) at room temperature. ETF: Fractionation by aqueous ethanol treatment (95 % ethanol) at 40 °C and then cooling at 0 °C.

Selective extraction of PC from deoiled lecithin can be carried out using supercritical mixtures with CO2 and ethanol as solvent (10 % ethanol). Extraction at 60 °C and 20.7 MPa resulted in 95 % selectivity to PC [27]. Although increased percentages of ethanol seem to favor selective extraction of PC, the increase in ethanol percentage and high temperature can cause denaturation of PLs.

3.6. Pure Phospholipids High purity PC is used for preparation of liposome, which encapsulate drugs and special food components for their controlled release, and can be also applied to synthesis of structured-PLs. Moreover PC normally contains linoleic acid (C18:2), an unsaturated fatty acid, considered as ―good fatty-acid‖ with respect to human health. For these reasons, pure PC demand is being increased in last years. Generally speaking, chromatographic separation is the method of choice to prepare polar lipids high in purity. Numerous methods have been published in scientific and patent literature and most of them are used for the quantitative analysis of PLs [18]. Good results in separation are usually achieved by combining different separation techniques, as for example solvent fractionation and column chromatography at low and medium pressure [18]. Alumina and silica play an outstanding role as adsorbents to produce large quantities of vegetable and egg PC. Nearly all the PC accompanying PLs are bound to

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adsorbent. Ethanol and other solvents can be used to recovery practically pure choline PLs in the effluent. The principal disadvantage of column chromatography is the retention in the products of lysophosphatidyl choline where fatty acid in 2-position has been hydrolyzed. High purity PC has been prepared by preparative HPLC (elution mode) from egg [28] and soybean [29] PLs. In these methods there were some inherent shortcomings, like low loading amount and high solvent consumption, which led to high production cost and price. To overcome these drawbacks, HPLC methods have been optimized [30, 31]. A simpler mobile-phase design (pure methanol) to develop an economic HPLC process has been also proposed [32]. In this way, was successfully separated PC from a mixture PC-PE (80 %-15 % w/w) with over 99 % purity and with 98 % yield. In last years, a method by displacement chromatography (High-Performance Displacement Chromatography, HPDC) has been developed for PC and PE separation [33]. Displacement chromatography usually includes four operation steps: equilibrium, loading, displacement and regeneration. The feed components are driven out of the column by the displacer that has stronger affinity to the stationary phase than any component in the feed. The feed components arranged themselves into a ―displacement train‖ of adjoining square wave concentration pulses of the pure substance according to the order of the affinity strength to the stationary phase. Compared with elution chromatography, displacement chromatography has some advantages such as large loading amount, high concentration of the product, little tailing, low solvent consumption and high efficiency of use of the stationary phase. Hence, it is appropriate to be used as preparative chromatography. In this HPDC method, dichloromethane-methanol (9:1, v/v) and ethanolamine are used respectively as carrier and displacer. PC (100 % purity) was obtained with 77.6 % of yield, and even when the purity of the PC product was 95.1 %, the yield of that was as high as 92.4 %.

Figure 7. Some reactions for chemical modification of phospholipids.

The displacement chromatography exhibits potentiality for application in the field of preparation of PLs in preparative scale. The recent development in this area seems to make it possible to transfer the separation into the large-scale purification of PLs. The separation of other pure PLs such as PI is of growing interest, and could be produced in a similar form than pure PC. More work is necessary at this respect.

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3.7. Chemical Modification of Lecithin Lecithin contains a number of functional groups that may undergo hydroxylation, acylation (acetylation), hydrolysis, hydrogenation, halogenation, phosphorylation, and sulfonation, when they are treated with different chemical reagents. These modified products have improved fluid properties, improved water dispersion, increased moisture retention, and enhanced o/w emulsifying properties, when they are compared with the same unmodified products. Only more important modifications are presented in this section, and a lot of them can not be applied to foods in countries where the use of some reagents does not regulated. In recent literature some publications discusses these modifications [34, 35]. Hydroxylation process essentially involves the addition of hydroxyl groups to double bonds in unsaturated fatty acids of PLs, using high concentrations of hydrogen peroxide (H2O2) in combination with organic acids, such as acetic acid or lactic acid (Figure 7). Crude lecithin hydroxylation is allowed to proceed until a 10 % reduction in iodine value occurs. Iodine value measures the amount of added iodine to double bonds. Hydroxyl groups give to lecithin the highest possible hydrophilic properties. The ethanolamine group is also modified during hydroxylation. Hydroxylated lecithin is primarily used in paints, cosmetic preparations and in leather treatment [34] and can only be used in foods in countries where hydroxylation is approved for foods [35]. In USA the use of lecithin and hydroxylated lecithin is permitted, and they can be applied to food coatings, for a variety of esthetic and protective purposes [36]. The fluidity and emulsifying properties of lecithin can be controlled by means of the acylation process, or acetylation process, when acetic anhydride is used. The reaction principally occurs on amino group of PE, and the product is an acetylated PE (Figure 7). The free amino-nitrogen content in the lecithin is substantially reduced when is acylated. A substituent group is introduced on the positively charged portion of the PE, and converts it to negatively charged lecithin. N-acetyl PE is very hydrophilic, is an effective emulsifier for o/w system and less sensible to heat than normal PE. Acetylated lecithin finds use in plastics and petroleum products [34] and is a superior release agent in food processing [35]. Crude lecithin is readily hydrolyzed with strong acids or bases. The hydrolysis must be stopped at an early stage, preferably just when one fatty acid is cleaved to generate useful lysophospholipids (Figure 7). When an o/w emulsion, is prepared with hard water containing calcium and magnesium ions, emulsion breaks and give a phase of hydrated lecithin and a phase of oil or fat on the surface of aqueous phase. Hydrolyzed lecithin eliminates this problem. Acid or basic hydrolysis is rather difficult to control, and for this reason, enzymes are preferred. Enzymatic hydrolysis of lecithin will be discussed in follow section of this chapter. Hydrolyzed lecithin has found application in foods and also in the manufacture of greases and lubricants and as ingredient for plastic formulations.

3.8. Enzymatic Modification of Lecithin The use of enzymes for processing offers possibilities of greater selectivity and can yield products that can not be made by chemical methods. The special value of enzymes is the additional control of the product composition through the differing specificities of the enzyme. Moreover enzymatic reactions are faster, are carried out under mild conditions, and generate a waste reduction. For these reasons, and in spite of some drawbacks, the enzymatic

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methods are gaining more and more space in the area of industrial processing. Mainly two groups of enzyme can be used with PLs as substrates, phospholipases and lipases. Phospholipases hydrolyze PLs in different positions of the structure (Figure 8). Phospholipase A1 (PLA1) cleaves a PL at the sn-1 position, and the enzyme that hydrolyzes sn-2 position, is called phospholipase A2 (PLA2). Phospholipase B (E.C. 3.1.1.5) is an enzyme with a combination of PLA1 and PLA2 activities; that is, it can cleave acyl chains from both sn-1 and sn-2 positions of the PLs. Phospholipase C (PLC) attacks on the phosphodiester bond at the glycerol backbone. If the enzyme hydrolyzes the phosphodiester side so that the phosphate group remains on the glycerol backbone, it is a phopholipase D (PLD). Lipases have triglycerides or triacylglycerols (TAGs) as natural substrates, hydrolyzing principally sn-1 and sn-3 positions. Lipases can also hydrolyze sn-1 fatty acid in PLs (Figure 8). These enzymes can also link a fatty acid in these positions depending on reaction conditions.

Figure 8. Cleavage sites of phospholipases and lipases acting on phospholipids molecules.

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3.8.1. Phospholipase A2 (PLA2)

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PLA2 from porcine pancreas, produced as by-product in the elaboration of porcine insulin, has been used to develop an enzymatic degumming process for vegetable oils with high phosphatide contents, known as ―Enzymax‖ process [4]. Lack of production of this enzyme and religious reasons by its animal origin have restricted the advance of this process. However, PLA1 from different microbial sources have been proposed for replace to PLA2 in oil degumming [5, 8]. PLA1 has been successfully assayed for enzymatic degumming in large scale (400 ton/day) [37]. Lysophospholipids (LPLs) can be obtained from lecithin by means enzymatic reaction with free PLA2 dissolved in the aqueous phase of a sludge lecithin/water. Drying the mixture, the hydrolized product can be processed. However the enzyme is not removed from the lecithin and there is a chance of continued hydrolysis on rewetting. This problem can be surpassed, by using PLA2 in mixed reversed micelles of lecithin/bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane, and separating products in a continuous membrane module [38] (Figure 9). PLA2 added to this reversed micelles is adsorbed onto the hydrophobic interface, and it would be fully activated by what is known as ―interfacial activation‖. The hydrolysis reaction quickly occurs and the membranes take advantage of the size differences between the enzyme and the reaction products. The enzyme can be trapped on one side of an ultrafiltration ceramic membrane, while the substrate is fed directly into the retentate side, where the reaction occurs. The LPLs are removed continuously from the reaction mixture, leaving the enzyme in the reaction zone for further reaction. Results demonstrate ≈ 100 % hydrolysis conversions with short reaction times needed to accomplish the complete hydrolysis of lecithin from egg yolks, effective enzyme retention, and simultaneous separation of fatty acids [38]. This catalytic system showed high stability in a long-term operation.

Figure 9. Continuous bioreactor with UF membranes for lecithin hydrolysis. A: reactor - B: ultrafiltration module - C: ceramic membrane - D y E: pumps - F: substrate reservoir G: product reservoir - H: back-pressure valve - I: pressure gauge.

PLA2 has different selectivity toward the PL polar heads in non-aqueous system compared to all-water media. In hexane for example, PC is hydrolyzed by PLA2 almost twice faster than PE, and PI in not hydrolyzed in this reaction [39].

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PLA2 has been also immobilized by adsorption on different supports, such as alumina and DEAE-Sephadex, to hydrolyze soy lecithin at laboratory scale [40] and a tubular batch reactor with a capacity of 90 cm3 [41]. Immobilized enzymes have as advantage the possibilities of separation and reuse of the enzyme, however enzymatic activities are reduced by immobilization. The hydrolytic activity of PLA2 adsorbed on alumina or DEAE-Sephadex, was lower than of the soluble enzyme, because the intrinsic properties are modified by immobilization [40, 42], and diffusion effects originated by substrate and product transport phenomena [42]. PLA2 immobilized on Celite has been used for hydrolyze soybean lecithin and water content was found to be the key parameter, recommending water activity greater than 0.24 but avoiding the presence of a bulk phase of water [39]. Lysolecithin (LL) can also be obtained from egg yolk lecithin with PLA2 immobilized in alginate-silicate sol-gel matrixes using buffer-alcohol systems [43]. The immobilization remarkably increased thermal stability of the enzyme that was successfully reused over 250 h. The problem of the LL production is mainly on the difficult to separate the enzyme from the product, when the reaction has been completed. These systems with immobilized enzyme are useful for catalytic performance of the enzyme and the easy purification of the product. Phospholipases also catalyze reactions characteristic from lipases such as acidolysis (direct transesterification) in organic media (Figure 10). These reactions involve the exchange of fatty acids in PLs structure in sn-1 position, if PLA1 is used as enzyme, and sn-2 position, with PLA2. Products are modified or structured PLs with a different fatty acid composition. Saturated fatty acids, including both medium chain and large chain, or polyunsaturated fatty acids (PUFAs) can be introduced in natural PLs. The interest in the incorporation of saturated fatty acids is mainly to improve the heat stability, emulsifying properties, and oxidation stability of the PLs, while the incorporation of PUFAs is due to the claimed health promoting effects.

Figure 10. Reactions catalyzed by 1,3-specific lipases on phospholipids as substrates.

In general, the incorporation of fatty acids into the sn-2 position of PLs by acidolysis with fatty acids, and transesterification with esters, is rather low and degree of hydrolysis is difficultly controlled (< 15 %) [44-46]. Free-solvent reaction systems can be more effective to introduce fatty acid with PLA2 immobilized on Celite [44]. The main fatty acids replaced

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with PLA2 were unsaturated acids, principally linolenic acid (C18:3) and linoleic acid (C18:2), and moreover, the hydrolysis could not be avoided. Recently, PLA2 has been successfully immobilized on non-ionic weakly polar resin, and assayed for synthesis of structured PLs under solvent-free conditions [47]. The reaction temperature and water addition had an inverse relationship between incorporation and recovery of soybean PC. Incorporation of caprylic acid (C8:0) into PC could reach 36 %, with a yield of 29 % of the PL fraction. Docosahexaenoic acid (DHA) and conjugated linoleic acid (CLA) were incorporated into PC with yield of 30 % and 20 %, respectively. Incorporation of acyl donor into lysophosphatidylcholine (LPC) was very low (< 4 %), which indicates that acyl migration is only a minor problem for PLA2 catalyzed synthesis reaction. Acyl migration is undesired reaction for lipases and phospholipases. PLA2 produces acyl exchange into sn-2 position as principal reaction, and hydrolysis in this position as secondary reaction. Hydrolysis reaction from PC produces 2-LPC. 1-LPC could be generated by acyl migration from sn-1 to sn-2 positions. Acyl group into sn-2 position converts to 1-LPC in PLA2 substrate for acyl exchange reaction. If this happened, LPC with acyl donor incorporated into sn-2 position, would have to appear between products.

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3.8.2. Phospholipase A1 (PLA1) PLA1 can be produced in extracellular form by cultivation of some bacteria from Fusarium and Serratia genera. LPLs can be obtained from soybean PLs by enzymatic hydrolysis using PLA1 [48]. Judged on the basis of productivity and the degree of hydrolysis, the yield of LPLs in a two-phase system was found to be better than that obtained in an emulsion system (PLs, water and surfactant agent). PLA1 showed the most efficient catalytic activity and stability in butyl acetate as solvent. PLA1 hydrolyzes fatty-acids in sn-1 position, which in PLs usually are saturated acid such as palmitic acid (C16:0) and stearic acid (C18:0), while unsaturated ones such as linoleic acid, predominantly in sn-2 position are not hydrolyzed. Hydroxylated PLs with hydroxy fatty acids can be produced by enzymatic interesterification between soybean and egg PC and esters from these acids. The presence of hydroxy fatty acids in the PLs imparts hydrophilic properties and improves moisture retention of lecithin with increased water dispersion. Hydroxylated PLs are useful in baking applications where it can improve the dispersion of fats and retard staling. Thus, ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid) can be introduced into sn-1 position of PC, using PLA1 immobilized on Celite and methyl ricinoleate, in hexane as reaction medium [49]. Soy and egg lecithin do not contain ricinoleic acid, by means this interesterification, PC with 50 % of ricinoleic acid was obtained. To establish the exclusive incorporation of ricinoleic acid into sn-1 position, PC was hydrolyzed with free PLA1, and the released fatty acid was found to be only ricinoleic acid. This clearly establishes that the sn-1 position of modified PC was completely replaced by ricinoleic acid. Essential fatty acids can not be constructed within an organism from other components (generally all references are to humans) by any known chemical pathways; and therefore must be obtained from the diet. Recently, some essential polyunsaturated fatty acids (n-3 fatty acids) from fish oil have been incorporated to soybean PC by means acidolysis reactions using PLA1 immobilized by adsorption on an ionic resin [50]. In a batch reactor, nearly 35 % of the total esterified fatty acid residues were n-3 species [eicosapentaenoic acid (EPA),

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docosapentaenoic acid (DPA), and DHA]. More study about the effects of water activity, is needed to facilitate design and operation of a packed bed reactor, for continuous acidolysis, minimizing the migration of acyl groups.

3.8.3. Phospholipase B (PLB) / Lysophospholipase PLB, which catalyzes complete deacylation of diacylglycerophospholipids can be produced extracellularly by fungi (Penicillum notatum and Cryptococcus neoformans) and yeast (Kluyveromyces lactis). This enzyme has also lysophospholipase activity catalyzing the deacylation of monoacyl glycerophospholipids. In C. neoformans, lysophospholipase activity is 10- to 20-fold greater than PLB activity, reducing the possibility of accumulation of LPL during hydrolysis reactions. Activities of lysophosphalipase transacylase were also identified in the enzyme preparations isolated from C. neoformans. Investigation about the use of PLB in PL modifications has not been reported in last years. Probably, the lack of availability of the enzyme at commercial scale has contributed to this.

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3.8.4. Lipases Lipases with 1,3-selectivity are extensively used for PL modifications due to their easy commercial availability as free and immobilized enzymes, and their spread use for modifications of their natural substrates the triglycerides. Figure 10 presents a summary of the reactions that can be catalyzed by lipases acting on PLs. In 2005, a review has been published presenting an extensive summary of works carried out with commercially available lipases, principally sn-1,3-lipases [51] . The preferably used substrate is PC from egg yolk or soybean. Lipases from Rhizomucor and Rhizopus genera are the most efficient lipases for PL modifications [52]. Lipases from Rhizomucor miehei (former Mucor miehei) and Rhizopus oryzae (former Rhizopus arrhizuz) are the most used. Lipases from fungus Aspergillus and Thermomyces are little active on PLs. For hydrolysis reactions is recommended a reverse micellar system (PC/AOT/isooctane) or an aw-controlled solvent system (hexane is commonly accepted in food industries) [51, 46]. Acidolysis reactions can be carried out with lipases in free-solvent systems to incorporate new fatty acids in sn-1-position (Figure 10). These reactions generally require high fatty acid /PC ratio, high enzyme concentrations, long reaction times, and low water content. Saturated fatty acids (caprilic acid, lauric acid, miristic acid), monounsaturated acids (oleic acid) and PUFAs (EPA and DHA from marine sources, CLA) can be incorporated with different efficiency (16-53 % of incorporated fatty acid) and yields (14-64 %) [46, 51]. Acidolysis catalyzed by lipase in a solvent system, can reduce the enzyme concentration and the reaction times, facilitating the mixing of enzyme and substrates, but hydrolysis reactions can be faster and the incorporation of fatty acids in the lysolecithin can be higher. Acidolysis in freesolvent systems can operate at higher temperatures, with higher reaction rate if lipase is not significantly affected in its enzymatic activity however the work temperature for acidolysis in solvent systems is limited by the boiling point of solvent. Alcoholysis of PLs by lipase can be used to simultaneously produce LPL and fatty acid esters (Figure 10). Soybean PC and short- and long-chain alcohols (methanol, ethanol, and C4, C8, C12, and C18 alcohols) have been used as substrates. Glycerol can be used as alcohol

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and the reaction is known as ―glycerolysis‖ [53]. Immobilized lipase from Rhizomucor miehei is significantly active on short-chain alcohols such as ethanol, while crude Thermomyces lanuginosa lipase catalyzed the alcoholysis in octanol, but it activity dropped significantly as the alcohol chain length decreased [54]. Later, the lack of selectivity of immobilized Rhizomucor miehei lipase with respect to fatty alcohol chain length was documented [55]. Enzymatic alcoholysis is characterized by high yields with values more than 70 % and up to 98% for the yield of LPLs and about or more than 80 % for yield of esters [54, 55]. Moreover the products should be of high quality due to the mild reaction conditions employed and simpler processing could be required to product separation. Small amounts of water can enhance significantly the reaction rate. Water can be added to reaction mixture [54] or provided with immobilized enzyme [55]. Enzymatic transesterification or interesterification between PC from soybean or egg-yolk, and methyl or ethyl esters of different fatty acids, have been also applied to change fatty acids in sn-1 position of PC (Figure 10). By means this process, capric acid (C10:0), lauric acid (C12:0) and myristic acid (C14:0) can be introduced in PC, using immobilized lipase from Rhizomucor miehei in a free-solvent system, to enhance the surface-active properties of soy lecithin [56]. C10:0, C12:0, and C14:0 were incorporated by 8.4 %, 14.1 % and 15.7 %, respectively. Lecithin modified with new saturated fatty acids lowers the interfacial tension against water and the degree of lowering the interfacial tension depends of the nature and amount of fatty acid incorporated. Moreover the percentage of linolenic acid (C18:3) was increased by about 10 units by tranesterification of PC with methyl esters from linseed oil, improving the nutritional quality of soy lecithin [56]. EPA and DHA were also incorporated in PC by transesterification with lipase leading to a product mixture of 39 % PC, 44 % LPC, and 17 % of sn-glycerol-3-phosphatidylcholine. The LPC fraction comprised 70 % EPA, whereas the PC component contained 58 % EPA [57]. The interesterification reaction was found to proceed at a comparable rate to the acidolysis reaction, and similar incorporation levels of EPA and DHA were obtained at equilibrium. The hydrolysis side reactions were much more extensive in the interesterification reaction than the acidolysis reaction (39 %). Transacylation of soybean PC with monoacylglycerols (MAGs) were about 20 % with C18:2MAG and C18:3MAG [58]. MAGs with C6 and C8 saturated acyl groups also gave very low transacylation and using diacylglycerols (DAGs) with those acyl groups transacylation was moderately improved. In last years, interesterification reactions between soy PLs and methyl esters of hydroxy and epoxy fatty acids, have been used to produce modified PLs using immobilized lipase [59]. As much as 65 % of hydroxyl-oleic acid, 57 % of hydroxyl-stearic acid, and 43 % of epoxy-oleic acid were incorporated. The corresponding incorporations of these acids were 51, 49, and 35 %, respectively, using sodium methoxide as chemical catalyst. The modified PLs showed distinct changes in surface properties such as effectiveness of interfacial tension reduction, critical micellar concentration, surface excess concentration, minimum area/ molecule, and free energy change of micellization. These modified PLs may find new applications as surface-active agent. Lipases catalyze the esterification mainly at the sn-1 position of a glycerophosphatidyl moiety (Figure 10). This process is a simple one-step esterification, where only two substrates, a free fatty acid (FFA) and a molecule with a glycerophosphatidyl group such as glycerophosphatidylcholine (GPC), and enzyme are needed for the reaction. Thus 1-acyl-LPC can be obtained from GPC. These reactions are water-producing reactions in non-aqueous

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media, reason why the water activity control is very important. Some approaches have been proposed to control water activity such as vacuum (20-30 mmHg of vacuum), or salt hydrate pairs [60] to remove water during the reaction but the best system is the use of a hydrophilic organic co-solvent such as dimethylformamide (DMF) [61]. The co-solvents known as ―water-mimicking solvents‖ act in a free-solvent system to keep the water activity low deprive enzyme particles of water. Conversion of 90 % was achieved using lipase from Rhizomucor miehei and operating window suggested for LPC production, including water activity of enzyme (0.33-0.53) and concentration of DMF (4-10 % vol/vol) as two parameters [61]. PA and 1-acyl-lysophosphatidic acid (1-acyl-LPA) can be produced by esterification of glycerol-3-phosphate (GP), and FFA or vinyl esters of fatty acids, using immobilized lipase from Rhizopus oryzae [62]. High total conversion (> 95 %) was observed for GP and lauric acid vinyl ester. Temperatures below melting point of the product favored precipitation and resulted in high final conversion and high product ratio [LPA/ (PA + LPA)]. Water consumption and volatile acetaldehyde formation during hydrolysis of vinyl esters shifted the reaction equilibrium to esterification. Lipase from Candida Antarctica has also been used to produce lyso-PC using vinyl esters as acyl donors, without PC formation during the proposed reaction period [63]. This lipase is non-.specific enzyme but has high selectivity towards sn-1 position during the first 24 hours of reaction. In a practical reaction system with enzymes like lipases, the acyl migration is a problem to consider that can be minimized but not avoided. The existence of side reactions of acyl migration is confirmed by the presence of 1-acyl-LPLs and 2-acyl-LPLs, between the products of transesterification and acidolysis reactions, for example. Several factors possibly influence acyl migration [64]. The reaction time is the most significant factor to control because acyl migration is increased when enlarged reaction times are used [64]. Increasing solvent polarity, or addition of water to non-polar solvents, causes lower rate of acyl migration, however, the high water activity favors hydrolysis reactions and thus results in lower yields. Excessive amounts of acyl donors have been applied to push the main reaction toward product formation. In a free-solvent system, to increase conversion higher enzyme load can be used however mixing will be extremely difficult. At elevated temperatures, the reaction rate for the acidolysis reaction becomes slower than acyl migration rates however the thermal resistance of the enzyme is limited. Certain support for the immobilization of enzymes can cause increased acyl migration in the reaction system as well. For these reasons, it is recommended that temperature, substrate ratio, and water addition should be low [64]. Acyl migration also has studied in reactions catalyzed by phospholipases [65]. In spite of many works has been carried out in laboratory scale, little effort has been made to scale up the modification of PLs by lipases to pilot plant or production scale, because of problems such as mass transfer limitations and side reactions, which result in low yield. For industrial applications, immobilized enzymes are preferred and lipases are commercially available in this form. Packed bed reactors (PBRs) were demonstrated to be advantageous over stirred tank reactors during lipase-catalyzed production of structured lipid, since the former had a much lower acyl migration [66, 67]. Moreover PBRs have high efficiency, low capital investment, ease of construction, continuous operation, and easy maintenance. In PBRs the substrate mixture is fed upward into the column containing packed enzyme by a pump (Figure 11). The heating is controlled with a water jacket or inside heating elements in large scales. The flow rate is the most crucial factor for the reactions in PBRs.

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Flow rate is measured by flow meter in the entrance of the reactor. When enzyme activity is reduced, the flow rate is regulated by means a valve to maintain the desired reaction progress.

Figure 11. Packed bed reactor for production of structured phospholipids with lipase.

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3.8.5. Phospholipase C (PLC) Phospholipase C is offered as an alternative to degumming process of vegetable oils because hydrolyzes the major PLs, PC and PE, found in crude vegetable oils, liberating as products DAG and the water-soluble phosphate esters of choline and ethanolamine, respectively [68]. Between the benefits of the use of this enzyme LPLs are not generated, significantly increases oil yields because produces DAG and no increases in FFA are observed. PLA1 and PLA2 produce FFA when they are used for enzymatic degumming of oils. Immobilized PLC from Bacillus cereus on different supports by covalent attached, can be repeatedly used for produce enantiomerically pure 1,2-DAGs, by PC hydrolysis (Figure 8) in a water-saturated organic solvent [69]. 1,2-DAGs can be recovered from organic phase and may be useful for synthesis of stereospecific compounds. Lipase-catalyzed hydrolysis of TAGs can only produce a mixture of sn-1,2 and sn-2,3 DAGs. Bacillus cereus secretes a series of isoenzymes with PLC activities being the most widely studied a PLC with high specificity toward PC. PE and phosphatidylserine (PS) are also good substrate for this PLC, while the degradation of other PLs is little or non significative. Organic phosphates such as dihydroxyacetone phosphate can be also manufactured by combination of PLC and PLD. Starting from natural PC, the substitution of the choline polar head for an alcohol donor is catalyzed by PLD (Figure 8); subsequent hydrolysis of the new

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PL obtained in the presence of PLC allows to obtainment of the phosphate of the alcohol donor [70].

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3.8.6. Phospholipase D (PLD) Phospholipase D catalyzes hydrolysis reaction indicated in Figure 9 remaining the phosphate group on glyceryl residue. In biphasic systems organic solvent/water PLD also catalyzes ―transphosphatidylation‖ to transfer the phosphatidyl residue from PLs to alcohols. PLD isoenzymes can be obtained from microbial sources (Streptomyces sp.) and from white cabbage. Primary alcohols are in general good substrates for PLD. Usually small watersoluble alcohols have a better incorporation such as, ethanol, serine, glycerol etc. Big primary alcohols with hydrophobic characteristics are more difficult to be incorporated. However, PLD from cabbage has shown activity inclusive on secondary alcohols [71]. A detailed list of transphosphatidylation reactions catalyzed by PLDs is presented in previous review [51]. Hydrolysis of head group is well known as a competitive reaction to transphosphatidylation. PLD from Streptomyces is the most active group of enzymes that act more on transphosphatidylation reaction instead of hydrolysis. Types of enzymes and acceptor alcohols are major reasons of the degree of hydrolysis. The hydrolysis level also depends on the polarity of the utilized organic solvent. Transphosphatidylation reactions can be used to produce PL analogs with N-heterocyclic head groups [71] and change head groups of natural PLs in soybean lecithin [72, 73]. Phosphatidylglycerol (PG) can be produced from soybean lecithin and glycerol, with the help of immobilized PLD [74]. PG is a surfactant with a greater hydrogen bonding capacity than PC, leading to better alveolus-stabilizing properties. Compared with controlled-pore glass, Amberlite XAD- and polyethyleneimine (PEI)-cellulose, the PLD immobilized with calcium alginate gel-enveloped PEI-glutaraldehyde, was most effective and a conversion of 87 % was observed [74]. Up to 15 batches, there was no significant decrease in the activity of immobilized PLD, and the half-life of conversion was 25 batches. When PC is used as substrate in a transphosphatidylation reaction choline produced as a byproduct easily accumulates in the vicinity of the enzyme, especially at high concentrations of PC, which inhibits the activity of enzyme, and results in lower conversion and difficult purification of products. Enzymatic oxidation of choline with choline oxidase and catalase [75] and cation-exchange resin as scavenger of choline [76] have been used to remove choline from reaction mixture. Another patented process to obtain PG from PC, firstly precipitates PG as calcium phosphatide and then converts the precipitate to its corresponding soluble salt by ion exchange in the organic solvent [77]. To avoid the use of solvents in transphosphatidylation reactions a pure aqueous suspension system has been developed using calcium sulfate to form aqueous suspension [78]. With this system lecithin can be completely converted to PS and modified PS can be easily extracted from the powder. Figure 12 shows the synthesis of two LPLs, 1-lauroyl-rac-glycerophosphate (1-LGP, lysophosphatidic acid) and 1-lauroyl-dihydroxyacetone phosphate (1-LDHAP), using a combination of two enzymes, PLC and PLD [79]. First reaction is a transphosphatidylation catalyzed by PLD, from PC and 1-monolauroyl-rac-glycerol (1-MLG) or 1-monolauroyldihydroxyacetona (1-MLDHA) as substrates. Conversions of 100 % can be achieved at low substrate alcohol concentrations. A two-phase system, diethyl ether /water, is used to easily

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recovery water insoluble complex PLs. The possibility of reutilization of the unreacted substrates makes the system very attractive. Both complex PLs are then hydrolyzed by PLC in a similar two-phase system in a very selective way.

Figure 12. Enzymatic two-step preparation of 1-lauroyl-rac-glycerophosphate (lysophosphatidic acid) and 1-lauroyl-dihydroxyacetonephosphate. PC: Phosphatidylcholine, 1-MLG: 1-Monolauroyl-rac-glycerol, 1-MLDHA: 1-Monolauroyldihydroxyacetone, 1-LPG: 1-Lauroyl-phosphatidylglycerol, 1-LPDHA: 1-Lauroylphosphatidyldihyroxyacetone, 1-LGP: 1-Lauroyl-rac-glycerophosphate, 1-LDHAP: 1-Lauroyldihydroxyacetonephosphate, X: Choline, DAG: Diacyl-glycerol, PLC: Phospholipase C, PLD: Phospholipase D, R1-R2: Fatty acids, R3: Lauric acid.

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3.9. Liposomes An important and interesting attribute of PLs is their ability to form special molecular structures called liposomes (Figure 13). When PLs are dispersed in aqueous medium, a large number of these molecules into a limited space, they will arrange themselves spontaneously to match their heads together and also their tails, forming spherical vesicles. The membranes of these liposomes are bi-layers of PLs, whose hydrophilic heads are oriented towards the external aqueous environment in one layer and towards hydrophilic core in the other layer. Hydrophobic hydrocarbon tails are oriented towards inside the membrane. Liposomes can encapsulate both hydrophilic and lipophilic materials. Water-soluble compounds can be entrapped in the water core and lipid soluble compounds aggregate in the lipid section. Liposomes resemble the lipid membrane part of cells. Liposomes can be unilamellar with only one bi-layer or contain hundreds of concentric bi-layers (multilamellar) attached like in the onion. With respect of the size of liposomes, small unilamellar vesicles have a size < 0.1 m, large unilamellar vesicles have a size range of 0.1-1.0 m, and large multilamellar vesicles can be up to 500 m in diameter. Liposomes can be prepared from soybean and egg yolk lecithin, modified lecithin, and pure PLs, principally PC and PE. Often, are also included in the membrane negatively charged lipids, such as PS and PI, ceramides such as sphingomyelin, and sterols (cholesterol,

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ergosterol, sitosterol, etc.). Recently, the use of synthetic, most often nonionic, are starting to increase. Liposomes can be created by sonication of PLs in aqueous medium, dehydrationrehydration, and newer methods such as extrusion, homogenization and microfluidization are also applied [80-82]. In the so-called ―pro-liposome method‖, an initial mixture containing vesicle forming amphiphiles, ethanol and water is converted into vesicles by a simple dilution step [82]. Liposomes are widely used as delivery vehicles for active agents in the pharmaceutical, cosmetic, food and nutrition industries, as well as in the coating industry. The substances encapsulated into liposomes are protected of thermal, oxidative and hydrolytic degradations. Thus, volatile flavor components can be entrapped in liposomes and used in baked products by microwave avoiding their degradation [83]. When these products are consumed flavor components are released in mouth and throat enhanced the sensorial quality of food. Enzymes such as proteases can be included in liposomes and released during cheese ripening [82]. Vitamins, antioxidants, antimicrobials, and high-value products like as nutraceuticals and dietary supplements can be also protected in liposomes [83, 84]. The high cost of equipment to produce industrially liposomes, and the use of solvents in some methods to make it, have contributed to reduce their use in food industries, favoring their application in cosmetic and pharmaceutical industries. Liposome formulations for pharmaceutical and cosmetic industries can be injected intravenously, intra-muscularly, subcutaneous (liquid suspensions), inhaled (aerosol of liposome suspension or lyophilized powder), applied to skin (suspension, cream, gel, ointment) or ingested (any of the physical forms). Many substances like as vitamins, drugs, and herbs, are degraded in stomach and poorly adsorbed in gastrointestinal tract. The administration of these substances in liposomes can increase the solubility and the adsorption into bloodstream enhancing their bioavailability. Liposomes with enzymes are also used for therapeutic treatments [82].

Figure 13. Unilamellar liposome structure.

Another interesting property of liposomes is their natural ability to target cancer [85]. The healthy blood vessels have endothelial cells in their walls with tight junctions however, tumor

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vessels, are diagnostically leaky. Tumor tissues have enhanced permeability and retention effect for liposomes. In healthy tissue vasculature liposomes are kept in the bloodstream. Liposome encapsulation can change spatial and temporal distribution of the encapsulated drug molecules in the body what may significantly reduce unwanted side effects and increase efficacy of the treatment. Liposomes can be used in gene therapy as a strategy to try to put a gene into the cells [86-88]. These liposomes are created as tiny fat bubbles around plasmids, which are small, circular pieces of DNA. Once injected into the body, the liposomes can fuse with cell membranes, emptying their plasmid DNA contents into the cells. The development of liposome-based vaccines has appeared in last years a new field for liposome applications. Liposomes containing proteins in their lipid bi-layers are referred as proteoliposomes, and those with viral proteins, as virosomes. For example, virosomes with influenza haemagglutinin as antigen have improved immune response and serve for efficacious vaccination for Hepatitis A and Influenza [89].

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4. CONCLUDING REMARKS In this chapter, are principally presented conventional and modern methods, to recovery and manufacture profitable products from sludge obtained in water degumming of crude vegetable oils. The lecithin manufacture by drying, deoiling and solvent fractionation is installed at large scale in industrial area, but more research is needed, on the advantages of the use of sludge different from soybean sludge, such as sunflower, canola, and rapeseed sludge. In this way, besides to obtain economic benefits, it will be contributed to the reduction of the volume of non eco-friendly wastes. The processes with supercritical fluids are not still industrially applied because to high investment and required equipment. In future, only is justified investigation about high-value products or compounds with special properties which can not be obtained using conventional methods. In the next years two areas offer interesting study opportunities, enzymatic modification of phospholipids and liposomes. Laboratory works have been developed to produce structured phospholipids, but more investigation is needed to scale up these proposals to pilot plant and industrial plants. Enzymatic bioreactor with immobilized enzymes may be studied, paying attention in the diffusion effects, and in unwanted reactions, like acyl migration. Immobilized phospholipases are not commercially available, reason why immobilization works of these enzymes at industrial level, could next be interesting. The study of new enzymes and structured phospholipids with new or enhanced properties may be also considered. With respect to liposomes, nowadays their use is developed in pharmaceutical and cosmetic industries, where the final price of the products can justify the high cost of the process. More experiences on liposome application in functional foods, dietary supplements, and premium quality products, which can be marketed with higher prices, would have to be undertaken.

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[7]

[8]

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[9]

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[16] Alasti, P. (2008). Artisan Rototherm® Dries Lecithin. Dec-28. Available from: http://www.artisanind.com/ps/docs/pa_lecithin.pdf [17] Van Nieuwenhuyzen, W. (1999). Fractionation of Lecithins. The European Food & Drink Review, Process Technology, 27-33. [18] Schneider, M. (1989). Fractionation and Purification of Lecithin. In B. F. Szuhaj (Ed.), Lecithins: Sources, Manufacture and Uses (pp. 109-130). Champaign, IL, AOCS Press. [19] Stahl, E. & Kirin, K. W. (1985). Deoiling of Crude Lecithin by Jet Extraction with Dense Carbon Dioxide. Fette Seifen Anstrichmittel, 87, 219-224. [20] Wagner, H. & Eggers, R. (1996). Extraction of Spray Particles with Supercritical Fluids in a Two-Phase Flow. AIChE Journal, 42, 1901-1910. [21] Peter, S. (1996). Supercritical Fractionation of Lipids in Supercritical Fluid Technology. In J. King, & G. R. List (Eds.), Oil and Lipid Chemistry (pp. 75-81). Champaign, IL, AOCS Press. [22] Teberikler, L., Koseoglu, S. & Akgerman, A. (2001). Deoiling of Crude Lecithin Using Supercritical Carbon Dioxide in the Presence of Co-Solvents. J. Food Sci., 66, 850-853. [23] Mukhopadhyay, M. & Singh, S. (2004). Refining of Crude Lecithin Using Dense Carbon Dioxide as Anti-Solvent. J. Supercritical Fluids, 30, 201-211. [24] Lack, E., Weidner, E., Knez, Z., Grüner, S., Weinreich, B. & Seidlitz, H. (2008). Particle Generation with Supercritical CO2. Dec-11. Available from: http://www.natex.at/ [25] Wu, Y. & Wang, T. (2004). Fractionation of Crude Soybean Lecithin with Aqueous Ethanol. J. Am. Oil Chem. Soc., 81, 697-704. [26] Nasirulla, H. (2005). Physical Refinning: Electrolyte Degumming of Nonhydratable Gums from Selected Vegetable Oils. J. Food Lipids, 12, 103-111. [27] Teberikler, L., Koseoglu, S. & Akgerman, A. (2001). Selective Extraction of Phosphatidylcholine from Lecithin by Supercritical Carbon Dioxide/Ethanol Mixture. J. Am. Oil Chem. Soc., 78, 115-119. [28] Amari, J. V., Brown, P. R., Grill, C. M. & Turcotte, J. G. (1990). Isolation and Purification of Lecithin by Preparative High-Performance Liquid Chromatography. J. Chromatogr. A, 517, 219-228. [29] Balazs, P. E., Schmit, P. L. & Szuhaj, B. F. (1996). High-Performance Liquid Chromatographic Separations of Soy Phospholipids. J. Am. Oil Chem. Soc., 73, 193-197. [30] Meeren, P. V., Vanderdeelen, J., Huys, M. & Baert, L. (1990). Optimization of the Column Loadbility for the Preparative HPLC Separation of Soybean Phospholipids. J. Am. Oil Chem. Soc., 67, 815-820. [31] Meulenaer, B. D., Meeren, P. V., Vanderdeelen, J. & Baert, L. (1995). Optimization of a Chromatographic Method for the Gram-Scale Preparative Fractionation of Soybean Phospholipids. Chromatographia, 41, 527-531. [32] Yoon, T. H. & Kim, I. H. (2002). Phosphatidylcholine Isolation from Egg Yolk Phospholipids by High-Performance Liquid Chromatography. J. Chromatogr. A, 949, 209-216. [33] Zhang, W. N., He, H. B., Feng, Y. Q. & Da, S. L. (2004). Separation of Phosphatidylcholine by Using High-Performance Displacement Chromatography. J. Chromatogr. A, 1036, 145-154. [34] Joshi, A., Paratkar, S. G. & Thorat, B. N. (2006). Modification of Lecithin by Physical, Chemical and Enzymatic Methods. Eur. J. Lipid Sci. Technol., 108, 363-373.

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[35] Van Nieuwenhuyzen, W. & Tomás, M. C. (2008). Update on Vegetable Lecithin and Phospholipid Technologies. Eur. J. Lipid Sci. Technol., 110, 472-486. [36] Baldwin, E. A., Nisperos, M. O., Hagenmaier, R. D. & Baker, R. A. (1997). Use of Lipids in Coatings for Food Products. Food Technol., 51, 56-64. [37] Yang, B., Zhou, R., Yang, J. G., Wang, Y. H. & Wang, W. F. (2008). Insight into Enzymatic Degumming Process of Soybean Oil. J. Am. Oil Chem. Soc., 85, 421-425. [38] Morgado, M. A. P., Cabral, J. M. S. & Prazeres, D. M. S. (1996). Phospholipase A2Catalyzed Hydrolysis in a Continuous Reversed-Micellar Membrane Bioreactor. J. Am. Oil Chem. Soc., 73, 337-346. [39] Doig, S. D. & Diks, R. M. M. (2003). Toolbox for Exchanging Constituent Fatty acids in lecithin. Eur. J. Lipid Sci. Technol., 105, 359-367. [40] Maroto, B., Camusso, S. & Zaritzky, N. (2001). Estudio Cinético de la Reacción de Hidrólisis de Lecitina de Soja Pura en Polvo con Fosfolipasa A2 Inmovilizada. Grasas y Aceites, 52, 33-37. [41] Maroto, B., Camusso, S. & Zaritzky, N. (2001). Estudio de la Distribución de los Tiempos de Residencia en un Reactor Tubular para la Hidrólisis de Lecitina de soja con Fosfolipasa A2 Inmovilizada. Grasas y Aceites, 52, 297-304. [42] Maroto, B., Camusso, S. & Zaritzky, N. (2004). Evaluación de los Efectos Difusionales sobre la Cinética de Hidrólisis de Lecitina de Soja con Fosfolipasa A2. Grasas y Aceites, 55, 148-154. [43] Kim, J., Lee, C. S., Oh, J. & Kim, B. G. (2001). Production of Egg Yolk Lysolecithin with Immobilized Phospholipase A2. Enzyme and Microbial Technology, 29, 587-592. [44] Aura, A. M., Forssell, P., Mustranta, A. & Poutanen, K. (1995). Transesterification of Soy Lecithin by Lipase and Phospholipase. J. Am. Oil Chem. Soc., 72, 1375-1379. [45] Park, C. W., Kwon, S. J., Han, J. J. & Rhee, J. S. (2000). Transesterification of Phosphatidylcholine with Eicosapentaenoic Acid Ether Ester Using Phospholipase A2 in Organic Solvent. Biotechnol. Lett., 22, 147-150. [46] Hossen, M. & Hernandez, E. (2005). Enzyme-Catalyzed Synthesis of Structured Phospholipids with Conjugated Linoleic Acid. Eur. J. Lipid Sci. Technol., 107, 730-736. [47] Vikbjerg, A. F., Mu, H. & Xu, X. (2007). Synthesis of Structured Phospholipids by Immobilized Phospholipase A2 Catalyzed Acidolysis. J. Biotechnol., 128, 545-554. [48] Kim, J. K., Kim, M. K., Chung, G. H., Choi, C. S. & Rhee, J. S. (1997). Production of Lysophospholipid Using Extracellular Phospholipase A1 from Serratia sp. MK1. J. Microbiol. Biotechnol., 7, 258-261. [49] Vikjeeta, T., Reddy, J. R. C., Rao, B. V. S. K., Karuna, M. S. L. & Prasad, R. B. N. (2004). Phospholipase-Mediated Preparation of 1-ricinoleoyl-2-acyl-sn-glycero-3phosphocholine from Soya and Egg Phosphatidylcholine. Biotechnol. Lett., 26, 10771080. [50] Garcia, H. S., Kim, I. H., Lopez-Hernandez, A. & Hill, C. G. Jr. (2008). Enrichment of Lecithin with n-3 Fatty Acids by Acidolysis Using Immobilized Phospholipase A1. Grasas y Aceites, 59, 368-374. [51] Zheng, G., Vikbjreg, A. F. & Xu, X. (2005). Enzymatic Modification of Phospholipids for Functional Applications and Human Nutrition. Biotechnol. Advances, 23, 203-259. [52] Hara, F., Nakashima, T. & Fukuda, H. (1997). Comparative Study of Commercial Available Lipases in Hydrolysis Reaction of Phosphatidylcholine. J. Am. Oil Chem. Soc., 74, 1129-1132.

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[53] Ghosh, M. & Bhattacharyya, D. K. (1995). Lipase-Catalyzed Glycerolysis Reaction of Soy Phospholipids. J. Oil Technol. Assoc. India, 27, 175-177. [54] Sarney, D. B., Fregapane, G. & Vulfson, E. V. (1994). Lipase-Catalyzed Synthesis of Lysophospholipids in a Continuous Bioreactor. J. Am. Oil Chem. Soc., 71, 93-96. [55] Ghosh, M. & Battacharyya, D. K. (1997). Enzymatic Alcoholysis Reaction of Soy Phospholipids. J. Am. Oil Chem. Soc., 74, 597-599. [56] Ghosh, M. & Battacharyya, D. K. (1997). Soy Lecithin-Monoester Interchange Reaction by Microbial Lipase. J. Am. Oil Chem. Soc., 74, 761-763. [57] Haraldson, G. G. & Thorarensen, A. (1999). Preparation of Phospholipids Highly Enriched with n-3 Polyunsaturated Fatty Acids by Lipase. J. Am. Oil Chem. Soc., 76, 1143-1149. [58] Hara, S., Hasuo, H., Nakasato, M., Higaki, Y. & Totani, Y. (2002). Modification of Soybean Phospholipids by Enzymatic Transacylation. J. Oleo Sci., 51, 417-421. [59] Das, S. & Bhattacharyya, D. K. (2006). Preparation of Surface-Active Properties of Hydroxy and Epoxy Fatty Acid-Containing Soy Phospholipids. J. Am. Oil Chem. Soc., 83, 1015-1020. [60] Han, J. J., & Rhee, J. S. (1998). Effect of Salt Hydrate Pairs for Water Activity Control on Lipase-Catalyzed Synthesis of Lysophospholipids in a Solvent-Free System. Enzyme Microb. Technol., 22, 158-164. [61] Kim, J. & Kim, B. G. (2000). Lipase-Catalyzed Synthesis of Lysophosphatidylcholine Using Organic Cosolvent for in situ Water Activity Control. J. Am. Oil Chem. Soc., 77, 791-797. [62] Virto, C., Svensson, I. & Adlercreutz, P. (1999). Enzymatic Synthesis of Lysophosphatidic Acid and Phosphatidic Acid. Enzyme Microb. Technol., 24, 651-658. [63] Virto, C. & Adlercreutz, P. (2000). Lysophosphatidylcholine Synthesis with Candida Antarctica lipase B (Novozymes 435). Enzyme Microb. Technol., 26, 630-635. [64] Vikbjerg, A. F., Mu, H. & Xu, X. (2006). Elucidation of Acyl Migration during LipaseCatalyzed Production of Structured Phospholipids. J. Am. Oil Chem. Soc., 83, 609-614. [65] Plückthun, A. & Dennis, E. A. (1982). Acyl and Phosphoryl Migrationin Lysophospholipids: Importance in Phospholipid Synthesis and Phospholipase Specifity. Biochem., 21, 1743-1750. [66] Xu, X. S., Balchen, C., Høy, E. & Adler-Nissen, J. (1998). Production of Specific Structured Lipids by Enzymatic Interesterification in a Pilot Continuous Enzyme Bed Reactor. J. Am. Oil Chem. Soc., 75, 1573-1579. [67] Vikbjerg, A. F., Peng, L., Mu, H. & Xu, X. (2005). Continuous Production of Structured Phospholipids in a Packed Bed Reactor with Lipase from Thermomyces lanuginosa. J. Am. Oil Chem. Soc., 82, 237-242. [68] Verenium Corporatium. Purifine Enzyme. 2008-Dec-20. Available from: http://www.verenium.com/ [69] Anthonsen, T., D‘Arrigo, P., Pedrocchi-Fantoni, G., Secundo, F., Servi, S. & Sundby, E. (1999). Phospholipids Hydrolysis in Organic Solvents Catalised by Immobilised Phospholipase C. J. Mol. Catal. B Enzymatic, 6, 125-132. [70] D‘Arrigo, P., Piergianni, V., Pedrocchi-Fantoni, G. & Servi, S. (1995). Indirect Enzymatic Phosphorylation: Preparation of Dihydroxyacetone Phosphate. J. Chem. Soc., Chem. Commun., 2505-2506.

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[71] Ulbrich-Hofmann, R., Haftendorn, R., Dittrich, N., Hirche, F. & Aurich, I. (1998). Phospholipid Analogs-Chemoenzymatic Syntheses and Properties as Enzyme Effectors. Fat. Sci. Technol., 100, 114-120. [72] Ulbrich-Hofmann, R. (2003). Enzyme-Catalysed Transphosphatodylation. Eur. J. Lipid Sci. Technol., 105, 305-308. [73] Doig, S. D. & Diks, R. M. M. (2003). Toolbox for Modification of the Lecithin Headgroup. Eur. J. Lipid Sci. Technol., 105, 368-376. [74] Wang, X. G., Qiu, A. Y., Tao, W. Y. & Shen, P. Y. (1997). Synthesis of Phosphatidylglycerol from Soybean Lecithin with Immobilized Phospholipase D. J. Am. Oil Chem. Soc., 74, 87-91. [75] Juneja, L. R., Taniguchi, E., Shimizu, S. & Yamane, T. (1992). Increasing Productivity by Removing Choline in Conversión of Phosphatidylcholine to Phosphatidylserine by Phospholipase D. J. Ferment. Bioeng., 73, 357-361. [76] Rich, J. O. & Khmelnitsky, Y. L. (2001). Phospholipase D-Catalyzed Transphosphatidylation in Anhydrous Organic Solvents. Biotechnol. Bioeng., 7, 2374-2377. [77] Tremblay, P. A., Marziani, F., Tino, J. A. F. & Pilkiewicz F. G. (1990). Enzymatic Synthesis of Soluble Phosphatides from Phospholipids. Assignee: The Liposome Co. Inc., WO Patent 91/16444. [78] Iwasaki, Y., Mizumoto, Y., Okada, T., Yamamoto, T., Tsutsumi, K. & Yamane, T. (2003). J. Am. Oil Chem. Soc., 80, 653-657. [79] Virto, C. & Adlercreutz, P. (2000). Two-Enzyme System for the Synthesis of 1-Lauroyl-rac-glycerophosphate (Lysophosphatidic acid) and 1-Lauroyl-dihydroxyacetonephosphate. Chem. Phys. Lipids, 104, 175-184. [80] Watwe, R. M. & Bellare, J. R. (1995). Manufacture of Liposomes: a Review. Curr. Sci., (India), 68, 715-724. [81] Sorgi, F. L. & Huang, L. (1996). Large Scale Production of DC-Chol Cationic Liposomes by Microfluidization. Int. J. Pharm., 144, 131-139. [82] Walde, P. & Ichikawa, S. (2001). Enzymes inside Lipid Vesicles: Preparation, Reactivity and Applications. Biomol. Engn., 18, 143-177. [83] Taylor, T., Davidson, P., Bruce, B. & Weiss, J. (2005). Liposomal Nanocapsules in Food Science and Agriculture. Crit. Rev. Food Sci. Nutr., 45, 587-605. [84] Keller, B. C. (2001). Liposomes in Nutrition. Trends Food Sci. Tech., 12, 25-31. [85] Rabasco-Alvarez, A. M. & Gonzalez-Rodriguez, M. L. (2000). Lipids in Pharmaceutical and Cosmetic Preparations. Grasas y Aceites, 51, 74-96. [86] Chonn, A. & Cullis, P. R. (1998). Recent Advances in Liposome Technologies and their Applications for Systemic Gene Delivery. Adv. Drug Del. Rev., 30, 73-83. [87] Suzuki, R., Takizawa, T., Negishi, Y., Hagisawa, K., Tanaka, K., Sawamura, K., Utoguchi, N., Nishioka, T. & Maruyama, K. (2007). Gene Delivery by Combination of Novel Liposomal Bubbles with Perfluoropropane and Ultrasound. J. Control. Release, 117, 130-136. [88] Ino, K., Kawasumi, T., Ito, A. & Honda, H. (2008). Plasmid DNA Transfection Using Magnetite Cationic Liposomes for Construction of Multilayered Gene-Engineered Cell Sheet. Biotechnol. Bioeng., 100, 168-176. [89] Zurbriggen, R., Novak-Hofer, I., Seelig, A. & Glück, R. (2000). IRIV-Adjuvanted Hepatitis A Vaccine: in vivo Absorption and Biophysical Characterization. Progr. Lipid Res., 39, 3-18.

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Chapter 3

A SURVEY OF METHODS FOR CHARACTERIZATION OF SULFATE-REDUCING MICROORGANISMS Bidyut R. Mohapatra*, W. Douglas Gould, Orlando Dinardo and David W. Koren CANMET Mining and Mineral Sciences Laboratory, Natural Resources Canada, 555 Booth Street, Ottawa, Ontario K1A0G1, Canada

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ABSTRACT Sulfate-reducing microorganisms (SRM) comprising of anaerobic bacteria and anaerobic archaea are an integral part of the global sulfur and carbon cycle for dissimilatory reduction of sulfate using sulfate as electron acceptor for the degradation of organic compounds with concomitant production of hydrogen sulfide. SRM are known to be ubiquitous in natural and engineering environments, including in the methanogenic and sulfidogenic sludge generated by municipal and industrial wastewater treatment facilities. Additionally, SRM are responsible for ca. 50% of the organic matter mineralization in wastewater treatment systems. SRM are detrimental to the safety, reliability and integrity of wastewater treatment facilities, and to the public and environmental health because hydrogen sulfide is highly corrosive, neurotoxic and malodorous. However, SRM have attracted significant industrial interest as potential biocatalysts for environmentally friendly remediation of acid mine and rock drainage, removal and reuse of sulfur compounds from waste effluents and off gases, recovery of heavy metals from wastewater and sludge, and biotransformation of petroleum- and hydrocarbon-containing sludge. Considerable efforts have been devoted for development of robust techniques to provide an early detection of SRM occurrence, to identify novel strains for bioremediation, and/or to evaluate their ecophysiological roles in the natural and engineering environments. This chapter provides an overview on the distribution and phylogenetic diversity of microorganisms associated with dissimilatory sulfate reduction, and the recent development of techniques used for the characterization of SRM in natural and industrial environments, including in the process of biological remediation of toxic wastewater and sludge.

* Corresponding address: Bidyut Ranjan Mohapatra, #203-Fantable Ninomiya-I, Ninomiya 1-10-19, Tsukuba, Ibaraki 305-0051, JAPAN, Fax: +81-29-861-6733, E. mail: [email protected] Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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1. INTRODUCTION Sulfur compounds with an oxidation state from –2 (completely reduced: S2–) to +6 (completely oxidized: SO42–) are ubiquitous in the environments (Suzuki, 1999). These compounds are mainly present as pyrite (FeS2) or gypsum (CaSO4) in rocks and sediments and as sulfate (SO42–) in seawater (Kellog et al., 1972). Sulfur compound-containing effluents are produced by several industries, such as mining, oil and natural gas exploration, petrochemical refineries, liquefied petroleum gas plant, tanneries, food processing, textile, pulp and paper manufacturing, and sewage treatment facilities (Oude Elferink et al., 1994; Colleran et al., 1995; Weijma et al., 2000). The global sulfur cycle responsible for the mineralization of sulfur-compound is mediated by chemical and microbiological processes (Kellog et al., 1972; Granat et al., 1976). The microbiological processes responsible for the transformation of sulfur compounds in nature consist of aerobic and anaerobic reactions (Peck and Legall, 1994). Sulfate, the highest oxidation state of sulfur, is chemically inert and nontoxic. Sulfate is reduced to sulfide by bacteria, fungi and plants for biosynthesis of sulfur containing amino acids and enzymes. This process is known as assimilatory sulfate reduction (Postgate, 1984). The sulfur cycle is also predominated by oxidative and reductive reactions (Canfield and Raiswell, 1999). The oxidation of sulfide in virtually all biotopes including alkaline, neutral and acidic environments is catalyzed by lithotrophic, phototrophic and heterotrophic microorganisms (reviewed in Mohapatra et al., 2008). The dissimilatory sulfate reduction is considered as the main anaerobic process in the biomineralization of organic matter in the natural and engineered environments, accounting for up to 50% of its degradation in marine, estuarine and freshwater lake sediments and aerobic wastewater treatment systems (Jørgensen, 1982; Jones and Simon, 1984; Sass et al., 1997; Okabe et al., 1999). The dissimilatory sulfate reduction is carried out by a group of phylogenetically diverse anaerobic microorganisms (SRM) belong to the domain bacteria and archaea (Gibson, 1990; Stackebrandt et al., 1995). SRM are ubiquitous in the environment and have pivotal role in the biogeochemical cycling of carbon and sulfur (Postgate, 1984; Rabus et al., 2006). In the dissimilatory sulfate reduction, the sulfate ion acts as terminal electron acceptor for the dissimilation of carbon substrates with concomitant production of hydrogen sulfide per mole of sulfate ion reduction (Eq. 1) (Berner, 1974). 2CH2O + SO42– → H2S + 2HCO3–

(1)

Generally, the dissimilatory sulfate reduction inside the microbial cells has been catalyzed by an array of enzymes, including ATP (adenosine triphosphate) sulfurylase (sta) (Eq. 2), APS (adenosine-5'-phosphosulfate reductase) (apr) (Eq. 3) and dissimilatory sulfite reductase (dsr) (Eq. 4). The biochemical details of the dissimilatory sulfate reduction pathways have been reviewed previously (Peck, 1993; Postgate, 1984). ATP + SO24 –

sta

APS + H+ + 2e–

APS + Pyrophosphate apr

HSO3– + AMP

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(2) (3)

A Survey of Methods for Characterization of Sulfate-Reducing …

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HSO3– + 6H+ + 6e–

dsr

HS– + 3H2O

65 (4)

SRM have a significant negative impact on natural and industrial environments due to production of sulfide. Sulfide is malodorous, neurotoxic, poisonous to industrial catalysts, and highly corrosive to metallic infrastructures (Roth et al., 1995; Hamilton, 1998; Mattorano and Merinar, 1999). According to United States Environmental Protection Agency, a maximum acceptable level of hydrogen sulfide for fish and other aquatic life is 2 µg/l. SRM are considered as the principal causative organism for the induction of microbiologically influenced corrosion (MIC) (Tatnall and Pope 1993; Droffelaar and Atkinson, 1995). A survey in United States has documented that the annual cost of corrosion is ca. 4–5% of the gross domestic product and it has been estimated that 30–40% of the corrosion failures are attributed to MIC (Pound, 1998; Virmani, 2002). SRM are a major concern to both offshore and onshore oil and natural gas industries where they cause severe problems including plugging of oil and gas reservoirs, souring of oil and gas deposits, and to the safety, reliability and integrity of production, refining, transmission, and storage facilities by causing MIC (Cord-Ruwisch et al., 1987; Herbert, 1987; Graves and Sullivan, 1996; Farthing, 1997; Magot et al., 2000; Zhu et al., 2003). In anaerobic microbial treatments of agro-industrial and municipal wastes for production of methane, SRM can compete with methanogenic microorganisms for available electron donors, such as acetate and hydrogen, and have the potential to inhibit the methanogenic decomposition and to accelerate souring of biogas with sulfide production (Gurijala and Suflita, 1993; Harvey et al., 1997). On the other hand, SRM have been emerging as efficient biocatalysts in the environmentally friendly treatment of acid mine and rock drainage (Gould and Kapoor, 2003; Johnson and Hallberg, 2005); removal and reuse of sulfur compounds from wastewater, sludge and off gases (Janssen et al., 2001; Rao et al., 2007); recovery of heavy metals including Cd, Co, Cu, Fe, Ni, and Zn from wastewater, sludge and groundwater by precipitation as metal sulfides (Hulshoff-Pol et al., 1998; Lens et al., 2007); and anaerobic biotransformation of recalcitrant organic pollutants like aliphatic hydrocarbons, BTEX compounds (benzene, toluene, ethylbenzene, and ortho-, meta- and para-xylene), phenol and polyaromatic hydrocarbons (Ensley and Suflita, 1995; Zhang and Young, 1997; Kniemeyer et al., 2007; Dou et al., 2008; Foght, 2008). The development of appropriate strategies to control and/or abate the detrimental effect of SRM and to enhance the biocatalytic efficacy of SRM for the bioremediation of toxic metals and organic pollutants in natural and industrial systems depends primarily on the compositions of the local SRM community (Lens et al., 2007). Robust methods for characterization of SRM community will not only provide information about their ecophysiological role in the environments but also help sustain the reliability and integrity of industrial settings by providing an early warning of their occurrence. In this review, we have provided an overview on the distribution and phylogeny of SRM in the environments and recent developments on the methods for the characterization of SRM.

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2. DISTRIBUTIONS OF SRM SRM are ubiquitous in the terrestrial and aquatic ecosystems. SRM have been isolated from diverse natural habitats, such as anaerobic biofilms (Raskin et al., 1996), marine, estuarine and freshwater sediments (Jørgensen, 1982; Jones and Simon, 1984; Devereux et al., 1996b; Sass et al., 1997), salt marsh (Devereux et al., 1996a; Rooney–Varga et al., 1997), hydrothermal vents (Jeanthon et al., 2002), mud volcanoes (Stadnitskaia et al., 2005), hypersaline pond and microbial mats (Minz et al., 1999), deep subsurface (Kovacik et al., 2006), oil and natural gas–field (Herbert, 1987; Nilsen et al., 1996), gas hydrates (Lanoil et al., 2001), uranium and petroleum hydrocarbon contaminated aquifer (Kleikemper et al., 2002a; Kleikemper et al., 2002b), acid mine drainage sites (pH < 2) (Gould and Kapoor, 2003) and soda lake (pH > 10) (Foti et al., 2007). Their presence have also been detected in several industrial environments: municipal and industrial wastewater treatment plants, high– pressure oil and natural gas transmission pipelines, offshore and onshore metallic infrastructures, cooling towers and sour whey digester (Zellner et al., 1989; Ramsing et al., 1993; Rao et al., 1993; Okabe et al., 1999; Okabe et al., 2003; Zhu et al., 2003). SRM are also detected in rhizosphere, rumen contents, termite guts, feces of humans and animals, and in gutless marine worm (Brauman et al., 1990; Lin et al., 1997; Hines et al., 1999; Dubilier et al., 2001; Loubinoux et al., 2002).

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3. PHYLOGENY OF SRM The sulfate reducers have been separated into two groups, such as incompletely– oxidizing SRM and completely–oxidizing SRM based on their nutrition (Holt et al., 1994). The typical substrates for incompletely–oxidizing SRM are lactate, hydrogen and propionate, and for completely–oxidizing SRM are fatty acids. The incompletely–oxidizing SRM catabolize organic compounds incompletely to acetate, which does not oxidize further. The completely–oxidizing SRM catabolize organic compounds completely to carbon dioxide. The comparative genomic analysis of the nearly complete 16S rRNA gene sequence of dissimilatory sulfate reducing microorganisms indicated that the SRM are polyphyletic and composed of seven phylogenetic lineages of Gram–negative and Gram–positive bacteria, and archaea (Muyzer and Stams, 2008). The bacteria occupied five lineages (deltaproteobacteria, clostridia, nitrospirae, thermodesulfobacteria and thermodesulfobiaceae), and the archaea occupied two lineages (euryarchaeota and crenarchaeota) in the phylogenetic tree (Muyzer and Stams, 2008).

3.1. Gram-negative Mesophilic SRM The mesophilic Gram-negative SRM belong to the delta subclass of Proteobacteria. These organisms can grow with temperature ranges from 20oC to 40oC with optimum ca. 30oC. The majority of bacteria characterized so far in the deltaproteobacteria group of SRM are mesophiles. These SRM, which consist of twelve genera, are the most ubiquitous microorganisms in the natural and industrial systems. Seven genera (Desulfoarculus,

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Desulfobacter, Desulfobacterium, Desulfococcus, Desulfomonile, Desulfonema and Desulfosarcina) are considered as the completely–oxidizing SRM. The other five genera (Desulfovibrio, Desulfobotulus, Desulfobulbus, Desulfohalobium and Desulfomicrobium) belong to incompletely–oxidizing SRM (Holt et al., 1994). Desulfobacter, Desulfobulbus, Desulfomicrobium and Desulfovibrio are reported to have the ability to fix nitrogen in the aquatic environments (Postgate et al., 1988; Widdel and Hansen, 1992). The species of the genera Desulfobacter, Desulfobacterium, Desulfohalobium, Desulfonema and Desulfosarcina were isolated from the marine and estuarine environments (Widdel and Bak, 1992). The presence of the species of the genera Desulfoarculus, Desulfobotulus, Desulfomicrobium, and Desulfomonile in the freshwater ecosystems have been documented (Widdel and Bak, 1992; Fauque, 1995).

3.2. Gram-negative Psychrophilic SRM

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The natural environment for most sulfate reducers is cold, since 90% of the sea floor has temperatures below 4oC. It is obvious that sulfate reducers should be able to grow at the low temperatures. However, only three psychrophilic genera (Desulfofrigus, Desulfofaba and Desulfotalea) have been reported so far from the sediment samples of Arctic Ocean (Knoblauch et al., 1999). The psychrophilic SRM belong to deltaproteobacteria. These bacteria usually grow in the temperature range from –1.8 oC to +19 oC with optimum growth at 7–10 oC. Desulfofrigus oceanense oxidizes the fatty acids (formate, acetate, butyrate and valerate) completely to carbon dioxide, whereas Desulfofrigus fragile, and the species of the genera Desulfofaba and Desulfotalea oxidize the fatty acids incompletely to acetate (Knoblauch and Jørgensen, 1999).

3.3. Gram–negative Thermophilic SRM The Gram–negative thermophilic bacteria are separated from the Gram–negative mesophiles by constituting 3 lineages: Nitrospirae (genus Thermodesulfovibrio), Thermodesulfobacteria (genus Thermodesulfobacterium) and Thermodesulfobiaceae (genus Thermodesulfobium) in the phylogenetic tree. Bacteria in these lineages were isolates from thermal environments including hot springs, hot oil reservoirs, and deep–sea hydrothermal vents (Zeikus et al., 1983; Henry et al., 1994; Nilsen et al., 1996; Mori et al., 2003). These bacteria can grow in the temperatures ranging from 45oC to 70oC with optimum ca. 60–65oC (Widdle, 1992a; Nilsen et al., 1996). These bacteria can grow with H2/CO2 produced during geothermal reaction and/or by incomplete oxidation of fatty acids to acetate (Widdel, 1992a).

3.4. Gram–positive SRM The Gram–positive SRM are spore–forming bacteria and occupy a separate phylogenetic lineage (Clostridia). The Clostridia lineage contains three genera of SRM, such as Desulfotomaculum, Desulfosporosinus and Desulfosporomusa (Widdel, 1992b; Stackebrandt et al., 1997; Plugge et al., 2002). Most of the species in this group belong to the genus

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Desulfotomaculum. The other two genera Desulfosporosinus and Desulfosporomusa contain three (Desulfosporosinus youngii, D. meridiei and D. orientis) and one species (Desulfosporomusa polytropa), respectively. This group of SRM contains heat–resistant endospore which may help in their survival in extreme environments by avoiding desiccation and oxic condition. Majority of Gram–positive SRM are mesophilic with growth temperatures from 25 oC to 45 oC. Some species can grow in moderately higher temperatures (40-65 oC) (Widdel, 1992b). Based on their capabilities of oxidizing organic substrates, the species of Gram–positive SRM can be classified into completely–oxidizing SRM and incompletely–oxidizing SRM by formation of the fermentation products carbon dioxide and acetate, respectively (Fauque, 1995). Unlike the mesophilic Gram–negative SRM, some species of Gram–positive SRM (Desulfotomaculum spp.) are capable of utilizing Fe (III) as the sole source of terminal electron acceptor for their growth and proliferation (Tebo and Obraztsova, 1998).

3.5. Archaeal SRM

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The cluster analysis of 16S rRNA sequences of the archaeal SRM produces two lineages (euryarchaeota and crenarchaeota). The phylogenetic lineage euryarchaeota contains three species Archaeoglobus profundus, A. veneficus and A. lithotrophicus. Two species Thermocladiun modestius and Caldivirga maquilingensis are clustered in crenarchaeota phylogenetic lineage. The euryarchaeons and crenarchaeons were isolated from the anoxic environments of submarine hydrothermal vent and acidic hot spring, respectively. These archaeons are usually extremophiles and grow over a temperature > 80oC, salinity > 35 ppt and pH > 2.3 (Burggraf et al., 1990; Stetter, 1992; Ito et al., 1998; Wagner et al., 1998; Ito et al., 1999). They are heterotrophs and oxidize the organic carbon substrates incompletely to acetate.

4. CHARACTERIZATION OF SULFATE-REDUCING MICROORGANISMS The diversity and presence of sulfate-reducers in virtually all anaerobic environments consider them as an important regulator of a variety of ecological processes including organic matter turnover, biodegradation of organic pollutants in anaerobic soils and sediments, and mercury methylation (Rabus, 2006). SRM are also a threat to the safety and reliability of metallic infrastructure by inducing corrosion (Droffelaar and Atkinson, 1995). In addition some SRM have been implicated in human diseases (Loubinoux et al., 2002). The functional and numerical importance of SRM in critical processes of ecosystem functioning, corrosion, diseases, inhibition and souring of biogas, and environmental remediation insist to develop robust tool to monitor the spatial and temporal changes in SRM community. Considerable efforts have been directed towards the development of rapid and accurate methods for detection and enumeration of SRM in natural and industrial environments. In general, these methods have been divided into four categories: microbiological, immunological, biochemical and molecular methods.

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4.1. Microbiological Methods 4.1.1. Broth bottle dilution This method is popularly known as RP-38 practice and was developed by American Petroleum Institute based on the most probable number (API, 1965). The RP-38 has been used as the standard practice for the detection and enumeration of SRM in industrial settings. The RP-38 method employs a serial dilution technique with a standardized medium, and especially estimates the density of SRM present in the sample. The broth medium contains sodium lactate as carbon and terminal electron acceptor. The broth is dispensed into the culture bottles and a small nail is incorporated inside the bottle for an iron source and to react with sulfide. After an incubation of four weeks the density of SRM in the culture bottles is evaluated by the formation of a black precipitate of iron sulfide. The modified versions of RP38 have been developed for specific environments including activated sludge, marine sediments, and samples from the oil and natural gas production facilities (Fedorak et al., 1987; Gibson et al., 1987; Tanner, 1989). Some studies have also used radiotracers by incorporating 35SO42- into RP-38 medium and estimate the growth of SRM by measuring the production of 35S-2 (Vester and Ingvorsen, 1998).

4.1.2. Agar deeps This method is a modification of broth bottle method by incorporation of agar into the medium to avoid dehydration. The agar deep is incubated for 5 to 8 days and is checked for blackening. Relative numbers of bacteria are estimated by noting the rapidity with which the blackening occurs (Tatnall and Pope, 1993).

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4.1.3. Melt agar This method is also a modification of broth bottle methods. This method involves placing the tubes in boiling water to liquefy the agar and then cooling the tubes until the medium reaches a temperature of 40 to 45oC before adding the sample. The tubes are then rapidly rolled over the frozen sponge sheet to form a thin coating of the agar medium around the walls of the tubes. The tubes are then incubated from 7 to 21 days. The numbers of SRM were enumerated by counting the black colonies around the walls of the tubes (Tatnall and Pope, 1993). Generally the distinct advantage of microbiological techniques is that they do not require special instruments and are sensitive to the well-identified SRM species. These methods are suitable for the enumeration of lactate-oxidizers, but not acetate-oxidizers. Attempts to prepare a universal medium for enumeration of SRM have not been successful because SRM are polyphyletic and the affinity towards the oxidation of carbon source is different. Moreover the incubation temperature is also selective. Although the RP-38 broth bottle dilution were most consistently accurate than agar deep and melt agar methods evaluated by Tatnall et al. (1988), 4 weeks incubation time and inconsistence results during 2- and 3- and 4-weeks restrict the application of RP-38 method for reliable detection of SRM.

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4.2. Immunological Methods The immunological method used for the characterization of SRM can be classified into four types: agglutination, immunodiffusion, immunofluoresence and ELISA (Enzyme Linked Immunosorbent Assay).

4.2.1. Agglutination Agglutination is a serological test in which the cell surface antigens of bacteria react with the specific antibody to form a clump. As agglutination is easily visible and each species of bacteria has a distinct type of surface antigens, this test has been considered as an important tool for differentiation of species of bacteria. Abbdollahi and Nedwell (1980) have evaluated cell surface antigens of 36 heterologous Desulfovibrio species using agglutination test and recorded a species-specific antigens for Desulfovibrio species. However, the later studies reported cross-reactivity among the species and genera (Singleton et al., 1984).

4.2.2. Immunodiffusion Immunodiffusion is a precipitin test in which cell surface antigens of bacteria and antibody are placed in a gel of agar or similar substance and allowed to diffuse towards one another to form a precipitate. This test was used by Abbdollahi and Nedwell (1980) for characterization of 36 strains of Desulfovibrio species. A genus specific internal antigen was detected in immunodiffusion test.

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4.2.3. Immunofluorescence In this method the antibody is conjugated to a fluorochrome and then allowed to react with the corresponding cell surface antigens of bacteria. The location of the antibody can thus be determined by microscopic observation of the resulting pattern of fluorescence. Norqvist and Roffey (1985) assessed cell surface antigens in the species of Desulfovibrio and Desulfotomaculum and found genus specific antigens for Desulfovibrio, and species-specific antigens for Desulfotomaculum. Similarly, Smith (1982) evaluated the immunofluroscence methods to discriminate 44 Desulfovibrio and Desulfotomaculum species and recorded highspecies specificity for both genera.

4.2.4. ELISA ELISA is an immunoassay method in which antibody or antigen is detected by the binding site of an enzyme either coupled to anti-immunoglobulin or to antibody specific for the antigen. The principle of the technique is similar to that of immunofluorescence except that the fluorochrome is replaced by an enzyme. Odom et al. (1991) examined the immunological diversity of adenosine 5'-phophosulfate (APS) reductases in the genera Desulfovibrio, Desulfotomaculum, Desulfobulbus, and Desulfosarcina using ELISA. APS reductase is a constitutive cytoplasmic enzyme present in all SRM species for dissimilatory reduction of sulfate (see Eq. 3). APS is believed to be not found in other microbial species.

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APS reductase was found to be immunologically different for the species of the genera Desulfovibrio, Desulfotomaculum, Desulfobulbus, and Desulfosarcina. The discrimination was highest for the species of Desulfovibrio. The strains of other genera were not distinguishable by the ELISA technique. APS reductase test has been used as a field detection kit for enumeration of SRM in industrial settings to access microbiologically influenced corrosion (Videla, 1996). In general, all the four immunological methods evaluated are good general detection methods for SRM. The lack of species specific reactions and the impact of cultivation medium in the expression of surface antigens restrict their application as an efficient tool for identification of SRM (Cloete and de Bruyn, 2001).

4.3. Biochemical Methods The biochemical methods used for the characterization of SRM have been divided into three types: hydrogenase test, cellular protein profiling and cellular fatty acid analysis. The hydrogenase test is used for the detection and enumeration of SRM. The cellular protein profiling and cellular fatty analysis are used for identification of the species of SRM.

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4.3.1. Hydrogenase test Hydrogen is associated with the metabolisms of SRM (Odom and Peck, 1984). In addition to utilizing molecular hydrogen directly as an electron donor for sulfate reduction, hydrogen may play a central role as an intermediate in the generation of a chemiosmotic gradient from the oxidation of organic molecules. Hydrogenase catalyzes the heterolytic cleavage of molecular hydrogen into protons and electrons (H2 = 2H+ + 2e) (Frey, 2002). This enzyme is ubiquitous in Desulfovibrio species and therefore uses as a marker for SRM detection (Matias et al., 2005). Hydrogenase test kit is manufactured by Caproco Ltd., Edmonton, Alberta, Canada for enumeration of SRB. Hydrogenase test is performed by placing samples in a vial containing the enzyme-extracting solution. After ca. 15 min the samples are filtered and placed in a clean vial in an anaerobic chamber. A gas generator provides an anaerobic atmosphere through the removal of oxygen by reaction with the generated hydrogen. In these conditions, the enzyme oxidizes the excess of hydrogen and reduces a specific dye in the solution. The hydrogenase activity is then related to the development of a blue color in less than 4 hours. The intensity of blue color is proportional to the specific numbers of SRM. This kit can detect a low detection threshold of 250 SRM cells/ml. As hydrogenase is also present in other groups of bacteria and archaea (Frey, 2002), and some species of SRM do not contain this enzyme, this method may not be suitable for accurate prediction of the density of SRM in the sample.

4.3.2. Cellular protein profiling A microbial cell expresses some 2000 different proteins which form a rich source of information for the characterization, classification and identification of microorganisms. The polyacrylamide gel electrophoresis (PAGE) of cellular proteins yields complex banding

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patterns which can be considered as highly specific fingerprints of the strains investigated. These protein electrophoregrams are highly reproducible provided strains are cultivated under reproducible conditions and standardized techniques are used. The value of cellular protein in microbial systematics has been well established (Vauterin et al., 1993). Depending on the protein electrophoretic variation within a given taxon individual strains can often be recognized by small, but specific and reproducible differences in part of their protein patterns (Kersters, 1985). Norqvist and Roffey (1985) examined the electrophoretic patterns of the envelop proteins of five strains of genus Desulfotomaculum and twelve strains of the genus Desulfovibrio by sodium dodecyl sulfate-PAGE. The protein profiles clearly distinguished the Desulfotomaculum spp. from Desulfovibrio spp. Within the genus Desulfotomaculum and Desulfovibrio, differences in the protein profiles among species as well as strains were observed. A study by Cloete and de Bruyn (2001) with the cultures of Desulfovibrio spp. (D. desulfuricans subsp. desulfuricans, D. africanus and D. gigas) and Desulfotomaculum spp. (D. nigrificans, D. orientis and D. guttoideum) reported that the expression of cellular proteins were significantly influenced by cultivation medium and therefore should be used in well characterized strains of SRM and in conjugation with other diagnostic methods.

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4.3.3. Cellular fatty acid analysis More than 300 fatty acids and related compounds have been found in the microbial cell membrane. The combination of these fatty acids is unique to the genera, species and subspecies and has been exploited for identification and characterization of microorganisms (MIDI, 2006). For the analysis of fatty acids, a bacterial culture is grown under standard cultivation conditions, and fatty acids are extracted from bulk acids. The fatty acids are then chemically modified to methyl esters. These volatile esters are identified by gas chromatography. Most of the short chain fatty acids (C9-C20) including the branched and hydroxy group containing fatty acids are used in the bacterial chemotaxonomy (MIDI, 2006). The cellular fatty acid profiles have provided valuable information for the classification of SRM. Vanshtein et al. (1992) compared the fatty acid profiles of 34 strains of Desulfovibrio species. Anteiso-15:0 defined a lineage containing D. gigas and related organisms (D. giganteus, D. fructosovorans, D. carbinolicus, D. sulfodismutans, Desulfovibrio sp. and D. alcoholovorans). Iso-17:1 and/or anteiso-15:0 fatty acids are specific characterization of Desulfovibrio species. Kohring et al. (1994) also observed the species-specific fatty acid profiles for Desulfovibrio species and a good correlation of fatty acid data with the 16S rRNA phylogenetic data. Other studies have proposed 16:0 and 17:1 cis 9 fatty acids as the biomarker for the genera Desulfobacter and Desulfobulbus (Edlund et al., 1985; Parkes and Calder, 1985; Dowling et al., 1986). Although the ability of fatty acid profiling for identification of SRM is remarkable, this method has several disadvantages including effect of the culture medium, difficulty in the interpretation of complex fatty acid profiles, and inability to discriminate at population level due to absence of distinctive fingerprints for some genera or even functional groups.

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4.4. Molecular Methods The molecular methods used for the characterization of SRM are cloning and sequencing of PCR-amplified genes, PCR with SRM-specific primers, DGGE (denaturing gradient gel electrophoresis), FISH (fluorescent in situ hybridization), T-RFLP (terminal restriction fragment length polymorphism), DNA microarrays and real-time PCR targeting the SRMspecific primers.

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4.4.1. Cloning and sequencing of PCR-amplified genes This method characterizes the microorganisms by targeting the small subunit 16S rRNA genes. The 16S rRNA genes have several advantages compared to other candidate genes, e.g. they are essential component of ribosomes, they are highly conserved, the sequence of 16S rRNA genes are taxon specific, and existence of extensive rRNA sequence reference databases for phylogenetic studies. The use of 16S rRNA gene-based analysis to characterize bacteria and archaea has increased significantly owing to the realization that >99% of the microorganisms in the environments are uncultivable under standard techniques (Amann et al., 1995; Kaeberlein et al., 2002). This method comprises of five steps: extraction of DNA from environmental sample, amplification of 16S rRNA genes by PCR, purification of PCRamplicons, screening for unique clones (insertion of target genes), sequencing of the clones by commercially available methods (ABI PRISM BigDye Terminator Cycle Sequencing Kit) and automated sequencer (ABI PRISM Sequencer) (Head, 1999). Taxonomic identification of the 16S rRNA gene sequence is performed by comparison with the available sequence databases using common search algorithm BLAST (basic local alignment search tool). Voordouw et al. (1996) used the 16S rRNA genes analysis to characterize the sulfate-reducers in western Canadian oil-field. Orphan et al. (2001) also evaluated the 16S rRNA to characterize the sulfate-reducing bacterial and archaeal communities associated with methane seep sediments from several different sites on the Californian continental margins. This method was also applied for assessment of phylogenetic diversity of SRM in a corrosive marine biofilm (Zhang and Fang, 2001) and in petroleum contaminated sediments in Guaymas Basin (Dhillon et al., 2003). Although PCR-clone-16S rRNA sequence analysis is a powerful culture-independent method for characterization of microbial population, identification of novel phylotypes and design of oligonucleotide primer for yet-to-cultured microorganisms, this method is labor intensive and time consuming. Furthermore, this method does not provide information about the link between the genetic identity of an uncultured microbe and its physiological and/or metabolic capability. It is therefore unlikely to be used as a routine environmental monitoring tool for characterization of SRM (Head, 1999). To elucidate the eco-physiological functions of metabolically active SRM in the environments, three functional genes: dsrAB (encoding the enzyme dissimilatory sulfite reductase), aprBA (encoding the enzyme dissimilatory adenosine-5'-phosphosulfate reductase) and [NiFe] hydrogenase gene (encoding the enzyme hydrogenase) have been used for the analysis (Wagner et al., 1998; Chang et al., 2001; Dhillon et al., 2003; Geets et al., 2006). However, the lateral gene transfer of dsrAB gene in certain environmental lineages of SRM and occurrence of aprBA gene in other groups of sulfur-oxidizing phototrophic and chemolithotorophic bacteria, and limited distribution of [NiFe] hydrogenase gene hinder their

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reliable application in SRM phylogentic studies (Wawer et al., 1997; Klein et al., 2001; Zverlov et al., 2005; Meyer and Kuever, 2007).

4.4.2. PCR with SRM-specific primers The design of oligonucleotide PCR primers for the diagnostic detection of microorganisms primarily depends on the presence of variable region in the 16S rRNA primary structure. The oligonucleotide PCR primers design for targeting the hypervariable regions of 16S rRNA sequence is found to be highly specific for identification of microorganisms at genus, species and subspecies level. Moreover, the presence of conserved 16S rRNA sequence helps to develop PCR primers specific for genus, subdivision and phylum. The 16S rRNA sequence-based PCR method has simple procedural steps, such as extraction of genomic DNA from environmental sample, amplification of 16S rRNA genes with specific oligonucleotide primers, and gel electrophoresis to identify the size(s) of the amplicon(s). The SRM-specific primers for specific amplification of 16S rRNA gene fragments are reported for five genera: Desulfotomaculum, Desulfobulbus, Desulfobacterium, Desulfobacter and Desulfovibrio; and a common primer set for three genera: DesulfonemaDesulfosarcina-Desulfococcus (Devereux et al., 1992; Daly et al., 2000) The major limitations of this method include the presence of multiple rRNA operons in environmental microorganisms, absence of target sequence due to inhibition of expression of target gene, and formation of chimeric PCR products. The authenticity of the results for identification of microorganisms in environmental samples using PCR-amplified 16S rRNA genes should be tested by cloning and sequencing (Head, 1999).

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4.4.3. DGGE DGGE is a technique that is used to resolve the PCR-amplified gene fragment on a denaturing gradient gel according to the differences in nucleotide sequences (Muyzer and Smalla, 1998). The experimental steps of this method include the extraction of genomic DNA, amplification of target gene using PCR with one of the specific primer set clamped with G-C, and electrophoresis of the amplicons on a special electrophoresis gel incorporated with a denaturing gradient (urea or formamide). When the amplicon (DNA) migrates from low to high concentration of denaturants, it starts to denature into single strands. Complete denaturation of double stranded DNA ceases the migration. The amplicon is unable to degrade completely due to the presence of G-C clamp in one of the primers. The migration of DNA amplicons primarily depends on the relative proportions of G-C and A-T contents. Since A-T bonds are more vulnerable to denaturation than G-C bonds, the differences in sequence between amplicons that result in differences in G-C content will cause DNA to migrate to different positions in the gel. DGGE is sensitive for identification a single nucleotide differences between the amplicons. The DNA present in the bands can be excised from the gel and sequenced for the phylogenetic studies. DGGE is considered as lesser labor intensive and lesser time consuming than the construction of a clone library. Furthermore, a broader spectrum of genetic diversity of microorganisms can be observed in DGGE gel owing to the absence of random process of picking up and sequencing of the clones. DGGE has been used extensively for studying the genetic diversity of SRM by targeting the 16S rRNA gene fragment (Muyzer et al., 1993; Santegoeds et al., 1998), [NiFe] hydrogenase gene (Wawer

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and Muyzer, 1995; Wawer et al., 1997), dsrAB genes (Dhillon et al., 2003; Dar et al., 2007) and aprBA genes (Meyer and Kuever, 2007). Most of the DGGE studies have used dsrA and dsrB genes as the functional marker for characterization of metabolically active SRM in environments. Dsr AB genes encode the α and β subunits of dissimilatory sulfite reductase enzyme (EC 1.8.99.1) that catalyzes the reduction of sulfite to sulfide (see Eq. 4). As dsrAB genes are highly conserved in sulfate-reducing bacteria and archaea, it is considered as a potential marker for the phylogenetic studies of SRM in different environments including eutrophic and pristine areas of Florida Everglades (Castro et al., 2002), petroleum contaminated sediments (Dhillon et al., 2003), deep-sea cold seep sediments (Fukuba et al., 2003), deep-sea hydrothermal sites (Nakagawa et al., 2004), water column and sediments of permanently frozen freshwater lake (Karr et al., 2005), sulfidogenic bioreactors (Dar et al., 2005), heavy metal contaminated groundwater treatment site (Geets et al., 2006) and uranium contaminated groundwater (Chang et al., 2001). Although DGGE is a successful tool for characterization of SRM, the banding patterns mainly represent the major constituents of the complex environmental communities (Heuer et al., 1997). Small SRM species that constitute Basicity (0.50) > other (0.35) > SiO2 (-0.04) > K2O (-0.55) > Na2O (-0.56) > Fe2O3 (-0.79) > P2O5 (-0.86) > MgO (-0.86) > Al2O3 (-0.87). Those of melting points were in the order: other (0.56) > SiO2 (0.49) > K2O (0.03) > CaO (0.01) > Na2O (0.01) > Basicity (-0.04) > Fe2O3 (-0.48) > Al2O3 (-0.50) > MgO (-0.53) > P2O5 (-0.62). Those of pouring points were in the order: other (0.54) > SiO2 (0.52) > K2O (0.12) > Na2O (0.10) > CaO (-0.08) > Basicity (-0.12) > Fe2O3 (-0.39) > Al2O3 (-0.40) > MgO (-0.43) > P2O5 (-0.52). Based on the results of R values, the selected input variables for softening, melting and pouring points are shown in Table 3.

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Table 2. The correlation coefficients between compositions of SSA and various temperatures of slag

SiO2 Al2O3 MgO P2O5 CaO Fe2O3 Na2O K2O other Basicity

Softening point -0.04 -0.87 -0.86 -0.86 0.65 -0.79 -0.56 -0.55 0.35 0.50

Melting point 0.49 -0.50 -0.53 -0.62 0.01 -0.48 0.01 0.03 0.56 -0.04

Pouring point 0.52 -0.40 -0.43 -0.52 -0.08 -0.39 0.10 0.12 0.54 -0.12

Table 3. The selected input variables for softening, melting and pouring points Temperature Softening point Melting point Pouring point

Compositions CaO, other, Basicity SiO2, CaO, Na2O, K2O, other SiO2, Na2O, K2O, other,

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Prediction of Softening Points

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The values of softening point fell within the range of 1126.2 – 1239.2 centigrade. Figure 2 (a) - (d) depict the prediction results of softening points using MFs of gaussmf, gbellmf, trimf and trapmf, respectively. All MAPE values are shown in Table 4. The 1st to 12th values were used for model training, 13st to 15th values were used to evaluate the fitness. As shown in Table 4, when training, MAPEs between the predicted and observed values of softening point were 0.0 %, but they were 4.3 % - 16.3 % when predicting. It revealed that ANFIS could predict the softening points of SSA during formation of slag precisely. Among all MFs, the MAPE when using trimf was the lowest, that using trapmf was the highest. They were in the order: trimf < gaussmf < gbellmf < trapmf.

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(d) Figure 3 Prediction results of melting point. (a) gaussmf, (b) gbellmf, (c) trimf, (d) trapmf.

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Table 4. Predicting performance using ANFIS with different MFs

Softening point (%) Melting point (%) Pouring point (%)

Training Testing Training Testing Training Testing

gaussmf 0.0 5.6 0.0 2.8 0.0 3.3

gbellmf 0.0 11.4 0.0 2.6 0.0 3.4

trimf 0.0 4.3 0.0 3.8 0.0 3.5

trapmf 0.0 16.3 0.0 7.3 0.0 7.6

Prediction of Melting Points The values of melting point lay between 1155.2 and 1295.4 centigrade. Figure 3 (a) - (d) show the prediction results of melting points using MFs of gaussmf, gbellmf, trimf and trapmf, respectively. As shown in Table 4, the MAPEs between the predicted and observed values of melting point were 0.0 % when training, but they were 2.6 % - 7.3 % when predicting. It represented that ANFIS could predict the melting points precisely. Among all MFs, the MAPE when using gbellmf was the lowest, that using trapmf was the highest. They were in the order: gbellmf < gaussmf < trimf < trapmf.

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Prediction of Pouring Points The values of pouring point fell within the range of 1160.7 – 1310.2 centigrade. The prediction results of pouring points when using MFs of gaussmf, gbellmf, trimf and trapmf, respectively, are shown in Figure 4 (a) - (d). The MAPEs between the predicted and observed values of melting point were 0.0 % when training, but they lay between 3.3 % and 7.6 % when predicting. It indicated that ANFIS could predict the pouring points precisely. Among all MFs, the MAPE when using gaussmf was the lowest, that using trapmf was the highest. They were in the order: gaussmf < gbellmf < trimf < trapmf.

Prediction Performance of Different MFs The MAPEs of gaussmf were 5.6 %, 2.8 % and 3.3 %, respectively, when predicting softening, melting and pouring point. The MAPEs of gbellmf were 11.4 %, 2.6 % and 3.4 %, respectively. The MAPEs of trimf lay between 3.5 % and 4.3 %. Among all MFs, trapmf showed the worst performance. When predicting softening, melting and pouring point, the MAPEs of trapmf were 16.3 %, 7.3 % and 7.6 %, respectively. The reason why the MAPEs of trapmf were the highest could be explained as follows. The trapmf revealed a trapezoidal shape in which top and bottom lines paralleled each other. When backpropagating the parameters associated with the input MFs and estimating the parameters associated with the output MFs, the unsmooth intervals of trapmf resulted in worse fitness. Therefore, trapmf showed the worst performance. Overall speaking, ANFIS‘s architecture consists of both ANN and fuzzy logic including linguistic express of MFs and if-then rules, so it can overcome the limitations of traditional

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5. CONCLUSIONS In this study, the compositions of SSA and the softening, melting and pouring points were determined during formation of slag. Then these measured data were used as input layer and output layer to train ANFIS. Finally, additional 3 sets of data were used to test the prediction of ANFIS. According to calculation, ANFIS could predict the softening, melting and pouring points of SSA during formation of slag precisely. The results can be drawn as follows. 





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

Different compositions of raw SSA were shown as follows: SiO2, 36.66-64.69 %; Al2O3, 5.78-10.37 %; MgO, 0.90-1.78 %; P2O5, 8.03-29.04 %; CaO, 2.54-7.79 %; Fe2O3, 6.64-14.26 %; Na2O, 0.99 % - 1.24 %; K2O, 1.69–2.09 %. The results revealed the positive correlation between the softening points and compositions of SSA including CaO, basicity and other. The relationship between the melting points and compositions including other, SiO2, K2O, CaO and Na2O revealed a positive correlation. The pouring points were positively correlated with compositions including other, SiO2, K2O and Na2O. The values of softening point fell within the range of 1126.2 – 1239.2 centigrade. The values of melting point lay between 1155.2 and 1295.4 centigrade. The values of pouring point fell within the range of 1160.7 – 1310.2 centigrade. The MAPEs for training were 0.0 % between the predicted and observed values when gaussmf, gbellmf, trimf and trapmf were adopted. The MAPEs of softening point were 4.3 % - 16.3 % when predicting. Those of melting point fell in the range between 2.6 % and 7.3 %. Those of pouring point lay between 3.3 % and 7.6 %.

REFERENCES Abu-Kaddourah, Z., Idris A., Noor M. J. & Ahmadun F. R. (2000). Effects of high temperature melting on the porosity and microstructure of slags from domestic sewage sludge. Water Science and Technology, 41(8), 99-105. Cakmakci, M. (2007). Adaptive neuro-fuzzy modelling of anaerobic digestion of primary sedimentation sludge. Bioprocess and Biosystems Engineering, 30(5), 349-357. Endo, H., Nagayoshi Y. & Suzuki K. (1997). Production of glass ceramics from sewage sludge. Water Science and Technology, 36(11), 235-241. Huang, Y. C. & Li K. C. (2003). Effect of reducing conditions on sludge melting process. Chemosphere, 50(8), 1063–1068. Jang, J. S. R. (1993). ANFIS: adaptive-network-based fuzzy inference system. IEEE Transactions on Systems, Man, and Cybernetics, 23(3), 665–685. Masaki, T., Nobuo, T. & Satoshi M. (1997). The behaviour of heavy metals and phosphorus in an ash melting process. Water Science and Technology, 36(11), 275-282. Okuno, N., Uriu, M., Horii, T. & Miyagawa, K. (1997). Evaluation of thermal sludge solidification. Water Science and Technology, 36(11), 227-233.

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Okuna, N. & Yamada, A. (2000). Evaluation of full scale thermal solidification processes implemented in Tokyo lightweight aggregate, slag and brick. Water Science and Technology, 41(8), 69-76. Okuno, N., Ishikawa, Y., Shimizu, A. & Yoshida, M. (2004). Utilization of sludge in building material. Water Science and Technology, 49(10), 225-232. Pai, T. Y., Ouyang, C. F., Su, J. L. & Leu, H. G. (2001a). Modeling the stable effluent qualities of the A2O process with Activated Sludge Model 2d under different return supernatant. Journal of the Chinese Institute of Engineers, 24(1), 75-84. Pai, T. Y., Ouyang, C. F., Su J. L. & Leu, H.G. (2001b). Modelling the steady-state effluent characteristics of the TNCU process under different return mixed liquid. Applied Mathematical Modelling, 25(12), 1025-1038. Pai, T. Y., Chuang, S. H., Tsai, Y. P. & Leu, H. G. (2004a). Development of two-stage nitrification/denitrification model (TaiWan Extension Activated sludge model NO.1) for BNR process. Journal of the Chinese Institute of Environmental Engineering, 14(1), 5160. Pai, T. Y., Tsai, Y. P., Chou, Y. J., Chang, H. Y., Leu, H.G. & Ouyang, C. F. (2004b). Microbial kinetic analysis of three different types of EBNR process. Chemosphere, 55(1), 109-118. Pai, T. Y., Chuang, S. H., Tsai, Y. P. & Ouyang, C. F. (2004c). Modelling a combined anaerobic/anoxic oxide and rotating biological contactors process under dissolved oxygen variation by using an activated sludge - biofilm hybrid model. Journal of Environmental Engineering, ASCE, 130(12), 1433-1441. Pai, T. Y., Chen, H. M., Chang, T. C., Chen, W. F. & Ouyang, C. F. (2006). Using ANN to predict the softening, melting and pouring points of sewage sludge ash during formation of slag. Proceediing of IWA Specialized Conference on Sustainable sludge management: state of the art, challenges and perspectives, May 29-31, Moscow, Russia, 373-377. Pai, T. Y. (2007). Modeling nitrite and nitrate variations in A2O process under different return oxic mixed liquid using an extended model. Process Biochemistry, 42(6), 978-987. Pai T. Y. (2008). Gray and neural network prediction of effluent from the wastewater treatment plant of industrial park using influent quality. Environmental Engineering Science, 25(5), 757-766. Pai, T. Y., Tsai, Y. P., Lo, H. M., Tsai, C. H. & Lin, C. Y. (2007). Grey and neural network prediction of suspended solids and chemical oxygen demand in hospital wastewater treatment plant effluent. Computers & Chemical Engineering, 31(10), 1272-1281. Pai, T. Y., Chuang, S. H., Ho, H. H., Yu, L. F., Su, H. C. & Hu, H. C. (2008a). Predicting performance of grey and neural network in industrial effluent using online monitoring parameters. Process Biochemistry, 43(2), 199-205. Pai, T. Y., Chuang, S. H., Wan, T. J., Lo, H. M., Tsai, Y. P., Su, H. C., Yu, L. F., Hu, H. C. & Sung, P. J. (2008b). Comparisons of grey and neural network prediction of industrial park wastewater effluent using influent quality and online monitoring parameters. Environmental Monitoring and Assessment, 146(1-3), 51-66. Pai, T. Y., Chungjen, C., Liaw, C. F. & Chang, J. H. (2008c). Treatment, recycling and disposal of solid wastes in Taiwan. Proceediing of 2008 Joint International Conference of USTB, CYUT, XAUAT and SCNU, October 29, Suncheon, Korea, F1-F12.

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Pai, T. Y., Chang, H. Y., Wan, T. J., Chuang, S. H. & Tsai, Y.P. (2009a). Using an extended activated sludge model to simulate nitrite and nitrate variations in TNCU2 process. Applied Mathematical Modelling, 33(11), 4259-4268. Pai, T. Y., Wan, T. J., Hsu, S. T., Chang, T. C., Tsai, Y. P., Lin, C. Y., Su, H. C. & Yu, L. F. (2009b). Using fuzzy inference system to improve neural network for predicting hospital wastewater treatment plant effluent. Computers & Chemical Engineering, 33(7), 12721278. Pai, T. Y., Wang, S. C., Chiang, C. F., Su, H. C., Yu, L. F., Sung, P. J., Lin, C. Y. & Hu, H. C. (2009c). Improving neural network prediction of effluent from biological wastewater treatment plant of industrial park using fuzzy learning approach. Bioprocess and Biosystems Engineering. (In press) Pai, T. Y., Wang, S. C., Lin, C. Y., Liao, W. C., Chu, H. H., Lin, T. S., Liu, C. C. & Lin, S. W. (2009d). Two types of organophosphate pesticides and their combined effects on heterotrophic growth rates in activated sludge process. Journal of Chemical Technology and Biotechnology. (In press) Pai, T. Y., Wang, S. C., Lo, H. M., Chiang, C. F., Liu, M. H., Chiou, R. J., Chen, W. Y., Hung, P. S., Liao, W. C. & Leu, H. G. (2009e). Novel modeling concept for evaluating the effects of cadmium and copper on heterotrophic growth and lysis rates in activated sludge process. Journal of Hazardous Materials, 166(1), 200-206. Ozaki, M., Watanabe, H. & Wiebusch, B. (1997). Characteristics of heavy metal release from incinerated ash, melted slag and their re-products. Water Science and Technology, 36(11), 267-274. Takaoka, M., Takeda, N. & Miura, S. (1997). The behaviour of heavy metals and phosphorus in an ash melting process. Water Science and Technology, 36(11), 275-282. Taruya, T., Okuno, N. & Kanaya K. (2002). Reuse of sewage sludge as raw material of Portland cement in Japan. Water Science and Technology, 46(10), 255-258.

Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

In: Sludge: Types, Treatment Processes and Disposal Editor: Richard E. Baily

ISBN: 978-1-60741-842-9 © 2009 Nova Science Publishers, Inc.

Chapter 12

SEWAGE SLUDGE TREATMENT AND RECYCLING SYSTEMS IN JAPAN: TRENDS, CHALLENGES AND FUTURE PERSPECTIVES Keishiro Hara Research Institute for Sustainability Science, Osaka University 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan, Tel & Fax: +81-6-6879-4140

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ABSTRACT Given the increasing volume of sewage sludge and limited capacity of the final disposal sites, building a sustainable sludge management system with proper treatment and recycling options has been and will be crucial for municipalities in Japan. This chapter aims to overview sewage sludge treatment and recycling practices in Japan with a particular focus on recent trends for the sewage sludge management. Besides material productions using the treated sludge, sludge utilization as an alternative energy source has been encouraged lately as a promising option amid the increasing attention to carbon neutral energy. Some innovative approaches addressing such options, which have currently been developed and promoted at the municipality level, are introduced. The chapter then briefly describes a unique sludge treatment and recycling system adopted in Tokyo where a shortage of final disposal sites for sludge has been serious and, thus, sludge incineration and recycling have been essential for reducing the volume of treated sludge conveyed to the disposal sites. The recycling options in Tokyo since the late 90s had included the productions of brick, slag, aggregate and RDF (Refuse Derived Fuel). An environmental assessment of these recycling options in Tokyo indicated that the processes of materials production were costly as well as energy consuming. Recently, different practices, such as sludge utilization for cement production and sludge carbonization, draw attention as promising options in Tokyo and they are found to be desirable from the viewpoints of cost, energy consumption and associated environmental loads. These technologies and practices about sludge treatment, recycling, and disposal in Japan shall provide useful lessons for other Asian countries where proper sludge treatment, disposal and recycling strategies are becoming very important amid the rapid urbanization and increasing sewage sludge generation as a consequence.

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Keishiro Hara

1. INTRODUCTION Constructing proper and sustainable systems for sewage sludge treatment, disposal and recycling has been a pressing issue for municipalities in Japan. With the incremental ratio of sewage coverage, the volume of sewage sludge discharged from wastewater treatment plants has been steadily increasing over the last few decades. The total coverage ratio of sewer systems in Japan as a whole increased from 55 % in 1996 to 67 % in 2004. Consequently, the volume of discharged sludge also expanded from 1,824 thousand DS (Dry Solid) tons in 1996 to 2,174 thousand DS tons in 2004 (Ministry of Land, Infrastructure, Transport and Tourism, 2006). The sewage sludge accounted for 18 % of the total volume of industrial wastes in Japan which was about 74.8 million tons equivalent in 2003. It has been of vital importance for the Japanese municipalities to treat and dispose of the increasing amount of sludge properly, or utilize the treated sludge efficiently. The chapter first overviews the situation of Japanese practices and policy orientations for sewage sludge treatment and management systems and then revisits a unique sludge treatment and recycling system applied in Tokyo‘s 23 wards as the representative case in Japan, especially focusing on the system in the late 90s when a drastic change in sludge recycling options took place. An evaluation of environmental conditions, such as the change in CO2 emission and energy consumption, associated with the operation of the system in Tokyo, is then demonstrated. This chapter concludes by commenting on the future prospects for sewage sludge treatment and management in Japan.

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2. SEWAGE SLUDGE TREATMENT AND RECYCLING IN JAPAN The volume of sewage sludge discharged from wastewater treatment plants has been steadily growing in Japan. Although the prospects of declining population in the Japanese society will likely change this situation in the future, the increase in sludge volume has been a clear trend over the past few decades. In the meantime, the remaining capacity of the disposal sites has been shrinking. Thus, it has been necessary for municipalities to reduce the sludge volume conveyed to the final disposal sites. In fact, the recent sludge management policies in Japan were formulated primarily for the purpose of reducing the sludge volume. In 1996, the Sewerage Law was revised in Japan and under the revised low it became obligatory for the municipalities to make efforts to reduce the sludge volume transported to the disposal sites by means of dewatering, digestion and incineration as well as possible recycling of the treated sludge. The revision of the Sewerage Law would have had some influence upon the sludge management practices at the municipality level in Japan and development of technologies pertinent to the proper treatment, such as incineration technologies, and recycling. Sludge recycling ratio—the percentage of the recycled sludge out of the total sludge volume discharged—has drastically increased from 15 % in 1988 to 67 % in 2004, almost reaching the set target of recycling ratio of 68 % which was to be met by the year 2007 (Ministry of Land, Infrastructure Transport and Tourism, 2006). In terms of the types of recycling, materials production from treated sludge accounted for 52 % of the total volume of discharged sludge by weight in 2004. These materials include bricks, slag, aggregate made from treated sludge and sludge utilization as raw material in cement production. Out of the 52

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% of materials recycling, utilization of sludge in the cement industry makes up for about 29 %, becoming a core option these days. The sludge use in the cement industry actually started after 1995 in Japan and became a competitive and promising option from the viewpoints of cost and smaller energy consumption additionally required. Sludge application in land and green areas, including composting, also accounts for about 14 % of the discharged sludge and the treated sludge disposed in the final disposal sites was about 31 % in 2004. It is certainly indispensable to make the treatment and recycling systems operate in a sustainable manner. These systems usually necessitate costs and additional energy in the series of treatment and recycling processes. Moreover, these processes cause associated environmental loads, such as green house gas (e.g. CO2) and acidification gas (e.g. SOx), due to the energy consumption and combustion of sludge itself in case of incineration. In Japan, the whole sewer system that includes wastewater treatment, pumping and sludge treatment processes are found to consume approximately 0.7 % of the yearly electricity consumption in the whole country, indicating that the sewer system is heavily energy consuming (Ministry of Land, Infrastructure, Transport and Tourism, 2007). Of the entire electricity consumption associated with the whole sewer system, the consumption in the sludge treatment processes, including dewatering and incineration, account for almost 20 %, making the processes the second largest electricity consuming after the water treatment processes whose consumption accounts for 45 %. Furthermore, the consumption of energy, including fuels, necessary for the operation of sewer systems, has been gradually growing over the past decades (about 20 % up between 1996 and 2003) and so has the energy consumption related to the sludge treatment processes. Coupled with the consumption of the fuels typically used for drying and incinerating the sludge, a large amount of energy is usually necessary for the sludge treatment. As argued later, sludge recycling processes, such as brick making, is also energy demanding and could cause the associated environmental loads. Hence, constructing energy efficient systems for sludge treatment and recycling is the key for sustainable operation. Lately, attention has been paid to the utilization of sewage sludge as a potential biomass which can be transformed to biogas through the anaerobic digestion process or even to electricity. This approach is considered an important option besides the material recycling, given the mounting attentions for the biomass as an alternative carbon neutral energy source in the era of global warming. Sewage sludge is estimated to account for nearly 30% of the potential biomass that exists within Japan by weight and, thus, there is a high potential of sludge utilization as an energy source, as long as socio-technical barriers are overcome. Biogas derived from sewage sludge has been already applied in Japan as a supplementary fuel in the incineration, heating and other processes. Apart from such application, new practices of the bioenergy utilization include, but are not limited to: 1) generation of electricity from sludge-based biogas, 2) production of carbonized sludge as a fuel and 3) utilization of sludge-based biogas as fuel for public vehicles. In Japan, the first introduction of facilities for electricity generation from the sludge biogas dates back in 1984 and 26 treatment plants with the facilities exist throughout Japan as of 2005. Morigasaki wastewater treatment plant located in Tokyo, which owns a series of sludge treatment processes within the plant, introduced the facilities for electricity generation with a generation capacity of 3,200 kW in 2004. Notably, the biomass-based electricity generation at the plant has been carried out through the mechanism of PFI or Private Finance Initiative. On the other hand, the carbonized sludge is produced by baking the sludge under the anaerobic condition after drying the dewatered sludge. For instance, the carbonized sludge has been provided partly as an

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alternative fuel to the fossil fuels in a thermal power plant in Aichi prefecture since 2008. The calorific value of the carbonized sludge is estimated to be about 13 MJ/kg. The application of this alternative fuel theoretically contributes to the reduction in CO2 emission, as it is counted as carbon neutral fuel. The bioenergy utilization in public vehicles—the third option —is now implemented in Kobe city. Kobe city successfully achieved a biogas production with a high density of 98 % Methane (CH4) composition. The biogas is now utilized as an alternative fuel for buses. It is also worth noting that the recovery and recycling of phosphorus contained in the sludge has been drawing attentions recently. Phosphorus is a limited and non-renewable resource and thus should be reused and recycled for its continuing usage. Based upon various studies, it is considered that the sewer system is a promising source of phosphorus recovery. Hara et al. (2000) argue that phosphorous derived from the sewer systems would be better utilized in the industrial sector in Japan, considering technological feasibility and societal conditions unique to Japan. The view that sewage sludge is not just a waste but a potential resource is now on the increase. Material productions from sludge and sludge utilization as an alternative energy source will constitute the core of sludge recycling practices in Japan and development of relevant technologies shall be further promoted. However, it is important that the recycling and treatment systems should work in a sustainable manner from the environmental and economic perspectives. In fact, the materials made from sludge, such as brick, tended to cost more than other commercialized bricks and innovative ideas to reduce the cost should be identified. To challenge this critical issue of sustainable operation, so-called ―LOTUS Project‖ was implemented in Japan between 2005 and 2008 with an aim to pursue the following two aspects: 1) development of recycling technologies which are cheaper in price than the cost needed for sludge disposal and 2) identification and development of technologies or systems for the biomass-based electricity generation in which the price of generated electricity is equal or even lower than that of bought electricity (Ministry of Land, Infrastructure, Transport and Tourism, 2006). The aspect of cost and energy reductions is a salient challenge encountered with the municipalities and should be tackled with a high priority while the municipalities develop highly advanced and innovative technologies.

3. SEWAGE SLUDGE MANAGEMENT IN TOKYO 3.1. Sludge Management and Recycling By 1994, the coverage of the sewer system reached 100% within Tokyo‘s 23 wards—the most urbanized area in Tokyo. As of 2001, about 8.3 million people who resided in the area were served by the sewer system. In accordance with the expansion of sewer system coverage, the volume of raw sewage sludge from the wastewater treatment plants located in Tokyo‘s 23 wards increased from 46,468,880 m3 in 1995 to 59,859,180 m3 in 2001 (Tokyo Metropolitan Government, 1995, 2001). As is the case with other large municipalities, Tokyo has also suffered from a severe shortage of final disposal sites for sewage sludge and it has become important to reduce the volume of treated sludge eventually conveyed to the final disposal site after its treatment. In this regard, incineration has been promoted to reduce

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sludge volume and stabilize the properties of treated sludge. Tokyo Metropolitan Government proposed in 1998 the following principles as a part of the future plan for sludge management policy: 1) promotion of a centralized and intensive sludge treatment system, maximizing the efficiency of the treatment, 2) achievement of 100 % incineration treatment to decrease the volume of treated sludge in the final disposal site and 3) utilization or recycling of treated sludge. Tokyo‘s recent sludge treatment and recycling systems were basically formulated based upon the principles. Since the incineration facility was first introduced at the Odai wastewater treatment plant in 1967, the incineration ratio has gradually increased. In the meantime, the Tokyo Metropolitan Government has promoted the recycling of treated sludge mainly for the purpose of reducing the sludge volume in the final disposal site and, at the same time, as part of promoting a recycle-based society which is the national policy orientation. The Nanbu sludge plant in Tokyo is a special sludge treatment plant equipped with recycling facilities, fabricating materials such as slag from the treated sludge. As of 1997, four types of materials including brick, aggregate, RDF (Refuse Derived Fuel) and slag were produced from the treated sludge, such as ash and dewatered sludge, at the plant. The brick, aggregates and sludge were mainly used for constructions and road pavement. The sludge recycling options in Tokyo drew attention at that time both domestically and internationally because of its unique approach and technologies used. In fact, the mass production system of such materials from sludge at the plant was very unique and distinctive. A drastic change in recycling policy, however, took place from the late 90s and the processes of Brick, RDF and slag production had been eventually scrapped from the recycling processes at the Nanbu plant. As argued in section 3.3, the abandoned options were very energy consuming as well as costly. Thus, these options failed to remain competitive in comparison with other commercialized materials.

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Table 1 Sludge flow in the treatment processes within Tokyo’s 23 wards (1997) Wastewater treatment plants

(Sources of raw sludge discharge) Mikawashima, Sunamachi Ariake Nakagawa Kasai Kosuge Nakano Ochiai Odai Shingashi Shibaura Shibaura (the rest) & Morigasaki

Plants for Dewatering

Plants for Incineration

Final destinations of sludge (disposal / recycling)

Sunamachi

Sunamachi

Final disposal site

Tobu plant

Tobu plant

Kasai

Kasai

Odai

Odai

Final disposal site Final disposal site, Others (e.g. Cement industry) Final disposal site Nanbu plant (Brick,

Shingashi

Shingashi

Shibaura Nanbu plant

Nanbu plant

aggregate)

Final disposal site Nanbu plant (Aggregate) Final disposal site Nanbu plant

(Brick, Aggregate, RDF)

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3.2. Sludge Flow within Tokyo’s 23 Wards

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The flow of treated sewage sludge in the treatment processes starting from each wastewater treatment plant to the final destinations within Tokyo‘s 23 wards has been historically very complicated. Based upon the actual situation in 1997, Table 1 demonstrates the actual flow of the sludge that originated from each of the twelve wastewater treatment plants, which existed then in Tokyo‘s 23 wards then, to the final destinations. Note that out of the whole treatment process, only the dewatering and incineration processes are highlighted in Table 1 for brief explanation purpose. It is shown in Table 1 that the raw sewage sludge from the wastewater treatment plants within Tokyo‘s 23 wards then was transported to five wastewater treatment plants equipped with sludge treatment facilities, such as dewatering and incineration processes, and at two special sludge treatment plants including the Nanbu plant, and then treated at those plants. For example, the sewage sludge discharged at the Nakano and Ochiai wastewater treatment plants respectively was transported to the Odai treatment plant in which the transported sludge was dewatered and incinerated, after being mixed with the sludge discharged at the Odai wastewater treatment plant. Subsequently, ash from the incineration process at Odai plant was then conveyed to the final disposal site while the rest of the ash was transported to the Nanbu plant for the production of recycling materials, such as brick. It is also important to note that the sludge was transported from one plant to another by means of the following three ways: (1) underground piping, (2) trucking and (3) shipping, depending on the locations of treatment plants and properties of the sludge. As an example, the raw sludge from the Nakano and Ochiai plants to the Odai plant was transported through the underground piping with the pressure force and the ash from Odai plant was transported to Nanbu plant by truck. Table 1 clearly illustrates the systematic but complex sludge flow in the processes of treatment and recycling within Tokyo‘s 23 wards.

3.3. Evaluation of Recycling and Treatment System Hara and Mino (2008) comparatively analyzed the four sludge recycling options—brick, slag, aggregate and RDF—from the energy consumption perspective and carried out environmental assessment of the whole sludge management system applied in Tokyo‘s 23 wards, looking into the actual practices from 1995 through 2001, when the treatment and recycling processes drastically changed. In addition, an environmental assessment about energy consumption and gas emissions was conducted for the same time period, targeting the operation of the whole system within Tokyo‘s 23 wards, including the processes of thickening, dewatering, anaerobic digestion, and incineration of all relevant treatment plants, together with the processes of final disposal, sludge transportation (piping, ship and truck) and sludge recycling at the Nanbu plant. The result of comparative energy analysis of four recycling options showed that the RDF production was the most energy consuming, whereas the aggregate production was the least energy consuming option and thus considered the most ideal from the viewpoint of energy efficiency. It was also identified that energy consuming options such as RDF and slag were costly. The RDF, slag and brick-making processes were scrapped from the Nanbu plant after 1997 and nowadays only the aggregate production process remains as a recycling process at the plant.

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The results of environmental assessment indicated that the whole sludge management system within the assessment boundary above was found to be less energy consuming in 2001, compared with 1995 despite the growing amount of treated sludge from the wastewater treatment plants. This is attributed mainly to a change in the sludge treatment as well as recycling systems, such as the shift of the recycling options from the energy intensive (RDF, slag) to the less energy consuming option (aggregate). The total CO2 emission associated with the operation of the whole evaluated system decreased during the period as well, mainly due to the reduction in energy (fuel) consumption in the system, whereas the total N2O emission, which has much larger global warming potential than CO2, increased from 1995 through 2001. N2O emission is mainly originated from the incineration of sludge itself. Hence, N2O emission tends to grow in accordance with the sludge volume in the incineration process. It is already known that the temperature control in the incineration process is the key for capping the N2O emission. This study identified that such green house gasses were mostly generated from the incineration process, in comparison with other processes analyzed in the study. The CO2 emission from the incineration process accounted for about 90% of all evaluated processes in 2001 and this is also true of the acidification gases, with NOx and SOx emissions from the process accounting for 83% and 87% of the total emissions from the whole evaluated processes in the same year, respectively (Hara and Mino, 2008). In general, the incineration process is the most energy consuming of all the sludge treatment processes and therefore tends to generate gas emissions intensively. These results indicate that it is highly essential to take appropriate measures in the incineration processes, such as temperature control and efficient fuel usage, for capping the green house gases and acidification gases emissions in pursuing an environmentally friendly sludge management system. This point will continue to be important, given the future prospects that the sludge incineration will likely remain as a core part of sludge treatment strategy in Tokyo and other large municipalities in Japan. In terms of recycling, sludge use in the cement industries has been recently promoted as a promising option and the ratio of this option has been growing steadily since 1997 in Tokyo. Carbonized sludge is also produced at some treatment plants as another new option. Importantly, both sludge utilization in the cement industry and the carbonized sludge are evaluated to be relatively energy efficient and less costly options compared with the conventional types of material recycling, such as RDF and brick production. Furthermore, biogas-based electricity is also implemented in the Morigasaki treatment plant as commented in section 2. These new approaches shall constitute the core of future sludge recycling options not only in Tokyo, but also other municipalities in Japan, beyond the conventional type of energy consuming materials recycling.

4. CONCULUSIONS This chapter overviewed the recent trends of policies and practices for sewage sludge treatment and recycling in Japan and then briefly summarized the sludge treatment as well as recycling systems in Tokyo, particularly looking into the aspects of environmental performances of the conventional sludge recycling options and the whole sludge management system applied in Tokyo‘s 23 wards. Given the limited capacity of the remaining final

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disposal sites, it is indeed indispensable to promote recycling or reuse of the treated sludge. As for material recycling, the energy consumption additionally required in the process of material production and the associated environmental loads, such as CO2 emission, are the primary concerns from environmental viewpoint. In fact, the environmental aspect is a critical point that necessitates urgent attentions. It is also important to make the material products made from sewage sludge competitive enough in the market by enhancing quality and cutting the cost. This aspect is particularly essential for keeping such recycling options. A hopeful option in coming decades is the utilization of sludge as an alternative energy source. Paying attention to the sewage sludge as an energy source, which is counted as carbon neutral, is significant amid the increasing concern of climate change. New attempts to transform the sludge to biogas and electricity for various purposes are now implemented in municipalities, including Tokyo. Accepting other types of biomass including food wastes for generating biogas shall be worth considering for attaining the merit of scale and efficient biogas production at a lower cost. At the experimental level, other innovative technologies, such as pyrolysis oil production from sewage sludge, are also under development with feasibility studies in Japan and they will be put in place as the actual sludge recycling practices in near future. Finally, it is stressed that the diversification of sludge recycling options is also crucial for securing the continuous and sustainable operation of sludge management systems. The experiences and lessons of sludge treatment and recycling practices in Japan shall be of help for other Asian countries where the expansion of sewer systems is resulting in the rapid increase in the volume of discharged sludge and handling the growing volume of sewage sludge is becoming critically important in the years to come.

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REFERENCES Hara, K. & Mino, T. (2008) Evaluation of Sewage Sludge Recycling Options and Management System in Tokyo, Waste Management, 28, pp.2645-2652 Hara, K., Morishita, Y., Nittami, T., Sato, T. & Mino, T. (2000) Estimation of Phosphorous Flow in Japan and a Study on its Recycling System, Proceedings of 28th Annual Meeting of Environmental Systems Research, Vol. 28, pp 107-112 , Japan Society of Civil Engineers (in Japanese) Ministry of Land, Infrastructure, Transport and Tourism (2007) Towards the Achievement of Resources Utilization (in Japanese) (Accessed on Dec 20, 2008) (URL: http://www.mlit.go.jp/crd/sewerage/shingikai-iinkai/shigen/sigen7th/02.pdf) Ministry of Land, Infrastructure, Transport and Tourism (2006) States and Challenges of Sewage Sludge Utilization (in Japanese) (Accessed on Dec 20, 2008) (URL: http://www.mlit.go.jp/crd/city/sewerage/gyosei/sigen1st/04.pdf) Tokyo Metropolitan Government (1995, 2001) Report on the sludge treatment system in Tokyo. Tokyo Metropolitan Government, Japan (in Japanese).

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INDEX

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A abiotic, 246 absorption, 3, 198, 255 Abundance, 83 acceptor, xi, 54, 63, 64, 68, 69, 170, 216 acceptors, 87, 165 accidental, 113 accidents, 146 accounting, 64, 167, 295 acetaldehyde, 52 acetaminophen, 269 acetate, 49, 65, 66, 67, 68, 69, 80, 102, 123, 227, 251, 257 acetic acid, xiv, 45, 131, 209, 222, 223, 224 acetone, x, 33, 36, 39, 40, 41, 249, 251, 255, 256, 257, 258, 259, 262 acetonitrile, 256, 257, 259, 262 acetylation, 45 achievement, 293 acid, x, xi, xiv, 33, 35, 36, 43, 44, 45, 46, 48, 49, 50, 51, 52, 54, 55, 62, 63, 65, 66, 71, 72, 78, 80, 81, 83, 87, 117, 119, 131, 173, 181, 205, 209, 212, 213, 217, 218, 222, 223, 224, 237, 238, 241, 251, 254, 256, 265, 269 Acid mine drainage, 82 acidic, 64, 68, 77, 83, 131, 196, 226 acidification, 224, 227, 291, 295 acidity, 190 activated sludge flocs, 4, 22, 27, 175 activation, 47, 215, 219, 220, 222, 223, 260 activation energy, 219, 220, 222, 223, 260 acute, xii, 90, 113, 114, 115, 263, 270 acylation, 45 additives, 38, 211, 268 adenosine, 64, 70, 73, 84, 91 adenosine triphosphate, 64 adhesion, 4, 168

adjustment, 148, 212 administration, 56, 109, 113 adsorption, 42, 48, 49, 56, 183, 195, 200, 215, 219, 246 aerobic, ix, 1, 3, 64, 85, 92, 105, 108, 120, 130, 163, 167, 168, 169, 172, 174, 180, 181, 182, 183, 185, 206, 213, 236, 246, 270 aerobic bacteria, 174, 182, 236 aerobic granulation, 169, 185 aerobic granules, 169, 180, 181, 185 aerosol, 56 Africa, 198 Ag, 113 agar, xiv, 69, 70, 229, 230 age, 12, 163, 166, 175 agent, x, 24, 33, 45, 49, 51, 189, 198, 216 agents, 11, 26, 56, 213, 268 agglutination, 70 agglutination test, 70 aggregates, 3, 16, 75, 86, 168, 196, 293 aggregation, 5, 15, 16, 19 agricultural, xiii, 115, 162, 183, 187, 188, 189, 190, 203, 204, 208, 211, 246, 247 agriculture, xii, 125, 126, 127, 129, 133, 134, 158, 161, 162, 175, 177, 193, 205, 210, 211, 212, 227, 246, 247, 268 air, xiv, 8, 11, 99, 128, 130, 132, 133, 139, 140, 142, 143, 144, 146, 148, 149, 150, 151, 152, 168, 178, 185, 190, 194, 209, 211, 213, 217, 219, 220, 221, 222, 223, 226, 227, 248, 268 air pollution, 211 air-dried, 194 Alberta, 71 alcohol, 42, 48, 50, 53, 54, 223, 241, 267 alcohols, 50, 54, 131 alcoholysis, 51 Aldrin, 266 algae, 10 Alginate, 10, 11, 19

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algorithm, 73 alkali, 99, 109, 110, 115, 120, 256 alkaline, xii, 42, 64, 89, 99, 182, 196, 213, 217, 224 alkaline hydrolysis, 217 alkenes, 218 alternative, xii, xiv, xv, 38, 40, 53, 115, 161, 162, 163, 169, 170, 175, 177, 179, 181, 189, 210, 218, 230, 236, 289, 291, 292, 296 alternative energy, xv, 289, 292, 296 alternatives, xii, xiv, 125, 175, 209, 213, 217, 218 aluminium, x, 2, 5, 20, 21, 29, 259, 279, 280 alveolus, 54 Amadori, 39 amendments, 82, 194, 196, 199, 200, 203 amino, 37, 38, 45, 64, 131, 218, 237, 238 amino acid, 37, 38, 64, 131, 218, 237, 238 amino acids, 37, 38, 64, 131 ammonia, xi, 89, 93, 94, 96, 97, 104, 105, 108, 116, 117, 118, 121, 124, 170, 175, 177, 180, 184, 199, 208, 235, 240 ammonium, 14, 24, 106, 123, 169, 170, 181, 182, 185, 196, 198, 234, 240, 241, 242, 251, 264, 268 ammonium chloride, 14, 24, 106 AMO, 93 Amsterdam, 207 amylase, 120 anabolism, 91, 163, 167, 168 anaerobe, 117 anaerobes, 117 anaerobic, xi, xii, xiii, 63, 64, 65, 66, 68, 71, 75, 86, 88, 91, 99, 101, 115, 116, 117, 118, 119, 122, 123, 124, 125, 130, 135, 136, 137, 138, 139, 144, 146, 148, 149, 150, 153, 163, 164, 165, 167, 169, 170, 171, 172, 173, 174, 175, 176, 177, 179, 180, 181, 182, 185, 186, 205, 209, 211, 213, 214, 215, 216, 217, 224, 225, 226, 227, 234, 240, 241, 242, 246, 267, 268, 285, 286, 291, 294 anaerobic bacteria, xi, 63 anaerobic digesters, 170, 175 anaerobic sludge, 164, 167, 169, 176, 182, 185, 216 analgesics, 269 analytical techniques, xiv, 245 Animal feed, 58 animal tissues, 247 animals, 66, 83, 112, 114, 165, 192, 194, 203, 206, 246, 249, 268 ANN, 274, 283, 286 annealing, 77 anoxic, xi, 68, 75, 78, 82, 84, 85, 86, 89, 91, 92, 94, 95, 96, 97, 98, 99, 104, 105, 108, 123, 163, 169, 184, 213, 286 antagonistic, 215 anthracene, 263

anthropogenic, 192, 269 antibiotic, 260 antibiotics, 260, 268, 269 antibody, 70 antigen, 57, 70 anti-inflammatories, 268, 269 API, 69, 78 application, xii, xiii, xiv, 32, 38, 44, 45, 56, 57, 69, 71, 74, 76, 77, 90, 97, 101, 123, 125, 126, 129, 132, 160, 161, 162, 168, 169, 172, 173, 175, 177, 178, 179, 181, 183, 184, 187, 188, 189, 190, 192, 194, 195, 196, 198, 203, 204, 207, 208, 210, 211, 230, 240, 241, 246, 250, 254, 256, 259, 291 aqueous suspension, 54 archaea, xi, 63, 64, 66, 71, 73, 75, 83, 85 Arctic, 67, 82 Arctic Ocean, 67 Argentina, 33, 187, 188, 190, 193, 198, 203, 208 aromatic compounds, 235, 236, 242 aromatic hydrocarbons, 80, 235, 263 aromatics, 235 Arrhenius equation, 219, 221, 222 ash, xiii, xiv, 111, 128, 133, 143, 148, 152, 155, 160, 162, 187, 189, 190, 191, 192, 193, 195, 196, 197, 198, 199, 201, 202, 203, 204, 205, 206, 207, 273, 274, 285, 286, 287, 293, 294 Asia, 76, 198 Asian, xv, 289, 296 Asian countries, xv, 289, 296 assessment, xv, 73, 121, 160, 183, 204, 205, 240, 289, 294, 295 assimilation, 90 asymptotically, 173 Atlas, 123 atmosphere, 71, 108, 162, 175 atmospheric pressure, 152, 255, 260 atomic force, 32 atomic force microscopy, 32 atoms, 223 ATP, 64, 91, 92, 167 attachment, 19, 256 attacks, 46 Australia, 159, 183, 198 authenticity, 74 autolysis, 174 automation, 248, 250 autotrophic, 169, 170, 185, 240 availability, ix, xi, xiii, 34, 50, 117, 185, 187, 188, 192, 196, 198, 199, 200, 203, 204, 207

B Bacillus, 53

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Index bacteria, ix, x, xi, xiv, 2, 3, 8, 10, 29, 49, 63, 64, 66, 67, 69, 70, 71, 73, 75, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 90, 93, 97, 112, 116, 120, 121, 130, 131, 165, 166, 167, 170, 174, 182, 183, 185, 188, 212, 216, 229, 230, 231, 233, 234, 235, 236, 240, 241, 242, 243 bacterial, x, 2, 8, 72, 73, 75, 79, 81, 82, 85, 86, 91, 97, 109, 231, 233, 235, 236, 237, 239, 242 bacterial cells, 91, 109 bactericides, 268 bacterium, xiv, 81, 82, 85, 87, 88, 184, 229, 230, 232, 234, 235, 241, 242 baking, 49, 291 barley, 204 barrier, 117, 120, 250 barriers, 291 basicity, 274, 275, 285 BED, 258 behavior, 105 behaviours, 206 Beijing, 209, 212, 215 Belgium, 97 bell, 276, 277 beneficial effect, 24 benefits, 53, 57, 99, 115, 166, 211, 246, 250 benzene, 65 benzoyl peroxide, 39 bias, 235 bi-layer, 55, 57 binding, 70, 77, 200, 215, 232 bioactive compounds, 41 bioavailability, 56 biocatalysts, xi, 63, 65 biodegrability, 216 biodegradability, 103, 169, 171, 172, 180, 186, 196, 213, 217, 225 biodegradable, xiii, 3, 99, 126, 133, 170, 173, 174, 175, 209, 212, 215 biodegradation, 3, 68, 80, 163, 171, 175, 219, 263, 268 biodiesel, 179, 181 bioethanol, 179 biofilms, 66, 75, 85, 86, 242 biogas, xii, xiii, 65, 68, 90, 116, 117, 118, 119, 120, 130, 131, 132, 135, 137, 139, 140, 144, 146, 149, 150, 151, 152, 153, 154, 155, 156, 160, 161, 171, 173, 174, 178, 179, 209, 212, 213, 214, 216, 217, 224, 225, 291, 295, 296 biogranulation technology, 182 biological nutrient removal, 99, 102 biological processes, 90, 91, 109, 181 bioluminescence, 94, 121 biomarker, 72

biomarkers, 80 biomass, ix, xiii, 1, 3, 98, 112, 120, 121, 163, 165, 166, 167, 168, 169, 175, 182, 183, 184, 187, 198, 199, 200, 202, 203, 206, 231, 240, 291, 292, 296 biomaterials, 91, 109 biomineralization, 64 biopolymer, x, 2, 4, 5, 21, 22, 29 biopolymers, xiii, 4, 26, 29, 209, 212 bioreactor, ix, 1, 3, 47, 57, 60, 61, 108, 120, 121, 123, 166, 181, 182, 183, 185, 186, 204 bioreactors, 75, 79, 85, 163, 166, 170, 182, 184, 185, 240 bioremediation, xi, 63, 65, 78, 241, 243 biosynthesis, 64, 90, 167 biotechnological, 267 biotechnology, 82, 84 biotic, 246 biotransformation, xi, 63, 65, 78 bisphenol, 251, 270 Black Sea, 86 bleaching, 39 blocks, 194 blood, 56 blood vessels, 56 bloodstream, 56, 57 blot, 231, 241 body weight, 113, 115 boilers, 131, 136, 149, 150, 151, 153 boiling, 50, 69, 145, 252, 256, 259, 260 bonding, 54, 200 bonds, 45, 74 Boston, 160 Brazilian, 159 breakdown, 131, 246, 267, 268, 274 breeding, 38 British Columbia, 81 Brno, 125 Brussels, 205, 249 bubbles, 57, 62, 173, 218 Buenos Aires, 187, 190, 191, 200, 208 buffer, 48, 75, 190 buildings, 131, 132, 146, 153 burn, 133 buses, 292 butyric, 131 bypass, 38, 167 by-products, 218

C Ca2+, 4, 12, 15, 16, 19, 20, 21, 22, 28, 194 cabbage, 54 cadmium, 204, 206, 215, 287

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caffeine, 269 calcium, x, 2, 5, 6, 8, 11, 16, 18, 19, 20, 21, 23, 24, 25, 26, 27, 28, 29, 36, 42, 45, 54, 211 Canada, 63, 71, 81 cancer, 56 Candida, 52, 61 capillary, 14, 23, 233 capital cost, 174 carbamide, 211 carbohydrate, 99, 100, 214 carbohydrates, 36, 40 carbon, xi, xiii, xv, 36, 40, 41, 59, 63, 64, 66, 67, 68, 69, 82, 85, 89, 98, 99, 102, 104, 105, 107, 108, 122, 123, 126, 127, 128, 130, 131, 138, 140, 145, 149, 163, 168, 170, 173, 175, 177, 179, 181, 184, 187, 193, 194, 195, 196, 197, 203, 205, 207, 223, 225, 234, 241, 243, 289, 291, 292, 296 carbon cycling, 243 carbon dioxide, 40, 41, 66, 67, 68, 126, 130, 131, 138, 140, 149, 163 carbon monoxide, 145 carbonization, xv, 289 carboxylates, 254 carcinogenic, 2, 263, 268 carrier, 44, 240, 254 case study, xii, 125, 135, 136, 145, 152, 155, 158 catabolic, 167, 236, 243 catabolism, 91, 163, 165, 167, 168 catalase, 54 catalyst, 51, 180 catalytic activity, 49 catalytic system, 47 catechol, 237, 241 cation, 5, 15, 21, 23, 24, 54 cattle, 123, 190, 203 cavitation, 173, 218 CEC, 188 cell, xiii, 4, 16, 57, 70, 71, 72, 75, 84, 90, 91, 93, 98, 99, 101, 102, 109, 112, 115, 117, 121, 123, 163, 165, 166, 168, 173, 183, 209, 212, 213, 215, 216, 217, 218, 226, 254, 259, 260, 261 cell membranes, 57 cell surface, 70, 183 cellulose, x, 2, 8, 10, 11, 19, 20, 22, 23, 24, 30, 54, 131, 259 cellulose fibre, 22 cement, xv, 126, 132, 133, 189, 207, 287, 289, 290, 295 centigrade, xiv, 273, 275, 281, 283, 285 ceramic, 47, 217 ceramics, 285 CH3COOH, 131 CH4, 90, 131, 140, 214, 292

cheese, 56 chelating agents, 35 chemical agents, 212 chemical composition, 11 chemical industry, 242 chemical oxidation, 172 chemical properties, x, xi, 2, 8, 27, 29, 34, 200 chemical structures, 268 chemicals, xi, 34, 114, 217, 246, 247, 263, 269 China, 185, 209, 210, 211, 214, 227 chloral, 218 chloride, 11, 15, 127, 211 chlorinated paraffin, 247 chlorpyriphos, 268 cholesterol, 55 chromatographic technique, xiv, 245, 247, 248 chromatography, xiv, 42, 43, 44, 72, 245, 248, 249 Chromium, 127 chromosome, 243 ciliate, 184 ciprofloxacin, 269 cis, 49, 72 classes, 251 classical, 5, 34, 35, 78, 163, 248, 250, 253, 255, 258, 259, 261, 262, 270 classification, 71, 72, 82, 87, 155, 235 clay, 190, 196, 200, 206, 208 clean energy, 115 cleaning, 133, 148, 159, 267 cleavage, 71, 231, 233, 235, 236, 242 climate change, 296 clone, 73, 74, 237, 238 cloning, 73, 74, 236 cluster analysis, 68, 242 Co, 41, 59, 62, 65, 171, 172, 180, 215, 216, 291 CO2, 40, 41, 43, 59, 67, 90, 99, 117, 118, 131, 140, 141, 145, 170, 171, 214, 218, 253, 254, 262, 290, 291, 292, 295, 296 coagulation, ix, 15, 170, 188 coal, 162, 235, 242 coatings, 45 cocoa, 42 cocoa butter, 42 coconut, 268 coding, 84 coefficient of variation, 10 coffee, 40 coke, xiv, 230, 235, 236, 237, 239 Coke, 235 colloids, 3, 26, 32 combined effect, 287 combustion, 128, 132, 133, 140, 141, 142, 143, 145, 146, 152, 160, 171, 189, 206, 208, 291

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Index communities, xiv, 73, 75, 77, 79, 81, 87, 88, 97, 229, 230, 241 community, xiv, 65, 68, 76, 77, 80, 82, 83, 85, 91, 97, 230, 235, 240, 241, 242, 243 compaction, 22, 24 competition, 185, 216 complexity, 11, 215, 248, 249, 252, 253, 270 compliance, 188 components, x, xi, 2, 3, 8, 22, 34, 36, 40, 43, 44, 49, 56, 111, 138, 142, 147, 163, 166, 190, 191, 200, 203, 208, 216, 223 composition, 3, 7, 11, 36, 45, 48, 76, 87, 115, 142, 143, 145, 169, 175, 215, 239, 247, 248, 279, 292 compost, 205, 206, 225, 267 composting, 126, 136, 153, 155, 156, 157, 158, 159, 204, 206, 246, 291 compounds, x, xi, 2, 4, 33, 34, 35, 41, 53, 55, 57, 63, 64, 65, 66, 72, 84, 87, 91, 138, 162, 164, 169, 173, 174, 178, 183, 192, 196, 200, 205, 213, 217, 219, 221, 235, 236, 237, 247, 249, 250, 251, 252, 253, 254, 255, 257, 259, 260, 261, 263, 264, 267, 268, 270 computation, 274 concentration, xi, xiii, 5, 10, 11, 12, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25, 27, 37, 44, 50, 51, 52, 74, 77, 89, 94, 96, 101, 103, 104, 107, 108, 109, 110, 115, 117, 118, 127, 166, 168, 179, 182, 183, 187, 189, 193, 200, 202, 203, 210, 211, 214, 215, 216, 217, 219, 222, 223, 224, 233, 246, 247, 248, 263, 267, 269 concrete, 130 condensation, 131, 152, 154 conditioning, x, 2, 7, 8, 11, 14, 24, 26, 29 conduction, 255 conductivity, 193, 194, 198, 205 conductor, 255 configuration, 11, 181, 226 conflict, 5 Congress, vi conjugation, 72 consciousness, 128 conservation, 36, 143, 236, 274 constraints, 90, 162 construction, 52, 74, 154, 155, 158, 168, 189, 237 construction materials, 189 consumption, xv, 38, 44, 52, 90, 103, 105, 146, 151, 165, 166, 175, 178, 203, 212, 217, 218, 250, 255, 270, 274, 289, 290, 291, 294, 295, 296 contaminant, 38, 210, 270 contaminants, xiv, 112, 120, 210, 215, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 256, 257, 259, 262, 263, 267, 268, 270

contamination, ix, xiii, 35, 77, 188, 211, 246, 247, 250 control, ix, xiii, 2, 3, 7, 38, 45, 52, 65, 78, 80, 90, 92, 94, 108, 113, 114, 116, 117, 120, 122, 128, 161, 174, 179, 187, 189, 194, 195, 200, 201, 203, 210, 211, 224, 246, 274, 275, 295 control group, 113 convection, 133 conversion, 52, 54, 85, 224 cooling, 38, 42, 43, 66, 69, 86, 218 copper, 204, 206, 207, 208, 215, 252, 253, 254, 258, 287 corn, 115 correlation, 72, 220, 275, 280, 285 correlation coefficient, 275, 280 corrosion, 65, 68, 71, 81, 86, 192 corrosive, xi, 63, 65, 73, 76 cosmetics, 35, 267, 268, 270 cost-effective, 90, 172, 231, 235 costs, ix, xii, xiii, 1, 7, 87, 90, 115, 130, 132, 149, 154, 155, 156, 157, 158, 159, 161, 162, 163, 166, 168, 172, 174, 178, 186, 188, 211, 217, 224, 291 cotton, 105 couples, 234 coupling, 84, 91, 179 covalent, 53 covering, 153 cracking, 38 CRC, 87, 183, 204, 205 crop production, xiii, 187, 189 crops, xiii, 179, 188, 200, 203, 204, 205, 212 crude oil, 219 Cryptococcus, 50 crystalline, 191 crystallization, 41, 177 cultivation, 49, 71, 72, 75, 78, 208, 230, 239, 240 cultivation conditions, 72 culture, xiv, 12, 14, 69, 72, 73, 75, 77, 79, 82, 92, 93, 94, 121, 229, 230, 234, 236, 239, 243 culture media, 12, 79 Cybernetics, 285 cycles, 77, 91, 102, 259, 260, 261, 262 cycling, 64, 79, 167, 243 cytochrome, 86 cytoplasm, 99 Czech Republic, 125, 128, 135, 153

D DAD, 255 dairy, 198, 204 Dallas, 78 data set, 239

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database, 75, 76, 77 death, 198 decay, 163, 165, 166, 167, 185 decomposition, 2, 65, 101, 130, 131, 148, 189, 196, 197, 198, 256 deep-sea, 75, 80, 82, 84 deficiency, 77, 207 definition, 198 degradation, xi, 38, 53, 56, 63, 64, 105, 108, 120, 124, 171, 174, 212, 216, 225, 236, 237, 239, 240, 242, 246, 253, 256, 257, 260, 270, 274 degradation mechanism, 174 degradation pathway, 236, 237 degradation process, 213 degrading, 236, 241, 243 degumming, x, 33, 34, 35, 36, 38, 39, 42, 47, 53, 57, 58 dehydration, 56, 69, 219 delivery, 56 denaturation, 43, 74, 77 denaturing gradient gel electrophoresis (DGGE), 73, 79, 84, 87 denitrification, xi, 89, 94, 96, 97, 99, 102, 103, 105, 107, 108, 122, 123, 169, 170, 175, 180, 181, 214, 225, 234, 286 denitrifying, 108, 240 density, 22, 69, 71, 77, 122, 195, 225, 254, 292 deoxyribonucleotides, 232 Department of Agriculture, x, 33, 34 deposition, 76, 246 deposits, 65, 188 derivatives, xi, 34, 237 desiccation, 68 desorption, 208, 246, 254, 259, 260 destruction, 115, 130, 162, 218 detection, xi, xiii, xiv, 63, 68, 69, 71, 74, 77, 78, 83, 87, 121, 187, 200, 203, 229, 230, 231, 232, 233, 234, 235, 240, 242, 243, 248, 249, 251, 252, 253, 262 detergents, 215, 247, 268 developed countries, 126, 133, 211, 214, 245, 268 developing countries, 162, 214 deviation, 114 diagenesis, 78 dibenzofurans, 206, 247 dibenzo-p-dioxins, 206, 247 dielectric constant, 255 diet, 38, 49 dietary, 38, 56, 57 differentiation, 70 diffraction, 14, 191 diffusion, 48, 57, 168, 254 digestibility, 198

digestion, ix, xii, xiii, 1, 3, 7, 76, 90, 91, 99, 101, 115, 116, 117, 118, 119, 122, 123, 124, 125, 130, 131, 132, 135, 136, 138, 139, 144, 146, 148, 149, 150, 153, 155, 156, 163, 169, 170, 171, 172, 173, 174, 175, 180, 181, 182, 186, 209, 211, 212, 214, 215, 216, 217, 224, 225, 226, 227, 246, 255, 256, 263, 267, 268, 285, 290, 291, 294 dimethylformamide, 52 dioxins, 133, 188, 206, 247, 270 discharges, xii, 161 discrimination, 71 diseases, 68 disinfection, 130 dispersion, 10, 15, 16, 25, 42, 45, 49, 250 displacement, 44 dissolved oxygen, 168, 169, 184, 286 distilled water, 14, 104, 106, 198 distribution, xi, 14, 17, 18, 41, 57, 63, 65, 73, 76, 78, 82, 84, 86, 155, 207, 249 diversification, 296 diversity, xi, 63, 68, 70, 73, 74, 76, 78, 79, 80, 82, 83, 84, 86, 87, 88, 240 DMF, 52 DNA, xiv, 57, 62, 73, 74, 76, 77, 102, 229, 230, 231, 232, 236, 237, 238, 242, 243 DNA sequencing, 238 Docosahexaenoic, 49 donor, 49, 53, 71, 108, 123 donors, 52, 65, 99, 102 dosage, 14, 25, 99, 179, 210, 217 dosing, 40, 42 double bonds, 45 draft, 205, 247 drainage, xi, 63, 65, 66, 78, 81, 82, 190 drugs, 43, 56 dry matter, 127, 128, 129, 132, 135, 138, 139, 140, 141, 144, 145, 146, 147, 148, 151, 152, 153, 154, 199, 210 drying, x, xii, 33, 38, 40, 57, 130, 132, 133, 136, 137, 140, 141, 144, 145, 146, 147, 148, 149, 150, 152, 153, 154, 156, 159, 161, 163, 208, 248, 259, 263, 291 drying medium, 132, 149 dumping, 90, 188 durability, 274 dust, 127, 145

E E. coli, 92, 232, 237 early warning, 65, 78, 233 earnings, 115 earth, 42, 139, 144

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Index ecological, 68 ecology, 80, 84, 88, 242 economics, 162 ecosystem, 68, 119 ecosystems, 66, 67 effluent, ix, 1, 3, 7, 8, 44, 94, 96, 97, 99, 105, 107, 108, 120, 166, 167, 170, 173, 186, 188, 189, 241, 246, 267, 286, 287 effluents, xi, 63, 64, 168, 169, 170, 180 egg, 38, 43, 44, 47, 48, 49, 50, 51, 55 eicosapentaenoic acid, 49 elaboration, 47 electric energy, 140 electrical conductivity, 198, 205 electricity, 159, 291, 292, 295, 296 electrolysis, 177, 180 electrolyte, 42 electron, xi, 63, 64, 65, 68, 69, 71, 84, 87, 91, 99, 102, 103, 108, 121, 123, 165, 170, 216, 249 electrons, 71 electron-transfer, 84 electrophoresis, 71, 73, 74, 76, 77, 79, 82, 84, 87, 98, 233, 235 ELISA, 70 elongation, 208 email, 1 emission, 127, 128, 145, 160, 163, 188, 196, 210, 290, 292, 295, 296 emulsification, 40, 42 emulsifier, 42, 45 encapsulated, 56, 57 encapsulation, 57 encoding, 73, 81, 83 endocrine, ix, 188, 260, 267, 268, 269, 270 endocrine system, 267 endothelial cell, 56 endothelial cells, 56 energy, xiii, xv, 84, 91, 92, 101, 109, 111, 119, 120, 130, 131, 132, 135, 137, 138, 140, 143, 148, 149, 152, 153, 154, 155, 156, 157, 158, 159, 161, 162, 163, 165, 167, 168, 169, 173, 174, 175, 178, 179, 181, 189, 209, 210, 211, 212, 213, 216, 217, 221, 222, 223, 224, 250, 255, 256, 274, 289, 290, 291, 292, 293, 294, 295, 296 energy consumption, xv, 178, 250, 274, 289, 290, 291, 294, 296 energy efficiency, 162, 294 energy recovery, xiii, 161 energy supply, 174, 189 energy transfer, 168 England, 14 enlargement, 168

303

environment, xii, 2, 22, 55, 64, 67, 81, 82, 83, 84, 115, 119, 125, 126, 131, 178, 211, 245, 246, 249, 250, 263, 268, 269 environmental conditions, 163, 171, 290 environmental contaminants, 80, 270 environmental contamination, ix, 35, 188, 246 environmental factors, xii, 161, 162 environmental impact, xiii, 81, 187, 189, 225 environmental policy, 206 environmental protection, 212 Environmental Protection Agency, 65, 126, 159, 247 environmental technology, 83 enzymatic, x, 12, 33, 35, 39, 45, 47, 48, 49, 50, 53, 57, 87, 237 enzymatic activity, 50, 237 enzymes, 45, 46, 48, 50, 52, 54, 56, 57, 64, 80, 109, 120, 174, 179, 215, 237, 243 EPA, 49, 50, 51, 208, 270 epoxy, 51 equilibrium, 44, 51, 52 ergosterol, 56 erosion, 246, 274 Escherichia coli, 233, 241 ESI, 249 ester, 52 esterification, 51, 52 esters, 48, 49, 50, 51, 52, 53, 72, 251, 270 estimating, 283 estradiol, 269 estrogens, 249, 267, 268, 269 estuarine, 64, 66, 67, 76, 79, 80 ethane, 224 ethanol, 39, 41, 42, 43, 44, 50, 54, 56, 59, 75, 120, 223, 251, 254 ethanolamine, 44, 45, 53 Ether, 60 ethers, 266, 268 ethyl acetate, 251, 257 ethylbenzene, 65 ethylene, 208 ethylene oxide, 208 EU, xii, 125, 126, 128, 129, 134, 158, 160, 161, 162, 181, 247 Europe, 76, 134, 198, 210 European Commission, 133, 159, 247 European Union, vii, xii, 125, 126, 210, 211, 245, 247 Eurostat, 128, 129, 159 eutrophication, 2, 99 evaporation, 75, 251, 257, 262 Everglades, 75, 79 evolution, 79, 195, 197, 199, 242, 243 expenditures, 115

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experimental condition, 252 exposure, 246 extraction, x, xiv, 33, 34, 38, 39, 40, 41, 43, 73, 74, 115, 204, 206, 235, 236, 245, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 270 extraction process, 254, 259 extremophiles, 68 extrusion, 56

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F failure, 158 family, 239, 249 farming, 129, 130, 132, 133, 188, 198 farmlands, 212 fasting, 180 fat, 45, 57, 111, 214 fats, 38, 49, 258 fatty acid, x, xi, 33, 36, 39, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 66, 67, 71, 72, 80, 83, 85, 87, 117, 131, 218, 224 fatty acids, x, xi, 33, 36, 39, 45, 47, 48, 49, 50, 51, 52, 66, 67, 72, 85, 117, 131, 224 February, 208 feces, 66, 83 feedback, 38 feeding, 20, 21, 95, 96, 112, 117, 119 feedstock, 189 fees, 155 fermentation, 68, 131, 224, 227, 246 fertility, 162, 195, 199, 207 fertilizer, 133, 160, 162, 170, 177, 200, 204, 205, 211, 212, 246 fertilizers, xiii, 187, 189, 211, 246 fiber, 111, 214 film, 38 filters, 23, 248, 251, 265, 269, 270 filtration, 41, 186, 259 financial support, 31 fingerprints, 72, 76 fish, 49, 65, 115 FISH, 73, 75 fish oil, 49 fitness, 281, 283 fixation, 75, 85, 227 flame, 247, 268 flame retardants, 247, 268 flavor, x, 33, 56 flavors, 40 flocculation, ix, x, 2, 4, 7, 11, 15, 16, 18, 19, 20, 21, 22, 26, 28, 29, 188

flow, xii, 7, 8, 11, 15, 38, 40, 41, 52, 125, 138, 139, 141, 233, 254, 293, 294 flow rate, 52 fluctuations, 16, 168, 204 flue gas, 85, 140, 145, 146, 150, 152, 153, 183 fluid, 41, 45, 133, 134, 250, 253, 254, 255, 259, 262 fluid extract, 250, 253, 259 fluidized bed, 133, 134, 160, 162 fluorescence, 70, 77, 232, 242, 252, 258 fluorescence in situ hybridization, 232, 242 fluoride, 127 fluorine, 263 fluoroquinolones, 260 focusing, 290 food, x, 12, 33, 35, 39, 40, 41, 42, 43, 45, 50, 56, 64, 115, 165, 167, 192, 198, 203, 205, 206, 214, 268, 296 food products, 268 Ford, 30 Forestry, 123 formamide, 74 fossil, xiii, 126, 132, 137, 153, 154, 209, 292 fossil fuel, xiii, 126, 132, 137, 153, 154, 209, 292 fossil fuels, 132, 137, 292 fouling, 186 fractionation, x, 33, 42, 43, 57 free energy, 51 free radical, 226 freezing, 99, 122, 225 freshwater, 64, 66, 67, 75, 76, 82 fuel, xiii, 90, 126, 128, 130, 136, 143, 149, 152, 153, 154, 179, 208, 209, 274, 291, 295 fungi, 50, 64 fungicides, 268 fungus, 50 furnaces, 133, 134 Fusarium, 49 fuzzy logic, 274, 275, 283

G gas, xiv, 41, 64, 65, 66, 69, 71, 72, 81, 83, 85, 88, 102, 103, 104, 105, 108, 115, 117, 128, 132, 133, 134, 140, 142, 143, 144, 145, 146, 147, 148, 149, 150, 152, 154, 159, 163, 170, 174, 176, 183, 210, 217, 218, 219, 221, 234, 245, 249, 259, 291, 294, 295 gas chromatograph, xiv, 72, 245, 249 gas exploration, 64 gas phase, 218 gases, xi, 2, 63, 65, 131, 189, 196, 235, 295 gasification, 126, 159, 181, 242 gastrointestinal, 56, 83, 192

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Index gastrointestinal tract, 56, 83, 192 gauge, 47 Gaussian, 276, 277 gel, 42, 54, 56, 70, 71, 73, 74, 76, 77, 79, 81, 84, 87, 251, 255, 257, 261, 262 gel permeation chromatography, 261 gelatin, 75 gels, 270 GenBank, 76 gene, xiv, 57, 66, 73, 74, 77, 80, 83, 84, 87, 88, 229, 230, 236, 238, 239, 240, 241 gene therapy, 57 gene transfer, 73, 88 generation, xv, 2, 71, 90, 95, 96, 101, 104, 105, 117, 119, 132, 146, 151, 152, 153, 154, 155, 159, 162, 183, 189, 218, 241, 289, 291, 292 genes, xiv, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 84, 85, 87, 94, 230, 235, 236, 237, 238, 239, 241, 242, 243 genetic diversity, 74 genome, 77, 239, 242 genomes, 240, 241 genomic, 66, 74, 76, 77, 236 genomics, 240 geothermal, 67 Germany, 158, 233, 267, 269 germination, 194, 199, 204, 208 glass, 54, 216, 254, 256, 285 glasses, 190 global warming, 291, 295 Global Warming, 178 globulin, 249 gloves, 249 glucose, 102, 124 glucosidases, 174 glutamate, 106 glutaraldehyde, 54 glycerol, 36, 46, 51, 52, 54, 55 glycogen, 165 glycol, 264, 268 glycolipids, x, 33, 34, 36 government, vi, 210 GPC, 51 grain, 112, 115 grains, 38, 39 Gram-negative, 66, 67, 83, 87, 88, 243 granules, 169, 180, 181, 185, 274 graph, 146, 150, 151 grass, 198 grasses, 207 grasslands, 207 gravity, ix, 1, 7 grazing, 163, 165, 167, 184, 194, 198, 203

Great Britain, 129 greenhouse, 126, 163, 188, 190, 194, 208 greenhouse gas, 126, 163, 188 greenhouse gases, 126 GRI, 85 gross domestic product, 65 groundwater, 65, 75, 79, 210 groups, xii, xiv, 5, 10, 42, 45, 46, 50, 51, 54, 66, 71, 72, 73, 75, 77, 90, 109, 112, 113, 114, 115, 120, 185, 221, 229, 230, 247, 261, 263, 268, 269 growth, xi, xiii, 2, 3, 4, 5, 14, 18, 67, 68, 69, 82, 89, 90, 92, 97, 98, 99, 120, 121, 122, 130, 131, 163, 166, 167, 175, 180, 184, 187, 189, 192, 198, 199, 200, 215, 231, 233, 239, 246, 287 growth rate, 82, 92, 199, 231, 233, 287 growth temperature, 68 Guangzhou, 215 guidelines, 10, 178, 205 Gulf of Mexico, 83 gums, x, 33, 34, 36, 37, 38, 39, 40, 42 gut, 79

H H2, 67, 71, 131, 214 half-life, 54 halogenated, 247 halogenation, 45 handling, ix, 1, 7, 36, 42, 121, 159, 160, 188, 249, 296 hardness, 267 harm, 210 harvest, 36, 200, 201, 202 hazardous substance, ix, 120, 188 hazardous substances, ix, 120, 188 hazards, 2, 126 health, ix, xi, 2, 38, 40, 43, 48, 63, 126, 162, 188, 246, 253, 268 health problems, 38 heat, xii, 45, 48, 68, 92, 120, 125, 126, 128, 130, 131, 132, 133, 135, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 217, 218, 224, 241, 250, 255, 262, 268 heat capacity, 145, 224 heat loss, 139, 146, 152 heat transfer, 132, 133, 139, 144, 250 heating, 52, 130, 132, 135, 136, 139, 146, 149, 150, 152, 153, 174, 217, 218, 224, 248, 255, 256, 259, 262, 291 heavy metal, xi, xii, xiii, 63, 65, 75, 78, 109, 112, 113, 114, 115, 123, 126, 127, 130, 133, 157, 161,

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162, 177, 187, 204, 205, 206, 210, 211, 212, 215, 241, 247, 285, 287 heavy metals, xi, xiii, 63, 65, 78, 109, 112, 113, 114, 115, 123, 126, 130, 133, 157, 162, 177, 187, 204, 210, 211, 212, 215, 241, 247, 285, 287 height, 13 hematite, 192 hemisphere, 207 Hepatitis A, 57, 62 heptane, 259 herbs, 56 heterogeneous, 3, 17, 75, 213, 232 heterotrophic, 64, 105, 169, 180, 287 heterotrophic microorganisms, 64 hexane, 39, 41, 47, 49, 50, 251, 252, 255, 256, 257, 258, 259, 262 high fat, 50 high pressure, 102, 105, 174, 218, 256, 259 high temperature, 42, 43, 213, 217, 250, 274, 285 high-speed, 216 high-value products, 56, 57 homogenous, 133 Honda, 62 horizon, 194 hospital, 286, 287 host, 235, 237 hot spring, 67, 68, 81, 84 hot water, 144, 149 household, 255, 270 households, ix, 188, 247 HPLC, 44, 59, 253 human, 2, 43, 68, 83, 162, 246, 268, 269 humans, 49, 66, 162, 192, 249 humidity, 131 hybrid, 178, 242, 286 hybridization, 73, 75, 77, 79, 82, 231, 232, 241 hydrate, 52, 61 hydrates, 66, 83 hydro, 45, 49, 52, 55, 65, 80, 82, 101, 162, 235, 247, 263, 270 hydrocarbon, xi, 55, 63, 66, 76, 82, 243 hydrocarbons, 65, 82, 270 hydrogen, xi, 39, 45, 54, 63, 64, 65, 66, 71, 80, 83, 86, 121, 131, 173, 174, 175, 177, 181, 184, 186, 216 hydrogen peroxide, 39, 45, 173, 174, 184, 186 hydrogen sulfide, xi, 63, 64, 65 hydrogenation, 45 hydrolysis, xiii, 45, 47, 48, 49, 50, 51, 52, 53, 54, 99, 115, 117, 122, 123, 130, 131, 170, 171, 172, 173, 174, 175, 181, 186, 209, 212, 216, 217, 225, 226, 255 hydrolyzed, 44, 45, 47, 49, 55, 102, 116

hydrophilic, 45, 49, 52, 55, 247 hydrophobic, 47, 54, 55, 101, 268, 269, 270 hydrophobic properties, 268 hydrophobicity, 270 hydrothermal, 66, 67, 68, 75, 82, 84 hydroxide, 42, 216, 256 hydroxides, 170 hydroxyl, 45, 51, 218 hydroxyl groups, 45 hydroxylation, 45 hygienic, 212, 249 hypothesis, 19, 239

I ibuprofen, 261, 269 id, 115, 200, 203 identification, xiv, 71, 72, 73, 74, 75, 78, 81, 97, 229, 240, 243, 292 identity, 73, 238 Illinois, 85 image analysis, 97 immobilization, 48, 52, 57, 183, 207 immobilized enzymes, 50, 52, 57 immune response, 57 immunofluorescence, 70 immunoglobulin, 70 immunological, 68, 70, 71, 78, 84 implementation, 134, 175, 178, 182, 186, 245 impurities, x, 33 in situ, 61, 73, 78, 82, 83, 90, 91, 109, 203, 240, 242, 243 in situ hybridization, 73, 82, 232, 242 in vivo, 62 inactive, 200 incidence, 115 incineration, xii, xv, 90, 125, 126, 127, 128, 129, 130, 132, 133, 136, 137, 140, 141, 142, 143, 144, 145, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 161, 162, 179, 183, 188, 189, 190, 192, 196, 197, 203, 204, 208, 210, 211, 246, 289, 290, 291, 292, 293, 294, 295 inclusion, x, 2, 30 incubation, 11, 69, 103, 104, 199, 232, 235 incubation period, 11 incubation time, 69, 199 India, 61, 62 indirect drying, 132 induction, 65 industrial, ix, x, xi, xiv, 1, 2, 3, 33, 46, 52, 57, 63, 65, 66, 68, 69, 71, 75, 78, 90, 93, 160, 169, 170, 173, 179, 204, 209, 210, 213, 215, 216, 223, 230, 240, 246, 263, 267, 268, 270, 286, 287, 290, 292

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Index industrial application, 52 industrial processing, 46 industrial production, 267 industrial wastes, 290 industry, 8, 56, 85, 88, 133, 180, 183, 242, 247, 291, 293, 295 inert, 40, 64, 91, 133, 164, 189, 254, 256, 259 inertness, 253 infectious, 126, 137, 145, 158, 249 inflammatory, 83, 265 influenza, 57 infrastructure, 68 inhibition, 68, 74, 76, 93, 94, 116, 118, 215, 216, 225 inhibitor, 105 inhibitors, 93, 121 inhibitory, xi, 89, 93, 96, 199, 215 inhibitory effect, xi, 89, 93, 96, 199 injection, 78, 207 injury, vi inorganic, 42, 84, 87, 91, 120, 138, 141, 173, 174, 189, 190, 196, 200, 213, 219, 230 inorganic salts, 42 insecticides, 268 insertion, xi, 34, 73 instruments, 69, 231 insulation, 139 insulin, 47 integration, 181 integrity, xi, 4, 63, 65, 78, 163, 166 interaction, 6, 185, 269 interactions, 5, 22, 232 interface, 13, 36, 47, 86, 174, 218 interfacial tension, 51 interference, 252 interval, 14 intestinal tract, 2 intravenously, 56 intrinsic, 48 investment, 52, 57, 155, 157, 159, 178, 179, 247, 250 iodine, 36, 45 ionic, 5, 49, 215, 253, 259 ionization, 249 ions, x, 2, 5, 8, 11, 16, 19, 20, 21, 23, 28, 31, 42, 45, 198, 255, 267 IPPC, 128 Ireland, 129, 210 iron, 5, 20, 21, 36, 69, 207, 279, 280 iron deficiency, 207 irradiation, 174, 181, 218, 226, 250, 255, 257 island, 135, 144 isoenzymes, 53, 54 isolation, 75, 88 isotope, 239, 242

J Japan, viii, xv, 182, 205, 229, 287, 289, 290, 291, 292, 295, 296 Japanese, 290, 296 Jefferson, 182 Jung, 82, 123, 185 jurisdiction, 188

K K+, 12, 194 kinetic energy, 216 kinetic model, xiv, 209, 219, 221, 222, 223 kinetic parameters, 222 kinetics, xiv, 209, 225, 226, 242, 259 King, 59, 205 Kobe, 292 Korea, 89, 123, 286

L labor, 73, 74 lactation, 111 lactic acid, 45, 131 lakes, 80 land, xii, xiii, 129, 130, 132, 133, 135, 161, 162, 163, 177, 178, 179, 183, 187, 188, 189, 190, 192, 203, 207, 208, 210, 211, 212, 246, 247, 270, 291 landfill, xii, 81, 90, 125, 158, 161, 162, 180, 210, 246, 274 landfills, 126, 163, 211 large-scale, xiii, 42, 44, 187, 190, 217 laser, 77 latex, x, 2, 8, 10, 11, 15, 28 law, 143, 163, 211 laws, 210, 212 leachate, 163, 180, 188 leachates, 205 leaching, 189, 204, 206, 212, 215, 246 learning, 275, 287 leather, 45 lecithin, x, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 45, 47, 48, 49, 51, 54, 55, 57, 59, 60 legislation, xii, 125, 126, 127, 129, 133, 134, 145, 146, 152, 155, 157, 158, 159, 210, 247 life cycle, 204, 205 light beam, 16 light scattering, 17 limitation, 109, 128, 169, 183, 185, 200, 210, 212, 218, 239

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limitations, xiv, 52, 74, 177, 229, 230, 247, 259, 274, 283 linear, 110, 172, 247, 267, 278 linguistic, 274, 276, 283 links, 239 linoleic acid, 43, 49 linolenic acid, 49, 51 lipase, 50, 51, 52, 53, 61, 120 lipases, xi, 34, 46, 48, 49, 50, 52, 174 lipid, 52, 55, 57, 58, 59, 60, 62, 80, 256, 265, 269 lipids, 4, 36, 43, 55, 131, 181, 258 lipophilic, 55, 247, 263 lipopolysaccharide, 80 lipoprotein, xi, 34 liposome, xi, 34, 43, 56, 57 liposomes, 55, 56, 57 liquid chromatography, xiv, 245, 248 liquid phase, 8, 173, 218, 255 liquids, 185 liquor, 4, 8, 12, 27, 177 liver, 38 livestock, 198 logistics, 132, 136, 149 London, 82, 87, 241 losses, 139, 144, 145, 146, 151, 152, 196, 251, 255, 257 low molecular weight, 199 low temperatures, 67, 173 LPG, 55 lubricants, 45 lysis, 4, 90, 98, 99, 163, 165, 166, 185, 287 lysophosphatidic acid, 52, 54, 55

M machinery, 192 magnesium, 5, 6, 36, 42, 45, 211 magnetic, vi, 250 MAGs, 51 maintenance, 52, 90, 111, 163, 165, 166, 168, 174, 178, 185 maltodextrin, 42 management, xi, xii, xiii, xv, 89, 91, 115, 119, 125, 135, 136, 137, 144, 146, 153, 154, 155, 157, 158, 166, 206, 209, 210, 211, 212, 213, 227, 286, 289, 290, 293, 294, 295, 296 management practices, 290 manipulation, 249 manpower, 174 manufacturing, 64, 115, 189, 235 manure, 204, 208 market, 115, 296 marsh, 66, 86

mass spectrometry, 248, 249 mass transfer, 52 matrix, 3, 5, 23, 174, 190, 203, 247, 248, 249, 250, 251, 253, 254, 255, 257, 259, 260, 261, 270 meals, 37 measurement, 36, 77, 233 measures, 45, 247, 295 meat, 38, 123, 214 media, 12, 47, 48, 52, 79, 87 medicine, 269 Mediterranean, 204, 206 melt, 69 melting, xiv, 52, 133, 273, 274, 275, 278, 280, 282, 283, 285, 286, 287 membership, 274 membranes, 47, 55, 166 mercury, 68, 79, 127, 179, 183 messenger RNA, 241 metabolic, xi, 73, 78, 89, 91, 92, 93, 97, 120, 121, 165, 168, 234, 241, 267 metabolic changes, 168 metabolic pathways, 267 metabolism, 4, 85, 88, 90, 98, 121, 165, 167, 168, 169, 185, 242 metabolite, 269 metabolites, 249, 267, 269 metagenomics, 78 metal content, 162, 210, 215 metal ions, 5, 267 metals, xii, 34, 36, 65, 90, 112, 130, 162, 196, 198, 203, 204, 205, 206, 208, 210, 212, 215, 246, 247, 261 metazoa, 120, 165, 167, 168 methane, xii, 65, 73, 75, 85, 86, 90, 115, 116, 117, 118, 120, 124, 126, 131, 132, 139, 140, 149, 163, 173, 174, 175, 214, 216, 217, 224, 225, 227 methanogenesis, 81, 88, 115, 117, 130, 131, 215, 216 methanol, 42, 44, 50, 88, 102, 105, 107, 109, 251, 253, 254, 256, 257, 258, 259, 260, 261 methionine, 38 methylation, 68, 79 metric, 190 Mg2+, 4, 12, 194 mica, 76 micelles, 47 microaerophilic, 85 microarray, 77, 83, 239, 243 microarray technology, 239 microbes, 5, 75, 242 microbial, xii, xiv, 3, 4, 47, 54, 58, 60, 61, 64, 65, 66, 70, 71, 72, 73, 75, 76, 77, 79, 80, 81, 83, 84, 85, 87, 88, 90, 91, 92, 97, 98, 102, 108, 109, 112, 115, 117, 120, 122, 168, 180, 182, 184, 196, 198,

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Index 206, 207, 209, 213, 215, 216, 217, 218, 225, 229, 230, 236, 237, 240, 241, 242, 243, 270, 286 Microbial activity, 207 microbial cells, xii, 64, 75, 90, 102, 108, 115, 117, 120, 216, 240 microbial communities, xiv, 79, 87, 88, 229, 230 microbial community, xiv, 76, 77, 91, 97, 230, 240, 241, 242, 243 microchip, 233 microcosms, 82 microelectrodes, 85 microflora, 38, 242 micronutrients, 246 microorganism, 75, 120, 131 microorganisms, ix, x, xi, xiv, 1, 2, 3, 4, 7, 8, 10, 22, 63, 64, 65, 66, 71, 72, 73, 74, 75, 77, 82, 84, 88, 99, 109, 112, 114, 120, 165, 167, 169, 175, 181, 184, 196, 229, 230, 235, 236, 239, 240, 242 microscope, 75 microscopy, 32, 75 microstructure, 285 microwave, 56, 174, 181, 186, 248, 250, 255, 256, 257, 258 microwave heating, 255, 256 microwave radiation, 256 microwaves, 255, 259 migration, 49, 50, 52, 57, 74 milk, 38, 112, 214 mimicking, 52 mine soil, 204 mine tailings, 206, 208 mineralization, xi, 63, 64, 85, 99, 122, 173, 184, 190, 196, 199, 203, 205, 206, 230 mineralized, 196, 197 mineralogy, 190, 191 minerals, 200 mining, 64, 91, 192 Ministry of Education, 31 MIP, 258 mixing, 8, 11, 14, 15, 16, 27, 40, 50, 52, 173, 197, 237 mobility, 203 modeling, 287 models, 78, 225, 274 moderates, 40 moieties, 269 moisture, 38, 45, 49, 75, 111, 143, 194, 196, 198, 219, 258 mole, 64 molecular markers, 79 molecular structure, 55 molecular weight, 184, 199, 213, 217 molecules, 36, 46, 55, 57, 71, 218

money, 235 montmorillonite, 190 morphology, 86 mortality, 113, 115 Moscow, 286 mouth, 56, 113 MRS, 135, 137, 138, 139, 146, 147, 148, 149, 157, 158 multiplexing, 78 municipal sewage, 109, 159, 160, 183, 184 municipal solid waste, 126, 136, 152, 205, 206, 211 municipal solid waste (MSW), 136, 144, 152, 153, 154, 155, 156, 157, 158, 159 mutagenic, 2, 263

N Na+, 5, 12, 194 Na2SO4, 251, 254, 258 N-acety, 45 NaCl, 42, 251 naphthalene, 88, 180, 252, 255, 263 National Academy of Sciences, 242 national policy, 293 National Research Council, 123, 206, 247 natural, xi, 5, 16, 34, 36, 39, 46, 48, 50, 53, 54, 56, 63, 64, 65, 66, 67, 68, 69, 75, 77, 78, 79, 82, 87, 119, 133, 189, 230, 236, 246, 267, 269 natural environment, 67, 82, 236, 269 natural gas, 64, 65, 66, 69 natural habitats, 66 natural selection, 230 neck, 7 negligence, 128 nervous system, 86, 192 Netherlands, 128 network, xiv, 20, 21, 22, 23, 132, 155, 158, 273, 274, 275, 284, 285, 286, 287 neural network, 274, 275, 284, 286, 287 neurochemistry, 86 neurotoxic, xi, 63, 65 neutral lipids, 40, 42 neutralization, 26, 217 New Jersey, 81, 84, 205 New York, v, vi, 30, 78, 80, 83, 85, 86, 88, 160, 183, 206, 207 New Zealand, 198, 206 Ni, 65, 113, 127, 212, 215, 216 nickel, 204, 215 Nielsen, 22, 30, 81, 183 nitrate, 80, 94, 96, 99, 100, 102, 103, 104, 105, 108, 165, 166, 169, 170, 176, 234, 286, 287 nitrates, 103

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nitrification, xi, 89, 91, 94, 96, 97, 99, 104, 105, 108, 121, 123, 170, 175, 180, 182, 185, 196, 197, 198, 214, 234, 286 Nitrite, 100, 103 nitrogen, xi, 7, 40, 45, 67, 78, 89, 90, 91, 93, 96, 97, 99, 102, 103, 104, 105, 107, 108, 109, 111, 117, 118, 120, 121, 123, 124, 127, 131, 133, 145, 162, 170, 175, 176, 180, 182, 185, 211, 213, 214, 225, 234, 235, 240, 243, 259 nitrogen compounds, 91, 97, 108 nitrogen dioxide, 127 nitrogen gas, 102, 103, 104, 105, 108, 117, 234, 259 nitrogen oxides, 121, 133 nitrosamines, 270 N-N, 214 non toxic, 40, 64 non-biological, x, 2, 4, 29 non-invasive, 15 nonionic, 56 non-renewable, 185, 189, 292 norfloxacin, 269 normal, 45, 105, 135, 139, 173, 192, 211 normal conditions, 135 North Africa, 198 North America, 76 NRC, 111, 123, 203, 206 nucleic acid, 4, 104, 105, 108, 109, 242 nucleotide sequence, 74 nutraceuticals, 56 nutrient, 37, 90, 99, 111, 189, 199, 210, 211, 213, 216, 227 nutrient imbalance, 199 nutrients, xiii, 2, 7, 12, 91, 99, 102, 111, 126, 163, 170, 174, 175, 178, 179, 187, 189, 195, 208, 230, 246, 247 nutrition, 56, 66

O obligations, 158 observations, 14 odorants, 247 offshore, 65, 66, 83 ofloxacin, 269 oil, x, xi, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43, 45, 47, 49, 51, 53, 64, 65, 66, 67, 69, 73, 79, 84, 87, 175, 182, 219, 220, 268, 296 oil production, 34, 79, 296 oil recovery, 34 oils, x, xi, 33, 34, 35, 36, 39, 40, 42, 53, 80, 268 oilseed, 34, 37 oligonucleotides, 77, 83, 242, 243 omeprazole, 260, 269

onion, 55 online, 159, 160, 185, 286 operator, 154, 155, 158, 191 opposition, 188, 199 optical, 15 optimization, 253, 256, 257, 261 oral, 109 ores, 189 organic chemicals, 247, 263 organic compounds, xi, 63, 66, 99, 103, 162, 203, 205, 219, 221, 247, 248, 250, 255, 268, 270 organic matter, ix, xi, xiii, 1, 2, 3, 63, 64, 68, 82, 131, 144, 145, 163, 167, 174, 189, 195, 196, 197, 199, 200, 205, 206, 207, 208, 209, 212, 217, 246, 259 organic polymers, 3 organic solvent, 41, 53, 54, 248, 251, 255, 261, 262 organic solvents, 251, 255 organism, 14, 23, 24, 49, 65 organophosphorous, 268 orientation, 293 OSA, 167, 180, 185 osmosis, 163 osmotic, 5, 121 oxidation, xiv, 36, 40, 48, 54, 64, 67, 69, 71, 82, 84, 86, 92, 93, 94, 99, 121, 163, 165, 167, 170, 171, 172, 180, 182, 186, 209, 213, 217, 218, 219, 221, 223, 226, 227, 234, 241, 243 oxidation products, 221 oxidation rate, 93 oxidative, 56, 64, 91, 92, 173, 183, 218 oxide, 208, 259, 279, 280, 286 oxides, 190, 196 oxygen, 3, 7, 40, 71, 90, 93, 94, 120, 130, 131, 140, 142, 146, 148, 152, 163, 165, 166, 168, 169, 175, 179, 183, 184, 218, 219, 222, 225, 234, 235, 286 oxygen consumption, 90, 175 ozonation, xi, 89, 100, 105, 108, 115, 120, 122, 123, 172, 173, 175, 180, 181, 183, 185, 213, 217 ozone, 99, 100, 121, 122, 123, 172, 173, 180, 181, 183, 184, 186, 218, 225

P Pacific, 84, 205 PAHs, 188, 247, 248, 249, 250, 251, 252, 254, 255, 256, 257, 259, 260, 261, 262, 263, 270 paints, 45, 268 pancreas, 47 PAO, 169 paraffins, 247 parameter, xiv, 12, 48, 144, 146, 209, 233, 253, 254, 277

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Index particles, x, 2, 3, 4, 5, 6, 8, 10, 11, 16, 20, 23, 29, 38, 41, 52, 190, 196, 260 particulate matter, 171, 189, 267 partition, x, 33, 39 pathogenic, 2, 112, 130, 133 pathogens, ix, 130, 174, 188, 189, 210, 212, 218, 246, 247, 249, 261 pathways, 49, 64, 236, 237, 243, 267 Pb, xiii, 113, 127, 187, 189, 192, 193, 200, 203, 207, 212, 215, 241 PCBs, 188, 247, 253, 256, 257, 258, 259, 261, 262, 268 PCP, 93 PCR, 73, 74, 76, 77, 78, 79, 87, 231, 235, 239, 240, 241 pentane, 251 percolation, 190 periodic, 247 permeability, 57, 166 permeabilization, 75 permeation, 260, 261 permit, 136, 158 peroxidation, 122 peroxide, 36, 38, 39, 218 perylene, 264 pesticides, ix, 188, 248, 251, 252, 259, 261, 267, 268, 270, 287 petrochemical, 64, 218, 219, 222, 223 petroleum, xi, 45, 63, 64, 66, 69, 73, 75, 76, 78, 82, 83, 192, 267 petroleum products, 45 pH, xii, xiii, 4, 11, 12, 16, 66, 68, 89, 100, 102, 110, 111, 117, 119, 130, 131, 163, 165, 171, 187, 190, 193, 194, 196, 197, 203, 204, 212, 213, 216, 217, 218, 257 pH values, 4, 197 pharmaceutical, x, 33, 40, 56, 57, 259, 261, 267, 268 pharmaceuticals, 247, 249, 260, 261, 268, 269 PHB, 181 phenol, 65, 226, 235, 240 phenolic, 260, 267 phenolic resins, 267 Philadelphia, 243 Philippines, 81 phosphate, 12, 36, 46, 53, 54, 61, 133, 160, 169, 177, 189, 204, 205, 211, 267, 270 phosphates, 53, 189, 196, 198 phosphatidic acid, x, 33, 36 phosphatidylcholine, x, 33, 36, 51 phosphatidylethanolamine, x, 33, 36 phosphatidylserine, 53 phospholipids, x, 33, 34, 44, 46, 48, 53, 57, 83 phosphor, 133

311

phosphorous, xiii, 7, 35, 78, 90, 99, 102, 108, 109, 123, 161, 162, 170, 175, 177, 183, 184, 211, 213, 225, 241, 285, 287, 292 phosphorylation, 45, 61, 91, 92, 121 photosynthetic, 85 phototrophic, 64, 73 phylogenetic, xi, 63, 66, 67, 68, 72, 73, 74, 76, 79, 81, 83, 235, 239 phylogenetic tree, 66, 67, 239 phylogeny, 65 phylum, 74, 75 physical properties, 189, 195, 225 physicochemical, 91, 109, 117 physiological, 65, 73, 184 physiology, 81 phytochemicals, 41 phytosterols, x, 33 phytotoxicity, 192 pig, 115, 207, 208 pigments, 39 pilot study, 183 pipelines, 66, 81, 88 plagioclase, 191 plants, ix, xii, xiii, 2, 37, 57, 64, 66, 90, 93, 109, 115, 119, 120, 126, 127, 128, 132, 133, 161, 162, 163, 172, 173, 175, 178, 180, 181, 186, 187, 188, 189, 192, 194, 200, 203, 205, 211, 212, 214, 215, 240, 242, 245, 246, 249, 267, 274, 275, 290, 291, 292, 293, 294, 295 plasmid, 57 plasmids, 57 plastic, 45, 194, 268 plastics, 45, 218 play, x, xiii, 2, 19, 21, 29, 38, 43, 71, 93, 188, 200, 203, 236, 239 PLC, 46, 53, 54, 55 PLD, 46, 53, 54, 55 poisonous, 65 poisons, 192 Poland, 128, 129 polarity, 52, 54, 251, 253, 254, 259 polarization, 255 pollutant, ix, 1, 7, 192, 204, 210 pollutants, ix, xi, xiv, 65, 68, 78, 89, 90, 91, 112, 128, 168, 188, 204, 230, 235, 240, 274 pollution, ix, xiii, 128, 163, 187, 188, 189, 210, 211, 212, 226 polyacrylamide, 71 polyaromatic hydrocarbons, 65 polybrominated diphenyl ethers, 268 polychlorinated biphenyls (PCBs), 259 polychlorinated dibenzofurans, 247 polycyclic aromatic hydrocarbon, 162, 247

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Index

polyelectrolytes, 25 polyethylene, 268 polyethyleneimine, 54 polyhydroxybutyrate, 169 polymer, x, 2, 6, 14, 20, 22, 24, 25, 26, 29, 258, 268 polymerase, 78, 84, 88, 241 polymerase chain reaction, 78, 84, 88, 241 polymeric materials, 5 polymerization, 258 polymers, 3, 4, 5, 14, 25 polymorphism, 73, 97 polymorphisms, 83 polysaccharide, 20, 21, 22 polysaccharides, x, 2, 4, 8, 109 polystyrene, x, 2, 8, 10, 11 polystyrene latex, x, 2, 8, 10, 11 polyunsaturated fat, xi, 34, 48, 49 polyunsaturated fatty acid, xi, 34, 48, 49 polyunsaturated fatty acids, xi, 34, 48, 49 polyurethane, 268 polyurethane foam, 268 pond, 66 pools, 191 poor, xiii, 7, 23, 96, 209, 218, 219, 230, 231, 253 population, 72, 73, 75, 97, 128, 167, 233, 239, 241, 274, 290 population size, 241 pore, 54 pores, 196, 260 porosity, 285 porous, 27 porphyrins, 39 Portugal, 267 positive correlation, 278, 285 potassium, 36, 211, 213, 214, 256 poultry, 37, 115, 208 powder, 54, 56, 254, 275 powders, 215 power, 103, 105, 109, 128, 132, 133, 140, 144, 146, 150, 151, 152, 153, 154, 155, 159, 183, 212, 217, 218, 253, 254, 256, 257, 292 power generation, 144, 146, 151, 152, 153, 155, 183 power plant, 133, 292 power plants, 133 powers, 257 precipitation, 41, 42, 52, 65, 86, 110, 111, 112, 119, 177, 190, 198, 215, 226, 258 predators, 120, 167 prediction, 23, 71, 275, 279, 281, 283, 285, 286, 287 prejudice, 127 premium, 57 preservatives, 268, 269

pressure, xi, 41, 43, 47, 66, 89, 102, 105, 120, 146, 152, 153, 154, 173, 174, 217, 218, 250, 253, 254, 255, 256, 258, 259, 260, 294 pressure gauge, 47 prevention, 87, 128 prices, 57, 153, 155 PRISM, 73 pristine, 75, 77, 79 private, 246 probability, 195, 197, 199, 201, 202 probe, 76, 232, 241, 252 processing variables, 36 producers, 158, 225 productivity, 49, 198, 206 profit, 155, 156 profitability, 153 profits, 155, 156 program, 80 prokaryotes, 79, 82, 83, 84, 85 proliferation, 68, 166 propane, 41 property, vi, 56, 126, 213 propionic acid, 117 proteases, 56, 104, 174 protection, xii, 83, 125, 126, 167, 203, 212 protective role, 190 protein, xii, 12, 37, 38, 71, 72, 78, 82, 89, 91, 100, 101, 102, 104, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 123, 175, 214, 217, 224 proteins, xii, 4, 38, 57, 71, 84, 89, 91, 99, 104, 105, 108, 109, 110, 115, 117, 119, 123, 131, 165, 237, 238 proteobacteria, 66, 243 proteolysis, 104 protocol, 42, 77 protocols, 248 protons, 71 protozoa, 120, 167, 168, 184 Pseudomonas, 184, 243 public health, 2 public opinion, 162 PUFAs, 48, 50 pulses, 44 pumping, 291 pumps, 47 purification, 44, 48, 54, 73, 230, 257 pyrene, 180, 263, 264 pyrimidine, 232 pyrite, 64 pyrolysis, 126, 159, 182, 235, 296

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Index Q quadrupole, 249 quartz, 190, 191, 254, 256 quaternary ammonium, 251, 268

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R R&D, 180 radiation, 101, 191, 252, 256, 270 radical mechanism, 226 radiolabeled, 76 rain, 194 rainfall, 190 random, 16, 74 range, xiv, 5, 15, 28, 39, 55, 67, 99, 101, 163, 170, 178, 203, 216, 217, 249, 258, 260, 270, 273, 281, 283, 285 rats, 113, 114 raw material, 115, 1RB, 216 reaction mechanism, 219 reaction medium, 49 reaction rate, 50, 51, 52, 219, 220 reaction temperature, 49, 219, 221 reaction time, 47, 50, 52 reaction zone, 47 reactivity, 70 reagents, 45, 165, 231 reclamation, xiii, 188, 203, 207, 209 reconstruction, xii, 125, 135, 144 recovery, xi, xii, xiii, 34, 40, 41, 44, 49, 55, 57, 63, 65, 89, 91, 109, 110, 112, 115, 120, 123, 161, 174, 175, 177, 182, 183, 184, 211, 226, 235, 250, 254, 256, 258, 259, 260, 261, 292 recovery processes, 112 recycling, xv, 90, 167, 171, 174, 177, 179, 188, 199, 205, 286, 289, 290, 291, 292, 293, 294, 295, 296 redox, 216 reductases, 70, 84, 87 refineries, 64 refining, 35, 39, 65, 192 refractory, 172, 221, 222, 235 regeneration, 44, 166 regulation, xii, 113, 125, 127, 163, 188, 192, 210, 247 regulations, 90, 127, 192, 270 regulators, 269 rehydration, 56 relationship, 20, 23, 49, 81, 196, 239, 253, 262, 274, 275, 285 relationships, 83, 86, 240 reliability, xi, 63, 65, 68, 78, 274

remediation, xi, 63, 68, 82 renewable energy, 132, 159 reproduction, 38 reserves, 167, 177 reservoir, 47 reservoirs, 65, 67, 83, 84, 198 residuals, 115 residues, 40, 49, 183, 204, 206, 246 resin, 49, 54, 241 resistance, 52, 136, 158, 268 resolution, 249, 253 resources, 3, 115, 182 respiration, 83, 87, 90, 98, 163, 166, 167, 168, 185 respiratory, 121 restriction enzyme, 76 restriction fragment length polymorphis, 73, 83, 97 retention, 12, 42, 44, 45, 47, 49, 57, 121, 165, 166, 167, 169, 174, 175, 183, 184, 190, 200, 203, 205, 212, 225 Reynolds, 130, 131, 160 RFLP, 73, 76, 97 rheological properties, 173 rhizosphere, 66, 81, 86, 207 Rho, 92, 93, 94, 95, 121 ribosomal, xiv, 79, 81, 83, 84, 229, 230, 240, 242 ribosomal RNA, xiv, 79, 81, 83, 84, 229, 230, 240 ribosomes, 73, 75 ribozyme, 232 ribozymes, 241 rice, 78, 87, 211, 212 rice field, 78, 87 Río de la Plata, 207 risk, ix, xii, xiii, 35, 40, 161, 188, 203, 206, 247 risk assessment, 206 RNA, 102, 231, 232, 233, 235, 242 room temperature, 36, 38, 42, 43, 235, 248, 258 root elongation, 208 runoff, 246 Russia, 286

S safety, xi, 63, 65, 68, 78, 249 saline, 80 salinity, 68, 198 Salmonella, xii, 90, 109, 112, 114, 115 salt, 15, 42, 52, 54, 66, 86, 219 salts, 42, 198, 199 sample, 3, 14, 16, 17, 28, 69, 71, 73, 74, 76, 77, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 275 sampling, xiv, 3, 12, 98, 195, 197, 199, 209 sand, 6, 133, 254, 280

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sanitation, 159, 217 SAS, 264, 268 saturated fat, 48, 51 saturated fatty acids, 48, 51 saturation, 18, 25, 141 savings, 155 SBR, 185 scattering, 14, 15, 17 scavenger, 54 schema, 159 Schmid, 234, 242 SD rats, 113, 114, 115 sea floor, 67 seafood, 180 search, 73 seawater, 64 sediment, 67, 79, 80, 86 sedimentation, ix, 96, 188, 231, 285 sediments, 40, 64, 66, 68, 69, 73, 75, 76, 78, 80, 82, 85, 86, 190, 256 seed, 12, 36, 199, 208 seeding, 12 seeds, x, 33, 38, 39, 194 selecting, 157, 158 selectivity, xiv, 43, 45, 47, 50, 51, 52, 209, 222, 224, 249, 250, 252, 253, 255, 258 Self, 148, 183 SEM, 14, 28, 191 semiarid, 204, 205 sensitivity, 76, 90, 155, 215, 249, 250 separation, 4, 32, 41, 43, 44, 47, 48, 51, 81, 166, 216, 235, 248, 251, 252, 253, 255 sequencing, 73, 74, 123, 179, 180, 182 sequencing batch reactors, 179, 182 serine, 54 services, vi Shanghai, 215 shape, 4, 168, 277, 283 shear, x, 2, 11, 16, 22, 27, 29, 101, 171, 185, 216 sheep, 198 shipping, 294 shortage, xv, 109, 289, 292 short-range, 5 short-term, 204 side effects, 57 signals, 278 signs, 16, 113, 115 silica, 42, 43, 251, 252, 255, 257, 258, 262 silicate, 48, 200 silicon, 78, 279, 280 similarity, 97, 236, 238 Singapore, 123 SiO2, 191, 274, 279, 280, 285

sites, xv, 46, 66, 73, 75, 84, 200, 211, 289, 290, 291, 292, 296 skills, 178 skin, 56, 268 slag, xiv, xv, 128, 273, 274, 275, 280, 281, 285, 286, 287, 289, 290, 293, 294, 295 Slovakia, 128, 129 Slovenia, 128 SO2, 141, 145, 196 soda lakes, 80 sodium, 5, 10, 11, 47, 51, 69, 72, 106, 259 software, 97 soil, xii, xiii, 79, 87, 88, 125, 126, 162, 183, 187, 188, 189, 190, 192, 194, 195, 196, 197, 198, 200, 202, 203, 204, 205, 206, 207, 208, 210, 211, 212, 237, 242, 246, 247, 256, 268 soils, xiii, 68, 77, 78, 80, 187, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 246, 247, 259, 261 solar, 132, 133 solar energy, 132 sol-gel, 48 solid waste, 126, 136, 152, 189, 204, 205, 206, 207, 210, 211, 286 solidification, 285, 286 solidification processes, 286 solubility, 38, 41, 42, 56, 217, 218, 254 solvent, x, 33, 34, 39, 40, 41, 42, 43, 44, 48, 49, 50, 51, 52, 57, 249, 250, 251, 252, 253, 255, 256, 258, 259, 260, 261, 262 solvents, x, 33, 38, 40, 41, 42, 44, 52, 54, 56, 250, 251, 253, 255, 256, 259, 260, 262 sorbents, 261 sorption, 190, 196 sorption process, 190 South America, 76, 198, 207 soy, 48, 51 soybean, x, xi, 33, 34, 35, 36, 37, 38, 39, 42, 43, 44, 48, 49, 50, 51, 54, 55, 57 Spain, 128, 129, 161, 210, 245, 267, 269 spatial, 57, 68, 78, 83, 85 speciation, 77, 206 species, 49, 67, 68, 69, 70, 71, 72, 74, 75, 76, 77, 79, 80, 87, 97, 190, 198, 199, 203, 216, 230, 232, 233, 239 species richness, 76 specific heat, 139, 145 specificity, 53, 70, 75, 78, 232, 236, 237 spectrophotometric, 248 spectrum, 74, 131, 248 speed, 14, 15, 16, 184, 216, 218, 259 spore, 67, 85 Sprague-Dawley (SD) rats, 109

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Index square wave, 44 SRT, 94, 165, 166, 168, 170, 227 stability, x, xi, xiv, 2, 5, 8, 10, 34, 39, 47, 48, 49, 90, 91, 99, 120, 169, 170, 206, 229, 230, 239, 241 stabilization, 38, 124, 130, 131, 137, 138, 139, 144, 146, 208, 226, 227, 246 stabilize, 5, 293 stages, 115, 131, 133, 134, 167, 216, 219 stainless steel, 259 standard deviation, 201, 252, 254, 255 standards, 3, 7, 112, 114, 115, 134, 157, 192, 193, 208, 210 Standards, 270 Staphylococcus, 241 Staphylococcus aureus, 241 starch, 214 starvation, 167, 182 STD, 202 steady state, 12, 13, 16, 169 steel, 217, 235, 259 steric, 77 sterile, 189 sterilization, 173 steroid, 269 sterols, 55 stock, 10, 14, 167 stomach, 56 storage, 36, 65, 126, 131, 248, 249 strain, 234 strains, xi, 63, 70, 71, 72, 85, 215, 239, 240 strategies, xv, 65, 78, 85, 87, 90, 91, 99, 121, 171, 178, 180, 289 streams, 79, 172, 175 strength, x, 2, 4, 5, 14, 27, 29, 44, 169, 179, 182, 253, 254, 278 Streptomyces, 54 stress, 171 subgroups, 79 substances, x, 2, 4, 8, 40, 56, 91, 101, 103, 105, 108, 109, 126, 127, 128, 130, 131, 133, 142, 157, 158, 159, 162, 174, 189, 198, 210, 211, 213, 216, 217, 218, 221, 222, 245, 255 substitution, 53 substrates, 46, 48, 50, 51, 54, 64, 66, 68, 91, 117, 164, 165, 166, 167, 196, 216, 241 sugar, 36, 214 sugarcane, 211 sugars, 34 sulfate, xi, 42, 54, 63, 64, 66, 67, 68, 70, 71, 72, 73, 75, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 123, 225, 226, 265, 266, 269 Sulfide, 65 sulfonamides, 269

sulfur, xi, 63, 64, 65, 73, 78, 79, 81, 82, 84, 85, 87, 88, 235 sulfur dioxide, 85 sulphate, 10, 79, 80, 82, 83, 84, 216, 259 sulphur, 133, 225, 252, 253, 254, 258 Sulphur dioxide (SO2), 127 Sun, 232, 242 sunflower, x, 33, 38, 42, 57 sunlight, 194 supercritical, x, 33, 40, 41, 43, 57, 181, 250, 253, 254 supercritical carbon dioxide, 40, 41 supercritical fluids, x, 33, 40, 57 supernatant, x, 2, 10, 20, 21, 22, 23, 29, 42, 102, 104, 105, 106, 110, 117, 171, 175, 177, 223, 251, 257, 286 supplements, 38, 56, 57 supply, 128, 133, 152, 153, 167, 200 surface area, 10 surface properties, 51, 183 surface water, 261, 269 surfactant, 49, 54 surfactants, 247, 248, 249, 250, 251, 253, 255, 256, 259, 267, 268, 270 surplus, 24, 122, 140 surveillance, 78 survival, 68 suspensions, 14, 15, 16, 25, 56 sustainability, 195 sustainable development, 120 Switzerland, 87, 129, 133, 158, 269 symptoms, 195, 199 synergistic, 215 synthesis, 43, 49, 53, 54, 75, 167, 255 systematics, 72

T Taiwan, 273, 274, 275, 286 tanks, 94, 95, 96, 97, 98, 130, 131, 132, 136, 139, 144, 146, 150, 155, 231 tar, 235 targets, xiv, 230 taste, 34, 40 taxonomic, 75, 76, 77 TCP, 256, 257, 258 technological change, 136 temperature, 38, 40, 41, 42, 43, 50, 52, 66, 67, 68, 69, 82, 84, 99, 128, 130, 131, 133, 139, 140, 141, 144, 145, 146, 147, 149, 150, 152, 153, 154, 163, 170, 171, 173, 174, 175, 181, 182, 190, 208, 213, 217, 218, 219, 222, 224, 242, 248, 250, 253, 254,

Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

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316

Index

256, 257, 258, 259, 260, 261, 274, 275, 278, 285, 295 temperature dependence, 222 temperature gradient, 84 temporal, 57, 68, 78 temporal distribution, 57 tension, 51, 184 tetracycline, 269 tetracyclines, 269 textile, 64 textiles, 267 thawing, 99, 122, 225 thermal decomposition, 101 thermal degradation, 257 thermal destruction, 189 thermal energy, 175 thermal resistance, 52 thermal stability, 48 thermal treatment, xiv, 99, 126, 133, 134, 135, 173, 175, 206, 209, 213, 217, 225 thermodynamic, 41 thermodynamic properties, 41 Thermophilic, 67, 88, 131, 144 threat, 68, 203, 268 threshold, 71, 203 throat, 56 thymus, 113 tight junction, 56 time consuming, 73, 74 tissue, xiii, 57, 187, 203 tocopherols, x, 33 Tokyo, xv, 205, 229, 286, 289, 290, 291, 292, 293, 294, 295, 296 toluene, 65, 251, 252, 255, 256, 258, 259, 260, 262 total organic carbon (TOC), xiv, 100, 127, 173, 209, 218 total product, 34 toxic, xi, 2, 63, 65, 112, 116, 120, 162, 189, 192, 198, 199, 215, 216, 240, 247, 250, 267, 268 toxic effect, 112, 199, 215 toxic metals, 65, 112, 198 toxic substances, 216 toxicities, 204 toxicity, xii, 90, 109, 113, 114, 115, 195, 198, 199, 215, 253, 263, 268 toxicological, 42, 246 toxins, 109, 112, 114 trace elements, ix, 188, 190, 192, 200, 205 trade, 259 training, xv, 273, 274, 275, 278, 281, 283, 285 trans, ix, 1, 7 transesterification, 48, 51, 52

transfer, 44, 54, 73, 88, 121, 132, 133, 139, 144, 150, 166, 168, 203, 216, 218, 250 transformation, 64, 130 translocation, 203 transmission, 65, 66, 81 transmits, 132 transparent, 256 transport, xii, 48, 91, 121, 137, 161, 163, 218, 249 transport phenomena, 48 transportation, 157, 188, 211, 294 treatment methods, 91, 171, 205 triacylglycerols, 46 trial, 5 triglycerides, 36, 40, 46, 50 triphenyltin, 268 trucking, 294 tubular, 48 tumor, 56 turbulence, 174, 216 turnover, 68, 196 two-dimensional, 249

U ultrasound, 180, 226, 252 uniform, 16, 199 United Kingdom, 1, 121 United Nations, 160 United States, x, 33, 34, 65, 80, 87, 123, 210, 242, 247 updating, 275 uranium, 66, 75, 79 urbanization, xv, 289 urbanized, 292 urea, 74, 106 USDA, x, 33, 34, 190 USEPA, 188, 192, 193, 208, 247, 259, 262, 263 UV, 241, 248, 251, 265, 269, 270 UV radiation, 270

V vaccination, 57, 249 vacuum, 38, 39, 52 values, xiv, xv, 4, 12, 24, 36, 51, 111, 112, 127, 128, 147, 192, 196, 197, 198, 201, 203, 209, 222, 223, 247, 273, 279, 280, 281, 283, 285 Van der Waals, 5 variability, 12, 24, 36 variables, 250, 277, 278, 280 variation, 4, 72, 77, 286 vasculature, 57

Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,

317

Index vector, 237 vegetable oil, 34, 35, 47, 53, 57 vegetables, 162 vehicles, 56, 291 velocity, 251 ventilation, 246 vesicle, 56 vessels, 57, 256, 257, 258 veterinary medicine, 269 Vietnam, 31 viruses, 165 viscosity, 10, 24, 39, 254, 260 visible, 16, 70, 199 visualization, 75 vitamins, 38, 56 volatile substances, 36 volatility, 251, 270 volatilization, 214, 246, 257

water-holding capacity, 195 water-soluble, 53, 54 weight changes, 114 weight gain, 112 WHC, 198 wheat, 214 whey, 66, 88 winter, 121, 131, 144, 146, 153 wood, 268 workers, 6 worm, 66, 80 WRC, 14 WTP, 93, 180

X xenobiotic, 205, 236, 243 X-ray diffraction (XRD), 191, 192 xylene, 65, 266, 269

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W war, 290, 292, 293, 294, 295 waste disposal, 91 waste incineration, 128, 133 wastes, 35, 65, 115, 159, 208, 286, 290, 296 wastewater treatment, ix, xi, xii, xiv, 2, 6, 7, 8, 12, 16, 31, 63, 64, 66, 75, 90, 91, 93, 115, 119, 120, 121, 122, 123, 125, 161, 162, 163, 167, 169, 175, 178, 179, 182, 185, 186, 188, 189, 205, 206, 210, 211, 212, 213, 214, 215, 218, 225, 229, 230, 239, 240, 249, 274, 286, 287, 290, 291, 292, 293, 294, 295 wastewaters, ix, 1, 81, 90, 97, 123, 226, 261 water absorption, 198 water quality, 134 water resources, 3 water vapor, 224

Y yeast, 50, 111, 112, 115 yield, xi, 38, 44, 45, 49, 51, 52, 82, 89, 92, 116, 117, 118, 119, 120, 131, 139, 163, 200, 201, 204, 211, 214, 225 yolk, 48, 50, 51, 55

Z zero-waste, 274 zeta potential, 10 zinc, 127, 192, 204, 207, 208, 215

Baily, Richard E.. Sludge : Types, Treatment Processes and Disposal, Nova Science Publishers, Incorporated, 2009. ProQuest Ebook Central,