Anaerobic Ammonium Oxidation: For Industrial Wastewater Treatment 9783110780093, 9783110779929

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Maulin P. Shah Anaerobic Ammonium Oxidation

Sustainable Water and Wastewater Treatment

Edited by Maulin P. Shah

Volume 3

Anaerobic Ammonium Oxidation For Industrial Wastewater Treatment Edited by Maulin P. Shah

Editor Dr. Maulin P. Shah Enviro Technology Limited Industrial Waste Water Research Lab Opp. Champapuri Jain Mandir A/103 Satsang Park Ankleshwar 393002 India

ISBN 978-3-11-077992-9 e-ISBN (PDF) 978-3-11-078009-3 e-ISBN (EPUB) 978-3-11-078018-5 ISSN 2747-4208 Library of Congress Control Number: 2023901114 Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at http://dnb.dnb.de. © 2023 Walter de Gruyter GmbH, Berlin/Boston Cover image: spanteldotru/E+/Getty Images Typesetting: Integra Software Services Pvt. Ltd. Printing and binding: CPI books GmbH, Leck www.degruyter.com

Contents List of contributing authors

VII

Sougata Ghosh, Bishwarup Sarkar, and Sirikanjana Thongmee 1 Anammox bacteria-mediated sewage treatment 1 Nilendu Basak, Atif Aziz Chowdhury, Taniya Roy, and Ekramul Islam 2 Diversity and distribution of ammonia-oxidizing archaea in engineered and natural environments 15 Bhaskar Deka, Nikita Chakraborty, and Bhisma Kumar Patel 3 Significant role of nitrogen cycle in wastewater treatment

31

Anne Bhambri, Santosh Kumar Karn, and Arun Kumar 4 Regulation and measurement of nitrification in terrestrial systems Djaber Tazdaït and Rym Salah-Tazdaït 5 Ammonia-oxidizing bacteria: their biochemistry and molecular biology 65 Sonia Saini, Sanjana Tewari, Jaya Dwivedi, and Vivek Sharma 6 Ammonia-oxidizing bacteria in wastewater 83 Aishwarya Das, Somakraj Banerjee, Ranjana Das, and Chiranjib Bhattacharjee 7 An overview of biochemical and molecular mechanism of ammoniaoxidizing bacteria and their potential application in wastewater treatment 105 Kashmira Gupta, Moulina Payra, Piyali Das, and Srijoni Banerjee 8 Diversity and environmental distribution of ammonia-oxidizing bacteria 121 Ritama Mukherjee, Mohana Baral, Srijoni Banerjee, and Piyali Das 9 Metabolism and genomics of anammox bacteria 139 Index

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List of contributing authors Chapter 1 Sougata Ghosh Department of Physics Faculty of Science Kasetsart University Bangkok Thailand And Department of Microbiology School of Science RK University Rajkot, Gujarat India [email protected] Bishwarup Sarkar College of Science Northeastern University Boston, MA USA Sirikanjana Thongmee Department of Physics Faculty of Science Kasetsart University Bangkok Thailand Chapter 2 Nilendu Basak Department of Microbiology University of Kalyani Kalyani Nadia 741235, West Bengal India Atif Aziz Chowdhury Department of Microbiology University of Kalyani Kalyani Nadia 741235, West Bengal India Taniya Roy Department of Microbiology University of Kalyani

https://doi.org/10.1515/9783110780093-203

Kalyani Nadia 741235, West Bengal India Ekramul Islam Department of Microbiology University of Kalyani Kalyani Nadia 741235, West Bengal India [email protected] Chapter 3 Bhaskar Deka Department of Chemistry Indian Institute of Technology Guwahati North Guwahati 781039, Assam India Nikita Chakraborty Department of Chemistry Indian Institute of Technology Guwahati North Guwahati 781039, Assam India Bhisma Kumar Patel Department of Chemistry Indian Institute of Technology Guwahati North Guwahati 781039, Assam India [email protected] Chapter 4 Anne Bhambri Department of Biochemistry and Biotechnology Sardar Bhagwan Singh University Balawala, Dehradun 248161 Uttarakhand India And Department of Biotechnology Shri Guru Ram Rai University Patel Nagar, Dehradun 248001 Uttarakhand India

VIII

List of contributing authors

Santosh Kumar Karn Department of Biochemistry and Biotechnology Sardar Bhagwan Singh University Balawala, Dehradun 248161 Uttarakhand India [email protected]

Vivek Sharma Department of Chemistry BanasthaliVidyapith Aliyabad 304022, Rajasthan India [email protected], viveksharma@ba nasthali.in

Arun Kumar Department of Biotechnology Shri Guru Ram Rai University Patel Nagar, Dehradun 248001 Uttarakhand India

Chapter 7 Aishwarya Das Chemical Engineering Department Jadavpur University Kolkata India

Chapter 5 Djaber Tazdaït Department of Natural and Life Sciences Faculty of Sciences Algiers 1 University Benyoucef Benkhedda Algiers Algeria

Somakraj Banerjee Chemical Engineering Department Jadavpur University Kolkata India

Rym Salah-Tazdaït Department of Environmental Engineering National Polytechnic School Algiers Algeria Chapter 6 Sonia Saini School of Earth Sciences Banasthali Vidyapith Aliyabad 304022, Rajasthan India Sanjana Tewari Department of Chemistry Banasthali Vidyapith Aliyabad 304022, Rajasthan India Jaya Dwivedi Department of Chemistry BanasthaliVidyapith Aliyabad – 304022, Rajasthan India

Ranjana Das Chemical Engineering Department Jadavpur University Kolkata India [email protected] Chiranjib Bhattacharjee Chemical Engineering Department Jadavpur University Kolkata India Chapter 8 Kashmira Gupta Department of Biological Sciences School of Life Science and Biotechnology Adamas University Kolkata India Moulina Payra Department of Biological Sciences School of Life Science and Biotechnology Adamas University Kolkata India

List of contributing authors

Piyali Das Department of Biological Sciences School of Life Science and Biotechnology Adamas University Kolkata India [email protected] Srijoni Banerjee Department of Biotechnology School of Life Science and Biotechnology Adamas University Kolkata India [email protected] Chapter 9 Ritama Mukherjee Department of Microbiology School of Life Science and Biotechnology Adamas University Kolkata India

Mohana Baral Department of Microbiology School of Life Science and Biotechnology Adamas University Kolkata India Srijoni Banerjee Department of Biotechnology School of Life Science and Biotechnology Adamas University Kolkata India [email protected] Piyali Das Department of Biological Sciences School of Life Science and Biotechnology Adamas University Kolkata India [email protected]

IX

Sougata Ghosh✶, Bishwarup Sarkar, and Sirikanjana Thongmee

1 Anammox bacteria-mediated sewage treatment Abstract: Removal of nitrogen from wastewater prior to discharge in water bodies is a vital process in order to maintain the quality of the natural water resources. Several conventional wastewater treatment plants therefore, oxidize ammonium to nitrite which is further oxidized to nitrate under aerobic conditions. However, such processes require significant amount of energy resources in the form of oxygen as well as organic matter. Hence, other sustainable treatment methods have been investigated out of which anaerobic ammonium oxidation (anammox) process was demonstrated to be an energy-neutral or energy-generating method of sewage treatment. Therefore, this chapter explains the application of anammox bacteria for biological nitrogen removal in sewage treatment. Anammox bacteria utilizes nitrite as an electron acceptor during anammox. There are several advantages of anammox over heterotrophic denitrification which includes low sludge yield, no requirement of organic carbon source, and high rate of denitrification. Various annamox bacteria such as Candidatus “Brocadia anammoxidans,” Candidatus Kuenenia, Candidatus “Brocadia fulgida,” and Candidatus “Anammoxoglobus propionicus” are reported to effectively remove nitrogen from wastewater samples. Thus, further investigation and large-scale operation of sewage treatment using anammox bacteria can be a sustainable and eco-friendly waste management strategy.

1 Introduction Excessive nitrogen in the natural water bodies can result in alteration in the water quality and may also interfere with the ecosystem [1]. Hence, biological methods are conventionally used for the removal of nitrogen from wastewater wherein, ammonium is oxidized to nitrite followed by the formation of nitrate under aerobic conditions. The Acknowledgment: Dr. Sougata Ghosh acknowledges Kasetsart University, Bangkok, Thailand for Postdoctoral Fellowship and Funding under Reinventing University Program (ref. no. 6501.0207/10870 dated 9th November 2021 and Ref. No. 6501.0207/9219 dated 14th September, 2022). ✶ Corresponding author: Sougata Ghosh, Department of Physics, Faculty of Science, Kasetsart University, Bangkok, Thailand; Department of Microbiology, School of Science, RK University, Rajkot, Gujarat, India, e-mail: [email protected] Bishwarup Sarkar, College of Science, Northeastern University, Boston, MA, USA Sirikanjana Thongmee, Department of Physics, Faculty of Science, Kasetsart University, Bangkok, Thailand

https://doi.org/10.1515/9783110780093-001

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Sougata Ghosh, Bishwarup Sarkar, and Sirikanjana Thongmee

nitrate produced is then reduced by a denitrifying microbe that utilizes organic carbon as its source of electrons [2]. This method is often used in conventional wastewater treatment plants, however, considerable amounts of energy are required by the ammonium- and nitrite-oxidizing bacteria along with an excessive requirement of organic matter for denitrification as well [2, 3]. Therefore, a sustainable approach for the removal of nitrogen from wastewater is required. Autotrophic nitrogen removal process such as anaerobic ammonium oxidation (anammox) is proposed to be one such technique that is an energy-neutral or even energy-generating process [4]. Anammox is a biological process that requires 60% less oxygen and no organic carbon as compared to biological nitrification and denitrification processes [1, 5]. Therefore, this chapter discusses the application of anammox bacteria for enhanced nitrogen removal during sewage treatment. Several anammox bacteria such as Candidatus “Brocadia anammoxidans,” Candidatus Kuenenia, Candidatus “Brocadia fulgida,” and Candidatus “Anammoxoglobus propionicus” are discussed in this chapter with respect to their utilization in enhanced nitrogen removal that is summarized in Table 1. Further evaluation of the efficacy for nitrogen removal by such anammox bacteria in presence of varying concentrations of salts, heavy metals, and other xenobiotic substances may provide meaningful insights for large-scale application of these anammox bacteria in wastewater and sewage treatment processes.

2 Anammox bacteria-mediated nitrogen removal from sewage Gong et al. [6] recently reported the contribution of anammox bacteria in the removal of nitrogen from domestic sewage as schematically represented in Figure 1. The aerobic stage in a sequencing batch reactor (SBR) demonstrated a high accumulation of nitrite (9.93 mg/L) without any removal of ammonium from the medium. The nitrite accumulation rate rapidly declined from 96.25% to 55.25% in the fourth phase. Moreover, the relative abundance of Nitrospira increased up to 0.54% during the fourth phase of the bioreactor operation which facilitated the reduction of partial nitrification. However, the addition of hydroxylamine resulted in high recovery of the nitrite accumulation rate that was around 90.19% after 170 days of operation. Further, the growth of anammox bacteria was detected in the third phase wherein Candidatus Brocadia demonstrated 0.44% abundance. Thereafter, the anammox bacteria utilized nitrite under anoxic conditions which resulted in a decrease in nitrite concentration from 10.59 mg/L to 4.08 mg/L. A maximum nitrogen removal efficiency of 82.21% was attained in the fifth phase of the reactor operation in which the partial nitrification was recovered. The average particle size of the sludge was 100 μm. The chemical oxygen demand (COD) and ammonium concentrations also decreased to 50.49 and 23.08 mg/L in the fifth phase under anaerobic conditions,

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1 Anammox bacteria-mediated sewage treatment

while 9.5 and 1.31 mg/L of nitrite and nitrate were produced, respectively. Ammonium, nitrite, and nitrate concentrations were reduced to 4.38, 1.64, and 0.13 mg/L, respectively. The anammox process then removed 2.44 and 5.02 mg/L of ammonium and total inorganic nitrogen (TIN), respectively, with a nitrogen removal efficiency of 88.8% after 192 days of the bioreactor operation. Isotopic labeling experiments further demonstrated specific anammox and denitrification activity of 1.48 and 1.22 μmolN/(g of volatile suspended solids [VSS] per h) that confirmed the synergistic action of denitrification and anammox process in the removal of nitrogen.

Stirrer

Feeding pump

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(a) pH/DO mater Feeding tank Effluent pump

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(b) Feeding Phase I (Day 1–19) II (Day 20–57) III (Day 58–133) 7min 30min

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4h

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5h

4h

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Figure 1: (a) Schematic diagram of the experimental setup and (b) operational mode for the sequencing batch reactor (reprinted with permission from Gong et al. [6]. Copyright © 2021 Elsevier B.V).

In another study, Wang et al. [7] reported enhanced nitrogen removal using anammox bacteria in the presence of graphene oxide (GO). A laboratory-scale anammox up-flow column reactor was used for the isolation of activated anammox bacteria. The majority of the anammox bacteria used in this study belonged to Candidatus

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Sougata Ghosh, Bishwarup Sarkar, and Sirikanjana Thongmee

Brocadia anammoxidans. The concentrations of NH4+-N and NO2−-N reduced from 120 and 150 mg/L to 30.1 and 31.3 mg/L after 42 h of incubation in the medium containing (NH4)2SO4 and NaNO2, respectively, along with the production of 23.4 mg/L of NO3−-N. The addition of 0.1 g/L of GO resulted in a decrease in the NH4+-N and NO2−-N concentrations to 21.0 and 19.3 mg/L, respectively, with a maximum NO3−-N production of 25.8 mg/ L. The average anammox activity of the bacteria was 4.19 mmol NH4+-N/g of VSS per day which increased to 4.62 mmol NH4+-N/g of VSS per day in the presence of 0.1 g/L of GO. It was further proposed that GO supplementation may act as a scaffold for the anammox bacteria for attachment which in turn, can facilitate exopolysaccharide (EPS) production. As expected, the total carbohydrate, protein, and EPS concentrations were increased to 42.5, 125.7, and 168.2 mg/g of VSS, respectively, in the presence of 0.1 g/L of GO. Additionally, transmission electron microscopy (TEM) results revealed packed anammox bacteria cells that were supported by the flake-like GO scaffold. In another recent study, Liu et al. [8] prepared a suspended sludge system using in situ enriching anammox bacteria that had enhanced nitrogen removal capability and could be potentially applicable for sewage treatment. The external excess sludge fermentation process was integrated with the simultaneous partial nitritation–anammox–denitrification reaction. This novel method was carried out in an anaerobic–aerobic–anoxic SBR that was inoculated with real sewage and sludge fermentation products. The TIN was reduced from 61.0 to 3.4 mg/L in the presence of 0.6 ± 0.2 mg/L of dissolved oxygen after operating the SBR for 110 days. Such high TIN removal efficiency of 94.56% was achieved due to the varying short and long hydraulic retention time (HRT) of 6.5 and 8 h, respectively. Moreover, a maximum nitrite accumulation ratio of 99.1% was attained after supplementation of the continuous sludge fermentation products wherein the external sludge ratio was reduced by 38.75% as well. Furthermore, the microbial community was analyzed using high throughput screening of 16S rRNA sequences that confirmed the in situ enrichment of Candidatus Brocadia and Candidatus Kuenenia. Additionally, the stoichiometric analysis suggested that 34.69% of TIN was removed because of concomitant nitrification and denitrification reactions along with anammox in presence of oxygen, whereas 35.21% of TIN was removed under anoxic conditions primarily because of anammox bacteria. Therefore, this study highlighted the novel method for the removal of TIN from real sewage samples using anammox bacteria. It is important to note that integration of anammox pathway into nitrogen removal reduced both energy consumption and organics requirement. As evident from Figure 2, the oxygen demand and COD requirements in partial nitritation–denitrification/anammox processes could be theoretically decreased by 37.03% and 76.52%, respectively. Organotrophic anammox bacteria were reported to be effective in nitrate reduction [9]. A comparison between the granular sludge reactor and a moving bed biofilm reactor (MBBR) was also carried out wherein both the reactors were operated at a temperature range of 18–25 °C with an aerobic/anaerobic time and COD/N ratios of 3 and less than 0.5 g of COD/gNH4-N, respectively. The biomass of both the reactors ranged from

1 Anammox bacteria-mediated sewage treatment

5

O2

1 mg/L

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NO2–

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/L mg

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0.726 mg/L

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3.43 mg/L 1 mg/L

2.16 mg/L 0.63 mg/L

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N2 (a) Nitritation-denitrification (Liu et al., 2017) Influent

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NH4+ Sludge Fermentation Products COD + Municipal Wastewater PO43–

NH4+ COD COD PAOs

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+

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NO2– N2

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Soluble COD Hydrolytic bacteria Sludge

(c) Schematic illustration of the potential conceptual mechanism for advanced nitrogen and phosphorus removal Figure 2: Comparison of (a) nitritation–denitrification and (b) partial nitritation–denitrification/anammox with respect to oxygen demand and COD saving, and (c) schematic illustration of the potential conceptual mechanism for advanced nitrogen and phosphorus removal in further research (reprinted with permission from Liu et al. [8]. Copyright © 2021 Elsevier B.V).

5.6 to 7.3 g of VSS/L, while the biomass of specific nitrogen removal capacity of both the reactors was also similar and ranged from 0.11 to 0.12 g of N/VSS/day. High-performance liquid chromatography (HPLC) results highlighted the presence of volatile fatty acids (VFAs) in the MBBR wherein 139 mg/L acetic acid was specifically observed along with the presence of succinate, propionic acid, butyric acid, methanol, and valeric acid that were present in very small concentrations. Additionally, the measured concentrations of nitrate in both the reactors were 40–68% lower than the expected concentrations. Fluorescent in situ hybridization (FISH) analysis then revealed the presence of Candidatus “Brocadia fulgida” in the biofilms of granular sludge reactor as well as MBBR. Moreover, Candidatus “Anammoxoglobus propionicus” was also present in the MBBR that contributed to the anammox process for nitrogen removal. Hu et al. [10] also reported the identification as well as quantification of anammox bacteria from eight different nitrogen removal reactors. The presence of ammonium and nitrite in the reactors facilitated the enrichment of anammox bacteria such

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Sougata Ghosh, Bishwarup Sarkar, and Sirikanjana Thongmee

that it became the dominant species in the reactor which was confirmed by fluorescent in situ hybridization (FISH) using an oligonucleotide probe Amx820 that was specific to anammox bacteria. Furthermore, the enumeration of anammox bacteria was also performed using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) wherein the abundance of anammox bacteria in seven out of eight reactors was 109 cells/mL. However, the reactor with a low copy number of anammox bacteria displayed a maximum per cell nitrogen removal rate of 42 pg/cell/day. Moreover, a novel anammox species identified in one reactor was named Candidatus Brocadia sinica. Additionally, several alterations in the temperature as well as influent wastewater, did not affect the activity of anammox bacteria in any of the reactors which highlighted their adaptability to varying environmental conditions. Wang et al. [11] demonstrated improvement of bacterial anammox activity after immobilizing the cells in polyvinyl alcohol and sodium alginate (PVA: SA) gel. The anammox gel beads exhibited a maximum nitrogen removal rate of 0.57 kg of N/m3/day by the end of the fourth phase along with more than 80% of nitrogen removal efficiency. The optimal PVA: SA gel bead ratio of 12%/2% displayed the maximum nitrogen removal rate that was attributed to its increased pore size. However, the mechanical strength of the optimized beads was poor. Interestingly, a shorter nitrogen loading time along with a higher specific anammox activity was observed in the upflow anaerobic sludge blanket (UASB) reactors inoculated with the optimized anammox beads as compared to the anammox granular sludge. A high-throughput screening using 16S rRNA sequencing then revealed a 37.96% abundance of Candidatus “Jettenia” as the dominant anammox species that was present in the beads. The specific anammox activity of the optimized beads with a 12%/2% ratio of PVA: SA was 0.365 g-N g/VSS/day. Liu et al. [12] also demonstrated the application of anammox bacteria in symbiosis with Nitrosomonas. A hollow cylinder composed of hydrophobic polypropylene foam was used in this study with a specific surface area and specific gravity of 1,500 m2/m3 and 0.98 g/cm3, respectively. The single-stage partial-anammox process was then performed in a laboratory-scale continuous stirred tank reactor at a temperature of 25 ± 1 °C wherein the reaction was carried out for 470 days that was divided into 7 stages. The maximum nitrogen removal efficiency and nitrogen removal of 81.1 ± 3.60% and 1.19 ± 0.15 kg-N/m3/day were achieved by anammox bacteria respectively with a nitrogen loading rate (NLR) of 1.5 kg-N/m3/day. Moreover, the start-up period of the process was 76 days with a NLR of 0.5 kg-N/m3/day while the maximum nitrogen removal potential was 2.5 kg/m3/day. Such efficient nitrogen removal rates were attributed to the enriched partial nitrification and anammox biomass, effective rates of nitrogen loading, improved mass transfer by the hollow functional carriers, the high specific activity of anammox bacteria, and the homogeneity of the reaction mixture in the reactor. Scanning electron microscopy (SEM) images then revealed biofilm formation on the carrier surface wherein the bacteria were attached to the inside as well as outside pores of the carrier surfaces. Moreover, the specific anammox activity of the biofilm sludge was 3.56 g-N/g-MLVSS/day in which anammox bacteria and ammonia-

1 Anammox bacteria-mediated sewage treatment

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oxidizing bacteria were suggested to form a symbiotic relationship. Thereafter, 16S rRNA sequencing was performed for the identification of the bacterial community present in the suspended flocs as well as carrier biofilms. Two genera of anammox bacteria namely, Candidatus Kuenenia and Candidatus Brocadia were identified in both cases wherein the abundance in the suspended flocs was 1.46% and 0.08% whereas it was 4.97% and 0.34% for carrier biofilms, respectively. In addition, the abundance of two obligate aerobic ammonia-oxidizing genera namely, Nitrosomonas and Nitrospira were present with an abundance of 5.68% and 0.26%, respectively. Further pyrogenic sequencing demonstrated the importance of symbiosis between the ammonia-oxidizing bacteria with anammox bacteria for effective nitrogen removal. A high nitrogen removal rate was achieved by Yokota et al. [13] wherein an UASB anammox reactor was used for continuous feeding of waste brine solution with a salinity of 3% and 180 mg-N/L of ammonium. During the initial phase of the reactor operation, the NLR was gradually increased by reducing the HRT and increasing the influent nitrite concentration, respectively. Hence, a nitrogen removal rate of 0.47 kg-N/m3/day was attained after 64 days of the reactor operation. Thereafter, the NLR was further increased to 10.3 kg-N/m3/day in the fourth phase. The ratio of NO2−/NH4+ was also increased from 1.12 to 1.23 after the addition of methanogenic granules in the UASB reactor. The size of the granules was 1,152 ± 308 μm after 296 days of the reactor operation, whereas the VSS concentration and anammox activity was increased from 13.6 to 18.9 g-VSS/L and 0.11 to 0.58 g-N/g-VSS/day, respectively, after 208 days of operation. The alterations in the structure of the microbial community were evaluated using highthroughput sequencing of 16S rRNA sequencing wherein only 4–8% of the abundance of Candidatus Scalindua was observed in the initial phase of the reactor operation prior to the inoculation of any granular biomass. However, after the addition of granular biomass, the relative abundance of the denovo31846 strain, which was phylogenetically identical to Ca. Scalindua increased to 30% and exhibited a maximum removal rate of nitrogen. These results were confirmed by qRT-PCR that showed an increase in the gene copy number of Ca. Scalindua after 119 days of the reactor operation. In addition, the anammox activity was reduced by 72% and 59% in the presence of 100 and 300 mg-N/L of NO2–, respectively. In a recent study, Ali et al. [14] reported the application of a novel anammox bacterium for efficient removal of nitrogen that could potentially be used for sewage treatment as well. A membrane bioreactor (MBR) was used for the cultivation of Candidatus Scalindua sp. AMX11 in the presence of Red Sea water that was supplemented with ammonium and nitrite. During phase I of the bioreactor operation which lasted for 30 days, a nitrogen removal rate of 0.35 ± 0.02 kg of N/m3/day. Later on, the salinity of the seawater was gradually reduced from 3.5% to 1.8% in the second phase of the reactor operation on day 55 showing similar nitrogen removal activity as that of the first phase. Moreover, the stoichiometric ratios of the amount of NO2− that was removed to the amount of NH4− removed ranged from 1.0 to 1.5, while the ratios of NO3− removed to NH4− removed had a range of 0.08–0.12 that was close to the theoretical anammox stoichiometric ratios.

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Sougata Ghosh, Bishwarup Sarkar, and Sirikanjana Thongmee

Therefore, nitrogen was primarily removed by the anammox bacteria during the first and second phases of the MBR operation. In the third phase of the reactor operation, the salinity of MBR was further decreased to 1.2% while 4 mM of acetate was added to the medium which resulted in a significant decline in anammox activity along with an increase in the NO2− concentration to 50 mg-N/L. The anammox activity was then recovered by reducing the acetate concentration to 2 mM wherein a stable nitrogen removal efficiency of around 92% was achieved. The average concentration of NO3− in the effluent was drastically reduced from 17 mg-N/L to 3 mg-N/L in the presence of 2 mM of acetate. However, the stoichiometric ratios indicated the involvement of other nitrogen removal processes such as heterotrophic denitrification and respiratory ammonification that may effectively contribute during the MBR operation in the presence of organic carbon. Moreover, isotope labeling experiments were carried out to identify the primary process involved in the removal of nitrogen wherein 2 mM of 5N-NO3− and 13CH3COOH were added as the organic carbon source. Also, 14N-NH4+ was also added to the medium which resulted in the production of 29N2 as the primary metabolized nitrogen product, whereas 30N2 was the main nitrogen gas in absence of the 14 N-NH4+ that was attributed to the respiratory ammonification by anammox bacteria. No change in NO3− reduction was observed after the addition of penicillin G which highlighted that anammox bacteria were mainly responsible for nitrogen removal as the heterotrophic bacteria were inhibited in the presence of the antibiotic. The oxidation rate of 13CH3COOH to 13CO2 by the anammox bacteria was 0.94 ± 0.02 nmol mg/protein/min. Similarly, the nitrate reduction rate was 2.1 ± 0.7 nmol mg/protein/min. Metagenomics and meta-transcriptomics analysis of the total DNA extracted from the MBR biomass further confirmed the primary contribution of anammox bacteria in the nitrogen removal process. Candidatus Scalindua sp. AMX11 was the most abundant anammox bacteria that also had the highest (93–96%) gene expression among the microbial community in the bioreactor during all three phases. The transcriptional activity was not altered with varying saline conditions in the bioreactor which highlighted the potential applicability of Candidatus Scalindua sp. AMX11 in various wastewater samples that may have different concentrations of salts. The nitrogen removal ability of marine anammox bacteria (MAB) was also demonstrated by Wei et al. [15] in the presence of extreme saline conditions. Seawater and freshwater media were used as the basal media supplemented with varying concentrations of NaCl. Initially, a rapid decline in the nitrogen removal efficiency from 86.9% to 67% was observed within a few days with a subsequent increase in the NaCl concentration from 30 g/L to 75 g/L, respectively. The efficiency of nitrogen removal was further reduced to 10% after 107 days of reactor operation in presence of 75 g/L of NaCl. The concentrations of NH4+ and NO3− were concomitantly increased with increasing time of reactor operation in presence of anammox bacteria, while NO2− was completely absent in the media. The efficiency of nitrogen removal was further recovered to 85% within 10 days of reducing the NaCl concentration to 30 g/L. Hence, the reversibility of the nitrogen removal efficiency of anammox bacteria was demonstrated within a short span of time. Moreover, after 41 days of reactor operating under normal

1 Anammox bacteria-mediated sewage treatment

9

conditions, the NaCl concentration was reduced to 0 g/L by changing seawater medium to freshwater medium. This also resulted in a drastic change in the average nitrogen removal rate from 87.3% to 46% within 7 days. Hence, it was highlighted that MAB can only effectively remove nitrogen in the presence of salts such as MgSO4.7H2O, KCl, NaBr, H3BO3, NaF, and KI. The maximum nitrogen removal efficiency of 91% was finally achieved after 182 days under normal operating conditions with a total nitrogen (TN) concentration of 210 mg-N/L. Further, denaturing gradient gel electrophoresis (DGGE) results identified two different MAB that was similar to Candidatus Scalindua wagneri and Chromatiales. Yin et al. [16] demonstrated effective nitrogen removal from wastewater samples using MAB. In this study, SBR was used for the investigation of nitrogen removal. A sharp decrease in the concentration of NH4+-N and NO2−-N was observed in presence of MAB at an ammonia removal rate (ARR) and nitrite removal rate (NRR) of 0.32 and 0.42 kg/(m3.d), respectively. On the other hand, NO3−-N concentration increased to 22.60 mg/L after 62 cycles of nitrogen removal which suggested anammox bacterial growth. A maximum ARR and NRR of 0.56 and 0.60 kg/(m3.d) were observed in presence of Fe(III) which was proposed to increase the enzymatic activity of MAB cells. The efficiency of ammonia and nitrite removal was then reduced gradually from 52.89% and 69.97% to 42.29%, respectively, with subsequent cycles of nitrogen removal. The activity of MAB was also significantly inhibited in the presence of high concentrations of nitrite. In addition, the stoichiometric ratios of nitrite consumption along with nitrate production to ammonium consumption were 1.32 and 0.26, respectively, in presence of high salt and low-temperature conditions. Hence, it was proposed that the marine Feammox process was the primary mechanism of nitrogen reduction wherein cell membranes of MAB consists of ferric reductase that couples NH4+-N oxidation to Fe(III) reduction. Analysis of the microbial community present in the sludge after 90 cycles revealed Candidatus Scalindua as the dominant genus present (17.07%) in the SBR which was further increased to 27.70% after supplementation of Fe(III). Other MAB genera were also present in the SBR such as Marinicella and Caldithrix that were suggested to consume NO3−-N for effective nitrogen removal. Inhibition kinetic analysis of MAB in presence of nitrogen shock loading further provided an inhibition coefficient of 90.81 mg/L using the Haldane model while the half-saturation constant was 15.15 mg/L. Hence, sufficient tolerance up to 224.4 mg/L of nitrite was observed wherein no significant metabolic inhibition of MAB was observed at 20 °C. Therefore, these MAB was proposed to be applicable for nitrogen removal from wastewater under high salinity and low-temperature conditions after Fe(III) supplementation. Van Duc et al. [17] also evaluated the nitrogen removal performance of MAB in continuous packed-bed columns having marine sediment samples. Complete removal of NO2− and NH4+ was achieved in the first phase (30 days) of operation in the presence of anammox bacteria. The efficiency of nitrogen removal was maintained at 94% even after 55 days of operation and with increasing concentrations of NH4+ and NO2− in the culture media. Moreover, during the third phase of column operation, 500 mg/L

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Sougata Ghosh, Bishwarup Sarkar, and Sirikanjana Thongmee

of KHCO3 was supplemented in the artificial seawater for promoting the growth of anammox bacteria. Thereafter, the nitrogen removal activity of anammox bacteria was further improved up to 90% in phase 4 with a considerable increase in the NLR of 1.0 ± 0.02 kg of N/m3/day. Three different groups namely, Planctomycetes, Proteobacteria, and Bacteroidetes comprised 60–90% of the total microbial community were found in the packed-bed columns. Further, Candidatus Scalindua was also present in the column with an abundance range of 21.3–56.4% while other anammox species such as Planctomycete UKU-1, Candidatus Jettenia asiatica, and Candidatus Brocadia were also reported to colonize the same niche. Huang et al. [18] used a laboratory-scale SBR for landfill leachate treatment wherein anammox reactions occurred after three months of inoculation of 2 L of activated sludge. The sludge was composed of 11.6 g/L of mixed liquid suspended solids and 9.3 g/L of VSS. The initial 83 days of reactor operation revealed a robust nitrogen removal rate that was primarily attributed to the denitrification process. Followed to this significant amounts of ammonia and nitrite were removed followed by concomitant production of nitrate because of anammox reactions. The first phase of the reactor operation displayed a nitrogen removal efficiency ranging from 84.5% to 90.0% for 93 days. In the later phases of the SBR operation, the HRT was reduced which resulted in a subsequent increase in the NLR to a maximum value of 2.05 ± 0.09 kg of N/m3/day at the fourth phase. Moreover, 85% of nitrogen removal efficiency was retained in all the phases of the SBR operation. The dissolved oxygen decreased from 0.8 to 1.0 mg/L in the first operation phase to 0.5–0.7 mg/L in the final phase of the reactor operation. However, there was no change in the performance of removal of nitrogen in the reactor due to alteration in the dissolved oxygen in the medium. In addition, the COD removal efficiency during the first phase of the bioreactor operation ranged from 37.1–49.7% to 62.5–65.1% in the fourth phase. A maximum COD removal rate of 0.48 kg COD/m3/day was achieved. The partial nitrification–anammox process was achieved at around 3 months in the presence of low dissolved oxygen levels of 0.5–1.0 mg/L. Additionally, efficient biomass was retained in the SBR with minimal biomass washout and a sludge volume index range of 35.7–54.5 mg/L. Moreover, the formation of biofilms in the SBR was suggested to provide ecological niches for anammox bacteria along with the low oxygen conditions and inhibition of free ammonia that further facilitated suppression of nitrite-oxidizing bacteria. Thereafter, 16S rRNA gene sequencing of the microbial community revealed the presence of unidentified anammox bacteria along with Nitrosomonas and other denitrifying bacteria. The abundance of the unidentified anammox bacteria was 20.05% as compared to the 4.02% abundance of Nitrosomonas. Zhang et al. [19] showed enhanced nitrogen removal from domestic wastewater using anammox bacteria that were enriched in a sequencing batch biofilm reactor (SBBR). Alternate modes of anaerobic, aerobic, and anoxic conditions were maintained in the reactor wherein a sponge was used as the carrier for inoculation of the sludge. During the first run of the reactor for 37 days, the average COD and TN were

1 Anammox bacteria-mediated sewage treatment

11

102.5 ± 11.5 mg/L and 36.8 ± 2.7 mg N/L with the organic loading rate (OLR) and NLR of 102.5 ± 11.5 mg COD/L/day and 36.8 ± 2.7 mg N/L/ayd, respectively. The efficiency of nitrogen removal was further improved within 7 days wherein the concentration of ammonium reduced from 15 mg/L to less than 1 mg/L. During the second run of the SBBR, the OLR and NLR increased to 149.2 ± 19.6 mg COD/L/day and 50.1 ± 3.1 mg N/L/day, respectively, which resulted in the improvement of the nitrogen removal efficiency from 47.5 ± 15.1% to 70 ± 8.0% due to reduction in the dilution ratio. The efficiency of nitrogen removal was further increased to almost 90% with a subsequent increase in the influent OLR and NLR. In addition, decreasing the SBBR temperature from 27 to 15.5 °C provided an efficiency of nitrogen removal of 88.2 ± 3.6% along with 9.5 mg N/L of TN that was present in the effluent. The biomass concentration in the SBBR then increased from 965 mg/L on the fifth day of operation to 4,500 mg/L after 124 days of operation of the SBBR. The abundance of anammox bacteria also increased from 1.11 × 104 to 1.69 × 106 gene copies/mL with a doubling time of 12–18 days. Further, a maximum anammox activity of 1.68 mg N · g/VSS/h was attained after 97 days of operation. In another similar study, Schmidt et al. [20] improved the nitrogen removal ability from granular sludge in a UASB reactor by inoculation of anammox bacteria. During the initial phase of the reactor operation, the concentration of nitrite and nitrate in the effluent was insignificant which suggested the presence of organic matter in the sludge samples that could be utilized for heterotrophic denitrification. Moreover, the ammonium concentration was negligible at the end of the experiment, while the nitrite concentration was also reduced significantly which highlighted the contribution of anammox in the removal of nitrogen from the reactors. FISH analysis using anammox specific AMX820 probe then further confirmed the presence of anammox bacteria in the reactors wherein high loading rates were attained up to 0.14 kg of NNH3/m3 reactor/day which resulted in 99% effective removal of ammonia from the reactor. Additionally, the kinetic studies revealed the perfect fit of the Monod kinetic model for explaining the anammox reactions for nitrogen removal. Table 1: Role of anammox bacteria in water treatment. Anammox bacterial species

Nitrogen removal efficiency

Reference

Candidatus Brocadia

. mg/L of NH+ and . mg/L of total inorganic nitrogen

[]

Candidatus Brocadia anammoxidans

. mg/L of NH+-N and . mg/L of NO−-N

[]

Candidatus Brocadia and Candidatus Kuenenia

.% of total inorganic nitrogen

[]

Candidatus Brocadia fulgida and Candidatus Anammoxoglobus propionicus

–

[]

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Sougata Ghosh, Bishwarup Sarkar, and Sirikanjana Thongmee

Table 1 (continued) Anammox bacterial species

Nitrogen removal efficiency

Candidatus Brocadia sinica

 pg of total N/cell/day

[]

Candidatus Jettenia

More than % of total nitrogen

[]

Candidatus Kuenenia and Candidatus Brocadia

. ± .% of total nitrogen

[]



Reference

Candidatus Scalindua

. kg-N/m /day of total nitrogen

[]

Candidatus Scalindua sp. AMX

% of total nitrogen

[]

Candidatus Scalindua wagneri and Chromatiales

% of total nitrogen

[]

Candidatus Scalindua, Marinicella sp., and Caldithrix sp.

.% of

[]

Candidatus Scalindua, Planctomycete UKU-, Candidatus Jettenia asiatica, and Candidatus Brocadia

% of NO− and NH+

[]

Unclassified anammox bacteria

.–.% of total nitrogen

[]

Unidentified anammox bacteria

% of total nitrogen

[]

Unidentified anammox bacteria

% of NH

[]

NH+-N

3 Conclusion and future perspectives Anammox bacteria have a greater potential for wastewater treatment due to their capability to alter the chemical composition of the effluent. Bacterial metabolism plays a pivotal role in the enzymatic remediation of the contaminants. However, optimization of the process parameters like the inoculum density, age of culture, duration of treatment, temperature, pH, aeration, and agitation will enable to exploit the anammox bacteria maximally for effective effluent treatment. Genetic engineering can help to develop recombinant strains of bacteria with higher capacity for remediation of nitrogen contaminated wastewater. Such bacteria can be encapsulated or immobilized on the surface of biomaterials like alginate so that they can be reused repeatedly. Inert nanomaterials like mesoporous silica nanoparticles and biopolymers can be used as carriers of the anammox bacteria. Various nanomaterials are reported to have strong catalytic activity which is used for degradation of the pollutants. Biologically synthesized nanoparticles are environmentally benign and biocompatible. Various bacteria, fungi, algae, and plants are reported to synthesize metal and metal oxide nanoparticles with exotic shape and size. Hence such biogenic nanoparticles should be used in combination with the anammox bacteria to develop more efficient wastewater treatment strategy [21]. Similarly, instead of single species of bacteria multiple species of bacteria or consortia in combination with cyanobacteria and algae can be used to design highly efficient water treatment process. Hereby, anammox

1 Anammox bacteria-mediated sewage treatment

13

bacteria mediated effluent treatment process seems to be one of the promising complementary and alternative strategies for controlling water pollution and ensuring clean environment.

References [1]

Ali, M., Okabe, S., 2015. Anammox-based technologies for nitrogen removal: Advances in process start-up and remaining issues. Chemosphere 141: 144–153. [2] Ma, B., Wang, S., Cao, S., Miao, Y., Jia, F., Du, R., Peng, Y., 2016. Biological nitrogen removal from sewage via anammox: Recent advances. Bioresour. Technol. 200: 981–990. [3] Luikham, S., Malve, S., Gawali, P., Ghosh, S., 2018. A novel strategy towards agro-waste mediated dye biosorption for water treatment. World J. Pharm. Res. 7(4): 197–208. [4] Kartal, B., Kuenen, J.V., Van Loosdrecht, M.C.M., 2010. Sewage treatment with anammox. Science 328(5979): 702–703. [5] Ghosh, S., 2020. Toxic metal removal using microbial nanotechnology. In: Rai, M., Golinska, P. (Eds.), Microbial Nanotechnology. CRC press, Boca Raton. eBook ISBN: 9780429276330. [6] Gong, Q., Wang, B., Gong, X., Liu, X., Peng, Y., 2021. Anammox bacteria enrich naturally in suspended sludge system during partial nitrification of domestic sewage and contribute to nitrogen removal. Sci. Total. Environ. 787: 147658. [7] Wang, D., Wang, G., Zhang, G., Xu, X., Yang, F., 2013. Using graphene oxide to enhance the activity of anammox bacteria for nitrogen removal. Bioresour. Technol. 131: 527–530. [8] Liu, J., Peng, Y., Qiu, S., Wu, L., Xue, X., Li, L., Zhang, L., 2021. Superior nitrogen removal and sludge reduction in a suspended sludge system with in-situ enriching anammox bacteria for real sewage treatment. Sci. Total. Environ. 793: 148669. [9] Winkler, M.K., Yang, J., Kleerebezem, R., Plaza, E., Trela, J., Hultman, B., van Loosdrecht, M.C., 2012. Nitrate reduction by organotrophic Anammox bacteria in a nitritation/anammox granular sludge and a moving bed biofilm reactor. Bioresour. Technol. 114: 217–223. [10] Hu, B.L., Zheng, P., Tang, C.J., Chen, J.W., van der Biezen, E., Zhang, L., Ni, B.J., Jetten, M.S., Yan, J., Yu, H.Q., Kartal, B., 2010. Identification and quantification of anammox bacteria in eight nitrogen removal reactors. Water Res. 44: 5014–5020. [11] Wang, J., Fan, Y.C., Chen, Y.P., 2021. Nitrogen removal performance and characteristics of gel beads immobilized anammox bacteria under different PVA: SA ratios. Water Environ. Res. 93: 1627–1639. [12] Liu, Y., Niu, Q., Wang, S., Ji, J., Zhang, Y., Yang, M., Hojo, T., Li, Y.Y., 2017. Upgrading of the symbiosis of Nitrosomanas and anammox bacteria in a novel single-stage partial nitritation–anammox system: Nitrogen removal potential and Microbial characterization. Bioresour. Technol. 244: 463–472. [13] Yokota, N., Watanabe, Y., Tokutomi, T., Kiyokawa, T., Hori, T., Ikeda, D., Song, K., Hosomi, M., Terada, A., 2018. High-rate nitrogen removal from waste brine by marine anammox bacteria in a pilot-scale UASB reactor. Appl. Microbiol. Biotechnol. 102: 1501–1512. [14] Ali, M., Shaw, D.R., Saikaly, P.E., 2020. Application of an enrichment culture of the marine anammox bacterium “Ca. Scalindua sp. AMX11” for nitrogen removal under moderate salinity and in the presence of organic carbon. Water Res. 170: 115345. [15] Wei, Q., Kawagoshi, Y., Huang, X., Hong, N., Van Duc, L., Yamashita, Y., Hama, T., 2016. Nitrogen removal properties in a continuous marine anammox bacteria reactor under rapid and extensive salinity changes. Chemosphere 148: 444–451.

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[16] Yin, S., Li, J., Dong, H., Qiang, Z., 2019. Enhanced nitrogen removal through marine anammox bacteria (MAB) treating nitrogen-rich saline wastewater with Fe (III) addition: Nitrogen shock loading and community structure. Bioresour. Technol. 287: 121405. [17] Van Duc, L., Song, B., Ito, H., Hama, T., Otani, M., Kawagoshi, Y., 2018. High growth potential and nitrogen removal performance of marine anammox bacteria in shrimp-aquaculture sediment. Chemosphere 196: 69–77. [18] Huang, X., Mi, W., Ito, H., Kawagoshi, Y., 2020. Unclassified anammox bacterium responds to robust nitrogen removal in a sequencing batch reactor fed with landfill leachate. Bioresour. Technol. 316: 123959. [19] Zhang, J., Zhang, L., Miao, Y., Sun, Y., Li, X., Zhang, Q., Peng, Y., 2018. Feasibility of in situ enriching anammox bacteria in a sequencing batch biofilm reactor (SBBR) for enhancing nitrogen removal of real domestic wastewater. Chem. Eng. Sci. 352: 847–854. [20] Schmidt, J.E., Batstone, D.J., Angelidaki, I., 2004. Improved nitrogen removal in upflow anaerobic sludge blanket (UASB) reactors by incorporation of anammox bacteria into the granular sludge. Water Sci. Technol. 49(11–12): 69–76. [21] Bhagwat, T.R., Joshi, K.A., Parihar, V.S., Asok, A., Bellare, J., Ghosh, S., 2018. Biogenic copper nanoparticles from medicinal plants as novel antidiabetic nanomedicine. World J. Pharm. Res. 7(4): 183–196.

Nilendu Basak, Atif Aziz Chowdhury, Taniya Roy, and Ekramul Islam✶

2 Diversity and distribution of ammoniaoxidizing archaea in engineered and natural environments Abstract: As the part of biogeochemical cycle microbe-driven nitrification is the important nitrogen (N) removal step from wastewater where ammonia is converted to nitrite (ammonia oxidation) and ultimately to gaseous nitrogen. Recent research shows ammonia-oxidizing archaea (AOA) also play significant role in nitrogen removal process in addition to the ammonia-oxidizing bacteria (AOB), especially in wastewater treatment plants (WWTPs). In WWTP amount of dissolved oxygen become critical which make life harder for AOB, while AOA could continued their activity in the oxygen limited environment. In line with activity of AOA in engineered system, although their role in natural environment is noteworthy, especially in driving N cycle, they are still unexplored in terms of diversity and function. While the cultivation and diversity of AOBs are well studied the same for archaeal counterpart is surprisingly overlooked. Although there is a bottleneck in the culture-based study of archaea, in recent years, however, with the development of molecular approaches such as “omic” technologies, the research on AOA has gained momentum. Environmental variables such as pH, nutrients, depth, organic matters act as determining factors for the diversity of AOA. This chapter summarizes the lesser-known part of the ecosystem – AOA, their diversity, distribution, and activity. overlooking the Archaea has been equivalent to surveying one square kilometer of the African savanna and missing over 300 elephants – Gary Olsen

1 Introduction The overall volume of discharge of wastewater continues to rise in tandem with the fast growth of urbanization. A substantial volume of municipal wastewater containing ammonium, in particular, is frequently released after treatment, accelerating eutrophication of water bodies and harming the natural environment and can pose a serious risk to aquatic lives as well as human health. As a result, an effective and consistent reduction in ammonia nitrogen effluent concentration is critical for eutrophication management [1]. ✶

Corresponding author: Ekramul Islam, Department of Microbiology, University of Kalyani, Kalyani, Nadia 741235, West Bengal, India, e-mail: [email protected] Nilendu Basak, Atif Aziz Chowdhury, Taniya Roy, Department of Microbiology, University of Kalyani, Kalyani, Nadia 741235, West Bengal, India https://doi.org/10.1515/9783110780093-002

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For the removal of nitrogenous chemicals from wastewater, a range of biological nitrogen removal techniques are available. The oldest and currently most common biological technique for eliminating nitrogen species from wastewaters has been nitrification/denitrification, which involves converting ammonia to nitrate and then organically reducing the nitrate to diatomic nitrogen. For the requirement of supplying oxygen for nitrification and organic matter for denitrification process, the conventional process is becoming costlier [2]. Production of sludge and greenhouse gas emissions during operation causes environmental harm. Anaerobic ammonium oxidation (anammox) is a unique method for reducing nitrogen in ammonium-rich wastewater that is now gaining popularity due to its low cost and energy consumption [3]. Nitrification entails ammonia oxidation by ammonia-oxidizing organisms to convert ammonia (NH3) to nitrite (NO2) and nitrite oxidation by nitrite-oxidizing species to convert nitrite to nitrate (NO3). Ammonia oxidation is essential to wastewater nitrogen removal and global nitrogen cycle because it frequently act as a rate limiting mechanism under a variety of condition. Ammonia oxidation reactions are as follows [4]: NH3 + O2 + 2e− + 2H+ ! NH2 OH + H2 O NH2 OH + 1=2 O2 ! NO2 − + 3H+ + 2e− 2NH3 + 3O2 ! 2NO2 − + 2 H2 O + 2H+

(1) (2) (3)

Autotrophic ammonia-oxidizing bacteria (AOB) associated with β- and γ-proteobacteria were assumed to be the only aerobic ammonia-oxidizing microbes in WWTPs for a long time. Since the discovery of ammonia-oxidizing archaea (AOA; currently attributed to the phylum Thaumarchaeota) in 2005, AOA has been found in a variety of settings and is widely regarded as the primary driver of nitrification in terrestrial, marine, and geothermal environments. This has dramatically expanded our understanding of global nitrogen and carbon cycles, as well as the ecology and evolution of the archaea domain. In the last decades, researchers have demonstrated the presence of archaeal amoA in wastewater treatment plants (WWTPs) [5]. It is difficult to generalize the contribution of amoA encoding archaea (AEA) toward ammonia oxidation, as the data is limited. For a better understanding, in this chapter, we have tried to audit the current information on the lesser-known driver of ammonia oxidation, AOAs in WWTPs and some natural environment, and their role in anaerobic ammonia oxidation.

2 History of archaea Archaea play an important role in the tree of life and account for a significant portion of microbial biodiversity. They are abundant in soils, ocean sediments, and the water column and play important roles in processes that mediate global atmospheric carbon and nutrient fluxes. The advent of culture-independent sequencing methods has

2 Diversity and distribution of ammonia-oxidizing archaea in engineered

17

enabled unprecedented access to genomic material from hitherto inaccessible archaeal lineages. This is changing our perception of the archaea’s diversity and metabolic capability in a wide range of habitats, which is a crucial step toward comprehending their ecological importance. Earlier bacteria and archaea were lumped into a single group, as all unicellular, non-eukaryotes were denoted as “Prokaryota” and later “Monera.” Advancements in molecular technology gave an insight into nucleic acid and phylogenetic analysis revealed their evolutionary relatedness by Carl Woese and his group. The idea was to divide the lives into three domains namely eukarya, eubacteria (later renamed as bacteria), and archaebacteria (archaea). Woese also proposed the closer relatedness of archaea to eukarya, than to bacteria [6]. Culture independent identification of broader environmental samples revealed the underlying complex diversity of the archaea, also referredto as “microbial dark matter.” Scientists are driven toward the survival mechanism of archaea for their ability to grow in extreme conditions. Advanced genomic technologies gives answer to the archaeal evolution. Archaea are more closely related to eukarya than bacteria, with most of the sequenced archaeal genome sharing similarity with 313 core conserved genes [7]. Conserved orthologs between studied archaea and bacterial genomes was observed using functional characterization and evolutionary reconstraction [8, 9]. Of the analyzed genomes of the halophilic archaea, 55.1% genes was transferred through horizontal gene transfer and 44.8% was from ancestral origin [8].

3 Archaeal metabolism and diversity Initially, archaea were thought to be thermophiles limited to methanogenesis and sulfur respiration, as most early works were related to this area [10]. Recent research has begun to broaden the known range of metabolic methods that archaea from various phyla may employ in a variety of settings. This expanded diversity sampling of archaeal genomes has also begun to allow for the tracing of evolving long histories of metabolic pathways among archaea. Estimating gene gain, loss, and transfer among lineages is used to predict the physiological capacity and model ancestral lineages. Using this method, Aigarchaeota showed to undergo significant loss of genes and at the same time incorporated genes for CO oxidation and dissimilatory sulfite reduction from bacteria and was evolved from thermophilic aerobes [11]. Archaeons range in diameter from 0.1 to 15 mm with a variety of morphology such as spheres, rods, spirals, and plates. Archaeal metabolism to some extent resembles bacterial counterpart, but still, they are very different in terms of metabolic behavior. Carbon fixation mechanism of Crenarchaeota utilizes 3-hydroxypropionate pathway or the Krebs cycle in the case of autotrophic mechanism, while AOBs use the Calvin cycle. Archaea differs from bacteria in terms of their adaptability to chronic

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energy stress which is supported by membranes with low permeability and certain catabolic pathways. Similar to the heterotrophs, Crenarchaeota can utilize dissolved organic matter as a source of carbon. Analysis of the genome also further established their mixotrophic potential by using CO2 and organic matter as carbon source [4]. Archaeal community in the mangrove rhizosphere, influenced by the root exudates and controls the bacterial community thereby affecting the plant growth and diversity [12, 13]. Depending upon the adaptation to the physicological needs in extreme conditions, archaea can be classified asthermophile (tolerate high temperature), methanogen (produce methane), halophile (tolerate high salt concentration). Based on 16S and 23S rRNA analysis archaeal domain can be classified in five phyla: Crenarchaeota (mostly hyperthermophiles), Euryarchaeota (most of the methanogens and halophiles), Korarchaeota (archaea from the underground aquifers), Thaumarchaeota (chemolithoautotrophic ammonia oxidizers), and Nanoarchaeota (single genus with a parasitic nature of living). Moreover, few more groups have been proposed namely Aigarchaeota, Parvarchaeota, Aenigmarchaeota, Nanohaloarchaeota, and Diapherotrites, though there existence is debatable [14]. A new kingdom was proposed as Proteoarchaeota, predicted on the study of ribosomal protein marker [15]. Anaerobic archaea adapts to high energy restriction. Methanogens of the anaerobic category and a few thermophilic archaea in this condition creates chemiosmotic gradient and thereby utilizes low energy substrate through oxidation–reduction reaction. Climatic and soil factors shape the archaeal community of agricultural ecosystem and archeal phyla Euryarchaeota and Thaumarchaeota functions in biogeochemical cycle [16, 17]. Euryarchaeota dominates wetland ecosystem and influences nitrogen turnover [18]. Ammonia and sulfur oxidation by autotrophic ammonia oxidizers and thermoacidophilic sulfur oxidizers grows in different ecosystem. Halophilic archaea from rhizosphere was reported to play a role phosphate solubilization [19]. Autotrophy was reported in Candidatus Nitrosopumilus maritimus through chemolithoautotrophic metabolism. The growth was further stalled by excreted organic matters from the autotrophs [20]. Ingalls et al. [21] reported through radiocarbon analysis that heterotrophs and autotrophs both are included in the archaea or they are a population of a single species with metabolism type – mixotrophy. AOA metabolic potential still has a lot of room to be explored.

4 Overview of nitrogen cycle and ammonia oxidation Nitrogen is the second most important constituent of life (~15%) after carbon (~50%) including amino acids and proteins. Nitrogen exists in several forms in the environment, varying from the most reduced (−3) form as organic proteinaceous material and ammonia (NH3) to the most oxidized forms: nitrite (NO2; +3) and nitrate (NO3; +5).

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19

Nitrogen is the most prevalent gas in the Earth’s atmosphere (approximately 78%), and it exists in its neutral form as dinitrogen gas (N2). It is recycled among the geosphere, biosphere, and atmosphere through a complicated cascade of reactions. Assimilatory and dissimilatory reactions are involved in this mechanism. Nitrogen cycling (Figure 1) also includes biotic redox transformation, as well as chemodenitrification [22].

Ocean

Feeding

Animals

Leaching

Death

Excretion

Decaying products

Death

Ammonification

Adsorption

Plants

Non-biological nitrogen fixation

Biological nitrogen fixation

Denitrification

Nitrogen

NO2Nitrosification

NO3-

Nitrification NH4+

Figure 1: Schematic diagram of nitrogen cycle.

To take part in the biological functions, atmospheric nitrogen must be fixed and made bioavailable. The fixation can be through natural (lightning), chemical (Haber process, later used as fertilizers for plants), or by biological (microbial fixation) process. Most of the microbes use ammonia, which can be naturally produced by ammonification, the mineralization of organic nitrogen. Two dissimilatory microbial activities recycle ammonium, which is created by N2 fixation or released during organic matter decomposition, to N2. (A) Nitrification is the aerobic oxidation of ammonium to nitrite and ultimately to nitrate by a particular type of microorganisms. (B) Denitrification is the anaerobic respiratory reduction of nitrate to N2 through nitrite, nitric oxide, and nitrous oxide, along with the oxidation of organic materials, hydrogen, or reduced iron or sulfur species.

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5 Ammonia-oxidizing archaea AOB has a lower affinity for ammonia than AOA, thus in WWTPs, AOB’s growth is favored. With low ammonia availability, AOA gets a growth advantage for their k-type strategy of growth and with a slow growth rate they can degrade complex substrates [23]. Though recently it is indicated due to archaeon surface area to volume ratio, the variation is observed [24]. Anammox is the chemolithotrophic process of anaerobic ammonium oxidation in which NH4+ is oxidized in an anoxic condition and finally produces N2. As anammox is considered as a new method, new organisms such as the mesophilic Crenarchaeota are being discovered. Several anammox genera have been identified to date from the phylum Planctomycetes, such as Candidatus Brocadia, Candidatus Kuenenia, Candidatus Scalindua, Candidatus Anammoxoglobus, and Candidatus Jettenia [25, 26]. Marine isolate Nitrosopumilus maritimus was shown to carry the genes of amoA, amoB, amoC, the three subunits of ammonia monooxygenase (AMO), through which ammonia oxidation is catalyzed [20].

5.1 Distribution Our bacteria-centric concept of nitrification was challenged by the revelation of archaeal ammonia oxidizers and points out the amazing variation of microbial role in biogeochemical cycles. AOB is been known for decades to oxidize ammonia (NH3) to nitrite (NO2−), the first step of nitrification. AMO, the enzyme involved in this step, widely used as a molecular marker, unveiled ammonia oxidation within archaea through amo-like genes in soil [27] and marine surface water [28]. Nitrosopumilus maritimus SMC1 (Group I.1a archaea), is the first isolated ammoniaoxidizing archaeon, widely distributed in coastal and open water, and constitutes 20–30% of marine microbes [20, 29]. The uncultivated Candidatus Cenarchaeum symbiosum, a marine sponge symbiont possesses the genes for NH3 oxidation and is considered anamoA-encoding archaeon [30]. Candidatus Nitrosoarchaeum limnia, group I.1a AOA, was cultivated from low salinity sediment, and the genome was reconstructed [31]. Through enriching agricultural soil and estuary sediment, Candidatus Nitrosoarchaeum koreensis and Candidatus Nitrosopumilus salaria genomes were derived [32, 33]. Thermophilic AOA Candidatus Nitrososphaera gargensis [34] and Candidatus Nitrosocaldus yellowstonii [35] of group I.1b archaea and from a lineage with several branches of high-temperature habitats (HWCG-III group) were characterized. AOA from freshwater sediments has also been reported [36]. The first report of Nitrososphaera viennensis EN76, a mesophilic AOA of group I.1barchaeon from soil [37] and group I.1a associated lineage, Candidatus Nitrosotalea devanaterra, an obligate acidophilic AOA [38], is of prime importance in future research and innovation. The diversity and distribution of AOA was demonstrated in Table 1 in different environments.

2 Diversity and distribution of ammonia-oxidizing archaea in engineered

21

5.2 Metabolism The first reported detection of amoA genes of AEA in WWTPs was in the activated sludge bioreactor of the USA [39]. AOA is highly specialized chemolithoautotrophs that obtain energy from ammonia oxidation using CO2 as their only carbon source [40], though they show quite a lot of metabolic flexibility. It is also reported that they can produce ammonia and CO2 from urea [41]. Moderate thermophilic Nitrososphaera gargensis and some other marine AOA were reported to use cyanate for the same purpose. Thaumarchaeota, an AEA from WWTP, showed no ammonia oxidation nor bicarbonate accumulation but proposed to use an alternative carbon source [42]. Organic substances can stimulate ammonia oxidation of archaea, which was linked with nonenzymatic decarboxylation for reactive oxygen species scavenging [40]. Some oligotrophic AOA possesses a higher affinity for ammonia than AOB, which otherwise dominates the ammonia-oxidizing community in the eutrophic condition of WWTP, though contradiction was also reported in the same review [41]. Table 1: Diversity and distribution of ammonia-oxidizing archaea in environment. Archaea

Environmental habitat

Location

Optimum growth temperature

Reference

Candidatus Nitrosocaldus cavascurensis

Hot spring

Ischia, Italy

 °C

[]

Candidatus Nitrosocaldus yellowstonensis

Hot spring

Yellowstone National park

 °C

[]

Candidatus Icelandic hot spring Nitrosocaldus islandicus biofilm

Graendalur valley, Iceland

 °C

[]

Nitrosomarinus catalina Temperate Pacific SPOT

Water column, nearshore California

– °C

[]

Candidatus Nitrosopelagicus brevis CN

Open sea

Water column, nearshore California

 °C

[]

Candidatus Nitrosopumilus salaria BD

Marine sediments

San Francisco Bay estuary

 °C

[]

Nitrosopumilus maritimus SCM

Tropical marine fish tank

Seattle Aquarium in  °C Seattle, Washington, USA

[]

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Table 1 (continued) Archaea

Environmental habitat

Location

Optimum growth temperature

Reference

Nitrosopumilus cobalaminigenes HCA

Dimly lit deep coastal waters

Hood Canal, Washington, – °C USA

[]

Nitrosopumilus oxyclinae HCE

Lower euphotic zone Hood Canal, Washington, – °C of coastal waters USA

[]

Nitrosopumilus ureiphilus PS

Surface sediment

Puget Sound estuary Seattle,Washington, USA

– °C

[]

Candidatus Nitrosopumilus adriaticus NF

Coastal surface waters

Northern Adriatic Sea

– °C

[]

Candidatus Nitrosopumilus DC

Northern Adriatic Sea

Piran, Slovenia

– °C

[]

Candidatus Nitrosoarchaeum koreensis MY

Rhizospheric soil of Caraganasinica

Chungbuk National University, South Korea

NA

Candidatus Nitrosocosmicus exaquare G

Municipal WWTPs

Guelph, Ontario,Canada

 °C

[]

Candidatus Nitrosocosmicus franklandus

Agricultural soil

Craibstone, Aberdeen, Scotland, UK

– °C

[]

[, ]

5.3 Anaerobic ammonia oxidation In the traditional concept of the nitrogen cycle, ammonium was thought to be the end product of anaerobic breakdown, which requires oxygen and aerobic AOB for further transformation. However, chemical data from oxygen-depleted marine waters and deep-sea sediments suggested that ammonium is converted anaerobically to N2 in these settings in the presence of nitrate and nitrite [22]. Anammox, performed mainly by anammox bacteria, is a short way of transforming ammonium and nitrate to gaseous nitrogen in the absence of denitrifiers. Simultaneous anammox and nitrification can occur at a low DO level. The anammox mechanism was in detail explained by Kartal et al. [52]. In the anammox process nitrate is the end product, whereas nitric oxide and hydrazine are the reactions intermediate. Though there is a lot to explore about involved protein. They proposed that the protein of annammoxosome membrane or within anammoxosome is

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involved in NirS-dependent nitrite and Nar enzyme-dependent transform of other nitrogenous compounds into nitric oxide and nitrate, respectively. Nitrate production is essential to keep the electron balance through the production or consumption of extra electrons [53].

5.4 Molecular methods in detection of AOA Given the advantages of culture-dependent techniques, such as cheap cost and the flexibility to integrate with other approaches, the availability of culture-based methods for environmental microbe studies drastically decreases research on microbial community structure in environmental conditions. Many of the restrictions involved with isolating and detecting AOA from natural and man-made systems have been overcome by modern molecular approaches. Information on the genes that encode enzymes engaged in nitrogen transformations is helpful not just for determining gene expression as well as for determining the variety of microorganisms participating in the global N cycle.

5.4.1 Immunofluorescence technology Fluorescent polyclonal antibodies (FPA) that recognize AMO function as functional immunofluorescences to detect ammonia-oxidizing microorganisms [54]. The availability of pure cultures is critical to FPA manufacturing. FPA was modified for species-specific counting of several Nitrosomonas spp supported by affiliated and suspended microorganisms [55].

5.4.2 Phylogenetic technology To describe the phylogenetic and functional diversity of microbial communities, culture-independent molecular techniques based on 16S rRNA genes and total DNA sequencing (metagenomic sequencing) have been developed. A metagenomic technique can help detect the microbiome structure of diverse settings, such as sludge and wastewater samples. Recently, integrated “omics” analyses have improved our understanding of the species and their roles in wastewater microbial communities [56]. High-throughput Illumina sequencing technique is in use for such analysis of microbial community that provides a better understanding of the underlying microbial consortia.

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5.4.2.1 PCR-based technology Amplification of conserved sequences through PCR is well-established method for the identification of microbial members of that specific natural habitat. With not being limited to that, microbial functions and behaviors can be determined with the amplification of responsible genes with a certain set of primers. Modification and advancement in PCR technology have given rise to other technologies where the expression of the genes can be measured at any certain point through quantitative PCR (q-PCR). DGGE is a molecular fingerprinting approach for determining microbial composition by separating PCR-generated DNA fragments. PCR products are exposed to increasing quantities of chemical denaturant as they move along a polyacrylamide gel during DGGE. Different DNA sequences (from varying microorganisms) denature at different denaturant amounts, resulting in a band pattern. Each band is thought to reflect a separate microbial population in the ecosystem. DGGE patterns, for example, show that nitrogen, phosphate, potassium, and organic matter fertilization leads to considerable alterations in the AOB community, not the AOA community. The combination of DGGE and real-time PCR (q-PCR) assesses the abundance of AOB and AOA under different fertilization procedures, as well as describes microbial community diversity, composition, and population movements. 5.4.2.2 Fluorescent in situ hybridization (FISH) Several molecular studies to detect the ammonia-oxidizing group of the β-proteobacteria subclass have been carried out, however, there are minimal FISH applications for estimating the quantity of nitrifying microorganisms in ecosystems. Newly developed techniques such as Raman-FISH, MAR-FISH, SIMS-FISH, stable isotope probing, and isotope array are employed to understand the mechanism of ammonia oxidation. Though the presence of AOBs in the consortium of WWTPs makes it harder to identify the role of AOAs [5]. Until recently, there has been minimal research on the ammonia-oxidizing group of Proteobacteria, which might be particularly useful for quantifying AOA and AOB groups in aquatic samples. Microarray technology is regarded as a high-throughput platform and useful tool for estimating potential candidate proteins and genes. DNA microarrays are widely used to study gene expression [57].

5.4.3 Omics technology As of lack of knowledge of variables that regulate microbial communities’ growth metabolism limits the application of traditional molecular methods. Advanced omics technology, which includes proteomics, genomics, metabolomics, and transcriptomics is gaining significant importance recently to understand the community functionality and diversity. These methods have accelerated the research over culture-based traditional

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methods. Omics technology helps the researcher to look into proteins, RNA, DNA, and metabolites from a total community as well as from single species. Generally, multiple omics approaches are used at a time to understand the underlying mechanism [58, 59].

5.5 Factors affecting ammonia-oxidizing archaea As revealed by the culture-based study of pure AOA, ammonia and oxygen plays as the electron donor and acceptor. Therefore, their concentration is a crucial parameter that determines the abundance and community structure of the AEA. On the contrary, both AOB and AOA coutilize these substrates and compete for them.

5.5.1 Ammonia Analysis of growth parameters by Limpiyakorn et al. [5] in WWTPs showed that both AOA and AOB have the maximum specific growth rate (μmax) of a similar range, but AOB has a higher affinity for ammonia (Ks). Thus, AOA thrives in ammonia-limiting conditions, whereas with ammonia concentration near saturation both AOA and AOB can co-dominate. Furthermore, at certain higher ammonia concentrations, the growth of many AOAs was reported to be declined. It is also supported by the abundance of amoA gene of AOA and AOBs in the wastewater. The determination of the nonparametric correlation coefficient of Spearman’s rank demonstrated that the amoA gene from AEAs negatively correlated with effluent wastewater’s ammonium level.

5.5.2 Oxygen AOA has slightly less Ks for oxygen than AOBs. An extremely low dissolved oxygen in the system might favor AEA growth, as amoA from AEA was found in the oxic–anoxic mode-operated activated sludge bioreactor [39]. Though, in contrast, the Spearman’s correlation coefficient has also shown no correlation among the amoA from AEA and dissolved oxygen [5].

5.5.3 pH and temperature The pH and temperature affect heterotrophic microbial growth. Though the optimal pH of denitrifying enzymes varies, thus affects the nitrate and nitrite reductase activity causing nitrite buildup. With the change in temperature, nitrite reductase is more susceptible to being affected than nitrate reductase, thus causing a decline in activity and accumulation of NO2–N in the system. The pH of the system affects the concentration

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of NH4+-N and NO2−-N, therefore, affecting the anammox reaction. Temperature more than 45 °C causes an irrecoverable decrease of anammox activity and lower than 15 °C causes unstable nitrogen removal [60]. The bioavailability of ammonia decreases through protonation at lower pH, therefore favoring AOAs growth over AOBs. While AOB struggles to survive at low acidic pH or high alkaline pH, AOAs can thrive easily. Therefore, they are suitable for application in WWTPs with acidic influent. AOAs adaptability to changed pH also aids it [61]. The temperature effect in ammonia oxidation is visible through the activity of AMO. AOAs vary in terms of temperature tolerance. Ammonia oxidation through AOA can be observed in deep seawater with temperatures as low as 0.2 °C to a hot spring of 74 °C in Yellowstone National Park [61]. Niu et al. revealed that AOB’s amoA gene abundance in biological active filtration changes significantly from summer to winter, whereas a slight change in AOA’s amoA was reported [62].

5.5.4 Carbon source and COD to NO3−-N (C/N) ratio The denitrification process is recorded to be affected by the type of carbon source because enzymatic activities are stimulated differently by changed electron donor [63]. Not only limited to these aforementioned factors but the process can also be affected by the type of reactor in use, the operation mode of the reactor, and the accumulation of toxic elements during operation are several among others that also affect the process [1].

6 Conclusion and future aspect The discovery of AOA defies the long-held belief that ammonia oxidation is solely carried out by AOB, contributing to a better understanding of the entire nitrogen cycle. AOA seems to be significant in nitrogen reduction from wastewater. As a result, the nitrogen cycle in a wastewater treatment system requires reconsideration because of the microbial community complexity, and inter- and/or intra-competitiveness at the species and community level. Advanced molecular methods have shown tremendous potential in archaeal research because of otherwise difficult culturing practices. As AOA’s better environmental flexibility than AOB’s and as microbial ammonia oxidation is impacted by multiple environmental factors more innovations in archaeal ammonia oxidation regimes are expected. Innovative efficient tools and technology have been evolving to feed the need of time and thus helping better understanding of the neglected biological factor of WWTPs and environment. It is of the need of this decade to exploit the archaeal repertoire to achieve the sustainable goals. And a better understanding of functional

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diversity and distribution of AOAs in WWTPs should be addressed in future studies so that they can be used for sustainable management of WWTPs. Moreover, sufficient consideration needs to be paid to cultivating more environmentally relevant model organisms with more traceability. More research is needed to completely understand archeal community diversity, distribution, and functioning, which may be utilized to design more effective techniques for WWTP management and the reduction of negative environmental consequences. Further research should concentrate on evaluating unclassified species from eubacterial and archaeal communities found in wastewater treatment facilities and natural extreme environments.

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Bhaskar Deka, Nikita Chakraborty, and Bhisma Kumar Patel✶

3 Significant role of nitrogen cycle in wastewater treatment Abstract: Reactive nitrogen compounds such as NH3, N2O, and NO3− are the major pollutants of wastewater due to their effect on the oxygen contents of water and their role in eutrophication. Excessive reactive nitrogen in the environment has the potential to cause a deleterious effect on our ecosystem. The reactive forms of nitrogen are mainly released into the environment by the wastewater treatment plants. Particular types of microorganisms play an essential role during nitrogen cycling in the removal of reactive nitrogen from wastewaters. In nitrifying and denitrifying processes, ammonia is converted to nitrate, and nitrate is converted to nonreactive dinitrogen gas by ammoniaoxidizing and nitrite-oxidizing bacteria. Ammonia is converted to nitrogen gas by using nitric oxide as an oxidant by anaerobic ammonia-oxidizing bacteria or anammox. The operational cost of this process is very low and is environmentally friendly.

1 Introduction Nitrogen is an indispensable building block for all forms of life. It is considered one of the most important elements for all plants and animals to survive. Nitrogen comprises an essential part of nucleic and amino acids, which make up an integral part of human and animal tissues, hormones, and enzymes. It plays a significant role in biological processes such as carbon and nitrogen metabolism, photosynthesis, and protein production [1, 2]. Atmospheric gases contain about ~78% of molecular nitrogen (N2) in our planet. The presence of a triple bond between the two nitrogen atoms in N2 makes it highly stable. This high stability of molecular nitrogen makes it unuseful for most organisms, and hence this nonreactive form must be converted to other reactive and useful forms. In this context, both biological and nonbiological processes play a vital role in converting unreactive nitrogen to reactive nitrogen. In biological processes, diazotrophs, a group of prokaryotes, help in converting N2 to NH3. Most of these prokaryotes are present as a symbiotic partner with plants and animals, and others are found in the free aquatic and terrestrial environment [3, 4]. Besides biological processes, lightening, industrial fixation, combustion, and biomass burning are some of the non-biological methods for fixation of nitrogen. ✶

Corresponding author: Bhisma Kumar Patel, Department of Chemistry, Indian Institute of Technology Guwahati, North Guwahati 781039, Assam, India, e-mail: [email protected] Bhaskar Deka and Nikita Chakraborty, Department of Chemistry, Indian Institute of Technology Guwahati, North Guwahati 781039, Assam, India https://doi.org/10.1515/9783110780093-003

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Lightning possesses sufficient energy required to break the nitrogen–nitrogen bond of N2. In the presence of atmospheric oxygen, lightning breaks the bond between N2 and forms NO2 (nitrogen dioxide) and HNO3 (nitric acid). Every day 10,000 tons of nitrate are produced by lightning and reach the surface through water droplets of rain. Then, this reactive form of nitrogen is utilized by the organisms. Industrial fixation of atmospheric nitrogen into fertilizers and valuable chemicals is important for agriculture. High temperature and pressure are used to produce ammonia from atmospheric nitrogen and hydrogen in these fertilizer plants. Though the Haber–Bosch process is used frequently by many industries for nitrogen fixation, it has some associated disadvantages. This arises the search for alternative ways of nitrogen fixation such as plasma synthesis and application of metal-complex catalyst. Moreover, the burning of biomass and cleaning of grassland and forests also release reactive forms of nitrogen into the environment. In some plants, the roots take up the reactive form of nitrogen and facilitate them to enter the food chain. However, most plants cannot utilize the ammonia form directly and demand the need to convert it into nitrate form. In such cases, a group of microorganisms called nitrifying bacteria helps to convert ammonia to nitrate. In this nitrification process, two types of aerobic bacteria are essential. Some fermentative microorganism helps in the mineralization process by converting organic nitrogen to ammonia and helps in the entry of reactive inorganic nitrogen back into the nitrogen cycle. The process of conversion of reactive nitrogen into molecular nitrogen is called denitrification or anammox. The nitrogen cycle is a biogeochemical cycle in which nitrogen is converted to other chemical forms including nitrates, nitrites, ammonia, etc. Preliminary nitrogen fixation processes include fixation, ammonification, nitrification, and denitrification. The nitrogen cycle is of immense importance to maintain ecological balance as it balances the nitrogen content in the environment. The importance of the nitrogen cycle is as follows: 1. The nitrogen compounds play a vital role in the synthesis of chlorophyll, which is an essential pigment for photosynthesis. 2. Nitrates are the major components in the nitrogen cycle and are very essential for the growth of plants. 3. During the process of ammonification, bacteria decompose dead and decayed organic matter to cleanse the environment and provide essential nutrients required by the soils. 4. Crucial components of living organisms such as proteins and nucleic acids are made of nitrogen. The main processes involved in the nitrogen cycle are presented in Figure 1.

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N2 R-NH2

Assimilation Mineralization

NH3/NH4+

N2H4

N2O

NO

NH2OH

NO2– NO3– Autotrophic nitrifying bacteria AOB

NOB

AMO

NH4+/NH3

NH2OH

HAO

O2

NO2–

NO3–

COMAMMOX Heterotrophic denitrifying bacteria

NO3–

Nitrate reductase

NO2–

–

NO3

Nitrite

NO2–

reductase

Nitrite Oxide reductase

Anammox bacteria NH4+/NH3 HH N2H2 NO

Nitrate reductase

Nitrate reductase

NO

N2 O

Nitrous oxide reductase

N2

HZO

Assimilation by microalgae Nitrite NO2 NH4+/NH3 reductase –

N2

Assimilation

Nitrogenase N2 Figure 1: The biological process of the nitrogen cycle.

1.1 Effects of excess reactive nitrogen in our environment There is no denying that the presence of reactive nitrogen is essential for the ecosystem. However, excess amounts of reactive nitrogen contribute to some serious environmental problems. Both human inventions and natural processes produce reactive nitrogen. Over the years, the increase in population and industries has tremendously contributed toward the release of reactive nitrogen forms in the environment, resulting in the

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Bhaskar Deka, Nikita Chakraborty, and Bhisma Kumar Patel

degradation of land, freshwater, and coastal zones. The industries associated with crop production contribute a significant amount of reactive nitrogen to the environment. Moreover, the combustion of fossil fuels, burning of biomass, and domestic sewage also play a substantial role in adding up reactive nitrogen in the environment. The reactive form of nitrogen can easily commute between air, water, and soils. This process of transportation is called a nitrogen cascade (Figure 2). Due to the nitrogen cascade process, a single molecule of nitrogen can contribute to severe effects on the environment. Excess nitrogen in the air leads to higher levels of ozone in the lower atmosphere, leading to respiratory diseases and vegetation damage. With acid deposition, reactive nitrogen can easily reach the earth’s surface and can have detrimental effects on different life forms. It also influences the ecosystem health and biodiversity by acidification of soil, uncontrol fertilization of trees and grasslands, increasing the unnatural growth rates and nutrient imbalance. From the ground, reactive nitrogen can easily pollute the groundwater and surface water which enhances the eutrophication in the coastal ecosystem and ultimately causes an imbalance in the biodiversity. The effects also include corrosion of bridges, buildings, and human-made structures. Excess reactive nitrogen in the environment causes a potential threat to human life. A high nitrate concentration in drinking water causes methemoglobinemia, commonly known as “blue baby syndrome.” To prevent this, the World Health Organization (WHO) has adopted standards for acceptable nitrate levels in drinking water (45 mg nitrate per liter). Nitrogen in the form of NOx boosts the formation of ozone and smog, which interact with the lungs, and long-term exposure can lead to asthma or even death. Moreover, reactive nitrogen is also responsible for many cardiopulmonary diseases and several types of cancers in humans.

2 Nitrification The sequential conversion of ammonium to nitrite and nitrate by a group of organisms is called nitrification. Nitrification is one of the essential processes in the nitrogen cycle in wastewater treatment plants. Nitrification is a two-step microbiological process. The first step in nitrification is the conversion of ammonium (NH4+) to nitrite (NO2−) in the presence of ammonium-oxidizing bacteria (AOB) [5]. In the next step, the nitrite form is quickly oxidized by the nitrite-oxidizing bacteria (NOB) to nitrate (NO3−). In nitrification, the first step is the most important and the rate-limiting step [6]. Nitrification is also known as a chemoautotrophic process, as the chemical energy released during the process is trapped by the bacteria. AOB are the driving force of this process. Various organisms such as autotrophic AOB, ammonia-oxidizing archaea (AOA), heterotrophic AOB, anaerobic AOB or anammox, NOB, and complete ammonia oxidizers or comammox are involved in the nitrification process.

Nr

Nr

Aquatic eutrophication

Natural ecosystems Nitrate in Stream waters

Soil acidification

Terrestrial eutrophication

Further emission of NOx and N2O carrying on the cascade

Ammonium nitrate in rain (NH4NO3)

Particulate matter and tropospheric ozone

Ammonia (NH3)

Livestock farming for food

Nr

Nitrous oxides (NOx)

Greenhouse gas balance

Leached nitrate (NO3-)

Nr in manure

Crops for food and animal feed

Nitrous oxide (N2O)

Figure 2: Nitrogen cascade diagram.

Crop biological nitrogen fixation

Fertilizer manufacture

High temperature combustion and industry

Atmospheric N2 fixed to reactive nitrogen (Nr)

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Bhaskar Deka, Nikita Chakraborty, and Bhisma Kumar Patel

The type of bacteria involved in nitrogen fixation is highly sensitive to acidity (pH). The rate of nitrification at pH less than 5.5 is very slow. Plants that grow in acidic medium use ammonium as the source of nitrogen nutrition due to the unavailability of nitrate form. The bacteria that are involved in nitrification are very sensitive to high temperatures. Nitrifying bacteria become inactive at temperatures above 140 °C, which results in slow nitrification.

2.1 Autotrophic ammonia-oxidizing bacteria Autotrophic AOB are involved in the first step of nitrification. This type of bacteria is cultivated in marine and thermal spring environments. Oxidation of ammonia by autotrophic AOB is associated with two key enzymes, an ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO) [7]. AMO helps to oxidize ammonia to hydroxylamine (Scheme 1a). It is composed of three subunits AmoA, AmoB, and AmoC. Hydroxylamine dehydrogenase (HAO) oxidizes hydroxylamine to NO2− (Scheme 1b). HAO is also made up of three identical subunits which contain eight c-type heme groups [8]. Out of all these eight, seven heme groups are typical c-type heme, and only one is P460-type heme.

Scheme 1: Oxidation of ammonia in presence of AMO and HAO.

The four electrons liberated during the oxidation process of NH2OH to NO2 are transferred from HAO to a ubiquinone pool through cytochrome CycA and cm552. Two of these four electrons are scattered in cytochrome oxidase, which are used to drive NADH production or generate a proton motive force (PMF) and the other two electrons are transferred back to AMO [9]. Autotrophic AOB can be divided into three categories: Nitrosomonas, Nitrosospira, and Nitrosococcus [8]. AOBs are found in natural aerobic environments such as freshwater, marine ecosystems, and soils, low-oxygen environments such as brackish water, and subsurface sediments [10]. AOBs can also be found in ammonium-rich environments which are subjected to anthropomorphic nitrogen sources such as industrial by-products, fertilizers, and wastewaters [6]. Some methanotrophic bacterial species can oxidize ammonia to nitrite by using the enzyme (particulate methane monooxygenase, pMMO) that is involved in methane oxidation [11]. The binding ability of pMMO with CH4 and NH3 is almost similar. Ammonia suppresses oxidation of methane by competing with methane at the pMMO active site [12].

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In various environments such as pristine, contaminated soils, and wastewaters, methanotrophic bacteria play an important role in the nitrification process [13].

2.2 Autotrophic ammonia-oxidizing archaea Autotrophic AOA are chemolithoautotrophic in nature. Most of the AOAs are not fully characterized. They are extremely difficult to obtain in pure form. AOA seems to be the dominant ammonia-oxidizing organism due to its high affinity for substrate at a low concentration of NH3. AOA is also capable of growing at low oxygen concentration, low pH, and at very high temperatures [14]. The high concentration of ammonia is not suitable for the growth of AOA. So, AOB is often found with ammonium oxidation in agriculture soils, fish aquaria, and wastewater treatment plants [15, 16]. The pathways used by AOA for oxidation of ammonia to nitrite are different from the ones that are used by bacteria. Apart from AmoA, AmoB, and AmoC, another AMO protein (AmoX) is involved in the case of archaea oxidation [17]. Moreover, many of the proteins from the bacterial ammonia oxidation pathway are not found in AOA [18]. Though HAO is not present in AOA, hydroxylamine is found to be an intermediate in Nitrososphaera maritimus [19]. The oxidation process of hydroxylamine to nitrite takes place in the (pseudo)periplasmic space [20]. Oxidation of hydroxylamine by AOA produces a proton gradient which drives ATP synthesis, and a significant amount of nitric oxide (NO) is also produced. Moreover, in this process, the production of cofactor F420 is also enhanced which could be responsible for the detoxification of reactive nitrogen species [21]. Till date, around 10 different genera of AOA have been identified in diverse environments (Table 1). Culture studies have been conducted in some of these organisms including Nitrosopumilus maritimus, Nitrosotalea devanaterra, and Nitrosocaldus yellowstonii.

2.3 Heterotrophic ammonia oxidizer Unlike chemolithoautotrophic ammonia oxidation where energy is released, heterotrophic ammonia oxidation does not produce energy [30]. The rate of heterotrophic nitrification is slower as compared to the autotrophic nitrification process. Nevertheless, heterotrophic ammonia oxidizer plays a vital role in the global nitrogen cycle due to their large populations in a variety of environment. A variety of bacteria from phyla contributes to nitrogen cycling in soils, wastewater treatment plants and aquaculture systems owing to their capability for heterotrophic ammonia oxidation [31]. The mechanism of ammonia oxidation by heterotrophic ammonia oxidizer is not fully characterized. It is assumed that the oxidation process is analogous to autotrophic nitrification. In the first step, NH3 is oxidized to hydroxylamine in the presence of AMO from Paracoccus denitrificans followed by hydroxylamine oxidation to

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Table 1: List of autotrophic ammonia-oxidizing bacteria and autotrophic ammonia-oxidizing archaea detected in wastewater. Species Autotrophic ammonia-oxidizing bacteria

Autotrophic ammonia-oxidizing archaea

References

Nitrosomonas europaea

[]

Nitrosomonas eutropha

[]

Nitrosomonas nitrosa

[]

Nitrosomonas oligotropha

[]

Nitrosomonas stercoris

[]

Candidatus Nitrosocosmicus exaquare

[]

Candidatus nitrosotenuis aquarius

[]

Candidatus nitrosotenuis cloacae

[]

Nitrosopumulis maritimus

[]

NO2− by hydroxylamine quinone reductase model. Various studies reveal that AMO is made up of two subunits (AmoA and AmoB) and they are activated by cupric ions [32]. AmoA subunit is present in Pa. denitrificans genome but the homolog for AmoB is unclear. Hydroxylamine is formed as an intermediate during heterotrophic nitrification process by Pa. denitrificans. Another probable change is that hydroxylamine is formed by nitrite reductase. It can reduce hydroxylamine to NH3. Studies have identified that Paracoccus also contain monomeric hydroxylamine oxidase protein (HAO) [33]. The genes coding of HAO and AMO are also found in the same chromosomal region. There is another enzyme known as pyruvic oxime dioxygenase (POD) which catalyzes nitrite formation from hydroxylamine [34]. First, hydroxylamine and pyruvate react to form pyruvic oxime and then pyruvic oxime is oxidized to nitrite and pyruvate in the presence of POD [35]. Nitrite reductase (NirBD) is also capable of converting ammonia to nitrite. POD and NirBD both are cytoplasmic proteins. So, ammonia and hydroxylamine have to be transferred into the cytoplasm. The genome of Pa. denitrificans contains a gene which is coded for ammonium transported protein into the cytoplasm. Heterotrophic nitrifiers involve another respiratory nitrate reductase (NarGHI), one of the bound molybdopterin oxidoreductase proteins [36]. The active site of Nar is also present in the cytoplasm. Therefore, NH3 has to be transported to the cytoplasm before the oxidation process.

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2.4 Autotrophic nitrite-oxidizing bacteria Chemolithoautotrophic NOB are present in large quantities in freshwater and marine ecosystems. This class of bacteria plays a vital role in the nitrogen cycling process [37]. Researchers have reported that 88% of the nitrate in oceans is contributed by chemolithoautotrophic NOB [38]. NOB is considered a critical component in many wastewater treatment plants. The energy released from the oxidation of nitrite to nitrate is consumed by chemolithoautotrophic NOB in nitrification. The oxidation reaction proceeds as shown in Scheme 2 [39].

Scheme 2: Oxidation of nitrite in the presence of NOB.

Nitrite oxidoreductase (Nxr), an iron–sulfur molybdoprotein, is involved in the first part of autotrophic nitrite oxidation. It is made up of three subunits known as NxrA, NxrB, and NxrC [40]. Nxr protein is divided into two types; one is detected in periplasmic space and another is in cytoplasm. These two Nxr proteins have some similarities with the DMSO reductase family protein. Periplasmic Nxr is present in Nitrospira and Nitrospina species and cytoplasmic Nxr is present in Nitrobacter [41]. In the case of periplasmic Nxr, protons gained from water contribute to PMF and in ATP synthesis [42]. But in cytoplasmic Nxr, protons have no contribution toward the PMF. For cytoplasmic Nxr, nitrite transported protein facilitates the transfer of nitrite across cytoplasmic membrane [43]. In the oxidation process of NO2 by Nxr oxidizers, two electrons are released which helps in the reduction of O2 to H2O. Cytochrome c550 proteins help to move the electron to cytochrome oxidase in aa3-type cytoplasmic Nxr, and in the case of periplasmic Nxr, electrons are transported to c-type cytochrome.

3 Denitrification A large number of microorganisms reduce the nitrate and nitrite forms present in the environment to gaseous forms of nitrogen (N2 and N2O). This process is carried out anaerobically and is termed as denitrification. The conversion from nitrate to dinitrogen is preceded by denitrifying bacteria in the following sequence: NO3– → NO2–→ NO → N2O → N2. This process is universal to terrestrial and aquatic ecosystems and is found in tropical and temperate soils, marine and freshwater environments, natural and intensively managed ecosystems, wastewater treatment plants, manure stores, and aquifers. It is an alternative for the treatment of wastewater that removes nitrate (NO3)

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but limits its efficiency by removing valuable nitrogen fertilizer from the soil and releasing the greenhouse gas N2O and the tropospheric pollutant NO. Denitrification is the only biotransformation process that removes nitrogen from the ecosystem, which otherwise cannot be reconverted due to its gaseous form. This process is restricted to specific environments, which are devoid of any oxygen. It is considered one of the most amplified metabolic pathways for the removal of nitrogen from the ecosystem [44]. It is the process that converts fixed nitrogen (i.e., nitrate and nitrite) to the inert N2 form. A variety of microorganisms are involved in the denitrification process. A large number of heterotrophic microorganisms are involved as denitrifiers in the heterotrophic denitrification process. The growth of heterotrophs is dependent on organic carbon. A huge number of denitrifiers are present in both marine and terrestrial ecosystems in all climatic zones. Most of the denitrifiers are considered facultative aerobes. These facultative aerobes are those species that gain energy by aerobic respiration. Though these types of bacteria require O2 as an energy source but they can also survive in a nitrogen environment when O2 exhausts. The enzymes that are used for denitrification get activated when the partial pressure of oxygen is low. In most cases, the final product of the denitrification process is N2. However, in some cases, N2O and NO are produced as the final product depending upon the environmental conditions and organism type. Similarly, NO3− is not always the terminal electron acceptor. Other intermediates are also used as the terminal electron acceptor. Bacteria such as Alcaligenes faecalis, Paracoccus denitrificans, and Pseudomonas stutzeri grow by using N2O as the electron acceptor. Some of the denitrifiers cannot complete the denitrification process by reducing nitrate to N2. In wastewater treatment, denitrifying bacteria accomplish the complete denitrification process. Those microorganisms which complete the reduction process of nitrites and nitrates to nitrogen gas are called true denitrifiers. Among the true denitrifiers, some of them are more effective than the others. Their effectiveness depends upon the activity of nitrate and nitrite reductases. Incomplete denitrifying microorganism only reduces nitrates to nitrites without any further reduction due to the absence of nitrite reductase enzymes. Denitrification can also be carried out by chemosynthetic organisms. In the case of chemosynthetic denitrifiers, inorganic compounds are used as a source of energy in nitrate reduction [44]. On the other hand, autotrophic denitrifiers use carbon dioxide or bicarbonate as the source of carbon. This type of nitrifier utilizes hydrogen, sulfur, sulfide, Fe(II), and so on as a source of electron [45].

4 Anammox (anaerobic ammonium oxidation) Anammox is another potential metabolism that is used in wastewater treatment plants. In this process, ammonia and nitrite are directly converted to molecular

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nitrogen and water. This process is considered as one of the most efficient pathways in wastewater treatment plants. However, its large-scale usage turns out to be complex in the field of design, operation, and maintenance. Anammox was first discovered in the early nineties in waste-water sludge. The discovery involved the treatment of ammonium-rich effluent from a methanogenic reactor in a fluidized bed reactor [46]. Using nitrite as the oxidant, the bacteria capable of working in this process oxidizes ammonia and resulting in molecular nitrogen (Scheme 3) [47].

Scheme 3: Nitrite as an oxidant in the presence of anammox.

Mostly, reactor systems follow a two-step process for biological nitrogen removal from wastewater. In contrast, anammox bacteria have the power of converting reactive nitrogen to nonreactive nitrogen in a single reactor which shortens the overall removal process. This process leads to 90% reduction of the operational costs [48]. To date, around 19 species of anammox bacteria are known [48]. These anammox bacteria are present in different anoxic ecosystems such as marine, freshwater, brackish, terrestrial environment, and hydrothermal vents. Anammox bacteria contribute up to 70% of N2 to the environment annually [44]. All the anammox bacteria belong to the bacterial phylum planctomycetes. The cell structure of planctomycetes contains intracytoplasmic membranes to compartmentalize the cell. This feature makes the cell structure of planctomycetes differ from other bacteria. In most of the anammox bacteria, three membrane system is present [47]. An outermost membrane, thin layer of peptidoglycan, is present in anammox bacteria and a second membrane layer is present surrounding the cytoplasm. The third membrane layer of anammox bacteria covers a large vacuolar cell organelle (anammosome) which is unique compared to other bacteria [49]. The anammoxsome membrane is less permeable than normal biomembranes due to the presence of ladderane lipids, but the first two membranes have similar characteristics to most biomembranes. Most of the enzymes that are accommodated in the anammoxosome and ammonium have to be transported into the organelle to oxidize the anammox process [50]. The whole process of anammox is completed in three steps [47]. In the first step, nitrite reductase (NirS) reduces NO2 into NO. Next, NH3 and NO form a highly reactive and volatile hydrazine intermediate by the hydrazine synthase. In the last step, hydrazine dehydrogenase oxidizes the hydrazine intermediate to N2 and releases four electrons. These four electrons provide energy in the early steps of the anammox process. The protons accumulated in the anammoxosome generate a PMF in the membrane of anammoxosome which drives the ATP synthesis. Table 2 lists all the anammox bacteria that are found in wastewater environment.

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Table 2: List of anammox bacteria found in a wastewater environment. Organism

Environment

Candidatus anammoxoglobus propionicus Candidatus Brocadia anammoxidants Candidatus Brocadia brasiliensis Candidatus Brocadia sinica Candidatus Jettenia moscovienalis Candidatus Jettenia caeni Candidatus Scalindua brodae Candidatus Scalindua wagneri

Bioreactor Bioreactor Municipal activated sludge Anammox reactor Semi-industrial bioreactor Membrane reactor Wastewater treatment plant Wastewater treatment plant

References [] [] [] [] [] [] [] []

5 Biological processes for removal of nitrogen Generally, wastewater treatment plants carry out several steps before releasing the wastewater into the environment. First, solids are separated and sludge is formed from wastewater after the removal of 50% solids. Next, sludge undergoes a biological and abiotic oxidation process. Three different treatment processes (filtration, aeration, and oxidation) are applied by wastewater treatment plants. Out of three, in the aeration process microorganisms are activated that help to remove phosphates and nitrogen present in water. Till now, there are many technologies and processes that have been developed and applied to remove nitrogen from wastewater [57]. Scientists are trying to improve the technologies to enhance the nitrification and denitrification processes in wastewater treatment plants. Herein through this chapter, we shall discuss a few of them.

5.1 Traditional biological technologies There are many traditional biological nitrogen removal processes such as bardenpho, predenitrification, postdenitrification, SBR, OD, etc. Among them, predenitrification process is more advantageous as compared to others such as short aerobic duration and more nitrogen removal efficiency. The conventional ways of the nitrogen removal process mainly depend on autotrophic nitrification and heterotrophic denitrification. First, ammonium is oxidized to nitrite by AOB via the formation of hydroxylamine. In these processes, an AMO and hydroxylamine oxidoreductase (HAO) are involved. Next, nitrite is oxidized to nitrate by the NOB with the involvement of membranebound nitrite oxidoreductase (Nxr) (Scheme 4).

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Scheme 4: Mechanism of the conventional ways of the nitrogen removal process.

In anoxic denitrification, various electron donors like methanol, acetate, and organic compounds present in wastewater reduce NO3– and NO2− to the molecular form of nitrogen (Scheme 5).

Scheme 5: Reduction of NO3− and NO2− to the molecular form of nitrogen.

Though many countries are still using the conventional nitrogen removal processes, but these processes have some limitations. In the nitrification and denitrification process, different microorganisms are involved, so they have to be designed and operated separately. Hence, a long retention time and a huge amount of wastewater are required to complete the nitrogen removal process cost-effectively. In nitrification and denitrification processes, high level of oxygen and carbon source is required [58]. Sometimes, an external carbon source must be added if the nitrogen concentration in wastewater is high, thereby increasing the operational cost.

5.2 Novel biological nitrogen removal processes 5.2.1 Simultaneous nitrification and denitrification (SND) In the simultaneous nitrification and denitrification process, both nitrification and denitrification take place in one compartment for the complete removal of nitrogen from wastewater [59]. Due to its highly effective and inexpensive nature, it is used frequently by wastewater treatment plants. SND has several advantages over conventional processes. The simultaneous occurrence of denitrification and nitrification in a single compartment reduces the cost and simplifies the overall process. There are two mechanisms for simultaneous nitrification and denitrification (SND). One is physical and the other one is a biological mechanism. In physical mechanism, SND occurs when dissolved oxygen (DO) concentration drops due to the diffusional limitation within the activated sludge flocs or biofilms. The nitrifiers in aerobic regions stay alive when dissolved oxygen (DO) concentration is 1–2 mg/L or higher.

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Bhaskar Deka, Nikita Chakraborty, and Bhisma Kumar Patel

But the denitrifiers present in anoxic region with less than 0.5 mg/L concentration of dissolved oxygen (DO). Microelectrode measurement and 15 N tracer techniques verify the oxygen concentration gradients in activated sludge flocs [60]. On the other hand, the biological SND is complicated and contradictory to autotrophic aerobic nitrification and heterotrophic anoxic denitrification process. Heterotrophic nitrifiers such as Alcaligenes sp., Acinetobacter sp., Coryne bacterium, Xanthomonas sp. Bacillus strains etc. are identified in wastewater and night soil treatment systems [61]. Thiosphaera pantotropha is identified both as an nitrifier as well as an denitrifier. Aerobic denitrificans such as Paracoccus denitrificans, Microvirgula, Thaurea mechernichensis, etc. are present in wastewater. SND is considered as the series of oxidation of ammonia and reduction of nitrate or nitrite in presence of heterotrophic nitrifiers and aerobic denitrifiers [62]. In SND process, various parameters like hydraulic retention time (HRT), sludge retention time (SRT), and pH have some major effects on the efficiency of the process. The growth of heterotrophic nitrifiers are rapid and has high tolerance capacity for acidity, therefore short SRT and acidic medium is favorable for the process. The efficiency of nitrogen removal in SND processes decreases with an increase in ammonium-loading rates. The major factors that influence the SND processes are the source of carbon, dissolved oxygen concentration, and floc size [60]. High biological oxygen demand (BOD) inhibits the capacity of autotrophic nitrifiers and a low concentration of BOD causes the deficiency of electron donor of denitrifiers. The BOD level of around 110–150 mg/L is perfect for the treatment of municipal wastewater. On the other hand, the dissolved oxygen concentration is very essential for SND process. It shows a two-fold effect. At low DO concentration, the rates of nitrification process are slow, and high DO concentration discourage the denitrification process. At around 0.5 mg/L, the rate of the nitrification and denitrification is similar and at this condition the SND is successful. Another important factor of SND is the size of the floc. A large size above 125 μm is preferable for SND processes due to the oxygen diffusion limitation.

5.2.2 Shortcut nitrification and denitrification The shortcut nitrification and denitrification process is also known as partial nitrification-denitrification. Complete nitrification-denitrification needs high energy and pH has to be maintained in the reactors for which alkaline compounds are needed to be added. In denitrification processes of complete nitrification-denitrification, electron donor organic compounds are required. But, in the case of shortcut nitrification and denitrification processes high energy and extra supplementations are not required. In this process, NO2- is formed as an intermediate after the oxidation of NH3 for which less energy is required for the aeration step. Next, the reduction of nitrite produces N2 which requires less electron donor compared to complete denitrification to nitrate.

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The shortcut nitrification and denitrification process is more favorable for wastewaters that contain high ammonium concentrations [63]. The shortcut nitrification and denitrification process perform better when AOB are present as dominant nitrifiers and NOB are absent in the system. The removal of NOB is very essential for this process because NOB convert partial nitrification to complete nitrification by oxidizing NO2– to NO3–. Factors such as dissolved oxygen concentration, temperature, and chemical inhibitor, affect the shortcut nitrification and denitrification process by inhibiting the NOB. In a low oxygen environment, AOB is generally present in higher quantity as compared to NOB. Hence, partial nitrification is better in low DO concentrations. Though a low concentration of dissolved oxygen is better for partial nitrification, but in low DO the rate of nitrification becomes slow. So, a perfect DO concentration of around 1.0–1.5 mg/L is needed to be maintained for shortcut nitrification and denitrification process [64]. Temperature is another factor that influences the rate of the partial nitrification process. At different temperatures, AOB and NOB have different growth rates. AOB has a higher growth rate compared to NOB at 20 °C. Below this temperature, NOB dominates AOB. For successful removal of nitrogen by the shortcut nitrification and denitrification process, wastewaters must be treated by keeping the concentrations of free ammonia and free nitrous acid at a high level in the reactor. Sometimes the presence of metals such as Ag, Ni, Zn, Cu, Hg, and Pd in wastewater suppresses NOB and enhances the partial nitrification process. This process can be very advantageous if the above factors are controlled properly. The major advantages of this process over traditional nitrification and denitrification are 1. Consumption of oxygen in the aerobic phase and requirements of electron donor in the anoxic phase is low. 2. Denitrification of NO2- is 1.5 to 2 times higher than NO3-. One of the famous shortcut nitrification and denitrification process is the single reactor system for the high ammonia over nitrite (SHARON) process. Initially, it was developed for removal of ammonia via the nitrite route [65]. Both autotrophic nitrification and heterotrophic denitrification occur in SHARON reactor. The SHARON reactor works perfectly at 35 °C, where AOB dominates NOB. It is dependent on temperature and thus it is not suitable for all wastewater systems. To enhance its effect, methanol is used in the reactor to maintain a neutral pH. The reactions of SHARON reactor are presented in Scheme 6.

Scheme 6: The reactions of SHARON reactor.

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Bhaskar Deka, Nikita Chakraborty, and Bhisma Kumar Patel

5.2.3 Anaerobic ammonium oxidation (anammox) systems The anaerobic ammonium oxidation process is a novel and very effective process used in wastewater treatment plants. It was first introduced in 1990s at Delft University of Technology [66]. It is considered one of the most cost-effective nitrogen removal methods. In this process, anaerobic AOB are used to oxidize ammonia to nitrogen. The main intermediate of this process is hydrazine and hydroxylamine. Here, an external source of carbon is not required as carbon dioxide acts as a carbon source for anaerobic AOB, and this in turn saves the chemical dosage cost. Moreover, the biomass yield is very low for anammox systems. These are some of the main advantages of this system. Two different strategies are used to supply anammox with nitrite. One is a single-step process and another is a two-step process. In the former process, nitrite is produced in the single-stage system, and in the two-step process, it is produced in a separate aerated reactor, and then it is supplied to anammox reactor. Though both these processes are successful but the onestep process is more cost-effective. The two-step process requires a higher cost but this process is more effective as it is more flexible and stable and can be controlled and optimized separately. The main drawback of this system is associated with the slower growth of the anammox bacteria. Sometimes nitrate and ammonia in anammox process create some problems at a concentration below 1,000 mg/L. Anammox system is not suitable in lowtemperature regions and with low concentrations of dissolved oxygen. Anammox bacteria are more effective at 26–28 °C [67]. Below 15 °C and above 40 °C the effectiveness of anammox bacteria drops rapidly. A comparison table of reactors involving different anammox is presented in Table 3. Table 3: Comparison of reactor involving anammox. Type

Single reactor system

Name

CANON

OLAND SBR

Biomass

Granular sludge

Rate (kgN/(md)) Ammonium conc. mg/L

SNAD

Aerobic denitrification

Two reactor system DEMON

NAS

SHARON anammox

Biofilm SuspenBiofilm Biofilm ded sludge

Suspended and granular sludge

SuspenGranular ded sludge sludge

.

.

.

.

.

.

.

.



,







,



,

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5.2.4 Complete nitrification-denitrification systems A complete nitrification-denitrification process is employed by municipal wastewater treatment plants for removal of reactive nitrogen from the water. In the first step, AOB and NOB oxidize ammonia to nitrate. Next, denitrification is done in an anoxic rector by anaerobic bacteria to form nitrogen gas. Mainly, sequencing batch reactor (SBR) and anoxic/oxic (A/O) reactors are used in this process. The overall nitrification reactions are performed by using autotrophic nitrifiers (Nitrosomonas and Nitrobacter) of AOB and NOB. In sequencing batch reactors, wastewater is first added and undesirable components are removed. Then wastewaters are added to biomass containing reactor and it accommodates the wastewater constituents. Next, aeration is done which enhances the nitrification of ammonia to nitrate. Finally, the reactor is made anoxic and nitrate converts to N2 through denitrification. An anoxic reactor has two parts; one is an anaerobic zone and another aerobic zone. Initially, wastewaters are passed through these zones and secondary clarifiers. Next, the activated sludge again passes through the anoxic reactor for denitrification. The efficiency of this anoxic reactor is less compared to the sequencing batch reactor (Figure 3).

5.2.5 Bioelectrochemical systems In bioelectrochemical systems (BES), microorganisms having electrochemically active and extracellular electron transferability are used. Electrons that are produced by pollutants oxidation are transferred to the anode and organic pollutant matters are removed. Based on the cathodic reaction, BES are divided into two types; microbial fuel cells (MFC) and microbial electrolysis cells (MEC). In MFC electrical power is produced, and the anodic oxidation is coupled to a cathode reducing an electron acceptor with high reduction potential. MEC needs electrical power because the anodic oxidation is coupled to a cathode which is operated at a low potential [68]. Denitrification at the cathode forms the basis of biological nitrogen removal in BES [68]. Here, denitrification is implemented in the cathodic compartment of BES either by H2 production or by accepting electrons from the cathode surface by denitrifying autotrophs. In addition, denitrification can also multiply its efficiency by applying a constant electric field in a continuous stirred tank reactor (CSTR), when the cathode potential is maintained constant around the standard potential value of the nitrate/nitrite redox couple [69]. The rate is subjective to applied potential and current. The method comes with added benefits like wastewater treatment, including removal of specific contaminants, stable operation at ordinary environmental temperatures, reduced environmental footprint, good processing speed, and is cost-effective due to cheap and self-propagating microorganisms. In addition, the process utilizes wastewater as a renewable source of

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Bhaskar Deka, Nikita Chakraborty, and Bhisma Kumar Patel

Sequential Batch Reactor

Fill

Sewage

Aeration

Decant

UV system

Sludge Discharge

Anoxic reactor

Anaerobic

Secondary clarifier Influent

Settle

Effluent

Aerobic

Return activated sludge

Waste activated sludge

Figure 3: Schematic diagram of sequencing batch reactor and anoxic reactor.

energy [70]. BES technique is also used for post-treatment of discharge which is released from other techniques. Some proteobacteria such as Geobacter, Acidithiobacillus, Shewanella, and Bacteroidetes, Chloroflexi, and Gram-positive bacteria Firmicutes, and Archaea show application in BES for wastewater treatment due to the ability of extracellular electron transfer [71]. Moreover, Phenazines produced by Pseudomonas or by chemical synthesis have pivotal role in this process. They serve as electron shuttles for extracellular electron transfer between electrodes and bacteria. QS systems regulate the Phenazine biosynthesis by the Pseudomonas species [72] and consequently, they are the reason for improving the efficiency of BES.

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6 Conclusion Nitrogen is a vital compound that is essential for the survival of life forms on earth. However, excessive reactive nitrogen in the environment has detrimental effects on both flora and fauna and disturbs the overall ecological balance. Hence, it is necessary to keep track of the free nitrogen content in the atmosphere, soil, and water bodies. In this context, biological wastewater treatment is a sustainable, environmentally benign, and economical way for removal of harmful nitrogen contaminants from wastewater before its discharge into the environment. This reactive nitrogen can be counterbalanced through various traditional biological processes such as nitrification and denitrification. Recently, several new biological nitrogen removal technologies including anammox, simultaneous nitrification and denitrification (SND), shortcut nitrification and denitrification, complete nitrification-denitrification systems, BES, and others have been at increase. All these different processes have been vividly discussed in this chapter. Considering the vastness of this field, only a brief description of the type of process, the microorganisms involved and their basic mechanism of action has been covered.

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Anne Bhambri, Santosh Kumar Karn✶, and Arun Kumar

4 Regulation and measurement of nitrification in terrestrial systems Abstract: Nitrification converts ammonia to nitrate by using bacteria which is an essential transformation in the terrestrial nitrogen cycle and there are various factors which regulates the nitrification process. The rate of nitrification process and their regulations are the crucial area for assessing the enhanced human’s impact on the terrestrial nitrogen cycle. Factors such as pH, water potential, oxygen concentration, and temperature have an adverse effect on the rates of nitrification of environmental controls. Over the past two decades, the trace nitrogen gas of soil production such as nitric oxide received a lot of attention as this nitrogen gas have some important adverse effects on the environment. This nitric oxide is relatively a short-lived trace gas which reacts with the O2 in the troposphere for the production of air pollutant ozone. In terrestrial geothermal ecosystems, research has been found on the nitrogen biogeochemical cycle which recently have been energized by the discovery of thermophilic ammonia-oxidizing bacteria. In this chapter, an in-depth study of factors, regulation and measurement of nitrification which affects the nitrification rate in the terrestrial system, is discussed.

1 Nitrification In nitrogen cycle, nitrification links the ammonia oxidation with the loss of fixed nitrogen in the dinitrogen gas by diverse groups of organisms such as ammonia-oxidizing bacteria, ammonia-oxidizing archaea, as well as nitrite-oxidizing bacteria which are mainly autotrophic as well as aerobes. A unique niche has been provided by their specialized metabolism, nevertheless, resulting in inefficient as well as slow growth. Anammox organisms

Acknowledgment: The authors are thankful to Gaurav Deep Singh, Chancellor, Sardar Bhagwan Singh University, Balawala, Dehradun, Uttarakhand, India, for providing facility, space, and resources to conduct this work successfully. ✶

Corresponding author: Santosh Kumar Karn, Department of Biochemistry and Biotechnology, Sardar Bhagwan Singh University, Balawala, Dehradun 248161, Uttarakhand, India, e-mail: [email protected] Anne Bhambri, Department of Biochemistry and Biotechnology, Sardar Bhagwan Singh University, Balawala, Dehradun 248161, Uttarakhand, India; Department of Biotechnology, Shri Guru Ram Rai University, Patel Nagar, Dehradun 248001, Uttarakhand, India Arun Kumar, Department of Biotechnology, Shri Guru Ram Rai University, Patel Nagar, Dehradun 248001, Uttarakhand, India https://doi.org/10.1515/9783110780093-004

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accomplish direct anaerobic oxidation of ammonia to nitrogen gas. It has been found that the conventional nitrification occurs in aquatic environments, in soils, as well as in sediments. In agriculture, it is very essential to regulate the availability of nitrogen fertilizers, whereas it participates in the excess nitrogen remediation from wastewater treatment systems [1]. In marine environment, nitrification determines the form of nitrogen that is accessible for the production in surface layer. In sediments as well as in low-oxygen waters, nitrification is coupled with denitrification which is an essential oxygen sink. Nitrifying bacteria involved in nitrous oxide as well as greenhouse gas production in both aquatic and terrestrial systems due to reductive pathways called as nitrifier denitrification. The rates of nitrification are determined by various environmental factors like pH, temperature, salinity, as well as oxygen in natural systems [2]. Due to the activity of nitrifying prokaryotes, the oxidation of ammonia to nitrate readily occurs in the oxic environments like drained soils and this process is essential for the fertility of soil as the nitrate is assimilated readily by plants. Nitrate is highly soluble in water and is denitrified or leached rapidly from the soils and helps to receive high rainfall. In wastewater treatment, nitrification is essential as it removes the ammonia that is highly toxic to many of the fishes [3]. Furthermore, in wastewater effluents, it decreases the NOD and also the first step in the nitrification/denitrification process in which ammonia converts to nitrite and then to nitrate [4]. In the nitrogen cycle, nitrification is a vital bacterial two-stage transformation as it is the only pathway where nitrate gets produced. By chemoautotrophic aerobic process, gaining energy is comparatively low as well as low rates as compared to the other processes of nitrogen cycle. Nevertheless, even with relatively low concentrations of ammonium, the process operates at low rates in many environments [5]. Therefore, low rates of nitrification require longer time for incubation as compares to other processes measure rates, specifically if the isotopes are not used. Although essential environmental factors influence the rate of nitrification such as O2, C:N ratio, pH, light, organic carbon availability, temperature, and ammonium availability [33–34]. Within the streams, nitrifying bacteria mostly get involved to substrata such as algae, fine particulate of organic matter as well as sediment consequently nitrification rates measurement comprise incubation of one or more of these materials. Nitrification completely converts the amount of ammonia to nitrate under the aerobic conditions. Gross rates estimate by the difference in the concentration of ammonia amongst the incubations in which nitrification constrains with the chemical nitrapyrin (2-chloro-6-[trichloromethyl]pyridine) and those in which nitrification allows to happens. Ammonia assimilation as well as mineralization is expected to be uninhibited in both incubations and enhance the ammonia in the incubations comprising nitropyrin which is a result of ammonia oxidation inhibition [6]. It is expected that the assimilation of ammonia as well as mineralization uninhibited in both incubations and enhanced the ammonia-containing nitrapyrin which results in inhibition of ammonium oxidation.

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The change in the availability of nitrate via time is the net nitrification as well as the difference between the assimilatory or dissimilatory nitrate reduction and gross nitrification. This net nitrification method is measured by the change in the concentration of nitrate in a substratum incubation in the stream water [7]. Due to the availability of the substrate, the autotrophic nitrification is primarily limited in the soil. In history, it has been described that the roots of plant are believed to reduce the nitrification rates by three possible mechanisms. Firstly, in rhizosphere soil, the zones of ammonia depletion occurs if the rates of root ammonia uptake exceed the rate of resupply, therefore, limiting the nitrification. Secondly, any factor that enhances the availability of carbon possibly increases the net ammonia immobilization into the bacterial bodies as the roots supply carbon content to the rhizosphere consequently again decreases the availability of ammonia to nitrifying bacteria. According to the statement that plants advantage from decreased nitrification, it has been assumed that plants chemically constrain the nitrifiers. Nevertheless, in rhizosphere soil, the unequivocal data represents the low total nitrification rates that have been lacking. In rhizosphere soil, the total nitrification rate is measured from Avena barbata, that is, a common yearly grass in California, using both field and the microcosm experiments [35–36]. Root uptakes the active ammonia in the area 8 to 16 cm from the root tip, the actual gross nitrification rate in the rhizosphere were zero according to the microcosm experiment, whereas nitrification rate indistinct from those of bulk soil in the area from which small amount of ammonia gets occur such as around 0–8 cm from the tips. Therefore, in the zones of active root uptake, there root competition for ammonia significantly decreases the nitrification. In the field experiment, it has also been found that in the presence of A. barbata roots, the total nitrification rate is higher as compares in the presence of complex plants community and therefore, their community composition impacts the nitrification rate. Whereas in microcosm study, it has been found that the potentials of nitrification is somewhat higher in soil adjacent to 0–8 cm zone as compares in bulk soil, whereas in the root zone, it is lower in soil from 8 to 16 cm [8].

2 Nitrogen cycle Nitrogen comprises around 79% of the Earth’s atmosphere in the form of dinitrogen gas that is biologically unavailable. This reserve is assessed 3.8 × 109 kg N and around 90% reserve worldwide, whereas crustal reserves contain remaining 10%. Nitrogen is an essential element to the extent that living organisms are concerned but the amount of nitrogen that is stored in the soil as well as in the biomass is small. Various physical as well as biological processes determined the global nitrogen that depends upon several environmental factors like texture of soil, other nutrients presence, solar energy, temperature, atmospheric concentrations of CO2, precipitation, and

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moisture in soil. These factors regulate the fluxes of nitrogen into and out of the vegetations as well as soils, thus inducing the nitrogen mass in these compartments and also its availability. Due to high rates of decomposition, soils of tropical forests display smallest amount of storage nevertheless due to the high production rates, the vegetation in tropical as well as temperate forests have higher storage of nitrogen. Generally, the activity of human tends to accelerate the nitrogen cycle by enhancing the rates of flux from one store to another. Nitrogen must be available in inorganic formal ammonia, nitrate, ammonium, and nitrite for the growth of plant. Nitrogen cycling processes of soil dominate the terrestrial nitrogen cycle having surface applications such as manure as well as fertilizers that provided most inputs of nitrogen. Nitrogen that is available in the soil gets produced by the breakdown of organic matter by bacteria. Nitrification, plant uptake, immobilization/mineralization, denitrification, as well as nitrate leaching then occurs. In water, nitrate gets soluble completely but not adsorbed to clay particles, it is susceptible to leached out of the soil by saturating irrigation water or rainfall. Usually, nitrogen movement occurs in three directions such as upward, downward, and lateral. Due to the anthropogenic activities, nitrogen cycle is strongly influenced. Since the twentieth century, it has been found that the land-use changes significantly disturb the natural nitrogen biogeochemical cycles including the activities of industries, fossil fuels combustion, intensive agriculture, energy production, deforestation, over-fertilization as well as burning of biomass. The growth rate of plants in natural ecosystem are relatively low, whereas its yearly uptake is relatively small. For sugarcane and wheat, the cultivated crops are highly demanding with the uptake of nutrients ranging 450 kg N ha/year and 100 kg N ha/year correspondingly. For livestock rearing, better grasslands typically involve 250 kg N ha/ year. Soil having capacity of mineralization that is inadequate in the maintenance of optimum growth. Consequently, for intensive agriculture, manures as well as chemical fertilizers are essential for the supply of nitrogen. At the regional, global as well as local scales, this resulted in the changes to long-term trends inside the nitrogen cycle [9].

3 Factor affecting nitrification 3.1 pH Aerobic transformation of nitrogen rate studied in six acid climax forest soils [10]. They also describe that in soil, the formation of nitrate did not take place under the natural pH 5; application of calcium carbonate enhanced the pH as well as initiated the formation of nitrate in the soils. In soil, accumulation of phosphorus has no effect on the nitrification.

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In soil, pH is the most important factor to regulate the process of nitrification and should range between 5.5 to about 10.0 with the optimum pH, that is, approximately 8.5. Nevertheless, Tisdale and Nelson [11] reported that nitrification occurs in soil with the pH as low as 3.8. De Boer and Kowalchuk [12] described that in acid soils, heterotrophs can contribute to the nitrification, whereas its involvement conclusively is hard to demonstrate.

3.2 Temperature Generally, a bell-shaped temperature curve has been followed by nitrification with optimum temperatures of 30–35 °C. On nitrification, the effect of temperature is dependent on the climate [13]. According to Sabey et al. [14], nitrification reached the maximum at about 25 °C after studying the ammonium sulfate nitrification in three Iowa soils with temperature ranging from 8 to 30 °C. In 1962, Justice and Smith [15] agreed and described that soil nitrification proceeds more quickly at 25 °C than at 35 °C, whereas according to Myers [16], the optimum temperature for nitrification is 35 °C after studying the nitrification in the tropical soil from Australia with temperature range of about 20–60 °C. The potential rates of nitrification at 20, 35, and 60 °C correspondingly in 21 days were 0.5, 4.8, and 0.25 mg/kg, which shows that the relation between nitrification and temperature differs from climate to climate. When compared with the soil in temperate regions, tropical soils have high optimum temperature for nitrification. Focht and Verstraete [17] reported the range of optimum temperature from 25 to 35 °C for nitrification in pure culture of nitrifiers. In 10 soils, in which 8 minerals and 2 histosols, nitrification has been studied by Sahrawat [18] having a range in organic carbon (12.2–227.0 g/kg), texture, total nitrogen (900–12,000 mg/kg), and pH (3.4–8.6). After the incubation of 4 weeks, the amount of nitrate-nitrogen has been produced at 30 °C which varied from 0 to 123 mg/kg soil. According to Sahrawat [18], organic soil having pH 5.6 produces only 5 mg nitrate-nitrogen Kg2 soil, whereas soils having pH less than 5.0 did not nitrify at all and soil with pH greater than 6.0 nitrifies at a fast rate as well as releases the nitrate-nitrogen ranging from 98 to 123 mg kg 21 soil. In the soil, the production amount of nitrate-nitrogen is extremely positively correlated with the pH of soil but not suggestively correlated to total nitrogen or organic carbon. Sahrawat [18] also described that the statistical analysis of data shows the formation of nitrate is not suggestively correlated with the pH of soils higher than 6. According to Sahrawat et al. [10], the study of nitrification shows that at natural soil of pH 5 in six acid climax forests from Wisconsin (USA), nitrification does not occur but soil limits enhance the pH as well as initiate nitrification in all soils. In the presence of oxygen, nitrification as well as nitrogen mineralization have been studied in two acid sulfate soils having pH of 3.4 as well as 3.7 for 2 weeks of incubation at 30 °C. Under these conditions, nitrification does not occur. Mineralized nitrogen has been entirely accumulated as

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ammonium nitrogen. A study of 4 years has been conducted by [37] to assess the importance of soil pH on nitrification of the fall-applied anhydrous ammonia in the Corn Belt region of the USA.

3.3 Oxygen In the soil matrix, the interaction between oxygen and moisture significantly impacts the formation of nitrate, nitrification, and their stability. Most of the pore spaces of soil has been occupied by the water under the high moisture of soil and this affects negatively to nitrification as well as aeration of soil. Optimum oxygen and moisture content are critical for nitrification of moisture that limits the diffusion of air into soil. Generally, moisture occupies the pore space present in the soil texture. Therefore, the optimum conditions for both aeration and moisture are dangerous for the nitrification to take place in the soil.

3.4 Carbon dioxide Sahrawat et al. [10] experimentally conducted the effect of carbon dioxide concentration on nitrification, denitrification, as well as production of nitrous oxide in silt loam. As the concentration of carbon dioxide enhance from 0.3% to 100%, it gradually slows down the rate of nitrification in the soil and found that no nitrification has been occurred at 100% carbon dioxide. As carbon dioxide enhances from 0% to 2.6%, nitrous oxide associated with nitrification also increases and tends to be higher as the concentration of carbon dioxide enhanced to 73%. It was also found that at 25 °C, no nitrification has been produced at 100% carbon dioxide during 7 days [19].

4 Regulation of nitrification process In many natural systems, nitrogen is the limiting nutrient. There are various mechanisms evolved to effectively utilize the nitrogen for stability, survival, as well as productivity and minimize the loss of nitrogen in grassland as well as in forest ecosystems [20, 21]. Direct absorption of organic nitrogen via the association of mycorrhizal or tannin complex of protein consequently bypasses the process of mineralization of nitrogen [22–24]. Due to organic compounds, nitrification inhibition occurred and also have regulatory effects on vegetation and the populations of nitrifier and nitrification in soils as well as their functioning [25]. Addition of carbon-rich litter stimulates the soil microbes to fix or stabilize the ammonia and nitrogen via immobilization process. Immobilization extension varies

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from 35% to 95% depending upon the species of plants as well as the type of soil [26]. In certain grassland as well as forest ecosystems, bacterial immobilization of ammonia protects the ammonium-nitrogen from denitrification as well as leaching [27]. NO2 immobilization via nitrosation, a chemical reaction of NO2 with the phenolics, forms organic nitrogen compounds [28].

5 Measurement of nitrification process The rate of nitrification term has at least three distinct meanings such as gross, net, and potential rate of nitrification. The net rate of nitrification is the rate of nitrate accumulation and is equivalent to the conversion rate of NH4+ þ/NH3− to NO3– which is generally a measured rate in both field conditions as well as in laboratory. Regardless of consumption, gross rate of nitrification is the actual conversion rate of NH4+ þ/NH3− to NO2− /NO3−. The use of isotopes (15N) gives distinct estimates of NO3− consumption as well as production to determine the gross rate of nitrification. In a mixed system, the potential rate of nitrification is the maximum rate with nonlimiting substrate supply (NH4+ þ/ NH3−). A clear view of nitrification process has been provided by the measurements of these different rates [29–32]. Figure 1 represents the Outline of regulation, measurement and factors involved in the nitrification in terrestrial system.

Regulation and Measurement of Nitrification in Terrestrial System

Factors

pH Temperature Measurement

Oxygen Carbohydrate

Nitrogen (N2)

Ammonia (NH3)

Nitrite (NO2–)

Nitrate (NO3–)

By Nitrifying Bacteria Figure 1: Outline of regulation, measurement and factors involved in the nitrification in terrestrial system.

6 Future prospect of nitrification For the terrestrial environment, regulation and measurement of nitrification rate is extended to both extremes of the spatial scale. Therefore, the improvement to manage as well as predict the nitrification is needful across the agricultural ecosystem which

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is a primary source of nitrogenous trace gases and leads to the climatic changes worldwide. It is still a challenging role that how the functional redundancy role in the nitrifying organisms relates with the controlling factors such as pH, temperature, oxygen, and carbon dioxide on the rate of nitrification in the environment.

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[18] Sahrawat, K.L., 1982. Nitrification in some tropical soils. Plant Soil 65: 281–286. [19] Sahrawat, K.L., Keeney, D.R., Adams, S.S., 1985. Rate of aerobic nitrogen trans-formations in six acid climax forest soils and the effect of phosphorus and CaCO3. Forest Sci. 31: 680–684. [20] Vitousek, P.M., Sanford, R.L. Jr, 1986. Nutrient cycling in moist tropical forest. Annu. Rev. Ecol. Syst. 17: 137–167. [21] Vitousek, P.M., Matson, P.A., 1988. Nitrogen transformations in a range of tropical forest soils. Soil Biol. Biochem. 20: 361–367. [22] Griffiths, R.P., Caldwell, B.A., 1992. Mycorrhizas in Ecosystems. Read. Lewis, D.J., Fitter, D.H.A., Alexander, I. CAB International, Walling ford: 98–105. [23] Nasholm, T., Ekblad, A., Nordin, A., Giesler, R., Hogberg, M., Hogberg, P., 1998. Boreal forest plants take up organic nitrogen. Nature 392: 914–916. [24] Kielland, K., 1994. Amino acid absorption by arctic plants: Implications for plant nutrition and nitrogen cycling. Ecology 75: 2373–2383. [25] Lata, J.C., Durand, J., Lensi, R., Abbadie, L., 1999. Stable co-existence of contrasted nitrification statuses in a wet tropical savanna system. Fun. Ecol. 13: 762–763. [26] Bengtsson, G., Bengtsson, P., Mansson, K.F., 2003. Gross nitrogen mineralization, immobilization, and nitrification rates as a function of soil C/N ratio and microbial activity. Soil Biol. Biochem. 35: 143–254. [27] Stark, J.M., Hart, S.C., 1997. High rates of nitrification and nitrate turnover in undisturbed coniferous forests. Nature 385: 61–64. [28] Dail, D.B., Davidson, E.A., Chorover, J., 2001. Rapid Abiotic Transformation of Nitrate in an Acid Forest Soil. Biogeoch. [29] Hart, S.C., Stark, J.M., Davidson, E.A., Firestone, M.K., 1994. Nitrogen mineralization, immobilization and nitrification. In: Weaver, R.W., Angle, S., Bottomly, P.D., Bezdicek,, Smith, S., Tabatabai, A. Wollum, A. (Eds.), Methods of Soil Analysis. Part 2, Microbiological and Biochemical Properties. Soil Science Society America, Madison, WI, 985–1018. [30] Murphy, D.V., Recous, S., Stockdale, E.A., Fillery, I.R.P., Jensen, L.S., Hatch, D.J., Goulding, K.W.T., 2003. Gross nitrogen fluxes in soil: Theory, measurement and application of N-15 pool dilution techniques. Adv. Agron. 79: 69–118. [31] Schmidt, E.L., Belser, L.W., 1994. Autotrophic nitrifying bacteria. In: Weaver, R., Angle, S., Bottomley, P.J., Bezdicek, D., Smith, S.J., Tabatabai, A. et al. (Eds.), Methods of Soil Analysis. Part 2, Microbiological and Biochemical Properties. Soil Science Society America, Madison, WI, 159–177. [32] Stark, J.M., 2000. Nutrient transformations. Methods in Ecosystem Science. Sala, O.E., Jackson, R.B., Mooney, H.A., Howarth, R.W. (Eds.). Springer-Verlag, New York: 215–234. [33] Strauss, E.A., Lamberti, G.A., 2000. Regulation of nitrification in aquatic sediments by organic carbon. Limnology and Oceanography. 45: 1854–1859. [34] Strauss, E.A., Mitchell, N.L., Lamberti, G.A., 2002. Factors regulating nitrification in aquatic sediments: effects of organic carbon, nitrogen availability, and pH.Canadian Journal of Fisheries and Aquatic Sciences. 59(3):554–563. DOI: 10.1139/f02-032 [35] Herman, D. J., Johnson, K.K., Jaeger, C. H., Schwartz, E., Firestone, M.K., 2006. Root Influence on Nitrogen Mineralization and Nitrification in Rhizosphere Soil. Soil Science Society of American Journal. 70(5). DOI: 10.2136/sssaj2005.0113 [36] Hawkes, C.V., Wren, I.F., Herman, D.J., Firestone, M.K., 2005. Plant invasion alters nitrogen cycling by modifying the soil nitrifying community. Ecology Letters. 8(9): 976–985. https://doi.org/10.1111/j. 1461-0248.2005.00802.x [37] Kyveryga,P.M., Blackmer, A.M., Ellsworth, J.W., Isla, R., 2004. Soil pH Effects on Nitrification of FallApplied Anhydrous Ammonia. Soil Science Society of American Journal. 68(2). DOI: 10.2136/ sssaj2004.0545

Djaber Tazdaït and Rym Salah-Tazdaït

5 Ammonia-oxidizing bacteria: their biochemistry and molecular biology Abstract: Nitrogen is naturally present in the environment in organic, inorganic, and gaseous forms. The reduced forms of nitrogen are used by nitrifying bacteria, which form a heterogeneous group comprising different genera and species highly specialized in nitrification. The ammonia oxidation by autotrophic nitrifying bacteria provides them with energy and produces nitrite, then nitrate. The best known and most studied representatives of nitrifying bacteria pertain to the genera Nitrosomonas, Nitrosococcus, and so on, which oxidize ammonia to nitrite, and Nitrobacter, Nitrospira, and so on, which oxidize nitrite to nitrate. Most of these microorganisms are chemolithoautotrophic, with some exceptions which are mixotrophic such as Nitrobacter, which can use acetate as a carbon source. Besides, there is an anammox process in which there is anaerobic oxidation of ammonia combined with nitrite reduction. This chapter summarizes the current knowledge on bacterial ammonia oxidation research and focuses on discussing the biochemical, genetics, and regulatory aspects of the bacterial metabolism of ammonia.

1 Introduction Nitrogen is the principal constituent of the atmosphere, representing 78% of the total atmospheric volume. It is the fourth component of living beings, in quantity, after carbon, hydrogen, and oxygen. It is found in the essential molecules of life, including almost all vitamins (thiamine, cobalamin, folic acid, etc.), nucleic acids, amino acids, and some lipids such as phosphatidylethanolamine and phosphatidylserine. Atmospheric nitrogen is very inert and can be used by living organisms only after transformation into an assimilable form (ammonia) through a process called nitrogen fixation involving a group of bacteria known as symbiotic nitrogen-fixing bacteria. The chemically and biologically active compounds of nitrogen (reactive nitrogen), which include ammonia, nitrate, nitrite, nitric oxide, nitrous oxide, nitric acid, nucleic acids, urea, and amino acids, can undergo a whole series of transformations in the environment (air, water, and soil) as well as within living beings, and return to their dinitrogen form through denitrification process.

Djaber Tazdaït, Department of Natural and Life Sciences, Faculty of Sciences, Algiers 1 University Benyoucef Benkhedda, Algiers, Algeria Rym Salah-Tazdaït, Bioengineering and Process Engineering Laboratory (BIOGEP), National Polytechnic School, Algiers, Algeria https://doi.org/10.1515/9783110780093-005

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Ammonia serves in most organisms for amino acid biosynthesis, which occurs through direct amination. Thus, for instance, the aspartase of many bacteria catalyzes the direct amination of fumarate to aspartate with ammonia, and the asparagine synthetase catalyzes the direct amination of aspartate with ammonia to yield asparagine. In cyanobacteria, bacteria, and to a fewer extent, yeasts, the assimilation of ammonium is mediated by the ATP-dependent glutamine synthetase/glutamate synthase system, which catalyzes the amination of α-ketoglutarate with ammonium to form glutamate. The latter serves as an amino donor to numerous central metabolic intermediates through transaminase reactions [1]. Besides, ammonia (or ammonia donors) is used in the first reaction of the bacterial pyrimidine nucleotides biosynthesis pathway in which the carbamyl phosphate synthetase uses ammonia, bicarbonate, and ATP to generate carbamyl phosphate [2]. On the other hand, many organisms’ primary metabolism, including microbes, generates ammonia as a waste metabolite from aerobic and anaerobic amino acids catabolism through deamination reaction. Ammonia is also released into the environment anthropogenically by diverse industries, including water treatment plants and fertilizer manufacturing plants [3]. Ammonia in all its forms (NH3 and NH4+) is toxic. It can cause if inhaled, several respiratory tract damages, which can ultimately cause death [4]. Ammonia is also responsible for eutrophication created by accumulating nutrients in aquatic media. In addition, when released into the atmosphere through volatilization, ammonia is responsible for much of the acidification of soil (acid soil) [5]. Ammonia-oxidizing bacteria form a group of microorganisms capable of accomplishing nitrification by oxidizing ammonia into nitrite and then into nitrate in the nitrogen cycle. This process occurs aerobically and involves microorganisms, which are either autotrophic or heterotrophic. Besides, the oxidation of ammonia can occur anaerobically through a process called anammox (anaerobic ammonia oxidation), in which ammonia is oxidized to form molecular nitrogen in the presence of nitrite or nitrate serving as the final electron acceptor [6]. In another anaerobic process, the autotrophic bacterium Anammoxoglobus sulfate was described as capable of catalyzing the conversion of ammonia to nitrogen gas parallel to the reduction of sulfate to sulfur [7]. This chapter aims to describe (1) the nitrogen cycle in the environment, (2) ammonia-oxidizing bacteria in the environment with a focus on their systematics and distribution, (3) the biochemistry and genetics of ammonia oxidation, and (4) the regulatory mechanisms of bacterial ammonia oxidation.

2 Bacterial nitrogen cycle in the environment The nitrogen cycle is a central issue in understanding environmental, agronomic, and health challenges and achieving sustainable development goals.

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The nitrogen cycle is the biogeochemical cycle that governs all nitrogen exchanges on the planet. It describes the succession of modifications undergone by the different forms of nitrogen (inorganic: N2, NO3–, NO2–, NH3, etc.; inorganic: proteins, nucleotides, lipids, etc.) through redox processes. The bacterial processes of the nitrogen cycle (Figure 1) take place both in the lithosphere and in the hydrosphere and involve specific bacterial groups that ensure the following processes:

2.1 Nitrogen fixation Nitrogen fixation is the conversion of atmospheric nitrogen into nitrogen (NH3) usable by plants and animals. This process, which is ATP-dependent, is performed by the enzyme nitrogenase that acts in both aerobic and anaerobic microorganisms with different trophic types (heterotrophic, autotrophic, and phototrophic) [8]. The implicated microorganisms live in soil or water either freely (cyanobacteria (Anabaena azollae), Klebsiella pneumoniae, Azotobacter chroococcum, Azotobacter vinelandii, etc.) or in symbiosis with plants (leguminous and non-leguminous). Symbiotic nitrogen-fixing species are significantly less numerous than free-fixing species. In the symbiotic fixation of nitrogen, the bacteria enter the roots through the channel of the root hairs, differentiate into bacteroid cells, and then induce a transformation of the plant’s tissues, resulting in the formation of plant nodules. The best-known nitrogen-fixing bacteria belong to the genera Rhizobium (R. lentis, R. alamii, R. etli, and R. leguminosarum), Bradyrhizobium (B. japonicum and B. elkani), Azorhizobium (A. caulinodans), and Sinorhizobium (S. fredii and S. meliloti) [9]. In this association, the plant provides the nitrogen-fixing bacterium with carbon substrates (oxaloacetate, fumarate, malate, and succinate) needed for nitrogen fixation, while the bacterium provides fixed nitrogen to the plant symbiote for its growth. It is worth noting that this association is not obligatory since the bacteria can be cultivated separately, and the plant can grow without its symbiote. The typical general chemical reaction of nitrogen fixation is N2 + 3H2 + 2H + ! 2NH3 + H2 ,

,

Δ G = −39.3 kJ=mol

(1)

Nitrogenase consists of the association of two proteins, namely dinitrogenase and dinitrogenase reductase. The first enzyme called MoFe protein is a heterotetramer of α2β2 structure, containing two Fe-S metalloclusters. The second enzyme called the Fe protein, is a dimer of two identical γ-chains with a single iron-sulfur (Fe4S4) cluster [10]. It is estimated that nitrogenase hydrolyzes 16 molecules of ATP per molecule of reduced N2 [11].

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2.2 Ammonification In the nitrogen cycle, ammonification usually refers to the release of ammonium by decomposition of dead organic matter in water or soil. It occurs in both aerobic and anaerobic environments. The microorganisms accomplish this process to derive their energy for growth from converting organic matter containing amine or amide groups into ammonium. They include aerobic (Bacillus, Pseudomonas, Neisseria, Achromobacter, Micrococcus, etc.) and anaerobic (Clostridium, Selenomonas, Wolinella, etc.) bacterial heterotrophs and fungi. Ammonium can be produced in the cells through the conversion of the amino acid glutamate into α-ketoglutarate catalyzed by glutamate dehydrogenase as follows: Glutamate + H2 O ! α-ketoglutarate + NH4 −

(2)

Ammonium can also be produced extracellularly, thanks to the hydrolysis of soluble/insoluble organic polymers, including DNA, proteins, chitin through extracellular enzymes produced by hydrolytic bacteria (respectively, nucleases, proteases, and chitinases) or by enzymes anchored to the outer cell membrane, to generate nitrogen-containing monomers of nucleotides, amino acids, and so on. These monomers and urea (present in the environment) undergo further degradation by extracellular enzymes (amino acid oxidases, urease, etc.), resulting in the release of ammonium [8, 12].

2.3 Bacterial ammonium oxidation 2.3.1 Nitrification The nitrification process refers to sequential oxidative reactions in which ammonia is aerobically converted into nitrate. The bacteria which perform this process are called nitrifiers and are grouped into (1) the ammonia-oxidizing bacteria (AOB), including Nitrosomonas, Nitrosococcus, Nitrosovibrio, Nitrosospira, and Nitrosolobus, which perform the two-step-oxidation of ammonia to hydroxylamine and nitrite by the enzymes ammonia monooxygenase and hydroxylamine oxidoreductase, respectively [13] and (2) the nitrite-oxidizing bacteria (NOB) (Nitrobacter, Nitrospira, Nitrococcus, etc.), which oxidize nitrite into nitrate by nitrite oxidoreductase [14]. The AOB and NOB groups are both chemolithoautotrophs, which means that they gain their energy from the oxidation of ammonium to nitrite and nitrite to nitrate, respectively, to fix inorganic carbon (CO2) used as their sole carbon source [8]. Certain bacterial species pertaining to the genus Nitrospira known as Commamox (complete ammonium oxidation), were reported to perform the entire ammonium oxidation reaction (from ammonium to nitrate) [15].

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The overall reaction of the process is as follows: 2NH3 + O2 ! 2NO3 − + 3H2 O,

,

ΔG = −349 kJ=mol

(3)

2.4 Anammox process Anammox (anaerobic ammonia oxidation) process is performed anaerobically by a particular group of bacteria, capable of oxidizing ammonia to form nitrogen gas by utilizing nitrite as a terminal electron acceptor to sustain their growth [6]. Nitrite is derived from either aerobic ammonium oxidation or the beginning of denitrification. The reaction of the process is given as follows: NO2− + NH4+ ! N2 + 2H2 O,

,

ΔG = −358 kJ=mol

(4)

The bacteria involved in this process are all strictly anaerobic chemoautolithotrophic and pertain to the phylum of Planctomyces [16] One of the metabolic models for anammox seems to occur in step reactions starting with reducing nitrite by nitrite oxidoreductase to yield nitric oxide, which acts by oxidizing ammonium into hydrazine through hydrazine hydrolase. Finally, the hydrazine is converted into molecular nitrogen by the enzyme hydrazine oxidoreductase [17]. Anammox process has been identified in different natural habitats and, interestingly, in wastewater treatment plants, it is cost and energy effective in removing nitrogen in comparison with the conventional two-step nitrogen removal process [18].

2.5 Nitrate reduction 2.5.1 Assimilatory nitrate reduction (nitrate immobilization) Nitrogen can only enter the metabolism in the ammoniacal form, and the use of nitrate as a nitrogenous source can only be done by a series of reductions according to the following overall reaction: NO3 − + 4H2 + 2H+ ! NH4 + + 3H2 O,

,

ΔG = −591 kJ=mol

(5)

In this process, which occurs in plants, algae, fungi, and bacteria, nitrate is reduced to ammonium via nitrite. This reduction pathway is called assimilatory nitrate reduction to ammonia (ANRA): ammonium will be incorporated into the metabolism mainly by the glutamine synthetase and glutamate synthase system [8]. The process is catalyzed by a cytosolic enzymatic system comprised of two enzymes, namely nitrate reductase (NR) and nitrite reductase (NiR), requiring two and six electrons, respectively):

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Nitrate reductase

Nitrate reductase

NO3 − + 2e − ! NO2 − + 4e− ! NH4 +

(6)

Cyanobacteria, such as Plectonema boryanum, also assimilate nitrate, and their nitrate reductase is partially associated to the thylakoid membrane.

2.5.2 Dissimilatory process In this case, nitrate is not a nitrogen source; it acts instead as a final electron acceptor in anaerobiosis and produces energy in the form of a membrane potential difference convertible into ATP. The dissimilatory process of nitrate includes two distinct pathways, which compete with each other: – Denitrification designates the reduction of nitrate through anaerobic respiration into nitrite. The nitrite formed serves as a respiratory acceptor and is reduced sequentially into the following gaseous compounds: NO, N2O, and N2, which accumulate in the atmosphere [8]. The sequential reactions involved in this process, along with their corresponding enzymes, are given as follows: Nitrate reductase

Nitrite reductase

Nitric oxide reductase

!NO2− !NO ! NO3− Nitrous oxide reductase

N2 O !N2

(7)

The overall reaction of the process is 2NO3 − + 5H2 + 2H+ ! N2 + 6H2 O,

–

,

ΔG = −1,121 kJ=mol

(8)

All stages of denitrification produce energy through ATP synthesis. Besides, since denitrification is an energy-producing mechanism, nitrate and nitrite consumption during this process is much higher than that required for nitrogenous molecules synthesis in the cell. Many bacterial species are denitrifying in nature and belong to very diverse genera such as Bacillus, Spirillum, Xanthomonas, Pseudomonas, and Thiobacillus. Aerobic bacteria can opt for nitrate respiration when deprived of oxygen. Denitrifying germs belonging to the genera Bacillus, Alcaligene, and Pseudomonas are abundant in the rhizosphere. Dissimilatory nitrate reduction to ammonium (DNRA), where nitrate is reduced into nitrite, which is, in turn, converted into ammonium. The latter is not necessarily assimilated, especially if the medium already contains a nitrogen source (for example, amino acids). Some steps of this pathway can be coupled to ATP synthesis, as in the case of denitrification. This process is observed in different bacterial genera, such as Bacillus, Pseudomonas, Clostridium, Escherichia, and Desulfovibrio. The reduction of nitrate to nitrite is mediated by nitrate reductase, while the conversion of nitrite into ammonium is catalyzed by a pentaheme cytochrome c nitrite reductase called NrfA [19].

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The key step in these dissimilatory processes is the reduction of nitrate to nitrite by molybdenum-dependent nitrate reductases. The bacterial dissimilatory nitrate reductases are categorized into dissimilatory water-soluble periplasmic nitrate reductase (Nap) and membrane‐anchored respiratory nitrate reductase (Nar). The Nar complex is found in many Gram-negative bacteria (Escherichia coli, Paracoccus pantotrophus, etc.) [20] and is implicated in denitrification and dissimilatory nitrate reduction to ammonium [21]. Nap complex is present in several bacteria and is involved in different physiological roles, including nitrate scavenging and denitrification [22]. The DNRA and denitrification processes are known to predominate under carbon-rich and carbonlimiting conditions, respectively [8, 21]. Besides, certain pesticides, such as Propiconazole inhibit denitrification at low doses [23]. Sulfide also has been shown to inhibit the process [24]. On the other hand, the nitrate reductase gene expression is repressed by oxygen but not by ammonium. This implies that both DNRA and denitrification are

Atmospheric nitrogen (N2)

Nitrous oxide

(N2O)

(5)

Nitric oxide (NO)

(1)

Nitrite

Nitrate (NO3–)

(6)

Organic nitrogen (nucleotides, amino acids, chitin, lipids, etc.)

(4)

(2)

Nitrite (NO2–)

Ammonia

(3)

(NH3)

(7)

Figure 1: Bacterial nitrogen cycle processes: (1) bacterial nitrogen fixation (Azotobacter, Bradyrhizobium, Azospirillum, cyanobacteria, etc.), (2) microbial ammonification (organic matter mineralization), (3) nitritation by ammonia-oxidizing bacteria (Nitrosomonas, Nitrosococcus, Nitrosovibrio, etc.), (4) nitration by nitrite-oxidizing bacteria (Nitrobacter, Nitrospira, Nitrococcus, etc.), (5) denitrification by denitrifying bacteria (Bacillus, Alcaligenes, Pseudomonas, etc.) and denitrifying fungi (Nectria, Trichoderma, Gibberella, etc.), (6) anammox process (Kuenenea, Anammoxoglobus, Scalindua, etc.), (7) dissimilatory nitrate reduction to ammonia (DNRA) (Citrobacter, Escherichia, Bacillus, Veillonella, Pseudomonas, etc.)/assimilatory nitrate reduction to ammonia (ANRA).

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inhibited in the presence of oxygen. However, denitrification can occur aerobically, as reported by many studies, such as the one conducted by [25] who reported that the expression of the nap operon encoding the Nap in Paracoccus pantotrophus was induced under oxic conditions and required the presence of highly reduced carbon substrates.

3 Ammonia-oxidizing bacteria in the environment (systematics and distribution) The analysis of the diversity of ammonium-oxidizing bacteria in the environment is carried out by the denaturing gradient gel electrophoresis (DGGE), the fluorescence in situ hybridization (FISH), the polymerase chain reaction (PCR), the quantitative PCR (qPCR), and the quinone profile techniques (reverse-phase HPLC, photodiode array detection, thin-layer chromatography, mass spectroscopy). The PCR consists of in vitro enzymatic amplification by an enzyme (DNA polymerase) of a short fragment of the genome of the agent sought. However, the qPCR consists of amplifying a DNA fragment, or complementary DNA (cDNA) for RNA, by duplicating it in an exponential and controlled manner. The DGGE consists of an amplification of a gene of interest in the sample to be analyzed. This amplification is carried out by “nested” PCR, which is based on two successive PCRs with two sets of primers and reduces nonspecific amplification of the DNA template. The FISH analyzes sections by microscopy and molecular imaging using probes with a fluorescent marker [61]. There are two groups, phylogenetically and biochemically distinct, of bacteria that oxidize ammonia: – Aerobic autotrophic ammonium-oxidizing bacteria represented by ammoniaoxidizing bacteria and comammox (complete ammonia oxidizers) bacteria (bacteria oxidize ammonia to nitrate). – Anaerobic oxidizing bacteria also called anammox (anaerobic ammonium oxidation) bacteria. AOB oxidize ammonia to nitrite. They remain in two phylogenetic groups: β-proteobacteria (Nitrosomonas, Nitrosospira, Nitrosovibrio, and Nitrosolobus) and γ-proteobacteria (Nitrosococcus) (Figure 2). In research today, Nitrosomonas europaea, isolated in 1890 by Sergei Winogradsky, is the model ammonia-oxidizing bacterium (Soliman and Eldyasti 2018). β-Proteobacterial AOB are generally found in soil environments, and wastewater treatment plants and the genus Nitrosospira tend to dominate soil AOB communities [27]. γ-proteobacterial AOB are naturally found in marine habitats; Nitrosococcus strain TAO100 is a notable terrestrial exception [28]. In soil, AOB dominate when ammonia concentration is high [29]. They have extensive intracytoplasmic membrane structures with a significant number of ammonia monooxygenase [30]. AOB are autotrophic; they fix atmospheric CO2 by the Calvin cycle [31].

5 Ammonia-oxidizing bacteria: their biochemistry and molecular biology

Domain

Bacteria

Phylum

Proteobacteria

Class

β-proteobacteria

γ-proteobacteria

Order

Nitrosomonadales

Chromatiales

Family

Nitrosomonadaceae

Genus

Nitrosomonas

Species

N. europaea

Nitrosolobus N. multiformis

N. eutropha

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Chromatiaceae

Nitrosovibrio

Nitrosospira

Nitrosococcus

N. tenuis

N. sp.

N. halophilus

N. inopinata

N. oceani

N. halophila N. mobilis N. communis N. sp. I

Ca. N. nitrificans Ca. N. nitrosa Ca. N. kreftii

N. sp. II N. nitrosa N. ureae

N. oligotropha N. marina N. sp. III N. aestuarii N. cryotolerans

Figure 2: Systematics of AOB species (blue boxes) and comammox species (bold green boxes) [Soliman and Eldyasti 2018, 26].

3.1 Comammox bacteria Comammox bacteria oxidize ammonia to nitrate. They belong to the bacterial genus Nitrospira [32]. Their diversity includes Candidatus species given in Figure 2. Nitrospira inopinata is the model comammox bacterium [33]. Comammox Nitrospira are found in neutral and acidic pH soils, volcanic soils, aquatic environments, hot springs, and engineered ecosystems [32, 34]. Comammox Nitrospira are adapted to low ammonia concentrations [30]. Comammox Nitrospira are autotrophic; they fix atmospheric CO2 by the oxygensensitive reductive tricarboxylic acid (rTCA) cycle [34].

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3.2 Anammox bacteria Anammox bacteria are affiliated with the order Brocadiales. They include Candidatus Scalindua, Candidatus Kuenenia, Candidatus Anaammoxoglobus, Candidatus Brocadia, Candidatus Anammoximicrobium, and Candidatus Jettenia (Figure 3), detected from anoxic environments that contain fixed nitrogen, like marine and freshwater sediments. In fact, these bacteria are well characterized but yet uncultured. Anammox bacteria are slow-growing, strictly anaerobic and chemolithoautotrophic. They oxidize ammonium to nitrogen gas using nitrite as a terminal electron acceptor. Catalytic reactions of the anammox pathway occur in a unique intracellular membrane-bound organelle, called the anammoxosome, surrounded by a membrane containing distinctive ladderane lipids. Anammoxosome comprises 50–70% of the cell volume [35–37].

4 Biochemistry and genetics of ammonia oxidation Ammonia monooxygenase (AMO) is the key enzyme of ammonia oxidation. This copperdependent membrane-bound enzyme belongs to the ammonia, methane, and alkane monooxygenases superfamily. In bacteria, AMO is formed by three subunits, A, B, and C, encoded by three genes called amoA, amoB, and amoC, respectively, organized in the operon amoCAB, often present in multiple copies, three copies in Nitrosomonas cryotolerans 49181, two copies in Nitrosospira tenuis NV-12 and Nitrosomonas eutropha C-91, for instance [38, 39]. The inactivation of one of the two copies results in slowed growth, a quantity of lower full-length AMO mRNA, and reduced AMO activity [40]. Thus, each copy has a metabolic function distinct but not essential for growth [41]. These three genes are co-transcribed into a single 3.5 kb mRNA [42]. The physical organization of this operon is different between the AOBs of the β-Proteobacteria class and those of the γProteobacteria class. In all cases, this operon consists of amoC followed by a space intergenic, then amoA and finally amoB. The size of the intergenic space between amoC and amoA is between 163 and 445 bp [39]. The distance between the different copies of the operon is not conserved in β-AOBs [43]. An intergenic space between amoA and amoB of 65 bp is observed only in γ-Proteobacteria AOB [44]. In Nitrosomonas europaea, AMO has an atomic mass of 283 kDa and appears to consist of different subunits: the α subunit (27 kDa), the β subunit (42 kDa) and the γ-subunit (24 kDa) [45]. However, in Nitrosospira sp. NpAV, the amoC gene, encodes a protein of approximately 31 kDa [46]. AMO oxidizes ammonia to hydroxylamine. Hydroxylamine is oxidized by hydroxylamine dehydrogenase (HAO, formerly known as hydroxylamine oxidoreductase) to nitric oxide (NO) in AOB and comammox bacteria [38]. HAO is a homotrimer of which each of the three subunits has a molecular weight of 64 kDa [47]. The hao gene encoding

5 Ammonia-oxidizing bacteria: their biochemistry and molecular biology

Domain

Phylum

Class

Order

Family

Genus

Species

Candidatus Anammoximicrobium Candidatus Anaammoxoglobus

Ca. A. propionicus Ca. B. anammoxidans Ca. B. carolinensis

Candidatus Brocadia

Ca. B. fulgida

Brocadiaceae

Brocadiales

Planctomycetia

Planctomycetes

Bacteria

Ca. B. sapporoensis Ca. B. sinica Ca. J. asiatica Candidatus Jettenia

Ca. J. caeni Ca. J. ecosi

Candidatus Kuenenia

Ca. K. stuttgartiensis Ca. S. arabica Ca. S. brodae Ca. S. erythraensis Ca. S. japonica

Candidatus Scalindua

Ca. S. rubra

Ca. S. pacifica Ca. S. profunda Ca. S. sorokinii Ca. S. wagneri Figure 3: Systematics of anammox bacteria [35–37].

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HAO consists of 1,710 bp. These genes and that of two cytochromes are generally organized in an operon denoted hao-orf2-cycAB. This operon is present in several copies in the AOB genome (Table 1). The gene cycB encoding cytochrome cm552 is sometimes absent in one of the operon copies [43]. Table 1: Diversity in number of identified copies of the hao gene [43, 48]. Strains Nitrosomonas europaea  Nitrosomonas eutropha C- Nitrosospira multiformis 

Number of copies of the hao gene

Copy number of complete hao-orf-cycAB operon

  

  

A third unidentified enzyme converts NO into nitrite in AOB and comammox bacteria [38]. However, a nitrite oxidoreductase (NXR) converts nitrite into nitrate in comammox bacteria [26]. The catalytic site of the NXR enzyme is a heterodimer consisting of the α and β subunits, encoded by nxrA and nxrB, respectively [49]. However, there are no nitric oxide reductase genes or nirA gene (encodes nitrite reductase) in the comammox genome, explaining the incapacity of comammox bacteria to use external nitrite as nitrogen and energy source [26, 50]. It should be noted that the operon nxrAB includes additional genes, but their function remains unknown or hypothetical [51]. In AOB catabolism, ammonium is the electron donor, and oxygen is the electron acceptor, as given in equations (3) and (9): NH4 + + 1.5 O2 ! NO2 − + 2 H + + H2 O

(9)

This reaction is divided into three steps: 1. The AMO enzyme oxidizes ammonium (NH4+) to hydroxylamine (NH2OH): NH4 + + O2 + 2 H + + 2 e ! NH2 OH + H2 O 2.

The HAO enzyme converts hydroxylamine (NH2OH) to nitric oxide (NO): NH2 OH ! NO + 3 H + + 3 e

3.

(10)

(11)

An identified enzyme converts nitric oxide (NO) to nitrite (NO2−).

All electrons appear to pass through cytochrome c554. Most electron flux passes through the ubiquinone pool. Thus, from c554, the easiest route to ubiquinone appears to be cytochrome cm552. Two of the electrons produced during reaction (11) are used to compensate for the consumption of reaction (10). The electrons not used for ammonia oxidation

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are directed toward O2 through the cytochromes bc1 (Complex III) and c552. Less electron flow is used for NAD+ reduction [52–54]. In comammox catabolism, ammonium is metabolized to nitrate (equation (12)) in four steps: the first three steps are the same found in AOB catabolism, and the fourth step is catalyzed by NXR converting nitrite into nitrate (equation (13)) [53, 54]: NH4+ + 2 O2 ! NO3 − + 2 H+ + H2 O

(12)

NO2 − + H2 O ! NO3 − + 2 H+ + 2 e

(13)

In anammox catabolism, ammonium and nitrite are converted without oxygen to dinitrogen (N2) (equation (12)). The anammox nitrogen metabolism is divided into three steps: 1. The nitrite oxidase (Nir) converts nitrite to nitric oxide (equation (14)) 2. The hydrazine synthase (HZS) converts nitric oxide and ammonium to hydrazine (N2H4) (equation (15)) 3. The hydrazine dehydrogenase (HDS) converts hydrazine to dinitrogen (N2) (equation (16)) NO2− + 2 H + + e ! NO + H2 O NO + NH4 + + 2H + + 3 e ! N2 H4 + H2 O N2 H4 ! N2 + 4 H + + 4 e

(14) (15) (16)

5 Regulatory mechanisms of bacterial ammonia oxidation The bacteria dealt with in this chapter are strongly influenced by various environmental parameters such as temperature, dissolved oxygen concentration, pH, the concentration of different forms of nitrogen, salinity, and concentration of sulfide or phosphate. Temperature, therefore, directly influences nitrification but can also intervene indirectly by acting on other parameters such as the concentration of free NH3 in the medium. For the same pH value, the free NH3 concentration is higher at 35 °C than at 30 °C. The partial pressure of oxygen has a direct influence on nitrification since oxygen is used as the final electron acceptor in aerobic bacteria. A lack of oxygen can cause the reaction to slow down or even stop. However, anaerobic bacteria can oxidize ammonium in the presence of pyruvate with nitrite as an electron acceptor. Nitrite, replacing oxygen as the terminal electron acceptor, is reduced to NO, N2O, and N2 [55]. Depending on the pH, nitrogenous substrates exist in different forms in equilibrium according to the following stoichiometric equations:

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NH4 + + OH− $ NH3 + H2 O

(17)

HNO2 + H2 O $ H + + NO2 −

(18)

Ammoniacal nitrogen is found in solution in the form of ammonium ion (NH4+) or ammonia (NH3), while nitrite (NO2–) can be in the form of nitrous acid (HNO2). A high pH (pH > 8) favors the ammonia (NH3) form and a low pH (pH < 6) that of nitrous acid (HNO2). However, free ammonia and nitrous acid have an inhibiting effect on nitrification above a certain concentration [56]. In addition, studies have suggested that hydroxylamine, an intermediate in the oxidation of ammoniacal nitrogen, may be a factor inhibiting nitrite oxidation [57]. AMO is a copper-dependent enzyme, so the copper chelator allylthiourea (ATU) inhibits all known ammonia oxidizers [38]. AMO is also inhibited by thiourea, diethyl-dithiocarbamate (DIECA), acetylene, and carbon monoxide. However, HAO is inhibited by hydrazine [58, 59]. The inhibitors of the different enzymes involved in the ammonium oxidation process can be classified into different groups: The first group targeting the enzyme ammonium monooxygenase acts by chelating the copper present at the enzyme’s active site. In this group, many sulfur compounds are included, such as thiosulfates (thiourea and ATU), thiocarbamates (DIECA) and xanthans (potassium ethyl xanthan). The second group includes acetylenic compounds, which are substrates for the AMO enzyme. Their oxidation transforms them into reactive products forming covalent bonds with the enzyme. The last group of nitritation inhibitors is formed by heterocycled nitrogen compounds such as nitrapyrine (2-chloro-6-trichloromethyl pyridine), whose mode of action remains unknown. The most frequently used nitritation inhibitor is ATU, which targets the active site of ammonium monooxygenase [60].

6 Conclusion The research conducted in the last four decades has greatly advanced the knowledge of biochemistry and genetics of ammonia-oxidizing bacteria. This body of knowledge continues to grow because of the increasing variety of bacteria capable of metabolizing nitrogen compounds in general and oxidizing ammonia in particular. Special attention should be given to the anammox process, which refers to the oxidation of ammonia into dinitrogen under anaerobic conditions in the presence of oxidant compounds. Indeed, this particular bacterial-mediated process plays a very interesting role in removing excess nitrogen (in its ammoniacal form) from sewage and industrial effluents in wastewater treatment processes.

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Sonia Saini, Sanjana Tewari, Jaya Dwivedi, and Vivek Sharma✶

6 Ammonia-oxidizing bacteria in wastewater Abstract: Numerous wastewater treatment facilities throughout the globe do not treat wastewater for reactive nitrogen before releasing it all out to the environment. Excessive reactive nitrogen level acts as a fundamental source of air and water pollution that eventually leads to serious health issues. To prevent the detrimental effects of too much reactive nitrogen in the environment, tertiary wastewater treatment methods that guarantee the removal of reactive nitrogen species must be used. Eco-friendly, safe, and cost-effective wastewater treatment by biological nitrogen removal techniques has gained significant attention in the last decades. These techniques include the use of particular microbial species that are involved in nitrogen cycling via conversion of ammonia to nitrogen gas to eliminate reactive nitrogen from reactor systems. One such bacterial species is ammonia-oxidizing bacteria (AOB) which plays a critical role in the process of turning ammonia into nitrite in wastewater treatment. Considering the role of AOB in wastewater treatment, this chapter aimed to highlight the key attributes, limitations, and associated mechanisms involved in the removal of water pollutants, along with its quantitative assessment in wastewater.

1 Introduction Wastewater treatment will become more challenging in the green twenty-first century because it will be harder to find materials and technologies that work well [1, 2]. Great progress has been achieved in this field up till now. Three main ways to clean water are physical, chemical, and biotechnological [3, 4]. Some examples of physical technology are membrane separation [5, 6] and adsorption [7]. Chemical technology includes chemical reduction [8], chemical oxidation [9], and electrolysis [10]. When it comes to removing majorly found types of pollutants from wastewater, physical treatment processes are typically inefficient and time-consuming. To get rid of high molecular compounds from water, chemical technology is often used. Chemical technology is frequently used to reduce chroma and high molecular compounds and turbidity, however, the creation of oxidizing chemicals and changes in ambient oxygen concentration, pH, or temperature are likely to damage the ecosystem. Biotechnology uses microorganism’s resources to remove pollutants from wastewater with minimal ✶

Corresponding author: Vivek Sharma, Department of Chemistry, Banasthali Vidyapith, Rajasthan 304022, India, e-mails: [email protected], [email protected] Sonia Saini, School of Earth Sciences, Banasthali Vidyapith, Rajasthan 304022, India Sanjana Tewari, Jaya Dwivedi, Department of Chemistry, Banasthali Vidyapith, Rajasthan 304022, India

https://doi.org/10.1515/9783110780093-006

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environmental impact. Biotechnology is less expensive for actual wastewater treatment than physical and chemical technologies, and it is more suited for contemporary eco-friendly development. Traditional biotechnologies are extensively used in today’s wastewater treatment facilities to provide compelling treatment outcomes, but they also face significant obstacles, like high sludge generation, huge land area requirement, and inefficiency lot of wastewater contaminants (e.g., endocrine disrupting compounds, azo dyes, pharmaceuticals, and personal care products). To address these issues, different integrated bio treatments and better biological approaches have been developed. Biodegradation still is the best way to get rid of trace organic chemicals (TrOC) because it’s cheap and can get rid of all of them [11, 12]. Due to the diverse metabolic process of nitrifying bacteria, such as ammonia-oxidizing bacteria (AOB), TrOC removal in biological processes is highly variable. Recently, it has been observed that TrOC elimination strongly correlated with increased ammonium oxidizing activities attained during bioremediation of wastewater [13]. Through cometabolic biodegradation, the nonspecific enzyme ammonia monooxygenase (AMO) may degrade a broad range of aliphatic and aromatic molecules [14]. Biodegradation of TrOCs by AOB was discovered in nitrifying activated sludge (NAS) when greater ammonia oxidizing activities were obtained, with a wide range of TrOCs being degraded. A chemical called allylthiourea (ATU) can slow down the process of nitrification [15]. This means that TrOCs were more likely to stay in the water when ATU was used. This suggests that this enzyme, through cometabolism, is the one that gets rid of TrOC. In order for AOB to make cometabolic biodegradation, there must be a growth source, like NH4 [16]. Heterotrophic bacteria have also been seen to degrade TrOCs, either alone or in combination with AOB, mostly by metabolism. AOB may change organic micropollutants (OMPs) via direct and indirect enzymatic processes. Biologically generated reactive nitrogen species (nitric oxide (NO), nitrite (NO2), and hydroxylamine (NH2OH)) may chemically change OMPs by hydroxylation, deamination, and nitration, and thus assist considerably to the noticed OMP transformations. OMPs with functional groups such as ether, aliphatic hydroxyl, alkyl, and sulfide as well as aromatic primary amines and replaced aromatic rings may be biotransformed by AMO. OMP biotransformation efficiencies and rates are greater in AOB-dominant microbial communities than in non-AOB-dominant microbial communities, especially in autotrophic reactors carrying out nitridation or nitrification. The following two main lines of evidence, the biotransformation of OMPs in wastewater treatment systems, may frequently be connected to ammonium (NH4+) removal: (i) the same transformation products – namely, disseminated, nitrogen – containing, and hydroxylated transformation products – found in AOB pure cultures are also present in wastewater treatment systems; (ii) for solitary OMPs in diverse systems with identical AOB and rNH4+ abundances, constant biotransformation of OMP (rbio, mol/g VSS/day) to elimination of NH4+ (rNH4+, mol/g VSS/day) rate ratios (rbio/rNH4+) is seen. We get to the conclusion in this chapter that AOB are the primary catalysts for OMP biotransformation during wastewater treatment operations. This data, together with other research,

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shows that nitrification-capable systems are more likely to be built and run than nonnitrifying systems that result in the abatement of certain pollutants [17].

2 Nitrification Nitrification, which is the biological oxidation of ammonia to nitrate from nitrite, is a cost-effective and long-term way to get rid of ammonia because it does not need chemicals [18]. When nitrifying, the first step is to make N–NO2. The second step is to make N–NO3 [19–21]: NH4 + + 1:5 O2 ! NO2 − + H2 O + 2H + −

NO2 + 1=2 O2 ! NO3

−

(1) (2)

Nitrification, or the two-step oxidation of ammonia into nitrate from nitrite, is a crucial step in the removal of biological nitrogen from wastewater in industrial and domestic wastewater treatment plants (WWTPs). AOB turn ammonia into nitrite, as the first step in the process of nitrification. In WWTPs, nitrification has long been thought of as a process that is both imprecise and uncertain because of how slow these bacteria grow and how easily they can be affected by things like weather and other things in the environment. Engineers and microbiologists have been studying how nitrification works and how AOB communities are structured for a long time now. It has been hard to study AOB’s physiology and ecology with cultivation-based methods. Because of advances in molecular methods, we’ve only learned a lot about these kinds of biological processes in the last 20 years. Many AOB 16S rRNA gene sequences from different places have been observed, which has made it possible to use rRNA-based techniques to figure out how AOB should be characterized in WWTPs. Another way to look for AOB was to look at the full-length sequence of the gene that makes the a-subunit of Nitrosomonas europaea’s ammonia monooxygenase (amoA) [22]. It has been determined that all AOB in wastewater belong to the b-proteobacteria based on phylogenetic analysis of amoA and 16S rRNA genes. Nitrosomonas oligotropha, Nitrosomonas europaea/Nitrosococcus mobiliz, and Nitrosomonas communis are the three most common Nitrosomonas species. Nitrosospira species can grow under certain conditions (like low temperature and low pH) and in industrial WWTPs [23, 24]. In situ hybridization with a set of fluorescently labeled hierarchical 16S rRNA-targeted AOB probes has also been used a lot to figure out composition of AOB in WWTPs [25–28]. There are usually only one or two AOB species in a WWTP, but some systems have a lot more AOB species than others. AOB in activated sludge flocs and biofilms has also been precisely measured using fluorescence in situ hybridization (FISH) [29, 30]. FISH is a good way to look for cells that have been labeled, but in materials that have a lot of self-fluorescence, it can be hard to see the labeled cells (e.g., industrial wastewater) [31]. Moreover, FISH has a very high detection threshold., requiring a concentration of 103–104 cells/mL to be effective for

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counts [32]. Quantitative polymerase chain reaction (PCR) has recently been used to figure out how many copies of 16S rRNA or the amoA gene there are of AOB [33]. This method is a reliable, sensitive, and quick tool that can be used in place of FISH, but it is limited by DNA extraction efficiency and PCR biases [34]. This method can be used in place of FISH. It has been very common to use standard PCR to make the 16S rRNA or amoA gene segments, then run them through denaturing gradient gel electrophoresis (DGGE) to get an AOB community fingerprint. The DGGE bands can also be sequenced for a comparison of AOB diversity, but the phylogeny may be skewed because the gel can separate only pieces shorter than 500 bp. This could make the phylogeny look a little different [35]. DGGE, on the other hand, can be used to look at time series and AOB population dynamics in a short amount of time because it can run and look at a lot of samples simultaneously [36].

NO3––N

CAO

nxr

B

hao

NO

– +H 2

NO 2

B

nxr NO2––N

e–

+ +2

H

– +2

NH

NO 3

2 OH

O→

amoA NH4+–N

amo

NH2OH

hao

NO2––N

NH4++O2+H++2e–→NH2OH+H2O NH2OH+H2O→NO2–+5H++4e– AOB Figure 1: Dissimilarity between comammox bacteria nitrification and traditional microbial nitrification [37].

Figure 1 depicts bacteria that may convert ammonium to nitrate inside a single cell. This fundamentally altered how we think about the conventional two-step nitrification procedure [37, 38]. Comammox bacteria have also been found in a variety of environments, including WWTPs, soil, lake sediments, natural wetlands and drinking water systems [39–41]. These bacteria are also found in soil. In contrast to other nitrifiers, their wide distribution has led to a lot of attention about how important they are to the environment. Despite the fact that comammox bacteria have been discovered in water treatment systems, it is unclear what function they play in the biological removal of nitrogen [42]. Comammox bacteria make up up to 100% of the amoA genes

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in some WWTPs [43]. They are important in water treatment systems for nitrification [44]. Comammox bacteria could be used to improve the efficiency and stability of residential wastewater treatment, cut the costs of sewage treatment, and reduce greenhouse gas emissions, among other things.

3 Ammonia-oxidizing microorganisms The ammonia oxidizers (AO), which are made by two main microbial groups: ammonia-oxidizing archaea (AOA) and AOB, are the first and most significant step in the nitrification process in a wide range of environments, like soil, water, and air. It is a crucial two-phase process for ammonia (NH4) to be converted into nitrate (NO3–) through nitrite (NO2) (Figure 2) [45]. Archaeal AMO genes are found in AOA, which are prokaryotes that can make AMO and oxidize ammonia. They have been found in activated sludge systems, where they can reduce ammonia and make AMO. Nitrosopumilus maritimus is one of the most common AOA species found in WWTPs [46], but there are many other species. It has been shown that Nitrosopumilus maritimus has genes that are thought to be from archaea. It has been postulated that the archaeal AMO genes in Nitrosopumilus maritimus are employed to transcribe the assumed archaeal AMO and carry out ammonia oxidation [47, 48]. Because it does not have the genes for the enzymes that make hydroxylamine oxidation, nitroxyl has been thought of as a unique archaeal way to get rid of ammonia [49]. In this scenario, nitroxyl might be formed by the sudden degradation of HNOHOH, produced by archaeal AMO inserting two oxygen atoms to ammonia. Nitroxyl oxidoreductase (NXOR) then converts nitroxyl to nitrite. When NXOR gets an electron, it sends it to the quinone pool. This creates the proton motive force for the ATP-making process. Also, NO has been thought of as an intermediate product in the ammonia oxidation process of archaea. During this process, NO is used as a “redox shuttle.” After ammonium was added to the culture of Nitrosopumilus maritimus, there was a short rise in the NO level [50]. The low Km value of Nitrosopumilus maritimus indicates that it has a strong affinity for ammonia, making it a viable choice for surviving in wastewater with low ammonia concentrations. In a moving bed bioreactor (MBBR) with low ammonia content, Roy et al. discovered that AOB was dominated by AOA [51]. This means that when ammonia levels are low, AOA contributes more nitrification than when ammonia levels are high. Retention of biomass is critical for superior performance because of AOA’s sluggish growth rate. Because biofilms may maintain bacteria with extremely sluggish growth kinetics, AOA is mostly used in biofilm procedures [51]. When regulating oxygen supply for selective suppression of NOB activity, in a single-stage membrane aerated biofilm reactor, it was shown that AOA was much more resilient than AOB (MABR). It is also recommended that MABR be operated in an oxygen-limited mode for improved

AOB

AOA

b

a

NH4+

NH4+

AMO

AMO

1890 the first report of autotrophic AOB (Winogradsky, 1890

1800

1872 the first report of nitrification in acidic soils (Houzean, 1872)

NH2OH or NOH

NH2OH

?

N2O

Hybrid formation

Nitrifier-denitrification

NO2–

N 2O

Nitrifier-denitrification

NO2–

Nitrification pathway

HAO

Nitrification pathway

1916 Increasing evidence of widespread occurrence of nitrification in acidic soils. (Fred and Graul, 1916; Noyes and Conner, 1919)

Nitrite oxidation

NO3–

N Fixation

NH4+

Ammomia oxidation

2000

2005 Unknow nitrifiers rather than AOB controlled the acidic soil nitrification. (Jordan et al., 2005)

N2

Anammox

N2O

Denitrification NO

NO2–

2001 Substantial evidence for AOB dominance in acidic soils was documented. (De Boer and Kiwalchuk, 2001)

1996 Detection of AOB 16S Rrna amd amoA genes from a variety of acidic soils. (Stephen et al., 1996, Laverman et al., 2001)

NO2– Nitrification

1977 Active nitrification under acidic conditions was attributed to heterotrophic nitrifiers. (Focht and Verstrate, 1977)

1974 Ammonia rather than ammonium was thought to be substrate for AOB. (Suzuki et al., 1974)

1990

1908 Nitrification in acidic soils was believed to take place in isolated calcium carbonate particales. (Hall et al., 1908

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Figure 2: (a) History of nitrification and ammonia oxidizing bacterium discoveries, and (b) the AOB/AOA route within nitrification is highlighted as one of the key stages in the nitrogen cycle [45].

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performance, which might reduce oxygen supply by 5% and result in a significant yearly operating saving of 2–3% when applied to the full WWTP. This is because of changes in oxygen and ammonia loadings, the AOA system has a much wider range of operating times for high-level total nitrogen removal [52]. AOA was more common in residential wastewater treatment systems than AOB was in industrial wastewater treatment systems [53–55], but the opposite was true in industrial wastewater treatment systems. Eight wastewater treatment systems which include industrial and domestic systems were evaluated in Beijing, and it was discovered that the concentration of AOB was about three magnitudes greater than that of AOA. The researchers found considerable abundance of AOA in four industrial wastewater treatment systems, with AOA presence in one of the systems being four orders of magnitude more than AOB. In another study, high quantities of spiramycin induced a considerable rise in the relative concentration of AOA in pharmaceutical wastewater treatment systems. Even though AOA has in recent years been made and enriched well in pure medium [56], there is very little information about how AOA is enriched in practical wastewater nitrogen removal systems. Sonthiphand and Limpiyakorn used an inorganic medium that had almost equal amounts of archaeal and bacterial amoA genes to boost the number of ammonia-oxidizing microorganisms in activated sludge [57]. As the cultivation time increased, AOA started to disappear from the ammonia-oxidizing collaborations in every reactor. A bigger number of bacteria can live attached to biofilms than can live suspended in floc activated sludge during long generation times. When researchers looked at biofilm samples from trickling filters and MBBRs that treat urban wastewater, they found that the archaeal amoA gene was more common than the bacterial amoA gene by 2–3 orders of magnitude. Biological aerated filters (BAFs) used to treat municipal waste have biofilms that behave in a similar way. Researchers used synthetic medium to grow a single AOA strain isolated from the filtering materials. It is unknown how AOB and AOA are distributed throughout various wastewater treatment systems, according to the literature we looked at. The ammonia content and organic load of the treated wastewater as well as the temperature and dissolved oxygen (DO) concentration may have an impact on the research results [58]. Numerous researches have been conducted to determine the evolutionary diversity of AOBs since their initial isolation in 1890. This is because of these studies: Five groups of AOB have been found and put in the Proteobacteria class. Out of these, a single Nitrosococcus cluster relates to the c-Proteobacteria subclass, whereas Nitrosolobus, Nitrosovibrio, Nitrosospira, and Nitrosomonas (including Nitrosococcus mobilis) belong to the b-Proteobacteria subclass. However, until the advancement of culture independent molecular techniques, investigating due to the difficulty and length of time involved in the production of these microorganisms, enumerating AOBs and determining their diversity and abundance in engineered systems or in their natural settings has remained a challenging task when employing standard culture-dependent techniques. Furthermore, owing to possible disruption in microcolonies and flasks, faulty cell suspension, or some cellular harm, culture-dependent approaches are likely

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to underestimate real cell counts. FISH, PCR, qPCR, DGGE, and quinone profile techniques, on the other hand, have made it easier to find and identify AOB communities without having to grow them in a lab. In order for an organism to be able to live in a certain environment, it has to have a certain ecology, phylogeny, and even morphology. All of these things play a part. How groups of bacteria that eat nitrogen, like nitrite-oxidizing bacteria (NOB) and AOB, work together can be found out with ecophysiological methods. Because the first two bacteria are phylogenetically distinct, each will have its own metabolic activities in terms of kind and productivity, which will vary based on the bacterial cluster and lineage. AOBs may be split into three classes, each with its own eco-physiological properties and favored environments, as shown in Figure 1. The Nitrosomonas genus, which is split into six lineages, is the first group. There are four species in the first lineage. These species are Nitrosomonas mobilis, Nitrosomonas halophila, Nitrosomonas eutropha, and Nitrosomonas europaea. This indicates that all need a lot of salt, since they have a substrate affinity of 30–61, implying that salt is required. As illustrated in Figure 1, it may be separated from sewage treatment facilities, eutrophic fresh water, and brackish water. The second Nitrosomonas lineage, which includes three species Nitrosomonas sp. II, Nitrosomonas sp. I, and Nitrosomonas communis, needs no salt, has no urease activity, and has a lower substrate affinity of (14–43) l M, and is found in nonacidic soils. There are Nitrosomonas species in the third branch that do not need salt, but they have positive urease activity and can get rid of waste. This branch is good at getting rid of waste. A lot of times, it can be found in fresh water that is rich in nutrients. Another lineage of Nitrosomonas is made up of two species, Nitrosomonas oligotropha and Nitrosomonas ureae. These two species are part of this fourth lineage. In this case, these two species don’t need salt, have positive urease activity, and prefer oligotrophic fresh water and natural salt as their favorite places to live. Nitrosomonas cryotolerans, Nitrosomonas aestuarii, Nitrosomonas sp. III, and Nitrosomonas marina make up the fifth and sixth lineages. They both need to be able to live in salt water, have positive urease activity, and be spotted in aquatic environments, and have the greatest affinity for substrate (50–52) and (42–44) l M for their food, respectively, which is why they make up the fifth and sixth lineages. There are two types of AOBs. There are a lot of Nitrosolobus multiformis and Nitrosovibrio tenuis and Nitrospira sp. I, which live in soil, rocks, and water, that have the same ecophysiological characteristics as Nitrosolobus multiformis and Nitrosovibrio tenuis. The final category includes the obligatory halophilic Nitrosococcus halophilus and Nitrosococcus oceani, both of which are found in marine environments with the former showing positive urease activity and the latter showing negative urease activity. There are a lot of different types of engineering systems that you can choose from; there are continuous stirred tank reactors (CSTR) and sequencing batch reactors (SBR). In addition, there are MBBR and single reactors for high activity ammonia removal over nitrite (SHARON) reactors that use moving bed biofilms to remove ammonia from the air (e.g., freshwater and raw wastewater). According to ammonia affinity, there are two types of AOB: (1) r-strategists, which have a large growth rate but a low ammonia affinity, and

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thus prevail in circumstances of high ammonia levels, and (2) k-strategists, which have a low growth rate and a high ammonia affinity, and thus prevail in circumstances when ammonia is limited. Tests with FISH in freshwater with low ammonia concentrations showed that N. marina was better than N. europaea at high ammonia concentrations, but N. europaea was the leads at low ammonia concentrations. AOB communities in 12 WWTPs were studied using DGGE. In 11 WWTPs with high ammonia concentrations, N. europaea and N. eutropha were the most common species in the communities of AOBs. When there were low ammonia concentrations in 11 WWTPs, N. ureae was the one that was most common. Furthermore, N. mobilis (a member of the Nitrosomonas genus) was shown to be prominent in salty wastewaters. Furthermore, it may be separated into two basic systems in terms of reactor configuration: suspended growth reactors and connected growth reactors. In the case of suspended growth systems, N. eutropha and N. europaea were discovered using DGGE fingerprinting and PCR amplification in a partial nitrification SBR treating landfill leachate with extraordinarily high nitrogen concentrations. A partial nitrification SBR also changed the shape of AOB communities, making them smaller and allowing more microcolony dispersal in flocks. However, qPCR analysis found that the dominating AOB cluster in a partial nitrification CSTR treating synthetic wastewater with higher ammonia levels and no organic carbon was N. europaea, indicating that it might be part of r-strategists. Furthermore, a 16S rRNA gene study indicated that N. eutropha was the dominant clone in the SHARON, accounting for 69% of the clones. In the context of attached growth systems, the variety of AOB communities was evaluated utilizing PCR analysis targeting 16S rRNA gene sequence combination with DGGE in a BAF and a trickling filter treating similar effluent. There was a disparity in the community structure between the two methods, with a greater variety of AOB in the trickling filter than in the BAF, despite the fact that N. mobilis predominated all of the samples examined in both reactors [59].

4 Parameters affecting AOB Partially nitrification has recently been popular as a first stage in both deammonification and nitrite shunt processes to remove nitrogen from wastewater efficiently and economically. In order to achieve effective partial nitrification, the first phase of nitrification must be stimulated while the second step is inhibited, resulting in an accumulation of AOB. Knowledge of their microbiological features and kinetics factors, as well as the primary parameters that might selectively restrict NOBs’ development or enable AOBs to outcompete them, is required for successful AOB accumulation. It was possible to achieve partial nitrification with bioreactors that had different topologies, like suspended and attached growth. Furthermore, in wastewater treatment facilities, a number of bioreactor designs, each with its own set of benefits and downsides have been used to partially nitrify the wastewater. Furthermore, a variety of bioreactor

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layouts have been employed to achieve partial nitrification in wastewater treatment facilities, each with its own set of benefits and downsides. These biological systems are either based on attached growth technologies, in which bacteria are grown in biofilms or suspended growth technologies, in which bacteria are grown in flocs (a collection of individual cells or tiny colonies that develops in a reactor under certain circumstances or when the medium has been treated with a certain agent). It is a complex, well-coordinated cellular products and structure of cells that may develop as big, thick particles or be connected to a suspended carriers or static solid surface. By collecting AOB and blocking NOB, effective partial nitrification might be obtained. The ability to accumulate AOBs relies on understanding the factors that influence their development. These variables might include the presence of free ammonia (FA) in the reactor, alkalinity, solid retention time (SRT), hydraulic retention time (HRT), pH, temperature, and DO concentration.

4.1 Dissolved oxygen concentration Managing the DO content in the reactor might help increase nitrite build-up and, as a result, AOB accumulation. It is based on the known discrepancies in the Monod oxygen saturation constants for NOB and AOB, which are 1.1 and 0.3 mg/L, respectively. It may be hard for NOB to grow when there isn’t enough oxygen. This is because AOB has a stronger desire for oxygen. Low levels of DO (0.5 mg/L) have negligible impact on the oxidation of ammonia. It was found that high levels of DO (2.5 mg/L) had the most impact on nitrite oxidation. During the time when DO levels were low, AOB grew faster, but NOB growth was not impacted. Even though there may be a lot of filamentous thickening and nitrification, there may be less DO in the water, which could make it less healthy for people to drink. Researchers, on the other hand, found that there is no apparent DO concentration for optimal nitrification. The impact of oxygen diffusion with the flocs, according to this research, is one probable cause for the vast variance in DO concentration found. This might justify the wide range of critical DO concentration values for partial nitrification control reported in the literature. There have been many ways to help nitrite build up in water with a wide range of DO concentrations like use of connected growth reactor systems and suspended growth. It was found that, there were 5.33 × 108 AOB cells/mL in suspended growth systems with a DO concentration of 0.4–0.5 mg/L and nitrites didn’t build up at all at 1.5–2.5 mg/L of DO in the water. This means that full nitrification had taken place and low levels of DO (0.2 mg/L) means that nitrite did not build up. The average nitrite build-up rate was just 1.7%. When there is a lot of DO in the water, it can slow down nitrite build-up. At a DO level of 2.0–4.0 mg/L, the rate of nitrite build-up in an SBR went from 95.43% to 3.09%. When the DO concentration was reduced to 0.8 mg/L, the rate of nitrite accumulation went down from 95.43% to 93.7%, which is a lot less than what it used to be. Another study says nitrification can take place at DO levels of 0.5–1.0 mg/L if the SRT is at a high enough level. At a

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lower SRT, more DO may be needed to get the same amount of oxygen. This was shown in a 3-day SRT test in a CSTR, where the concentration of DO required to sustain partial nitrification came to be 1.54 ± 0.87 mg O2/L. Often, the transport of oxygen from a liquid to a biofilm limits the conversion rate in biofilm systems. It took about 1.5 mg/L of DO for a biofilm airlift suspension reactor to get a lot of nitrite and good ammonia conversion at the same time. It takes longer for ammonia oxidation to happen when there is less than 1 mg/L of DO. This means there are less nitrites in the effluent. In addition, when the reactor’s DO level reached 2.5 mg/L, all of the ammonia in the reactor was converted to nitrate, so there was no more ammonia in the reactor. In a static sequencing batch worm reactor, NOB was similarly shown to be blocked by a DO dose of 1.5 mg/L. The higher ammonia rate of oxidation at low oxygen levels compared to the nitrite oxidation rate may be attributed to NOB localization in the inner area of biofilm as well as the low oxygen half-saturation constant for AOB.

4.2 Ammonia level The concentration of ammonia in the atmosphere has a substantial effect on the development of AOB and AOA because ammonia is a common substrate (nitrogen source) for both types of AOB. Because AOA has a stronger affinity for ammonia than AOB, the inhibitory concentration for AOA is smaller. AOA may experience the suppressed condition sooner than AOB if exposed to a greater ammonia concentration. It was observed that when the quantity of ammonia in a municipal WWTP increased, the proportion of AOA amoA gene in rotating biological contactors decreased, showing that low ammonia levels suited AOA well. At high ammonia levels, AOB was more competitive than AOA, and the abundance of AOB increased with increasing ammonia levels. The amount of AOA did not change significantly at different ammonia nitrogen concentrations (14, 56, and 140 mg/NL). It was also observed that AOA reduced considerably when the quantity of ammonia in the nitrification tank for salty wastewater treatment was raised from 200 to 300 mg/L, but the abundance of AOB remained constant. Furthermore, AOB dominated the ammonia oxidation process in a landfill leachate treatment system with a high amount of ammonia concentration (2,180 ± 611 mg/NL). Changes in the microbial community structure of AOB and AOA were shown to be caused by changes in ammonia levels, which were influenced by the kind of wastewater.

4.3 Temperature Ammonia can be oxidized in partial nitrification systems if the temperature is too hot or too cold. This happens more quickly in the summers, but it can also happen at any time of year. The temperature effect on the elimination of nitrogen from batch biofilm

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reactors was looked into, and it was found out that Ammonia was easier to get rid of when the temperature in the reactor went up. A temperature of 25 °C removed more than 80% of the ammonia from the water. At 15 °C and below, less than 30% was removed. Furthermore, it was discovered that activated sludge facility was used to develop nitrite, particularly during the summer. It is found that at high temperatures, NOB is going to grow slower than AOB. During very cold weather, NOB is much more likely to grow than AOB. At temperatures between 10 and 20 °C, Nitrobacter, a species which is known to dominate during nitrification, was found to be active. This means that the temperature effect of Nitrobacter activation won out over the effect that FA inhibition had. Other than that, when the temperature was raised to between 20 and 25 °C, it made the ammonia oxidation process work better as shown in graph below. In addition, a decrease in nitrate content at the reactor’s outflow was seen when the temperature of an inverse turbulent bed reactor was raised from 30 to 35 °C. If the right conditions were in place, some partial nitrification could be done at lower temperatures if AOB was able to beat NOB. Temperature also impacts the chemical balance of free nitrous acid (FNA) and FA, which affects partial nitrification efficacy. FA concentrations rose to 122.92 ± 27.23 mg N–NH3/L on average at 35 °C from 20.76 ± 4.23 mg N–NH3/L on average at 25 °C in an SBR, but FNA levels decreased to 0.12 ± 0.02 mg N–HNO2/L from 0.47 ± 0.09 mg N–HNO2/L. Partial nitrification reactors are often operated at temperatures between 30 and 35 °C.

4.4 Free nitrous acid (FNA), free ammonia (FA), and pH Many times, pH control is used to make partial nitrification happen because the balance of FA and FNA, which block both AOB and NOB, changes with the pH. There have been reports of NOB inhibition at 0.1–1.0 mg FA/L. AOB inhibition takes 10–150 mg FA/L. Nitrite oxidizers seem to be more delicate to FA than other types of oxidizers. In a similar fashion, NOB was suppressed in batch reactors at concentrations ranging from 0.1 to 4.0 mg FA/L in other studies. In other experiments, NOB was suppressed at concentrations ranging from 1 to 5 mg FA/L in anaerobic–aerobic treatment of high-strength ammonium wastewater, but AOB was inhibited at values more than 7 mg/L and halted at 20 mg/L and 0.1–4.0 mg FA/L in batch reactors. One thing that has been proven is that FA does not kill NOB, nitrite oxidizers can adapt to high levels of FA and reactivate after long periods of cultivation, so they can work again. The following is the link between FA concentration and pH: FA ðmg=LÞ = 17=14 ✶ NH3 − N ✶ 10pH =10pH + exp ð6, 344= 273 + TÞ Nitrite oxidizers are much more reactive to FNA than ammonia oxidizers, in addition to FA concentration. FNA levels of 0.42–1.72 mg N/L reduced AOB activity by 50%, whereas lower concentrations of 0.011–0.07 mg N/L began to inhibit NOB, and 0.026–0.22 mgN/L

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could totally hinder NOB. Furthermore, FNA functioned as an uncoupler in an activated sludge by donating a proton within the cell, thus interfering with the transmembrane pH gradient necessary for ATP generation, resulting in inhibition. The following is the connection between pH and FNA: FA ðmg=LÞ = 46=14 ✶ NO3 − N =10pH ✶ exp ð−2, 300= 273 + TÞ As a result, most research says that a pH of 7.5–8.5 is best for reducing NOB. When it comes to Nitrobacter species, the ideal pH is between 7.2 and 7.6. Nitrosomonas species, on the other hand, need a pH between 7.9 and 8.2, while Nitrobacter species need between 7.2 and 7.4. A pH of 7.5–7.8 was also indicated to facilitate partial nitrification. Another thing to note is that, in a nitrifying biofilm activity, nitrite began to build up above pH 7 and rose to 85% at pH 8. Another study found that a pH greater than 7.5 was needed to inhibit NOB and build up nitrite.

4.5 Hydraulic retention time (HRT) and solid retention time (SRT) Because the minimum doubling time of AOB is 7–8 h less than that of NOB (which is 10–13 h), HRT and SRT alterations might cause changes in the microbial population in wastewater treatment facilities, resulting in the washing out of NOB populations. However, connected growth systems or biomass recycling that boost the SRT relative to HRT have been explored to produce decoupled SRT and HRT partial nitrification reactors. Several researches have looked at the impact of HRT on partial nitrification reactors. If we talk about suspended growth systems, a nitrite accumulation rate of 96% was attained in an activated sludge after 9.1 h of HRT. In addition, the impact of HRT on partial nitrification in an SBR utilizing precultured aerobic granules in a continuous flow reactor was examined. Both the ammonia elimination efficiency and the nitrite build-up rate approached 90% at HRTs of 12 and 7.2 h. At a HRT of 2.4 h, the ammonia removal efficiency declined and varied (20–56%). As compared to that, the best HRT in an SBR handling acrylic fiber wastewater was 20 h. The ammonium clearance rate was 97% at this HRT, with an 87% nitrite build-up. Ammonium was turned into nitrite in an MBR as long as the HRT was kept at 10 h. Biofilms were also looked at in a plastic SHARON bioreactor that was used to make a submerged bio filter with PVC carriers. In addition, the effect of HRT on biofilms was looked at. People found that a HRT of 12 h turned all the ammonia into nitrite. A HRT of 9.6 h only turned 60% of the ammonia into nitrite. A lot of Nitrosomonas species, which can make nitrite from ammonia, formed biofilms after the 12 h HRT. Operating the reactor at a 9.6 h HRT, on the other hand, aided the production of heterogeneous biofilms, allowing for a tighter ammonium-to-nitrite ratio. If you want to grow more AOB in a hybrid moving bed biofilm reactor, you need a high turnover rate of 9.5 h. The SRT significantly affects bacterial communities in partial nitrification reactors. Based on observations in a CSTR, controlling the reactor at an SRT of 3 days led to NOB

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washout in suspended growth systems. On the other hand, it was discovered that sludge that had been around for a while responded well to partial nitrification. With a nitrite accumulation rate of 95%, an SRT of 10–3 days was shown to be more appropriate for AOB accumulation in an activated sludge process. Additionally, continuous partial nitrification deteriorated with shorter SRTs while the nitrite build-up rate was decreased from 93% to 37% when the SRT was increased to 16 days.

4.6 Organic loading The development of AOB is objectively influenced by organic materials. AOB are autotrophic bacteria, while AOA are not sure whether they are purely autotrophic or mixotrophic. Organic compounds were shown to have a considerable inhibitory impact on the development of several AOA strains, including Nitrosocaldus yellowstonii and Nitrosopumilus maritimus SCM1, according to various researches. The newest research discovered that adding organic molecules to AOA strains HCA1 and PS0 might boost their development, demonstrating their mixotrophic features. It was also shown by genome sequencing that certain AOA strains have the capacity for both autotrophic and heterotrophic metabolism. These AOA strains possessed the 3-hydroxypropionic acid/4-hydroxybutyric acid cycle (autotrophic metabolism) and the tricarboxylic acid cycle (heterotrophic metabolism). AOA and AOB have different capacities for oxidizing ammonia because AOA may have more complex metabolic pathways than AOB and display distinctive metabolic characteristics depending on the carbon source.

5 Technique used for detection and quantification of AOB One of the main goals of microbiology is to be able to quickly and accurately identify bacterial populations in their native habitats. Because methods based on culture take a long time and can be too picky, especially for hard-to-culture or unknown bacteria, they do not show how mixed bacterial communities or microbial diversity are made up. PCR and hybridization or sequencing, as well as FISH, have changed many aspects of microbiology in the last few years, making it easier to find and identify bacteria with great precision [60].

5.1 Fluorescence in situ hybridization (FISH) FISH is a molecular method used to detect and visualize specific microorganisms in both artificial and natural settings. Hybridizing fluorescent probes to complementary

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target sequences on microscope slides is the first step in this method. Then analyzing the fluorescence signals to figure out which nucleic acid sequences (16S rRNA) are paired with the probes is the next step. However, since bacterial cells are normally frozen and hybridized on microscope slides rather being processed in solutions, this procedure is impractical for processing several samples in parallel. However, since bacterial cells are normally frozen and hybridized on microscope slides rather being processed in solutions, this procedure is impractical for processing several samples in parallel. Furthermore, cell fixation and hybridization for particular environmental samples and Gram-positive cells may be difficult.

5.2 Denaturing gradient gel electrophoresis (DGGE) and polymerase chain reaction (PCR) Techniques that do not require bacteria to be isolated have become more popular and are now used more often to identify bacteria in both natural and man-made setups. There are many steps in many molecular procedures that start with the extraction of nucleic acids from microbial populations, such as DNA and RNA, to start with. Many ways have been suggested to reach this goal. The DNA that was found in the bacteria is used as a target in a PCR to make some genes amplified. When we use a technique called reverse transcriptase-PCR, we convert RNA into cDNA and then use the PCR to make more of the RNA, which can then be used to make more DNA. In both circumstances, the final product happens to be a combination of DNA fragments collected from various cultures. In another research, researchers looked at complicated combinations of microorganisms in a new way; they found another way to look at them that was based on molecules. They did not put the PCR results into E. coli and then sequence random clones. They used a method called DGGE to tell which PCR products came from different species. DGGE can be used to find DNA fragments that have different base-pair sequences but the same length, which can help you find them. The speed at which PCR-amplified DNA fragments move through polyacrylamide gels with a linearly increasing gradient of denaturants is used to separate them from each other. Furthermore, by comparing the sequences to known 16S rRNA sequences and sequencing the individual bands of the DGGE gel, the phylogenetic affiliation of the discovered bacteria may be deduced. In an intermittent aeration sequencing batch process (SBR, plant A) and 12 additional livestock WWTPs, bacterial populations and beta proteobacterial AOB communities were investigated seasonally. The Nitrosomonas ureaeoligotropha-marina cluster was prominent in two plants with low quantities, whereas the Nitrosomonas europaea-eutropha cluster amoA sequences were dominant in 11 plants with high ammonia-nitrogen concentrations in the raw wastewater. 16S rRNA gene sequence analysis was used to identify undeveloped Nitrospira-like bacteria in several activated-sludge and biofilm samples. In this study, at least four different types

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of Nitrospira have been found. The Nitrospira phylum of bacteria were found for the first time in wastewater treatment systems using PCR and cloning. A competitive PCR analysis revealed that Nitrosomonas oligotripha-like cells were outnumbered by Nitrospira sp. cells in the municipal WWTP by more than 62 times. It was suggested by researchers to use Nitrosomonas marina-like AOB instead of the Nitrosomonas europaea– Nitrosococcus mobilis cluster AOB to start nitrifying in aquatic systems. The community, diversity, and DGGE structure of the betaproteobacterial AOB in two full-scale treatment reactors – a trickling filter and a BAF-receiving the same wastewater were studied using PCR of 16S ribosomal RNA (rRNA) gene fragments using AOB-selective primers. The community structure of AOB was discovered to be varied in various portions of each reactor, as well as variances between all AOB-like sequences and the reactors observed within the genus Nitrosomonas. Although the AOB most closely linked to Nitrosococcus mobilis seemed to predominate in all samples investigated, trickling filters showed a greater range of AOB than the BAF. In the study tests on samples from 12 different sewage plants showed that Nitrosomonas oligotropha members were found in all of them, even though the plants used different ammonia removal and treatment strategies over the course of three different years. Ammonia oxidizer numbers changed from time to time, but the number of ammonia oxidizers did not change very much at all.

5.3 Next-generation sequencing (NGS) Succeeding sequencing (NGS) enables both massively parallel nucleotide sequencing over a target RNA or DNA sections or complete genome. This technique has been widely used to the study of environmental microbiomes. There are many ways to use NGS to learn more about the microbial communities in drinking water and how they work. It can also be used to keep an eye on the growth of bacteria and pollution in drinking water. The four primary phases in a typical NGS procedure are: (i) library preparation; (ii) amplification; (iii) sequencing; and (iv) data processing. In the first step, the sample’s target DNA is extracted, and a library is created by fragmenting the target DNA and adding specialized connectors to both ends. The matching sequences in these adapters enable the DNA fragments to attach to the flow cell. To boost the signal, the library is amplified in the second stage utilizing clonal amplification techniques and PCR. In the third phase, a sequencing tool is used to sequence all of the DNA in the library. It creates a large quantity of data, which the instrument software interprets. The NGS approach has a better sensitivity for detecting low-frequency variations, which is a significant benefit. The NGS approach, on the other hand, generates a large quantity of data that requires professional analysis to deliver a succinct conclusion.

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5.4 Flow cytometry method (FCM) For measuring bacterial concentrations in drinking water, flow cytometry provides a quick, reproducible, and reliable approach. It employs a laser light source to generate optical signals that are scattered from particles or produced by auto-fluorescing cells or fluorescently tagged cells for the purpose of identifying and quantifying them. The sample, which may include cells or particles, is suspended in a sheath fluid and allowed to pass through the center of the focused laser beam in a single file in order to analyze its optical characteristics. Dye-specific fluorescence, side-scattered light, and forwardscattered light signals are all measured by the detector, which are then translated into electrical signals and processed by a computer to collect data. Flow cytometry employs a range of fluorescent reagents. Fluorescent proteins, fluorescently coated antibodies, nucleic acid dyes, DNA-binding dyes, and viability dyes are some of these reagents. About 0.1% 2-phenoxyethanol, HEPES-buffered saline and phosphate-buffered saline are popular sheath fluids that aid to lower surface tension. Hydraulic retention duration and total bacterial cell counts determined by FCM in drinking water samples have been shown to be highly correlated. FCM has the advantage of allowing you to describe each cell in a population without averaging it. One may gather and analyze the cells of interest that have passed through the flow cytometer using an FCM with fluorescenceactivated cell sorting capabilities. The FCM device, on the other hand, may be rather complicated and prone to microfluidics system obstructions. It is ineffective for cells that are prone to clumping together.

5.5 Cell mass counting A hemocytometer or a counting chamber may be used to count the number of cells while the quantity of nitrifiers is being assessed under a microscope. The intermediate region between the gridlines may be measured using a hemocytometer, which is a microscope slide with several gridlines of predefined size. By multiplying the number of grid cells per unit area by a conversion factor that depends on the chamber capacity and dilution factor, one may calculate the bacterial concentration. Contrarily, the direct cell count technique has a number of disadvantages, such as the possibility of missing microscopic cells that are not visible under a microscope and the fact that it just counts total cell numbers without differentiating between live and dead cells. Nitrifiers and a specific bacterial group are equally challenging to find in a mixed culture. This strategy is suitable when nitrifying bacteria outnumber other bacteria in a sample. Optical density or turbidity measurements may also be used to determine the biomass content. According to recent research, there is a linear correlation between dry matter concentration and optical density. However, few occurrences of postnitrification turbidity increase were noted, but most often it remained steady.

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5.6 Fluorescent antibody test The fluorescent antibody (FA) approach may be used to see and identify bacteria that are difficult to separate or cultivate in environmental samples. Nitrifier strains may be identified by looking for a specific antigen (typically a specific protein on the surface of bacteria). Different FAs are required to identify different nitrifier strains. To see the target distribution under a fluorescent microscope, fluorescent molecules (fluorophore) are attached to the constant region of an antibody. Direct FA (DFA) and indirect FA (IFA) are two types of FA methods. To stain the target protein, the DFA approach employs a single antibody that has been directly coupled to a fluorophore. The IFA test employs two antibodies, one of which binds the target protein and the other of which is detected using a conjugated secondary antibody. One drawback of this strategy is that FAs may bind to EPS in nonspecific ways.

6 Conclusion Since it is engaged in both aerobic and anaerobic processes, nitrite occupies a unique place in the nitrogen cycle. Its accumulation is determined by the balance of its formation and transformation rates. The factors that influence these equilibriums are numerous and diverse. The discovery of AOA defies the long-held belief that ammonia oxidation is solely carried out by AOB, advancing our understanding of the global nitrogen cycle. AOA appears to be crucial in the removal of nitrogen from wastewater. As a result, the nitrogen cycle in a wastewater treatment system must be reconsidered. In practical wastewater nitrogen removal systems, more research into the collaborative, competitive, and inhibitive dynamics in microbial communities is needed. Various environmental variables impact AOB, and AOA has greater environmental flexibility than AOB, allowing for the creation of innovative nitrogen removal systems with AOA dominating ammonia oxidation under extreme environmental conditions (such as low temperature and low oxygen level). The reason that nitrite accumulation is not the norm in aquatic systems, despite delicate balances, is due to the various properties of nitrite-producing and nitrite-removing species. Recent technologies, on the other hand, employ the presence of nitrite as an intermediary in wastewater treatment or other fields. The right management of operational parameters, such as maintaining a high temperature (>25 °C), pH at 7.5–8.5 °C, and DO at 0.5 mg/L, can aid in the formation of nitrite in the system. When compared to traditional denitrification, this concentration of nitrite can be used through a nitrite shunt, which can save money on aeration and reduce or eliminate the requirement for organic carbon input.

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Aishwarya Das, Somakraj Banerjee, Ranjana Das✶, and Chiranjib Bhattacharjee

7 An overview of biochemical and molecular mechanism of ammonia-oxidizing bacteria and their potential application in wastewater treatment Abstract: Anammox bacteria are responsible for carrying out the process of nitrification under anoxic conditions. They are the group of lithoautotrophic bacteria discovered in wastewater plants, but are subsequent inhabitants of all natural environments. Anaerobic ammonia oxidation process occurs in a specific lipid bilayer membrane-bound compartment known as anammoxosome, located in cytoplasm. The membranes are rich in ladderane lipids, unique to biological membranes. The bacterial enzyme ammonia monooxygenase (AMO) initiates the process of anaerobic ammonia oxidation. As, supported by the molecular and biochemical studies, the enzyme model comprises of three subunits with metal centers of copper and iron. The application of this biogenic nitrogen removal process in treating ammonia-rich industrial wastewater is promising and cost effective. This chapter review discusses an overview of the molecular mechanism of the anammox bacteria, the biochemical studies of the bacterial enzyme in oxidizing the ammonia to nitrogen compounds. This chapter also presents a comprehensive review on the technological application of the anammox process exhibited by the bacteria in treating the industrial wastewater.

1 Introduction Ammonia-oxidizing bacteria (AOB), a group of chemolitho-autotrophic bacteria, play the key role in transforming ammonia to nitrite in wastewater treatment plants. They are ubiquitous in nature and inhabit soil, freshwater, marine systems, engineered ecosystems, and also human skin. AOBs are responsible for the rate-limiting step of the nitrification process in the environment, rendering them important in the global cycling of nitrogen. They possess the unique ability to convert ammonia to nitrite and use it as their sole energy source. Besides, AOBs use CO2 as the sole carbon source and O2 as an electron acceptor. Five known AOB genera have been listed and categorized in the ✶

Corresponding author: Ranjana Das, Chemical Engineering Department, Jadavpur University, Kolkata, India, e-mail: [email protected] Aishwarya Das, Somakraj Banerjee, Chiranjib Bhattacharjee, Chemical Engineering Department, Jadavpur University, Kolkata, India https://doi.org/10.1515/9783110780093-007

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Proteobacteria category up to current revisions on ammonia-oxidizing bacteria (AOB). Four of them, including the genera Nitrosomonas (including Nitrosococcus mobilis), Nitrosovibrio, Nitrosolobus, and Nitrosospira, are members of the β-proteobacteria (-AOB) subgroup. The only known species of the Gamma-proteobacteria (AOB) are Nitrosococcus oceani and Nitrosococcus halophilus [1–4]. AOB has been detected in nearly a wide range of habitat in marine and terrestrial environment. AOB also thrive in extreme climatic conditions like hypersaline areas and hot springs. The diversity of AOB in the ecosystem depends upon the type of environmental conditions like pH, temperature, DO, salinity, and ammonia content. Anaerobic ammonium oxidation (anammox), which was initially identified in a laboratory reactor inoculated with wastewater from a wastewater treatment plant, is a process that breaks down nitrogen into nitrate and nitrite. By employing nitrite as an electron acceptor and an anoxic environment, the anaerobic ammonia oxidation (anammox) process turns ammonia into N2 gas. The anammox process has become an efficient and feasible alternative to traditional nitrification/denitrification process. The uniqueness of the process is its ability to convert ammonia directly in nitrogen anaerobically. This makes the process efficient in terms of energy use, a 60% decrease in oxygen demand, a 100% decrease in need for organic carbon, and a 90% reduction in sludge production during wastewater treatment [5–8]. Anthropogenic waste generation and its release into the water bodies are alarming. The nitrogen rich effluents that enter the water bodies are the reason for occurrence of eutrophication and depletion of oxygen. Wastewater loaded with extremely concentrated NH4+ is often treated using chemical, physicochemical, and biological approaches. The conventional biological treatment method used for treating the effluent is activated sludge system, which comprises two processes, nitrification and denitrification [9]. High energy utility, emission of greenhouse gases, and also the operation cost signified that, in the long term this process will not stay sustainable [10, 11]. This method can be used to treat wastewater in place of traditional treatment systems since it is crucial for removing fixed nitrogen from both engineered and natural systems. Anammox is more affordable and environmentally friendly than traditional denitrification since it produces fewer greenhouse gases (such as N2O and CO2) and doesn’t need aeration or organic carbon inputs. In the 1990s the discovery of the anammox process marked the search of related microbial entities and development of alternative energy efficient and feasible technology. Anammox bacteria’s discovery offers a fresh biochemical route for the concept and technological advancement of inorganic nitrogen removal. The anammox bacteria mediate the anammox process, and as of right now, five different genera of these bacteria are recognized as belonging to the phylum Planctomycetes. These five Candidatus genera include Brocadia, Kuenenia, Scalindua, Anammoxoglobus, and Jettenia [12–14]. Studies also state that the abundance of AOB is affected by the seasonal variation rather than the diversity. AOB are the first group of microorganisms to carry out the nitrification reaction in the biogeochemical cycle of nitrogen. In recent times, researchers are paying attention to the anammox process in much detail. The advancement in developing an efficient and cost effective alternative to conventional biological nitrogen removal process is the

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insight of current research. With the discovery of the process named anammox, there came an alteration in the conventional concept of biological nitrogen cycle (Figure 1). The process of anammox is mediated in a specific compartment called anammoxosome, a single membrane bound structure. The overall equation for reaction mechanism of anammox is as follows: NH4 + + NO2− = N2 + 2H2 O ðΔG = −357 kJ=molÞ This review intends to study the biochemical pathway of anammox process along with a comprehensive study on the key enzyme used for the process and also its application in treating effluents rich in nitrogen in wastewater plants. This chapter also briefly covers a critical analysis on the future research on anammox. N2 atmosphere

Nitrogen fixing bacteria and archea Nitrogen from amino acids and other reduced nitrogen

Ammonia (NH4+) Anammox bacteria

Denitrifying bacteria /fungi

Nitrifying bacteria and archaea Nitrite NO2–

Nitrate NO3– Nitrifying bacteria

Figure 1: Nitrogen cycle.

2 Ecology and distribution of AOB AOB were considered as the sole organism to carry out the first step in the process of nitrification. Their distribution and diversity is detected in more or less every ecosystem. The diversity of AOB in extreme climatic conditions has been reported according to previous studies. Habitats like terrestrial and marine have been investigated for the thriving of AOB. High temperature and hypersaline conditions have effects on the abundance of the species. Previous research has shown a varied distribution of salttolerant and halophilic species of AOB. There are several reported species isolated and enumerated from this extreme climate. The experiment based studies have stated the distribution of the species based on the molecular analysis and phylogenetic studies. Few hypersaline ecosystems have been investigated in previous studies in search of diversity of the microbial communities. Monolake has been identified with the domination of communities of Nitrosomonas europea and Nitrosomonas eutropha in the study of Carini and Joye [15]. Nitrosomonas halophila, Nitrosomonas marina, and

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Nitrosomonas communis species are reported to be detected in Salae de Huasco by the scientists [16]. Nitrosomonas species have been reported to be found in acidic hot springs, hydrothermal vents. Researchers have detected the growth of AOB species at 46 °C and the species diversity happened to be noticeable [17]. Molecular analysis and studies by the scientists have revealed the stability of the enzymes of AOB in high temperature and thus their existence. Phylogenetic evidence based on 16s rRNA sequencing has revealed the existence of AOB in marine ecosystems. They are found in all possible seas and oceans like Arctic Ocean, Antarctic Ocean, and Mediterranean Sea, besides oceans AOB are also present in estuaries, fresh water, brackish water, and anoxic sediments [18, 19]. A study by Christman et al. [20] stated that the abundance of the bacterial enzyme β-AOB AmoA is more than the archaeal enzyme. Presence of AOB in saline water is predominant in previous studies conducted by the researchers. In the marine ecosystem, diversity of AOB is in contradiction to the presence of AOA, that is, the archaeal species. Some scientists reported in their studies that the majority of archaeal species are found in the marine ecosystem, which are responsible for the ammonia oxidation. Others have different opinions as per their investigation which clearly states the presence of AOB is higher [21–23]. The abundance of AOB is higher in saline waters than in the freshwater regions. Nevertheless, the dominance of AOB is largely dependent on the different types of habitats and geographical locations [24]. Separation of niches, geographical area, salinity, and abundance of ammonia has been marked as parameters for the distribution of AOB and also drive their growth in marine ecosystems. Presence of AOB species has been studied in different ecological conditions, among which soil micro biota of AOB has been studied vividly. The distribution of the AOB community primarily depends upon the availability of ammonia. Ammonia-rich regions contribute to the growth and survival of AOB. In research there is evidence of co-existing of both AOB and AOA [25, 26]. AOB primarily regulates the rates of nitrification in soil. AOB have been seen to be more active in fertilized paddy fields or agricultural soil, while both AOB and AOA have been found in alkaline soil. Both soil characteristics and environmental factors, such as fertilizers (trophic status and levels of ammonium), soil conditions, concentration of oxygen, moisture, salinity, particle size, and organic carbon content, may have a significant impact on the abundance, distribution, and community structure of AOB [27]. The diverse distribution of anammox bacteria in soil was conditioned to high nitrogen content. The evidence for the existence of anammox reaction in terrestrial environments was assembled from the soil which was receiving slurry manure for 25 years [28] or N-loaded peat soils [29]. Based on the studies of these two scientists it was revealed that the highest diversity of anammox species is detected in soil rather than in any other niche. More than four genera of anammox bacteria (i.e., Brocadia, Kuenenia, Anammoxoglobus, and Jettenia) were commonly detected. The widespread distribution of anammox bacteria and the process in the natural environment have encouraged scientists to investigate more about the process, as well as studies have been conducted on isolating the species and their metabolism and also their contribution to the biogeochemical N cycle.

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Nitrogen transformation in wastewater plants is mediated by microorganisms. In the first part of the nitrification reaction, ammonia oxidation is carried out by the ammonia-oxidizing microorganisms AOB and AOA and the oxidation of nitrite to nitrate is mediated by nitrite oxidizing bacteria. In a wastewater treatment plant, there is a coexistence of both AOB and AOA. Studies regarding the abundance of specific groups show that in some of the municipal wastewater treatment plants the AOB are major players [13, 14]. In some systems, AOA has outnumbered AOB. The different process parameters of the wastewater plants are responsible for this variation in diversity. As per the studies conducted by previous researchers, nitrification reactors show the abundance of AOB with increased amount of ammonia concentration [30]. Nitrosomonas and Nitrosospira were observed as the most important genus of AOB in activated sludge system [31]. In various bioreactors, Nitrosomonas is found to be one of abundant genus. Evidence from previous studies portray that nitrification is majorly driven by AOB in the engineered systems [32]. The aerobic sludge system with high amount of nitrogen contains more number of AOB than AOA. Low temperature and abundance of ammonia are key parameters for the diversity and distribution of the AOB in engineered systems.

3 Anammoxosome: the reaction center for anammox In 1999, Strous and his colleagues had discovered the missing lithotrophs as novel planctomycetes in a wastewater treatment plant enrichment culture reactor [33]. Anammox is the name given to these bacteria because of their unique metabolic metabolism. These anammox bacteria have a number of unique characteristics, including the ability to synthesize and metabolize hydrazine, a highly toxic chemical, in the form of a catabolite intermediate, the biosynthesis of ladderane lipids, and the presence of an internal compartmentalization structure known as an anammoxosome. Anammoxosome is a compartment where anammox reaction is carried out. It is a lipid bilayer structure found inside the cytoplasm. Anammox bacteria belong to the group Planctomycetes. Planctomycetes lack the peptidoglycan layer; instead they possess a proteinaceous cell wall [34]. The membrane consists of a combination of lipids as found in bacteria (ester-linked) and in archaea (ether-linked). The compartment is bounded by a single membrane. The catabolism reaction takes place in this compartment. Anammox bacteria cytoplasm consists of three compartments which are separated by single bilayer membranes: (1) the outer region paryphoplasm; (2) the riboplasm, which contains the nucleoid; and (3) the inner ribosome-free compartment, the anammoxosome, which is bounded by the anammoxosome membrane. Anammoxosome contains many unconventional lipids which are known as ladderane lipids. In chemical science these lipids are defined as an organic molecule that is composed of two or more cyclobutane rings.

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The structure of the lipid is like a series of cyclobutane rings fused together. The structure represents a ladder. The presence of these lipids in the biological system is unique and the natural forms are a component of the membrane of anammoxosome. Previous studies have revealed the structure of ladderane lipids in bacterial species Brocadia anammoxidan and Kuenenia stultagartiensis. The investigation reports about the abundance of lipid content, which is more than 50% of membrane lipid. The unique lipid structure found in the anammox bacteria is composed of different ring systems in combination of one, two, or both namely X and Y. the ring system of the ladderane consists of three cyclobutane moieties and one cyclohexane moiety that are substituted with an octyl chain, which is ether-bound to the glycerol unit at its final carbon atom. Five linearly concatenated cyclobutane rings are substituted with a heptyl chain containing a methyl ester moiety at its ultimate carbon atom in the ring-system Y. Cis-ring junctions fuse all rings in ring systems X and Y, giving in a staircase-like configuration of fused rings known as ladderane. Membrane lipids with ladderane moieties X and Y are prevalent in anammox bacteria. In Candidatus “Brocadia anammoxidans,” they account for 34% of total lipids [35]. The proton motive force necessary for ATP production is produced and maintained by the anammoxosome membrane [36].

4 AMO: the key enzyme in the functioning of the anammox process AMO is a membrane-bound, copper-dependent enzyme that catalyzes the first step of nitrification in AOB, converting ammonia to hydroxylamine by reductively inserting an O atom from a dioxygen-derived O–H bond. The copper-dependent AMO catalyzes the first step of nitrification, the oxidation of NH4+ to hydroxylamine (NH2OH), which is followed by the formation of nitrite (NO2) catalyzed by the iron-dependent hydroxylamine oxidoreductase (HAO), and finally by the formation of nitrate (NO3) [37]. The active form of the AMO enzyme is challenging to purify, and little is known about its structure and biology. A wide variety of hydrocarbon co-oxidation substrates are used by the related bacterial AMO and particulate methane monooxygenase (pMMO). By contrasting the responses of the archaeal AMO, a bacterial AMO, and pMMO to inhibition by linear 1-alkynes and the aromatic alkyne, phenylacetylene, this study sheds light on the AMO of previously unstudied archaeal taxa. The archaeal AMO may have a limited hydrocarbon substrate range than the bacterial AMO, as previously observed for other genera of AOA, based on its reduced sensitivity to inhibition by bigger alkynes. Multimeric transmembrane copper-dependent enzyme AMO belongs to the CuMMO, which also includes the ammonia, methane, and alkane monooxygenases [38–40]. The CuMMO family contains a diverse variety of substrates, and it has been proposed that following metabolic processes determine the functional role of CuMMO-

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containing microorganisms. Whole-cell studies examining AMO’s interaction with both reversible and irreversible inhibitors have substantially provided insights into the structure and function of the protein. For instance, the copper chelator allylthiourea (ATU) inhibits the bacterial AMO, which clearly suggests that it is a copper-dependent enzyme. Both AMO and pMMO are effectively inhibited by acetylene. Acetylene works as a suicide substrate in Nitrosomonas europaea, and cells must synthesize new AMO proteins from scratch in order to resume NH3-oxidizing activity. H191 in the AmoA was covalently bonded by the ketene product of acetylene activation. The genes for AmoA, AmoB, and AmoC produce three protein subunits that make up the AMO enzyme [41]. Despite the possibility for genetic research utilizing all three AMO genes, only a small portion of AmoA functions as an AOB and AOA gene, which, according to Arp et al. [42], accelerate the first reaction in ammonia oxidation, and have been used frequently as a biomarker gene to evaluate the diversity and AOB in plenty, the gene AmoA, which creates a product with the AMO active site and provides the energy is most important for AOB growth with a well-established unlike AmoB and AmoC databases [43].

5 Biochemical process of ammonia oxidation by anammox by bacteria The major question in research regarding this process was how ammonia was oxidized, considering its inert nature. Since the discovery of Kueneria stuttagartienesis and its genome sequence, the reaction mechanism of ammonia oxidation was framed keeping in context the substrates. The development of a minimal set of reactions by which the two substrates of anammox bacteria, ammonium and nitrite, were transformed into the end product N2 was made possible by preliminary physiological and biochemical research. Van de Graaf et al. [44] proposed two potential routes for the anammox process; one is oxidation of the ammonium ion to hydroxylamine, which then interacts with nitrite to decrease it to nitrogen. The second pathway involves hydroxylamine (NH2OH), which is produced by the partial reduction of nitrite, and interacts with ammonium to produce hydrazine (N2H4). Nitrogen is created by further processing of hydrazine. For the initial reduction of nitrite, this oxidation would provide the necessary reducing equivalents. The most likely explanation for the anammox mechanism has been put forth by Kuenen and Jetten [45]. Hydroxylamine is produced as a result of nitrite reduction by a nitrite reducing enzyme. Ammonia and hydroxylamine are transformed into hydrazine by an unidentified hydrazine hydrolase (HZS), which is then transformed into nitrogen by HAO/HZO. For the initial reduction of nitrite, this oxidation would provide the necessary reducing equivalents. By effectively consuming protons in the riboplasm and producing them inside the anammoxosome, a phenomenon known as separation of

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charges, the anammox reaction creates a proton gradient in the biochemical model. According to the studies of Schouten et al. [46] based on isotopic analysis of carbon, Candidatus Scalindua sorokinii and Candidatus Brocadia anammoxidans use the same carbon fixation pathways – possibly the Calvin cycle or the acetyl coenzyme pathway to fix carbon in their environment (Figure 2) [47]. In 1997, 15N labeling experiments were initially conducted to comprehend the potential metabolic pathway for anammox [44]. These tests demonstrated that hydroxylamine, which is most likely produced from nitrite, served as the main electron acceptor in the biological oxidation of ammonium. It is hypothesized that the reaction producing the electron equivalents for the reduction of nitrite to hydroxylamine is the conversion of hydrazine to dinitrogen gas. Two potential reaction mechanisms were generally covered [45]. Ammonium and hydroxylamine are first converted to hydrazine by a membrane-bound enzyme complex, and then hydrazine is oxidized to dinitrogen gas in the periplasm. The same enzyme complex that is responsible for hydrazine oxidation also reduces nitrite to hydroxylamine at the cytoplasmic location at the same time. The following is a conclusion for a further potential mechanism for the anammox process: A membrane-bound enzyme complex converts ammonium and hydroxylamine to hydrazine, which is then oxidized in the periplasm to dinitrogen gas. The electrons produced by this process are then transferred via an electron transport chain to a nitrite reducing enzyme in the cytoplasm, where nitrite is reduced to NH2OH. Three successive, coupled redox processes are used to convert the substrates, and two extremely hazardous intermediates, NO and hydrazine, are produced in each reaction. The current working hypothesis’s initial step, the nitrate reductase converts nitrite to nitric oxide in the first phase (NirS), the first step of the reaction. HZS then mediates the second step by combining ammonium with nitric oxide to create hydrazine. Hydrazine/hydroxylamine oxidoreductase (HDH) converts hydrazine to dinitrogen gas as the last step. The overall equation can be broken down into redox half reactions that are carried out inside the anammoxosome: NO2− + 2H+ + e− = NO + H2 O ðE = +0.38 VÞ NO + NH4+ + 2H+ + 3e− = N2 H4 + H2 O ðE = +0.06 VÞ N2 H4 = N2 + 4H + + 4e− ðE = −0.75 VÞ

(1) (2) (3)

This procedure would entail a novel biochemical process: hydrazine synthase (HZS) is an enzyme, and based on investigations of the genome, a specific multi-heme protein was proposed as the contender HZS. Hydrazine is oxidized to N2 in the final stage (eq. (3)), which releases four electrons to power steps in the first reaction of the mechanism. It has been observed that condensation of NO or HNO and ammonium on an enzyme associated with the ammonium monooxygenase family could play a possible role for NO or HNO in anammox. The HAO enzyme might then transform the created hydrazine or imine into dinitrogen gas, and the reducing equivalents generated during the reaction are needed to combine NO or HNO with ammonium or to reduce nitrite to NO.

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Studies on environmental genomics of the species Candidatus Kuenenia stuttgartiansis proposed NO as the intermediary in place of hydroxylamine (Figure 2) [33]. However, this theory also acknowledged that hydrazine was a crucial metabolic intermediary. Two enzymes that are exclusive to anammox bacteria are found in this pathway (Figure 2): hydrazine hydrolase (hh) and hydrazine dehydrogenase (hd). Nitric oxide and ammonium are used by the hh to create hydrazine, and the hd then moves the electrons from hydrazine to ferredoxin. A small number of novel genes were found, including some known genes for fatty acid biosynthesis and S-adenosylmethionine radical enzymes [33], which contain domains involved in electron transfer and catalysis.

NO2– NH2OH

Hydrazine hydrolase

NO3–

5 H+

Nitritereducing enzyme

Cell cytoplasm

4e–

Hydazine– oxidizing enzyme NH3

N 2 H4

Anammoxosome

N2 4H+

Figure 2: Enzymes involved in anammox process and the metabolic pathway (with permission from Young-HoAhn [47]).

6 Application of anammox bacteria in treating ammonia-rich industrial wastewater Nitrogen-containing contaminants in wastewater are of prime concern worldwide in today’s time. The most frequent nitrogen-based contaminant introduced into aquatic habitats is ammonia. In natural conditions, the amount of ammonia in groundwater and surface water is less than 0.2 mg/L [48]. Municipal and industrial wastewater outflow, as well as agricultural activities like use of agrochemicals, has all contributed to a significant rise in ammonia concentrations in water [49]. Ammonia accumulation in water facilitates excess growth of cyanobacteria, green algae, and so on. These reduce the dissolved oxygen in water bodies and release toxins causing harm to aquatic life [50]. Strict environmental regulations and legislations

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are being forged to push industries to look for sustainable solutions to removal of ammonia from wastewater streams. Various technologies have been employed to treat ammonia-rich wastewater like physical, chemical, and biological technologies. Membrane separation, ion exchange, oxidation by ozone, precipitation and stripping are some of the physicochemical technologies used so far [50]. Excessive cost of operation and need for skilled labors have necessitated the search for better alternatives to these processes. Anaerobic ammonium oxidation (anammox) is a biological process of nitrogen removal that utilizes the direct oxidation of ammonium to create nitrogen gas using anammox bacteria (N2). This technology has gained popularity in recent times as a cost-effective and ecologically benign way to treat ammonia-rich wastewater [51]. In general, biological nitrogen removals processes constitute nitrification and denitrification using microbes. Nitrification is a two-step process catalyzed by obligate chemolithoautotrophic prokaryotes such as, nitrite-oxidizing bacteria (NOB) and ammonia-oxidizing archaea (AOA) or ammonia-oxidizing bacteria (AOB) [52]. The AOBs and AOAs oxidize ammonia to nitrite followed by oxidation by NOBs to form nitrate. As autotropic bacteria, anammox bacteria use ammonium as the energy source through its anaerobic oxidation and facilitate direct reduction of nitrite into N2 yielding nitrate as a byproduct. HCO3- and CO2 usually fulfill the carbon requirements of anammox bacteria. The metabolism follows the Calvin cycle or the Acetyl-coA path to use CO2. In a way this process of wastewater treatment reduces the carbon footprint. The reactions involved are: NH4+ + 1:32NO2− + 0:066HCO3− + 0:13H + ! 1:02N2 + 0:26NO3− + 0:066CH2 O0:5 H0:15 + 2⋅:03H2 O NH4+ + NO2− ! N2 + 2H2 O 0:26NO2− + 0:066HCO3− ! 0:26NO3− + 0:066CH2 O0:5 H0:15 The most ubiquitous anammox bacteria are Kuenenia stuttgartiensis. Other microorganisms employed in treatment of ammonia-rich wastewater has suffered from inhibition due to presence of high concentration of ammonia while anammox bacteria remains mostly unaffected by that. That’s why anammox process is perfect for removal of low and high concentration of ammonia in water with equal effectiveness. Heterotrophic ammonia-oxidizing bacteria (HOAB) are another interesting approach toward ammonia removal. Although from the works of [53, 54] it can be seen that HAOB activity is inhibited by 1 ppm Cu2+ concentration, while anammox bacteria can work with up to 19.3 ppm concentration. Recently, partial nitration (PN) or partial anammox (PN-A) processes are being used for high temperature and concentrated ammonia-rich wastewater. Partial nitration-anammox (PNA) saves energy by reducing the demand for aeration by more than half [55]. This necessitates higher usage of organic carbon.

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A concerning downside of the anammox process is the production of nitrate as one of the reaction products which, in turn, increases the total nitrogen in the wastewater. Anammox process coupled with partial denitrification (PDN) has been thought of as a remedy for this in recent studies. From the observations of [56], PDN produces nitrite from nitrates, with a strong preference for nitrate as an electron acceptor. It has also been found out that, increase in the pH of denitrification reaction considerably minimizes the yield of N2O, a greenhouse gas. From the work of [57], it can be said that biofilms are the most effective way to retain anammox biomass. Research is also going on to study nitration-anammox systems. A pre-treatment for organic capture along with mainstream nitration-anammox has been found to be more energy effective for wastewater treatment [58]. The works of [49] show that membrane bioreactors loaded with anammox and n-DAMO microorganisms yield excellent nitrogen removal [51]. We were recently studying a two-step anammox process where nitrogen loading rates in two reactors were increased while reducing the hydraulic retention time. The process also showed Candidatus Kuenenia as the prevalent anammox bacterium. The findings of these works have shown the path to develop more processes for high concentration ammonium wastewater treatment under various physical conditions. Further study should include assessments of the influence of high NO2–N concentrations on anaerobic ammonium oxidizing bacteria, loading rate, and total effectiveness of the hybrid process.

7 Conclusion Anaerobic AOB are found in all types of niches. The diversity and distribution of the anammox microbial community depends on the availability of ammonia. The proposed biochemical pathway of anammox process is carried out in a specialized compartment known as Anammoxosome. The enzymatic process involves AMO as the key functional enzyme to carry out the process. Through a variety of interactions with the expanding global population, anthropogenic activities contribute to nitrogen emissions. Environmental problems like soil acidification and eutrophication are brought on by excessive nitrogen flows into natural streams. Diverse biological techniques and treatment plans are used to remove ammonia from wastewater because it is a dangerous nitrogen-containing compound with many negative effects on the environment and human health. Application of anammox process in treating ammonia-rich waste water has provided new opportunities to explore the anaerobic AOB communities. This chapter summarizes the diversity and distribution of AOB, along with their metabolic pathway, role of the enzyme. This chapter also includes the application of anammox bacteria in wastewater treatment.

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8 Future trends of research in anammox process: a critical analysis on the limitations and challenges The anammox processes have drawn interest from all over the world due to a variety of factors, including their molecular metabolism, environmental impact, and wastewater treatment applications. It will be essential to create anammox procedures for the treatment of municipal wastewater based on the knowledge gleaned from several investigations. More municipal wastewater treatment operators may decide to fund the technology or identify the key areas for further optimization with the aid of information on nitrogen removal performance and mechanism. Anammox bacteria are being used more and more frequently in wastewater treatment, especially for the removal of municipal wastewater with low levels of polluted nitrogen, a large proportion of organic matter, and low temperature. However, its technical use frequently faces numerous difficulties, necessitating the development of custom solutions based on understanding of the anammox bacteria’s metabolic mechanism. Further research is required to provide answers to a number of significant issues like (i) the absence of pure cultures of anammox bacteria is a significant problem that will cause some research to be postponed. For instance, this would inevitably require mass biomass if the amount of daily treated wastewater in the mainstream was large in terms of amount. Current culture techniques could be used to address this problem, but their effectiveness is still severely constrained by the expansion of anammox organisms, necessitating additional study. The faster growth of anammox bacteria will open a new chapter into the potential for novel designs of anammox treatment when pure anammox bacteria cultivation, especially at room or low temperatures, is created. (ii) Studies on ultrastructure of the anammox bacteria at this time is conducted solely by electron microscope which is a limitation in much of recent research. Application of metagenomics and metaproteomics in studies on ultrastructure of anammox bacteria can provide current research with an alternative method. The implementation of the anammox process in sewage biological treatment will be guided by an examination of the differences in anammox ultrastructure and function under various water quality and operational conditions. More research is needed to better understand the energetics, enzymology, and cell biology (iii) another challenging issue in municipal wastewater treatment is assessing anammox activity and locating the relevant organism in distinct ecological niches. Anammox activity in municipal wastewater treatment should be measured using new technologies or extremely sensitive sensors, which should be created and put into use. Further studies are still needed to learn more about the interacting mechanisms of functional bacteria, such as anammox organisms and nitrifiers, denitrifiers, and dissimilatory nitrate reducers (iv). The reaction conditions and other parameters required for the anammox metabolic pathway is not properly established. The importance of the conditions

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on diversity of the microbial community is not well explained. It is crucial to understand the reaction mechanisms of anammox under various abiotic circumstances in order to improve its functional stability and make the engineered application easier. The immense potential of the anammox process for industrial application requires comprehensive study of the anammox system. Some of the issues needed to be addressed are (a) engineered applications for mainstream anammox, operating performance, and process control methods for partial nitration (PN) in conjunction with anammox, which need to be adapted, concerning factual application, (b) monitoring the performance of anammox in removal of micropollutants from anammox treatment system, and (c) investigation of the robustness of the anammox process against shock loads and any potential detrimental impacts of urban wastewater compositions in order to further improve the efficacy of the anammox process to be deployed in municipal wastewater treatment process.

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Kashmira Gupta, Moulina Payra, Piyali Das✶, and Srijoni Banerjee✶

8 Diversity and environmental distribution of ammonia-oxidizing bacteria Abstract: In agricultural soil, the biological oxidation of ammonia to nitrite and then to nitrate is known as nitrification. In the nitrogen cycle, nitrification plays an impressive role. In nitrification, the first step is ammonia oxidation where ammonia-oxidizing bacteria (AOB) oxidize ammonia to nitrate. AOB produce nitric and nitrous oxides in low oxygen levels in the environment. Nitrosococcus, Nitrosomonas, and Nitrosospira are three main cultures of AOB. The AOB populations have also been detected in biofilms and quantified by 16s rRNA molecule by PCR amplification. AOB have been developed in many environments (like sediments, soils, estuaries, and wastewater treatment plants). The marine organisms consist of both the Betaproteobacteria and the Gammaproteobacteria, whereas the Betaproteobacteria is limited to terrestrial AOB organisms only. Thus, in marine environment a consortium of Nitrosococcus, Nitrosomonas, and Nitrosospira along with archaea have been developed that play a promising role with their contribution in regulating the microbial diversity, nitrification process, and biogeography. Therefore, regardless of extensive researches on ammonia oxidation for the past decades, this chapter mainly emphasizes on recent advances of ammonia oxidation and also discusses upcoming discoveries of ammonia-oxidizing microorganisms.

1 Introduction In 1877, Schloesing and Muntz first found out in their research that the biological process is more preferable than the chemical process in ammonia oxidation [1]. They also exhibited that chloroform inhibited soil-based nitrification [1]. In 1890, ammoniaoxidizing bacteria (AOB) was first cultured by Frankland and Frankland [1]. After that for further study, Sergei Winogradsky isolated Nitrosomonas europaea which is used till day. From 1890s to 1980s, the nitrifying bacteria were placed in Nitrobacteraceae

Note: Kashmira Gupta and Moulina Payra Authors equally contributed. ✶ Corresponding author: Piyali Das, Department of Biological Sciences, School of Life Science and Biotechnology, Adamas University, Kolkata, India, e-mail: [email protected] ✶ Corresponding author: Srijoni Banerjee, Department of Biotechnology, School of Life Science and Biotechnology, Adamas University, Kolkata, India, e-mail: [email protected] Kashmira Gupta, Moulina Payra, Department of Biological Sciences, School of Life Science and Biotechnology, Adamas University, Kolkata, India

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family. The genera were recognized by their cell morphology and the properties of ammonia or nitrite oxidation [1]. In the early 1990s, the restructuring of phylogeny was mainly dependent on 16s ribosomal RNA [2]. Three types of aerobic autotrophic microorganisms present in environment that can oxidize ammonia are: (1) AOB, (2) ammonia-oxidizing Archaea (AOA), (3) comammox bacteria (complete oxidation of ammonia to nitrate). This study mainly discussed about AOB. The process (in agriculture soil) of converting ammonia to nitrite and then to nitrate is known as nitrification [3]. Nitrification plays an important role in nitrogen cycle. There are two steps of this process: the first step of nitrification is ammonia oxidation. In this step AOB oxidize ammonia (NH3) to nitrite (NO2–). These bacteria released nitric and nitrous oxide in low oxygen levels. Anammox bacteria can oxidize ammonia under anaerobic conditions. There are two phylogenetic groups of AOB: (1) Betaproteobacteria (Nitrosomonas and Nitrosospira) and (2) Gammaproteobacteria (Nitrosococcus). In marine systems both Gammaproteobacteria and Betaproteobacteria are found although in most of the marine environment large numbers of archaeal ammonia oxidizers are found. The selection of this environment mainly depends on the tolerance of temperature and salinity. Both the Betaproteobacteria and the Gammaproteobacteria consist of marine organisms whereas Betaproteobacteria is limited to terrestrial AOB organisms. In marine system, Nitrosococcus (class Gammaproteobacteria, order Chromatiales, family Chromatiaceae) spread out extensively. In a betaproteobacterial AOB, Nitrosococcus mobilis is not a usually represented name. Gammaproteobacteria is associated with genus Nitrosococcus. The Nitrosococcus winogradskyi is the main strain of genus Nitrococcus, which has been extinct. 16S rRNA gene sequences, amoA sequences, and ecophysiological characteristics are identified as having at least six lines of descendants of Nitrosomonas. There are many types of strain that are not publicly available and also many of them are not documented. The high level determination of 16s RNA has created problem for Nitrosospira. If 16s–23s rRNA intergenic spacer region or amoA genes full length is used as additional marker, it can be a solution. In freshwater systems, AOA have salinity gradients toward the marine end, whereas AOB have salinity gradients from marine to fresh water. In waste water treatment plants, ammonia treatment is highly managed. In the primary stages nitrification is enhanced and nitrifying biomass is maintained. In secondary stages excess ammonia is extracted. In this system, AOB is studied thoroughly. In terrestrial system some common AOB are mainly observed, such as: Nitrosospira clusters 3, 2, and 4. Ecophysiological traits and phylogenic clusters are corrected [2]. On 16S rRNA Nitrosospira clusters are dependent. Primary succession occurs in sand dunes and in lava flows to name a few. During succession in nitrogen cycle, nitrogen availability resists plant establishment and growth. To colonize new primary substrate, the ability of AOB must be examined. Local site conditions are controlled by the colonization of new substrate. On soil, the microbial culture is exhibited during secondary succession. Sometimes for this reason diversity and number of microorganisms may be affected. The rates of nitrification and nitrate in soil have been enhanced during the 4 years postfire. From

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a long period of time Nitrosospira’s cluster 3 has been transferring to cluster 4 without fertilization. For this reason, in soil (calcareous grassland in Netherland), fertilization was discontinued in the initial stage of secondary succession. If Nitrosospira’s cluster 3 would have been fertilized for 100 years then the sequence of clusters 4 and 2 would be contiguous with the native soil. In pasture soils the fertilization takes 10–20 years and the AOB helps to develop the fertilization. Here fertilization in Nitrosopira clusters 2 and 3 was mutual. In undeveloped land; clusters 3, 7, 2, and also novel groups can be found. AOB can be found in all countries and oceans. In acidic and extreme alkaline conditions, AOB has been discovered. AOB provides basic metabolic oxygen and ammonia. Conditions similar to their habitat of origin have been created in the laboratory to culture them. AOB, AOA, and comammox Nitrospira – these three groups are autotrophic. They all have different pathways of inorganic carbon assimilation. By Calvin cycle, AOB can fix atmospheric carbon dioxide (CO2), and comammox Nitrospira can fix atmospheric carbon dioxide (CO2) by reductive tricarboxylic acid whereas by the hydroxypropionate-hydroxybutyrate cycle, AOA can fix nitric acid (HCO3–). This carbon dioxide (CO2) and nitric acid (HCO3–) concentration is based on pH stability. If pH level decreases, then the concentration of HCO3– also decreases. This means high affinity is present in acidophilic AOA which helps to grow in low pH level. In AOA and comammox structures, only plasma membrane is present. In ammonia oxidation membrane bound is the key enzyme, so the volume of membrane is significant. A single AOB cell might have large number of ammonia monooxygenase (AMO) than AOA and comammox cell. This has never been observed previously. AMO oxidizes ammonia to hydroxylamine during ammonia oxidation. AMO enzyme participates in all three groups of ammonium-oxidizing microorganisms [2]. It is also a copper-dependent enzyme. In both bacterial and archaeal ammonia oxidation, hydroxylamine and nitric oxide are found. Bacteria and archea mainly are differentiated by enzymology. Hydroxylamine is oxidized both in AOB and comammox by using hydroxylamine dehydrogenase [2].There are two pathways in bacterial ammonia oxidation. At first AMO oxidizes ammonia to hydroxylamine; and then HAO (hydroxylamine oxidoreductase) oxidizes hydroxylamine to nitrite. By this information it is assured that the reaction product of bacterial HAO is NO, and not nitrite. After discovering NO, there are now three steps of bacterial ammonia oxidation. In the third step, NO is converted into nitrite [2]. In further studies, the ecology, biochemistry, classification of AOB, and environmental factors play an important role in AOB (Ammonia Oxidizing Bacteria) [2].

2 The ecology We can find ammonia-oxidizing microorganism in various ecosystems like soil, wastewater treatment plants, fresh-water and marine habitats, rivers with high inputs and

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outputs of sewage, and even human skin [1]. They can also be distributed geographically [4]. AOB produces N2O gas (greenhouse gas) in huge amounts [1]. They can survive in specific environmental changes as they are very sensitive and more resistant to drought [3]. In wastewater treatment and also in soil numerically AOB is outrun by AOA. The growth of AOB is influenced by the concentration of salt. In case if we isolate ammonia oxidizer from marine system it needs a requirement of salt at seawater concentration. On the other hand, Nitrosococcus halophilus needs high concentration of salt after isolating from salt lakes and salt lagoons. Moreover, some isolated strain cannot tolerate salt, but also found in marine system like Nitrosospira [5]. Temperature is also influenced by the diversity and population structure of AOB as we know ammonium is a primary energy source for ammonia oxidizers [6]. The pH is also connected with AOB. There are two forms of ammonia. One is protonated and another one is nonprotonated. They are dependent on pH equilibrium. AOB are unable to grow in acidic condition because in acidic condition the amount of ammonia is low and N. europaea uses ammonia rather than ammonium. If we use urease, then AOB can grow in acidic conditions. From the recent study, the ammonium salinity and pH concentration are almost same as the aquaria. They are also considered as important factor to determination of AOB structure [6]. In marine system, AOB prefer low ammonia concentration but in soil high amount of ammonia concentration is present [1]. Ammonia oxidizer was first found in 16S rRNA and the amoA. AMO, which is an important enzyme, present in all three ammonia oxidizer groups [1]. Previously it was discussed that there are two phylogenetic group present in AOB: (1) Betaproteobacteria (Nitrosomonas and Nitrosospira) and (2) Gammaproteobacteria (Nitrosococcus).

2.1 AOB of Betaproteobacteria The genera of Betaproteobacteria AOB are made up of Nitrosomonas and Nitrosospira. But Nitrosococcus mobilis is not a validly represented name. The arrangement of Nitrosomonas cryotolerans and Nitrosomonas sp. is complicated. It is not possible to retain the classification of Nitrosomonadaceae in two or more genera based on phylogenic taxonomy. We can more specifically know about the boundaries for the genera by the current gene sequence [7].

2.1.1 Nitrosomonas The 16s rRNA gene sequence, amoA sequence defined the family of Nitrosomonas. Till now there are six lines of genus present in Nitrosomonas. Many species names are not enlisted or identically same species having several names in various nations and some are not publicly available. So, there is a chance of removal from the valid group list and it is dangerous for those species. In distinct environment, the family of

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Nitrosomonas is distributed as per the salinity, ammonia concentration, and pH, and is defined by crucial environment. Ammonia-sensitive strains are found in fresh water, terrestrial system, and also in estuaries. Nitrosomonas oligotropha represented the cluster 6A. Nitrosomonas aestuarii and Nitrosomonas marina (cluster 6B) can tolerate high salinity including marine systems. Nitrosomonas europaea, Nitrosococcus mobilis, and Nitrosomonas eutropha (cluster 7) can tolerate the concentration of high ammonia. Nitromonas communis and Nitrosomonas nitrosa (cluster 8) show diverse ecophysiological features [8].

2.1.2 Nitrosospira 16S–23S rRNA are used as markers to analyze the group. High levels of 16s rRNA created problem for Nitrosospira. From the DNA homology and genome sequencing of Nitrosospira, we can determine the value. Cluster 1 has no pure culture representative. On the other hand, clusters 0, 2, 3, and 4 have representative isolate culture. The distribution (of Nitrosospira) is dependent on the pH and salt tolerance, urea reactivity. Clusters 2, 3, and 4 have been found in various environment like soils, freshwater, and marine systems but cluster 0 is mainly found in soil and fresh water, and cluster 1 is found in marine water [9].

2.2 AOB of Gammaproteobacteria In Nitrosococcus (genus), Gammaproteobacteria is present. Till now we can determine two gammaproteobacterial species, N.oceani and Nitrosococcus halophilus. N.oceani is also known as purple sulfur bacteria. It is found in marine systems and was also determined by the presence of 16S rRNA gene and amoA sequences. On the other hand, N.halophilus is found only in saline ponds. Gammaproteobacterial AOB was determined by 16S rRNA gene sequences and the observation was confirmed by amoA [10]. In marine systems, both Betaproteobacteria and Gammaproteobacteria of AOB are found. N. cryotolerans, N. marina, Nitrosomonas sp. strain Nm143, and some salttolerant clusters for example Nitrosomonas ureae (cluster 6) were detected from surface and layer of the water and marine system. Clusters 1 and 3 of Nitrosospira were also isolated from marine systems. In freshwater systems the changes of nitrogen can affect the AOB communities [11].Commonly N. oligotropha and Nitrosospira were found in the fresh water system. Ammonia levels are treated in wastewater system. Nitrification is enhanced in the primary stage and the excess ammonia was removed in the secondary stage. From sewage we can isolate N. eutropha and N. nitrosa strain. Nitrosospira clusters 3, 2, and 4 are generally found in terrestrial environments [2].

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3 Biochemistry The AMO plays an important role in ammonia oxidation. This enzyme oxidized ammonia to hydroxylamine and it is the only enzyme being shared by all three groups [12]. The amoA is the gene encoding of subunit A of AMO [13]. It is used as a functional marker which can determine the ecology of ammonia oxidizer. At active site on the PmoB subunit a di-nuclear copper center is present in particulate methane monooxygenase (pMMO) which binds with zinc. At the interface of PmoB and PmoC, it gets localized. In N. europaea copper inflamed its activity. Copper chelator allylthiourea resisted the all known ammonia oxidizer. Without any additional subunits M. capsulatus (Bath) is activated by PmoB subunit. The AMO active site can determine the condition of enzyme which is very important for it. There is also an extracellular active site present but the exact place of activation site is not known properly [1]. amoC

amoA

amoB

Figure 1: Organization of the AMO gene clusters in bacteria.

Both in bacterial and archaeal ammonia oxidation, hydroxylamine and nitric oxide (NO) are intermediates whereas they are different with respect to their enzymology. Hydroxylamine is oxidized by AOB and comammox organisms using hydroxylamine dehydrogenase. Because of a novel enzyme, hydroxylamine oxidation step takes place. There are two step pathways of ammonia oxidation bacteria. In the first step, AMO oxidizes ammonia into hydroxylamine and in the second step, HAO (hydroxylamine oxidoreductase) oxidizes it into nitrite. The bacterial product of HAO is NO, but not nitrite. Like AOB, anammox bacteria perform oxidation of hydroxylamine to NO. Nitric oxide (NO) is intermediate of AOB. There are three steps of the ammonia oxidation pathway, and in the final step NO is converted into nitrite. Caranto and Lancaster thought that NirK was the missing enzyme. NirK was presumed as nitrite reductase. But the deletion mutant (NirK) of N. europaea can still reduce nitrite. NirK has shown the oxidation of NO into nitrite and also reductase of NO into nitrite in vitro. For the deletion of mutants, the rate of ammonia oxidation becomes lower and the N2O production rate becomes higher than the wild type. The phylogeny of NirK can determine multiple evolutionary origins in AOB. Therefore, there are different enzymes which play different roles in the oxidation of NO. In many AOB, red copper proteins nitrosocyanin can be found by the encoded gene nycA. It is not proved that the ammonia oxidation pathway is preserved between AOB and comammox Nitrospira like genes encoding for AMO. The NirK and HAO in AOB are preserved in the published genomes of comammox Nitrospira. If NO inhibits AOA it may create a problem with the evidence of NO as an intermediate in the

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AOA pathway, but not in AOB. AOB can also be inhibited by NO scavengers although AOB is present at higher concentrations than AOA [1].

3 e– 2 e– + NH3

NH2OH

Respiration

NO –

NO3– 2 e–

e– NO

HAO

2 e–

NirK?

AMO

NXR

Comammox only Figure 2: Ammonia oxidation pathway in bacteria.

There are many discoveries over the time. In a recent study, hydroxylamine is oxidized using a multi-copper oxidase (MCO1), though MCO1 is not present in the core genome of Nitrosotalea [6]. In the presence of NO if the NO-oxidizing enzyme still needs to be induced, the reaction is dependent on oxygen. Nitrite and nitrous acid are in a pH-dependent equilibrium. Both in hypothetical models of the archaeal ammonia, oxidation pathways are probable in that they would describe all the monitoring made so far. And also in both models the result is in net accession of two electrons. If NirK is not present, then it is difficult to explain the purpose of NO for the hydroxylamine oxidation. There is a new three-step model, the net yield being one rather than two electrons in the absence of NirK, which would be energetically viable [1].

4 Classification of AOB as per environmental diversity There are two types of classification of AOB reckoning on environmental diversity: 1. Soil AOB and 2. marine AOB.

4.1 Soil ammonia-oxidizing bacteria It cannot be emphasized enough how important ammonia-oxidizing microorganisms are to soils. This analysis in distinct types of soil is centered on nitrification, on the exertion of AOB, and the fundamentals impacting the cornucopia and addresses about the significance of ammonia-oxidizing microbes.

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4.1.1 Autotrophic nitrification Autotrophic nitrifying microorganisms and bacterial ammonia oxidizers regulate nitrification in the soil. AMO converts ammonium into hydroxylamine (NH2OH), which is then changed into NO2 in the process of oxidizing ammonia/ammonium by autotrophic bacteria [14]. A relatively small number of bacteria only engage in chemoautotrophic nitrification [14]. The main enzyme involved in chemolithoautotrophic nitrification is AMO. Three subunits, amoA, amoB, and amoC, make up the membrane-bound enzyme AMO. Environmental microbiology has employed the amoA gene as a molecular proxy since it is a metabolic marker for autotrophic nitrifying microorganisms [14].

4.1.2 Heterotrophic nitrification It has been proposed that the presence of nitrification in soils with a low pH is a sign of heterotrophic nitrification [14]. However, according to [35], acid-tolerant autotrophic nitrifiers may also serve these purposes [14]. Heterotrophic nitrifiers can use both inorganic and organic substrates. Since some heterotrophic nitrifiers also have the ability to denitrify and accumulate very little to no nitrate, the real signs of heterotrophic nitrification may have been misunderstood [14]. Fungus account and an investigation that could create nitrate were provided by [36]. The oxidation of various nitrogen molecules in culture has now been linked to numerous new heterotrophic species. The significance of heterotrophic nitrification has been demonstrated by numerous researches. Because it is not connected to cellular growth, heterotrophic nitrification differs significantly from autotrophic nitrification [14].

4.2 Diversity of ammonia oxidizers Ammonia can be oxidized by a wide variety of microorganisms, including bacteria, archaea, and even fungi. In the soil, AOB are widely dispersed. Chemolithoautotrophic AOB regulates the first and rate-limiting phase of nitrification, the conversion of ammonia to nitrite, and these organisms have the capacity to efficiently utilize this process as their source of sole energy. Numerous bacteria, such as Nitrosospira, Nitrosomonas, and Nitrosococcus, have been identified as ammonia oxidizers. The Proteobacteria’s subdivision has a monophyletic group that includes the genera Nitrosospira and Nitrosomonas. The genus Nitrosococcus belongs to the γ-subdivision of the Proteobacteria and individuals of this genus only oxidize ammonia in marine environments. Heterotrophic nitrifying bacteria include Thiosphaera pantotropha, Paracoccus denitrificans, Pseudomonas putida, and Alcaligenes faecalis. Through complementing metagenomic analyses of seawater and soil, it was discovered that uncultivated species of Crenarchaeota include the amoAgene. The first AOA discovered was Nitrosopumilus maritimus,

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which was isolated from the rocky substrate of a tropical marine aquarium tank. It is the first mesophile and archaeal chemolithoautotrophic nitrifier discovered in the marine group I Crenarchaeota, according to study. Members of the new phylum Thaumarchaeota include AOA. They were initially recognized as Crenarchaea members. The Cenarchaeum symbiosum, Candidatus Nitrosocaldus yellowstonii and Candidatus Nitrososphaeragargensis are other archaeal nitrifiers. It was once thought that only bacteria and archaea were responsible for nitrification. But in this process, Aspergillus flavus, Penicillium, and Absidia cylindrospora, among other fungus, have been linked [14].

4.3 Marine ammonia-oxidizing bacteria The initial step of nitrification, the sequential oxidation of ammonium (NH4+) to nitrite (NO2) and nitrate (NO3), the core process of biogeochemical nitrogen cycling, is mediated by AOA and AOB. It has also been discovered that a third group of aerobic ammonia oxidizers mediates the entire aerobic ammonia oxidation to nitrate (comammox). In a variety of habitats, such as soil, freshwater streams and lakes, and the marine environment, AOA vastly outnumber their bacterial counterparts. However, in some coastal, estuarine, and open ocean habitats where NH4+ supply is sufficient to allow AOB growth, NH4+ growth kinetics cannot fully explain the dominance of AOA. Furthermore, the cohabitation of AOB and AOA in marine settings, even at low AOB abundances, raises the possibility that AOB occupy a unique chemical niche that enables them to survive despite having a weak affinity for NH4+. The presence of many copper-rich plastocyanin and halo-enzymes in the marine AOA isolate Nitrosopumilus maritimus SCM1 instead of the Fe-dense cytochromes generally found in AOB19 points. The requirements for cupric ion (Cu2+) and unchelated Fe for optimal growth in N. maritimus were discovered to be higher than in marine environment typically those measured (0.01–0.1 pmol/ L Cu2+ and 1 pmol/L Fe), suggesting that marine nitrification in the open oceans may be constrained by these metals. Other processes involved in the marine N-cycle, such as nitrogen fixation, NO3 assimilation, and denitrification, have been shown to be constrained by Fe or Cu. An accurate evaluation of the role of these metals in regulating marine ammonia oxidation rates and defining the niche separation between AOA and AOB is not yet possible due to the lack of an analogous physiological investigation of marine AOB which is iron and copper requirements for growth. According to the theory that existing microbes have cellular fingerprints of changing trace metal, comparison of Fe and Cu needs also offers a fresh viewpoint on the age-old subject of the relative development of AOB [15].

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4.4 Trace essence share disquisition of cells Prior to trace element analysis, reagents and all samples were processed in a clean lab utilizing trace metal clean procedures and acid-washed plastic wares to remove weakly bound surface metals. The N. oceani and N. maritimus cells at “max” were collected in the middle of the exponential phase by centrifugation at 5,000g for 25 min. The following method was used to digest the cells in acid-cleaned Teflon: overnight refluxing at 100 °C with H2O2. Then the liquid was allowed to dry out and reach a 2% q.d. concentration. To the resuspended dried materials, HNO3 was added and refluxed for an hour. A technique tailored for high-salt matrices was used to determine the amounts of Fe, P, and Cu utilization [15].

4.5 AOB in Chinese paddy soil With nearly a quarter of the world’s rice paddy farming area, China is one of the greatest rice producers in the world [15]. Through the porous tissue of rice, oxygen is carried from the atmosphere into the roots, creating an oxic environment that can promote nitrification. The underlying microbial contributions to the nitrification process are poorly known, despite the extensive study of nitrification in paddy soil. A study on acidic field paddy soils with various long-term fertilization treatments or on various paddy soil types further verified the numerical preponderance of AOA over AOB in paddy soils. By measuring the number of amoA gene copies, it was found that rice farming significantly increased AOA abundance but not AOB abundance. It is yet impossible to determine if soil pH is a selection factor for the niche separation of AOB in this wet and high N-input soil environment given the dearth of studies examining nitrifier communities in rice paddy soil. It is obvious that more investigation is needed on nitrification in rice paddy soils [16].

5 Environmental factors 5.1 Level of ammonia In the environment, the amount of ammonia has a substantial impact on the growth of AOB. AOB has a higher inhibitory concentration because it has a stronger affinity for ammonia [17]. AOA may experience the suppressed condition earlier than AOB if exposed to greater ammonia content. In high ammonia concentrations, AOA were less competitive than AOB, and the abundance of AOB increased as ammonia concentration increased according to Gao et al. [17]. At varied ammonia-nitrogen levels (140 mg N/L), there was likewise little variation in the abundance of AOA. Ye and Zhang noted

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that the nitrification tank which consisted of the abundance of AOB, when treated with the salty wastewater, the ammonia quality increased from 200 to 300 mg/L. Additionally, AOB dominated the ammonia oxidation process in the system treating of landfill leachate with a high ammonia concentration (2,180 ± 611 mg N/L) [18]. It was possible to draw the conclusion that the variation between the microbial community structure of AOB and AOA were due to the ammonia level. It was influenced by the types of wastewater [14].

5.2 Organic loading The growth of AOB is objectively impacted by organic materials. While AOA may be mixotrophic or purely autotrophic, AOB are characterized as autotrophic microbes. According to certain research, organic compounds such as Nitrosocaldus yellowstonii and Nitrosopumilus maritimus SCM1 significantly inhibited the growth of various AOA strains [19]. AOA and AOB may have varying ammonia oxidation capacities due to differences in their various metabolic features and more sophisticated metabolic pathways as compared to AOB [14].

5.3 Temperature The main way that temperature has an impact on AOB is through how it affects AMO activity [20]. The AOB that have currently been discovered are mesophiles, but the AOA have a very wide range of adaption temperatures. One could notice that active ammonia oxidation by AOA take place at 0.2 °C in the deep waters of the North Japan Sea and 74 °C in the hot spring in Yellowstone National Park [19]. He et al. discovered that during summer AOB (water temperature from 21 to 25 °C), and AOA during winter (water temperature from 3to 4 °C) were the predominant ammonia oxidation bacteria in the layer of sediments along the Shandong Peninsula [21]. Niu et al. discovered that the AOB and amoA genes’ abundances changed slightly in the winter (4.6–5.5 °C is the water temperature) compared to the summer (17.7–28.6 °C is the water temperature) [21]. For wastewater treatment in the engineered marsh and system, Sims et al. similarly noted that in low temperatures, AOB were more susceptible than AOA [22]. As a result, temperature is not that much affected on the occupation of AMO, giving AOA with a competitive edge in an environment with highest level of temperatures. The ability of AOA to adapt to temperature changes is inseparable from the unique structure of glycerol ether in the cell membrane [16].

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5.4 Level of oxygen The nitrification process requires oxygen as a reaction substrate. The oxygen content will have an impact on the nitrification process since different nitrifying microbes have different affinities for oxygen (AOA > AOB > nitrite-oxidizing bacteria). In hypoxic environments like deep soils, deep oceans, and layer of sediments, where too little oxygen is present, high oxygen affinity makes less competitive AOB [23]. Large volumes of AOA with low dissolved oxygen levels (0.2 mg/L) were identified in the outer ditch of an Orbal oxidation ditch. Park et al. also discovered that simultaneous denitrification and nitrification took place there [24]. According to Li et al. simultaneous denitrification and nitrification could be sped up to remove nitrogen heterotrophic denitrifying bacteria, and AOA may be combined in one activator by lowering the aeration pressure to restrict the action of nitrite-oxidizing bacteria [24]. Additionally, at low amount of dissolved oxygen levels (dissolved oxygen is from 0.3 to 1.5 mg/L) in the system for treating landfill leachate, real-time PCR was employed by Yapsakli et al. to determine the presence of nitrite-oxidizing bacteria, AOA, and anammox bacteria [24]. According to a calculus-based model developed by Liu et al., combined AOA nitrification with anammox was highly efficient at removing nitrogen from autotrophic plants than coupled AOB with anammox in a high range of concentrations of ammonia nitrogen (30 to 500 mg/L), with low level of oxygen consumption and a powerful inhibitory outcome on nitriteoxidizing bacteria occupation [25]. The management of dissolved oxygen level to develop the community formation might be used to achieve to remove nitrogen by the common AOA, AOB, and anammox bacteria or denitrifying bacteria. It is also anticipated to offer fresh perspectives on how to create nitrogen removal processes for wastewater that are both highly effective and resource-efficient [16].

5.5 pH The AOA SAT1 strain enhanced from activated sludge was reported to have a pH range of 5.0–7.0, and with the ideal pH being at 6.0. It is designating that the SAT1 strain was neutrophilic [26]. The ammonia bioavailable can be decreased by the adding of a proton of ammonia when pH drops. It might be highly beneficial for the development of AOA from the standpoint of substance use. Now research has shown that AOA dominates ammonia oxidation in acidic soils, while AOB struggles to survive at low pH levels and is primarily answerable for nitrification in alkali soils [27]. However, it was also claimed that Candidatus Nitrosotalea devanaterra (AOA), which demonstrated remarkable adaptation to pH fluctuation, could grow in alkaline soil [28]. The differences between these two types of ammonia oxidation bacteria that are impacted by environmental pH are still up for debate. Similar knowledge exists regarding how pH effects the distribution of AOB in treating wastewater systems. However, treating wastewater systems with acid affluent may be able to use the AOA strain due to its high flexibility to pH fluctuations [16].

AOB dominant

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Organic Loading

DO Temperature Ammonia Level

Low

Environmental Factors

High

Figure 3: According to the varied environmental parameters (ammonia, organic loading, oxygen level, and temperature) and proposed AOB (partially based on Guo et al.) [29].

5.6 Salinity The presence of mariners in water or soils is known as “Salinity.” By their causes, salinity is of three types – primary saltiness is caused by natural process so it’s also called natural saltiness; where groundwater situations rise, secondary saltiness is caused there, so it’s called dry land saltiness, and in case of tertiary saltiness it’s also called irrigation saltiness. Littoral metropolises are used in seawater. From life conditioning, saline and hypersaline wastewater is generated. By domesticating freshwater actuated sludge, the treatment of saline wastewater has been observed by some experimenters in saline surroundings. The influence of saltiness covers in their ammonia oxidizing microorganisms’ subunit A and messenger ribonucleic acid. Ammonium plays an important role in AOB. Saline water was treated by a nonstop reactor nitrification. Saline water was operated under low level of dissolved oxygen situations (0.15–0.5 mg/ L) for nearly one time. The high level of nitrogen loadings were occurred in four phases (0.26–0.52 kg N/(m3 day)). In AOB, diversity in the cloning anatomized quantitative polymerase chain reaction (qPCR) and terminal restriction scrap length polymorphism (T-RFLP) occurred. The result of all the four experimental phases showed one dominant AOB species in the reactor. The ammonium loadings and dissolved oxygen situations were changed by the AOB population. In the nitrification reactors, AOB plays an important role [30]. The eutrophication of many ecosystems in recent decades has led to an increased interest in the ecology of nitrogen transformation. Chemolithoautotrophic AOB are responsible for the ratelimiting step of nitrification in a wide variety of environments, making them important

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in the global cycling of nitrogen. These organisms are unique in their ability to use the conversion of ammonia to nitrite as their sole energy source. Because of the importance of this functional group of bacteria, understanding of their ecology and physiology has become a subject of intense research over recent years. The monophyletic nature of these bacteria in terrestrial environments has facilitated molecular biological approaches in studying their ecology, and progress in this field has been rapid. The βsubclass Proteobacteria helps to progress the knowledge of the distribution, diversity, of molecular microbial ecology [15].

5.7 Other implicit factors At low level pH, AOB growth is allowed by urease exertion. N. viennensi plays an important role in AOB and participate numerous consecutive impediments of nitrification. Implicit factors are situated in AOB. In agrarian soils the ratio of AOB is treated with impediments. The over pollution of air has operated by nitrous oxide, which is made up of AOB. In marine surroundings, N2O manufacture accompanies the growth of N. maritimus. Then, lower amount of N2O is induced by AOB [31].

5.8 Exemplifications In most of the cases, observers noticed that in situ exertion does not reflect on dominance of AOB. Carbon dioxide assimilation of growth is showed by Lehtovirta-Morley et al. Neutral agrarian soil is suitable place for growing AOB. Acidic agrarian soil is also ideal environment for growing AOB [32].

6 Conclusion and outlook Exciting new ammonia oxidation-related creatures have been discovered recently, and the ecology and genomes of these organisms have been thoroughly examined. Although many of the ideas derived from genomic analysis have not yet been validated, the mechanisms behind the distribution and activity of these microorganisms in the environment are still largely unknown. Another idea that has to be tested in future research is the potential for a unique ammonia oxidation pathway in AOA, which has been highlighted in this study. To explore how AOB (AOA and comammox Nitrospira) function, recently discovered culture models of microorganisms adjust to and react to their surroundings [1]. For a thorough and systematic knowledge of these mechanisms and how they influence ammonia oxidizing communities, there needs to be better coordination of research on the ecology, physiology, and biochemistry of ammonia

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oxidation. The mechanistic understanding of ammonia oxidation could also be quickly improved by new methodological breakthroughs, such as mutagenesis systems for AOA and comammox Nitrospira, which would help to close the gap between the biochemistry and ecology of ammonia oxidation. We can better understand the diversity, quantity, and activity of ammonia oxidizers as well as how they contribute to nitrogen losses and greenhouse gas generation in various contexts by elucidating what occurs at the cellular and molecular level [33]. In wastewater treatment facilities, AOB are essential for the conversion of ammonia to nitrite. AOB are challenging to isolate from environmental samples, necessitating the employment of genomic techniques for the characterization of AOB communities in such systems. An effective method for identifying and measuring AOB populations in biofilms and activated sludge flocks is fluorescence in situ hybridization, which uses fluorescently labeled probes that target 16S rRNA molecules. Using primers that either amplify the 16S rRNA or amoA genes, real-time qPCR can also be used to detect the quantity of AOB [34].

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Ritama Mukherjee, Mohana Baral, Srijoni Banerjee✶, and Piyali Das✶

9 Metabolism and genomics of anammox bacteria Abstract: Anammox is a biological process through which anaerobic ammonium is oxidized to dinitrogen gas by using nitrite as the electron acceptor conducted by anaerobic ammonium-oxidizing bacteria. The process removes N2 from wastewater and the global N cycle. These bacteria have the unique metabolic ability to combine ammonium and nitrite or nitrate to form nitrogen gas, hence contributing to the loss of about 50% of nitrogen from the marine environment. The importance of the anammox metabolism process lies in its ability to remove ammonium from wastewater and can be contributed to the loss of fixed nitrogen from the ocean; furthermore, as these bacteria contribute havoc to the global nitrogen cycle, the study of the regulation and mechanism of CO2 sequestration will contribute to our understanding of the global climate change. This unique ability has a huge role in environmental and industrial microbiology. Among the special properties of the bacteria, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids, and the role of cytoplasm differentiation are unique and hence of great interest among biologists. The genomic study of anammox is conducted to expose the genetic blueprint of the organisms’ special properties and the evolutionary history of Planctomycetes. Moreover, extensive research in the recent past consisting of anammox bacteria and its structural and metabolic properties have been carried out and are helping the scientific community with a better understanding of its nature at the proteomics and genomics level. Thus, this chapter emphasizes the physiology, cell biology, and metabolism of anammox bacteria studied mostly in the recent past and concludes with an outlook of undone research and emerging urgent issues that need to be addressed further.

Acknowledgments: The authors wish to express their sincere thanks to Honorable Vice Chancellor of Adamas University, India; the Dean of School of Life Science and Biotechnology, Adamas University for their support and motivation. The author(s) received no financial support for the research, authorship, and/or publication of this chapter. Note: Translated from German by Deborah Bowen. ✶ Corresponding author: Piyali Das, Department of Microbiology, School of Life Science & Biotechnology, Adamas University, Kolkata, India, e-mail: [email protected]; ✶ Corresponding author: Srijoni Banerjee, Department of Biotechnology, School of Life Science and Biotechnology, Adamas University, Kolkata, India, e-mail: [email protected] Ritama Mukherjee, Mohana Baral, Department of Microbiology, School of Life Science and Biotechnology, Adamas University, Kolkata, India

https://doi.org/10.1515/9783110780093-009

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1 Introduction Since centuries the possibility of oxidation of ammonium under anaerobic conditions remained unnoticed until in 1977 physicist Broda proposed the existence of lithotrophic organisms that derived their energies for proliferation through the oxidation of ammonium and reduction of nitrate or nitrite producing dinitrogen as the end product. The basis of this discovery was the notion that inert nitrogen requires activation through oxygen as the first step toward oxidation. However, attempts to isolate these microbes have only provided negative results mostly due to a lack of knowledge of cultural approaches. In the mid-1960, the observation of a significant loss of ammonium under the anoxic condition from a highly stratified anoxic fjord during the analysis of nitrogen balance pointed toward the existence of these bacteria. A similar phenomenon was also observed three decades later in Delft, Netherlands in a denitrifying bioreactor. On the basis of these observations, the adoption of anammox in wastewater treatment was suggested, mostly due to the fact that the use of these microbes can reduce costs of ammonium removal and also reduce CO2 emission [1]. Anammox belongs to a group of Planctomycetes bacteria five of which have been discovered so far: Kuenenia, Brocadia, Anammoxoglobus, Jettenia, and Scalindua are among those. Thirty-seventy percent of the bacterial population in the Annamox consortium belongs to the phylum Chloroflexi, Chlorobi, Proteobacteria, Acidobacteria, and Bacteroidetes, respectively. Though these microbes share a symbiotic relationship, their potential roles are yet to be determined. However, it is assumed that this relationship might benefit the microbes in metabolism, supply of growth factors, or scavenging of anammox metabolites [2]. Initially, it was postulated that anammox are only experts in chemolithoautotrophic activities; however, in recent findings, it was observed that these microbes are capable of using a wider variety of elements like ferrous ions and other organic compounds like carboxylic acids as electron donors. The use of nitrate is of greater curiosity due to the fact that during denitrification in nature the compound is converted into dinitrogen gas whereas anammox uses a different route of converting ammonium and nitrite to dinitrogen forming nitric oxide and hydrazine as intermediates. The study of annamox is not only an interesting subject from an ecological perspective but also as a cell biologist. These bacteria have similarities with both Archaea and Eukarya and they also do not have the conventional prokaryotic cell plan; rather the single bilayer membrane is divided into three compartments with different characteristics. The innermost compartment consists of the anammoxosome which is the unit for energy metabolism; the middle compartment consists of riboplasm which houses the nucleoid and ribosomes with the functionality of genetic information processing; last, the outermost compartment is called paryphoplasm whose function is yet to be discovered. Further, it is hypothesized that the cell wall of anammox lacks peptidoglycan and outer membrane; moreover, the anammox membranes are single-

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layered and made of special types of proteins called ladderines. Through molecular modeling, it was revealed that due to the presence of these proteins the membrane lacks certain degrees of rotational freedom, thus limiting its permeability [3]. Initially, the use of annamox faced skepticism due to its prolonged growth rates. However, developing technologies such as SBR or sequence batch reactor has been implemented to mimic anammox’s natural habitat in pertaining high biomass capacity at the constant low substrate. The reason for this low growth rate can be rationalized by their low metabolic activity (15–80 mmol of N2 formed/g dry weight of cells/ min). The energy for growth is highly associated with the 1:1 conversion of ammonium and nitrite into nitrogen. Due to the use of inorganic energy sources as substrates these organisms are classified as chemolithotrophs. These organisms have a high affinity toward their substrates, ammonium and nitrite are utilized to very low levels and hydrogen carbonates serve as the major source for the synthesis of biomass (CH2O0.5N0.15), making the organisms autotrophs. The reduction of cell carbon dioxide accounts for the cell carbon fixation. These reducing equivalents are derived from the oxidation of nitrite to nitrate making nitrite formation a critical point for anammox growth. The secret of the anammox mechanism lies in the genomics of the organism, thus decoding it has been a priority of the researchers. At present, the genomic of only one species of anammox Candidatus K. stuttgartiensis has been sequenced through metagenomic approaches from a laboratory-based synthetic wastewater reactor. Through DNA sequencing along with a combination of shotgun method and fosmid analysis and bacterial artificial chromosome libraries, the genome was assembled into five contigs; the mean read coverage over the contigs was determined to be 22-fold. However, the assembled genome was estimated to be 95% complete suggesting 60 missing genes. The mostly complete anammox genome was further determined to be 4.2 megabases coding for 4,663 ORFs, 70.3%, or 3,279 genes depicted similarities with other genes whereas only 1,385 of them are predicted to be functional. These encoded genome data suggest that anammox microbes are sufficiently more versatile than were initially predicted to be [4]. This chapter intends to deal precisely with the ongoing studies related to anammox processes and their application in real life. The significance of studying anammox and its potential use in the industry will further be discussed.

2 Cell biology of anammox bacteria 2.1 The anammox cell Since the beginning of the chapter, it was clear that the bacteria of interest are indeed enigmatic and this enigma onsets from its fascinating cell biology. When observed

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under the microscope these bacterial cells show a coccoid shape with a diameter of 1 µm, and some of the anammox species also display pilus-like appendages. However, no outer membrane or peptidoglycan is observed in these bacterial cells making it difficult to classify them either as Gram positive or Gram negative bacteria hence grouping them into Planctomycetes (a group of microorganisms lacking peptidoglycan) [5]. When compared with Planctomycetes, anammox differs from them due to the presence of a third membrane. Another interesting anomaly is observed in the constituents of the lipid membrane of these microbes, both ether-linked, and esterlinked fatty acids are present. The presence of ether-linked fatty acid is anomalous as it is a characteristic of the archaeal cell. Apart from this, a special kind of membrane lipid called ladderanes, deriving the name from its ladder-like shape, is also possessed by these bacteria (Figure 1). Anammox cells constitute three compartments. The outermost membrane called the paryphoplasm is free of ribosome, the middle compartment, riboplasm, enclosed by paryphoplasm, contains DNA and ribosome and the central organelle anammoxosome. In order to understand the anammox bacteria, we must try deciphering these organelles [6].

2.2 Anammox cell organelles 2.2.1 The anammoxosome Around 50–70% of the cell volume is constituted by anammoxosome. It is the site where all metabolic activities take place (similar to mitochondria in eukaryotes), and the anammox reaction occurs here. The anammoxosome constitutes many arches, the function of which can be selective protein binding creating small pockets where selected ion channels can be stored. These arches can also be useful during membranebound metabolic processes as they increase the surface area to a sufficient amount. Through transmission electron microscopy (TEM), it was visualized that the anammoxosome has no link with any other cell membrane and contains tube-like structures, and it was further ascertained that these structures contain iron particles. Cytochrome C proteins, which show involvement in anammox reaction, are observed inside this organelle. Using immunogold localization hydroxylamine oxidoreductase (HAO), another enzyme taking part in anammox reaction and ATPase was detected in the anammoxosome membrane [7]. The energy for the growth of anammox is derived by anaerobically oxidizing ammonium with nitrite as an electron acceptor to dinitrogen gas. This reaction can be elaborated into three steps: (i) reducing nitrite to nitric oxide; (ii) condensing nitric oxide and ammonium to hydrazine; and (iii) oxidizing hydrazine to dinitrogen gas. Four electrons are released as a result of oxidizing the hydrazine, and these electrons are assumed to be transported to a membrane-bound cytochrome bc1 complex through

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cytochrome c-type proteins being used as an intermediate electron carrier, one among those electrons commutes to reducing nitrite and the other three are utilized in the synthesis of hydrazine. The electron transport helps in enhancing the displacement of protons by the bc1 complex across the anammoxosome membrane generating a proton motive force (pmf), and this force is in turn utilized by the membrane-bound ATPases in order to produce ATP. Last, this organelle harbors ladder-like lipids called the ladderines, and these are unique to anammox and are assumed to help reduce permeability. They form a diffusion barrier which helps to reduce the loss of energy from passive diffusion of the pmf and reaction intermediates over the anammoxosome membrane [8].

2.2.2 The riboplasm Anammox riboplasm is similar to the cytoplasm of any regular bacteria, housing ribosome, and nucleoids. It is assumed that transcription and translation take place here. It is also observed that anammox bacteria have the special capability of transporting proteins from riboplasm to either the paryphoplasm or anammoxosome, although which one of them remains unclear. Moreover, how this transportation occurs is also unanswered. Signal peptides for different compartments could not be detected; however, it is assumed that protein sorting is achieved by both sec (secretory pathway for both the paryphoplasm and anammoxosome) and by twin-arginine translocation pathway (for anammoxosome) the presence of chaperones in order to achieve specificity and facilitate separate translocation routes is also being postulated. Additionally, in their riboplasmic compartment traces of stored glycogen were observed. There are several hypotheses to this, one being that glycogen acts as an energy and carbon storage compound, stored in order to supply the cell with energy and carbon in case of stress and starvation whilst the others being due to the presence of excess carbon and deficit of nitrogen in the medium, or perhaps the glycogen might be beneficial in the formation of biofilm [9].

2.2.3 The paryphoplasm The outermost compartment of the anammox cell is the paryphoplasm, very less is known about this organelle. From the current knowledge, it is known that paryphoplasm constitutes 20% of the cell volume but it is yet to be determined as a cytoplasmic compartment of a periplasmic space. When observed through TEM this was the most electron light compartment observed. Some RNA might be present in this region but is devoid of ribosomes and DNAs. Further through TEM, an electron-dense, bracketshaped structure was observed which is determined to be the dividing ring. However, this cell division ring was not observed in the metagenome of K. stuttgartiensis but the gene that encoded this protein, kustd1438 was observed [10].

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Anammoxasome Membrane

Nucleoid

Anammoxosome

Cell wall

Riboplasm Cytoplasmic Membrame Paryphoplasm

Intracytoplasmic membrane

Figure 1: Cellular organelle of anammox bacteria.

3 Growth of annamox bacteria A major criticism faced during the adoption of anammox bacteria was its prolonged doubling time. It was observed that these bacteria when kept at 30–40 °C take 10–14 days to divide. Analyzing the mass balance, it was observed that in order to produce biomass (CH2 O0.5N0.15), carbon from carbon dioxide is utilized as a source. The nitrite functions both as an electron acceptor for the oxidation of ammonium and as an electron donor for reducing carbon dioxide. The anammox reaction can be stoichiometrically depicted as follows: NH4 + + 1:32NO− 2 + 0:066HCO− 3 + 0:13H + ! 1:02N2 + 0:26NO− 3 + 0:066CH2O0.5 N0.15 + 2:03H2 O Also, certain optimum conditions are required for the growth of these microbes. Some of which is the concentration of oxygen must be kept 0.5% below air saturation, above which the growth ceases. Further, it must be noted that anammox metabolism diminishes with the nitrogen concentration reaching above 10 mM and its growth ceases on reaching a nitrogen concentration above 20 mM. Also, anammox are obligate anaerobes [11]. Though the generation time can be reduced by providing optimum environmental conditions providing which is impossible due to the composition of wastewater. Further, these microbes are very sensitive to environmental conditions; hence in

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order to cultivate these microbes, mimicking their natural environment was necessary. In order to mimic the natural environment, the biomass is supposed to be retained during low substrate concentration. SBR or sequencing batch reactors are therefore used to serve the purpose. Anammox cells are chosen on the basis of their settling properties through continuously filling the cells, settling the biomass, and extracting the supernatant. After this, the cell is kept in the bioreactor for a very long time [9]. SBR was accepted because of its ability to retain biomass efficiently, distribute substrates, products, and biomass aggregates homogeneously all over the reactor, its ability to operate for a year and being able to function under stable substrate limiting conditions. In spite of all these benefits served by SBR the major loophole to this process was that SBR used activated sludge as an inoculum and using SBR it took around 4 months to start the process. Hence, to start the anammox process sooner membrane bioreactor or MBR was developed. The limitations of other techniques were overcome using MBR. MBR is used as a membrane that does not allow microbial cells to pass through it, hence retaining the complete biomass. It must further be noted that MBR unlike SBR is not based on the settling of biomasses. With the advent of MBR, it was possible to cultivate these slow-growing microbes by retaining biomass. With the help of a stirrer, the biomass and substrate are distributed homogenously. This helps in achieving good growth of the anammox cells [12].

4 Anammox metabolism The anammox reaction can be stoichiometrically depicted as follows: The main substrates present in the anammox process are ammonium, nitrite, and bicarbonate. The catabolic reaction consists of the coupling reaction between the nitrogen atom present in the ammonium and the nitrogen atom present in the nitrite. Both combine to form the dinitrogen gas (N2). The catabolic reaction can be depicted as follows: NH + + NO2− ! N2 + 2H2 O

(1)

Anammox bacteria are autotrophic in nature. HCO3− is utilized as the carbon source for the production of biomass during anabolism. Electrons are generated during the oxidation of nitrite to nitrate. These electrons are further required in the reduction of HCO3−. This reaction can be depicted as follows: HCO3 − + 2.1NO2 − + 0.2NH4 + + 0.8H + ! CH1.8 O0.5 N0.2 + 2.1 NO3 − + 0.4H2 O

(2)

Anammox reaction can be summarized through the above reactions; however, an indepth explanation of the whole process must be studied in this paper. It must be noted that ammonium is inert in nature and to get activated oxygen is required to oxidize it to hydroxylamine (NH2OH). But anammox are anaerobic bacteria, making the requirement of oxygen in the process intriguing; apparently, it is

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hypothesized that the oxidative power of NO is utilized to perform this task [13]. In another instance, while culturing the anammox cells, the cells kept getting deactivated from time to time. However, the cells could be restored by adding catalytic amounts of hydroxylamine. The underlying process behind this resurrection was the conversion of hydroxylamine (NH2OH) to hydrazine (N2H4). Through this experiment, it was concluded that hydroxylamine and hydrazine act as intermediates in the anammox reaction. Further, a three-step pathway was proposed which consists of (1) reduction of four electrons of nitrite, the opposite of what is done by HAO during aerobic ammonium oxidation; (2) formation of hydrazine by the condensation of hydroxylamine and ammonium; (3) oxidation of hydrazine to get dinitrogen gas as the end product. Four other electrons are released in order to reduce nitrite. Examining the genome of K. stuttgartiensis 200 genes responsible for anammox catabolism and respiration were detected. However, the gene responsible for coding nitrite, HAO, was missing. Instead, few genes coding for nitrite, nitric oxide oxidoreductase [cytochrome cd1 nitrite reductase (NirS)] and other proteins, were observed. Through this, it was concluded that nitric oxide (NO) is yet another intermediate in the anammox process [14]. Compiling the genomic data and other evidence three reactions were postulated that could be the referred to as the central anammox metabolism. The reactions are: N2 H4 ! N2 + 4H + + 4e ðE0 ′ = −0:75 VÞ NO + NH4 + + 2H + + 3e ! N2 H4 + H2 O ðE0 ′ = + 0.06 VÞ NO2 − + 2H + + e ! NO + H2 O ðE0 ′ = + 0.38 VÞ

(3) (4) (5)

In eq. (3) four electrons of hydrazine are getting oxidized by protein hydrazine dehydrogenase (HDH) and this further generates the dinitrogen gas. These electrons are further utilized to reduce nitrite by NirS. This is depicted in eq. (5). Last, the hydrazine synthesis has been depicted in eq. (4). Further, the genome analysis of K. stuttgartiensis revealed the presence of quinol: cytochrome c oxidoreductase (bc1, complex III) and an ATPase complex pointed toward the proposal of a mechanism that is chemiosmotic in nature to conserve the energy obtained from the anammox reaction as ATP. The electrons obtained from the oxidation of hydrazine are passed on to the bc1 complex using quinone. The bc1 complex helps to perform two functions namely (a) distribution of the electrons in order to reduce nitrites as depicted in eq. (5) and the synthesis of hydrazine as depicted in eq. (4); (b) according to the proton motive Q cycle in order to generate a pmf protons keep getting displaced across the membrane system, this phenomenon helps the synthesis of ATP. The transfer of electrons intermediately is carried on by a number of cytochrome c proteins. K. stuttgartiensis genome consists of three bc1 complexes and four ATPases [15]. The energy generated from catabolism is utilized by the bacteria for their autotrophic growth. Observing the composition of the cell carbon it was postulated that the

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acetyl-coenzyme A pathway is followed by anammox to fix carbon. Inside the genome of K. stuttgartiensis, the acetyl-CoA pathway could be observed completely. In order to carry out the acetyl CoA pathway, electrons are utilized at very low redox potential (−0.41 V); however, anammox gains their electrons through the oxidation of nitrite to nitrate (+0.43 V). While following this pathway electrons derived from hydrazine which consisted of high energy are translocated to the acetyl-CoA synthetase/CO dehydrogenase with the help of ferredoxin in order to compensate for the loss of hydrazine that was invested during the carbon fixation. This process requires reverse electron transport (Figure 2). While studying the process two unique enzymes were detected. One of them was hydrazine hydrolase, and this enzyme helps to produce hydrazine from nitric oxide and ammonium. Another enzyme involved was HDH, and this enzyme helps to transfer an electron from hydrazine to ferredoxin [16].

5 Metabolic versatility of anammox Throughout the context of this chapter, we have known that anammox bacteria are capable of converting ammonium to nitrite. But the metabolic activity of anammox is not just restricted to that “Brocadia,” “Anammoxoglobus,” and “Kuenenia” are some of the anammox genera bacteria; these bacteria are capable of metabolizing the fatty acids propionate, acetate, and formate. These fatty acids are oxidized to CO2, and this oxidation is followed by the reduction of nitrate to ammonium via nitrite. The ammonium required to carry out standard catabolism is produced by the bacteria by the above-mentioned process. Further, it must be noted that the fatty acids metabolized do not add up to the biomass but get converted to CO2 completely. How anammox is able to do the above function is yet an unanswered question as the CO2 fixation performed by anammox is a very energy exorbitant mechanism [17]. Other than fatty acids Kuenenia stuttgartiensis are capable of converting Fe2+ to Fe3+ through oxidation using nitrate as the electron acceptor. Further, they can also reduce Fe3+ to Fe2+ and Mn4+ to Mn2+ using formate as the electron donor. In order to study the metabolic versatility and effect of organic compounds on anammox certain experiments were conducted. Two SBRs were inoculated with activated sludge which consisted of Candidatus Brocadia anammoxidans and Candidatus K. stuttgartiensis. Propionate and acetate in the concentrations 0.8 and 1 mM were supplied to them, respectively, and also ample amount of ammonium and nitrate was supplied to the reactor along with a trace amount of nitrite. After a certain time as the bacteria in the reactor proliferated the concentration of influent nitrite and ammonia increased from 2.5 to 45 mM; however, their effluent concentration remained undetected until their concentrations were increased up to 15 and 30 mM, respectively [12].

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According to the competition model, it was postulated that a co-culture will be obtained containing 30–40% of anammox if the organic compounds present are utilized by the heterotrophic denitrifiers. However, after 4 months about 80% anammox was observed in the cell mass, indicating the fact that anammox is able to metabolize organic compounds with greater affinity. Further, it must be noted that in the propionate reactor Candidatus Anammoxoglobus propionicus were predominant and in the acetate reactor Candidatus Brocadia fulgida were predominant. Both the species of bacteria could oxidize organic substrates [18].

6 Genomics of annamox bacteria 6.1 Interpretation of the Candidatus K. stuttgartiensis The anammox species that has been sequenced is Candidatus K. stuttgartiensis. Genomes were collected from a laboratory reactor maintained with wastewater. While sampling 74% of the total microorganisms present consisted of K. stuttgartiensis, twenty-eight taxonomic units were present other than Kuenenia sp. Among these six bacterial phyla along with two lineages of bacteria that were not cultured were detected. By combining shotgun and fosmid analysis the DNA sequences were derived. DNA sequences were also collected from bacterial artificial chromosome libraries. Combining all this information the genome is organized into five contigs. The result suggested that around 60 genes are missing, and the genome obtained is 98.5% complete [1]. The nearly complete genome codes for 4,663 ORFs. When compared with other gene databases 3,279 genes showed similarity but 1,385 genes showed function. The size and the number of encoded proteins of the genome are surprising as initially anammox bacteria were thought to be lithotrophic. The ecophysiological study described the organism as more versatile. Through genomic information, new functions regarding energy metabolism and anabolism can also be discovered. The genome sequence of anammox bacteria also reveals how these bacteria harbor so many unique anammox properties [1]. Physiological studies indicated that the reduction of nitrite through ammonium oxidation to produce N2 forms intermediate hydroxylamine and hydrazine. Five ammonium transporters, five nitrite/formate transporters, and two nitrite/nitrate transporters were found responsible for uptaking the substrates in the K. stuttgartiensis genome. The genomic study of anammox supports the notion that through chemiosmotic mechanism anammox are able to synthesize their ATP [1]. Anammox microorganisms are most likely the uttermost down-the-line extension to the biogeochemical nitrogen cycle. These microorganisms use their energy to convert ammonium and nitrite into dinitrogen gas. This reaction takes place in the deficiency of oxygen. These

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microorganisms are related to the Planctomycetes. Anammox organisms are depicted by a compartmentalized cell design which includes a central cell compartment, the “anammoxosome.” So far one of kind “ladderane” lipid particles have been recognized as a component of their layer framework encompassing the various other cell compartments. Nitrogen development includes the middle portion arrangement of hydrazine, an exceptionally receptive and harmful compound. Utilizing a metagenomic approach the genome of the anammox species K. stuttgartiensis was collected. This was collected from a local area filled with a sequencing clump reactor [4]. The gathered gene sequences helped the in silico reproduction of anammox digestion and were probably associated with the cycle. The present anammox pathway known to us is the only one steady with an ample amount of accessible exploratory information, thermodynamically and biochemically plausible and predictable with Ockhams’s razor. The presence of anammox microbes has been in numerous oxygen-restricted marine environments and also in freshwater frameworks, in which seas, estuaries, bogs, streams, and large water bodies like lakes are included. In the marine water ecosystem, more than half of the dinitrogen gas generated is produced by the anammox bacteria. Utilizing the anammox process offers an alluring opportunity to clean wastewater in contrast to the conventional wastewater treatment framework for the evacuation of alkali nitrogen. In the present times about five full-scale reactor frameworks are functional [2].

6.2 Deciphering the evolution and metabolism of an anammox bacterium from a community genome Anammox has been widely utilized in the domains of oceanography study and wastewater treatment. Therefore, the anammox process can be referred to as the biogeochemical nitrogen cycle’s major biochemical enigma. Some of the important highlights of this process include the event of formation of hydrazine as an unconfined moderate of catabolism, the making of ladderane lipids, and the function of cytoplasm separation, all of which are special in science [2]. In order to understand the genomics of the bacteria better the natural genomics of the bacteria was utilized. The genomic information of the bacteria was first collected from the climate and deciphered. First, the genome of the crude anammox bacterium K. stuttgartiensis was gathered from a complex bioreactor belonging to a local area. The genomic information obtained helped to bring the developmental history of Planctomycetes into light and further permitted to discover the hereditary outline of the unique properties harbored by the anammox bacteria. Most essentially, the genomic study of the organism helped to recognize the up-andcomer qualities liable for ladderane biosynthesis and organic hydrazine digestion; also, it helped to discover the metabolic adaptability of anammox [19].

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What is happening inside the anammoxosome? ADP + ATP 3H

Cytoc home Bc-1

ATPase Anammoxosome lumen

Q 6 H+

cyt

cyt

1 electron NirS Enzyme NO

Anammoxosome Membrane

+

NO

cyt

3 electrons HZS Enzymes

NH

4 electrons HDH

N2H4

N2

Enzymes

+

Figure 2: Metabolic activity inside anammoxosome.

7 Role of anammox bacteria in the environment Various methods have been formulated for the detection of anammox bacteria. Sequences from various studies were collected, and their activities in a natural ecosystem as well as in a manmade ecosystem were studied. These bacteria are found in marine, brackish, terrestrial, and various other anoxic environments. Anammox was also found in the natural environment recently [27]. Habitat-specific studies show that various factors are affecting the distribution of anammox in various habitats. Around 6,000 anammox genes were investigated through 16S rRNA sequencing. The anammox bacterial population can also be found in harsh climatic conditions. The anammox growth rate is very slow. Their growth rate is only 15 generations in a year. These bacteria have genus-specific and species-specific habitats. From the dry soil and wet soil, in fact, anammox samples could also be isolated from groundwater, and samples were further gathered globally across 10 countries to explore the distribution of anammox bacteria [20]. To generate a community profile, a high-throughput amplicon sequencing method was applied to the functional gene hydrazine synthase. Results show that from a global perspective, Candidatus Brocadia is the most abundant genus. They consist of about 80–90% of the collected sequences [21]. The second most abundant sequence is Jettenia accounting for more than 20% of the observed sequences in all over the place. In order to culture an organism, a suitable temperature condition is required for growth. In the laboratory, the anammox organism grows at 43 °C whereas in the sea ice in Greenland it can grow in temperatures as low as −2 °C. The diversity found in the snow sample is

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more discrete, whereas in dry places, wet soil and groundwater most relatable community of anammox is present [28]. The diversity of the anammox community is highly dependent upon organic matters like carbon and nitrite. In the dry lands, wetlands, and groundwater ecosystem almost a similar type of anammox community can be observed. However, the snow samples turn out to be quite different [21]. Specific ecological terms and conditions must be maintained for the anammox process. Anammox bacteria have proved to be quite versatile.

7.1 Anammox – terrestrial ecosystem To test whether anammox bacteria are present in soil and to determine their diversity PCR technique is used. Very recent inventions showed that 16S rRNA sequences can be extracted from soil samples and groundwater. This shows soil samples contain more genus-based diversity than the marine ecosystems and the terrestrial ecosystem is an efficient habitat for the anammox organisms (Gonzalez-Martinez et al., 2018). Despite the huge phylogenetic diversity found in the terrestrial system, the anammox activity there is generally much lesser than in marine systems. Soil with high nitrogen content promotes anammox diversity. The soils of wetlands, lake shores, permafrost soil, and agricultural soil show a huge diversity of anammox, it must also be taken into account that anammox is mainly present in wetlands which generally consists of high nitrogen content. The difference in soil properties and depth reflects a diversity of microniches of anammox bacteria within terrestrial habitats. The global environment plays a very important role to promote the enrichment of the bacteria [22].

7.2 Anammox – marine ecosystem The key nutrient that might affect the primary productivity of anammox is inorganic nitrogen. The energy source utilized is ammonium during the oxidation of nitrite and nitrate. The global biogeochemical nitrogen cycle is extensively dependent upon anaerobic ammonium oxidation, which is observed in marine water. In anammox bacteria, diversity salinity plays a very important role. The highest anammox diversity is present in the freshwater environment on the other hand the lowest anammox diversity is observed in marine water [23]. In marine sediments which is at a huge depth from the sea level the temperature is very low, this is probably the main reason of the abundance of anammox organism there. Around 23–30% of nitrogen loss from the marine environment is contributed by anammox organism. In various marine ecosystems, anammox bacteria are a major or even only sink for fixed nitrogen. Anammox bacteria play a key role in the global N cycle [21]. The abundance of anammox bacteria in marine sediments was positively correlated with marine water depth.

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Table 1: Contribution to biogeochemical N2 production. Type of ecosystem Groundwater Paddy soils Lakes Estuaries

Contribution to N production –% –% –% –%

There are five known anammox genera with 16 species proposed. The Ca. Scalindua genus consists of nine proposed species, six of which were discovered in marine environments. Ca. Scalindua Arabica originated in the Arabian Sea and the Peruvian oxygen minimum zone. From the Bohai Sea Ca. Scalindua Pacifica was found in the marine sediment of Swedish fjord Ca. Scalindua profunda were retrieved. Observations of anammox bacterial diversity have demonstrated that Ca. Brocadia, Ca. Kuenenia, and Ca. Anammoxoglobus were commonly found in nonsaline environments. Marine anammox bacteria could be a promising alternative for the treatment of NH4+ rich saline industrial and municipal wastewater.

7.3 Anammox – freshwater ecosystem In the freshwater ecosystem anammox is a hard-to-find species. The first direct anammox process in an ecosystem was associated with the lakes in the suboxic water layer at 100–110 m depth. Extreme environments like very high ambient temperature or a very low ambient temperature, acidic pH or a basic pH, and eutrophic anammox organisms were investigated predominantly. In freshwater ecosystems, Brocadia, Kuenenia, and Anammoxoglobus genera were mainly found. In the contaminated groundwater, Ca. Brocadia is the major anammox phylotype that has been found. Amplification in anammox organism has been observed in places where river and sea interact. Also, the zones where the freshwater and marine environment meet, considered transitional zone like an estuary, and mangrove sediment are some of the dynamic habitats where the anammox process is found (Bagnoud et al., 2020). Pearl estuary is a subtropical estuary. Organic matter found here is highly enriched. The anthropogenic activities here elevate the inorganic nutrient content and are a matter of concern. When 16S rRNA and HZO genes were experimented within the anammox bacterial population discovered in Pearl estuary as well as the surface sediments. Quantitative PCR techniques were used to investigate the presence of anammox organisms in the water and also in the terrestrial environment. With the change in salinity gradient, the dominant species changed from Candidatus Brocadia to Candidatus Scalindua, thus community shift was detected [20]. To upgrade the conventional process of wastewater treatment plant (WWTP) anammox process is being utilized. Applying the anammox process the sewage treatment can

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be turned into resources and it can, in turn, be utilized as an energy-saving mechanism. Sewage can prove to be liquid gold from a cost-effective, energy-saving, and resourcelimiting point of view. Ammonium and nitrite can be converted to dinitrogen gas by some of the anammox bacteria. Various organic and inorganic compounds can also be converted [24]. Formate, acetate, and methylamine oxidation are also performed by some of the anammox species. They can use iron as an electron donor and can perform nitrite respiration. Also using nitrite as an electron acceptor, they oxidize organic acids. Anammox bacteria have proven to be very important as they are very less cost demanding and do not put a bad impact on the environment as well as arena efficient substitute for the wastewater treatment process that is generally done. Greenhouse gases are a major matter of concern nowadays, applying anammox process the N2O and CO2, which are considered to be greenhouse gases can be reduced. Anammox organisms reside in the environment not by competing with the other nitrogen cycle microbes but by maintaining a smooth interaction with them. The role of these organisms varies. Sometimes their contribution is highly effective or it can be negligible sometimes. Removal of fixed nitrogen highly changes from estuarine to coastal sediments. Distribution, abundance, and activity mostly in salinity play a very important role in anammox bacterial patterns [2]. Physiological and phylogenetic knowledge about anammox species is much limited to date. We need to enrich our knowledge to enhance the use of this process in technological and environmental systems. Significant progress has been achieved. Nitrogen removal performance and system stability need to be gradually improved by increasing experimental evidence [2].

8 Applications of annamox bacteria A study of the annamox organism would only be helpful if it could be implemented in day-to-day life. Since the discovery of annamox, it was realized that these microbes have huge efficiency for the removal of highly contaminated ammonium from wastewater and waste gases. Hence Arnold Mulder and colleagues patented the process immediately; later it was made possible to grow the culture reproducibly and hence Annamox was implemented in ammonium removal along with the Paques company [25]. On the other side, Mark van Loosdrecht and colleagues came up with a large-scale process SHARON which transforms ammonium to nitrite, not nitrate. This proved to be ideal to produce equal ratio mixture of nitrite and ammonium needed for the anammox reaction further coupling of NH4+ and NO2− biochemically to contribute N2 gas [26]. This process saves costs as less energy for aeration is needed and also other electron donor is not required. Anammox are autotrophic bacteria so they save cost on chemical dosage as they do not require sources which have organic carbon in them. Production of excess sludge is reduced and CO2 emission is much less. If this works out

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efficiently it can prove to be an outstanding technique for treating municipal wastewater perfectly. In order to make a stable effluent for the discharge, temperature, pH, DO, and carbon source content are required; moreover, nitrogen loading should be maintained in exact terms [23]. Anammox bacteria achieve maximum productivity at high environmental temperatures. To increase the application of these anammox organisms, an intensive work is going on the metabolism of the bacteria at low temperature by metagenomic analysis. Black water, brown water, urine water, or yellow water can be treated through this bacterium. When various reactions are done within a single tank, the SHARON process takes advantage of the incomplete nitrification under oxygen limitation in the other different tanks by aerobic ammonium oxidizing organisms [8]. This process is itself enough to take the place of the general nitrification/denitrification that is done in large-scale treatment plants. Anammox contributes significantly to the global marine and terrestrial nitrogen balance. Anammox reaction changes more than 50% nitrogen compound into nitrogen gas. As it is a new technology anammox reactor operation requires knowledge. Biotechnology has been so much developed these days that there is a long list of techniques based on the anammox reaction that does the removal of nitrogen in various industrial and civic effluents with maximum effectiveness, low rate, and also least damage to the environmental balance (Gonzalez-Martinez et al., 2018). The cost of wastewater treatment can be decreased by 60% by the effective application of the anammox process. An in-depth study of microbial population, various chemical and morphological parameters, and operational conditions are needed for much good understanding and proper implementation of the process. The first anammox reactor was built in the Netherlands. In Germany, on another wastewater treatment plant, anammox activity coincidentally happened. The Netherlands has three fullscale processors. Among them one is municipal and two are industrial. The disadvantage of utilizing anammox is their slow doubling time which makes it difficult to grow enough sludge. Also, the recovery time is longer. The optimum pH level is 8 so it also needs to be maintained and adjusted by adding caustic [22]. A further limitation to the anammox application is the long start-up time and high sensitivity of the bacteria toward oxygen and nitrite concentration.

9 Conclusion This review intends to provide a perception about environmental importance and necessity of anammox in global biogeochemical cycles, and it is outlined by worldwide research development. Anammox processes play a huge role in the sustainable administration, significantly in the nitrogen cycle. These organisms grow slowly but show diverse phylum, which is distributed among various environments and conditions. The conventional nitrogen removal process is a very costly technology, and the anammox process, on the other hand, is much more cost-effective and environmental friendly; it

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directly contributes to a balance in the environment and economy. The activities of anammox organism can be increased by providing a properly balanced environment that must be carefully maintained while treating real wastewater. The research on anammox nurtures the pathway understanding and genomic blueprint understanding. Interactions among various microbes promote the function of anammox bacteria and increase the biological nitrogen removal efficiency. Sustainable management of wastewater can be done by proper use of the anammox process. Integrated process design, scale-up, and full-scale case studies can enhance the use of anammox efficiency. Gaseous emission should be operated minutely during the anammox process. Worldwide special attention is being given to the anammox process. Industrial operators can optimize their point of focus by increasing information about this process although many challenges are faced during its application. Therefore, many fundamental questions are already raised during the application which needed further investigation. One of the examples is when a huge amount of wastewater is required to be refined these need huge biomass. This challenge can be solved by various cultural methods. As anammox is a very slow grower, if possible, fast growing anammox can open more possibilities. The recent research is confined to electron microscopy; if it could be studied by metagenomics it can open so many paths in anammox application. Under various water quality how anammox organisms are working should also be verified. So many applied studies are needed for efficient anammox application. Anammox offered a great possibility from the industrial point of view. Further study, research, and more applications will enhance the use of this process.

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Index (pseudo)periplasmic space 37 16S rRNA 4, 150 abiotic oxidation 42 acetate 8 Achromobacter 68 aerobic 32 aerobic zone 47 agricultural 61 agriculture 58 Aigarchaeota 17 ammonia 57, 66, 74, 130 ammonia monooxygenase 36, 110 ammonia removal rate (ARR) 9 ammonia-oxidizing archaea 34 ammonia-oxidizing bacteria 105–107, 121, 123–124, 134 ammonification 8, 32 ammonium 68 ammonium-oxidizing bacteria 34 AMO 74, 76, 78 amoA encoding archaea 16 anaerobic 72 anaerobic conditions 140 anaerobic zone 47 anaerobically 39 anammosome 41 anammox 2, 16, 20, 22, 55, 69, 74 anammox bacteria 106, 109, 116 anammox reactor 154 Anammoxoglobus 66 anammoxosome 107, 109, 111–112 anammoxsome 41 anoxic 47 anoxic rector 47 anoxic/oxic 47 anthropomorphic 36 AOB 68, 72, 74, 76–77 Archaea 16 archaea oxidation 37 atmosphere 57 autotrophic 34 autotrophic ammonia-oxidizing bacteria 16 Azorhizobium 67 Azotobacter 67

https://doi.org/10.1515/9783110780093-010

Bacillus 68, 70 Betaproteobacteria 122, 124 biogenic nanoparticles 12 biogenic nitrogen removal 106 biogeochemical cycles 58 biogeochemical nitrogen cycling 129 biological 19 biological oxygen demand 44 biomass 145, 155 bioreactor 149 biotransformation 40 blue baby syndrome 34 Bradyrhizobium 67 Candidatus 73–74 Candidatus Brocadia anammoxidans 4 carbon dioxide 60 carbon source 46 catabolism 149 cathodic reaction 47 chemical oxygen demand 2 chemiosmotic mechanism 148 chemoautotrophic 34, 56 chemolithoautotrophic 37 chemolithoautotrophic nitrite-oxidizing bacteria 39 chemolithoautotrophs 21 chemosynthetic 40 climate 59 Clostridium 68, 70 comammox 34 comammox bacteria 122 commamox 68 community profile 150 consumption 61 continuous packed-bed columns 9 continuous stirred tank reactor 47 conventional 2 Crenarchaeota 17–18 cyanobacteria 70 cytochrome C 142 decomposition 58 denaturing gradient gel electrophoresis (DGGE) 9 denitrification 16, 19, 56, 61, 70

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Index

denitrifiers 40 denitrifying 2 Desulfovibrio 70 DGGE 24 diazotrophs 31 dissimilatory 70 dissolved oxygen 43 DNA sequence 148 dynamic habitat 152 ecophysiological study 148 efficiency 155 effluent 154 energy 4 energy exorbitant 147 environment 61 Euryarchaeota 18 exopolysaccharide 4 facultative 40 fatty acids metabolism 147 fermentation 4 ferric reductase 9 FISH application 24 fixation 32 fluorescent in situ hybridization (FISH) 5 Fluorescent polyclonal antibodies 23 fosmid analysis 141 Gammaproteobacteria 122, 125 genomic technologies 17 Gram-positive bacteria 48 granular sludge reactor 4 graphene oxide 3 Haber–Bosch process 32 hao 74, 76, 78 heterotrophic 34 high-performance liquid chromatography (HPLC) 5 high-temperature habitat 20 horizontal gene transfer 17 hydrazine 147 hydroxylamine dehydrogenase 36 hydroxylamine oxidoreductase 42 hydroxylamine quinone reductase 38 immobilization 60 isotopic 3

kinetic 11 Korarchaeota 18 k-type 20 ladderane 109 ladderines 141 landfill leachate 10 manures 58 marine ammonia-oxidizing bacteria 127 marine N-cycle 129 marine sediment 151 marine sponge 20 membrane bioreactor (MBR) 7, 145 mesoporous silica 12 metagenomic 149 metagenomics 8 meta-transcriptomics 8 methanogen 18 methanogenic granules 7 methanotrophic 36 methemoglobinemia 34 methylamine 153 Microarray technology 24 microbial electrolysis cells 47 microbial fuel cells 47 microcosm 57 microniches 151 mineralization 56 mineralized 59 moisture 60 Monera 17 moving bed biofilm reactor (MBBR) 4 multiple environmental factor 26 mycorrhizal 60 Nanoarchaeota 18 nanomaterials 12 nitrate 56 nitrification 10, 16, 19, 56–57, 59, 61–62, 121–122, 127–134 nitrifying 32 nitrite 69, 77 nitrite oxidoreductase 39, 42 nitrite reductase 25, 38, 41 nitrite removal rate (NRR) 9 nitrite-oxidizing bacteria 34 nitrite-oxidizing bacteria (NOB) 90

Index

Nitrobacter 39, 68 Nitrococcus 68 nitrogen 18, 57–58, 65, 67, 69 nitrogen cascade 34 nitrogen cycle 55 nitrogen loading rate (NLR) 10–11 nitrogen removal efficiency 9 nitrogenase 67 nitrogenous chemical 16 Nitrosococcus 36, 68, 72 Nitrosolobus 68, 72 Nitrosomonas 10, 36, 68, 72, 74, 76, 124 Nitrososphaera maritimus 37 Nitrosospira 36, 68, 72, 74, 76, 122, 125 Nitrosovibrio 68, 72 Nitrospina 39 Nitrospira 2, 39, 68, 73 NOB 68 nucleic acids 32 obligate anaerobes 144 oceanography 149 omics 23 omics technology 24 optimum conditions 144 optimum pH 154 organic loading rate (OLR) 11 organotrophic 4 oxic–anoxic 25 oxygen 60, 132 oxygen minimum zone 152 Pa. denitrificans 38 paddy soil 130 Paracoccus 71 Paracoccus denitrificans 37 PCR 24 PCR technique 151 pH fluctuation 132 pH range 132 phylogenetic 151 phylum 41 Planctomyces 69 Planctomycetes 10 Planctomycetes 20, 41, 142 polypropylene foam 6 polyvinyl alcohol 6 predenitrification 42

prokaryotes 31 propionate reactor 148 proteins 32 Proteoarchaeota 18 proteobacteria 48 proton motive force (pmf) 146 Pseudomonas 48, 68, 70 quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) 6 redox potential 147 rhizobium 67 rhizosphere 57 saline conditions 8 salinity 8, 133, 151 scanning electron microscopy (SEM) 6 sensitivity 154 sequencing batch biofilm reactor (SBBR) 10 sequencing batch reactor 2, 47 sequencing batch reactors (SBR) 145 SHARON 45, 153 slow grower 155 sodium alginate 6 soil ammonia-oxidizing bacteria 127 Spearman’s rank 25 statistical 59 stoichiometric ratios 7 suboxic water 152 subtropical estuary 152 suspended flocs 7 sustainable management 27 sustainable management of wastewater 155 symbiotic partner 31 symbiotic relationship 7 temperature 26, 62 terrestrial 151 Thaumarchaeota 18 thermal spring environments 36 thermophile 18 Thiosphaera pantotropha 44 three domain 17 total nitrogen (TN) 10 trace element analysis 130 trace organic chemicals (TrOC) 84 transformation 58

161

162

Index

transitional zone 152 transmission electron microscopy (TEM) 142

wastewater 15 wastewater treatment 109, 114 wastewater treatment plant (WWTP) 152

upflow anaerobic sludge blanket (UASB) 7 xenobiotic 2 versatile 151 volatile fatty acids (VFAs) 5 volatile suspended solids 3

About the series Sustainable Water and Wastewater Treatment discusses the recent advances in water and wastewater treatment research and processes covering bio-remediation, bio-degradation, molecular approaches, and electro-biochemical strategies. It also evaluates the possible applications of these corrective strategies to remove toxic pollutants from the environment. This series describes the limitations and challenges of wastewater treatments. In addition, it covers various advanced and innovative technologies to remove toxic and hazardous pollutants present in wastewater in a sustainable manner. Moreover, it considers the application of mathematical modelling and different emerging molecular tools to wastewater treatment. Particular attention is given to water reuse and recovery of added-value products from wastewater since it would contribute to a circular and bio-based economy. Editor-in-Chief Maulin P. Shah, Enviro Technology Ltd., India Please send any book proposals to Maulin P. Shah.

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