Studies on the Biological Treatment of Wastewater from Starch Industry for Pollution Control

Table of contents

PART A:
Certificate
Statement of thesis preparation
Declaration
Dedication
Acknowledgement
Table of Content
List of figures
List of tables
Nomenclature and abbreviations
Abstract
List of publication

PART B:
1. INTRODUCTION
1.1. Water- the basis of civilization
1.1.1. Water pollution control- the needs and necessities
1.1.1.1. Physical water treatment methods
1.1.1.2. Chemical water treatment methods
1.1.1.3. Biological water treatment methods
1.1.2. Regulation of water pollution
1.1.3. Wastewater- a good resource for energy recovery
1.1.4. Corn starch industry wastewater pollution
1.1.5. The current scenario in the treatment of corn starch wastewater
1.1.6. Problem statement and justification
1.1.7. The objective of this research
2. LITERATURE REVIEW
2.1. Corn starch production and market statistics
2.2. Wastewater generation and characterization
2.3. Understanding the biologics of starch effluent
2.3.1. Overview of the anaerobic digestion process
2.3.2. Microbiology of anaerobic digestion process
2.3.3. Critical influential factors for anaerobic digestion process
2.3.3.1. Temperature
2.3.3.2. pH
2.3.3.3. Substrate characteristic
2.3.3.4. Loading rate
2.3.4. Review on anaerobic treatment technologies
2.3.5. Overview of the nitrogen removal process
2.3.5.1. The general concept of the nitrogen cycle
2.3.5.2. Nitrification
2.3.5.3. Denitrification
2.3.5.4. Anaerobic ammonium oxidation
2.3.5.5. Analysis of limitations of present technologies in nitrogenous waste treatment
2.3.5.6. Scope of new technologies for the effective treatment of starch industry effluent

3. CHAPTER 3: MATERIALS AND METHODS
3.1. Sample collection
3.2. Inoculum collection
3.2.1. Leaf debris sample
3.2.2. Anaerobic sludge sample
3.2.3. Cow dung sample
3.2.4. Anammox sample collection
3.3. Sample enrichment
3.4. Batch reactor setup
3.5. Continuous reactor set-up
3.6. Analytical methods
3.6.1. pH assessment
3.6.2. Temperature assessment
3.6.3. COD assessment
3.6.3.1. Reagent preparation
3.6.3.2. Procedure
3.6.3.3. Calibration
3.6.4. BOD assessment
3.6.4.1. Required reagents
3.6.4.2. Procedure
3.6.4.3. Calculation
3.6.5. Ammoniacal nitrogen assessment
3.6.5.1. Required reagents
3.6.5.2. Procedure
3.6.6. Nitrite- nitrogen assessment
3.6.6.1. Required reagents
3.6.6.2. Procedure
3.6.7. Nitrate assessment
3.6.7.1. Reagent preparation
3.6.7.2. Procedure
3.6.8. Total nitrogen assessment
3.6.8.1. Required reagents
3.6.8.2. Procedure
3.6.9. Phosphate assessment
3.6.9.1. Required reagents
3.6.9.2. Procedure
3.6.10. Sulfate assessment
3.6.10.1. Required reagents
3.6.10.2. Procedure
3.6.11. VFA assessment
3.6.11.1. Required reagents
3.6.11.2. Procedure
3.6.12. Biomass (as VSS) assessment
3.6.13. Biogas assessment
3.7. Research design
4.1.1. Screening of potential inoculum for anaerobic treatment of starch industry wastewater
4.1.1.4. Selection of best potential biomass for inoculum preparation
3.7.1. Screening of potential inoculum for anaerobic treatment of starch industry wastewater
3.7.2. Screening and confirmation of low pH tolerant methanogenic bacteria
3.7.3. Optimization of reduction potentiality of COD by leaf debris microflora
3.7.3.1. Optimization strategy
3.7.4. Optimization of methane production by leaf debris microflora from starch effluent
3.7.5. Screening of anammox activity by microflora isolated from different habitat
3.7.6. Screening of anammox bacteria by the 16S ampicon study
3.7.6.1. Isolation and qualitative and quantitative analysis of gDNA
3.7.6.2. Preparation of libraries for 2 x 250 bp Run Chemistry
3.7.6.3. Cluster Generation and Sequencing
3.7.6.4. Bioinformatics analysis
3.7.7. Enrichment of anammox biomass in a continuous reactor
3.7.8. Studies on the requirement of carbon source and its effect on anammox activity
3.7.9. Studies on the requirement of nitrogen source and its effect on anammox activity
3.7.10. Optimization of pH and temperature for optimal nitrogenous waste removal by anammox culture
3.7.11. Effect of salinity and antibiotic activity on reduction of nitrogenous waste and community interaction in anammox reactor
3.7.12. Development of novel acidophilic pilot scale methane-bioreactor and anammox system for treatment of starch industry wastewater

4. CHAPTER 4: RESULTS AND DISCUSSIONS
4.1. Enrichment of low pH tolerant methanogens and anammox bacteria from different natural resources
4.1.1.1. Effect of leaf debris microflora for COD reduction and biomass increase
4.1.1.2. The response of anaerobic sludge microflora for COD reduction and biomass increase
4.1.1.3. The response of cow dung microflora for COD reduction and biomass increase
4.1.2. Screening and confirmation of low pH tolerant methanogenic bacteria
4.1.3. Screening of anammox activity by microflora isolated from different habitat
4.1.3.1. Analysis of ammonia removal potentiality
4.1.3.2. Analysis of nitrite removal potentiality
4.1.4. Enrichment of anammox biomass in a continuous reactor
4.1.4.1. Analysis of biological change during operation
4.1.4.2. Analysis of biological community and changes by 16S amplicon analysis
4.2. Studies on the effect of physicochemical parameters on the growth of enriched methanogen and anammox bacteria
4.2.3.1. Effect of different salts of nitrogen on anammox
4.2.3.2. Effect of combination of different salts of nitrogen on anammox
4.2.5.1. Effect of pH and temperature on the removal of ammonia
4.1.3.3. Selection of biomass
4.1.3.4. Confirmation of anammox bacteria by 16S amplicon studies
4.2.1. Optimization of COD reduction by leaf debris microflora
4.2.1.1. Evaluation of biomass generation
4.2.1.2. Sensitivity analysis
4.2.1.3. Process optimization
4.2.2. Studies on the requirement of carbon source by anammox bacteria
4.2.2.1. Estimation of limit of tolerance for carbon in media
4.2.3. Studies on requirement of nitrogen source by anammox bacteria
4.2.3.3. Effect of ammonia concentration on anammox
4.2.3.4. Effect of nitrite concentration on anammox
4.2.4. Media optimization for anammox bacteria
4.2.5. Optimization of pH and temperature for anammox bacteria
4.2.5.2. Effect of pH and temperature on the removal of nitrite
4.2.5.3. Optimum point analysis for of nitrite removal
4.2.5.4. Optimization of the process
4.2.6. Effect of salinity on the growth of anammox bacteria
4.2.7. Effect of antibiotics on anammox activity
4.3. Studies on biodegradation of starch industry wastewater for pollution control using enriched species under optimized condition in shake flask
4.3.1.2. Evaluation of COD removal potential by utilizing response surface methodology
4.3.1.3. Effect of pH on methane production
4.4. Design and optimization of laboratory scale wastewater treatment system for treatment of carbonaceous and nitrogenous waste
4.3.1. Optimization of methane production by leaf debris microflora
4.3.1.1. Effect of pH on COD reduction
4.3.1.4. Optimization of process
4.4.1. Analysis of methane bioreactor
4.4.2. Analysis of anammox bioreactor

5. CHAPTER 5: CONCLUSION AND FUTURE SCOPE OF WORK
5.1. Conclusion
5.2. Future scope of work

6. CHAPTER 6: REFERENCES

NATIONAL INSTITUTE OF TECHNOLOGY

DURGAPUR

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Certificate

It is certified that the work contained in the thesis entitled “Studies on the biological treatment of wastewater from starch industry for pollution control” has been carried out by Shubhaneel Neogi (14/BT/1501) under the guidance of Prof. (Dr.) Apurba Dey and Dr. Pradip Kumar Chatterjee. The data reported herein is original and that this work has not been submitted elsewhere for any other Degree or Diploma.

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This is to certify that the above declaration is true.

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Prof. (Dr.) Apurba Dey Dept. of Biotechnology National Institute of Technology Mahatma Gandhi Avenue Durgapur 713209, India

Dr. P. K. Chatterjee Ex. Chief Scientist and Head Thermal Engineering group CSIR-Central Mechanical Engineering Research Institute

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STATEMENT OF THESIS PREPARATION

1. Title: Studies on the biological treatment of wastewater from starch industry for pollution control
2. Degree for which the thesis is submitted: Doctor of Philosophy
3. Thesis guide was referred to for preparing the thesis: Yes
4. Specifications regarding thesis format have been followed: YES
5. The content of the thesis have been organized based on the guidelines: Yes
6. The thesis has been prepared without resorting to plagiarism: YES
7. All sources used have been cited appropriately: Yes
8. The thesis has not submitted elsewhere for a degree: I agree

Name: Shubhaneel Neogi

Registration No.: NITD/PhD/BT/2016/00703

Department: Department of Biotechnology

Declaration

I hereby declare that the thesis entitled “Studies on the biological treatment of wastewater from starch industry for pollution control” was carried out by me in the Department of Biotechnology, National Institute of Technology, Durgapur under the guidance of Dr. Apurba Dey, National Institute of Technology, Durgapur and Dr. Pradip Kumar Chatterjee, CSIR-Central Mechanical Engineering Research Institute, Durgapur and this project work is submitted in the partial fulfillment of the requirement for the award of the degree of Doctor of Philosophy. I also declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged and that it has not been previously or concurrently submitted for any other degree or any other institution.

Shubhaneel Neogi

Roll No.: 14/BT/1501

Registration No.: NITD/PhD/BT/2016/00703

Department: Department of Biotechnology

National Institute of Technology

West Bengal- 713209, India

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Acknowledgment

It gives me immense pleasure to acknowledge, convey my heartiest thanks and express my gratitude to those persons whom I met during my journey of the doctoral program.

I am extremely grateful to my Supervisor Prof. (Dr.) Apurba Dey, Department of Biotechnology, National Institute of Technology (NIT), Durgapur and Dr. Pradip Kumar Chatterjee, Former Chief Scientist & Head, Thermal Engineering Group, CSIR- Central Mechanical Engineering Research Institute (CMERI), Durgapur and who has endlessly encouraged, guided and motivated me throughout this research work. Both of their superior knowledge, wisdom, and willingness to give complete freedom enriched me immensely as a researcher.

I offer my sincere appreciation and gratitude to Dr. Suravi Chowdhury, Professor and Head, Department of Biotechnology, National Institute of Technology Durgapur, for her help, valuable advice and support during my research program.

I offer my sincere appreciation and gratitude to all my DSC (Doctoral Scrutiny Committee) members, Dr. Dalia Dasgupta Mandal, Dr. Kaustav Aikat, Dr. Nibedita Mahata, Dr. Tamal Mandal, National Institute of Technology Durgapur, for their important suggestion and advice during my research program.

I am grateful to all the faculty members in the Department of Biotechnology, National Institute of Technology, Durgapur, for their valuable suggestion, advice, and timely help throughout my research program.

I wish to thank all the staff members in the Department of Biotechnology, NIT Durgapur.

I also extend my heartfelt thanks to all my colleagues from CSIR-CMERI, Dr. Amit Ganguly, Scientist, CSIR-CMERI, Preeti Singh, Shouvik Saha, Anamika Bhattacharya, and

laboratory staff. I also thank Dr. Subashish Dutta, Priyanka Sarkar, Rohan Yadhav from NIT Durgapur including all members.

I would specially thank my teacher from my graduation college, Mrs. Aparupa Khan and Mrs. Priyanka Sen Kundu who had constantly monitored and motivated during my PhD. I would also like to express gratitude to my supervisor from IPCA Laboratories Ltd., Mr. Bhaskar Pal, who personally identified my potentiality, motivated to pursue research and financially assisted at my difficult times.

I must thank my present employer, the Sukhjit Starch Industries and Mr. Puneet Sardana, MD for having faith in me and to allow me to pursue this research on real-time problems of starch industries. I also thank all the colleagues and laboratory assistants for their constant support in every activity.

I also would like to thank my family members and friends for their moral support. I hope this effort may bring an aura of a vibrant smile to my late father’s face, wherever he is. I also express gratitude to my very supportive brother and my mother.

Date.

Shubhaneel Neogi

Roll No.: 14/BT/1501

Department of Biotechnology

National Institute of Technology Durgapur

West Bengal- 713209, India

List of Figures

Fig. 1.1 World water stress assessment report 1995-2025

Fig. 2.1 Typical hypothetical configuration of maize kernel

Fig. 2.2 Maximum maize producing countries

Fig. 2.3 The end-use fate of corn, Indian Market

Fig. 2.4 Step of maize starch production & relative derivatives

Fig. 2.5 Steps involved in the anaerobic digestion process

Fig. 2.6 Nitrogen transformation pathways mediated by various organisms

Fig. 2.7 The biological concept of the nitrification process

Fig. 2.8 Biological concept of de-nitrification process

Fig. 2.9 The biological concept of ANAMMOX process

Fig. 3.1 Batch reactor setup

Fig. 3.2 Flow diagram of the lab scale effluent treatment system

Fig. 3.3 Lab-scale effluent treatment system in operation

Fig. 3.4 Calibration curve for COD

Fig. 3.5 Experimental set-up for the screening of potential anaerobic bacteria

Fig. 3.6 Quality checking of gDNA. Lane 1- Red cluster, lane M- HindIII 64 marker

Fig. 3.7 Reactor set up for continuous treatment of starch industry effluent

Fig. 3.8 Pictorial representation of the reactor set-up

Fig. 3.9 Test set-up to estimate the role of carbon

Fig. 4.1 Reduction of starch wastewater (COD) by leaf debris microflora

Fig. 4.2 Increment of biomass by leaf debris microflora

Fig. 4.3 Reduction of starch wastewater (COD) by anaerobic reactor 73 sludge

Fig. 4.4 Increment of biomass by anaerobic industrial sludge inoculum

Fig. 4.5 Reduction of starch wastewater (COD) by cow dung microflora

Fig. 4.6 Increment of biomass by cow dung microflora

Fig. 4.7 Comparison of COD reduction efficiency by different microflora

Fig. 4.8 Comparison of biomass generation by different microflora

Fig. 4.9 Comparison of growth in broth by different microflora at pH 5.0- 77 9.0 from left to right (A. Leaf debris inoculum, B. Anaerobic sludge inoculum, C. Cow dung inoculum)

Fig. 4.10 Plate cultures of leaf debris microflora from 0- 15 days (1. Blank, 78 2. At pH 3.0, 3. At pH 5.0, 4. At pH 7.0, 5. At pH 9.0)

Fig. 4.11 Growth of bacteria in slant culture

Fig. 4.12 Microscopy of colonies from leaf debris inoculum

Fig. 4.13 Ammonia reduction by different microflora

Fig. 4.14 Nitrite reduction by different microflora

Fig. 4.15 Comparative analysis of potentiality of reduction by 83 different microflora

Fig. 4.16 Amplicon analysis of microbial communities present in 85 the anammox cluster

Fig. 4.17 Performance of batch reactor under various operational condition

Fig. 4.18 A. Sludge after 40 day’s enrichment, 87 B. 60 day’s old reactor sludge, C. 120 day’s old sludge, D. 150 day’s old sludge with significant anammox granules

Fig. 4.19 Analysis of microbial composition and changes during operation

Fig. 4.20 Effect of the operating parameter on COD removal

Fig. 4.21 Effect of the operating parameter on biomass generation

Fig. 4.22 Sensitivity analysis by perturbation method for the RSM model

Fig. 4.23 Optimized operational parameter determination

Fig. 4.24 Reduction of different carbon sources by anammox

Fig. 4.25 The efficiency of carbon reduction for the different load

Fig. 4.26 Reduction of different nitrogen source by anammox bacteria

Fig. 4.27 Reduction of nitrogen in a combination with ammoniacal 96 nitrogen and nitrite

Fig. 4.28 Reduction of nitrogen in the combination with ammoniacal 96 nitrogen and nitrate

Fig. 4.29 Reduction of nitrogen in the combination with nitrite and nitrate

Fig. 4.30 Reduction trend of ammonia (A) and nitrite (B) for various N- 98 loading

Fig. 4.31 Reduction trend of ammonia (A) and nitrite (B) for various NO2- 98 loading

Fig. 4.32 Effect of pH and temperature in ammonia removal

Fig. 4.33 Effect of pH and temperature in nitrite removal

Fig. 4.34 Optimum point analysis for ammoniacal nitrogen removal

Fig. 4.35 Optimum point analysis for nitrite removal

Fig. 4.36 Removal trend of ammonia in different salinity

Fig. 4.37 Effect of salinity on anammox activity

Fig. 4.38 Assessment of the effect of salinity on the biological community 106 of anammox cluster

Fig. 4.39 Effect of pH for COD removal and biogas generation in 108 reactor 1(R1)

Fig. 4.40 Effect of pH for COD removal and biogas generation in 109 reactor 2(R2)

Fig. 4.41 Effect of pH for COD removal and biogas generation in 109 reactor 3(R3)

Fig. 4.42 Optimization of COD removal process

Fig. 4.43 Effect of pH on methane production for different 111 biomass load

Fig. 4.44 Methane production model

Fig. 4.45 Operational conditions of the methanogenic reactor

Fig. 4.46 Conversion of biomass against COD reduction

Fig. 4.47 The efficiency of biogas and methane production by the lab-scale 116 reactor

Fig. 4.48 Removal of ammonia and nitrite in anammox reactor

List of Table

Table 1.1 Comparison of regulatory norms by different regulatory bodies

Table 2.1 Nutritional value of maize grain.

Table 2.2 Global maize production 2013-2019

Table 2.3 Water balancing and wastewater generation in corn star production

Table 2.4 Characteristic of effluent in various stages of starch processing

Table 2.5 Wastewater characterization of few leading corn starch industries of

Table 2.6 Wastewater characterization of few leading corn starch industries of

Table 2.7 Classifications of methanogen

Table 2.8 Industrial application of different bioreactor for methane production

Table 3.1 Analysis of average daily wastewater production by Sukhjit Starch Industries, Malda, India

Table 3.2 Composition of methanogenium medium

Table 3.3 Composition of synthetic anammox media

Table 3.4 Composition of methanogenium agar medium

Table 3.5 Primers used in the present study for anammox bacteria identification

Table 4.1 Analysis of average daily wastewater production by Sukhjit Starch Industries, Malda, India

Table 4.1 Biochemical characterization of leaf debris colonies

Table 4.2 Optimum point analysis of feed C: N ratio

Table 4.3 The final composition of anammox media based on optimization

Table 4.4 Optimum point prediction

Table 4.5 Effect of antibiotic on anammox activity

Table 4.6 Kinetic evaluation of methane production at different pH

Table 4.7 Optimum point analysis of variables

ABBREVIATIONS

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Abstract

The present research work was undertaken for the biological treatment of starch industry wastewater for pollution control. Starch industry effluent generates a large volume of wastewater with strong acidity and enormous carbon and nitrogen pollutants. In the present study, the development of low pH methanogens and anammox microorganisms were given special attention. Low pH methanogenic reactor and anammox reactor were fabricated for the biological treatment of corn starch industry effluent. In the first part of the work, low pH tolerant methanogens were isolated from peat bog of leaf debris. It showed the presence of Gram-negative, non-spore forming, and cocci with creamish black colour colonies. The leaf debris inoculum was taken for optimum COD removal under anaerobic conditions. Influential parameters were treatment time, pH and biomass loading. It was found that 95% COD removal was achieved at biomass loading of 15% at a pH of 5.0 and biomass concentration of 13.4 g/L. treatment time of 72 h was kept for optimum efficiency and lesser biomass generation. In the next step leaf debris peat bog inoculum was utilized for biogas production. It was found that under acidic pH the rate of the reaction was faster, which removed 99% COD and 2980 ml of biogas was generated from 8283 mg/L of COD, at a treatment time of 62 hours and F/M ratio of 0.82.

Identification and isolation of anammox group of bacteria were performed using enrichment and 16S rRNA amplicon analysis. The objective of the study was to remove ammonia under the anaerobic condition from starch industry effluent. The media optimization using carbon and nitrogen source was undertaken which is required for anammox enrichment. The physical growth factors like temperature and pH were optimized. The optimized temperature and pH were 39 0C and 7.2 respectively. Some restrictive growth factors like salinity and antibiotic concentration were also studied which were found insignificant in our study.

Finally, the overall process was scaled up in the laboratory reactor. The reactor study revealed that in the anaerobic reactor 1.16 kg COD/kg VSS/d was removed producing 42-46% methane as output gas. The anammox reactor was able to reduce 82% nitrogen load from starch industry effluent at the rate of 1.5 kg/m[3]/d after 568 days of initiation.

Thus the overall aim of the study was the treatment of starch industry wastewater for pollutant reduction and simultaneous yield of biogas which can be utilized as alternate source energy, in the present day of energy crisis and nitrogen removed by anammox group of bacteria can be used as fertilizer.

List of Publications

Journal Publications:

[1] Neogi S, Dey A, Chatterjee P.K, Neogi, Microflora from leaf debris is suitable for treatment of starch industry wastewater. Eng Life S ci. 2016; 16(8):683-689. doi:10.1002/elsc.201500086
[2] Neogi S, Dey A, Chatterjee P.K. Corn starch industry wastewater pollution and treatment processes- A review. J Biodivers Environ Sci. 2018; 12(3):283-293. http://www.innspub.net/jbes/corn-starch-industry-wastewater-pollution-treatment- processes-review/.
[3] Neogi S, Saha S, Jeon B, Dey A, Chatterjee P.K. Low pH treatment of starch industry effluent with bacteria from leaf debris for methane production. Water Environ Res. 2019; 91(5):377-385. doi:10.1002/wer.1033

Book Chapters:

[1] Neogi S, Dey A, Chatterjee P.K. Studies on Rapid Initiation of Anammox Process for Starch Industry Effluent Treatment. In: Ghosh SK, ed. Waste Water Recycling and Management. Singapore: Springer Singapore; 2019:97-110. doi: 10.1007/978-981-13- 2619-6
[2] Shubhaneel Neogi[1], Priyanka Sarkar[1], Pradip K Chatterjee[2] Apurba Dey[1]*, ANAMMOX technology for food industry nitrogenous wastewater treatment, The Future of Effluent Treatment Plants-Biological Treatment Systems, Ed. Maulin P Shah, Elsevier (Under Publication).
[3] Shubhaneel Neogi[1], Pradip K Chatterjee[2]*, Acidophilic methanogenesis will be the most suitable technology in food processing industries, Handbook of Advanced Approaches towards Pollution Prevention and Control, Volume 2: Ed. Rehab O. Abdel Rahman, Elsevier (Under Publication).

Conference Proceedings:

[1] S. Neogi, A. Dey, Chatterjee PK. Investigation on reduction potentiality of corn starch industry effluents by different microbial population and their relativity with physic-chemical growth factors, In: 7th International Congress of Environmental Research ICER-14. Vol 3. ; 2014:90-100.
[2] Dey A, Neogi S, Chatterjee P.K. Studies on Optimization of Physical Growth Factors for Rapid Initiation of Anammox. In: PABE-19 Sept. 5-7 2019 Paris (France). Excellence in Research & Innovation; 2019:38-43. doi:10.17758/EIRAI6.F0919218
[3] Apurba Dey[1]*, Shubhaneel Neogi[1], Pradip K. Chatterjee[2], The scope and challenges of Anammox in treatment for starch industry wastewater, International Conference On Chemical, Bio & Environmental Engineering, Chembioen-2020, February 13-14, 2020, National Institute of Technology, Jalandhar- 144011, India.

Chapter 1 : INTRODUCTION

1. Introduction

1.1. Water- the basis of civilization

Water is the basis of life and contributes about 75% of any biological system. Although, two thirds of the globe contains water, very less of it is potable [1]. In the history of human civilization, there is strong evidence of the role of water that promoted the peak development of most civilizations. Water security is always a key issue behind the development of civilization but has been downplayed in recent history, especially the post-industrial revolution [2]. Among the 97% water coverage of the earth, 97.6% is in the sea and cannot be used directly due to high salt content. The freshwater shares only 0.8% as groundwater and 0.1% as surface water. Also, about 1.5% of water is trapped in glaciers and polar ice reserves, which is now depleting at a rapid rate [3].

The pollution from the beginning of the 21st century became a severe concern for future due to continuous population growth, heavy industrialization, rapid urbanization, reducing forestland, food production practices, and poor water usage and wastewater management strategies [4]. The increasing deoxygenated dead zones in oceans across the globe is mainly due to the contribution of untreated water to river water bodies from various sources starting from municipal to industrial. The fourth world water development reported that only 20% of globally produced wastewater receives treatment before disposal to water 84% rural population bodies [5]. The problem is even severe with developing and underdeveloped countries which contributes only 8% treatment capacity against high-income countries (70%) [6]. The inadequate wastewater treatment system, loopholes in regulatory criteria, lack of awareness increasingly posing a higher risk of sustainability to human and other biotic health, wellbeing and economic activity [7]. Every recent survey critically highlights the requirement of clean water. Also, considering wastewater as a resource is required and must be dealt with effective management to avoid further damage to the sensitive ecosystem and the aquatic environment [8][9]. So considering the current scenario every country whether developed or developing or underdeveloped is coming under the global regulatory radar to respond to global norms to control pollution [10] [11]. Water pollution by food production and processing are a severe concern especially in developing countries where small and medium industries are failing regulatory norms due to lack of knowledge and adequate treatment facilities [12][13][14]. The rapid deterioration of wetland, lakes, and rivers by eutrophication in past few decades resulted in a reduction in biodiversity severely across China, Europe, Japan, South Asia and Southern Africa [15]. Recent surveys predict that around 74% of water is used for agriculture and irrigation, 8% in domestic purpose and 18% for industrial utilization [16]. Without a clean source of water crop production will definitely reduce in future and preserving it will be more difficult [17]. The impact of urban groundwater utilization and pollution discharge to river streams affects around 84% of the rural population badly, who do not have access to clean water.

The report from Population Action International suggested that more than 2.8 billion people in 48 countries will face severe water stress in terms of quality deterioration and availability by 2025 [18]. Especially the West Asian countries will be the first to be hit by water scarcity due to the heavy population increase and increasing demands of water in the next two decades. 40% of the total projected population by 2050 i.e., 9.4 billion across 54 countries is projected to be facing more stress [19]. The predicted global status of water scarcity from 1995 to 2025 by UNEP is represented in Fig. 1.1.

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Fig. 1.1: World water stress assessment report 1995-2025 [20].

The global demand for water has been predicted to increase from 4600 km[3] per year to 5500-6000 km[3] per year with a rate of 20-30% increase by 2050 from now. The consumption in industrial water will also increase across all regions of the world, except North America, Western and Southern Europe, by six folds [21]. The manufacturing sector will see a 400% increase in water demand from the year 2000-2050 according to OCED (2012) [22].

Most of our water bodies have been negatively impacted due to water transportation activities, the release of untreated waste into streams feeding the sea, uncontrolled addition of industrial chemical waste, heavy oil spillages, bio-dumping of urban waste, etc. the reduction of potable water globally in recent few years became severe [23]. The pollution in the sea has also increased causing the death of several important species and increasing dead zones [24]. The micro-plastic is another severe issue damaging the ecosystem of the coral reef and also inhibiting the life of several micro and macro-fauna across the globe [25][26].

1.1.1. Water pollution control- the needs and necessities

Environmental protection is influenced by three interconnected factors: ethics, education, and legislation. There are some ancient cultures like the Indus valley civilization, ancient Greek culture, Mesopotamian civilization, Chinese cultures, where several texts depict the importance of the protection of nature and some of them made them mandatory for the people of that culture to practice that by connecting them to religious mythology [27]. Despite that, the three physical bases of life i.e., the air, water and soil out of the five dimensions of life is severely damaged now and mainly because of human activities.

The pollutants of water can broadly be categorized as physical, chemical and biological [28]. The biophysical contamination of water happens mainly due to massive construction activity where wood, rock, concrete blocks and non-degradable materials disturb the aquatic ecosystem when discharged [29]. The chemical contamination is a more severe issue contributed from various sources from industries, agricultural activity, sewage and other human activities [30]. The biological contaminants also add from slaughterhouses, food processing units, sludge from industrial discharge and hospital wastes, which severely affects human health and other life forms [31].

The contaminants of water can be reduced by various means starting from physical, chemical and biological methods.

1.1.1.1. Physical water treatment methods

The elimination of unwanted materials from water is the main aim of any water treatment method. The physical method of water treatment is generally done by screening or filtration. There are several types of filtration methods available that can reduce pollutants to different degrees.

- Screens: This is basically used as a pre-treatment method to remove larger particles [32].
- Sand filtration and multimedia filtration: This type of filters assist in removal of suspended solids from water, however minute solids and dissolved solids can pass through these types of filter and thus requires secondary treatment [33].
- Microfiltration: This system uses membrane which can eliminate 0.1 to 10-micron particles along with suspended solids, bacteria, and certain viruses, but fails to remove dissolved solids. This technology is mainly helpful in upstream of the reverse osmosis plant to generate feeds [34].
- Ultrafiltration: This is a more advanced technology that allows filtration up to 0.005 to 0.01-micron size by utilizing pressured water stream to separates solids via a membrane. This technology also cannot remove dissolved solids but is very useful for removing most suspended solids and microbes from contaminated water [35].
- Reverse osmosis: This is the most advanced and effective technology and more commonly used across industries and households water treatments. This technology uses semipermeable membrane and water pressure that allows selective passage of water molecules through the membrane, discarding, most of the ions, bacteria, viruses, and other contaminants up to 0.005 to 0.0001-micron size [36].

1.1.1.2. Chemical water treatment methods

The chemical treatment methods are in an application for a long time and are usually utilized for disinfection purposes. But with advanced chemical engineering now it is available for multidimensional purposes like coagulation, chemical precipitation, chemical oxidation, ion exchange, neutralization, and stabilization.

- Chemical precipitation: This is the most effective method especially for metals and ions, where a precipitating agent is added to the contaminating water and later filtering the precipitation out [37].
- Chemical and advanced oxidation: In chemical oxidation, the state of pollutants are modified by donation of an electron by the oxidizing chemicals making them less reactive. This is especially helpful for removing organic pollutants from water [38].
- Chemical stabilization: This is a similar process like oxidation used for large volumes and used mostly in biological processes where the oxidants react to a large volume of sludge restricting their growth or making them ineffective. These processes are also used to reduce odour from a large volume of wastewater [39].

1.1.1.3. Biological water treatment methods

Biological treatment of water is a popular choice since long due to easy operation and cost-benefit. The oldest known biological treatment found was septic tank which dates back to 1860 by a French physicist John Mouras [40]. Later several technologies were developed to handle biologically degradable waste on a large scale efficiently by aerobic or anaerobic treatment.

- Aerobic treatment: This process involves degradation of biologically degradable waste in the presence of oxygen by several groups of bacteria. It is rapid, efficient and can reduce up to 98% pollutants to yield cleaner water. There are several types of aerobic treatment such as trickling filter, activated sludge process, aerated lagoon, and oxidation pond. The activated sludge process can efficiently remove both carbon and nitrogen pollutants efficiently and is a popular choice across various industries [41].
- Anaerobic treatment: This method is popular for treating high strength wastewater, where, in the absence of oxygen, anaerobic methanogenic bacteria reduces waste to produce biogas. This process is typically slow but also produces very less quantity of waste sludge. This process has limitations to treat nitrogenous pollutants and needs further subsidiary treatment before discharge, yet it is popular because it can provide-40-60% recovery of waste [42].
- Aerated lagoon: this is also another type of aerobic treatment where large lagoon of wastewater is mechanically aerated to reduce waste. The main drawback of this technology is the produced sludge is in great quantity and requires further treatment [43].
- Oxidation pond: this is a system with a mix population of bacteria, algae and other organisms where the rate of reduction is very slow and the requirement of the area is very high. This is applicable where the pollutant load is in low quantity.
- Activated sludge process: This is an advanced process of treatment comprising a series of the primary filter, anaerobic treatment, primary clarifier, aerobic treatment and secondary clarifier to reduce biological waste at a very high rate. This is popular and utilized among most industries, especially food processing industries. The main benefit of this system is that on a small space compact treatment of most pollutants whether it is carbonaceous or nitrogenous, can be treated at a very low cost and recovery of waste is also achieved as biogas [44].
- Trickling filter: This kind of filter contains multiple layers of rocks, coke, gravel, ceramic, plastic media that forms a mess trap with increasingly low porosity from top to bottom and provides good filtration for large volumes of water treatment. However, the rate of filtration of these kinds of filters are very low and it has limitation for high load wastewater [45].

It was found that the treatment of wastewater is also quite successful due to -

a) Very low installation and operation cost.
b) The energy requirement is comparatively low considering the efficiency since most biological treatment systems operate on ambient temperature and pressure.
c) Also, these processes contribute to minimal secondary environmental pollution during operation.
d) The operational complexity and design are less as compared to other methods.

Amongst various biological treatments, the activated sludge process is most popular as it provides a complete solution for nutrient removal from wastewater and waste recovery.

1.1.2. Regulation of water pollution

Controlling pollution to water sources, especially around civilization is very necessary. The sources of water pollution differ with locations and are contributed by industrial effluents, sewer drainage, wastewater treatment plants, rainwater runoff, etc. The increasing loss of natural resources bounds global authorities to consider enforceable environmental laws and regulation by state-level governmental authorities. The World Health Organization (WHO) took the pledge to set a basic guideline to assist the development of a national standard for reducing health hazard [46]. However, the guideline recommended by WHO is not absolutely mandatory, rather it is given to assist the local and international base of standards from where it should be applied. The environmental, social and cultural values must be taken into consideration before setting any feasible pollution control norms [47]. A comparison of the quality standard adopted by several international bodies is represented in Table 1.1.

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1.1.3. Wastewater- a good resource for energy recovery

The majority of wastewater (~80%) produced globally due to various human activities are generally released to water bodies, leading to eutrophication, disease generation, greenhouse gas emission in the form of nitrous oxide (NO) and methane (CH4) [52]. With the advancement of technologies, the opportunity to recover the water, energy, nutrients and other materials from wastewater is significantly higher today [53]. So a paradigm shift from “treatment and disposal” to “recycle and resource recovery” has happened in the industrial domain in the last decade [54]. The perception of considering wastewater as a burden is changing slowly with lucrative opportunities to use it as a resource which further benefits additional profit in the whole margin amongst industries [55]. Up to now large scale centralized wastewater treatment was a popular solution covering nearly 60% sewer system user globally, however, it largely avoids rural area coverage. But with passing time more localized technology will be required with lower operational cost and maintenance [56]. Advanced treatment system resists the loss of resources from the production system and enhances the overall profitability of industries [57]. The management and control of wastewater can be understood in four different interconnected steps-a. Prevention of volume of wastewater generation.

b. Removal of contaminants from wastewater.
c. Reutilization of wastewater.
d. Recovery of resources that have some economic value.

1.1.4. Corn starch industry wastewater pollution

Starch is a common resource used widely in food, pharmaceutical, paper and in the textile industry. Among several resources, maize is the highest produced cereal crop that is used for starch production on an industrial scale [58]. Production of starch from corn is carried out either by dry roasting or wet milling process. Wet milling is a common practice and generates a huge quantity of wastewater, usually per ton of maize grinding generates 5 m[3] of wastewater [59]. The biochemical profile of corn starch shows acidic wastewater (pH 4.2-5.3) which contains around 6000-19000 mg/L COD and 4500-8000 mg/L BOD, processed from the high content of carbohydrate, cellulose, hemicellulose, protein, etc. [60]. So it becomes necessary to treat the wastewater before discharging to water bodies [61].

1.1.5. The current scenario in the treatment of corn starch wastewater

The wastewater generated from starch wet milling industries are highly biodegradable in nature, thus biological treatment of wastewater and waste to energy recovery is widely practiced [62]. The primary goal of any biological treatment system is to remove carbonaceous and nitrogenous compounds through the anaerobic and aerobic pathway and to make it suitable for utilization for irrigation purpose or river stream disposal, and application of more advanced technologies like reverse osmosis, which can make this water potable after treatment [63]. The age-old technologies which are in use to treat corn starch wastewater were based upon general treatment system and rely on neutral pH operation while the effluent generated by starch plants are highly acidic in nature. Thus neutralization of the effluent before introduction to the treatment facility becomes compulsory and involves a significant increase in operational cost and overall hydraulic retention time [64]. Most of the anaerobic reactor system like upflow anaerobic sludge bed reactor (UASBR), expanded granular sludge bed reactor (EGSBR), anaerobic filter bed reactor (AFBR), anaerobic fixed film moving bed bioreactor (AFFMBR), anaerobic digestion ultrafiltration (ADUF)are popular technologies to treat carbonaceous pollutants and to recover waste from water as methane gas, are designed for operation between pH 6.8-7.6 [65]. The starch industries employ two-phase nutrient removal systems, on the first phase complex materials are broken down anaerobically and waste is recovered as methane gas. The degradation of carbonaceous material mostly happens in the first stage and in second stage nitrogen compounds are being degraded by a nitrification-denitrification pathway involving auxic-anoxic phase in the cycle [66]. Nitrogen in effluent appears in a wide degree of complex forms, from protein to ammonia, ammoniacal, nitrate, nitrites, salt of ammonia, amino acids, etc., where most of them contribute to the formation of essential & non-essential amino acids for the microbial cell after bioconversion [67]. Bioconversion of nitrogenous wastes mediates through various oxidation-reduction states via nitrogen cycle, mediated by several groups of bacteria, algae & few higher organisms. For the corn starch industry, the activated sludge process containing aerobic- anoxic systems are very popular due to its short HRT and high load tolerance. Several other technologies are also in the application in various industries like an aerated lagoon, anammox process, etc. [68].

The aerobic treatment is generally not a first line choice among starch industries as no valuable products can be recovered by this process. But it is generally used to treat the nitrogenous content after recovery of carbon pollutants as biogas.

1.1.6. Problem statement and justification

Food processing industries, especially corn starch industries have to spend a significant amount of their profit to treat waste. The acidic water involves an additional cost of neutralization. The starch industry effluent contains high carbonaceous and nitrogenous load, of which both require treatment before disposal. The conventional methods of anaerobic treatments are expensive and critical in operation. Treatment of wastewater under the acidic condition is promising as the presence of acidophilic methanogen is reported widely in past years. The novelty lies in the isolation and development of low pH tolerant methanogen from natural resources and evaluating its effectiveness in response to starch wastewater pollutant removal and energy recovery ability in terms of methane.

The anammox process is the nascent technology which is highly cost-efficient against the nitrification-denitrification process due to low power consumption, low retention and high efficiency. But, there is no defined method available to grow the bacterial community onsite at variable climate condition, neither the sources of bacteria are properly defined. While a few of reactor are in operation in various industries, surprisingly all of them are developed from the same mother culture from where the bacteria was first cultivated in the lab. So the ambiguity of availability and culture condition of the bacteria, moreover real-time failure incidents after installation of large scale plant leaves enough opportunity to explore the biological characteristic, nutrition mode, growth pattern and influential parameters on growth.

1.1.7. The objective of this research The objective of this present study is–

1. To enrich low pH tolerant methanogens and anammox bacteria from different natural resources.
2. To study the effect of physicochemical parameters on the growth of enriched methanogens and anammox species.
3. To study the biodegradation of starch industry wastewater for pollution control using the enriched species under optimized conditions in shake flask.
4. Design & optimization of laboratory scale wastewater treatment system for treatment of carbonaceous and nitrogenous waste.

Chapter 2: LITERATURE REVIEW

2. Literature review

2.1. Corn starch production and market statistics

Maize, also known as the “queen of cereals”, is the second most important crop [69]. Corn starch production is a long practiced process and developed over 150 years. Corn wet milling primarily engaged in producing starch, syrup, oil, sugar, and by-products such as gluten feed, and meal [70]. In the corn wet milling process, the corn kernel is separated into 3 principle parts-

- The outer skin called the bran or hull,
- The germ, containing most of the oil, and
- The endosperm comprises gluten and starch mostly [71].

A hypothetical structure of a longitudinal sectional view of the corn kernel is represented in Fig. 2.1. An average bushel of corn weighing 25 kg produces approximately 14 kg of starch, along with 6.6 kg of feed and feed products, and 0.9 kg of oil and water as remainder [72].

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Fig. 2.1: Typical hypothetical configuration of maize kernel [199]

Around 598 million metric ton of maize yield over 139 million hectares of cultivated land is contributed worldwide in 2014, among them major producers are USA (240 mMT), China (125 mMT), European Union (39 mMT), Brazil (37 mMT), Mexico (19 mMT), Argentina (14 mMT) and India (11 mMT) [73]. The overall share of major maize production by different countries represented in Fig. 2.2.

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Fig. 2.2: Maximum maize producing countries

Global maize production as per 2016-2017 global survey report has touched approx. 1040 mMT [74]. Four major types of corn are mostly used in industrial allocation in US & China, are dent corn (Zea mays var. indentata), flint corn (Zea mays var. indurata), popcorn (Zea mays var. evarta), sweet corn (Zea saccharata or rugosa), besides some other variations like waxy corn (Zea mays var. ceratina), amylo-maize (Zea mays), pod corn (Zea mays var. tunicate), striped maize (Zea mays var. japonica) [75]. In India, Corn has no such segregation done due to ignorance on the part of farmers. Types of corn available are generally different types of hybrid seeds used for growing corn [76].

Maize is the only food cereal crop that can be grown in different seasons, due to its high tolerance to changing weather. The diverse use of maize as food, feed, and fodder and source of more than 3500 processed products, make it highly promising cereal crop [73]. End-use of average utilization of maize in the Indian market is represented in Fig. 2.3.

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Fig. 2.3: End-use fate of corn, Indian Market. The nutritional value of maize is shown in Table 2.1.

Table 2.1: Nutritional value of maize grain.

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Corn starch wet milling process is carried out by five basic steps [77]. Initially, corn is collected, investigated and cleaned. Then it is steeped for 30-40 hours to begin breaking the starch and protein bonds. Sequentially the process involves a coarse grinding to separate the germ from the rest of the kernel. The remaining slurry consisting of fiber, starch, and protein is finely ground and screened to separate the fiber from starch and protein. The starch is separated from the remaining slurry from hydro cyclones. The starch then can be converted to syrup or it can then be converted to several other products through various fermentation processes [78]. The entire process is represented as a flow diagram as shown below in Fig. 2.4.

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Fig. 2.4: Step of maize starch production & relative derivatives

The growth of the Maize Starch industry has doubled in the last 5 years in terms of grinding capacity of maize [79]. The fast-growing global market of maize in the financial year 2013 -2019 is represented in Table 2.2.

Table 2.2: Global maize production 2013-2019 [80]

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2.2. Wastewater generation and characterization

Corn starch wet milling involves a huge amount of water utilization starting from maize cleaning, steeping, grinding, and process cleaning [81]. Corn starch processing in most recent trends follows close loop technology to reduce wastewater generation by inter-circulation of process water at various steps like multistage steeping, gluten separation, fiber separation, etc.[82]. The condensate during the steeping process and process wash water are main contributing waste from any corn starch industry. Other sources vary with the diversity of product manufacturing. The wastewater from corn starch industry contains a high amount of volatiles, precipitation of modified starch, dissolve chemicals used in the modification, impurities from corn syrup & dextrose, etc. are characterized as high strength [83]. The COD of wastewater is very high, which is mostly from soluble and biodegradable components [84]. Typical wet milling process uses almost 5-11 m[3] water for per ton maize processing [85]. Corn starch industry effluents are mostly highly acidic in nature [86]. The steeping process involved in starch solubilization by acidic hydrolysis thus produces a high amount of acidic wastewater ranging from pH 3.0 to 5.5. Volumetric estimation of wastewater generation in each step of corn starch production for a standard 150 MT maize crushing plant is shown in Table 2.3.

The wastewater generated in every step contains a high amount of suspended solids as a biodegradable material, COD, sulfate, phosphate, and chloride load. A trend analysis of process water chemical characteristics is represented in Table 2.4.

Also, a brief status of corn starch industries water pollution in Indian environment [61][87][88][89] is represented in Table 2.5 and 2.6.

Table 2.3: Water balancing and wastewater generation in corn starch production [77]

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Table 2.4: Characteristics of effluent in various stages of starch processing [90][91][92].

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Table 2.5: Wastewater characterization of a few leading corn starch industries of India-1.

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Table 2.6: Wastewater characterization of a few leading corn starch industries of India-2.

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2.3. Understanding the biologics of starch effluent

The basic constituent of starch industry effluent contains a large amount of carbohydrate and protein. The fate of carbon and nitrogen in industrial wastewater is usually modulated through anaerobic and aerobic biological treatment processes [42]. Mesophilic aerobic digestion of carbon and nitrogen offers 50% conversion of organic compounds by external physical co-factors and convert 50% into sludge which requires additional expenses prior to disposal [93]. While the anaerobic system is capable of reducing COD up to 90% and generates methane biogas, it serves as a fuel [94]. Starch industry in practice involves a multistep process wherein the first step the higher molecule is broken down to a simpler molecule in anaerobic digestion phase then consumed by methanogenic bacteria to produce biogas [95]. The nitrogenous compounds are more difficult to be treated and require additional treatment in the second phase wherein nitrification-denitrification pathway the ammoniacal compounds are broken down to nitrogen gas [96]. 2.3.1. Overview of the anaerobic digestion process It has been known from centuries that combustible gas generates from the wasteland, peat bog, stagnant sewage. Historically in the 7th century, Van Helmont recorded flammable gas generation from decaying organic materials in piles [97]. Later in 1776, Volta proposed a relationship between the amount of organic material degradation and the amount of gas production. John Dalton and Humphrey Davy (1804-1808) finally established the fact that this combustible gas was methane by independent investigation [98]. Bachamp (1868), suggested that the process was microbiologically mediated and later was confirmed by Omelianski (1890) when he was able to isolate hydrogen, acetic acid, and butyric acid-producing microbes during methane production from cellulose as substrate [98]. The concept of the septic tank in 1881, was developed by Mouras and is considered as the first wastewater treatment facility. The large scale application credit though goes to India for building first-ever anaerobic digester degrading human waste to produce methane that was used to light streets [99]. Successful large scale biogas plant from manure was also first built by Indian microbiologist S.V. Desai from Indian Agricultural Research Institute (IARI), formerly known as Imperial Agricultural Research Institute.

Anaerobic digestion is a multistep process and is economically beneficial due to the production of biogas as a by-product of the fermentation of complex molecules. This process is mediated by several groups microflora that exists in the same environment and shows synergistic syntrophic relationship [100]. The whole process can be segregated into four sequential steps- hydrolysis, acidogenesis, acetogenesis, methanogenesis, and are accompanied by different groups of bacteria, predominantly archaea and methanogenic bacteria [101][102]. In the first step complex, organic material is broken down to simpler forms of sugar, amino acids and other volatile fatty acids like formic, ethanoic, propionic, and butyric acids by fermentative anaerobic bacteria. Then these hydrolyzed products are converted to simple organic acids, CO2, and hydrogen by acid formers. In the last step, methanogenic bacteria converts volatile organic acids and derived alcohols to methane and carbon dioxide [103]. The whole process is represented in Fig. 2.5. In order to the successful removal of nutrients, a dynamic equilibrium is required between acidogens, acetogens, and methanogens [104]. In an anaerobic digestion environment, syntrophic existence of nonmethanogenic obligate and facultative acidogens which produces hydrogen is utilized by strict anaerobic methanogens. In a steady state, a dynamic equilibrium exists between two groups [105].

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Fig. 2.5: Steps involved in the anaerobic digestion process.

2.3.2. Microbiology of anaerobic digestion process

Several studies were conducted throughout globe to understand complex ecology of anaerobic digestion process [106], Though presence of a highly diversified microbial composition was observed ranging from bacteria, fungi, protozoa, rhizopods, nematodes, ciliates flagellates, etc., but the microbial activity of anaerobic digester largely depends upon hydrolytic, fermentative acidogenic, acidogenic, and methanogenic bacteria.

- Hydrolytic microbial consortia:

Some bacteria are capable of breaking down higher bio-molecules like protein, lipid, and carbohydrate with the help of secreted extracellular enzymes like protease, lipases, cellulases to yield soluble monomers (e.g. - glucose, amino acids, fatty acids, etc.) [107]. Some facultative & obligate anaerobic group of hydrolytic Clostridium sp., Fibrobactor sp. (F. succinogenes), Corynebacterium sp., Actinomycetes sp, Staphylococcus sp. and Escherichia species are found predominant carrying on this kind activity within the anaerobic mixed microbial consortium.

- Fermentative acidogenic bacteria:

Acidogenesis converts sugars, amino acids, fatty acids to organic acids, alcohols and ketones, acetates, carbon dioxide and hydrogen [108]. The end product of the biological reaction is highly dependent upon bacterial species involved, temperature, pH, redox potential, etc. Several Clostridia sp. & other species mediate the above kind reaction.

- Acetogenic bacteria:

Acetogenic bacteria convert fatty acids and alcohols into acetate, hydrogen and carbon-di-oxide, the food for the methanogens. But the reaction is negatively affected by partial pressure imparted by hydrogen. On higher hydrogen partial pressure rather acetate, conversion bacteria start generating carbonic acids (propionic, butyric acid, etc.). A symbiotic relationship relies on between methanogens & acetogens. Methanogens help to achieve low hydrogen tension [109]. E.g. of these groups of bacteria are- Syntrobactor wolinii, Syntrophomonas wolfei, etc.

- Methanogens:

Methanogens are fastidious bacteria belonging to the kingdom eukaryote within the archaea domain, found naturally in a diversified habitat, from deep sediment to the ruminating stomach of herbivores. Methanogens are very slow growing strict anaerobic bacteria that lack peptidoglycan in the cell wall. Also interestingly the cell membrane of methanogens are made of branched hydrocarbon chains attached to glycerol by an ether linkage. Methanogens have ribosomal RNA sequence that is quite different from bacteria and eukaryotes. In the methanogenesis, process bacteria converts fermentation end products (e.g. hydrogen, formate, acetate) to methane and carbon dioxide [110]. The reaction is catalyzed by specific coenzymes F420, which act as an electron carrier in metabolism. Based on substrate utilization methanogens are subdivided into two major groups:

- Hydrogenotrophic methanogens; these are hydrogen utilizing chemolithotrophs, capable of converting hydrogen and carbon dioxide into methane (as shown in Eq. 2.1). A large group of Methanococcales and Methanobacteriales comes under this group [111].

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- Acetotrophic methanogens; these groups of bacteria convert acetate into methane and carbon dioxide [112]. The main genuses that comes under these groups are- Methanosarcina, Methanothrix, an d Methanosaeta.

A detailed phylogeny of anaerobic methanogen is represented in Table 2.7.

Table 2.7: Classifications of methanogens [113] [114].

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2.3.3. Critical influential factors for anaerobic digestion process

Anaerobic digestion is a biologically mediated process and is highly dependent on several factors that affect the growth and activity of bacteria. The influence of their parameters are presented as follows-2.3.3.1. Temperature The temperature has a great significance in any biological reaction. Production of methane gas is widely recorded from 0 [0]C to 97 [0]C [115], but for industrial use based on application generally, two groups of methanogen are given importance. The mesophilic methanogens which show the active reaction between 32 [0]C - 42 [0]C [114] and the thermophiles which work best beyond 45 [0]C to 70 [0]C [116]. The most industrial reactor uses mesophilic bacteria, a few also is in an application with thermophilic methanogens. However, thermophiles are more sensitive toward temperature alteration and rapidly lose activity if not maintained properly [117].

2.3.3.2. Acidity / alkalinity (pH)

Industrial reactors mostly use cow dung as starter culture [118], thus most of the bacteria obtained to generate methane are capable of showing activity between pH 6.7-8.2. Acidic pH greatly impacts the activity of methane bacteria. However, there are several species reported like Methanosarcina sp., which are capable of showing activity in acidic pH but are not in an application for large scale utilization yet [119][120].

2.3.3.3. Substrate characteristic

The character of substrate regulates the rate of reaction in any complex biological system [121]. The higher the presence of long chain polymers the higher the time requires to break it down to monomers before it enters into the methanogenic reaction. Microorganisms also require carbon and nitrogen in a proper ratio for assimilation. A ratio of C:N:P:S is reported 500-1000: 15-20: 5: 3 respectively [122]. Maintaining nutrient ratio for anaerobic reactor has great significance because if the C/N ratio is too small it will hamper the production of methane by producing ammonia [123]. The ratio of COD: N: P is reported to be ideal near 800: 5:1 [124]. Higher C/N ratio can also affect methane generation due to deficiency of the formation of energy derived from the protein.

2.3.3.4. Loading rate

Loading rate or organic loading rate is the amount of biodegradable load introduced to the reactor for certain treatment time and is a critical factor for optimal efficiency. For the continues system, OLR depends upon the design of the reactor, hydraulic retention time (HRT) and COD of the wastewater. The formula to determine HRT and OLR is as represented in Eq. 2.2 and Eq. 2.3 respectively- Vol.of reactor (in[3])

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2.3.4. Review on anaerobic treatment technologies

Anaerobic treatment of industrial wastewater considers few important aspects of treatments like maximal COD removal, feasible operational cost, minimum waste sludge production, lesser risk of failure [125]. First full-scale industrial application of anaerobic digestion was reported on the Netherlands in 1978, was implemented to treat effluent from the beverage industry, and later on vigorously developed into a broader area like distilleries, food processing industries, paper, sugar, fermentation, starch production industries, petrochemical industries, etc. [126]. Later on, wide application and modification of reactor design were performed to meet the biochemical profile of wastewater from various food production and processing industries [127][128][129]. ]. Few industrial applications of different bioreactors for methane production are given in Table 2.8.

Table 2.8: Industrial application of different bioreactor for methane production.

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Starch industry effluent is a rich source of carbohydrate, gluten, mono-sugars with an average COD load of 20000 mg/L and a total nitrogen load of 500 mg/L. An average corn starch processing unit with an average COD load of 20000 mg/L [137] produces around 30 ton of COD and 18 ton of BOD per day. UASBR is the first-line choice from long past for anaerobic digestion of carbon waste from the starch industry as it operates at low cost.

2.3.5. Overview of the nitrogen removal process

Role of nitrogenous substances along with carbon sources in water bodies recognized as a main significant reason for eutrophication from long back, even from the 20th century [138]. Higher nutrient availability leads to enhancement in the growth of aquatic phytoplankton, which leads to deterioration of water quality. Water became turbid & leads to loss of value in many objects including higher treatment cost & loss of aesthetic enjoyment [139]. Recent advancement on wastewater research also revealed that hazardous toxin production by several groups of cyanobacteria also enhances with nitrogen availability in water bodies [140]. Thus the limitation of the contribution of nitrogen wastes should be a prime factor for ecological bio-control of the environment [28].

2.3.5.1. The general concept of the nitrogen cycle

Nitrogen is the largest part of the atmosphere and is recycled between biosphere, geosphere, and atmosphere naturally by several complex pathways and medium. While in nature both assimilatory and dissimilatory processes are involved to regulate gaseous nitrogen to nitrate, nitrite, ammonia, and cellular components, the similar process is also mimicked and mechanized to remove wastes generated by industries in large quantity every day [141].

The process in nature starts with nitrogen assimilation bacteria (e.g. - Rhizobium sp.) which converts di-nitrogen gas from air to ammonia. This fixed ammonia then converted to nitrite compounds by chemolithotrophic bacteria. This is a multistep process where ammonia oxidizing bacteria (AOB) with the help of ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) enzyme fix aerobic nitrogen into ammoniacal compound within the cell. The chemolithotrophic process then converts ammonia to nitrogen dioxide with hydroxylamine as an intermediate. The nitrite oxidizing bacteria (NOB) then oxidize nitrite to nitrate with the help of nitrite oxidoreductase (NOR) [142]. More recent research on the process found heterotrophic ammonia oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) which are also capable of ammonia conversion [143]. In this case, heterotrophs use organic carbon sources as an electron acceptor under the anoxic condition to convert ammonia to nitrogen gas via NO3, NO2, NO and N2O [144]. Dissimilatory nitrate reduction to ammonium (DNRA) is another pathway where produced ammonia from nitrate is used as a substrate for growth under anoxic or anaerobic condition [145]. In recent years the presence of several species of bacteria found that are capable of reducing ammonia in the presence of nitrite under anaerobic condition (ANAMMOX) [146]. Several heterotrophs are also capable of carrying out ammonification, where organic nitrogen is hydrolyzed to NH3 or NH4+. The cyclic relationship of nitrogen from nature to various microorganisms is represented in Fig. 2.6.

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Fig. 2.6: Nitrogen transformation pathways mediated by various organisms [147]

Nitrogen in effluent appears in a wide degree of complex forms, from protein to ammonia, ammoniacal, nitrate, nitrites, salt of ammonia, amino acids, etc., where most of them contribute the formation of essential & non-essential amino acids for the microbial cell after bioconversion. Bioconversion of nitrogenous wastes mediates through various oxidation-reduction states via nitrogen cycle, mediated by several groups of bacteria, algae & few higher organisms.

2.3.5.2. Nitrification

Biological degradation of ammonium biomolecules into nitrate via nitrite is known as nitrification. This is a two-step process mediated by chemoautotrophic bacteria. The first step is mediated mostly by genera of Nitrosomonas, which enzymatically converts ammonia to nitrite in the presence of oxygen. In the second step genera of Nitrobactor transform nitrite to nitrate [148]. The overall phenomena are represented in Eq. 2.4 and Eq. 2.5.

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The released energy in the above reaction is uptaken by nitrifying organisms in the presence of inorganic carbon sources such as CO2, HCO3- and carbonate compounds.

- Microbiology of nitrification

Nitrification is a complex process, comprises a huge group of microbial flora that exists on a symbiotic syntrophic relationship within aerobic biological degradation process of wastewater. The nitrification process is observed to be carried out mostly through two main groups of chemo-litho-autotrophic proteobacteria; the ammonia oxidizing bacteria [149], that belongs to β-subclass of proteobacteria and are monophyletic [150], and the nitrite oxidizing bacteria, belongs to α, γ, δ subclass of proteobacteria [151][152]. Oxidation of ammonia in this process is mainly carried out by Nitrosomonas and Nitrosococcus groups of bacteria. Though a wide variety of other bacteria’s is also capable of carrying out a similar reaction, e.g. -Nitrosolobulas multiformis, Nitrosospira briensis [153]. Formation of nitrite is mediated by Nitrobactor & nitrocystis genera. e.g. - N. agilis, N winogradsky, Nitrosococcus mobilis, Nitrospira gracilis [154].

- Role of enzymes

Two different microbial consortia, i.e. the nitroso group, which catalyzes ammonium to nitrite, and the nitro group, which converts nitrite to nitrate, involves specific utilization of enzymes in subsequent steps and are been well studied [155]. The first step of the above depicted metabolic pathway is catalyzed by ammonia monooxygenase (AMO), and the second step is by hydroxyl-amine oxidoreductase (HAO) [156]. AMO generates hydroxylamine by oxidizing ammonia, HAO converts hydroxylamine to nitrite and yield 2 electrons, which is then used by first step AMO. The oxygen atom in the derived nitrite is contributed, one atom by water and one by oxygen [157]. Formation of nitrite yields totals four electrons, out of which two electrons are transferred via cytochrome C554 either to AMO or diverted to electron transport chain [158]. In the next step of the nitrification process (Fig. 2.7), nitrate oxidase (NO) catalyzes the oxidation of nitrite to nitrate. Both the reaction is high energy-yielding that converts ADP to ATP.

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