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BIOREACTOR CONFIGURATION FOR TREATMENT OF MULTI METAL CONTAINING WASTEWATER

DEEPAK GOLA

CENTRE FOR RURAL DEVELOPMENT & TECHNOLOGY INDIAN INSTITUTE OF TECHNOLOGY DELHI

MAY 2018

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©Indian Institute of Technology Delhi (IITD), New Delhi, 2018

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BIOREACTOR CONFIGURATION FOR TREATMENT OF MULTI METAL CONTAINING WASTEWATER

by

DEEPAK GOLA

CENTRE FOR RURAL DEVELOPMENT & TECHNOLOGY Submitted

in fulfilment of the requirements of degree of Doctor of Philosophy to the

Indian Institute of Technology Delhi

MAY 2018

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CERTIFICATE

This is to certify that the thesis entitled “Bioreactor configuration for treatment of multi metal containing wastewater”, being submitted by Mr. Deepak Gola to the Indian Institute of Technology Delhi for the award of “Doctor of Philosophy” is a record of bonafide research work carried out by him. He has worked under our guidance and supervision and has fulfilled the requirements for the submission of this thesis. To the best of our knowledge the results contained in this thesis have not been submitted in part or full to any other university or institute for award of any degree or diploma.

Date:

Place:

Dr. Anushree Malik Dr. Shaikh Z. Ahammad Professor Assistant Professor Centre for Rural Development Biochemical Engineering and Technology and Biotechnology

Indian Institute of Technology Delhi Indian Institute of Technology Delhi New Delhi- 110016 New Delhi- 110016

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ACKNOWLEDGEMENTS

I express my sincere gratitude and indebtedness to my supervisors Prof. Anushree Malik Professor, Centre for Rural Development and Technology and Dr. Shaikh Z. Ahammad,

Assistant Professor, Biochemical Engineering and Biotechnology, IIT Delhi, for their guidance and valuable suggestions throughout the research work and consistent encouragement, support and co-operation. Their dedication and keen interest above all their overwhelming attitude to help their students had been solely responsible for completing my work. Their timely advice, meticulous scrutiny, scholarly advice and scientific approach have helped to a very great extent to accomplish this work.

I also express my gratitude to my SRC members- Prof. Satyawati Sharma, Prof. T.R.

Sreekrishnan, Prof. A.K Nema and other faculty members of CRDT for their useful feedback, insightful suggestions and comments in research presentations from time to time. I am extremely thankful to Dr. Neelam Patel, Water Technology Centre, IARI, Delhi for providing help in field studies. I would also extend my sincere gratitude to Prof. Hitendra Malik, Physics Department, IIT Delhi for his word of encouragement and motivation.

I would also like to acknowledge my seniors Dr. Megha Mathur, Dr. Nitin Chauhan, Dr. Prachi Kaushik, Dr. Pushap Chawla, Dr. Abhishek Mishra, Dr. Amit Tyagi and Dr. Sanjeev Kumar Prajapati for their guidance in planning and execution of experiments and their valuable feedback.

A special thanks to Mr. Maneesh Namurath for providing unconditional help in bioreactor studies. I wish to acknowledge my lab members Poonam Choudhary, Sumit Kumar, Arghya Bhattacharya, Pushpender Kumar, Priyadarshini Dey for extending their help during the period of experimentation and thesis preparation. I thankfully acknowledge the members of Metagenomics Lab and Waste Treatment Lab, Biochemical Engineering and Biotechnology

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for providing help and guidance in instrumentation to conduct microbial community studies. I thank Mr. Vinod Kumar and Mr. Sabal Kumar for their kind help and co-operation throughout my experimentation study.

I am extremely thankful to my friends Pragati Sharma, Reecha Sahu, Abhishek Acharya, Vinnet, Siddharth and Abhishek Shina for making the journey more memorable.

My parents, brother and sister’s blessings, inspiration, help in adverse situations and consistent encouragement to carry out my study have been the supreme force behind my academic career in general and this dissertation in particular. Above all, I thank almighty God for bestowing good health to me throughout the course of the study.

Deepak Gola

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iv ABSTRACT

The objective of the present study was to assess the quality of drain water in terms of heavy metal contamination and to develop an on-site treatment facility for the removal of heavy metals from drain wastewater prior to irrigational use in Delhi region. Water quality of Najafgarh and Loha mandi drain was evaluated in terms of spatial variations in the physicochemical characteristics (TDS, pH, DO and COD) as well as heavy metal concentrations (Cd, Cr, Cu, Zn, Pb and Ni). Drain water monitoring was done for the period of two years (July 2012-March-2014) representing three seasons: pre-monsoon, monsoon, and post- monsoon. Varied concentration of multi heavy metals were found due to the extensive discharge of industrial effluent into the drain water. Punjabi bagh of Najafgarh drain was the most polluted sampling point with highest contamination of Zn (12.40 mg L-1) followed by Cr(2.43 mg L-1) and Cu(2.16 mg L-1). The presence of multi heavy metal ions above the FAO permissible limits indicated that drain water was not suitable for irrigational purposes, hence adequate measures are required to remove the heavy metal load from the drain water. A potential remediation technology for multi heavy metals removal from contaminated water was developed. For this, Beauveria bassiana (non-pathogenic and entomopathogenic fungi) was examined for its heavy metal removal ability in individual and multi metal solutions. The results depict that under single metal exposure, maximum removal was observed for Niand Cu (74%-75%), while the minimum removal was observed with Pb (58%). Interestingly, highest total metal removal (83.3%) was observed in presence of multi metal ion, with maximum removal of Pb, Cr(99%) and minimum removal of Zn (57%).

Further spectroscopic and microscopic analysis (FTIR, AFM, TEM-HAADF and SEM-EDX) elucidated the ultrastructural changes acquired by the fungal cell as a toxicity response. The present study delivers first report using High Angle Annular Dark Field (HAADF) that revealed the precise location of different heavy metals inside the fungal cell in presence of

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multi metal ion. Interestingly, the specific bioaccumulation site of each metal did not change when the fungus was exposed to individual or multiple metal stress. Multi metal removal efficiency was also investigated at different pH, temperature, salt concentration and nitrogen sources in order to judge the effect of environmental conditions on bioremediation. To ease the process of storage and transportation, fungal spores were formulated into three different products. A pioneering attempt was made to design the myco-capsules with a shelf-life of 12 months for multi metal removal. However, inability of B. bassiana to outcompete the native microbes was noticed during continuous reactor runs. Hence, native microbial consortia (aerobic and anaerobic) was developed from the Loha mandi drain and was tested for its metal removal ability from actual wastewater as well as with elevated metal ion concentration (considering the metal concentrations fluctuations) in lab scale (4 L) aerobic and anaerobic bioreactor. Up to 23-100% metal removal was observed for selected metals even at elevated initial concentrations. Further, to evaluate the performance of native microbial consortium under field conditions, on-site bioreactor systems (5 L) were developed and fed with actual as well as metal spiked wastewater. Consistent multi metal removal was recorded in both unspiked and spiked 5 L bioreactor system over a period of 60 days, proving the compatibility of reactor system to handle shock load. To meet the demand of experimental irrigational field, the process was gradually scaled up to an on-site 50 L and then 500 L bioreactor. The 500 L bioreactor was able to remove up to 26-57% of metal from the actual wastewater and metal concentrations in the treated water were within FAO permissible limits.

The results showed that bioreactor system was perfectly compatible with heavy metal concentration fluctuations and seasonal variations [5 L (pre-monsoon), 50 L (monsoon) and 500 L (post-monsoon)] in wastewater quality. Untreated as well as treated water from bioreactor was used for irrigation. Significantly, less metal accumulation was observed in plants irrigated with treated water as compared to wastewater irrigated plants, indicating the suitability of treated water for irrigation.

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सार

वर्तमान अध्ययन का उद्देश्य भारी धार्ु प्रदूषण के मामले में नाली के पानी की गुणवत्ता का आकलन करना और ददल्ली क्षेत्र में अपदिष्ट जल द िंचाई िंबिंधी उपयोग र्था भारी धार्ुओिं को हटाने के दलए ाइट पर उपचार ुदवधा

दवकद र् करना था। नजफगढ़ और लोहमािंडी नाली के की जल गुणवत्ता का भौदर्क र ायन दविेषर्ाओिं (TDS, pH, DO and COD) में भारी बदलावोिं के ाथ- ाथ भारी धार्ु ािंद्रर्ा (Cd, Cr, Cu, Ni, Pb और Zn) में स्थादनक बदलावोिं के िंदभत में मूल्ािंकन दकया। र्ीन ाल (जुलाई 2012-माचत -2014) की अवदध के दलए पानी की दनगरानी

दो ाल की अवदध के दलए की गई थी: पूवत मान ून, मान ून और मान ून के बाद। नाली के पानी में औद्योदगक प्रदूषण के व्यापक दनवतहन के कारण बहु भारी धार्ुओिं की दवदभन्न ािंद्रर्ा पाई गई। नजफगढ़ नाली का पिंजाबी

बाग ब े प्रदूदषर् नमूना दबिंदु था दज में Zn (12.40 दमलीग्राम एल -1) के उच्च प्रदूषण के ाथ Cr (2.43 दमलीग्राम एल -1) और Cd (2.16 दमलीग्राम एल -1) था। एफएओ अनुमर् ीमाओिं के ऊपर बहु भारी धार्ु आयनोिं की

उपस्स्थदर् े िंकेर् दमलर्ा है दक नाली का पानी द िंचाई प्रयोजनोिं के दलए उपयुक्त नहीिं था, इ दलए नाली के पानी

े भारी धार्ु भार को हटाने के दलए पयातप्त उपाय की आवश्यकर्ा है। दूदषर् जल े बहु भारी धार्ुओिं को हटाने

के दलए िंभादवर् उपचार र्कनीक दवकद र् दकया गया था। इ के दलए, बेउवेररया बाद याना (एिंटोमोपाथोजेदनक कवक) की जािंच एकल और बहु धार्ुओिं के दमश्रण े भारी धार्ु हटाने की क्षमर्ा के दलए की गई थी। पररणाम दिातर्े हैं दक एकल धार्ु एक्सपोजर के र्हर्, दनवारण हटाने को Ni और Cu (74% -75%) के दलए देखा गया था, जबदक न्यूनर्म दनवारण Pb (58%) के ाथ देखा गया था। ददलचस्प बार् यह है दक बहु धार्ु आयन की उपस्स्थदर्

में उच्चर्म धार्ु हटाने (83.3%) पाया गया था, दज में अदधकर्म Pb, Cr (99%) और न्यूनर्म दनवारण के दलए Zn (57%) का था। आगे स्पेक्ट्रोस्कोदपक और ूक्ष्म दवश्लेषण (एफटीआईआर, एएफएम, टीईएम-HAADF और ए ईएम-ईडीएक्स) ने दवषाक्तर्ा कोदिका द्वारा दवषाक्तर्ा प्रदर्दिया के रूप में अदधग्रदहर् अल्ट्रास्ट्रक्चरल पररवर्तनोिं को स्पष्ट दकया। वर्तमान अध्ययन हाई एिंगल एनुलर डाकत फील्ड (HAADF) का उपयोग करके पहली

ररपोटत प्रदान करर्ा है दज ने खुला ा दकया मल्ट्ी मेटल आयन की उपस्स्थदर् में कवक कोदिका के अिंदर दवदभन्न भारी धार्ुओिं का टीक स्थान। ददलचस्प बार् यह है दक कवक को व्यस्क्तगर् या एकादधक धार्ु र्नाव के िंपकत में आने पर प्रत्येक धार्ु की दवदिष्ट जैव िंचय ाइट नहीिंबदली। मल्ट्ी धार्ु हटाने की दक्षर्ा की जािंच दवदभन्न पीएच पर भी की गई थी, बायोमेदडएिन पर पयातवरण की स्स्थदर् के प्रभाव का न्याय करने के दलए र्ापमान, नमक

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एकाग्रर्ा और नाइटरोजन स्रोर्। भिंडारण और पररवहन की प्रदिया को कम करने के दलए, फिंगल स्पोर र्ीन अलग- अलग उत्पादोिं में र्ैयार दकए गए थे। बहु धार्ु हटाने के दलए 12 महीने के िेल्फ जीवन के ाथ मायको-कैप्सूल को दडजाइन करने के दलए एक अग्रणी प्रया दकया गया था। हालािंदक, दनरिंर्र ररएक्ट्र रनोिं के दौरान मूल ूक्ष्मजीवोिं को बाहर दनकालने के दलए बी बाद याना की अक्षमर्ा देखी गई थी। इ दलए, मूल माइिोदबयल किं ोदटतया (एरोदबक और एनारोदबक) लोहमािंडी नाली े दवकद र् दकया गया था और प्रयोगिाला पैमाने (4 एल) एरोदबक में ऊिंचे धार्ु आयन एकाग्रर्ा (धार्ु ािंद्रर्ा में उर्ार-चढ़ाव पर दवचार करने) के ाथ वास्तदवक अपदिष्ट जल े धार्ु हटाने की क्षमर्ा के दलए परीक्षण दकया गया था और एनारोदबक बायोरेक्ट्र। उन्नर् प्रारिंदभक ािंद्रर्ा

पर चयदनर् धार्ु ेवन के दलए 23-100% धार्ु हटाने को देखा गया था। इ के अलावा, क्षेत्रीय स्स्थदर्योिं के र्हर्

देिी माइिोदबयल किं ोदटतयम के प्रदितन का मूल्ािंकन करने के दलए, ाइट बायोरेक्ट्र द स्ट्म (5 एल) को

दवकद र् दकया गया था और वास्तदवक और ाथ ही धार्ु के स्पाइक दकए गए अपदिष्ट जल के ाथ स्खलाया गया

था। अ िंगर् और र्ेज 5 एल बायोरेक्ट्र दोनोिं में बहुउद्देिीय बहु धार्ु हटाने को दजत दकया गया था िॉक लोड को

िंभालने के दलए ररएक्ट्र द स्ट्म की िंगर्र्ा ादबर् करने, 60 ददनोिं की अवदध में प्रणाली। प्रयोगात्मक द िंचाई क्षेत्र की मािंग को पूरा करने के दलए, प्रदिया को धीरे-धीरे 50 लीटर और उ के बाद 500 लीटर बायोरेक्ट्र र्क बढ़ा ददया गया था। 500 लीटर बायोरेक्ट्र वास्तदवक अपदिष्ट जल और धार्ु े 26-57% धार्ु को हटाने में क्षम था इलाज दकए गए पानी में ािंद्रर्ा एफएओ अनुमर् ीमाओिं के भीर्र थी। नर्ीजे बर्ार्े हैं दक बायोरेक्ट्र प्रणाली

अपदिष्ट जल की गुणवत्ता में भारी धार्ु एकाग्रर्ा में उर्ार चढ़ाव और मौ मी बदलाव [5 एल (पूवत मान ून), 50 एल (मान ून) और 500 एल (मान ून के बाद)] के ाथ पूरी र्रह े िंगर् थी। बायोरेक्ट्र े उपचार न दकए गए पानी के ाथ ही द िंचाई के दलए इस्तेमाल दकया गया था। महत्वपूणत बार् यह है दक अपदिष्ट जल द िंचाई वाले पौधोिं

की र्ुलना में इलाज दकए गए पानी े द िंदचर् पौधोिं में कम धार्ु िंचय देखा गया था, जो द िंचाई के दलए इलाज दकए गए पानी की उपयुक्तर्ा का िंकेर् देर्ा है।

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TABLE OF CONTENT

CERTIFICATE………...………i

ACKNOWLEDGEMENTS…….………...……..….ii

ABSTRACT………....iv

TABLE OF CONTENT……….vi

LIST OF FIGURES………..…xii

LIST OF TABLES……….…xviii

Chapter 1-Introduction and Literature review………1

1.1 Introduction……….……….1

1.2 Literature Review……….………...4

1.2.1 Metal pollution scenario in water bodies and drain……….……….4

1.2.2 Impact of wastewater irrigation on quality of agricultural produce.……….8

1.2.3 Wastewater Treatment techniques (pre and on farm) prior to irrigation……….11

1.2.4 Biological methods for heavy metal remediation………..…………..12

1.2.4.1 Mechanism for heavy metal uptake by microorganism……….13

1.2.4.2 Heavy metal removal by fungi………..……15

1.2.4.2.1 Heavy metal removal by fungus (single metal ion)………15

1.2.4.2.2 Heavy metal removal by fungus (Multi metal ion)………19

1.2.4.3 Heavy metal removal by bacteria……….……….25

1.2.4.3.1 Heavy metal removal by bacteria (single metal ion) ……….25

1.2.4.3.2 Heavy metal removal by bacteria (multi metal ion)………..………29

1.2.4.4 Limitation using isolated micro-organism for heavy metal removal……….……37

1.2.4.5 Native microbial consortium for metal removal ……….…….39

1.2.4.6 Bioreactor configuration for multi metal removal ………..………..41

1.2.5 Challenges in technology development and implementation………..……….45

1.2.6 Conclusion………...46

1.2.7 Scope of work………..47

Chapter 2-Assessment of drains water used for irrigation in rural and peri-urban settings of Delhi region……….50

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2.1 Material and Methods……….………50

2.1.1 Description of selected sampling sites………..50

2.1.2 Collection of water samples and characterization………53

2.1.3 Reagent and standard preparation………53

2.1.4 Analytical techniques………...54

2.1.4.1 Chemical oxygen demand ………...………….54

2.1.4.2 Heavy metal and other element concentration in wastewater………...54

2.1.5 Statistical analysis………54

2.2 Results and discussion………55

2.2.1 Assessment of physicochemical parameters (TDS, pH, DO and COD in collected wastewater samples from selected sites………....55

2.2.1.1 pH ………...……..56

2.2.1.2 Total Dissolved Solids (TDS)………...………56

2.2.1.3 Chemical oxygen demand (COD)………....56

2.2.2 Assessment of heavy metal (Cr, Cu, Cd, Zn, Pb and Ni) concentrations in collected wastewater………...……….57

2.3 Inference ………..…………..65

Chapter 3-Performance evaluation of entomopathogenic fungi Beauveria bassiana for multi metal removal………...……….66

3.1 Materials and methods……….…………..………….66

3.1.1 Microorganisms, growth conditions and minimum inhibitory concentration (MIC)…..66

3.1.2 Metal solutions preparation……….….67

3.1.3 Effect of metal on growth kinetics and metal removal mechanism………….…………67

3.1.4 Development of fungal formulation (myco-granules, myco-tablets and myco- capsules)...68

3.1.4.1 Granulation, tabletting and capsulation techniques………..…………68

3.1.4.2 Physical characterization of tablets and capsules……….………….69

3.1.4.3 Evaluation of shelf-life and multi-metal removal capacity ………..70

3.1.5 Effect of process parameters………71

3.1.5.1 Effect of initial heavy metal concentration………..….71

3.1.5.2 Effect of increment in metal complexity ……….72

3.1.5.3 Effect of pH and temperature on metal removal in multi metal mixture …………..….72

3.1.5.4 Effect of different salt concentration on metal removal in multi metal mixture……...73

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3.1.5.5 Effect of different nitrogen sources on metal removal in multi metal mixture………..73

3.1.6 Reactor studies………..…..74

3.1.7Analytical techniques……….…..…74

3.1.7.1 Measurement of residual metal concentration……….…….………...74

3.1.7.2 Estimation of glucose content………...……...74

3.1.7.3 Biomass production……….………75

3.1.7.4 FTIR (Fourier Transform Infrared Spectroscopy)………..….75

3.1.7.5 TEM-HADDF (Transmission Electron Microscopy- High-angle annular dark field)……….75

3.1.7.6 AFM (Atomic Force Microscopy)………...76

3.1.7.7 SEM-EDX (Scanning Electron Microscopy-Energy Dispersive X-Ray Spectroscopy)………...76

3.1.8 Statistical analysis………..……….77

3.2 Results and discussion………....………77

3.2.1 Minimum Inhibitory Concentration (MIC)………..77

3.2.2 Effect of metal on growth kinetics and metal removal mechanism……….…..78

3.2.3 Morphological analysis………85

3.2.3.1 FTIR (Fourier Transform Infrared Spectroscopy)………....85

3.2.3.2 AFM (Atomic Force Microscopy)………..………..88

3.2.3.3 TEM-HAADF (Transmission Electron Microscopy-High angle annular dark field)………...90

3.2.3.4 SEM-EDX (Scanning Electron Microscopy-Energy Dispersive X-Ray Spectroscopy)………...96

3.2.4 Development of fungal formulation (myco-granules, myco-tablets and myco- capsules)……….………101

3.2.4.1 Physical characterization of myco-tablets and myco-capsules………..…101

3.2.4.3 Viability (cfu count)………102

3.2.4.4 Shelf life and heavy metal removal efficiency of formulations ……….…104

3.2.5 Effect of process parameters………..107

3.2.5.1 Effect of initial heavy metal concentration……….………107

3.2.5.2 Effect of incremental metal complexity………..110

3.2.5.3 Effect of pH and temperature on metal removal in hexa metal mixture………..113

3.2.5.4 Effect of initial salt concentration on metal removal in multi metal mixture……….114

3.2.5.5 Effect of different nitrogen source on metal removal in multi metal mixture……….117

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3.2.5.6 Reactor studies………121

3.3 Inference………..……….123

Chapter 4-Development and performance evaluation of native microbial consortium for multi metal removal in aerobic and anaerobic bioreactor……….…..125

4.1 Material and methods………125

4.1.1 Development of microbial consortium (aerobic and anaerobic) from wastewater under multi metal stress………...…….125

4.1.2 Experimental set up………....126

4.1.3 Bioreactors………...…..126

4.1.4 Feeding tank………...127

4.1.5 Wastewater composition for bioreactor……….127

4.1.6 Experimental procedure……….128

4.1.7 Analytical techniques……….………..……..129

4.1.7.1 Residual heavy metal concentration………...….129

4.1.7.2 COD (chemical oxygen demand) estimation ……….………129

4.2 Results and discussion………..129

4.2.1 Heavy metal removal……….129

4.2.1.1 First phase (batch mode)-R1 and R3 [R1 (aerobic bioreactor) and R3 (anaerobic bioreactor) fed with actual drain wastewater]……….129

4.2.1.2 Second phase (continuous mode)-R1and R3 [R1 (aerobic bioreactor) and R3 (anaerobic bioreactor) fed with actual drain wastewater] ………132

4.2.1.3 First phase (batch mode)-R2 and R4 [R2 (aerobic bioreactor) and R4 (anaerobic bioreactor) fed with actual drain wastewater amended with 30mg L-1 multi metal mix]…...135

4.2.1.4 Second phase (continuous mode)-R2 and R4 [R2 (aerobic bioreactor) and R4 (anaerobic bioreactor) fed with actual drain wastewater amended with 30mg L-1 multi metal mix]……137

4.2.2 COD removal………...144

4.3 Inference………...………148

Chapter 5-Performance evaluation of native microbial consortium in on-site bioreactor and community dynamics assessment……...………..149

5.1 Material and methods………150

5.1.1 Site description………..…………150

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5.1.2 First stage-Batch and continuous operation in 5-litre CSTR………..151

5.1.2.1 Experimental set-up………151

5.1.2.2 Bioreactors……….………….151

5.1.2.3 Feeding tank………152

5.1.3 Second stage-Batch and continuous with 50-litre CSTR………..….152

5.1.3.1 Experimental set-up………..……..152

5.1.3.2 Bioreactor………...152

5.1.3.3 Feeding tank………....153

5.1.4 Third stage-Batch and continuous with 500-litre CSTR………...………..153

5.1.4.1 Experimental set-up………153

5.1.4.2 Bioreactor………..…….154

5.1.4.3 Feeding tank………..….154

5.1.5 Wastewater composition………...…154

5.1.6 Experimental procedure……….155

5.1.7 Microbial community analysis………..155

5.1.7.1 Sampling for DNA extraction………155

5.1.7.2 Amplification of 16s rRNA gene………..……….156

5.1.7.3 DGGE (Denaturing gradient gel electrophoresis)………..156

5.1.7.4 Phylogenetic analysis………...…..157

5.1.8 Heavy metal accumulation in Solanum lycopersicum irrigated using untreated and treated wastewater ………..……..158

5.1.8.1 Irrigation field site description...158

5.1.8.2 Irrigation schedule and sampling………..………158

5.1.9 Analytical techniques……….……..158

5.1.9.1 Residual heavy metal concentration in wastewater………...158

5.1.9.2 Heavy metal concentration in sludge……….159

5.1.9.3 Heavy metal concentration in plant………...………159

5.1.9.4 COD (Chemical oxygen demand)………...………..159

5.1.9.5 Nitrate estimation……….…….160

5.1.9.6 Phosphate estimation………...…..160

5.1.10 Volatile solids……….………...………….160

5.1.11 Statistical analysis………...……161

5.2 Results and discussion……….161

5.2.1 First stage-batch and continuous experiment with 5-liter CSTR……….161

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5.2.2 Second stage-batch and continuous experiment with 50-liter CSTR………...…169

5.2.3 Third stage-batch and continuous experiment in 500-liter CSTR……….….174

5.2.4 Overall conclusion of bioreactor scale up……….….180

5.2.5 Concentration of heavy metal in sludge………..………..…….…181

5.2.6 Heavy metal accumulation in Solanum lycopersicum irrigated using untreated and treated wastewater ……….…183

5.2.7 Microbial community assessment of different bioreactor……….186

5.3 Inferences ……….195

Chapter 6-Summary and Conclusion………..196

6.1 Assessment of drains water used for irrigation in rural and peri-urban settings of Delhi region………..…196

6.2 Performance evaluation of entomopathogenic fungi Beauveria bassiana for multi metal removal………...197

6.3 Development and performance evaluation of native microbial consortium for multi metal removal in aerobic and anaerobic bioreactor………..198

6.4 Performance evaluation of native microbial consortium in on-site bioreactor and community dynamics assessment………...199

6.5 Benefits and Scope for Future Research………199

References……….………....201

Annexure 1………...….231

Bio-Data………234

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LIST OF FIGURES

Figure 1.1 Current and future water usage by different sectors in India……….1

Figure 1.2 Heavy metal concentration in various industrial wastewater……….3 Figure 1.3 Different mechanism for heavy metal removal by microorganism………..13 Figure 2.1 Location of sampling sites on Najafgarh drain and Loha mandi drain ………..…….51 Figure 2.2 Sampling sites selected for wastewater collection: (A1) Nilothi; (A2) Keshopur; (A3) Punjabi Bagh; (A4) Daryai Nala; (A5) Nehru Vihar and (B) Loha Mandi Drain………….……52 Figure 3.1 Growth kinetics and biomass production by B. bassiana with time in absence of

metal………..78

Figure 3.2 shows the pH profile, rate of glucose utilization and biomass produced by B.

bassiana...…….79 Figure 3.3 (A) Growth kinetics and removal of heavy metal by B. bassiana in presence of 30 mg L-1 hexa-metal mix (5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and Cu) and (B) Concentration of individual heavy metal removed by B. bassiana in presence of 30 mg L-1 hexa-metal mix (5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and Cu) ……….………..……...82 Figure 3.4 Passive and active heavy metal uptake by B. bassiana in presence of 30 mg L-1 hexa- metal mix (5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and Cu) ………..…….….…..…84 Figure 3.5 Infrared spectra of B. bassiana in absence of metal………..86 Figure 3.6 Infrared spectra of B. bassiana (A) at 30 mg L-1 of Cu; Zn; Cd and Ni; (B) at 30 mg L-

1 of Cr; Pb and multi metal mix………..………...……..87 Figure 3.7 AFM of B. bassiana (A) In absence of metal; (B) at 30 mg L-1 of Cd; (C) at 30 mgL-1 of Cu; (D) at 30 mg L-1 of Cr ;(E) at 30 mg L-1 of Zn;(F) at 30 mg L-1 of Ni; (G) at 30 mg L-1 of Pb; and (H) at 30 mg L-1 of Multi Metal Mix……….………89 Figure 3.8 TEM micrograph of B. bassiana with EDX in absence of metal………..……91 Figure 3.9 (A) TEM of B. bassiana in presence of 30 mg L-1 Cd; (D) HAADF micro-graph indicating Cd localization and (C) EDX micrograph………..92 Figure 3.10 (A) TEM of B. bassiana in presence of 30 mg L-1 Cu; (D) HAADF micro-graph indicating Cu localization and (C) EDX micrograph………..92

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Figure 3.11 (A) TEM of B. bassiana in presence of 30 mg L-1 Ni; (B) HAADF micro-graph indicating Ni localization and (C) EDX micrograph………..……….93 Figure 3.12 (A) TEM of B. bassiana in presence of 30 mg L-1 Pb; (D) HAADF micro-graph indicating Pb localization and (C) EDX micrograph……….……….93 Figure 3.13 (A) TEM of B. bassiana in presence of 30 mg L-1 Zn; (D) HAADF micro-graph indicating Zn localization and (C) EDX micrograph……….…….………95 Figure 3.14 (A) TEM of B. bassiana in presence of 30 mg L-1 Cr; (D) HAADF micro-graph indicating Cr localization and (C) EDX micrograph………..……...…..95 Figure 3.15 TEM –HADDF of B. bassiana in presence of 30 mg L-1 hexa-metal mix (5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and Cu): (A) TEM micrograph; (B) localization of Cd (C) localization of Cr; (D) localization of Cu ;(E) localization of Pb;(F) localization of Zn; (G) localization of Ni

and (H) EDX micrograph……….………..…....97

Figure 3.16. Scanning Electron micrographs of B. bassiana (A) In absence of metal; (B) at 30 mg L-1 of Cd (C) at 30 mg L-1 of Cu (D) at 30 mg L-1 of Cr ;(E) at 30 mg L-1 of Zn;(F) at 30 mg L-1 of Ni; (G) at 30 mg L-1 of Pb; and (H) at 30 mg L-1 of multi metal mix………..….99 Figure 3.17. EDX spectra of B. bassiana pellet (A) In absence of metal;(B) at 30 mg L-1 of Cd (C) at 30 mg L-1 of Cr ;(D) at 30 mg L-1 of Cu ;(E) at 30 mg L-1 of Ni;(F) at 30 mg L-1 of Zn; (G) at 30 mg L-1 of Ni (H) at 30 mg L-1 of multi metal mix………..………100 Figure 3.18 Different formulations of B. bassiana: (A) Mycogranules; (B) Mycotablets and (C)

Mycocapsules………...101

Figure 3.19 Comparison of biomass production by different formulation with storage time in absence and presence of multi metal ion……….104 Figure 3.20 Multi metal removal (%) with time by myco-granules, myco-capsules and myco-

tablet………....106

Figure 3.21 Residual individual heavy metal concentration with different metal combinations by B. bassiana. (30 °C; 150rpm; pH: 6.5±0.2)………...111 Figure 3.22 Biomass production in control (Xc) and multi metal (Xmm) by B. bassiana in presence of 30 mg L-1 hexa-metal mix (5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and Cu) at different initial pH[4- 10] (30 °C; 150rpm; pH: 6.5±0.2)………..………...113

6

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Figure 3.23 Biomass production in control (Xc) and multi metal (Xmm) by B. bassiana in presence of 30 mg L-1 hexa-metal mix (5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and Cu) at different incubation

temperature[20°C-40°C] (150rpm; pH: 6.5±0.2)……….…………114

Figure 3.24 Glucose consumption and metal removal by B. bassiana at different salt concentration (0-4%) in presence of 30 mg L-1 hexa-metal mix (5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and Cu) (30 °C; 150rpm; pH: 6.5±0.2)………...………..116

Figure 3.25 Residual individual heavy metal concentration at different salt concentration (0-4%) by B. bassiana in presence of 30 mg L-1 hexa-metal mix (5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and Cu) (30 °C; 150rpm; pH: 6.5±0.2)……….……...117

Figure 3.26 Glucose consumption and metal removal by B. bassiana with different nitrogen sources in presence of 30 mg L-1 hexa-metal mix (5 mg L-1 each Cr, Cd, Pb, Ni, Zn and Cu) (30 °C; 150rpm; pH: 6.5±0.2)………..………..………….119

Figure 3.27 Residual individual heavy metal concentration with different nitrogen sources by B. bassiana in presence of 30 mg L-1 hexa-metal mix (5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and Cu) (30 °C; 150rpm; pH: 6.5±0.2)………..………..…..120

Figure 3.28 Lab scale reactor set-up……….…121

Figure 3.29 Residual heavy metal concentration in reactor system………...122

Figure 4.1 Schematic diagram of aerobic bioreactor (R1 and R2)………126

Figure 4.2 Schematic diagram of anaerobic bioreactor (R3 and R4)………...126

Figure 4.3 Outlet heavy metal concentration in bioreactor R1 (aerobic) during batch operation………..130

Figure 4.4 Outlet heavy metal concentration in bioreactor R3 (anaerobic) during batch operation………..…131

Figure 4.5 Outlet heavy metal concentration in bioreactor R1 (aerobic) during continuous operation ……….133

Figure 4.6 Outlet heavy metal concentration in bioreactor R3 (anaerobic) continuous operation………..134

Figure 4.7 Outlet heavy metal concentration in bioreactor R2 (aerobic) fed with heavy metal spiked (30 mg L-1) wastewater during operation [5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and Cu]………136

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Figure 4.8 Outlet heavy metal concentration in bioreactor R4 (anaerobic) fed with heavy metal spiked (30 mg L-1) wastewater during batch operation [5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and

Cu] ………...……….……...136

Figure 4.9 Outlet heavy metal concentration in bioreactor R2 (aerobic) fed with heavy metal spiked (30 mg L-1) wastewater during continuous operation [5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and Cu]………...……….……138

Figure 4.10 Outlet heavy metal concentration in bioreactor R4 (anaerobic) fed with heavy metal spiked (30 mg L-1) wastewater during continuous operation [5 mg L-1 each of Cr, Cd, Pb, Ni, Zn and Cu] ………138

Figure 4.11 Average COD value with removal percentage in aerobic bioreactors (R1 and R2) and anaerobic bioreactor (R3 and R4) in batch mode……….144

Figure 4.12 Average COD value with removal percentage in bioreactor [R1 (aerobic) and R3 (anaerobic)] in continuous mode with actual wastewater……….146

Figure 4.13 Average COD value with removal percentage in bioreactor [R2 (aerobic) and R4 (anaerobic)] in continuous mode with multi metal spiked wastewater……….147

Figure 5.1 Schematic overview of experimental site………...…150

Figure 5.2. Field experimental site: (A) Loha mandi drain and bioreactor installation site; (B) Front view of Bioreactor installation site and (C) Experimental field……….150

Figure 5.3 Schematic diagram of aerobic bioreactor (5 L)………..…151

Figure 5.4 Schematic diagram of aerobic bioreactor (50 L)………....152

Figure 5.5 Schematic diagram of aerobic bioreactor (500 L)………..153

Figure 5.6 Heavy metal concentration (Zn, Cd and Cr) in inlet and outlet effluent in 5 L bioreactor operated with actual wastewater under ambient conditions (continuous mode 15-60 days)……….…163

Figure 5.7 Heavy metal concentration (Cu, Ni and Pb) in inlet and outlet effluent in 5 L bioreactor operated with actual wastewater under ambient conditions (continuous mode 15-60 days)……….163

Figure 5.8 Heavy metal concentration in inlet and outlet effluent of 5L bioreactor, spiked with 30 mg L-1 multi metal mixture under ambient conditions (continuous mode 60-122 days)…….…165

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Figure 5.9 COD concentration in inlet and outlet effluent with COD removal percentage in 5 L bioreactor operated with actual wastewater and wastewater spiked with multi metal ion under ambient conditions (continuous mode 15-122 days)………...………166 Figure 5.10 Phosphate concentration in inlet and outlet effluent in 5 L bioreactor operated with actual wastewater and wastewater spiked with multi metal ion under ambient conditions

(continuous mode 15-122 days)……….……..167

Figure 5.11 Nitrate concentration in inlet and outlet effluent in 5 L bioreactor operated with actual wastewater and wastewater spiked with multi metal ion under ambient conditions (continuous

mode 15-122 days)……….…..167

Figure 5.12 VSS concentration in inlet and outlet effluent in 5 L bioreactor operated with actual wastewater and wastewater spiked with multi metal ion under ambient conditions………168 Figure 5.13 Heavy metal concentration (Zn, Cd and Cr) in inlet and outlet effluent in 50 L bioreactor operated with actual wastewater under ambient conditions (continuous mode 15-150

days)……….171

Figure 5.14 Heavy metal concentration (Cu, Ni and Pb) in inlet and outlet effluent in 50 L bioreactor operated with actual wastewater under ambient conditions (continuous mode 15-150

days)……….172

Figure 5.15 COD concentration in inlet and outlet effluent with COD removal percentage in 50 L bioreactor under ambient conditions (continuous mode 15-150 days)………....172 Figure 5.16 Phosphate concentration in inlet and outlet effluent in 50 L bioreactor under ambient conditions (continuous mode 15-150 days)……….173 Figure 5.17 Nitrate concentration in inlet and outlet effluent in 50 L bioreactor under ambient conditions (continuous mode 15-150 days)……….…174 Figure 5.18 Heavy metal concentration (Zn, Cd and Cr) in inlet and outlet effluent in 500 L bioreactor operated with actual wastewater under ambient conditions (continuous mode 15-74 days)……….177 Figure 5.19 Heavy metal concentration (Cu, Ni and Pb) in inlet and outlet effluent in 500 L bioreactor operated with actual wastewater under ambient conditions (continuous mode 15-74

days)……….…177

Figure 5.20 COD concentration in inlet and outlet effluent with COD removal percentage in 500 L bioreactor under ambient conditions (continuous mode 15-74 days)………178

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Figure 5.21 Phosphate concentration in inlet and outlet effluent in 500 L bioreactor under ambient conditions (continuous mode 15-74 days)………179 Figure 5.22 Nitrate concentration in inlet and outlet effluent in 500 L bioreactor under ambient conditions (continuous mode 15-74 days)………...…179 Figure 5.23 DGGE (Denaturing Gradient Gel Electrophoresis) of sludge samples from different

bioreactor ………187

Figure 5.24 Phylogenetic tree for microbial community present in 5 L bioreactor ….………...189 Figure 5.25 Phylogenetic analysis of microbial community present in 5 L bioreactor spiked with 30 mg L-1multi metal ion...……….…….191 Figure 5.26 Phylogenetic analysis of microbial community present in 50 L...……….…….193 Figure 5.27 Phylogenetic analysis of microbial community present in 500 L bioreactor...……194

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LIST OF TABLES

Table 1.1 Concentration of heavy metals in river water in different parts of the world……...…5 Table 1.2 Guidelines for maximum permissible limits of heavy metal in irrigational water by different countries and agencies……….………...….6 Table 1.3 Safe limit of heavy metals by different agencies in food products………...7 Table 1.4 Concentration of various heavy metal in the vegetables, crop and fruits irrigated from contaminated water………..…………...9 Table 1.5 Heavy metal removal by fungal strain from single metal ion solution………...16 Table 1.6 Heavy metal removal by fungi in multi metal ion solution………..20 Table 1.7 Heavy metal removal by different bacterial strain from single metal ion solution….26 Table 1.8 Heavy metal removal by bacteria in multi metal ion solution………..30 Table 1.9 Various recently developed biological method for heavy metal removal from wastewater………..………..…42 Table 2.1 Average value of physico-chemical parameters of wastewater………...…55 Table 2.2 Copper (Cu) concentrations in wastewater at various sampling sites of Najafgarh

Drain……….58

Table 2.3 Chromium (Cr) concentrations in wastewater at various sampling sites of Najafgarh

Drain……….58

Table 2.4 Lead (Pb) concentrations in wastewater at various sampling sites of Najafgarh

Drain……….60

Table 2.5 Zinc (Zn) concentrations in wastewater at various sampling sites of Najafgarh

Drain……….…60

Table 2.6 Cadmium (Cd) concentrations in wastewater at various sampling sites of Najafgarh

Drain……….…61

Table 2.7 Nickel (Ni) concentrations in wastewater at various sampling sites of Najafgarh

Drain……….62

Table 2.8 Heavy metal concentrations in wastewater at various sampling sites of Loha Mandi

Drain……….…63

Table 2.9 Descriptive statistics of the heavy metal concentrations in wastewater of Najafgarh drain and Loha Mandi drain………..64 Table 3.1 MIC of heavy metal for B. bassiana……….77

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Table 3.2 The effect of heavy metal on the specific growth rate and percentage of metal removal by B. bassiana at 30mgL-1 initial heavy metal concentration (30 °C; 150rpm; pH:

6.5±0.2)...80 Table 3.3 Characterization of myco-tablets………103 Table 3.4 cfu count of different formulation with storage time………..103 Table 3.5 Bioaccumulation of Cu, Ni, Cd, Zn, Cr, Pb and multi metal mixture in composite media by B. bassiana (at 30ºC, 150 rpm and 120h)……….……….……….108 Table 3.6 Effect of initial salt concentration on metal removal and biomass production by B.

bassiana (initial hexa-metal concentration of 30 mg L-1; 30 °C; 150 rpm; pH: 6.5±0.2)…..115 Table 3.7 Effect of various nitrogen sources on metal removal and biomass production by B.

bassiana (initial hexa-metal concentration of 30 mg L-1; 0 °C; 150 rpm; pH: 6.5±0.2)…..118 Table 4.1 Inlet and outlet heavy metal concentration in bioreactor R1 (aerobic) and R3

(anaerobic) after treatment……….132

Table 4.2 Inlet and outlet heavy metal concentration in bioreactor R2 and R4 after

treatment……….………140

Table 4.3 Comparison of present study with recently developed various biological methods for heavy metal removal from wastewater……….………..143 Table 5.1 Chemical constituent of Denaturing Gradient Gel Electrophoresis (DGGE) gel………..……….157 Table 5.2 Performance of field bioreactor (5L) in batch mode operated with actual wastewater

(0-14 days)………..………161

Table 5.3. Average inlet and outlet concentration of heavy metal in bioreactor (5 L) during continuous mode operated with actual wastewater (15-60 days)…………..………..162 Table 5.4. Average inlet and outlet concentration of heavy metal in bioreactor (5 L) during continuous mode operated with wastewater spiked with multi metal ion (60-122 days)…...164 Table 5.5 Performance of field bioreactor (50L) in batch mode operated with actual wastewater

(0-14 days)………..169

Table 5.6 Average inlet and outlet concentration of heavy metal in bioreactor (50 L) during continuous mode operated with actual wastewater (15-150 days)……….………170 Table 5.7. Performance of field bioreactor (500L) in batch mode operated with actual

wastewater (0-14 days)……….………..175

Table 5.8. Average inlet and outlet concentration of heavy metal in bioreactor (500 L) during continuous mode operated with actual wastewater (15-74 days)………176

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Table 5.9 Average heavy metal concentrations in sludge of bioreactor system in comparison

with EPA standard………..……182

Table 5.10 Average heavy metal concentration in plant irrigated with wastewater and treated water………...184

Annexure Table: Table 1 TDS value of wastewater at various sampling sites of drain………231

Table 2 pH value of wastewater at various sampling sites of drain………...231

Table 3 DO value of wastewater at various sampling sites of drain………231

Table 4 COD value of wastewater at various sampling sites of drain………232

Table 5 Concentration of heavy metal in inlet and outlet of CETP’s in Delhi………...………233

References

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