SUPPRESSION OF SULFATE REDUCING

BACTERIA DURING ANAEROBIC TREATMENT

by

SK. ZIAUDDIN AHAMMAD DEPARTMENT OF BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY

Submitted

in fulfillment of the requirements of the degree of

DOCTOR OF PHILOSOPHY

to the

INDIAN INSTITUTE OF TECHNOLOGY DELHI NEW DELHI - 110016, INDIA

JULY 2009

ti\J2.41 kA,(.

I. L., ill

Acc.

Certificate

This is to certify that the thesis entitled "Suppression of Sulfate Reducing Bacteria

during Anaerobic Treatment" being submitted by Sk. Ziauddin Ahammad is worthy of

consideration for the award of the degree of Doctor of Philosophy. The thesis has been

prepared under my supervision and guidance in conformity with the rules and regulations

of Indian Institute of Technology Delhi and is a record of the original bonafide research

work. The results presented in this thesis have not been submitted in part or full to any

other universities or institutes for the award of any other degree or diploma.

Dr. T.R. Sreekrishnan Dr. James Gomes Professor Associate Professor Department of Biochemical Engineering and Biotechnology School of Biological Sciences Indian Institute of Technology Delhi Indian Institute of Technology Delhi

Acknowledgements

I am extremely grateful to my supervisors Prof. T. R. Sreekrishnan and Dr. James Gomes

for their expert guidance and constant encouragement throughout my project. It would not

have been possible at all to complete my thesis successfully, without their continuous support

and motivation. I am highly indebted to them for their patience and devoting their valuable

time throughout my work.

I am very thankful to Prof. G. P. Agarwal, Prof. J. K. Deb, Prof. Sunil Nath, Dr. P.K.

Roychoudhuri, Dr. A.K. Mittal and Prof. Mukesh Khare for their valuable suggestions and

encouragements.

My indebted thanks to Dr. Russell Davenport, Mrs. Fiona Read, Prof. Thomas Curtis of

CEG, Newcastle University for their expert guidance and teaching, which helped me to learn

molecular biology techniques applied in Environmental Engineering.

I am very much thankful to Mr. J. A. Khan, Mr. D.B. Sharma for their technical support and

cooperation.

I am grateful to the authorities of Indian Institute of Technology, Delhi for the facilities

received, to the All India Council of Technical Education, New Delhi for financial assistance

through National Doctoral Fellowship to carry out the research work.

My heartfelt thanks to my lab mates Dr. Mukesh Goel, Mr. Ramesh Motipalli, Mr. Dileep

P.V. for their ever-helping hands. I express my gratitude to my friends Mr. A.Remesh, Mr.

Mohit Naresh, Ms Meenu Chhabra, Mr. Muthu M., Mr. Javed Equbal, for their wholehearted

support throughout my stay in IIT.

It is not possible to express in word the support and love I received from my parents and

other family members. ‘x .21'444444- A-kuwb.A31 Dated, IIT Delhi

(Sk. Ziauddin Ahammad) Hauz Khas, New Delhi, The 8th July, 2009

ii

Abstract

In anaerobic wastewater treatment processes, the sulfate-reducing bacteria (SRB), which

coexist with methanogens, produces hydrogen sulfide (H2S). Though several techniques

are being used to remove H2S from biogas to obtain H2S-free biogas, none of those are

cost effective. This study was taken up to attempt the suppression of SRB in anaerobic

reactors using modifications in process as well as reactor operating conditions.

The effects of different parameters like temperature, volatile fatty acids (VFA)

concentration and reactor dilution rate on the growth of SRB in these reactors were

studied. Complete suppression of SRB with H2S free biogas containing 72 and 76%

methane was achieved at 55°C in batch reactor and in continuous stirred tank reactor

(HRT 3.8 d) respectively. The effect of immobilization on the suppression of SRB was

also studied with different support materials. Bio-film immobilized on nylon granules

produced H2S free biogas containing 68% methane in an anaerobic fluidized bed reactor

(HRT 0.55 d). Anaerobic RBC having acrylic discs favored the methanogens over SRB

to form bio-film on the disc resulting in H2S free biogas containing 53 % methane. SRB

suppression was found to be independent of VFA concentration in both batch and

continuous processes. A mathematical model has been developed and simulated with the

experimental observations to predict the interaction behavior of SRB and methanogens in

the mixed culture system.

The results of this research show for the first time that it is possible to eliminate SRB

from anaerobic cultures to produce H2S-free methane by manipulating the operational

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parameters. Consequently, the process technology based on these findings will be placed

economically at a significantly advantageous position compared to existing technologies

for the removal of H2S from biogas.

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CONTENTS Page No.

Certificate

Acknowledgements ii

Abstract iii

Contents v

List of Figures x

List of Tables xviii

Nomenclature xx

1 INTRODUCTION AND OBJECTIVES 1-4 1.1. Introduction 1

1.2 Objectives 3

2 LITERATURE REVIEW 5-38 2.1 Background 5

2.2 Anaerobic wastewater treatment process 7

2.3 Biogas- a degradation byproduct 10

2.4 Characteristics of methanogens and SRB 12

2.4.1 Methanogens 12

2.4.2 Sulfate reducing bacteria (SRB) 14

2.5 Competition between sulfate reducers, methanogens

and acetogens 17

2.5.1 Hydrogen 17

2.5.2 Acetate 18

2.5.3 Propionate 18

2.5.4 Butyrate 19

2.6 Commonly used reactors in anaerobic process 20

v

2.7 Parameters affecting the reactor performances

during anaerobic treatment 21

2.7.1 pH 22

2.7.2 Waste composition 23

2.7.3 Temperature 23

2.7.4 Loading rate 24

2.7.5 Retention time 24

2.7.6 Toxicity 24

2.7.7 Granule deterioration 27

2.8 Problems associated with SRB 27

2.9 Industries prone to MIC by SRB 29

2.10 Containment of H2S to control MIC 31

2.10.1 Growth inhibition of SRB in the treatment system 32

2.10.1.1 Growth inhibition using biocides 32

2.10.1.2 Prevention of anaerobic activity by changing

terminal electron acceptor 33

2.10.2 Removal of H2S from the biogas produced in the

treatment system 34

2.10.2.1 Biological oxidation of H2S formed 34

2.10.2.2 Chemical oxidation of H2S formed 35

2.10.2.3 Chemical or biological scrubbing 35

2.10.2.4 Slow release solid-phase oxygen (Mg02/Ca02) 36

2.10.2.5 Microbial fuel cells (MFCs) 37

3 MATERIALS AND METHODS 39-59 3.1 Materials 39

3.1.1 Medium composition 39

3.1.1.1 Glucose medium composition 39

3.1.1.2 Defined volatile fatty acids medium composition 39

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3.1.1.2.1 Trace element solution 41

3.1.1.2.2 Vitamin solution 42

3.1.2 Acclimatization of anaerobic culture 42

3.1.3 Experimental Setup 43

3.1.3.1 Batch reactor 43

3.1.3.2 Continuous reactor 45

3.1.3.2.1 Suspended cell systems 45

3.1.3.2.2 Attached cell systems 48

3.1.3.2.2.1 Anaerobic Rotating Biological

contactor (ARBC) 48

3.1.3.2.2.2 Anaerobic Fluidized Bed

Reactor (AFBR) and Hybrid

Anaerobic Reactor (HAR) 49

3.2 Experimental Methods 51

3.2.1 Treatment for SRB suppression 51

3.2.1.1 Effect of temperature 51

3.2.1.2 Effect of VFA concentration 51

3.2.1.3 Effect of dilution rate 52

3.2.1.4 Preferential immobilization of methanogens 52

3.2.2 Estimation of kinetic parameters of methanogens and

SRB interaction 53

3.3 Analytical Methods 53

3.3.1 Measurement of VFA concentration 53

3.3.2 Analysis of Gas composition 54

3.3.3 Determination of sulfate ion concentration 54

3.3.4 Measurement of sulfide ion concentration 55

3.3.5 Bio-film morphology 55

3.3.6 Measurement of Zeta Potential 56

3.3.7 SRB detection test 56

3.3.8 SRB detection using fluorescence in-situ hybridization 58

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4 RESULTS AND DISCUSSION 60-131 4.1 Effect of temperature 60

4.2 Effect of higher concentration of VFA 70

4.3 Effect of Dilution rate 73

4.4 Effect of immobilization 77

4.4.1 Effect of substrate and inert support on

preferential immobilization of methanogens 77

4.4.2 Conditions and material suitable for preferential

immobilization of methanogens 94

4.4.3 Studies on continuous attached cell system to

achieve SRB free methanogenic bio-film 95

4.4.3.1. Anaerobic fluidized bed reactor 96

4.4.3.2. Anaerobic rotating biological contactor 101

4.5 Development of mathematical model to determine

the interaction between methanogens and SRB 105

4.5.1 Background, motivation and premise 105

4.5.2 Model Development 107

4.5.2.1 Assumptions for model 107

4.5.2.2 Model equations 108

4.5.2.2.1 Biomass balance 108

4.5.2.2.2 Substrate balance 109

4.5.2.2.3 Product balance 110

4.5.3 Experimental design 111

4.5.4 Solution procedure of the model 112

4.5.5 Statistical validation of model 115

4.5.6 Model prediction 125

4.5.7 Conclusion 131

viii

5 SUMMARY AND CONCLUSIONS 132-134 5.1 Summary and Conclusions 132

REFERENCES 135-159

APPENDICES 160-175 Appendix I 160

Appendix II 172

AUTHOR'S RESUME 176-178

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