Biological sulfate reduction in sulfate rich industrial wastewaters by anaerobic fluidized-bed reactors: effect of electron donors

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Content
BIOLOGICAL SULFATE REDUCTION IN SULFATE-RICH INDUSTRIAL
WASTEWATERS BY ANAEROBIC FLUIDIZED-BED REACTORS: EFFECT
OF ELECTRON DONORS

by

Atosa Vahdati Nikzad

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(ENGINEERING)

December 2007

Copyright 2007 Atosa Vahdati Nikzad

ii

EPIGRAPH

Life is too short
Spend it doing something you love
With people who make you happy
iii

DEDICATION

To my parents

Feridoon Vahdati Nikzad and Mehri Motaheddin
&
To my husband

Farbod Fotovat

For their unconditional love and support

iv

ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my advisor, Professor
Massoud Pirbazari, for his guidance, support, encouragement, and patience from the
very first day to the end of my Ph.D. study at USC. My special thanks go to
Professor Teh Fu Yen and Professor Katherine S. Shing for being members of my
Ph.D. dissertation committee, and to Professor George V. Chilingar and Joseph S.
Devinny for guiding me in my qualifying exam and for their valuable suggestions.
This work would have not been possible without the assistance of
Varadarajan Ravindaran, who supported me with his broad knowledge all through
the years. In addition, I thank Erick J. Hernandez and Lance E. Hill for their
technical assistance during my lab work.
I would like to thank the Metropolitan Water District of Southern California
and the United States Environmental Protection Agency for their financial support on
the fluidized bed reactor (FBR) studies with hydrogen and carbon dioxide gas.
No words of thanks can express my deep appreciation for the constant love,
encouragement, and trust of my father Feridoon Vahdati Nikzad, my mother Mehri
Motaheddin, and my husband Farbod Fotovat throughout the years.
v

TABLE OF CONTENTS

Epigraph ii
Acknowledgements iv
List of Figures viii
List of Tables xii
List of Acronyms xv
Abstract xvii
Chapter 1: Introduction 1
Chapter 2: Background 4
2.1 Sulfur Cycle 4
2.2 Sulfate Sources 7
2.2.1 The Water Quality-Based Approach to Pollution 9
2.3 Rational Behind Choosing Biological Methods 16
2.4 Problems Associated with Anaerobic Treatment of High Sulfate
Wastewaters
18
2.5 Reasons for the Choice of FBR with Recycle and GAC as the Solid
Support
19
2.6 Feasibility Studies and Laboratory-Scale Studies in Biological Sulfate
Reduction
21
2.7 Mechanism of Sulfate Reduction 24
2.8 Selection of an Electron Donor for Biological Sulfate Reduction 25
2.9 Choice of Electron Donor Based on Free Energy 27
2.10 Thermodynamics and Calculation of Energy Yields of Metabolic
Processes
28
2.10.1 Comparison of the Reaction Free Energy for Different Carbon
Sources
29
2.10.2 Optimum Temperature, and pH 36
2.10.3 Determination of Biokinetic Parameters in a CMBR 36
2.11 H
2
S Health Standards and Methods of Removal 41
2.11.1 Air Pollution Standards for H
2
S 41
2.11.2 Water Pollution Standards for H
2
S 42
2.11.3 Chemical, Physical, and Biological Methods for Hydrogen Sulfide
Removal from Water
42
2.12 Anaerobic and Aerobic Biofilters for Treatment of H
2
S Off-Gas from
FBR System
44
vi

2.13 Methanogenesis Phenomena in Fluidized-Bed Reactors 53
2.13.1 The Inhibitory Effect of Hydrogen Sulfide on MPB and SRB 62
Chapter 3: Research Objectives and Scope 64
3.1 Research Objectives 64
3.2 Research Scope

65
Chapter 4: Materials and Methods 67
4.1 Materials 67
4.1.1 Synthetic Wastewater and Constituents 67
4.1.2 Electron Donors 68
4.1.3 Granular Activated Carbon 70
4.2 Analytical Methodologies 70
4.2.1 Sulfate, Acetate, and Lactate Measurement 70
4.2.2 Measurement of Alkalinity 71
4.2.3 Measurement of Hydrogen Sulfide 71
4.2.4 Measurement of Suspended Solids, Volatile Suspended Solid 71
4.2.5 Analysis of Attached Volatile Solids 72
4.2.6 Analysis of the FBR Column Off-Gases, Methane and H
2
S 73
4.2.7 Scanning Electron Microscopy of the Bacterial Growth on GAC 73
4.2.8 Measurement of Ethanol 74
4.3 Experimental Methodologies 75
4.3.1 Preparation of the Synthetic Wastewater and Electron Donor
Solutions
75
4.3.2 Microbial Culture 75
4.3.3 CMBR Studies 76
4.3.4 Fluidized Bed Reactors 78
4.3.5 FBR System with Hydrogen and Carbon Dioxide 81
4.3.6 Hydrogen Sulfide Sparging System 83
4.3.7 FBR System with Lactate and Acetate 85
4.3.8 FBR System with Effluent Mixing 87
4.3.9 Biofilters for H
2
S Off-Gas from FBR Column 89

Chapter 5: Results and Discussion 91
5.1 CMBR Studies 91
5.1.1 CMBR Studies with Acetate as Electron Donor 91
5.1.1.1 Different pHs 92
5.1.1.2 Different Temperatures 95
5.1.1.3 Different C/S Ratios 97
5.1.2 CMBR Studies with Lactate as Electron Donor 100
5.1.2.1 Different pHs 100
5.1.2.2 Different C/S Ratios 102
5.1.2.3 Different Temperatures 104
5.1.2.4 Determination of Biokinetic Parameters 108
vii

5.1.3 Batch Test with Hydrogen Gas as Electron Donor 115
5.1.4 Molasses, Citric Acid, and Formic Acid as Electron Donors 116
5.2 Biological Sulfate Reduction in Fluidized Bed Reactors 120
5.2.1 Sulfate Removal Using Hydrogen Gas as Electron Donor 120
5.3 Hydrogen Sulfide Production in FBR Reactor with Hydrogen Gas as
Electron Donor
125
5.4 Hydrogen Sulfide Stripping from FBR Aqueous Effluent 126
5.5 Fluidized Bed Reactor Studies with Acetate as the Electron Donor 130
5.6 Fluidized Bed Reactor Studies with Lactate as the Electron Donor 136
5.7 Sulfur Mass Balance 142
5.7.1 Significance of Sulfur Mass Balance Analysis 142
5.7.2 Mass Balance in Fluidized Bed Reactors 143
5.7.3 Mass Balance in Anaerobic Biofilter 160
5.8 Managing the Produced H
2
S and Production of Hydrogen Fuel 162
5.9 Scanning Electron Microscopy of the Fluidized Bed Reactors GAC 165
5.10 Managing the BOD Byproducts in the FBR Effluent 167
Chapter 6: Summary, Conclusions and Recommendations 170
6.1 Summary and Conclusions 170
6.1.1 CMBR Studies 170
6.1.2 Anaerobic FBR Studies 172
6.2 Recommendations 174
References 176
Appendixes 190
Appendix A 191
Appendix B 193
Appendix C 195
Appendix D 208
Appendix E 232
viii

LIST OF FIGURES

Figure 2.1 A Simplified Sulfur Cycle 5
Figure 2.2 Pathway in Anaerobic Degradation of Polymeric Compounds by
SRBs 28

Figure 2.3 Glyoxylate Cycle for Microorganisms Grown on Acetate, or Other
Substrates Methabolized through a 2-Carbon Intermediate 33

Figure 2.4 Krebs Cycle 34
Figure 2.5 Lactate Oxidation by Sulfate –Reduing Archaeon Archaeoglobus 35
Figure 2.6 Typical Reactions in Methanogenesis 55
Figure 4.1 Citric Acid Structure 69
Figure 4.2 Schematic of Experimental Setup for Completely Mixed Batch
Reactor Studies 77

Figure 4.3 Schematic of the FBR System with Hydrogen and Carbon Dioxide
Gas 82

Figure 4.4 Schematic for Hydrogen Sulfide Sparging System 84
Figure 4.5 Schematic of the FBR System with Lactate or Acetate as Electron
Donor 86

Figure 4.6 Schematic of the FBR System with Effluent Mixing 88
Figure 5.1 Sulfate Reduction Rates at Different pHs with Acetate as Electron
Donor 94

Figure 5.2 Acetate Utilization Rates at Different pHs 94
Figure 5.3 Sulfate Reduction Rates at Different Temperatures with Acetate as
Electron Donor 96

Figure 5.4 Acetate Utilization Rates at Different Temperatures 97
Figure 5.5 Sulfate Reduction Rates at Different C/S Ratios with Acetate as
Electron Donor 98
ix

Figure 5.6 Acetate Utilization Rates at Different C/S Ratios 99
Figure 5.7 Sulfate Reduction Rates at Different pHs with Lactate as the
Electron Donor 101

Figure 5.8 Lactate Utilization Rates at Different pHs 102
Figure 5.9 Sulfate Reduction Rates at Different C/S Ratios with Lactate as the
Electron Donor 103

Figure 5.10 Lactate Utilization Rates at Different C/S Ratios. 104
Figure 5.11 Sulfate Reduction Rates at Different Temperatures with Lactate as
the Electron Donor 105

Figure 5.12 Lactate Utilization Rates at Different Temperatures 106
Figure 5.13 Maximum Specific Growth Rate (μ ˆ ) with Lactate as the Electron
Donor 108

Figure 5.14 Maximum Specific Growth Rate (μ ˆ ) with Acetate as the Electron
Donor 109

Figure 5.15 Growth Yield for Sulfate with Lactate as the Electron Donor 110
Figure 5.16 Growth Yield for Sulfate with Acetate as the Electron Donor 110
Figure 5.17 Growth Yield for Lactate as the Electron Donor 111
Figure 5.18 Growth Yield for Acetate as the Electron Donor 112
Figure 5.19 Specific Utilization Rate Graph for Sulfate Utilizing Lactate
(mg substrate/mg biomass-time) 113
Figure 5.20 Specific Utilization Rate Graph for Lactate
(mg substrate/mg biomass-time) 113

Figure 5.21 Specific Utilization Rate Graph for Sulfate Reduction with Acetate
(mg substrate/mg biomass-time) 114

Figure 5.22 Specific Utilization Rate Graph for Acetate
(mg substrate/mg biomass-time) 114

x

Figure 5.23 CMBR Studies with Citric Acid 116
Figure 5.24 Sulfate Reduction Rates (Zero-Order Kinetic) in CMBR with
Citric Acid and Acetate as Electron Donors 117

Figure 5.25 Sulfate Reductions with Hydrogen and Carbon Dioxide Gas
(Days 0-91) 122

Figure 5.26 Sulfate Reductions with Hydrogen Gas as Electron Donor
(Days 91-166) 124

Figure 5.27 H
2
S Production Profile for FBR with Hydrogen Gas as the
Electron Donor 125

Figure 5.28 Schematic of a Gas Stripping Column 126
Figure 5.29 Hydrogen Sulfide Stripping with Nitrogen Gas at Different pHs 128
Figure 5.30 Hydrogen Sulfide Stripping with Nitrogen Gas at Different
Column Heights 129

Figure 5.31 H
2
S Sulfide Stripping with Nitrogen Gas at Different Liquid Flow
Rates 130

Figure 5.32 Sulfate Reduction Results in FBR with Acetate as the Electron
Donor 132

Figure 5.33 Acetate Utilization Results in FBR with Acetate as the Electron
Donor 134

Figure 5.34 H
2
S Production in FBR with Acetate as the Electron Donor 135
Figure 5.35 Sulfate Reduction Results in FBR with Lactate as Electron Donor 137
Figure 5.36 Lactate Utilization Results in FBR with Lactate as the Electron
Donor 139

Figure 5.37 H
2
S Production in FBR with Lactate as the Electron Donor 141
Figure 5.38 A Simplified Schematic of the Sulfur Balance in FBR 143
Figure 5.39 Percentages of H
2
S and HS
-
in Aqueous Form at Different pHs 148
xi

Figure 5.40 Mass Balance for FBR with Acetate with Effluent H
2
S Removing
Bioreactor 152

Figure 5.41 Mass Balance for FBR with Acetate and Effluent H
2
S Stripping 153
Figure 5.42 Mass Balance for the FBR with Lactate with Effluent H
2
S
Removal from the Bioreactor 158

Figure 5.43 Mass Balance for the FBR using Lactate with Effluent H
2
S
Stripping 159

Figure 5.44 Liqui-Cel Membrane Contactor System 163
Figure 5.45 SEM micrograph demonstrating morphologically different SRBs
in the GAC biofilm 165

Figure 5.46 SEM micrograph demonstration the GAC biofilm thickness 166
Figure 5.47 A typical SEM micrograph showing the heterogeneous nature of
biofilm 167

Figure A1. Gas-Liquid Separator Installation 191

xii

LIST OF TABLES

Table 2.1 Sulfate Concentrations in Wastewaters from Different Industries 9
Table 2.2 2002 CWA Section 303 (d) List of Water Quality Limited Segment
in Los Angeles Area 11

Table 2.3 An Example Sulfate Loads Analysis for Calculation of Sulfate
TMDL 15

Table 2.4 Reported Reactor Types, Carbon and Electron Sources, HRTs, and
Reduction Rates for Anaerobic Sulfate Reduction Process. 17

Table 2.5 Energetic Data and Growth Yield for SRB Utilizing Different
Substrates 23

Table 2.6 Representative Genera of Sulfate- and Sulfur-Reducing Bacteria 26
Table 2.7 Observed Free Energy in kJ for Sulfate Reduction Reactions with
Different Electron Donors 32

Table 2.8 Research Performed on the Usage of Biofilters for Hydrogen Sulfide
Removal 47

Table 2.9 Some of the Microorganisms Used for H
2
S Removal 49
Table 2.10 Specifications of H
2
S Utilizing Bacteria 52
Table 2.11 Sulfur Oxidation Mechanism in Different Sulfur Bacteria 52
Table 2.12 Sulfate-Reducing, Methanogenic and Acetogenic Reactions with
Organic Matters 58

Table 4.1 Substrate Composition in the Synthetic Wastewater 67
Table 4.2 Trace Nutrient Composition 68
Table 4.3 Characteristics of Fluidized Bed Reactors 80
Table 4.4 Hydrogen Sulfide Stripping System Specifications 83
Table 4.5 Jaeger Rings Packing Properties 84
Table 4.6 Physical Properties of the H
2
S Biofilter 89
xiii

Table 4.7 Operation Conditions of the H
2
S Biofilter 89
Table 5.1 Experimental Conditions in CMBR Studies with Acetate 92
Table 5.2 Kinetic Results at Different pHs with Acetate as Electron Donor 93
Table 5.3 Kinetic Results at Different Temperatures with Acetate as Electron
Donor 96

Table 5.4 Kinetic Results at Different C/S Ratios with Acetate as Electron
Donor 98

Table 5.5 Kinetic Results at Different pHs with Lactate as Electron Donor 101
Table 5.6 Kinetic Results at Different C/S ratios with Lactate as Electron
Donor 103

Table 5.7 Kinetic Results at Different temperatures with Lactate as Electron
Donor 105

Table 5.8 CMBR Kinetic Results with Different Electron Donors 107
Table 5.9 Maximum Specific Growth Rates, Growth Yields, and Specific
Consumption Rates Results 115

Table 5.10 CMBR Studies Results with Ethanol as Electron Donor 119
Table 5.11 Summary of Operational Changes in Fluidized Bed Reactor 123
Table 5.12 Stripping Ratio Calculations 127
Table 5.13 List of the Parameters in the FBR with Acetate as the Electron
Donor 136

Table 5.14 List of the Parameters in the FBR System with Lactate as the
Electron Donor 141

Table 5.15 Percentages of Aqueous H
2
S and HS
-
at Different pHs 147
Table 5.16 The sulfur Mass Balance between Liquid and Gas Phase in the
FBR with Acetate 151

Table 5.17 The Sulfur Mass Balance between Liquid and Gas Phase in the
FBR with Lactate 157
xiv

Table C1. CMBR Data for Acetate as the Electron Donor (Different pHs) 196
Table C2. CMBR Data for Acetate as the Electron Donor
(Different Temperatures) 199

Table C3. CMBR Data for Acetate as the Electron Donor (Different pHs) 202
Table C4. CMBR Data for Lactate as the Electron Donor (Different pHs) 204
Table C5. CMBR Data for Lactate as the Electron Donor
(Different Temperatures) 207

Table D1. Sulfate Data in FBR with Hydrogen Gas as Electron Donor 209
Table D2. Hydrogen and Carbon Dioxide Flow Data in FBR with Hydrogen
Gas as Electron Donor 213

Table D3. Hydrogen Sulfide Production Data in FBR with Hydrogen as
Electron Donor 217

Table D4. Sulfate Data in FBR with Acetate as Electron Donor 218
Table D5. Acetate Utilization Data in FBR with Acetate as Electron Donor 221
Table D6. Sulfate Data in FBR with Lactate as Electron Donor 224
Table D7. Lactate Utilization Data in FBR with Lactate as Electron Donor 226
Table D8. Hydrogen Sulfide Production Data in FBR with Lactate as Electron
Donor 229

Table D9. Hydrogen Sulfide Production Data in FBR with Acetate as Electron
Donor 230

Table E1. H
2
S Stripping Data at Different pHs 232
Table E2. H
2
S Stripping Data at Different Liquid Flow Rates 234

xv

LIST OF ACRONYMS

AB: Acetogenic Bacteria
APS: 5’-phosphosulphate
ATP: Adenosine Triphosphate
AVS: Attached Volatile Solids
BOD: Biological Oxygen Demand
CAAQS: California Ambient Air Quality Standard
CFR: Code of Federal Regulations
CMBR: Completely Mixed Batch Reactor
CMFR: Completely Mixed Flow Reactor
COD: Chemical Oxygen Demand
cREF: chronic Reference Exposure Level (OEHHA)
CWA: Clean Water Act
DO: Dissolved Oxygen
EGSB: Expanded Granular Sludge Bed
EPA: Environmental Protection Agency (U.S.)
FID: Flame Ionization Detector
FBR: Fluidized Bed Reactor
GAC: Granular Activated Carbon
GAR: Gas Lift Anaerobic Reactor
GSB: Green Sulfur Bacteria
HAP: Hazardous Air Pollutant
HRT: Hydraulic Retention Time
MCL: Maximum Contamination Level
MGD: Million Gallons per Day
MOS: Margin of Safety
MPB: Methane-Producing Bacteria
NAD
+
: Nicotinamide Adenine Dinucleotide
NPDES: National Pollutant Discharge Elimination System
NTC: Numerical Target Concentration
OEHHA: Office of Environmental Health Hazard Assessment
ORP: Oxidation Reduction Potential
POTW: Publicly Owned Treatment Works
REL: Reference Exposure Level
RF: Radio Frequency
RfC: Reference Concentration (U.S. EPA)
RO: Reverse Osmosis
SEM: Scanning Electron Microscopy
SRB: Sulfate-Reducing Bacteria
SS: Suspended Solids
TCA: Tricarboxylic Acid
xvi

TDS: Total Dissolved Solids
TMDL: Total Maximum Daily Load
UAFF: Up-Flow Anaerobic Fixed Film
UASB: Up-Flow Anaerobic Sludge Blanket
VFA: Volatile Fatty Acids
VOC: Volatile Organic Carbon
VSS: Volatile Suspended Solids
WRP: Water Reclamation Plant
XRD: X-ray Diffraction
xvii

ABSTRACT

High-sulfate wastewaters are a major problem in industry because they
increase the total dissolved solid content and interfere with methanogenesis, resulting
in a decrease in the production of methane, which is a valuable fuel. A large variety
of industries, including pulp and paper production, molasses fermentation, seafood
processing, potato-starch factories, and tanneries, that produce wastewaters with a
high sulfate concentration, have major problems in discharging their wastewaters.
The reason for this is that the discharge of industrial wastes into water bodies is
governed by National Pollutant Discharge Elimination System (NPDES) program,
which limits the amount of pollutants, especially chemical oxygen demand (COD),
received by the surface waters. Unfortunately, high sulfate content in the wastewater
limits the usage of anaerobic methanogenesis for COD reduction.
Biological sulfate reduction is an effective means of removing sulfate from
wastewater. Sulfate-reducing bacteria can adjust effectively to different
environments, and the production of biofilm protects the bacteria from the toxic
environment. The ability of the bacteria to acclimate to different pH levels, along
with the possibility of toxic metal precipitation by hydrogen sulfide, have made this
method a very attractive treatment alternative for wastewaters containing heavy
metals.
This research has investigated the effectiveness of biological reduction in the
removal of high concentrations of sulfate from wastewater. An anaerobic fluidized
xviii

bed reactor (FBR) with recycle was chosen, and thermodynamic and kinetic
parameters were used to evaluate best electron donors. Completely mixed batch
reactor (CMBR) studies with different electron donors were conducted to investigate
the feasibility of biological reduction with each electron donor. In addition, the
effects of pH, temperature and carbon to sulfur ratio on sulfate reduction have been
evaluated in several CMBRs. The results of the batch biokinetic studies rationalized
directly to the fluidized bed bioreactor studies to perform the biological sulfate
reduction from wastewater with a high level (2000 mg/L) of sulfate.
High sulfate removal efficiencies, as high as 96%, were observed, without any
inhibition by produced H
2
S. Production of hydrogen fuel from the by-products of the
experiment is proposed as a promising technology. Anaerobic biofilters have been
introduced for effective removal of H
2
S as well as an effective alternative for
producing elemental sulfur from the produced H
2
S. Finally, the FBR systems not
only mange to remove sulfate with very high efficiencies, but this method can have a
significant financial return from production of valuable products including methane,
hydrogen fuel, and elemental sulfur from the by-products of the system.
1

Chapter 1

INTRODUCTION

Sulfate naturally exists in soil and rocks and it can easily dissolve in water
and find its way into surface water or groundwater. Sulfate is a secondary pollutant
in the atmosphere. Combustion of fossil fuels and emitted gases from most industries
release large quantities of sulfur dioxide into the atmosphere. Sulfur dioxide can
follow different atmospheric pathways and oxidize to sulfate which has a high
solubility in water. Sulfate can dissolve in raindrops and be deposited along with
them (wet deposition), or it can precipitate in dry form. In both cases, the result is an
increase in concentration of sulfate, which will eventually find its way into water
bodies. Sulfate is the oxidized form of sulfur; therefore, volcanic activity, biological
degradation, and minerals that release sulfur can be other major sources of sulfate.
Runoff from fertilized agricultural lands also contains high concentrations of sulfate
(Cooney et al., 1996).
Sulfate in drinking water currently has a secondary maximum contaminant
level of 250 mg/L, based on aesthetic effects (taste and odor). Sulfate has a laxative
effect, in which is of more concern for sensitive groups of the population and infants.
Tourists are also more sensitive to high concentrations of sulfate. Discharges of
industrial wastes into water bodies are governed by the National Pollutant Discharge
Elimination System (NPDES) program, which limits the amount of chemical oxygen
demand (COD) discharged into surface water. Industries with high sulfate
2

concentrations in their wastewaters must follow the best available technologies to
reduce their COD discharge into water bodies. High sulfate wastewaters present a
major problem for industries because high sulfate content limits the usage of
anaerobic methanogenesis for production of methane gas, a valuable fuel for power
generation. The use of anaerobic methanogenesis becomes highly limited when
hydrogen sulfide is produced in the system (Mizuno et al., 1994).
High sulfate concentration can increase the salt content in the receiving water
and therefore disturb the ecology of the receiving ecosystem. In addition, high
sulfate concentration can unbalance the natural sulfur cycle by altering the
biodegradation pathways and decomposition rates (Lens et al., 1998).
High sulfate wastewaters are major problem in many industries. Chemical
and biological oxidation of sulfidic ores during and after mining activity can produce
wastewaters with high concentrations of sulfate. Industrial wastewaters from pulp
and paper production, molasses fermentation, seafood processing, potato-starch
factories, tanneries, edible oil refineries, pharmaceutical production, petrochemical
processing, and wine distilleries contain high concentrations of sulfate, and therefore
discharge of these wastewaters is problematic (Lens et al., 1998).
This research is organized into six chapters. Chapter 1 is an introduction to
sulfate-rich wastewaters and biological methods. Chapter 2 discusses the background
for wastewaters with high sulfate concentration, the problems associated with these
wastewaters, and the use of anaerobic biological fluidized bed reactor (FBR) as a
viable technology for sulfate reduction. The research objectives and scopes are
3

presented in Chapter 3, and Chapter 4 explains the materials and methods used in
this study. Chapter 5 discusses the results, and finally Chapter 6 presents the
conclusions and recommendations.
4

Chapter 2

BACKGROUND

2.1 Sulfur Cycle
Sulfur is the tenth most abundant element in the earth’s surface, and it has an
essential role in biological reactions. Sulfur is required by organisms for synthesis of
the amino acids cysteine and methionine, and it is necessary for synthesis of
vitamins, hormones, and coenzymes. Sulfur follows complex chemical pathways,
and it affects the cycling of other elements (Maier et al., 2000). Sulfur enters the
atmosphere through volcanic activity, deep-sea venting, release by bacteria in
marshes and wetlands, industrial emissions, soil dust, and production of aerosols at
the surface of the ocean. In the atmosphere, sulfur oxidizes and returns to the earth
by wet or dry deposition (Ricklefs and Miller, 2000).
Sulfate-reducing bacteria (SRBs) fall into that part of the sulfur cycle that
uses the sulfate but does not necessarily incorporate it into the cell. The pathway of
SRBs in the sulfur cycle includes dissimilatory sulfate reduction, as shown in Figure
2-1. Genera of Desulfobacter, Desulfobulbus, Desulfococcus, Desulfonema,
Desulfocarcina, Desulfotomaculum, and Desulfovibrio can use sulfate as the terminal
electron acceptor. SRBs are mostly chemoheterotrophs that can utilize low-
molecular-weight compounds such as acetate, lactate, and methanol as their electron
donors (Maier et al., 2000). In this research, dissimilatory sulfate reduction was used
to reduce the sulfate content in the high-sulfate-content wastewaters.
5

Assimilatory sulfate reduction occurs in plants and most microorganisms,
which incorporate sulfide into amino acids or other sulfur-containing molecules.
Assimilatory sulfate reduction is an energy requiring reduction.
The release of sulfur from its organic form is called sulfur mineralization, and
it occurs under both aerobic and anaerobic conditions.

Figure 2-1: A Simplified Sulfur Cycle
(Adapted from Robertson and Kuenen, 1991)

Biological oxidation
with O
2
or NO
3
-
Mineralization
processes
SO
4
2- S
2-
Organic
Sulfur
Compounds
S
Sulfate
Reserves
(Seawater)
Sulfidic
Minerals
(e.g. pyrite)
Sulfur
Deposits
Assimilatory
sulfate
reduction
Dissimilatory
sulfate
reduction
Biological
oxidation with
O
2
or NO
3
-
Anaerobic
oxidation by
phototrophic
bacteria
Dissimilatory
sulfur reduction
Spontaneous
oxidation
Biological
oxidation with
O
2
or NO
3
-
Anaerobic
oxidation by
phototrophic
bacteria
6

Sulfur oxidation can happen by a group of chemoautotrophic bacteria under
strictly aerobic conditions. In addition, Thiobacillus denitrificans, which is a
facultative anaerobic organism, can use nitrate as the terminal electron acceptor, and
oxidize sulfur into sulfate, as shown in Reaction 2-1.
2 4 3 3
0
N CaSO CaCO NO S + → + +
−
(2-1)
A number of aerobic heterotrophic microbes, including bacteria and fungi,
can also oxidize sulfur to thiosulfate or sulfate. Sulfur oxidation can also happen by a
group of phototrophic bacteria under strictly anaerobic conditions (Maier et al.,
2000). These phototrophic bacteria fix carbon dioxide by utilizing light energy and
oxidize sulfide to sulfur, as presented in Reaction 2-2.
+ → +
0
2 2
S S H CO Fixed carbon (2-2)

Note that in this research, the phototrophic bacteria were used in the biofilters
to remove hydrogen sulfide from the FBRs off-gases.
Discharge of high concentrations of sulfate into water bodies can unbalance
the natural sulfur cycle. Different oxidized forms of sulfur (sulfate, sulfite, or
thiosulfate) can be present at the same time in the wastewater, and each can undergo
several reduction paths with different available organic sources. These organic
sources are intermediates of the mineralization process. Differences in each
reaction’s free energy, along with the variety of the SRBs, lead to complete or partial
dissociation of the organic carbon sources into CO
2
, and complete or partial
reduction of sulfate, sulfite, or thiosulfate. In addition, the amount of available
7

sulfate could affect the methane producing bacteria (MPB) or sulfate reducing
bacteria (SRB) reactions in the natural system (Lens et al., 1998).

2.2 Sulfate Sources
Sulfate exists naturally in the environment as insoluble salts (e.g., gypsum) or
in dissolved form in water bodies. Percolated rainfall can dissolve the sulfate salts
and increase the sulfate concentration in the groundwater, where under anaerobic
conditions sulfate can be biologically changed into sulfide.
Water bodies can receive high concentrations of sulfate through wastewater
discharges from industries or by acid deposition of sulfur compounds from the
atmosphere. Kettuen and Rintala (1995) reported that leachate from oil shale
contains high concentrations of sulfate. Studies by Nedwell and Reynolds (1996)
showed that leaching from landfills also produces wastewaters with a high
concentration of sulfate. Similarly, bioleaching from solid wastes can produce
leachate with very high concentrations of sulfate (Tichy et al., 1998).
High-sulfate wastewaters, with sulfate in the range of 1.5 to 50 g/L, are a
major problem in industry because they increase the total dissolved solids (TDS) in
receiving water bodies (discharge limit is between 600 and 1000 mg/L). Table 2-1
lists some of the major industries with high-sulfate wastewaters. High sulfate
concentration can unbalance the natural sulfur cycle by altering the biodegradation
pathways and kinetic rates (Lens et al., 1998). When methanogenesis is the
technology used for COD reduction from wastewater, sulfate reduction produces
hydrogen sulfide, which in turn interferes with methanogenesis (Speece, 1996).
8

In addition, production of hydrogen sulfide in wastewater treatment plants
causes severe corrosion in pipes and other fixtures used in the transport line. In cases
where reverse osmosis is used for brine treatment, high concentrations of sulfate will
cause membrane fouling and will increase the frequency of backwashes. In turn, an
increase in the number of backwashes will decrease the recovery and increase the
volume of the brine water (Williams et al., 2002).
Textile processing is another major source of high-sulfate-content
wastewaters (2690 mg/L SO
4
2-
). Its effluent has a wide spectrum of sulfur
compounds (S
2-
to SO
4
2-
) (Kabdasli et al., 1995).
The electroplating industry also produces high-sulfate wastewaters. The basic
constituent for Watts-nickel-plating solution is nickel sulfate, which has sulfate
concentrations of 195-262 g/L (EPA, 2003). This concentration range produces
wastewaters with an average sulfate concentration of 2000 mg/L (Song et al., 1998).
In addition, in the production of sulfur-containing xenobiotics, sulfate, or
sulfite is usually used to add functional groups of sulfonate or sulfate to an aromatic
or aliphatic compound (alkyl sulfate detergents). As a result, the effluent contains
high concentrations of sulfur compounds, which can easily biodegrade to produce
sulfate (Lens et al., 1998). Table 2-1 lists some of the major industries with high
sulfate wastewaters.
Serious consideration has been given to membrane technology for
reclamation and reuse of agricultural drainage water for two reasons: first,
9

reclaiming the water reduces the quantity of imported water, and second, the volume
of drainage water will significantly decline.

Table 2-1: Sulfate Concentrations in Wastewaters from Different Industries
Industry Sulfate Concentration in
Wastewater (g/L)
Source
Mining 1.9- 2.1 Glombitza (2001)
Alcohol Production 2.9 Lens et al. (1998)
Citric Acid Production 2.5-4.5 Colleran et al. (1995)
Pulp and Paper 1-2 Colleran et al. (1995)
Textile Processing 2.7 Kabdasli et al. (1995)
Fatty Acid Production 40-50 Colleran et al. (1995)
Seafood Processing 2.1- 2.7 Lens et al. (1998)
Tannery 2- 3 Galiana-Aleixandre et al. (2005)
TNT Manufacturing 5.4 Lens et al. (1998)

High levels of calcium sulfate and bicarbonate ions in agricultural drainage
water can cause precipitation in the membrane; therefore, control of calcium sulfate
is a major challenge in the development of membrane processes for desalination
(Yann et al., 2002).

2.2.1 The California Water Quality-Based Approach to Pollution
The water quality-based approach to pollution protects the overall quality of
water bodies and controls the amount of pollution entering a water body based on the
real conditions of that body of water and the standards that protect it. The steps that
usually are taken for this approach are:
10

- Identification of the water bodies that, after implementation of the
technology-based controls, do not meet the quality standards. These water
quality-limited waters still need total maximum daily load (TMDL)
controls
- Ranking and prioritizing of the water quality-limited waters
- Development of TMDLs under section 303 (d) of the 2002 Clean Water
Act (CWA)
- Implementation of the control actions
- Assessment and evaluation of the water quality-based control actions
Based on the 2002 CWA 303 (d) list for impaired waters, several water
bodies must be controlled for sulfate. Some of these water bodies in the Los Angeles
area are listed in Table 2-2 (2002 CWA Section 303 (d), Los Angeles Regional
Water Quality Control Board).
To regulate the amount of pollutant discharge, each facility must obtain a
National Pollution Discharge Elimination System (NPDES) permit. The goal is to
control and eliminate pollutants that are harmful to receiving waters. Industrial
discharges will be controlled for priority toxic pollutant discharge by obtaining an
NPDES permit. The NPDES program applies to point sources, which can be an
industry or publicly owned treatment works (POTW) that need to directly discharge
into a water body. The NPDES permit, once issued, includes effluent limitations for
pollutants that have the potential to exceed a water quality standard and affect the
beneficial use of the water body.
11

Table 2-2: 2002 CWA Section 303 (d) List of Water Quality Limited Segment in Los
Angeles Area
Region Type Name Cal. Water
Watershed
Pollutant/
Stressor
Pollutant
Source
TMDL
Priority
Estimated
Size
Affected
4 R Calleguas
Creek
Reach 4
40311000 Sulfates Non-point
Source
Medium 7.2 Miles
4 R Calleguas
Creek
Reach 6
40362000 Sulfates Non-point
/Point
Source
High 15 Miles
4 R Calleguas
Creek
Reach 7
40367000 Sulfates Non-point
Source
High 14 Miles
4 R Calleguas
Creek
Reach 8
40366000 Sulfates Non-point
/Point
Source
High 7.2 Miles
4 R Calleguas
Creek
Reach
9A
40312000 Sulfates Non-point
/Point
Source
High 1.7 Miles
4 R Calleguas
Creek
Reach 9B
40363000 Sulfates Non-point
/Point
Source
High 6.2 Miles
4 R Calleguas
Creek
Reach 10
40364000 Sulfates Non-point
/Point
Source
High 3 Miles
4 R Calleguas
Creek
Reach 11
40365000 Sulfates Non-point
/Point
Source
High 8.7 Miles
4 R Calleguas
Creek
Reach 12
40364000 Sulfates Non-point
/Point
Source
High 5.5 Miles
4 R Calleguas
Creek
Reach 13
40368000 Sulfates Non-point
/Point
Source
High 17 Miles
4 R Fox
Barranca
40362000 Sulfates Non-point
Source
High 6.7 Miles
4 R Hopper
Creek
40341000 Sulfates Non-point
/Point
Source
Low 13 Miles
4 R Wheeler
Canyon/
Todd
Barranca
40321000 Sulfates Non-point
Source
Low 10 Miles
4 R Pole
Creek
40331000 Sulfates Non-point
Source
Low 9 Miles
R: Rivers and Streams, 4: Los Angeles Area
Source: USEPA

12

Beneficial uses include municipal and domestic water supply, irrigation and
stock watering agricultural supply, industrial process and service supply, contact and
non-contact water recreation, freshwater habitat for both warm and cold water
species, migration waters for both warm and cold water species, spawning for warm
and cold water species, wildlife habitat, and navigation (California Water Resource
Board, personal communication, 2007).
Marine and estuarine water protection happens through the U.S.
Environmental Protection Agency’s (EPA’s) National Coastal and Marine Policy,
issued in January 1990. Through that EPA’s goals are established as:
- To recover full use of the nation’s shores, beaches, and water
- To restore the nation’s shell fisheries and saltwater fisheries
- To minimize the use of coastal and marine water for waste disposal
- To improve and expand coastal science
- To support international efforts to protect coastal and marine resources
EPA’s program for protection of oceans, coastal waters, and great lakes from
nutrient and toxic pollutants follows the Clean Water Act and Marine Protection,
Research, and Sanctuaries Act (Ocean Dumping Act). Total maximum daily load can
be used to manage the marine and estuarine waters. TMDLs for sulfate in Los
Angeles area is under approval.
The Clean Water Act (40 CFR 130.2 and 130.7) and EPA guidance (1991)
defined TMDLs as the “sum of the individual waste load allocations for point
sources and load allocations for non-point sources and natural background” in a way
13

that the loading capacity of the water body is not exceeded. For this reason, TMDLs
should account for seasonal variations and must include a margin of safety. States
are responsible to submit TMDLs to the EPA for approval. If the EPA disapproves a
TMDL submitted by a state, then the EPA should establish a TMDL for that water
body.

A TMDL should contain these elements:
Explanation of the environmental problem, and beneficial uses
Presentation of the proper numerical target to attain the water quality
An inventory of the sources for concerned pollutant
A link between the pollutant input and the environmental response
Identification of the TMDL allocations for each source
Description of the margin of safety and its basis
Estimation of the economic and population growth and their effects on water
quality
The implementation and monitoring strategies of the TMDL

TMDL can be calculated by using Equation 2-3:

TMDL = LC = Q × NTC × MOS × F (2-3)

14

where
LC: Loading capacity (lbs/day)
Q: In-stream flow at critical condition (MGD)
NTC: Numerical target concentration (mg/L)
MOS: Margin of safety
F: Conversion factor (8.34 (lb/day)/ (MGD-mg/L)

The standard for the water body controls the TMDL. For example, E.V.
Spence Reservoir has a standard of 450 mg/L for sulfate (TNRCC, 2000). The total
load allocation for Wilson Slough, part of the lower Arkansas basin, is 450 mg/L,
according to the EPA TDML. TMDL for Cross Bayou, part of the Red River Basin
in Louisiana is 12.86 tons/day for sulfate (FTN Associates Ltd., 2006).

Table 2-3 presents an example of sulfate loads used for calculation of sulfate
TMDL for Calleguas Creek Watershed (Larry Walker Associates, 2006). Different
point and non-point sources were considered to assess the total load of sulfate in the
Calleguas Creek. These data, with consideration of the 250-mg/L sulfate standard
along with the safety factor, are used to calculate the TMDL for sulfate.

15
Table 2-3: An Example Sulfate Loads Analysis for Calculation of Sulfate TMDL
Water Source
Sulfate Load from
Water Supply (lb/day)
Urban Wastewater
Sulfate Load (lb/day)
POTW Salt Load*
Sulfate Load
(lb/day)
Dry Weather Urban
Load from Sub-
watershed
Sulfate Load
(lb/day)
Sulfate Dry Weather
Agricultural Loads
from Sub-watershed
(lb/day)
Sulfate Load from Wet
and Dry Deposition
(lb/day)
Sulfate Load from Base
Flow (exfiltration) from
Sub-watershed (lb/day)
Sulfate Load from Simi
Sub-watershed
Ground-water
Pumping (lb/day)
State Water
Project
53075.76 Res.
use
4809 SVWWTP 1717
5
Simi 695 3913 90 10400 29939
Santa Clara
River

475.38 Co
m.
use
5379 Hill
Canyon
WWTP
1172
9
Las Posas 63 12880
Groundwater
from
Las Posas
Basin
32876.28 Ind.
use
4217 CSD WRP 6569 Conejo 630 4540 9559
Groundwater
from
Pleasant Valley
Basin
33593.52 ---- ----- Camarillo 29 362
Total Load 161829.36
(=161000)
1b/day
14405
lb/day
Pleasant
Valley
(Calleguas)
60 1864 24399
Revolon 126 44199
Total Load 161829.36
(=161000)
1b/day
14405
lb/day

1603 67758 90 44358 29939
Total Load= 319153 lb/day

*SVWWTP: San Vicente Wastewater Treatment Plant; WWTP: Wastewater Treatment Plant; WRP: Water Reclamation Plant; Res.: Residential; Com:
Commercial; Ind.: Industrial (Adapted from Larry Walker Associates, 2006)

16
2.3 Rationale behind Choosing Biological Methods
Several chemical and physical processes can be used to remove sulfate from
water and wastewater, including evaporation, chemical precipitation, crystallization,
and ion exchange (Williams et al., 2002). In evaporation and crystallization
processes, high amounts of energy are needed for heating. In chemical precipitation
and ion exchange, a large amount of chemicals must be used and large volumes of
sludge must be treated. High initial and operational costs are the problem with the
more advanced methods including membrane treatment. For example, reverse
osmosis membranes are subjected to severe fouling in such applications.
Biological sulfate reduction is an effective means of removing sulfate from
water or wastewater. Sulfate-reducing bacteria can adjust very well to different
environments, and production of the biofilm can isolate the bacteria from the toxic
environments, therefore making sulfate reduction possible even in unsuitable
conditions. Furthermore, the ability of bacteria to acclimate to different pHs, along
with the possibility of toxic metal precipitation by hydrogen sulfide, make this
method a good choice for treating wastewaters containing heavy metals (Steed et al.,
2000).
Anaerobic processes have lower biomass yield in comparison to aerobic
processes; therefore, they produce less sludge, which makes anaerobic processes
more cost effective (Rittmann and McCarty, 2001). Anaerobic reduction of sulfate
can be accomplished in ambient temperature without substantial need for heating;
therefore, it has lower energy consumption, which decreases operation cost.

17
Lower energy consumption, low sludge production, and strong adaptation
capabilities make anaerobic biological sulfate reduction a good choice for high
sulfate removal from wastewaters.
Different types of reactors have been tested for biological reduction of
sulfate. Table 2-4 presents a list of reactors used for high sulfate reduction. The
results indicate that the FBR systems achieved the highest sulfate reductions.

Table 2-4: Reported Reactor Types, Carbon and Electron Sources, HRTs, and
Reduction Rates for Anaerobic Sulfate Reduction Process.
Reactor
Type
a

Carbon and
Electron Source
Sulfate
Concentration
HRT Reduction Reference
FBR Ethanol,
Propionate
160-1200 mg/L 4.2 hr 85% COD Zitomer et al. (2000)
GAR CO
2
gas (+ H
2
) 0.035-0.05 M
(3.36-4.8 g/L)
Variable 80-%100 Van Houten et al.
(1994)
CMFR Glucose, Sodium
acetate
1-10 kg/m
3

(1-10 g/L)
1-10 day 60-82% Moosa et al. (2002)
EGSB Methanol 4.318 g/L
2.75 g/L
10 hr
3 hr
55%
50%
Weijma et al. (2000)
UASB Acetic acid 5-5.2 g/L 8.4 days 75% Steed et al. (2000)
UAFF

Acetate 25 g/L 20 hr 14% El Bayoumy et al.
(1999)
a.
Refer to List of Acronyms
Because anaerobic sulfate reduction is a very slow reaction compared to
aerobic reaction, design of the system must improve this deficiency (Rittmann and
McCarty, 2001). Use of solid supports to facilitate the development of biomass and
biofilm is of major importance. In addition, design of a robust system to develop
biomass with high settling ability and high density is very important in producing a

18
clean effluent. A high sheer configuration will also break the flocs, which require
weeks to develop; therefore the design must provide an even flow with minimum of
sheer to provide good settleable flocs. An FBR with solid packing is one of the
systems that can very well respond to these problems.

2.4 Problems Associated with Anaerobic Treatment of High-Sulfate
Wastewaters
Sulfate is the most oxidized and therefore the most stable form of sulfur in
contact with the atmosphere. Generally, it is considered as non-toxic, but at very
high concentrations (higher than 10 g/L); it has an inhibitory effect on
methanogenesis. Anaerobic sulfate reduction can also produce sulfide, which is the
most reductive form of sulfur and the most reactive form of it. Hydrogen sulfide
(H
2
S) is highly toxic to microorganisms, plants, and animals. It is fatally toxic to
humans, causing death within 30 minutes at gaseous concentrations of 800-1000
ppm (Speece, 1996). Higher concentrations are instantly fatal, and death occurs even
faster than for exposure to hydrogen cyanide. Hydrogen sulfide also paralyzes the
respiratory center and at high concentration paralyzes the olfactory nerves (Speece,
1996). In low concentrations, it has an inhibitory effect on methanogenesis.
Consequently, the methane-producing bacteria (MPB) cannot utilize the wastewater
COD, resulting in high COD in effluent, and therefore less methane production.
Sulfide is also very corrosive and can cause crown corrosion in pipes.

19
2.5 Reasons for the Choice of FBR with Recycle and GAC as the Solid Support
The FBR can achieve superior performance in comparison to completely
mixed and fixed-bed reactors because the biofilm is evenly distributed throughout
the reactor while the liquid regime has plug-flow characteristics (Rittmann, 2004).
FBR can maximize pollutant removal and minimize sludge production (Rodgers et
al., 2003). In FBRs, the media provides a large surface for biological attachment and
growth, so the biomass concentration is very high. This large concentration of
biomass results in a smaller reactor volume. In addition, an FBR can have a
relatively high loading rate and small reactor size; therefore, its application is more
common in wastewater treatment (Zitomer et al., 2000); Nagpal et al. (2000 a) used
FBR with low liquid recirculation flows and could achieve sulfate reduction rates up
to 6.33 g sulfate/L-day at a hydraulic retention time (HRT) of 5 hours. Yoda et al.
(1987) studied the competition between MPBs and SRBs for acetate in the biofilm
using FBR. They reported that at low organic flow rates, SRBs could out-compete
the MPBs.
The advantages of the FBR process, as explained by Iza et al. (1991) are:
It can provide high concentrations of biomass, which are attached to the solid
support and cannot be easily washed out from the reactor.
It provides a very large surface area for biomass attachment.
Dilution also reduces the substrate concentration (important for high COD
wastes) and reduces the shock effect of toxic wastewaters.
It has high mass transfer properties (high solid retention time).

20
Fluidization prevents plugging, channeling, or gas hold-up (expansion of the
bed keeps the solid packing in suspension; therefore, clogging is not a
problem, and the uniform flow prevent channeling or gas hold-up).
It has the ability to control and optimize biofilm thickness.
Biomass support can be tailored to a specific application to enhance
performance.

A number of investigators have used FBR for methanogenic treatment of
wastewater due to the possible high loading rate, and the smaller required reactor
size (Heijnen, 1988). Zitomer et al. (2000) used FBR for treatment of high COD and
high sulfate wastewater using aerated methanogenesis. Polanco et al. (2001) used
granular activated carbon FBR-GAC for simultaneous anaerobic removal of organic
nitrogen and low concentrations of sulfate. Other researchers used anaerobic fixed-
bed reactors and packed bed reactors (Dvorak et al., 1992; Steed et al., 2000;
Muthumbi et al., 2001), but they faced clogging and channeling problems, especially
in packed columns (Kolmert and Johnson, 2001). The effects of hydraulic retention
time and sulfide toxicity on acidic metal-containing wastewater have also been
studied by Kaksonen et al. (2004) in a sulfate- reducing FBR.
In this research, the FBR with GAC as the solid support were used to
evaluate the highest attainable sulfate removal from a high-sulfate-content
wastewater. Solid support for biological reduction must (1) withstand physical
abrasion caused by the fluidization, (2) provide a large surface area for bacterial

21
growth, (3) minimize the required velocity for fluidization, (4) enhance the diffusion
and mass transfer, and (5) provide a shielded irregular surface for biomass protection
(Speece, 1996). In addition, the desirable adsorption properties of GAC can help
reduce high concentrations of toxic compounds that could otherwise be toxic to
bacteria. This, in turn, will aid further growth of the biofilm and its ability to
synthesize the enzymes that would be helpful in stabilizing the toxic compounds.
Furthermore, GAC adsorption capacity helps to retain the inhibitory or less
biodegradable compounds, and therefore gives more time to bacteria to biodegrade
the less biodegradable compounds (Speece, 1996). Hydrogen sulfide controlling
methods are discussed in Sections 2.11 and 2.12.

2.6 Feasibility Studies and Laboratory-Scale Studies in Biological Sulfate
Reduction
Feasibility of biological sulfate reduction relies on the possibility of the
biological process and the practicality of the method. In order to make a decision
regarding the possibility of the reaction, the thermodynamics of the reactions and the
possible efficiencies, along with the inhibition effect of the products, and the
production of the by-products must be considered as first step. The thermodynamics
of the possible electron donors are discussed in Section 2.10, indicating that lactate,
acetate, and hydrogen gas produce more energy during sulfate reduction. Production
of water and hydrogen sulfide as the only products from utilizing hydrogen as
electron donor make that electron donor a good candidate for future study, whereas

22
production of acetate puts a constraint on considering other electron donors, such as
ethanol and malate.
The practicality of biological sulfate reduction, on the other hand, relies on
the cost of the electron donor needed for sulfate reduction during the anaerobic
process, and the process products. The formation of undesirable by-products such as
acetate, which could decrease the removal efficiency by decreasing the pH and
accumulation, needs to be minimized. Utilization of different forms of electron
donors, including organic wastes from sewage sludge, wastewater from breweries,
dairy whey, molasses, chemical gases such as hydrogen or a mixture of hydrogen
and CO
2
, and pure chemicals such as lactate and acetate, must be analyzed with
regard to applicability.
Organic wastes have the advantage of low cost, but the complex composition
of these wastes makes the prediction of system behavior and control of the system
very difficult. In addition, production of intermediates during degradation of organic
wastes could promote the undesirable growth of methanogens (Goorissen, 2002).
Utilization of pure chemicals provides better control of the system, and study
of the feasibility and kinetics of the sulfate reduction is possible by using pure
chemicals, but the price for these chemicals must be considered.
Anaerobic sulfate reduction is a cost-effective method because it has a lower
biomass synthesis rate (5-20% of the aerobic system), and consequently produces
less sludge. In addition, lower nutrient requirements (5-20% of the aerobic system)
in anaerobic processes result in less chemical usage, which makes this method more

23
cost efficient. Biomass resilience for months or years enables the system to deal with
unfavorable conditions because bacteria can very well tolerate the shock in the
system (Speece, 1996).
All of these benefits are attainable with lower initial and maintenance costs.
Overall, the feasibility of the biological sulfate reduction process is due to a number
of factors, including energetic of the electron donor, price of the used chemicals, rate
of sulfate and electron donor utilizations, inhibitory effect of products, price and
maintenance of the system, and treatment of the process by-products. A clear
understanding of these factors is obtainable by considering the effect of influencing
environmental parameters, including temperature, electron donor, and electron
acceptor. These parameters can be evaluated by conducting completely mixed batch
reactor (CMBR) studies. Table 2-5 presents a summary of the energetic data, and the
growth yield for sulfate reducing bacteria utilizing different electron donors.

Table 2-5: Energetic Data and Growth Yield for SRB Utilizing Different Substrates
Electron
Donor
Δ Δ Δ ΔG, Kcal/mole
(electron donor)
Biomass Yield
GDW/mole (electron donor)
References
H
2
-9.10 3.05 Badziong and Thauer
(1978)
Acetate -11.35 4.75- 4.8 Widdle et al. (1981)
Ethanol -15.91 0.75- 0.93 Nagpal et al. (2000 b)
Propionate -9.00 4.3-5.5 Widdle et al. (1982)
Lactate -18.87 6.96 Cooney et al. 1996
Lactate -18.87 2.66-2.84 Nagpal et al. (2000 b)

24

In this research, the kinetics of anaerobic sulfate reduction was studied by
performing CMBR studies with different electron donors. The results of these
CMBR studies were used in the design of laboratory- scale FBR experiments.

2.7 Mechanism of Sulfate Reduction
Sulfate is abundant in soil, and plants and microorganisms can directly take
up the sulfate for production of amino acids (e.g. cysteine), and other sulfur
containing chemicals, a process called assimilatory sulfate reduction.
Sulfate reduction in SRBs follows dissimilatory sulfate reduction, and in spite
of their name, SRBs cannot reduce sulfate. In fact, sulfate must be activated by
adenosine triphosphate (ATP). After sulfate is transported into the cell, ATP is used
to convert the sulfate into the adenosine 5’phosphosulfate (APS). The reaction of
ATP sulfurylase is not favorable toward formation of APS; therefore, the reaction is
pulled to the right by an inorganic pyrophosphatase, which hydrolyses the
pyrophosphate to phosphate. This enzyme in SRBs can be activated by a reducing
agent. The next reaction is the reduction of adenosine phosphate by the APS-
reductase enzyme, which reduces APS to sulfite and adenosine monophosphate
(AMP). The sulfite reductase system, or the enzyme complex which transfers sulfite
to sulfide, is often associated with soluble enzymes. Different mechanisms, including
the cyclic reaction mechanism, have been suggested (Postgate, 1984; Garrett and
Grisham, 2005). Reactions 2-4 to 2-8 present the suggested mechanism for
dissimilatory sulfate reduction.

25

Sulfate (outside the cell)     → 
transport active
Sulfate (inside the cell) (2-4)

ATP + sulfate     → 
e sulfurylas ATP
APS + PP
i
ΔG
0
= +46 KJ/mole

(2-5)
adenosine phosphosulfate pyrophosphate

PP
i
+H
2
O     → 
atase Pyrophosph
2 P
i
ΔG
0
= -22 KJ/mole (2-6)

APS+ 2e     → ←
−reductase APS
AMP + SO
3
2-
ΔG
0
= -69 KJ/mole (2-7)

0.5 HSO
3
-
+ 0.5 SO
3
2-
+ 3 H
2
+H
+
    → 
reductase sulfite
0.5 HS
-
+ 0.5 H
2
S+ 3H
2
O
ΔG
0
= -174 KJ/mole (2-8)
The fate of the sulfate in the biological sulfate reduction is governed mostly
by dissimilatory sulfate reduction, and it can happen autotrophically by using
hydrogen as the energy source, or heterotrophically by using low molecular weight
carbon sources.

2.8 Selection of an Electron Donor for Biological Sulfate Reduction
Selection of an electron donor must be based on a complete knowledge of the
favorable biological reaction and the needs of the system. The following parameters
must be considered in choosing the right electron donor:
Price and availability
Safety based on the usage of the system effluent (for example, toxic chemicals
cannot be used if the goal is to remove sulfate from drinking water)
Reaction products (whether the products are safe—if not, they need to be
removed)

26
Effective use of electron donor by bacteria
Thermodynamic considerations (whether the reaction has enough energy to
go)Kinetic considerations (how fast the reaction goes, whether it has a direct
impact on reactor size)
In this research, all the above-mentioned parameters were considered in order to
provide the best electron donor for biological sulfate reduction from high sulfate
containing wastewaters. Table 2-6 presents the characteristics of different genera of
SRBs. Each genus is capable of utilizing specific electron donors, and each can use
specific forms of sulfur oxides as electron acceptors.

Table 2-6: Representative Genera of Sulfate- and Sulfur-Reducing Bacteria
Genus Cell shape Electron Donor Electron Acceptor
Desulfovibrio Vibrio Lactate, ethanol SO
4
2-
, SO
3
2-
, S
2
O
3
2-

Desulfococcus Coccus Lactate, ethanol SO
4
2-
, SO
3
2-
, S
2
O
3
2-

Desulfosarcina Ovoid; aggregate Lactate, ethanol SO
4
2-
, SO
3
2-
, S
2
O
3
2-

Desulfobacter Rod; vibrio Acetate SO
4
2-
, SO
3
2-
, S
2
O
3
2-

Desulfobulbus Oval Lactate, acetate SO
4
2-

Desulfomonile Rod Pyruvate, benzoate SO
4
2-
, SO
3
2-
, S
2
O
3
2-

Desulfomicrobium Rod Lactate SO
4
2-
, SO
3
2-
, S
2
O
3
2-

Desulfobotulus Vibrio Longer-chain fatty acids SO
4
2-
, SO
3
2-
, S
2
O
3
2-

Desulfobacterium Rod; vibrio Lactate; acetate, longer-chain
fatty acids
SO
4
2-
, SO
3
2-
, S
2
O
3
2-

Desulfurella Rod Acetate S
0

Desulfuromonas Oval; curved rod Acetate, propionate S
0

Reference: Perry et al. (2002)

27
These data can also be used interchangeably for choosing the right genus to utilize
the sulfate using a specific electron donor, or to help provide the growth condition
for specific bacteria.

2.9 Choice of Electron Donor Based on Free Energy
Bacteria need a carbon and electron donor for cell synthesis. SRBs are
heterotrophs, and they can use simple organic compounds as electron donors.
Different electron donors follow different pathways, and therefore release various
amounts of energy. The most energetic reaction is the most favorable for bacteria. In
addition to a carbon source, bacteria need energy for growth and maintenace. Energy
could be obtained from light (phototrophs) or from a chemical reaction
(chemotrophs). SRBs can use hydrogen or a simple organic molecule as the electron
donor. The electron acceptor is sulfate, sulfite, or thiosulfate (Rittmann and McCarty,
2001, Maier et al., 2000). Figure 2-2 shows the major steps in the anaerobic
degradation of polymeric materials in sulfate-reducing environments. During the first
step, large molecules break down into smaller molecules. Following that, the low
chain, easily available electron donors are used up by the SRBs, converting sulfate to
elemental sulfur, or sulfide during the process.
In this research, the uptake of simple organic molecules such as acetate and
lactate by SRBs were evaluated in order to employ the most efficient electron donor
for biological sulfate reduction from sulfate-rich industrial wastewaters.

28

Figure 2-2: Pathway in Anaerobic Degradation of Polymeric Compounds by SRBs
(Michiko and Zuber, 2004)

2.10 Thermodynamics and Calculation of Energy Yields of Metabolic Processes
Microorganisms, including SRBs, need energy for growth and maintenance,
and they obtain their energy from oxidation-reduction reactions. Although,
phototrophs obtain their energy from sunlight, they use oxidation-reduction reactions
to convert the light energy into ATP and Nicotinamide Adenine Dinucleotide
(NADH). In chemotrophs, oxidation-reduction reactions between the electron donor
Hydrolysis and Fermentation
Fatty Acids
Alcohols
H
2
+ CO
2

Formate
Acetate

Polysaccharides
Proteins
Lipids
SO
4
2-

Sulfate

Reducers
S
0

CO
2

29
and electron acceptor provide the energy needed for catabolism and anabolism. More
energy production relates to more bacterial yield, and thus faster growth rates.
Understanding which bacterial processes predominate under a given set of
circumstances requires knowledge of the energetic of dissimalatory metabolism and
growth, including chemical thermodynamics and kinetic constraints on chemical
reactions. The Gibbs free energy expresses the energetic of the reaction, but the
kinetics is only attainable through CMBR studies.

2.10.1 Comparison of the Reaction Free Energy for Different Electron Donors
Reactions 2-9 to 2-24 provide the energy required from complete dissociation
of the electron donor into carbon dioxide, hydrogen sulfide, and water (Rittmann,
2004). In reality, depending on the classification of the bacteria, the degree of
progress in the reactions is different. Sometimes the reaction is not complete, and the
accumulation of the intermediate compound, along with the inhibitory effect of
hydrogen sulfide, will stop the reaction before it reaches complete dissociation.
Acetate:
1/8 CH
3
COO
-
+ 3/8 H
2
O → 1/8 CO
2
+ 1/8 HCO
3
-
+H
+
+e
-
ΔG
0
= -27.40 KJ/e
-
eq (2-9)
1/8 SO
4
2-
+ 19/16 H
+
+ e
-
→ 1/16 H
2
S + 1/16 HS
-
+ ½ H
2
O ΔG
0
= 20.85 KJ/e
-
eq (2-10)
1/8 CH
3
COO
-
+3/16 H
+
+ 1/8 SO
4
2-
→1/8 CO
2
+ 1/8HCO
3
-
+1/16 H
2
S + 1/16 HS
-
+1/8 H
2
O
ΔG
0
= -6.55 KJ/e
-
eq
2CH
3
COO
-
+3H
+
+ 2SO
4
2-
→ 2CO
2
+ 2HCO
3
-
+H
2
S + HS
-
+2H
2
O (2-11)
ΔG
0
= -52.4 KJ/mole acetate
ΔG
0
= -12.52 Kcal/mole acetate

30
Hydrogen and CO
2
:
½ H
2
→ H
+
+e
-
ΔG
0
= -39.87 KJ/e
-
eq (2-12)
1/8 SO
4
2-
+ 19/16 H
+
+ e
-
→ 1/16 H
2
S + 1/16 HS
-
+ ½ H
2
O ΔG
0
= 20.85 KJ/e
-
eq (2-13)
1/8 SO
4
2-
+ ½ H
2
+3/16 H
+
→1/16 H
2
S + 1/16 HS
-
+ ½ H
2
O ΔG
0
= -19.02 KJ/e
-
eq
2SO
4
2-
+ 8 H
2
+3 H
+
→ H
2
S + HS
-
+ 8 H
2
O ΔG
0
= -38.04 KJ/mole H
2
(2-14)
1/5 CO
2
+ 1/20 NH
4
+
+ 1/20 HCO
3
-
+ H
+
+e
-
→ 1/20 C
5
H
7
O
2
N + 9/20 H
2
O (2-1)

The cell synthesis reaction does not have a ΔG
0
because the reduced species
are not chemically defined (Rittmann and McCarty, 2001).
Glucose:
1/24 C
6
H
12
O
6
+ ¼ H
2
O → 1/4 CO
2
+ H
+
+e
-
ΔG
0
= -41.35 KJ/e
-
eq (2-16)
1/8 SO
4
2-
+ 19/16 H
+
+ e
-
→ 1/16 H
2
S + 1/16 HS
-
+ ½ H
2
O ΔG
0
= 20.85 KJ/e
-
eq (2-17)
1/24 C
6
H
12
O
6
+1/8 SO
4
2-
+3/16 H
+
→1/4 CO
2
+1/16 H
2
S + 1/16 HS
-
+ 1/4 H
2
O
ΔG
0
= -20.5 KJ/e
-
eq
C
6
H
12
O
6
+3 SO
4
2-
+4.5 H
+
→6 CO
2
+1.5 H
2
S + 1.5 HS
-
+ 6 H
2
O ΔG
0
= -492 KJ/mole glucose (2-18)
ΔG
0
= -117.59 Kcal/mole glucose
Ethanol:
1/12 CH
3
CH
2
OH + ¼ H
2
O → 1/6 CO
2
+ H
+
+e
-
ΔG
0
= -31 KJ/e
-
eq (2-19)
1/8 SO
4
2-
+ 19/16 H
+
+ e
-
→ 1/16 H
2
S + 1/16 HS
-
+ ½ H
2
O ΔG
0
= 20.85 KJ/e
-
eq (2-20)
1/12 CH
3
CH
2
OH +1/8 SO
4
2-
+3/16 H
+
→1/6 CO
2
+1/16 H
2
S + 1/16 HS
-
+ 1/4 H
2
O
ΔG
0
= -10.15 KJ/e
-
eq
2CH
3
CH
2
OH +3SO
4
2-
+4.5 H
+
→ 4CO
2
+1.5 H
2
S + 1.5HS
-
+ 6H
2
O (2-21)
ΔG
0
= -121.8 KJ/mol ethanol
ΔG
0
= -29.11 Kcal/mol ethanol

31
Lactate:
1/12 CH
3
CHOHCOO
-
+ 1/3 H
2
O → 1/6 CO
2
+1/12 HCO
3
-
+ H
+
+e
-
ΔG
0
= -32.29 KJ/e
-
eq (2-22)
1/8 SO
4
2-
+ 19/16 H
+
+ e
-
→ 1/16 H
2
S + 1/16 HS
-
+ ½ H
2
O ΔG
0
= 20.85 KJ/e
-
eq (2-23)
1/12 CH
3
CHOHCOO
-
+1/8 SO
4
2-
+3/16 H
+
→1/6 CO
2
+1/16 H
2
S + 1/16 HS
-
+ 1/6 H
2
O
ΔG
0
= -11.44 KJ/e
-
eq
2CH
3
CHOHCOO
-
+3 SO
4
2-
+9/2 H
+
→4 CO
2
+3/2 H
2
S + 3/2 HS
-
+ 4 H
2
O (2-24)
ΔG
0
= -137.28 KJ/mole lactate
ΔG
0
= -32.81 Kcal/mole lactate

These reactions present the energy required from complete dissociation of the
electron donor into carbon dioxide, hydrogen sulfide, and water. The degree of
progress in the reaction depends on the classification of the bacteria and the
accumulation of the intermediate compound, along with the inhibitory effect of
hydrogen sulfide. In most cases, the reaction will stop before it reaches complete
dissociation. Table 2-7 represents a list of the thermodynamic data for some of the
reported biochemical reactions of sulfate-reducing bacteria. Based on the data
presented in Table 2-7, pyruvate has the most energetic reaction; therefore, it is the
most favorable reaction for bacteria, and could be the best electron donor based on
energy.
In contrast, acetate and formate have the least by-products because these two
energy sources can completely dissociate into the inorganic compounds and
therefore no accumulation of the organic compound prevents the reaction from going
to completion. Hydrogen and carbon dioxide gases are very good choices because

32
they produce only water and sulfide ion and the reactions are very energetic. Lactate
reaction produces more energy than hydrogen gas, but it has acetate as a by-product.

Table 2-7: Observed Free Energy in kJ for Sulfate Reduction Reactions with
Different Electron Donors
Reactions Δ Δ Δ ΔH
0
Δ Δ Δ ΔH
0
/H
2
Δ Δ Δ ΔG
0
Δ Δ Δ ΔG
0
/H
2

4 H
2
+ SO
4
2-
→ S
2-
+ 4 H
2
O -196.46 -48.91 -123.98 -30.93
Acetate + SO
4
2-
→CO
2
+ HCO
3
-
+ S
2-

+H
2
O 48.07 12.12 -12.41 -3.09
CH
4
+ SO
4
2-
→ CO
2
+ H
2
O + S
2-
40.55 10.11 16.72 4.18
2Ethanol + SO
4
2-
→ 2 aceticacid

+ 2H
2
O + S
2-
-55.59 -13.92 -59.36 -20.06
4 Formate
-
+ SO
4
2-
→ 4 HCO
3
-
+ S
2-
-213.6 -53.5 -182.67 -45.56
4 Pyruvate
-
+ SO
4
2-
→ 4 Acetate
-
+ 4 CO
2
+ S
2-
-351.12 -87.78 -331.06 -82.76
2 Lactate
-
+ SO
4
2-
→ 2 Acetate
-
+ 2CO
2
+2 H
2
O + S
2-
-79.42 -19.85 -140.45 -35.11
2 Malate
-
+ SO
4
2-
→ 2 Acetate
-
+ 2CO
2
+ 2HCO
3
-
+ S
2-
154.66 38.66 -181.00 -45.35
Source: Postgate (1984)

Ethanol also has acetate as a by-product and is a less efficient reaction and
the energy of the reaction is less than lactate and hydrogen. Therefore, based on the
above observations, carbon dioxide appears to be the most appropriate carbon
source. Carbon dioxide and hydrogen gas, lactate, acetate, citric acid, molasses and
formic acid are the carbon and electron sources that were tested in this research.
Pathways of acetate and lactate utilization in bacteria are presented in Figures
2-3 to 2-5 (Perry et al., 2002). The gluoxylate shunt (Figure 2-3) is a variation of the
tricarboxylic acid (TCA) cycle, and it is of great importance for bacteria growing on

33
acetate. As the intermediate compounds are removed by anabolic reaction in the
TCA cycle, the bacteria that grow on 2-carbon substrates, such as acetate, must have
a mechanism for replenishing oxaloacetate for continuous operation of the cycle.
When the bacteria grows on acetate, it does not have the 3-carbon intermediate that
is needed (e.g., phosphophenolpyruvate) to form oxaloacetate. Therefore, organisms
that grows on substrates such as acetate use an inducible glyoxylate shunt, through
which isocitrate is cleaved by the inducible isocitrate lyase, producing succinate and
glyoxylate. The produced glyoxylate reacts with one molecule of acetate to produce
malate. With this method, the organism will have an unlimited supply of 4-carbon
intermediates for the TCA cycle (Perry et al. 2002).

Figure 2-3: Glyoxylate Cycle for Microorganisms Grown on Acetate, or Other
Substrates Methabolized through a 2-Carbon Intermediate
(Perry et al., 2002)

34

Figure 2-4 presents the TCA cycle, or Krebs cycle. One turn of the TCA
cycle oxidizes one molecule of acetate and produces NADH and CO
2
.

Figure 2-4: Krebs Cycle (Perry et al., 2002)

35
In the anaerobes, the enzyme α-ketoglutarate dehydrogenase is not
synthesized. Succinyl-CoA is formed by reduction of oxaloacetate, and malate, as
demonstrated in Figure 2-3, is produced during glycolate cycle, or by carboxylation
of phosphoenolypyruvate (Perry et al., 2002).
Figure 2-5 presents the lactate oxidation in a sulfate-reducing Achaean (Perry
et al., 2002). Oxidation of lactate by Archaeoglobus proceeds through pyruvate to
acetyl-CoA. Acetyl-CoA later oxidizes to CO
2
and methytetrahydromethanopterin
(Methyl-H4MPT). Methyl-H4MPT is then oxidized to methane and H4MPT through
a pathway similar to methylotrophic methanogens (Perry et al., 2002).

Figure 2-5: Lactate Oxidation by Sulfate –Reduing Archaeon Archaeoglobus

36
(Perry et al., 2002)
2.10.2 Optimum Temperature and pH
Sulfate reducing bacteria work in the mesophilic area, and the proper
temperature for them is around 30
o
C, although they can tolerate up to 42
o
C
(Postgate, 1984). However, above 42
o
C, the mesophilic bacteria cannot survive; and
therefore, it is essential to conduct CMBR studies to determine the optimum
temperature.
Sulfate reducing bacteria can tolerate pH values in the range of 5-9.5
(Postgate, 1984), but they are most active in the pH range of 6.5-8. Therefore, it is
necessary to conduct CMBR studies to determine the optimum pH.

2.10.3 Determination of Biokinetic Parameters in a CMBR
Mass balance in a CMBR can be expressed by Equation 2-25.
x
i
r J
t
C
+ −∇ =
∂
∂
r
(2-25)
and since 0 = ∇ − J
r

XV
dt
dX
V μ − = 0 (2-26)
where
X
dt
dX
μ = − (2-27)
so that:
t
X
X
μ ˆ ) ln(
0
− = (2-28)

37
In the above equations, the following notations are observed:
r
x
= rate of growth, mg/ L-hr
μ= specific growth rate coefficient (per hr)
X= biomass concentration, mg/L
Therefore, the rate of bacterial growth is proportional to the number of bacteria and
the specific growth rate according to equation 2-27.
The relationship most frequently used to represent bacterial growth kinetics is
the Monod equation, (Equation 2-29) that relates the bacterial growth rate to the
concentration of the substrate (Rittmann and McCarty, 2001). The bacterial growth
or degradation can be very well described by four parameters namely, i) the
maximum specific growth rate (μ ˆ ), ii) the substrate affinity constant (Monod half
saturation constant, K
S
), iii) the growth yield (Y), and ix) the minimum substrate
concentration (S
min
).

S K
S
dt
dX
X
S
a
a
+
= = μ μ ˆ )
1
( (2-29)
where
μ= specific growth rate , time
–1

μ ˆ = maximum specific growth rate, time
–1

S = concentration of growth-limiting substrate, mass/unit volume
K
S
= Monod half-saturation constant, mass/unit volume

38
At low substrate concentrations (S << K
S
), Equation 2-29 can be simplified to
Equation 2-30, which presents a linear dependence of growth rate to substrate
concentrations.
S
a
a
K
S
dt
dX
X
μ μ ˆ )
1
( = = (2-30)
At high substrate concentrations (S >> K
S
), Equation 2-29 can be simplified to
Equation 2-31, showing the independence of growth rate from substrate
concentrations (Maier et al., 2000). This is the case in CMBR studies, in which
bacterial growth is at its maximum specific rate. Integration of Equation 2-31 results
a relationship similar to that of Equation 2-28, and therefore a plot of ln(X) versus
time yields a slope equivalent to the maximum growth rate. In the case of SRBs, the
K
S
is very small and its value is negligible in comparison to substrate concentrations
(Cooney et al., 1996), so that the use of Equation 2-28 in CMBR studies becomes
justifiable. The microbial growth rate can be described by the reaction:

μ μ ˆ )
1
( = =
dt
dX
X
a
a
(2-31)
The above equation can be integrated as

∫ ∫
− =
t X
X a
a
dt
X
dX
0
ˆ
0
μ
To yield the relationship
t
X
X
μ ˆ ) ln(
0
− =

39
The rate of substrate utilization can be expressed as:
r
ut
= -
a
X
S K
S q
+
ˆ
(2-32)
where
r
ut
= rate of substrate utilization (M
S
L
-3
T
-1
) (mass/unit volume-time)
q ˆ = maximum specific growth rate of substrate utilization (M
S
M
X
-1
T
-1
)
(mass
S
/mass
X
-time)
Substrate utilization and biomass growth are connected by the reaction:
μ ˆ = q ˆ Y (2-33)
where
Y= true yield for cell synthesis (M
S
M
X
-1
) (mass
S
/mass
X
)
The true yield represents the amount of biomass that can be produced in the
absence of cell maintenance, and it can be expressed as the net rate of biomass
production (dX/dt) per rate of substrate consumption (-dS/dt).

dS
dX
dt dS
dt dX
Y
−
=
−
=
/
/
(2-33)
The true yield can be estimated in a system where bacterial growth follows
the maximum growth rate, μ ˆ , and that the substrate utilization for maintenance is
relatively small in comparison to the amount used for growth. This condition is met
in CMBR; where substrate and nutrient levels are high (Maier et al., 2000);
therefore, a plot of biomass produced as a function of substrate utilization will

40
demonstrate a linear relationship, where in the slope denotes the true yield. This
method has been used in this work for estimating the true microbial yield.
The net yield is less than Y, because part of the energy obtained from
substrate utilization is used for cell maintenance. The net growth rate is equal to the
difference between the growth from substrate utilization minus decay due to
endogenous respiration. Equation 2-35 presents the net yield calculation.
dt dS
X
b Y
dt dS
dt dX
Y
n
/ /
/
−
− =
−
= (2-35)
When the rate of substrate utilization per unit mass of cells is sufficiently
low, the net yield becomes zero. In such cases, the substrate utilization rate is just
sufficient for maintenance. In a CMBR, this situation can be observed towards the
end of the experiment. When Y
n
approches zero, Equation 2-35 will simplify to:
m
Y
b
X
dt dS
= =
− /
(2-36)
Parameters such as specific growth rate (μ), yield coefficient (Y), and Monod
half saturation constant (K
S
) are measures of how fast the bacteria will utilize the
substrate and therefore how fast the reaction goes, how much biomass and therefore
sludge could be expected from the reaction, and how fast the reaction reaches the
saturation level, respectively. All of these parameters are obtainable from batch
studies.
Cooney et al. (1996) used the following procedures to obtain the growth
parameters from CMBR experiments in their studies. This research adopted similar
procedures to estimate the kinetic parameters.

41
Equation 2-34 was used to estimate the growth yield, Y (mg/mg). For this
purpose, the concentration of biomass against the concentration of substrate
was plotted. The growth yield is the slope of this graph during exponential
growth.
Equation 2-28 was employed to estimate the specific growth rate, μ ˆ (per hr)
.

For this purpose, the natural logarithm of biomass concentration versus time
was plotted, and the slope of the graph during the exponential growth phase
was used to determine the maximum specific growth rate.
Specific consumption rate, Q
S
(mg/mg-hr) is the slope of the graph of
substrate to biomass changes versus time during exponential growth
(dS/dX-t).

2.11 H
2
S Health Standards and Methods of Removal
2.11.1 Air Pollution Standards for H
2
S
Based on the Air Resource Act of 1967, the Air Resource Board divided
California into air basins with specific air quality standards (Health and Safety Code
Section 39606). The California EPA’s statewide ambient air quality standard
(CAAQS) for H
2
S is 0.03 ppm (30 ppb, 42 μg/m
3
), averaged over a 1-hour period.
The Office of Environmental Health Hazard Assessment (OEHHA) in 1999
specified 30 ppb as the acute reference exposure level (REL) to evaluate the peak
concentration emitted from industry. In 2000, the OEHHA adopted a chronic
reference exposure level (cREF) of 8 ppb (10 μg/m
3
) to asses long-term emissions.

42
At the federal level, U.S. EPA did not classify H
2
S as a criteria air pollutant or
hazardous air pollutant (HAP). The chronic reference concentration (RfC)
established by the U.S. EPA for H
2
S was 1 μg/m
3
.

2.11.2 Water Pollution Standards for H
2
S
Hydrogen sulfide is not regulated in primary and secondary drinking water
standards, because a concentration high enough to be a drinking health hazard also
makes it unpalatable. The H
2
S odor in the water is detectable in concentrations as
low as 0.5 ppm, therefore for drinking water to be palatable the H
2
S concentration
must be lower than this limit. H
2
S concentrations less than 1 ppm give the water a
swampy odor, and in higher concentrations of 1-2 ppm it has the rotten odor and is
very corrosive to the piping and mechanical system.

2.11.3 Chemical, Physical, and Biological Methods for Hydrogen Sulfide Removal
from Water
For removal of hydrogen sulfide from wastewater, one of the following
methods could be used: gas stripping, chemical precipitation, oxidation (aeration,
chlorination, ozonation, potassium permanganate treatment, or hydrogen peroxide
treatment), or biological reduction to elemental sulfur (Kohl and Nielsen, 1997).
Chemical precipitation of hydrogen sulfide includes reactions with iron
oxide, zinc oxide, or sodium hydroxide and precipitation in the form of metal

43
sulfides. Reactions 2-37 to 2-39 present some of the possible reactions (Kohl and
Nielsen, 1997).
Fe
2
O
3
+ 3H
2
S → Fe
2
S
3
+ 3H
2
O (2-36)
ZnO + H
2
S → ZnS + H
2
O (2-37)
2NaOH + H
2
S → Na
2
S +2H
2
O (2-38)
Biological oxidation of sulfide to elemental sulfur or sulfate by using
chemolithoautotrophic bacteria, Thiobacillus genus, is reported in the literature
(Janssen et al., 1999) according to Reactions 2-40 and 2-41.
− −
+ → + OH S O HS 2 2 2
0
2
ΔG
0
= -169.35 kJ/mol (2-40)
+ − −
+ → + H SO O HS 2 2 4 4
2
4 2
ΔG
0
= -732.58 kJ/mol (2-41)
Since sulfate production is more energetic, in order to get elemental sulfur the
oxygen must be added in controlled amounts (Janssen et al., 1999).
Very few investigations have used oxidation reduction potential (ORP) to
control the toxicity of hydrogen sulfide in their biological sulfate reduction studies.
Khanal and Huang (2003) used ORP to control the oxidation of hydrogen sulfide in a
chemostat. A gas/liquid separator was used in that research to return the biogas back
into the chemostat, and oxygen was introduced into the biogas return line (Khanal
and Huang, 2003).
Glombitza et al. (2001) used biological sulfate reduction to treat wastewater
from an acid lignite mine. The hydrogen sulfide produced in an anaerobic FBR was
used to precipitate out the heavy metals, and the remaining dissolved sulfide in the
effluent was oxidized to elemental sulfur by using hydrogen peroxide.

44
In this study, the FBR effluent stream containing dissolved hydrogen sulfide
was passed through an anaerobic reactor. Nitrate was added to the influent stream to
act as the electron acceptor, while H
2
S was the electron donor. The concentration of
H
2
S was measured periodically and the concentration of the nitrate was adjusted
accordingly.

2.12 Anaerobic and Aerobic Biofilters for Treatment of H
2
S Off-Gas from FBR
System
Biofilters have been originally designed for odor control in wastewater
treatment plants and composting operations. The first attempts to use biologically
active biofilters for H
2
S control occurred in 1923. Biofilters use microorganisms to
biodegrade the pollutants. Biofilters are a three-phase bioreactor (gas, liquid, solid)
made from a packed filter with high porosity, high buffer capacity, high nutrient
ability, and high moisture retention capacity to ensure the growth of microorganisms
(Elias et al., 2002). Biofilters have low maintenance and operation costs, high
removal efficiency, and less secondary pollution (Bohn, 1992). The pollutant gas
continuously enters the biofilter, while the nutrient solution is periodically added.
Microorganisms that utilize these pollutants are attached to the surface of a solid
support material such as soil, peat, compost, wood bark, or synthetic materials. The
process is catalyzed by enzymes, takes place at ambient conditions, and requires
little energy for operation and maintenance. Biofilters can work for years without the
need for replacement, and they are capable of biodegrading the pollutant completely.

45
Biological oxidation of mineral compounds (such as sulfide) supplies energy to the
microbial cells and produces odorless compounds such as elemental sulfur or sulfate,
in addition to CO
2
, water, and new biomass (Janssen et al., 1999).
The use of support media in the biofilter provides a large surface area for
bacterial attachment and growth. Production of biofilm on the surface of the support
media can isolate and protect the bacteria from the harsh environment and inhibitory
compounds (Ng et al., 2004). Many industries and utilities are among the main
producers of H
2
S. These include wastewater treatment, food processing, pulp and
paper manufacturing, fuel treatment, petroleum refineries, and sanitary landfills
(Chung et al., 1996).
H
2
S removal from wastewater treatment plant airstreams by using compost
and GAC biofilters have been reported and the bacteria were acclimated to low pH in
such a way that 99% removal efficiencies for H
2
S were observed (Webster et al.,
1997). Different solid support materials and various microorganisms have been
studied to obtain the maximum removal efficiencies, as summarized in Table 2-8.
Hydrogen sulfide is the product of anaerobic reduction of sulfate, and in the
pH range of 7-7.5, it dissociates almost equally in the liquid phase according to
Equations 2-42 to 2-44 (Rittmann and McCarty, 2001).

H
2
S⇔ H
+
+HS
-
04 . 7 ,
] [
] ][ [
1 1
2
= =
− +
a a
pK K
S H
HS H
(2-42)

HS
-
⇔ H
+
+S
2-
9 . 12 ,
] [
] ][ [
2 2
2
= =
−
− +
a a
pK K
HS
S H
(2-43)

46

H
2
S (aq)⇔ H
2
S (g) ) 35 ( / 13
)] ( [
)] ( [
2
2
C mol atm K
aq S H
g S H
o
H
= = (2-44)

Hydrogen sulfide in the biogas of anaerobic digesters is product of
biodegradation of proteins and other sulfur-containing compounds in the organic
feedstock to the digester.
H
2
S is very corrosive and, along with its unpleasant odor, it is highly toxic.
Biological methods are more efficient and more economical than physiochemical
methods, if proper operational conditions are maintained. The desirable bacteria to
convert H
2
S to S
0
in a bioprocess should have these qualifications: reliable capacity
to produce elemental sulfur from H
2
S, minimum nutrient requirement, and easy
separation of S
0
from biomass (Syed et al. 2006).
Large varieties of reactions are possible under aerobic and anaerobic
conditions, and either can proceed, depending on the availability or absence of
oxygen. Dissimilatory sulfur oxidation happens under aerobic and anaerobic
conditions for the domains Archaea and Bacteria. In Eukarya, dissimilatory sulfur
oxidation is mediated by lithotrophic bacteria endosymbionts. In the domain
Archaea, dissimilatory sulfur metabolism exists in the Crenarchaeota and
Euryarchaeota (Friedrich et al., 2001).
In the domain Bacteria, reduced inorganic sulfur compounds (hydrogen
sulfide, polysulfide, elemental sulfur, sulfite, thiosulfate, or polythionates) act as
electron donors for anaerobic or aerobic photolithotrophic growth.

47

Table 2-8: Research Performed on the Usage of Biofilters for Hydrogen Sulfide
Removal
Solid Support Material H
2
S Concentration Bacteria References
Cell–laden, Ca-aliginate 5-100 ppm H
2
S T. thioparus Chung et al. (1996)
Compost/hog fuel 10- 450 ppm H
2
S Indigenous (in sludge) Wani et al. (1999)
Cell–laden, Ca-aliginate 60-120 ppm H
2
S Pseudomonas putida Chung et al. (2001)
Pig manure + sawdust 10-45 g H
2
S m
-3
h
-1
Indigenous Elias et al. (2002)
Structured plastic
packing
60-155 ppm H
2
S --- Cox and Deshusses
(2002)
Wood chips, GAC 30-450 ppm H
2
S T. thioparus Kim et al. (2002)
Wood-based medium 1.07 mg H
2
S m
-3
Indigenous Shareefdeen et al.
(2002)
Granulated sludge 5-25 ppm H
2
S ----- Malhautier et al.
(2003)
GAC 1-10 ppm Mixed Webster et al. (1997)
Lava rock, compost,
GAC, woodchips
0.01-30 ppmv ------- Chitwood and Devinny
(1999)
Manure compost 50 ppm H
2
S Indigenous Morgan-Sagastume et
al. (2003)
Peat 355-1400 ppm H
2
S T. thioparus Schieder et al. (2003)
Pellet-activated carbon 10-125 ppmv Indigenous Duang et al. (2006)
Activated carbon 500, 700, 900 ppm Thiomonas sp. Ng et al. (2004)
Lava rock 50-300 ppm Acidithobacillus
ferrooxidans (ATCC
19859) and
Acidithiobacillus
thiooxidas (ATCC
19877)
Li et al. (2005)
Synthetics (hydrophilic
mineral cores coated with
hydrophobic sorption
material)
0-40 ppm ----- Shareefdeen et al.
(2003)
Porous ceramic 200-2200 ppmv Acidithiobacillus
thiooxidans AZ11
Lee et al. (2006)
Peat 0-250 ppm Thiobacillus thioparus
DW44
Cho et al. (1991)

48
Other forms of electron donors including hydrogen, carbon monoxide, and
ferrous iron, and other metals could also be used in both aerobic and anaerobic
environment. Different energy metabolisms can be observed in one strain, which
could be an alternative mode of life (Friedrich, 1998).
Photolithotrophic and chemolithotrophic bacteria in aerobic or anaerobic
environments can be used for H
2
S oxidation. Some of the microorganisms that have
been used for H
2
S removal are listed in Table 2-9. Biodegradation of H
2
S by
chemolithotrophs can be done in aerobic conditions by utilizing oxygen as the
electron acceptor or in anaerobic conditions with an alternative electron acceptor
(e.g., nitrate), depending on the type of bacteria (Prescott et al., 2003).
hiobacillus denitrificans is a chemolithotroph that can use S
0
, S
2
O
3
2-
, and H
2
S
as the electron donors, and O
2
or NO
3
-
as the electron acceptor, while utilizing CO
2

as the carbon source. The possible reaction with Thiobacillus denitrificans as
reported by Kleerebezem (2002) is presented in Reaction 2-45.

O H S N N O CH H HCO NH NO HS
2 2 2 . 0 5 . 0 8 . 1 3 4 3
4 . 27 5 . 14 5 . 2 3 . 20 2 . 0 5 5 . 14 + + + → + + + +
+ − + − −

(2-45)

Another anaerobic photosynthetic bacterium is Chlorobium
thiosulfatophilium, which utilizes CO
2
as the carbon source and obtains energy from
light to oxidize H
2
S (Basu et al. 2006). Total conversion of H
2
S and the sulfur
recovery was reported to be near the theoretical yields.

49

Table 2-9: Some of the Microorganisms Used for H
2
S Removal
Microorganism Optimum
pH
Optimum
temperature
Energy
Sources
Oxygen
Requirement
Sulfur
Deposit
Reference
Thiobacillus
thiooxidans
2.0-3.5 28-30 Hydrogen
sulfide,
polithionates,
elemental
sulfur
Strictly aerobe ----- Takano et
al. (1997)
Thiobacillus
novellas
7.0 30 Hydrogen
sulfide,
methyl
mercaptan,
dimethyl
sulfide,
dimethyl
disulfide
Strictly aerobe ------ Cha et al.
(1999),
Kelly et al.
(2000)
Thiobacillus
thioparus
7.5 28 Thiosulfate,
sulfide
Strictly aerobe Extracellular Vlasceanu
et al.
(1997)
Thiobacillus
denitrificans
6.8-7.4 28-32 Sulfide,
thiosulfate,
tetrathionate,
thiocyanate,
elemental
sulfur
Facultative
anaerobe
------ Kelly and
Wood
(2000)
Thermothrix
azorensis
7.0-7.5 76-78 Hydrogen
sulfide,
thiosulfate,
tetrathionate,
elemental
sulfur
Strictly aerobe Intracellular Odintsova
et al.
(1996)
Thioalkalispira
microaerophila
10 ---- Sulfide,
polysulfide,
elemental
sulfur,
thiosulfate
Strictly aerobe
and
microaerophile
Intracelluiar Sorokin et
al. (2002)
Thiomicrospra
frisia
6.5 32-35 Sulfide,
thiosulfate,
tetrathionate,
sulfur
Strictly aerobe Extracellular Brinkhoff
et al.
(1999)
Xanthomonas
sp. Strain
DY44
7.0 30 Hydrogen
sulfide
Aerobe ------ Cho et al.
(1991)

Aerobic sulfur-oxidizing prokaryotes belong to several genera, including
Acidianus, Acidithiobacillus, Aquaspirillum, Aquifex, Bacillus, Beggiatoa,
Methylobacterium, Paracoccus, Pseudomonas, Starkeya, Sulfolobus,
Thermithiobacillus, Thiobacillus, and Xanthobacter, which are mainly mesophilic

50
(Friedrich et al. 2001). Ecologically, they can live in transition zones between
aerobic and anaerobic conditions, where both oxygen and hydrogen sulfide are
available.
There have been limited studies on anaerobic photolithotrophs for H
2
S
removal; the reason could be that the number of bacteria that can anaerobically
utilize H
2
S is limited. Research on microbial ecology of phototrophic bacteria has
shown that one type of green sulfur bacteria (GSB), Chlorobium limicola, is a good
candidate for sulfide removal. Chlorobium limicola, strictly anaerobic bacteria, can
oxidize sulfide to elemental sulfur by utilizing light, CO
2
, and inorganic nutrients for
growth. GSBs are non-motile and deposit elemental sulfur extracellularly; therefore,
they can be used for recovery of elemental sulfur from sulfide-containing
wastewaters. Therefore, Chlorobium limicola is suitable for use in an anaerobic
biofilter to remove H
2
S. The overall photochemical reaction of GSB through which
they oxidize S
2-
to S
0
while reducing CO
2
to carbohydrates is presented in Van Niel
Reaction 2-46 (Syed et al., 2006).
O nH O CH nS nCO S nH
n
h
2 2
0
2 2
) ( 2 2 + + →  +
ν
(2-46)
Vetter (1985) suggested that sulfur stored in bacteria is an energy reservoir
and not a method for detoxification of excess hydrogen sulfide to prevent inhibition.
As long as H
2
S is available, sulfur is stored in the globules, but when the H
2
S in the
environment is depleted, the stored sulfur is oxidized to produce energy. The X-ray
diffraction (XRD) on intracellularly produced sulfur globes has shown that the
structure of fresh sulfur globes is a liquid or amorphous form of sulfur, which

51
converts into crystalline orthorhombic sulfur after drying or aging (Janssen et al.,
1999).
Studies on sulfur globules produced by bacteria extracellularly revealed that
these sulfur compounds can be separated from the bacteria very easily. In the
suggested model proposed by Steudel (1987) for the sulfur globules excreted by
Acidithiobacillus ferrooxidans, the globules consisted of sulfur nucleus (mainly S8
ring), and long-chain polythionates on the surface. In another model, Steudel et
al.(1989) proposed that the produced sulfur consists of vesicles of a polythionate
membrane.
H
2
S oxidation can happen in light or dark environments, using inorganic or
organic electron donors, and in an aerobic environment (utilizing oxygen as the
electron acceptor) or anaerobic environment (using forms of electron acceptors other
than oxygen). Different combinations of environmental conditions favor different
bacteria for H
2
S oxidation.
Phototrophic anaerobic sulfur-oxidizing bacteria are mainly neutrophilic and
mesophilic and belong to several genera, including Allochromatium (formerly
Chromatium), Chlorobium, Rhodobacter, Rhodopseudomonas, Rhodovulum, and
Thiocapsa. Chemolithoautotrophic growth in the dark has been discussed for
Thiocapsa roseopersicina, Allochromatium vinosum, and other purple sulfur bacteria
(Friedrich et al. 2001). Table 2-10 presents specifications of some of the H
2
S-
utilizing bacteria. Table 2-11 lists the sulfur oxidation mechanism in different
bacteria.

52
Table 2-10: Specifications of H
2
S Utilizing Bacteria
Sulfur Bacteria
Appearance
Environmental Condition Example
Colorless sulfur bacteria Aerobic Beggiatoa
Green sulfur bacteria Anaerobic, phototrophic Chlorobium
Purple sulfur bacteria Anaerobic, phototrophic Chromatium

Table 2-11: Sulfur Oxidation Mechanism in Different Sulfur Bacteria
Class Microbial
Nutrition
Electron
Donor
Product Mechanism Members
Cyanobacterium Oxygenic
photosynthesis,
using (PS)I
Hydrogen
sulfide
Elemental
sulfur
CO
2

assimilation via
pentose-
phosphate
cycle
Oscillatoria
limmetica
Green sulfur
bacteria
Anoxygenic
photosynthesis,
(Strictly anaerobic
and obligatory
phototrophic)
Hydrogen
sulfide, and
elemental
sulfur
Sulfate, and
elemental
sulfur
CO
2

assimilation via
reductive citric
acid cycle
Chlorobium
limicola
Phototrophic
Proteobacteria,
(gamma division of
purple bacteria)
Anaerobic
phototrophic
Hydrogen
sulfide, and
elemental
sulfur
Sulfate Sulfur first
accumulated
inside the cell,
and then further
oxidized when
H
2
S depleted.
Includes families
of Chromatiaceae,
Ectothiospiraceae,
and
Rhodosprillaceae
Rhodosprillaceae Anaerobic
photolithotrophic

Hydrogen
sulfide
Elemental
sulfur
Sulfide:quinone
reductase yield
elemental
sulfur in vitro
Rhodobacter
capsulatus,
Rhodobacter
sphaeroides,and
Rhodosprillum
rubrum
Non-phototrophic
bacteria, (colorless
thiobacteria)
Facultative or
Obligate
lithotrophic
Hydrogen
sulfide
------- CO
2

assimilation via
ribulose 1,5-
bisphosphate
cycle
Thiobacillus
thiooxidans, T.
ferrooxidans, and
T. neapolitanus
Sources: Friedrich (1998); Maier (2000)

Prokaryotes can oxidize hydrogen sulfide, sulfur, thiosulfate, and
polythionates under acidic, neutral, or basic conditions (Friedrich et al., 2001).

53
The chemolithoautotrophs can grow and produce new cells by using
inorganic carbon (CO
2
) as a carbon source while reducing H
2
S as an energy source.
In contrast, chemoorganotrophic heterotrophs can grow by using reduced organic
carbon sources (e.g. glucose and amino acids) while utilizing inorganic compounds
such as H
2
S as the energy source (Prescott et al., 2003).
In this research, utilization of H
2
S from the FBR reactor off-gas and
production of elemental sulfur was tested in an anaerobic biofilter. In the biofilter, a
mixed group of anaerobic bacteria, possibly green sulfur bacteria, were used to
utilize hydrogen sulfide as the energy source. Inorganic nutrients were added to the
biofilter periodically to support bacterial growth. The presence of CO
2
, along with
H
2
S in the off gas from the FBR reactors provides the proper environment for growth
of the GSB bacteria (equation 2-46).

2.13 Methanogenesis Phenomena in Fluidized-Bed Reactors
In the absence of oxygen, organic material can be mineralized to carbon
dioxide by fermentation or anaerobic degradation. Anaerobic degradation requires
the availability of an electron acceptor such as an organic compound, or an inorganic
electron acceptor. In methanogenic condition, some of the electron acceptors in order
of a decrease in electron affinity are nitrate, manganese, iron, sulfate, and carbonate
(Maier et al., 2000).
Methane sources in the atmosphere are mostly from industry and landfill gas
emissions (10
7
metric tons methane/year). In nature, methanogens are the obligatory

54
anaerobic archaebacteria that are responsible for methane production
autothrophically via Equation 2-47 (Maier et al., 2000).

4H
2
+ CO
2
→ CH
4
+ 2H
2
O ∆G
o
’= -130.7 kJ (2-47)

In this reaction, CO
2
is the terminal electron acceptor, and hydrogen is the electron
donor. Methanogenesis can happen heterotrophically by using simple organic
substrates, including acetate, methanol, and formate (Maier et al., 2000). Hydrogen
and acetate are the key precursors for methane production by methane producing
bacteria (MPB), whereas SRB use them as energy, electron, and carbon sources for
sulfate reduction. To produce these electron donors, more complex polymers must be
degraded. Figure 2-6 presents the typical anaerobic reactions in methanogenesis.
If the main carbon and electron sources are polymers, they first degrade to
simple sugars (e.g., glucose) and common fermentation products such as lactate,
ethanol, and volatile fatty acids (propionate, butyrate, and acetate). Intermediate
bacteria later convert these compounds to acetate and H
2
. MPB and SRB compete for
available hydrogen and acetate. Acetate and H
2
are the major substances for
methanogenesis.
The free energy changes for acetate and hydrogen utilization are –59.9 and –
151.9 kJ, respectively for SRB, and they are –31.0 and –135.6 kJ for MPB
(McCartney, 1991).

55

Cellulose
Cellobiose
+
Glucose
Lactate
+
Ethanol
Acetate H
2
+CO
2
Clostridium
thermohydrosulfuricum,
Thermoanaerobacter
ethanolicus
Desulfotomaculum
nitrificans
Methanosarcina
thermophila
Methanobacterium
thermoautotrophicum
CH
4
+CO
2
Clostridium
thermocellum
Cellulolytic
microorganisms
degrade cellulose to
glucose and
cellobiose
Intermetabolic
microorganisms
convert the sugars
to lactate, ethanol,
acetate, H
2
, and CO
2
Methanogenic
microorganisms
convert acetate, H
2
,
and CO
2
to methane

Figure 2-6: Typical Reactions in Methanogenesis
(Perry et al., 2002)

In reality, actual free energy depends on the activities of the reactants and
products in each reaction, therefore MPB will out-compete SRB in high electron-
donor concentrations, whereas SRB out-competes MPB at high sulfate

56
concentrations. In addition, the SRB reactions are pH dependent, and their reactions
are less favorable at high pH values (McCartney and Oleszkiewicz, 1993).
In anaerobic sulfate-utilizing bioreactors, both sulfate reduction and
methanogenesis can happen because SRB have the capability to utilize most of the
intermediate products of the methanogenesis. Figure 2-7 presents the pathways of
competition between acetogenic, methanogenic and sulfate-reducing bacteria during
anaerobic degradation of organic matter (Kalyuzhnyi et al., 1998).

Figure 2-7: Pathway of Competition between AB, SRB, and MPB
(Kalyuzhnyi et al., 1998)
FB
Polymers
Monomers, Fatty
acids, sugars,
amino acids
Acetate
CO
2
and
H
2
S
CH
4
and
H
2
S
H
2
and
CO
2

Fermentation
Intermediates
FB
SRB
SRB
SRB
MB
MB
AB
HAcB
SRB
SRB

57
Because of limited metabolism of methanogens, organic acids are degraded
by associations of acetogenic bacteria and methanogenic archaea. Acetate,
propionate, and butyrate are major components of most wastewaters. Complete
mineralization of propionate and butyrate requires the availability of acetogenic
bacteria, and two types of methanogenic archaea (Stams et al., 2005). On the
contrary, a single species of SRB in the sulfate-rich environments can directly utilize
the intermediate organic acids, including propionate and butyrate.
Kinetic properties of SRB, methanogenes, and acetogens can be used to
predict the outcome of the competition (Oude Elferink et al., 1994; Stams et al.,
2003). Table 2-12 presents different reactions that SRB, methanogenes, and
acetogens follow.
In general, in biological reactors, there is a substrate competition between
SRB and acetogenic bacteria (AB) for VFA and ethanol, and competition between
SRB and MPB for acetate and hydrogen. Monod kinetics data for SRB, AB, and
MPB indicate which specie out-competes the other ones (Oude Elferink et al., 1994).
Other studies also have reported that two main mechanisms control the
reactions that occur in the anaerobic system: (1) the competition between SRB and
MPB, and (2) the concentration of produced sulfide by SRBs (Yoda et al., 1987;
McCartney and Oleszkiewicz, 1991). The inhibitory effect of sulfate salts on
methanogenesis was first reported in 1932 (Lawrence et al., 1966). Later McCarty
(1964) reported that concentrations above 200 mg/L of sulfide are quite toxic for
anaerobic treatment.

58
Table 2-12: Sulfate-Reducing, Methanogenic and Acetogenic Reactions with
Organic Matters
Reaction
∆G
o
(kJ/reaction)
Acetogenic Reactions
Propionate
-
+ 3 H
2
O → Acetate
-
+ HCO
3
-
+ H
+
+ 3H
2

Butyrate
-
+ 2 H
2
O →2 Acetate
-
+ H
+
+ 2H
2
2Propionate
-
→ Acetate
-
+ Butyrate
-

+76.1
+48.3
0
Methanogenic Reactions
4 H
2
+ HCO
3
-
+ H
+
→ CH
4
+3 H
2
O
Acetate
-
+ H
2
O → CH
4
+ HCO
3
-

-135.8
-31.0
Sulfate Reducing Reactions
4 H
2
+ SO
4
2-
+ H
+
→ HS
-
+ 4 H
2
O
Acetate
-
+ H
2
O → 2HCO
3
-
+ HS
-

Propionate
-
+ ¾ SO
4
2-
→ Acetate
-
+ HCO
3
-
+ ¾ HS
-
+ ½ H
+

Butyrate
-
+ ½ SO
4
2-
→2 Acetate
-
+ ½ HS
-
+ ½ H
+

-151.9

-47.6
-37.7
-27.8
Homoacetogenic Reactions
4 H
2
+ 2HCO
3
-
+ H
+
→ Acetate
-
+ 4H
2
O

-104.6
Sources: Stams et al. (2005), and Thauer et al. (1977)

59
In the competition between SRB and MPB, in a substrate-limiting
environment, the most efficient microorganism will be predominant, whereas in
conditions where there are no substrate limitations, the fast-growing bacteria will be
dominant. Thermodynamic data, discussed previously, show that SRBs are
producing more energy and are more efficient than MPBs, and that they can grow
very well in low electron donor environments; however, the previously reported
batch studies with SRB showed very slow kinetics (Bhattacharya et al., 1996). It
must be noted that MPB out-grow SRB in carbon-nutrient-rich environments, as
could be expected from the Monod kietics.

The Monod equation for substrate utilization is presented in Equation 2-48:

S K
SX q
dt
dS
r
ut
+
− = =
ˆ
(2-48)
where
r
ut
: Rate of substrate utilization (M
S
L
-3
T
-1
)
S: Substrate concentration (mg/L)
T: Time
X: Concentration of active biomass (mg/L)
q ˆ : Maximum specific rate of substrate utilization (M
S
M
X
-1
T
-1
)
K: Concentration at half of the maximum rate (M
S
L
-3
)

X q
Y
X
YS
XS
S K Y
XS
dt
dS
m
m m
ˆ
) (
= =
−
=
+
−
= μ
μ μ
(2-49)

60
X
S
XS
S K
XS
dt
dX
m
m m
μ
μ μ
= =
+
= (2-50)
where

Y: True yield for cell synthesis (M
X
M
S
-1
)
μ
m
: Maximum specific growth rate (T
-1
)

In an environment with high substrate concentrations, according to Equation
2-49, the substrate utilization is independent of K, and depends on the maximum
specific rate of substrate utilization ( q ˆ ) and the biomass concentration. In the same
environment, the growth rate (Equation 2-50), is independent of K, and depends on
the maximum specific growth rate and the biomass concentration. The Monod
kinetic parameters K

and q ˆ obtained in a chemostat for SRB are reported as 102 mg
acetate/L and 2.4/d respectively, whereas for MPB, the corresponding values are 116
mg acetate/L and 3.2 /d (Bhattacharya et al., 1996). Furthermore, the growth yield
parameters for SRB and MPB were calculated as 0.0602 and 0.0346 mg cells/mg
acetate, respectively (Bhattacharya et al. 1996). On the basis of these data it can be
seen that at high substrate concentrations the microorganism with the higher q ˆ
value, namely MPB, shall be predominant. In contrast, at low substrate
concentrations, the microorganism with a low K
S
value, such as SRB, shall
predominate. It has been reported that MPBs will out-compete SRBs at a volumetric
loading of 18 g acetate/L-d, but at a volumetric loading of 1.3 g acetate/L-d, the SRB

61
became dominant (Yoda et al., 1987). Reactions 2-51 to 2-53 present the sulfate
reduction reaction to produce H
2
S along with organic material oxidation to produce
methane and CO
2
.

Organics + SO
4
2-
→ Cells + H
2
S + CO
2
+ Alkalinity (2-51)
Organics → Cells + H
2
S + CO
2
(2-52)
Organics + SO
4
2-
→ Cells + H
2
S + CH
4
+ CO
2
+ Alkalinity (2-53)

Reaction 2-51 generally precedes Reaction 2-52. The concentration of the
electron donor (e.g., acetate) is usually expressed as COD in anaerobic systems.
Stochiometrically, 64 g COD reduces 96 g of sulfate. This means that methane
producers could not grow at COD/SO
4
2-
ratios of less than 0.67. Within the
COD/SO
4
2-
ratios of 1.7 to 2.7, active competition between MPBs and SRBs are
observed, whereas in COD/SO
4
2-
ratios of less than 1.7, SRBs are predominant (Choi
and Jay, 1991). McCartney and Oleszkiewicz (1991) also reported that the lactate
degradation pathway was dependent upon the COD: SO
4
2-
ratio (g/g) in the feed
solution. A ratio of 3.7 g/g resulted in a pathway that had propionate and acetate as
products, but there was not significant sulfate reduction. They also reported that the
COD:SO
4
2-
ratios less than or equal to 1.6 g/g resulted in an SRB pathway that had
acetate as a product and resulted in sulfate reduction. Similar studies reported an
increase from 13% to 67% COD reduction due to an increase in sulfate level from
COD/SO
4
2-
of 0.7 to 1.1 (Shin et al., 1997).

62

Kinetic studies by a number of researchers including, Widdle (1988),
Robinson and Tiedje (1984), and McCartney (1991) predicted that SRB would out-
compete MPB for hydrogen. Tursman and Cork (1989) reported that in the MPB the
hydrogenase enzyme system is located in the cytoplasm, whereas in the SRB it is in
the periplasmic space. Thus the SRB’s higher affinity for hydrogen could be due to a
less osmotic barrier inside the cell.
Isa et al. (1986) and Yoda et al. (1987) reported that MPBs grow better in
biofilm, whereas SRBs prefer suspended growth. Koster et al. (1986) suggested that
the presence of a pH or sulfide gradient in the biofilm may limit the hydrogen sulfide
concentration inside the biofilm, enabling MPBs to grow better. Further, McCartney
(1993) reported that increasing the hydraulic retention time of the reactor increases
the SRB’s contributions to electron flow.

In this study, C/S ratios of 1 to 2 in accordance with an SRB pathway have
been used. Nonetheless, some degree of competition with MPB may be present.

2.13.1 The Inhibitory Effect of Hydrogen Sulfide on MPB and SRB
Neutrally charged hydrogen sulfide is the most toxic form because it can
penetrate into the cell membrane, and once it enters the cytoplasm, H
2
S might
denature the proteins through formation of sulfide and disulfide cross-links between
the polypeptide chains (Conny et al., 1987). H
2
S might also interfere with various

63
coenzyme activities (Stouthamer, 1988). Table 2-13 lists some of the reported H
2
S
concentrations that inhibit methanogenesis.

Table 2-13: Some of the Reported Data on Inhibitory Effect of H
2
S on
Methanogenesis
Degree of Inhibition H
2
S Concentration Reference
100 % 15.2 mM (500 mg/L) as
TS
Parkin et al. (1990)
50 % 1.7 mM (55 mg/L) as H
2
S Kroiss and Wabnegg
(1983)
50 % 7.4 mM (250 mg/L) as
H
2
S
Koster et al. (1986)
50 % 3.13- 7.0 mM (100-224
mg/L) as TS
Karhadkar et al. (1987)
MPB inhibition 120 –140 mg/L as TS Choi and Jay, (1991)

In the hydrogen sulfide production range of 200 to 1200 mg/L related to
sulfate-rich wastewaters, the inhibition of methanogenesis is of major concern. In the
system used in this research, the short retention time, as well as high hydrogen
sulfide transfer rate from solution to gas phase (fluidization action), minimized the
H
2
S inhibitory effect on bacteria.

64
Chapter 3

RESEARCH OBJECTIVES AND SCOPE

3.1 Research Objectives
The primary objectives of this research were to achieve the removal of sulfate
at high concentrations from wastewaters using biological sulfate reduction
technology, and demonstrate the feasibility and effectiveness of the technology in
laboratory-scale bioactive anaerobic fluidized bed reactor systems. These objectives
were accomplished by conducting a series of laboratory-scale investigations,
including completely-mixed batch reactor (CMBR) biokinetic studies and fluidized-
bed reactor (FBR) studies to evaluate biological sulfate removal from a synthesized
wastewater using different electron donors, and evaluating the effect of biological
sulfate process parameters, including different influent sulfate concentrations,
carbon-to-sulfur ratios, and pH conditions. Anaerobic biofiltration studies were
conducted for removing the hydrogen sulfide that evolved in the bioactive fluidized
bed reactors systems, and for achieving sulfide oxidation to elemental sulfur. These
investigations were supported by mass balance analyses to determine the fate of
sulfur in the process. Hydrogen and possibly methane (energy sources) produced in
the biological system were proposed as promising source of energy for future
considerations.

65
3.2 Research Scope
The scope of the research intended to achieve the objectives mentioned above could
be briefly outlined as follows:
1. Perform an in-depth analysis of the possible reactions and associated
thermodynamics pertaining to biological sulfate reduction to identify the
best electron donors.
2. Conduct CMBR biokinetic studies and evaluate the effect of various
environmental parameters, such as electron donor, temperature, pH, and
carbon-to-sulfur ratio for desulfurization of the wastewater.
3. Estimate the biokinetic parameters for optimal process conditions from
CMBR studies, including the Monod maximum substrate utilization rate
and the yield coefficient, Y, and specific growth rates. Based on these
results, choose the best electron donor among the following: acetate,
lactate, hydrogen, citrate, ethanol, and molasses.
4. Perform anaerobic sulfate-reduction FBR experiments to evaluate the
process efficiencies under different initial sulfate concentrations for a
number of electron donor including acetate, lactate, and hydrogen. These
initial sulfate concentrations would vary from medium (700-1100 mg/L)
to high (1100-2000 mg/L) levels, covering a spectrum of different types
sulfate-rich wastewaters.

66
5. Conduct comprehensive analyses of the experimental data with reference
to sulfate removal, electron donor utilization (in gas or liquid form),
hydrogen sulfide production, and any by-product formation.
6. Conduct anaerobic biofiltration experiments in specially designed biofilter
systems to determine the conversion of hydrogen sulfide produced in the
FBR system to elemental sulfur, and to check for any methane production.
7. Perform mass balance analysis to determine the fate of sulfur in the FBR
system.
8. Conduct economical analysis to identify the most efficient and cost-
effective electron donor for achieving biological sulfate reduction in FBR
systems.
9. Identify operational and design problems in the overall treatment process
system, and suggest changes in process designs for the potential
implementation and operation of pilot-scale systems.
10. Investigate the potential economically valuable products including
hydrogen fuel, methane, and elemental sulfur, from the products of the
biological sulfate reduction.

67
Chapter 4

MATERIALS AND METHODS

4.1 Materials
4.1.1 Synthetic Wastewater and Constituents
Synthetic wastewater was used as the feed solution for all experiments. The
sulfate concentration range represents a number of industrial wastewaters. The
constituents and concentrations of species in the synthetic wastewater are listed in
Table 4-1.

Table 4-1: Substrate Composition in the Synthetic Wastewater
Constituent Value
pH 7.0
TDS (mg/L) 3300-3500
Sodium, Na
+
(mg/L) 370-500
Calcium, Ca
2+
(mg/L) 250-350
Magnesium, Mg
2+
(mg/L) 110-150
Chloride, Cl
-
(mg/L) 420-470
Sulfate, SO
4
2-
(mg/L) 700-2000
Alkalinity (mg/L CaCO
3
) 90-400

68
Trace nutrients and amino acids were added to the synthetic wastewater. The
trace nutrient composition is presented in Table 4-2. Amino acid concentration was
maintained at 17 mg/L. All chemicals were of reagent-grade quality and were
purchased from VWR Scientific (West Chester, PA).

Table 4-2: Trace Nutrient Composition
Constituent Concentration
CoCl
2
.2 H
2
O (mg/L) 190
MnCl
2
.4 H
2
O (mg/L) 100
ZnCl
2
(mg/L) 70
H
3
BO
3
(mg/L) 62
Na
2
MoO
4
.2 H
2
O (mg/L) 36
NiCl
2
.6H
2
O (mg/L) 24
CuCl
2
.2H
2
O (mg/L) 17
FeCl
2
.4H
2
O (mg/L) 500
HCl (37%, ml/L) 7

4.1.2 Electron Donors
Lactate, was one of the candidate for electron and carbon sources for biological
reduction of high sulfate wastewaters. To produce lactate, lactic acid or 2-
hydroxypropanoic acid, with the chemical formula of CH
3
CHOHCOOH is used, It is a

69
monoprotonic acid and dissociate according to Reaction 4-1 to produce lactate. Lactate
was added in the concentration range of 700-2000 mg/L, depending on the sulfate
concentration.
+ −
+ → H CHOHCOO CH CHOHCOOH CH
3 3
(4-1)

Another candidate was acetate, which used in the form of sodium acetate,
Sodium acetate dissolved easily to produce acetic acid, which dissociates to acetate
according to Reaction 4-2. Acetate was added to the reactor in the concentration
range of 500-1700 mg/L, depending on the sulfate concentration.
+ −
+ → H COO CH COOH CH
3 3
(4-2)

Citrate and ethanol C
2
H
6
O

were among the other electron donors that have
been tested in this research. Ethanol (90%) provided from Alfa Aesar (Ward Hill,
MA). To provide citrate, citric acid, C
6
H
8
O
7
.H
2
O, was used, which can further
dissociate into citrate. Citric acid was provided from Mallinckrodt Baker Inc.
(Phillipsburg, NJ) in the granular form. Figure 4-1 presents the chemical structure
for citric acid.

Figure 4-1: Citric Acid Structure
O
O
HO
OH
OH
OH
O

70
Molasses was purchased from Mott’s U.S.A., an associate of Cadbury
Beverage, Inc. (Alpharetta, GA). It contained 12 g sugar per 15 ml concentrated
molasses. Formic acid was also used as electron donor in this research. It was used in
the sodium salt form (sodium formate), and it was purchased from the EMD
chemicals (San Diego, CA) with 99% purity.

4.1.3 Granular Activated Carbon
Granular activated carbon (GAC) was used as supporting media in the
anaerobic fluidized bed reactors. GAC with effective carbon particle size of 1.68-
2.00 mm (US Standard 8/10 mesh size) was purchased from Carbon Activated
Corporation (Compton, CA). In order to remove carbon fines and ashes, it was
thoroughly washed with distilled water and dried at 105
o
C overnight. After
desiccation, the carbon was stored in an airtight container.

4.2 Analytical Methodologies
4.2.1 Sulfate, Acetate, and Lactate Measurement
Solution samples were filtered through a 0.2-μm membrane disc filter (VWR
Scientific, West Chester, PA) to remove microorganisms and suspended particles,
and stored in a refrigerator at 4
o
C prior to analysis. Sulfate, acetate, and lactate were
measured by using an ion chromatograph (DIONEX DX-100, Dionex Corp.,
Sunnyvale, CA) equipped with the Dionex AS4A anions specific column, and a
conductivity meter detector. The eluent solution consisted of mixture of sodium
carbonate, and sodium bicarbonate. This solution was maintained at a flow rate of

71
1.2 mL/min. Nitrogen gas was used for eluent pressurization. Samples diluted to
appropriate concentration ranges before injection into the chromatograph.

4.2.2 Measurement of Alkalinity
Alkalinity was measured by the titration method (Method 2320B) as
described in Standard Methods (1998). In this procedure, 50 ml of unfiltered sample
titrated against standard sulfuric acid solution (0.1, 0.025, and 0.01 N H
2
SO
4
) to an
end-point pH of 4.5 or 4.3.

4.2.3 Measurement of Hydrogen Sulfide
Hydrogen sulfide was measured using the iodometric method (Method 4500
F) as outlined in Standard Methods (1998). A quantity of 200 ml of the sample was
acidified by using 2 ml of 6N hydrochloric acid. Standardized iodine solution with
0.1 N concentrations was added and then back titrated with 0.025 N standardized
thiosulfate solutions.

4.2.4 Measurement of Suspended Solids, Volatile Suspended Solid
The gravimetric method (Method 2540, Standard Methods, 1998) was used to
measure the amount of suspended solids (SS) and volatile suspended solids (VSS).
Filter papers were dried at 105
o
C and after cooling in a desiccator, their weight was
recorded. A 50-ml volume of samples was then filtered through 0.2-micron glass
fiber filters (Whatman GF/C, VWR Scientific, West Chester, PA). The filters

72
containing biomass were dried at 103-105
o
C for 1 hour and cooled to room
temperature in a desiccator. The difference in the weight of the filter paper and the
sample after drying were recorded as suspended solids concentration. The filter was
then heated to 550
o
C for 15 minutes to volatilize any organic contents. The filter was
subsequently cooled in a desiccator, and its weight was measured. The difference in
filter paper weight after drying at 105
o
C and after combustion was recorded as VSS
concentration.

4.2.5 Analysis of Attached Volatile Solids
Typical biomass samples were obtained from the FBR-GAC column and
measured in terms of attached volatile solids (AVS) per gram of dry GAC, according
to Standard Methods (1995). The bioparticles obtained from the reactor were washed
with water to remove any nonattached biomass, dried at 105
o
C for 24 hours, cooled
in the air, and weighed. The bioparticles were then combusted at 550
o
C for 30
minutes, cooled, and re-weighed. After washing the biomass-free particles with weak
acid to remove the ash content, they were dried at 105
o
C for another 24 hours,
cooled, and weighed. The difference in the weights yielded the amount of attached
biomass. The dried GAC was weighed and used in the calculation of biomass in
terms of attached biomass per gram of dried GAC.

73
4.2.6 Analysis of the FBR Column Off-Gases, Methane and H
2
S
Gas samples from the FBR columns were collected in sealed gas sampling
bags, and the sampling times were measured. The gas samples were injected into a
GC-FID (HP-6890 with DB-1 column) for analysis of methane.
To measure H
2
S, a zinc acetate solution consisting of 1g/500 ml distilled
water and 0.5 ml of 6N NaOH was prepared. The FBR column effluent line was
inserted into the zinc acetate solution glass bottle, and the time duration was
recorded. The bottle was immediately sealed with a stopper without any air space,
and the solution was mixed slowly. The reaction between zinc acetate and hydrogen
sulfide is presented in Reaction 4-3.

Zn AC + H
2
S → ZnS + HAC (4-3)

The off-gas volume was measured by the water replacement method.
Appropriate amount of NaOH was added to the zinc acetate solution to produce a pH
of 9, and the precipitate was filtered through a filter paper, mixed with 250 ml
distilled water, and the amount of H
2
S was measured by iodometric titration
Standard Methods (1998).

4.2.7 Scanning Electron Microscopy of the Bacterial Growth on GAC
GAC samples from the FBR column were fixed for SEM observation by the
method of Karnovsky (1965). The GAC particles were immersed for 2 hours in a 2%

74
paraformaldehyde/2.5% glutaraldehyde solution in 0.1 M sodium phosphate buffer at
a pH of 7.3. A 3-hour post-fixation followed, using 1% OsO
4
in 0.1 M sodium
phosphate buffer at a pH of 7.3. Subsequently, dehydration was performed by rinsing
the GAC particles in graded ethanol (50, 70, 80, 90, and 100%). GAC samples were
prepared for critical point drying in a 1:1 ethanol-amylacetate solution, and stored
overnight in amylacetate. A DCP-1 critical point dryer was used for drying the fixed
particles. Later, the particles were mounted on aluminum stubs and coated with 100
Angstrom gold (in a glow-discharge coater) to minimize charging and to increase the
conductivity of the biological material (Pirbazari, et al., 1990).

4.2.8 Measurement of Ethanol
A Perkin Elmer gas chromatograph with a flame ionization detector (FID)
was used for the analysis of ethanol. A glass column with a height of 6 feet and a 2
mm inside diameter, packed with GP4 5% Carbowax 20 M on 60/80 Carbopack B
(Supelco, Bellefonte, PA) was employed. Helium was used as the carrier gas, and its
flow rate was maintained at 20 mL/min. The injector, detector, and oven
temperatures were maintained at 150
o
C, 250
o
C and 85
o
C, respectively.

75
4.3 Experimental Methodologies
4.3.1 Preparation of the Synthetic Wastewater and Electron Donor Solutions
Electron donor and the wastewater solutions were prepared in two different
storage tanks to prevent biological growth. The solutions were prepared by passing
de-ionized and air stripped water through two ion exchange cartridges in series
(USFilter, USA. Each solution tank later underwent oxygen stripping with nitrogen
gas. The alkalinity and concentration of some metallic species (sodium, calcium,
magnesium, and chloride) were increased accordingly as sulfate concentrations in
FBR influent were increased from 700 to 2000 mg/L. Electron donors such as
lactate, hydrogen, and acetate were fed with varying concentrations according to
different carbon to sulfur ratios. In CMBR studies the alkalinity and metallic species
concentrations were prepared according to 700 mg/L sulfate concentration.

4.3.2 Microbial Culture
Anaerobic bacteria were obtained from the Terminal Island wastewater
treatment plant digester. The sludge contained a mixed culture of sulfate reducing
bacteria. The bacteria were acclimated to the electron donors (acetate and lactate,
citrate, ethanol and molasses) in a batch, and in the case of CO
2
and H
2
in a semi-
batch system. These acclimated bacteria were used in the CMBR studies. In addition,
they were used to coat the granular activated carbon in the fluidized bed reactors.

76
4.3.3 CMBR Studies
Batch biokinetic studies were conducted in a completely mixed batch reactor
to evaluate optimal reaction conditions as well as the biokinetic rate coefficients for
different temperatures, pHs, and carbon to sulfur ratios. The CMBR consisted of a
1.5 liter of glass reactor equipped with a sampling port and a biogas discharge port.
All glassware was sterilized in an autoclave to eliminate the possibility of microbial
contamination. The working volume of the glass reactor was 1.2 liter. After the
addition of the nutrient solution and electron donor, the solutions were purged with
nitrogen gas to remove the dissolved oxygen, and then 10 ml of concentrated
biomass was added. For each set of experiments, the bacteria were acclimated to the
electron donor for 2 to 4 weeks. The biomass concentrations in each batch reactor
were kept at a concentration range of 70-80 mg/L. The reactors were sealed with
rubber stoppers to maintain anaerobic conditions. A submersible heater and
thermometer were used to control the water bath temperature. Figure 4-2 presents the
experimental setup used in these experiments.
The content of CMBRs was mixed with a magnetic stirrer. The biogas from
the reactor was passed through a zinc acetate solution in a glass reservoir to remove
the generated hydrogen sulfide. Samples were periodically withdrawn from the
reactor, and the concentrations of sulfate and electron donor were measured.

77

7
3
2
4
5
1
6

1. CMB Reactor 5. Sampling Port
2. Water Bath 6. Gas Effluent
3. Zinc Acetate Scrubber Solution 7. Magnetic Stirrer
4. Hydrogen Sulfide Gas Exhaust Line

Figure 4-2: Schematic of Experimental Setup for Completely Mixed Batch Reactor
Studies

78
4.3.4 Fluidized Bed Reactors
Different anaerobic FBR systems were employed to investigate the extent of
biological sulfate reduction, as outlined below:

• FBR I: System with hydrogen gas as the electron donor, and carbon dioxide
as the carbon source (sulfate concentration of 1000-1100 mg/L).
• FBR II: System with acetate as carbon and electron source (sulfate
concentration 1000-1100 mg/L).
• FBR III: System with acetate as carbon and electron source (sulfate
concentration of 2000 mg/L).
• FBR IV: System with lactate as carbon and electron source (sulfate
concentration of 1000-1100 mg/L).
• FBR VI: System with influent-effluent mixture, utilizing lactate as carbon
and electron source (sulfate concentration of 2000 mg/L).

The fluidized bed reactors were constructed from Plexiglas with an inner
diameter of 1.5 inches and the height of 42.5 inches. They had an empty volume of
1.23 L. The FBR outer layer had a water jacket connected to a constant temperature
water bath. The working volume of each reactor under non-fluidized conditions was
about 900 ml, with a hydraulic retention time of 2.5 hours, corresponding to an
influent flow rate of 6 ml/min. The recirculation pump was adjusted to 1.5 L/min to
provide 32-inch active reactor height. The operating conditions of the anaerobic

79
fluidized bed reactors are summarized in Table 4-3. Three pumps were used to
provide the flow into the fluidized bed reactor. The first two pumps supplied the
synthetic feed solution with a flow of 4 ml/min, and the electron donor with a flow of
2 ml/min. The third pump automatically controlled the pH. A recirculation flow rate
of 1.55 L/min was produced by a centrifugal pump (Cole-Palmer, Vernon Court, IL).
Granular activated carbon (GAC) was used as the supporting media. In order to have
a uniform distribution in the influent flow, glass beads with 5 mm diameter were
placed on a perforated tray placed at the entrance port to produce a uniform flow
distribution. Bacteria-coated GAC were charged into the FBR column and the
system was operated in the batch mode for one day to promote biofilm growth. The
nutrient medium was subsequently fed to the reactors, and the pH was adjusted
automatically by injecting an acid or base solution.

Samples from the fluidized bed reactors were tested for sulfate, lactate or
acetate, and hydrogen sulfide content. The sulfate concentration was gradually
increased with time to optimize the bacteria acclimation, and prevent any shock from
sudden increase in hydrogen sulfide production. A number of investigators employed
an oxidation reduction potential (ORP) control system to oxidize the produced H
2
S
into elemental sulfur in a controlled oxidative environment (Khanal and Huang,
2003). In this study, however, there was no need to use an ORP oxidation system to
eliminate the H
2
S inhibition on the sulfate reduction.

80
Table 4-3: Characteristics of Fluidized Bed Reactors
Characteristics

Value
Influent Flow Rate (ml/min)
Feed (synthetic wastewater +Nutrients)
Electron Donors
6
4
2
Hydraulic Retention Time (HRT) (hr) 2.5
Expansion Rate (%) 40
Active Volume (L) 0.9
Temperature (
o
C) 30
Packing Medium GAC (330 g, US mesh size 8-10)
Recirculation Rate (L/min) 1.55
pH 7.5

Biofilter and effluent polishing bioreactors were used to remove hydrogen
sulfide from the FBR off-gas and FBR effluent solution by biologically converting
sulfide to elemental sulfur.

81
4.3.5 FBR System with Hydrogen and Carbon Dioxide
Evaluations of hydrogen gas as an electron source and carbon dioxide as a
carbon source were conducted as part of this research. Utilization of hydrogen as
electron donor is a thermodynamically favorable reaction (ΔG
o
= -124 kJ/mole H
2
)
as shown in Table 2-7. The products of the hydrogen gas oxidation and sulfate
reduction are only sulfide and water (equation 2-13). This makes hydrogen a good
electron donor candidate for treatment of sulfate-rich wastewaters, where discharge
of the extra COD to the water bodies is of major concern.
In this study, hydrogen and CO
2
were introduced inside an 800-ml liquid-gas
contactor by two sets of ultra-fine diffusers. The H
2
/CO
2
solution mixture was then
injected into the FBR. Gas flow meters measured the gas flow rates. The operation
conditions are presented in Table 4-3. The average hydrogen and carbon dioxide
flow rates were 44.8 ml/min and 8.9 ml/min, respectively. Figure 4-3 presents the
schematic diagram for the FBR system with hydrogen and carbon dioxide gas.

82
3
8
1
2
9
1
10
4
5
6
7
12
11
15
13 14
16
17

1. Fluidized Bed Reactor 10. pH Controller
2. Water Jacket 11. Anaerobic Biofilter
3. Water Bath 12. Air Vent
4. Feed Solution 13. Carbon Dioxide Cylinder
5. Acid Solution 14. Hydrogen Gas Cylinder
6. Base Solution 15. Flow Meter
7. Pump 16. Gas-Liquid Contactor
8. Recirculation Pump 17. Bioreactor for Removing Effluent H
2
S
9. Effluent
Figure 4-3: Schematic of the FBR System with Hydrogen and Carbon Dioxide Gas

83
4.3.6 Hydrogen Sulfide Sparging System
Biological sulfate reduction produces hydrogen sulfide. Part of the hydrogen
sulfide will remain in the solution as the soluble forms of HS
-
and S
2-
, while part of it
transfers into a gas phase as H
2
S gas. The hydrogen sulfide toxicity necessitates its
removal in the FBR off-gas, and from the FBR effluent solution. Biofilters manage
to effectively control the produced hydrogen sulfide in the off-gas. In order to
remove the dissolved hydrogen sulfide, H
2
S was sparged out of the system and the
off-gases were passed through a biofilter.
A counter-current hydrogen sulfide sparging system was employed in these
experiments. A packed column of plastic rings (Jaeger Rings, Jaeger Production
Inc., Houston, TX) was used to increase the gas/liquid contact. Table 4-4 lists the
stripping column specifications. The specification of the packing material is
presented in Table 4-5.

Table 4-4: Hydrogen Sulfide Stripping System Specifications
Characteristics Value
Diameter (in) 2.5
Area (m
2
) 0.0032
Column Height (in) 26
Gas Flow Rate (L/min) 1, 3, 5, 7
Liquid Flow Rate (ml/min) 0, 5, 6, 10, 15, 20, 25
pH 6, 6.5, 7, 7.5
Packing Material Jaeger Rings

84
Table 4-5: Jaeger Rings Packing Properties
Properties Value
Size (in) 5/8
Geometric Surface Area (ft
2
/ft
3
) 108
Packing Factor (ft
-1
) 97
Void Space (%) 86
Bulk Density (lb/ ft
3
) 7.75

9
2
1 8
4
6
7
5
3

Figure 4-4: Schematic for Hydrogen Sulfide Sparging System

1. Effluent from FBR
2. Pump
3. Wastewater Influent
4. Packed Column
5. Gas Effluent
6. Support Plate
7. Wastewater Effluent
8. Gas Flow meter
9. Nitrogen Gas Cylinder

85
Effluent from the FBR column was introduced into the sparging reactor from
the top, and nitrogen gas was delivered to the bottom of the reactor through a fine
diffuser. Samples from the influent and effluent were taken periodically and
analyzed for hydrogen sulfide concentration. Figure 4-4 shows the schematic for the
H
2
S sparging system.

4.3.7 FBR System with Lactate and Acetate
Lactate or acetate solution was introduced into the FBR from a feed reservoir.
Nutrients and electron donor solutions were pumped into the FBR from two separate
feed reservoirs in order to prevent bacterial growth in the feed reservoir. These sets
of experiments had similar operational conditions as presented in Table 4-3. The
experimental setup is shown in Figure 4-5.

86

3
8
1
2
13
12
10
4
5
6
7
11
14
9

1. Fluidized Bed Reactor 8. Recirculation Pump
2. Water Jacket 9. Effluent
3. Water Bath 10. pH Controller
4. Feed Solution 11. Anaerobic Biofilter
5. Acid Solution 12. Air Vent
6. Electron Donor Solution 13. Sampling Port
7. Pump 14. Bioreactor for Removing H
2
S from Effluent
Stream

Figure 4-5: Schematic of the FBR System with Lactate or Acetate as Electron Donor

87
4.3.8 FBR System with Effluent Mixing
In the FBR with lactate, a new strategy was employed. In these experiments,
part of the effluent was pumped into the influent line to dilute the influent and to
control the influent sulfate concentration. The effluent was first filtered through a
membrane filter, and then stored in a 20-L glass container. It was later pumped into
the influent line.
This method avoids bacteria from receiving shock due to high sulfate loads,
and furthermore, less biomass is produced in the FBR reactor. In addition, the system
produces less H
2
S, and utilizes the extra electron donor in the effluent line by
recycling, which helps the system to work more efficiently. Figure 4-6 presents the
experimental setup for FBR with effluent mixing.

88

1. Fluidized Bed Reactor 10. pH Controller
2. Water Jacket 11. Anaerobic Biofilter
3. Water Bath 12. Biofilter Effluent
4. Feed Solution 13. Effluent Stream Storage Tank
5. Acid Solution 14. Membrane Filter
6. Electron Donor Solution 15. Sampling Port
7. Pump 16. Bioreactor for Removing H
2
S from
8. Recirculation Pump Effluent Stream
9. Effluent

Figure 4-6: Schematic of the FBR System with Effluent Mixing
4
5
6
7
1
3
2
8
13
14
10
12
11
9
16
15
15
15
15
15

89
4.3.9 Biofilter for H
2
S Off-gas from FBR Column
A laboratory-scale biofilter was used in this study. It consisted of a Plexiglas
column 26 inches long and 3 inches in diameter. GAC was employed as support
material, and packed inside the Plexiglas tube to a height of 17.5 inches, yielding 3 L
of packing volume. The GAC was then coated with activated sludge microbial
culture obtained from a wastewater treatment plant. The inlet system ensured
uniform gas distribution across the bed. The composition and physical properties of
the biofilter are presented in Table 4-6. Operation conditions for the biofilter are
listed in Table 4-7.

Table 4-6: Physical Properties of the H
2
S Biofilter
Physical Properties
Diameter of column (inches) 3 (7.62 cm)
GAC mesh size (US Standard) 8-10
Height of the column (inches) 26 (66 cm)
Height of the GAC packing (inches) 17.5 (44.45 cm)
Volume of porous area (ml) 450

Table 4-7: Operation Conditions of the H
2
S Biofilter
Operation Parameter
Gas retention time (min) 0.9
Inlet H
2
S concentration (mg/L) Varied (200-600)
Gas flow rate (ml/min) 0- 400

90
The H
2
S produced in the anaerobic FBR columns entered the biofilter from
the bottom of the column. A nutrient solution (Table 4-2) was introduced into the
column weekly. The nutrient solution was purged with nitrogen gas to eliminate
oxygen before usage in the biofilter. Gas samples were taken from the inlet and exit
sampling ports. No H
2
S was detected in the biofilter effluent throughout the
experiments.

91
Chapter 5

RESULTS AND DISCUSSION

5.1 CMBR Studies
CMBR studies were employed to determine the optimum conditions for
sulfate reduction. Two different sets of biokinetic studies were conducted in CMBR
systems using acetate and lactate as electron donors. For each electron donor, the
investigations were carried out under different pH conditions, carbon/sulfur ratios,
and temperatures. Sulfate, acetate, lactate, citrate, molasses and biomass were
measured in regular intervals, and the results were utilized to calculate the biokinetic
parameters. The sulfate reduction and electron donor utilization concentration
profiles as functions of time were determined, and the rate constants were estimated.
The results of these studies are depicted in Figures 5-1 through 5-12, and the
estimated zero-order rate constants for sulfate reduction and electron donor
utilization are presented in Tables 5-2 through 5-8.

5.1.1 CMBR Studies with Acetate as the Electron Donor
The biokinetic studies using acetate as the electron donor were conducted
under the conditions discussed previously. In these experiments, acetate was used as
the sole electron donor. Different environmental parameters, including pH,
temperature, and carbon/sulfur ratio were tested. In each set of experiments, one
main parameter was tested while the others were kept constant. The results of each

92
parameter optimization were used in the next parameter optimization to finally arrive
at the optimum conditions. These studies were performed with an initial sulfate
concentration of 700 ppm, and the nutrient composition listed in Table 4-2. Table 5-1
presents the experimental conditions for the CMBR studies. In the presentations of
the kinetic data, the initial lag phase and the final stationary phase have been
omitted, and the growth kinetics have been calculated for the logarithmic growth
phase.

Table 5-1: Experimental Conditions in CMBR Studies with Acetate
Parameters Variable Experimental Condition
pH 6.0, 7.0, 7.5, 8.0 Temperature: 30
o
C, C/S= 1
Sulfate Concentration: 700 mg/L
C/S Ratio 0.8, 1.0, 1.2, 1.4 Temperature: 30
o
C, pH=7
Temperature
(
o
C)
20, 25, 30, 35 C/S= 1, pH= 7
Sulfate Concentration: 700 mg/L

5.1.1.1 Different pHs

The first set of experiments was conducted under different pHs, namely 6.0,
7.0, 7.5, and 8.0 using a constant C/S ratio of 1.0, and a temperature of 30
o
C. In these
experiments the sulfate utilization, and acetate uptake followed zero-order reaction
rates. The sulfate and acetate concentration profiles are presented in Figures 5-1 and
5-2, respectively. Table 5-2 summarizes the zero-order rate constant values for
sulfate reduction and acetate utilization as well as percentages. The sulfate reduction

93
and acetate utilization concentration profiles as well as reduction rates denoted by
their rate constants indicate that the optimal pH was about 7.0.

Table 5-2: Kinetic Results at Different pHs with Acetate as Electron Donor
Parameters Range Rate
Constant k
(mg /L/hr )
for Sulfate
Reduction
Rate
Constant k
(mg /L/hr )
for Acetate
Utilization
Sulfate
Removal
(%)
Acetate
Utilization
(%)
pH 6.0
7.0
7.5
8.0
5.60
5.07
3.92
2.53
6.50
6.30
4.24
2.98
48.9
38.5
73.6
63.6
100
100
100
100

The pH determines the degree of dissociation of hydrogen sulfide, which
corresponds to the degree of inhibition for SRB growth; therefore, pH is an
important factor and must be controlled for a successful experiment. Figure 5-2 and
Table 5-2 reveal that acetate utilization is faster at pHs of 6.0 and 7.0. The kinetic
results with respect to sulfate reduction show, that pHs of 8.0 and 7.5 have zero-
order rate coefficients (k) of 2.53, and 3.92, respectively, which are lower than the
kinetic rate of 5.07 corresponding to pH 7.0. The results for pH 6.0 show a rate
coefficient of 5.60, which is close to the k value for the pH of 7.0. For the pH of 6.0,
the sulfate removal was incomplete, and a 350-mg/L sulfate remained at the end of
the CMBR studies, whereas at pH of 7.5 the sulfate residual was only 220-mg/L,
although it has significantly a smaller k value. The reaction kinetics for a pH of 8.0
was very slow. Based on these observations, pH of 7.5 was the best pH for acetate
utilization, with the most sulfate removal efficiencies.

94
0
100
200
300
400
500
600
700
800
0 20 40 60 80 100 120 140 160 180
Time (hr)
Sulfate Concentration (mg/L)
pH= 8
pH= 7.5
pH=7
pH=6

Figure 5-1: Sulfate Reduction Rates at Different pHs with Acetate as Electron Donor
0
50
100
150
200
250
300
350
400
450
500
550
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Time (hr)
Acetate Concentration (mg/L)
pH=8
pH=7.5
pH=7
pH=6

Figure 5-2: Acetate Utilization Rates at Different pHs

95
5.1.1.2 Different Temperatures

The effects of reaction temperature with respect to the biokinetics of sulfate
reduction and acetate utilization are presented in Figures 5-3 and 5-4, respectively. In
this set of experiments, the pH and the C/S ratio were maintained at 7.0 and 1.0,
respectively, while the reaction temperature was varied from 20
o
C to 35
o
C in
increments of 5
o
C.
These studies qualitatively showed that reaction rates improved with
temperature up to 35
o
C. It was found that sulfate removal drastically dropped at 40
o
C
and beyond. In calculation of the removal efficiencies for sulfate, a temperature of
35
o
C showed the fastest reaction rate (6.39 mg/L-hr) with residual sulfate
concentration as low as 60 mg/L. Nonetheless, it is important to note that reaction
rate constants for sulfate reduction and acetate utilization are marginally larger at
35
o
C than at 30
o
C, indicating that an increase in reaction temperature beyond 30
o
C
did not result in a significant advantage in process kinetics, although it entailed
higher energy costs from an operational standpoint.
Table 5-3 lists the rate constant values, sulfate removal rates, and acetate
utilization rates. The rate constant (k) for a temperature of 20
o
C was very low (3.23
mg/L-hr), and sulfate reduction was not efficient (440 mg/L sulfate residual). Acetate
utilization at different temperatures shows the fastest reaction rate at 35
o
C (k= 7.06
mg/L-hr), but 30
o
C was selected as the optimum temperature for the studies.

96
Table 5-3: Kinetic Results at Different Temperatures with Acetate as Electron
Donor
Parameters Range Rate Constant k
(mg /L/hr ) for
Sulfate
Reduction
Rate Constant
k
(mg /L/hr ) for
Acetate
Utilization
Sulfate
Removal
(%)
Acetate
Utilization
(%)
Temperature
(
o
C)
20
25
30
35
3.23
3.27
5.67
6.39
4.93
2.72
6.51
7.06
38.60
75.69
43.93
82.97
100
100
100
100

0
100
200
300
400
500
600
700
800
0 20 40 60 80 100 120 140 160
Time(hr)
Sulfate Concentration (mg/L)
T=35
T=30
T=25
T=20

Figure 5-3: Sulfate Reduction Rates at Different Temperatures with Acetate as
Electron Donor

97
0
100
200
300
400
500
600
700
0 20 40 60 80 100 120 140 160 180 200 220
Time (hr)
Acetate Concentration (mg/L)
T=35
T=30
T=25
T=20

Figure 5-4: Acetate Utilization Rates at Different Temperatures

5.1.1.3 Different C/S Ratios
For these sets of CMBR studies, the pH and temperature were maintained at
7.0 and 30
o
C, respectively, and the C/S ratio was varied from 0.8 to 1.4 in
increments of 0.2. The sulfate and acetate concentration profiles are presented in
Figures 5-5 and 5-6, respectively. Table 5-4 presents the reaction rate constants for
sulfate reduction and acetate utilization as well as the associated removal and
utilization percentages.
The rate constants for sulfate reduction show a steady increase with
increasing C/S ratios, from 2.17 to 7.50 mg/L-hr. At a C/S ratio of 1.2, almost the
entire sulfate was removed. However, at lower C/S ratios of 0.8 and 1.0, after
complete utilization of the acetate, some sulfate remained in the reactor. This

98
indicates that the electron donor (acetate) could be rate limiting. The results indicate
that the higher C/S ratio of 1.4 did not manifest any significant advantage.
Nevertheless, it appears that a C/S ratio of 1.0 represents a near-optimal condition, as
reflected by the reaction rate constants presented in Table 5-4.

Table 5-4: Kinetic Results at Different C/S Ratios with Acetate as Electron Donor
Parameters Range Rate Constant
k
(mg /L/hr ) for
Sulfate
Reduction
Rate Constant
k
(mg /L/hr ) for
Acetate
Utilization
Sulfate
Removal
(%)
Acetate
Utilization
(%)
Carbon to
Sulfur Ratio
0.8
1.0
1.2
1.4
2.17
4.84
7.42
7.50
3.18
6.18
6.53
8.50

48.65
49.70
94.68
98.48
100
100
82.03
97.82

0
100
200
300
400
500
600
700
800
0 20 40 60 80 100 120
Time (hr)
Sulfate Concentration (mg/L)
C/S=1.4
C/S=1.2
C/S= 1
C/S=0.8

Figure 5-5: Sulfate Reduction Rates at Different C/S Ratios with Acetate as Electron
Donor

99
The efficiencies of sulfate reduction at high carbon to sulfur ratios of 1.2 and
1.4 were 95% and 98%, respectively. However, at lower carbon to sulfur ratios of
0.8, and 1.0, the efficiencies reduced to near 49% and 50%.

0
100
200
300
400
500
600
700
800
900
0 20 40 60 80 100 120
Time (hr)
Acetate Concentration (mg/L)
C/S=1.4
C/S=1.2
C/S=1
C/S=0.8

Figure 5-6: Acetate Utilization Rates at Different C/S Ratios

These investigations showed that the near-optimal pH, C/S ratio, and
temperature for biological sulfate reduction using acetate as the electron donor were
7.0, 1.0, and 30
o
C, respectively.

100
5.1.2 CMBR Studies with Lactate as the Electron Donor

Batch biokinetic studies for lactate as the electron donor were performed to
investigate the sulfate reduction at different pHs, C/S ratios, and temperatures. A
temperature of 20
o
C did not yield sufficient microbial growth for anaerobic sulfate
reduction; therefore, the data for this temperature have not been reported. As a first step,
different pHs were tested to obtain the optimum pH. The results were used in the next
sets of experiments to investigate different C/S ratios, and finally the optimum pH and
C/S ratio were employed to find the optimum temperature. The results of CMBR
biokinetic investigations for lactate are presented in Figures 5-7 through 5-12.

5.1.2.1 Different pHs

In order to evaluate the effect of pH on reaction kinetics, CMBR experiments
were conducted at different pHs, namely 6.0, 6.5, 7.0, and 7.5 while maintaining a
C/S ratio of 1.0, and temperature of 30
o
C. The sulfate and lactate concentration
profiles are presented in Figures 5-7 and 5-8, respectively. Table 5-5 shows the
values for rate constants, sulfate removal rates, and acetate utilization rates. The table
also demonstrates that in all of these studies the reduction did not go to completion. For
example, for maximum lactate utilization (71%), sulfate reduction was only 35%. These
results indicate that lactate was rate limiting and that a C/S ratio of 1.0 was too low. In
addition, the results show that when the concentration of lactate reached a minimum of
100 mg/L, the sulfate reduction stopped. Figure 5-7 shows that the final sulfate
concentrations ranged between 450 and 500 mg/L.

101

Table 5-5: Kinetic Results at Different pHs with Lactate as Electron Donor.
Parameters Range Rate Constant k
(mg /L/hr ) for
Sulfate
Reduction
Rate Constant
k
(mg /L/hr ) for
Lactate
Utilization
Sulfate
Removal
(%)
Lactate
Utilization
(%)
pH 6.0
6.5
7.0
7.5
4.50
3.24
9.28
4.22
4.91
4.50
9.01
5.05

27.5
22.0
34.7
34.4
65.5
68.3
71.5
64.7

The sulfate and lactate concentration profiles as well as reduction rates
denoted by their rate constants indicate that the optimal pH was 7.0. Therefore, a pH
of 7.0 was used in the next set of experiments.

0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
0 2 0 4 0 6 0 8 0
T im e (h r)
Sulfate Concentration (mg/L)
p H = 7 .5
p H = 7
p H = 6 .5
p H = 6

Figure 5-7: Sulfate Reduction Rates at Different pHs with Lactate as the Electron
Donor

102

0
50
100
150
200
250
300
350
400
0 10 20 30 40 50 60 70
Time (hr)
Lactate Concentration (mg/L)
pH=7.5
pH=7
pH=6.5
pH=6

Figure 5-8: Lactate Utilization Rates at Different pHs

5.1.2.2 Different C/S Ratios

In these sets of CMBR studies, a pH of 7.0 and temperature of 30
o
C were
utilized. In the next set of experiments, carbon to sulfur ratios of 1.0, 2.0, 2.5 and 3.0
were used and the results are reported in Figures 5-9 and 5-10 and Table 5-6. The
results indicate that the higher C/S ratios of 3.0 and 2.5 did not manifest any
significant advantage; a C/S ratio of 2.0 represented a near-optimal condition, as
reflected by the reaction rate constants presented in Table 5-6. Therefore, a carbon-
to-sulfur ratio of 2 was chosen in these experiments.

103

Table 5-6: Kinetic Results at Different C/S ratios with Lactate as Electron Donor
Parameters Range
Rate Constant
k
(mg /L/hr ) for
Sulfate
Reduction
Rate Constant
k
(mg /L/hr ) for
Lactate
Utilization
Sulfate
Removal
(%)
Lactate
Utilization
(%)
Carbon/Sulfur
Ratio
1.0
2.0
2.5
3.0
9.29
9.99
9.94
9.94
5.73
14.40
14.69
19.91
34.7
96.0
96.2
97.7
71.5
70.2
70.6
77.2

0
100
200
300
400
500
600
700
800
0 20 40 60 80 100
Time (hr)
Sulfate Concentration (mg/L)
C/S=1
C/S=2
C/S=2.5
C/S=3

Figure 5-9: Sulfate Reduction Rates at Different C/S Ratios with Lactate as the
Electron Donor

104

0
500
1000
1500
2000
2500
0 50 100 150
Time (hr)
Lactate Concentration (mg/L)
C/S=1
C/S=2
C/S=2.5
C/S=3

Figure 5-10: Lactate Utilization Rates at Different C/S Ratios.

5.1.2.3 Different Temperatures
In these experiments, the effect of temperature on the biokinetics of sulfate
reduction (Figure 5-11), and lactate utilization (Figure 5-12) were investigated. The
pH and the C/S ratio were maintained at 7.0 and 2.0, respectively, while the reaction
temperature was varied from 25
o
C to 35
o
C in increments of 5
o
C. The results for 20
o
C
were not included in these figures due to the slow reaction kinetics. Table 5-7 lists
the rate constants for sulfate reduction and lactate utilization and the corresponding
removal efficiencies.

105
0
100
200
300
400
500
600
700
0 20 40 60 80
Time (hr)
Sulfate Concentration (mg/L)
T=25oC
T=30oC
T=35oC

Figure 5-11: Sulfate Reduction Rates at Different Temperatures with Lactate as the
Electron Donor

Table 5-7: Kinetic Results at Different temperatures with Lactate as Electron Donor
Parameters Range Rate Constant
k
(mg /L/hr ) for
Sulfate
Reduction
Rate Constant
k
(mg /L/hr ) for
Lactate
Utilization
Sulfate
Removal
(%)
Lactate
Utilization
(%)
Temperature 25
30
35
11.90
14.69
20.35
9.729
13.101
18.004
86.45
96.13
84.66
77.14
70.22
64.33

These studies qualitatively showed that reaction rates improved with
increasing temperature. Table 5-7 demonstrates that the fastest sulfate reduction rate
is achieved at 35
o
C (k = 20.35 mg/L/hr), and the slowest at 25
o
C (k = 11.90
mg/L/hr). Figure 5-12 compares the lactate utilization rate profile for different
temperatures of 25
o
, 30
o
, and 35
o
C. Again, the highest temperature of 35
o
C shows

106
the fastest kinetic (k=18.0 mg/L/hr) and the lowest temperature, 25
o
C, has the
slowest kinetic (k= 9.7 mg/L/hr). Based on the above observations, it appears that
35
o
C would be the optimum temperature for sulfate reduction. Nonetheless, for
economical considerations (energy saving), 30
o
C was chosen in these studies. Based
on the above investigation, the near-optimal pH, C/S ratio, and temperature for
sulfate reduction using lactate as the electron donor were 7.0, 2.0, and 30
o
C,
respectively.

0
200
400
600
800
1000
1200
1400
1600
0 25 50 75 100
Time (hr)
Lactate Concentration (mg/L)
T=25oC
T=30oC
T=35oC

Figure 5-12: Lactate Utilization Rates at Different Temperatures

107
The results of the CMBR studies with acetate and lactate are summarized in
Table 5-8.

Table 5-8: CMBR Kinetic Results with Different Electron Donors

Acetate as
Electron Donor
Parameter
Range
Rate Constant k
(mg /L/hr ) for
Sulfate Reduction
Rate Constant k
(mg /L/hr ) for
Acetate
Utilization
Carbon/Sulfur
Ratio
0.8
1.0
1.2
1.4
2.17
4.84
7.42
7.50
3.18
6.18
6.53
8.50

Temperature
(
o
C)
20
25
30
35
3.23
3.27
5.67
6.39
4.93
2.72
6.51
7.06

pH 6.0
7.0
7.5
8.0
5.60
5.07
3.92
2.53
6.50
6.30
4.24
2.98
Lactate as
Electron Donor
Parameter
Range
Rate Constant k
(mg /L/hr ) for
Sulfate Reduction
Rate Constant k
(mg /L/hr ) for
Lactate
Utilization
Carbon/Sulfur
Ratio
1.0
2.0
2.5
3.0
9.29
9.99
9.94
9.94
5.73
14.40
14.69
19.91

Temperature
(
o
C)
25
30
35
11.90
14.69
20.35
9.73
13.10
18.00
pH 6.0
6.5
7.0
7.5
4.50
3.24
9.28
4.22
4.91
4.50
9.01
5.05

108
5.1.2.4 Determination of Biokinetic Parameters

In this research, determinations of the biokinetic parameters were carried out
according to the equations and procedures discussed in Section 2.10.3.
Equation 5-1, a repeat of Equation 2-28, was used to calculate the maximum
specific growth rate μ ˆ
.
To accomplish this, the natural logarithm of biomass
concentration versus time was plotted, and the slope of the exponential growth was
used to determine the maximum specific growth rate. Figures 5-13 and 5-14 present
the maximum specific growth rate for lactate and acetate utilization, respectively.
t
X
X
μ ˆ ) ln(
0
− = (5-1)
R
2
= 0.97
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60 70
Time (hr)
Ln (Biomass Concentration mg/L)

Figure 5-13: Maximum Specific Growth Rate (μ ˆ ) with Lactate as the Electron Donor
μ ˆ = 0.0434 hr
-1

109
R
2
= 0.98
4.4
4.45
4.5
4.55
4.6
4.65
4.7
4.75
4.8
0 20 40 60 80 100
Time (hr)
Ln(Biomass Concentration, mg/L)

Figure 5-14: Maximum Specific Growth Rate (μ ˆ ) with Acetate as the Electron Donor

As presented in Table 5-9, the maximum specific growth (μ ˆ ) with lactate is
seven times larger than with acetate (0.0434 vs 0.0062 hr
-1
)

Equation 5-2, taken from Equation 2-34, was used to calculate the growth
yield, Y (mg/mg). To accomplish this, the concentration of biomass was plotted
against the concentration of substrate.
dS
dX
dt dS
dt dX
Y
−
=
−
=
/
/
(5-2)
μ ˆ = 0.0062 hr
-1

110
The growth yield is the slope of the line during exponential growth. Figures
5-15 through 5-18 present the graphs for the estimation of growth yields for sulfate
reduction and electron donor utilization using lactate and acetate as electron donors.
R
2
= 0.94
0
100
200
300
400
500
600
700
800
0 200 400 600
Sulfate Concentration (mg/L)
Biomass Concentration (mg/L)

Figure 5-15: Growth Yield for Sulfate with Lactate as the Electron Donor

R
2
= 0.92
0
20
40
60
80
100
120
140
300 400 500 600 700
Sulfate Concentration (mg/L)
Biomass Concentration (mg/L)

Figure 5-16: Growth Yield for Sulfate with Acetate as the Electron Donor
Y= 0.9536 mg biomass/mg sulfate
Y= 0.1146 mg biomass/mg sulfate

111
The growth yield for sulfate reduction when utilizing lactate as the electron
donor is 0.9536 mg biomass/mg sulfate (Figure 5-15), and when utilizing acetate it is
0.1146 mg biomass/mg sulfate (Figure 5-16). The yield for lactate utilization is
0.7808 mg biomass/mg lactate (Figure 5-17), and the yield for acetate utilization is
0.0974 mg biomass/mg acetate (Figure 5-18).

R
2
= 0.96
0
100
200
300
400
500
600
700
200 300 400 500 600 700 800 900 1000
Lactate Concentration (mg/L)
Biomass Concentration (mg/L)

Figure 5-17: Growth Yield for Lactate as the Electron Donor
Y= 0.7808 mg biomass/mg sulfate

112
R
2
= 0.96
0
20
40
60
80
100
120
140
0 100 200 300 400 500
Acetate Concentration (mg/L)
Biomass Concentration (mg/L)

Figure 5-18: Growth Yield for Acetate as the Electron Donor

Specific consumption rate, Q
S
(mg/mg-hr), is the slope of the graph of
substrate to biomass changes versus time during exponential growth, as reported
by Cooney et al., (1996) and expressed in Equation 5-3.
Q
S
= dS/dX-t (5-3)
Figures 5-19 through 5-22 present the graphs for the estimation of specific
consumption rates. The specific consumption rates for sulfate with lactate (Figure
5-19) or acetate (Figure 5-21) as electron donors are 0.2714 and 0.0979 (mg/mg-
hr), respectively. Specific consumption rates for lactate (Figure 5-20) and acetate
(Figure 5-22) are 0.3900 and 0.0834 (mg/mg-hr), respectively.
Y= 0.0974 mg biomass/mg sulfate

113
R
2
= 0.93
0
2
4
6
8
10
12
0 10 20 30 40 50
Time (hr)
Sulfate/Biomass

Figure 5-19: Specific Utilization Rate Graph for Sulfate Utilizing Lactate (mg
substrate/mg biomass-time)
R
2
= 0.91
0
2
4
6
8
10
12
14
16
0 20 40 60
Time (hr)
Lactate/Biomass (mg/mg)

Figure 5-20: Specific Utilization Rate Graph for Lactate (mg substrate/mg biomass-
time)

114

R
2
= 0.96
0
1
2
3
4
5
6
7
8
9
10 15 20 25 30 35 40 45 50 55 60
Time (hr)
Sulfate/Biomass (mg/mg)

Figure 5-21: Specific Utilization Rate Graph for Sulfate Reduction with Acetate (mg
substrate/mg biomass-time)

R
2
= 0.99
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 10 20 30 40 50 60 70
Time (hr)
Acetate/Biomass (mg/mg)

Figure 5-22: Specific Utilization Rate Graph for Acetate (mg substrate/mg biomass-
time)

115
Table 5-9 presents a comparison of the biokinetic data obtained from using lactate
and acetate as the electron donor.

Table 5-9: Maximum Specific Growth Rates, Growth Yields, and Specific
Consumption Rates Results
Kinetic Parameter

Sulfate Reduction with
Lactate as Electron Donor
Sulfate Reduction with
Acetate as Electron
Donor
Maximum Specific
Growth Rate, μ ˆ (hr
-1
)
0.0434 0.0062

Kinetic Parameter Sulfate Lactate Sulfate Acetate
Growth Yield, Y,
(g biomass/g substrate)
0.9536 0.7808 0.1146

0.0974
Specific Consumption
Rate, Qs, (mg/mg-hr)
0.2714 0.3900 0.0979 0.0834

5.1.3 Batch Test with Hydrogen Gas as the Electron Donor
No information was available in the literature regarding CMBR studies for
sulfate reduction with hydrogen gas and carbon dioxide. Attempts were made to
employ the standard CMBR tests for gaseous system (hydrogen and carbon dioxide),
however they all failed to yield satisfactory results. Further attempts to construct and
operate a semi-batch system resulted in surmountable operational difficulties.

116
5.1.4 Molasses, Citric Acid, and Formic Acid as Electron Donors
Molasses, citric acid, and formic acid were among the other electron donors
studied in this research. CMBR studies were conducted for formic acid at a C/S ratio
of 1.0, temperature of 30
o
C, and pH of 7.0. No bacterial growth in the CMBR system
was observed, possibly due to the toxicity effect of the formic acid on SRBs.
CMBR studies were also performed for citric acid with a C/S ratio of 1.0,
temperature of 30
o
C, and pH of 7.0. results indicated that the kinetics of the citric
acid utilization was controlled by a two step processes; first citric acid dissociated
into acetate and then the acetate was utilized by the SRB. As can be observed in
Figure 5-23, the first reaction was slow, and it took four days for the dissociation to
reach completion.
0
100
200
300
400
500
600
700
800
900
1000
0 50 100 150 200 250 300
Time (hours)
C o n c e n tra tio n (m g /L )
Sulfate
Acetate

Figure 5-23: CMBR Studies with Citric Acid

117
However understandably, the acetate utilization and the associated sulfate
reduction were similar to those of earlier studies reported for acetate (Table 5-8 and
Figure 5-24). As a result, the CMBR studies with citric acid were discontinued.

Figure 5-24: Sulfate Reduction Rates (Zero-Order Kinetic) in CMBR with Citric
Acid and Acetate as Electron Donors

For molasses as electron donor, the CMBR studies were initially performed
with a C/S ratio of 1.0, temperature of 30
o
C, and pH of 7.0. However, with a C/S
ratio of 1.0, no bacterial growth was observed. At C/S ratio of 1.2, sulfate was
initially reduced from 820 to 590 mg/L, but soon after reduction stopped. Other
k (sulfate)=4.87 ( mg/L-hr)
k (Acetate)=5.27 (mg/L-hr)
0
100
200
300
400
500
600
700
800
900
1000
0 50 100 150 200 250
Time (hours)
Concentration (mg/L)
Sulfate
Acetate
Linear (Sulfate)
Linear (Acetate)

118
CMBR studies with molasses at different pHs and temperatures, did not result
significant reduction. The low sugar level in molasses was considered to be the main
reason for such observations.

5.1.4 CMBR with Ethanol as the Electron Donor
Ethanol was also tested as electron donor. The CMBR biokinetic studies
demonstrated fast sulfate reduction. However, the reduction was incomplete, and
yielded a significant amount of acetate byproduct. Accumulation of acetate when
treating industrial wastewaters with a high sulfate concentration, can be of major
environmental concern from the viewpoint of oxygen depletion in the receiving
water bodies. Table 5-10 presents the CMBR study results with ethanol as the
electron donor (Pirbazari et al., 2006).

119

Table 5-10: CMBR Studies Results with Ethanol as Electron Donor
Parameters Value Sulfate Reduction Rate
(mg /L/hr )
Acetate Utilization Rate
(mg /L/hr )
Carbon/Sulfur Ratio 0.8
1.0
1.2
1.4
10.63
11.94
13.44
15.66
12.53
15.46
17.03
17.48
Temperature (
o
C) 20
25
30
35
4.96
8.67
11.71
13.55
8.54
14.41
19.63
20.69
pH 6.5
7.0
7.5
8.0
13.36
13.82
16.98
----
14.88
14.61
14.81
-----

120
5.2 Biological Sulfate Reduction in Fluidized Bed Reactors
5.2.1 Sulfate Removal Using Hydrogen Gas as the Electron Donor
An anaerobic fluidized bed reactor with recycle was tested to investigate the
effect of carbon dioxide and hydrogen as carbon source and electron donor,
respectively, on the sulfate removal efficiency. Initially membrane contactors were
employed to introduce the gases into the fluidized bed reactors. However, due to the
low aqueous solubility of hydrogen, dissolution of the gas with membrane contactors
was not possible, and high pressure was required to maintain the hydrogen in
solution. A new approach was therefore employed for effective diffusion, mass
transfer, and dissolution of hydrogen and carbon dioxide, by which the gases were
directly injected into the column by means of a micro-porous diffuser, and their
flows were monitored and controlled by standard flow meters.
The hydrogen and carbon dioxide were injected into the reactor at flow rates
of 50 and 14 ml/min, respectively. After an initial acclimation phase of the reactor,
high sulfate reduction efficiencies of 95% were observed on day 28, as shown in
Figure 5-25. Subsequently, the gas flow rates were reduced on day 33. The decrease
in gas flow rates initially resulted in a decrease in the sulfate reduction efficiency,
but subsequently, the reactor experienced a gradual and steady performance
recovery. The sulfate reduction efficiency increased to 94% on day 35, and remained
steady until the influent sulfate concentration was increased from 900 to 1000 mg/L.
On day 40, the reactor efficiency dropped to 62%, possibly as a result of the increase
in sulfate concentration.

121
The higher influent sulfate concentration, along with continuous clogging and
biomass loss during the cleaning operations, warranted a reactor system redesigning.
On day 60, biomass loss due to cleaning decreased the removal rate to 38%. On day
60, two sets of ultra-fine diffusers were introduced directly in the column. As a
result, better sulfate removal rates were observed during the next few days. However,
the ultra-fine diffusers experienced frequent clogging. To circumvent this problem,
ultra-fine diffusers were installed inside a small cylindrical container, and placed in
the recirculation line to enhance the diffusion mass-transfer and dissolution of the
gases in the aqueous phase (day 80). A summary of the operational changes in the
FBR with hydrogen is presented in Table 5-11. The gas flow rates for hydrogen and
carbon dioxide were reduced to 7.4 ml/min and 34 ml/min, respectively. These gas
flow rates were apparently sufficient for the process operation due to the improved
mass-transfer and gas dissolution in the aqueous phase. The reactor responded
favorably to the design changes, and the sulfate reduction efficiency progressively
increased from 62% to 96% over the next 6 days (between days 85 and 91).

122
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
Time (Days)
Sulfate Concentration (mg/L)
0
10
20
30
40
50
60
70
80
90
100
Removal (%)
Influent Effluent Removal (%)

Figure 5-25: Sulfate Reductions with Hydrogen and Carbon Dioxide Gas
(Days 0-91)

On day 92, however, a sudden drop in hydrogen gas injection drastically
decreased the sulfate removal efficiency, as shown in Figure 5-26. Further, reactor
and diffuser cleaning decreased the removal efficiencies (days 111, 124 and 134). A
sulfate reduction efficiency of 64% was observed on day 161, when influent sulfate
concentration was increased to 1500 mg/L. The column operation was stopped on
day 165 due to time limitation.

123

Table 5-11: Summary of Operational Changes in Fluidized Bed Reactor
Time
(Day)
Operational Changes
21 The flow meters were repaired and the flow rates were adjusted
23 Feed problems were encountered
28 New flow meters were installed with these gas flow rates, H
2
, 50
ml/min; CO
2
, 14 ml/min
35 Sulfate concentration increased from 900 to 1000 mg/L
Gas flow rates increased to these values: H
2
, 44.8 ml/min; CO
2
, 8.9
ml/min
40 Sulfate concentration increased from 900 to 1000 mg/L
Gas flow rates increased to these values: H
2
, 50 ml/min; CO
2
, 10 ml/min
45 Influent lines and gas diffusers were cleaned
57 Reactor and diffusers were cleaned
69 New diffusion system was installed with ultra-fine diffusers
80 Hydrogen injection was stopped
81 Gas/liquid contactor was installed in the recirculation line
82 Gas flow rates decreased to these values: H
2
, 7.4 ml/min; CO
2
, 1.8
ml/min
111 Influent lines and gas diffusers were cleaned
124 Influent lines and gas diffusers were cleaned
149 A leak in the gas/liquid contactor was observed
161 Sulfate increased from 1000 to 1500 mg/L

124

0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
88 93 98 103 108 113 118 123 128 133 138 143 148 153 158 163 168
Time (Days)
Sulfate Concentration (mg/L)
0
10
20
30
40
50
60
70
80
90
100
Removal (%)
Influent Effluent Removal (%)

Figure 5-26: Sulfate Reductions with Hydrogen Gas as Electron Donor
(Days 91-166)
It is important to note that the hydrogen and carbon dioxide system operation
was an arduous task due to mass-transfer and solubility limitations of the hydrogen
gas, which necessitated frequent monitoring and maintenance. Nonetheless, the
results were encouraging from two standpoints, namely, achieving high process
efficiency for sulfate reduction, and maintaining low organic residuals in the treated
effluent. More studies are required to maintain consistent system operation.

125
5.3 Hydrogen Sulfide Production in FBR Reactor with Hydrogen Gas as the
Electron Donor
The hydrogen sulfide concentration as a function of time for the FBR column
is presented in Figure 5-27. As expected, hydrogen sulfide production follows a
pattern similar to that of sulfate reduction, with concentrations ranging from 52 to
188 ppm (or mg S
2-
/L) for the duration of the operation.
0
50
100
150
200
250
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
Time (Days)
Hydrogen Sulfide Concentration (mg/L S2-)

Figure 5-27: H
2
S Production Profile for FBR with Hydrogen Gas as the Electron Donor

As can be observed in Figure 5-27, the sulfide concentrations reached
maximum peaks of 166 ppm and 188 ppm on days 36 and 90 respectively. These
values correlated well with the high sulfate removal efficiencies of 92% and 91%,

126
respectively. More experimentation is needed to draw a definite conclusion on the
performance of FBR sulfate reduction systems using hydrogen as the electron donor.

5. 4 Hydrogen Sulfide Stripping from FBR Aqueous Effluent Stream
Hydrogen sulfide stripping tests were performed on the FBR column effluent
stream. Hydrogen sulfide stripping usually employs air, but in this research, nitrogen
gas was used to prevent re-oxidation of sulfide into sulfate. Jaeger rings were used as
the packing material in the hydrogen sulfide sparging system. Properties of the
Jaeger rings packaging are presented in Table 4-5 in the previous chapter.
The stripping ratio (R) was calculated by using Equation 5-4 and following
the gas stripping schematic presented in Figure 5-28.

Figure 5-28: Schematic of a Gas Stripping Column

L
L
G
G

127
t
LP
HG
R=
(5-4)
where:
H: Henry’s constant (atm)
G: Loading rate of gas (KM/m
2
-sec)
L: Loading rate of liquid (KM/m
2
-sec)
P
t
: Total pressure (atm)

In the hydrogen sulfide stripping system, Jaeger rings were used as the
packing material. Properties of the Jaeger rings packing are presented in Table 4-5.
Table 5-12 presents the data for hydrogen sulfide stripping tests. Different liquid
flow rates and loading rates, as well as gas flow rates and loading rates, are
summarized in this table. Table 5-12 also demonstrates the stripping ratio, R, which
is calculated from Equation 5-4.

Table 5-12: Stripping Ratio Calculations
Liquid Flow
Rate (ml/min)
Q
L
(L/m
2
-sec)
Liquid loading
Rate, Q
L
/A
(L/m
2
-sec)
L
(Kmol/m
2
-sec)
Gas Flow Rate
(L/min)
Q
g
(L/m
2
-sec)
Gas Loading
Rate, Q
g
/A
(L/m
2
-sec)
G (Kmol/m
2
-
sec) GLR
/(22.4*1000*27
3/293)
Stripping
Ratio, R
(H=515 atm)
5 8.33E-05 0.0263 0.00146 5 0.083 26.33 0.00109 385.59
6 0.0001 0.0316 0.00175 5 0.083 26.33 0.00109 321.32
10 0.00017 0.0526 0.00292 5 0.083 26.33 0.00109 192.79
15 0.00025 0.0790 0.00438 5 0.083 26.33 0.00109 128.53
20 0.00033 0.1053 0.00585 5 0.083 26.33 0.00109 96.40
25 0.00042 0.1316 0.00731 5 0.083 26.33 0.00109 77.12

128

0
10
20
30
40
50
60
70
80
90
100
0 64 128 192 256 320 384 448 512
Stripping Ratio (R)
Removal Rate (%)
pH = 6
pH =6.5
pH = 7
pH = 7.5
Stripping gas : N
2
Liquid flowrate : 6 mL/min
pH = 6, 6.5, 7, and 7.5
0 L/min 1 L/min 3 L/min
5 L/min
7 L/min

Figure 5-29: Hydrogen Sulfide Stripping with Nitrogen Gas at Different pHs

Figure 5-29 demonstrates that a pH of 6 is most efficient in stripping out
hydrogen sulfide at low gas flow rates. This is consistent with the results reported by
other investigators for hydrogen sulfide stripping in different wastewater treatment
plants (Metcalf and Eddy 2003).

129

Figure 5-30: Hydrogen Sulfide Stripping with Nitrogen Gas at Different Column
Heights

Figure 5-30 presents the results for hydrogen sulfide removal as a function of
packing height in the stripping column. The results reveal that with a 5-inch packing
height, 68% removal was obtained. Increasing the packing height to 10 inches
resulted in 82% removal. Beyond a 15-inch height, the removal efficiency remained
constant at about 86%.
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20 25 30
Column Packing Height (in)
Hydrogen Sulfide Removal (%)
Stripping gas: N2
Gas flow rate (G): 5 L/min
Liquid flow rate (L): 6 mL/min
Liquid pH: 6.5

130
Figure 5-31 presents hydrogen sulfide removal as a function of liquid flow
rate. As is evident from the figure, the maximum sulfide removal can be achieved at
a liquid flow rate of 5 ml/min.

Figure 5-31: H
2
S Sulfide Stripping with Nitrogen Gas at Different Liquid Flow Rates

5.5 Fluidized Bed Reactor Studies with Acetate as the Electron Donor
Acetate was the second electron donor tested for biological sulfate reduction.
During the experiment, acetate concentration was kept at a C/S ratio of 1, which
corresponds to the results obtained from the batch biokinetic studies. The acetate
concentration was increased gradually according to sulfate concentration. The FBR
started as the batch mode, and after two days it was transferred into a continuous
70
72
74
76
78
80
82
84
86
88
0 5 10 15 20 25 30
Liquid Flow (ml/min)
Hydrogen Sulfide Removal (%)
Stripping gas: N 2
Gas flow rate (G): 5 L/min
Liquid flow rate (L): 5, 6, 10, 15, 20, 25 ml/min
Liquid pH= 6.5

131
mode. As alkalinity was produced in the column, it was deemed necessary to adjust
the pH. The pH was automatically maintained at 7.5, based on the results from the
earlier CMBR studies. Figure 5-32 presents the sulfate removal profile in the FBR
with acetate as the electron donor. On day one, the influent sulfate concentration was
850 mg/L. On day 10, sulfate concentration was increased to 950 mg/L. Following
each incremental increase in sulfate concentration, removal decreased presumably
due to shock to the SRBs, but the recovery was quite fast, and the removal rate
increased within a few days. The same pattern was observed on the next incremental
sulfate concentration increases to 1000 mg/L on day 18, and later to 1100 mg/L on
day 26. From day 26 to 70 the sulfate concentration was kept around 1150 mg/L.
Biomass production in the system caused the GAC particles to cement together. As a
result, on day 49, the GAC particles accumulated in the recirculation pump which
necessitates cleaning. Also, carbon particles caused the clogging of the screen mesh
located in the bottom of the reactor, which needed cleaning. Reduction of the
removal in days 49 to 50 was due to biomass washout during the cleaning process.
After day 58, removal rates exceeded 90%. On days 70 and 79, influent sulfate
concentration was increased to 1200 and 1300 mg/L, respectively. Further increases
in influent sulfate concentration to 1400, 1500, 1600, and 1700 on days 88, 98, 105,
and 110, respectively achieved higher than 90% removal efficiencies. At this stage,
excess biomass was observed in the system, causing agglomeration of carbon
particles. On day 110, sulfate concentration was increased to 1800 mg/L. To prevent
GAC particles from sticking to each other, medium height on day 113 was increased

132
from 31.1 to 31.3 inches. On day 117, sulfate influent concentrations were increased
to 1900 mg/L and removal rates as high as 96% were observed. On day 125, sulfate
concentration increased to 2000 mg/L, and removal rates of 95% ± 3 were observed.
Due to the presence of excess biomass in the system, the operation was terminated
on day 130.

Figure 5-32: Sulfate Reduction Results in FBR with Acetate as the Electron Donor

The pattern of acetate utilization followed that of sulfate removal, with
acetate removals higher than 90% being observed in the system (Figure 5-33).
During the experiment, the C/S ratio was kept at 1.0. As evident from Figure 5-33,
Sulfate Concentration (mg/L)
0
500
1000
1500
2000
2500
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
Time (Days)
0
10
20
30
40
50
60
70
80
90
100
Influent Effluent Removal
Removal Efficiency (%)

133
the acetate utilization efficiencies increased quickly from day 1 to 10, until it reached
82% on day 10. Increasing the sulfate influent concentrations on days 10 and 18
resulted in a small decrease in acetate utilization (similar to the pattern shown in
Figure 5-32 for the sulfate profile), but as the system recovered very fast, the acetate
utilization increased accordingly, and on day 26 it reached 87%. The reason for
observing this cyclic pattern is that before each influent sulfate increase, the biomass
concentration is in balance with sulfate concentration, but after each increase more
substrate would be available to biomass to utilize. The decrease in sulfate reduction
actually happens during, the time when the SRB needs to produce more biomass and
provide a steady state balance between sulfate utilization and biomass production.
This pattern again was observed on day 33 after a further increase in sulfate
concentrations. On day 41, agglomeration of the GAC particles caused the FBR
system to stop for a short period, during which a decrease in acetate utilization as
well as drop in sulfate reduction were observed. More increase in sulfate
concentration on days 61 and 65 followed a similar pattern, and a small decrease in
acetate utilization was followed by fast recovery in the acetate utilization
efficiencies. On day 77, sulfate concentrations increased to 1300 mg/L, and a similar
decrease in acetate utilization from 95% to 83% was observed. The system recovered
quickly, and the acetate utilization efficiency increased to 87% on day 82. A further
increase in influent sulfate concentrations on days 88, 98, 105, and 110 followed
similar patterns, with a decrease in utilization efficiencies followed by fast recovery.
The acetate utilization efficiencies were between 82% and 92% during this time.

134
Increase in expansion rate to 31.3 inches on day 113, increased acetate utilization
from 80% to 82%. Further increases in sulfate concentrations to 1900 and 2000 mg/L
on days 117 and 125, respectively, followed a similar decrease and fast increase in
acetate utilization. The reactor was stopped on day 130, at which time the acetate
utilization efficiently reached 88%.

0
200
400
600
800
1000
1200
1400
1600
1800
2000
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
Time (Days)
Acetate Concentration (mg/L)
0
10
20
30
40
50
60
70
80
90
100
Removal Efficiencies (%)
Influent Effluent Removal

Figure 5-33: Acetate Utilization Results in FBR with Acetate as the Electron Donor

Figure 5-34 presents the H
2
S profile in FBR with acetate. H
2
S production in
the liquid effluent of the FBR column was measured from day 18 to 127. As shown

135
in Figure 5-34, the concentration of H
2
S increased progressively from 200 to 600
mg/L. The largest amount of H
2
S was observed on day 122, with 608 mg/L S
2-
. As
discussed earlier, the biomass accumulation caused agglomeration of the GAC
particles, and cleaning of the system resulted in a decrease in the dissolved H
2
S from
day 124 to 129.

0
100
200
300
400
500
600
700
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
Time (Days)
Hydrogen Sulfide Concentration (mg/L)

Figure 5-34: H
2
S Production in FBR with Acetate as the Electron Donor

It is important to note that during these studies, high H
2
S concentration did
not inhibit system performance and sulfate removal as high as 98% was obtained

136
without any need for H
2
S control. The parameters used in the FBR with acetate as
the electron donor are listed in Table 5-13.

Table 5-13: List of the Parameters in the FBR with Acetate as the Electron Donor

Parameters Value
C/S 1
pH 7.5
Temperature (
o
C) 30
HRT (hr) 2.5
Bed Length (in) 31
Recirculation Ratio (L/min) 1.55

5.6 Fluidized Bed Reactor Studies with Lactate as the Electron Donor
Lactate was the third electron donor tested in the FBR system to investigate
sulfate reduction. Lactate was added in the form of lactic acid, and the pH was
adjusted by adding NaOH 0.15 N. The system pH and C/S ratio were maintained at
7.0 and of 2.0, respectively, based on the results of batch biokinetic studies. Figure 5-
35 presents the sulfate reduction results in these sets of experiments.
This FBR experiment was performed in two stages: in the first stage, sulfate
concentration increased gradually to 1100 mg/L. In the second stage, the
concentration of the sulfate input was increased gradually from 1100 to 2000 mg/L;

137
however, a dilution strategy was pursed. In this procedure, the influent sulfate
concentration of 2000 mg/L was mixed with the FBR effluent stream in order to
maintain an FBR column influent concentration between 1200-1350 mg/L. Before
mixing, however, the effluent stream was filtered with a 0.45 μ membrane filter to
exclude all biosolids.
0
200
400
600
800
1000
1200
1400
1600
0 10 20 30 40 50 60 70 80 90
Time (Days)
Sulfate Concentration (mg/L)
0
10
20
30
40
50
60
70
80
90
100
Removal (%)
Influent Effluent Removal

Figure 5-35: Sulfate Reduction Results in FBR with Lactate as Electron Donor

As before, the system was started in a batch mode with an initial sulfate
concentration of 750 mg/L, and after two days was changed to continuous mode.
Originally, the C/S ratio of 1.0 was chosen, but on day 4 the C/S ratio increased to
2.0, and an immediate increase in the sulfate reduction was observed. On day 12, the
sulfate concentration was increased to 800 mg/L, and a decrease in sulfate removal

138
was observed for one day, but the system recovered very quickly, and on day 25
sulfate removal reached 75%. On day 26, sulfate concentration was increased to
1000 mg/L, and again a small decrease in sulfate removal was observed but the
system quickly recovered and sulfate removal reached 76% on day 33. On day 34,
sulfate concentration was increased to 1200 mg/L and maintained until day 43, at
which time a sulfate removal of 93% was observed. On days 43 and 50, the FBR
column influent sulfate concentrations were increased to 1250 and 1320 mg/L,
respectively. As evident from Figure 5-35 the sulfate efficiency dropped from 94%
to 71%, but the system recovered very fast and on day 54, sulfate removal reached
92%. As a result of excessive growth on GAC particles, the media expanded from 31
inches to 34 inches.
On day 63, it was deemed necessary to adjust the column height back to 31
inches by removing GAC particles from the upper portion. On day 64, the column
influent concentration was increased to 1370 mg/L. Removal dropped to 78% as a
combined effect of increase in influent concentration, biomass washout, and carbon
withdrawal. However, the system again recovered quickly, and on day 75 reached a
steady state removal efficiency of 98% with an effluent sulfate concentration of less
than 55 mg/L.
In these experiments, the amount of lactate in the influent was progressively
increased from 1000 to 2000 mg/L to maintain a C/S ratio of 2.0. It is important to
note that the length of the reactor was increased about 27 inches, to increase the

139
settling time of the biomass, and to prevent active biomass loss during the cleaning
activities.
The “dilution strategy” was deemed advantageous from the following
perspectives.
i) Reducing shock to the system due to high sulfate concentration
ii) Increasing bacterial enzymes in the FBR system by recycling the
effluent
iii) Reducing excess lactate in the FBR effluent stream
0
500
1000
1500
2000
2500
0 10 20 30 40 50 60 70 80 90
Time (Days)
Lactate Concentration (mg/L)
0
10
20
30
40
50
60
70
80
90
Removal (%)
Influent Effluent Removal

Figure 5-36: Lactate Utilization Results in FBR with Lactate as the Electron Donor

140
As presented in Figure 5-36, lactate utilization followed the same pattern as
sulfate reduction. It should be noted that each increase in lactate concentration was
followed by a decrease in reduction rate and consequently an increase in the effluent
lactate concentration.
Figure 5-37 presents the hydrogen sulfide production profile in the aqueous
effluent stream of the FBR column. As shown in the figure, gradual increase in H
2
S
production corresponds to an increase in sulfate influent concentration and the
associated sulfate removal. Biomass washout from the system decreased the sulfate
reduction rate, and correspondingly a decrease in H
2
S production was observed. As
shown in Figure 5-37, H
2
S concentration progressively increased from 200 to 450
mg/L during these experiments. A sulfur mass balance in the system is presented in
ensuing section.
It is important to note that high concentrations of H
2
S (as high as 450 mg/L)
did not manifest any inhibitory effect in the system. Also, the system did not require
backwashing because of lower biomass production. The parameters used in the FBR
system with lactate are listed in Table 5-14.

141

Table 5-14: List of the Parameters in the FBR System with Lactate as the Electron
Donor

Parameters Value
C/S 2
pH 7.0
Temperature (
o
C) 30
HRT (hr) 2.5
Bed Length (in) 31
Recirculation Ratio (L/min) 1.55

0
50
100
150
200
250
300
350
400
450
500
0 10 20 30 40 50 60 70 80 90
Date
Hydrogen Sulfide Concentration
(mg/L)

Figure 5-37: H
2
S Production in FBR with Lactate as the Electron Donor

142
5.7 Sulfur Mass Balance
5.7.1 Significance of Sulfur Mass Balance Analysis
Performing a sulfur mass balance is of major importance in the FBR reactor
discussed in this research due to the following reasons.
i) Mass balance reveals the degree of hydrogen sulfide dissociation in the liquid,
and it clarifies whether it can inhibit the treatment process.
ii) It suggests the degree of sulfide oxidation into elemental sulfur.
iii) It determines whether and in which condition, sufficient sulfide containing
biogas would be generated to support economical elemental sulfur
production.
iv) The ratio of sulfur recovery from sulfate can be identified through performing
a mass balance.
v) Sulfur mass balance could be used to elucidate the sulfur cycle in the
biological FBR system.

The continuity equation, equation 5-5, represents the temporal and spatial
distribution of sulfur (S).

= - +

(5-5)
Net rate of
accumulation of
sulfur within the
control volume
Rate of mass
input of sulfur
across the
control volume
boundaries
Rate of mass
output of sulfur
across the control
volume
boundaries
Rate of chemical
reaction of sulfur
within the control
volume

143
In a biological FBR, the reactor acts as a plug flow reactor. A schematic of
the flow in and out of reactor is presented in Figure 5-38. In order to perform the
mass balance, flow and sulfur concentrations in the influent and effluents were
measured.

S
L
G

Figure 5-38: A Simplified Schematic of the Sulfur Balance in FBR

5.7.2 Mass Balance in Fluidized Bed Reactors

An important aspect of FBR studies is the fate of sulfate in the system. This
section provides details on sulfate conversions and the separation of the products in
gas and liquid phases. The discussion addresses the sulfur mass and speciation as
sulfide due to the hydrogen sulfide equilibrium between the gaseous and aqueous
phases. The sulfate reduction in biological FBRs was accompanied by the production
S: Sulfur mass in the influent (mg)
L: Sulfur mass in the aqueous effluent (mg)
G: Sulfur mass in the off-gas (mg)

144
of hydrogen sulfide, and its subsequent conversion to soluble sulfide forms including
HS
-
, and S
2-
. Measurements of total sulfides in the effluent provided an indication of
the biological activity and the extent of sulfate conversion within the reactor system.
The concentrations of HS
-
and H
2
S in the reactors were determined theoretically, and
compared with the experimental results. Additionally, the effects of temperature,
TDS, and pH on the H
2
S dissociation constant are discussed here, beside of the H
2
S
and HS
-
concentrations in the liquid phase. The results of these studies are presented
in Tables 5-15 and Figures 5-39 to 5-43.
The sulfur mass balance was based on the data from acetate FBR on day 125,
when the system reached the steady state. The sulfate flow rate into the FBR was
360 ml/hr, with a concentration of 2000 mg/L, and the sulfate concentration out of
the reactor was 75 mg/L at the same flow rate of 360 ml/hr. The amount of dissolved
H
2
S in the effluent liquid was estimated at 520 mg/L at the flow rate of 360 ml/hr.
The gas flow in the off-gas line was measured by the liquid displacement technique.
The hydrogen sulfide was removed from the system through a precipitation reaction
with zinc acetate, and was measured by iodometric titration. The gas flow in the off-
gas line was 200 ml/hr, and the H
2
S concentration was 120 mg/L S
2-
for the FBR
using acetate. Similarly, the flow of gas in the off-gas line was 200 ml/hr, and the
concentration was 170 mg/L S
2-
for the FBR using lactate. All the mass balance
calculations were based on the sulfur components in the system.
The equilibrium, or saturation concentration, of the volatile compounds and
their dissociation between the gas and liquid phase is generally a function of the type

145
of the compound and the partial pressure of it in the atmosphere. The relationship
between concentrations of gas in the liquid and gas phase follows Henry’s law, as it
presented in Equation 5-6.

g
T
g
x
P
H
p = (5-6)
where

p
g
= mole fraction of gas in air, mole gas/mole air
H= Henry’s law constant, atm (mole gas/mole air)/(mole gas/mole water)
P
T
= total pressure, usually 1 atm
x
g
= mole fraction of gas in water, mole gas/mole water

The Henry’s law constant can be estimated for each gas by knowing the ΔH
0
f

(energy of formation) and the temperature, and the value for H
2
S can be estimated
from Equation 5-7 as shown below:

Log H = 809 . 2 88 . 5
) 30 15 . 273 )( / 987 . 1 (
/ 10 85 . 1
3
= +
° +
× −
K kmol kcal
kmol kcal
(5-7)

so that H = 644 atm

In the anaerobic digestion process, sulfate is reduced to sulfide, which in turn
is distributed as follows: H
2
S in the gas phase; H
2
S, HS
-
, and S
2-
in the aqueous
phase; and insoluble metallic sulfides in the solid phase. The molar equilibrium

146
between H
2
S in the gas phase and free H
2
S in solution is governed by Equation 5-8
where H represents the Henry’s law constant in dimensionless form.
[H
2
S]
g
= H
u
[H
2
S]
aq
(5-8)

It must be noted that at any temperature, 1.0 mole of air is equal to 0.082T
liter of air, where T is given in Kelvin. Therefore, the dimensionless form of Henry’s
law can be calculated as shown below:



















=
water mole
L
L T
air mole
water mole gas mole
air mole gas mole atm
H H
u
6 . 55 082 . 0 ) / (
) / (







=
T
H
H
u
559 . 4
(5-9)

At 30°C, the dimensionless Henry’s law constant is estimated as: H=483 @
20°C (Metcalf and Eddy, 2003):
35 . 0
) 30 15 . 273 ( 559 . 4
483
=






+
=
u
H

It must be noted that H
2
S is the only form of sulfide that transfers into the gas
phase through Reaction 5-10; while the other dissolved forms (HS
-
, S
2-
) do not
account for the transfer into the gas phase.

g aq
S H S H
2 2
→ (5-10)

147
The dissociation of H
2
S species in water follows the Reactions 5-11 and 5-12 as
shown below.
−
+
+
  → ← HS H
k
S H
1
2
'

(5-11)

−
+
+
 → ←
− 2
2
S H
k
HS
(5-12)

At low pH values the dissolved H
2
S is more dominant than HS
-
. Therefore, at a pH
of 7.5 the first reaction is predominant and 22.5% of the total dissolved sulfide is in
the form of aqueous H
2
S, whereas at a pH of 7.0, 47.7% of the total dissolved sulfide
is in the form of H
2
S (see Table 5-15).

Table 5-15: Percentages of Aqueous H
2
S and HS
-
at Different pHs
pH Percentage of H
2
S Percentage of HS
-

4 99.9 0.1
5 98.9 1.1
6 90.1 9.9
7 47.7 52.3
7.5 22.5 77.5
8 8.3 91.7
8.5 2.8 97.2
9 0.89 99.1

The mass balance calculations were performed for the FBR systems using
acetate and lactate under steady state conditions. Based on the analyzed total sulfide
concentration of H
2
S mg/L S
2-
in each FBR, the concentrations of HS
-
and H
2
S at
different FBR temperatures and pHs were theoretically determined and are presented
in Figure 5-39.

148

0
20
40
60
80
100
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
pH
Percentage (%)
Aqeuous H2S
HS-
22.5% H
2
S
77.5% HS
-
pH = 7.5
47.7% H
2
S
52.3% HS
-
Aqueous H
2
S
HS
-

Figure 5-39: Percentages of H
2
S and HS
-
in Aqueous Form at Different pHs

The operating conditions for an FBR using acetate were the following: a pH
of 7.5 and a temperature of 30°C. The estimations of the ionization constant at FBR
salinity, and temperature are explained below. The conditional ionization constant is
as presented in Equation 5-13:
] [
] ][ [
'
2
1
S H
HS H
K
− +
= (5-13)

The dissociation constant for zero ionic strength pK
1
at T = 30°C (303.15K) is
presented below:

149
pK
1
= 32.55 + 1519.44/T – 15.672 logT + 0.02722T (5-14)

= 32.55 + 1519.44/303.15 – 15.672 log303.15 + 0.02722×303.15

= 6.9214

The ionic strength can be calculated from Equation 5-15 as follows:

I = TDS × 2.5 × 10
-5
(5-15)

The TDS for the FBR with acetate 21,000 mg/L is; so that

I(acetate) = 21000 × 2.5 × 10
-5
= 0.5

The Debye-Huckel parameter “A “ can then be estimated as shown below:
A = 0.7083 – 2.277 × 10
-3
T

+ 5.399 × 10
-6
T
2
(5-16)
= 0.7083 – 2.277 × 10
-3
× 303.15 + 5.399 × 10
-6
× 303.15
2

= 0.5142

The ion activity coefficient parameter defined by Equation 5-17 can be estimated as
shown below:








−
+
= I
I
I
A pf
m
3 . 0
1
(5-17)
1358 . 0 5 . 0 3 . 0
5 . 0 1
5 . 0
5142 . 0
,
=








× −
+
=
Acetate m
pf
Therefore, the conditional ionization constant for acetate is
K′
1
= 10
-pK1+2pfm
(5-18)
[H
+
] = 10
-pH+pfm
(5-19)

150
so that
K′
1
= 10
(-6.9214+2×0.1358)
= 1.64 × 10
-7

[H
+
] = 10
(-7.5+0.1358)
= 4.32 × 10
-8

and

] [
'
1
] [
1
2
+
+
=
H
K
S
S H
T
(5-20)
The influent and effluent sulfate concentrations of the sample obtained from
sulfide analysis under steady-state from the FBR using acetate are as follows:

Influent SO
4
2-
concentration: 2000 mg/L
Effluent SO
4
2-
concentration: 75 mg/L
SO
4
2 -
removal: 2000-75= 1925 mg/L SO
4
2 -

6 . 642
07 . 96
07 . 32 1925
=
×
mg S
2-
/L

The concentration of aqueous forms of H
2
S in the FBR with acetate at 30
o
C and pH
of 7.5 is as follows:
Total sulfide concentration:
520 mg/L S
2-
= 520 × 10
-3
/ (32.07) = 0.016 M (S
2-
)
003 . 0
10 32 . 4
10 64 . 1
1
016 . 0
] [
8
7
2
=
×
×
+
=
−
−
S H M = 107 mg/L as S
2-

and

151
[HS
-
] = 520-107 = 413 mg/L as S
2-

The H
2
S concentration in the gas phase was measured by precipitation of zinc
sulfide due to reaction between H
2
S gas with zinc acetate and back-titration of the
solution phase. The H
2
S concentration in the gas phase was 120 mg/L as S
2-
. The
sulfur mass balance between the gas and liquid phase is presented in Table 5-16.

Table 5-16: The sulfur Mass Balance between Liquid and Gas Phase in the FBR with
Acetate
Influent to FBR Effluent from FBR
Unit SO
4
2 -
SO
4
2 -
H
2
S HS
-
H
2
S (gas)
mg/L 2000 75 107 413 120
mmol/L 21 0. 8 3 13 3.7
mg/L (S
2 -
) 667 25.6 107 413 120

The fate of 99.8% of the sulfur in the system was theoretically accounted for
in the main balance analysis. The observed error (0.2%) could be attributed to minor
errors in measurement of sulfur species. In the laboratory-scale FBR studies a second
reactor was used to convert H
2
S dissolved in the aqueous phase into elemental sulfur
while utilizing nitrate as electron acceptor. In the pilot-scale system the use of a H
2
S
sparging column with nitrogen gas was recommended to effectively transfer of H
2
S
into the gas phase and use the gas to produce elemental sulfur and hydrogen biofuel.
Therefore, the mass balances for both laboratory-scale and pilot-scale systems are as
presented in Figures 5-40 and 5-41, respectively. The H
2
S sparging data were used to
calculate the H
2
S levels in the FBR with a sparging column.

152

Over 99.9% removal of gaseous
H 2S
(complete conversion)
Gaseous effluent less
than 0.01% of the total
sulfur
Biofilter
ST: 120 mg/L
18% ST remained as
elemental sulfur
Acetate FBR
(96% SO4
2-
removal)
Influent: 360 ml/hr
SO4
2-
: 2000 mg/L
SO4
2-
: 21 mmol/L
ST: 667 mg/L
H 2S-S: 120 mg/L
H 2S-S: 3.7 mmol/L
Gaseous H 2S
SO4
2-
: 75 mg/L Total H 2S: 520 mg/L S
2-

SO4
2-
: 0.8 mmol/L H 2S-S: 16 mmol/L
(SO4
2-
-S: 26 mg/L) H 2S: 107 mg/L S
2-
(3 mmol)
ST: 546 mg/L S
2-
HS
-
: 413 mg/L S
2
(13mmol) C/S ratio: 1.0, pH: 7.5,
Temperature: 30
o
C
H
2
S removing
reactor
(99.8% conversion to
elemental sulfur)
78%
ST
82%
ST
18% S T
E ffluent: 360 mg/L
SO4
2-
: 75 mg/L H 2S: 0.2 mg/L
SO4
2-
: 0.8 mmol/L HS
-
: 0.8 mg/L
(SO4
2-
-S: 26 mg/L) ST: 27 mg/L
4% ST
S: 519 mg/L
S: 16 mmol/L
ST: 519 mg/L

Figure 5-40: Mass Balance for FBR with Acetate with Effluent H
2
S Removing Bioreactor

153

C/S ratio: 1.0, pH: 7.5,
Temperature: 30
o
C
Acetate FBR
(96% SO4
2-
removal)
Biofilter
ST: 588 mg/L
88% ST remained as
elemental sulfur
Influent: 360 ml/hr
SO4
2-
: 2000 mg/L
SO4
2-
: 21 mmol/L
ST: 667 mg/L
SO4
2-
: 75 mg/L Total H 2S: 520 mg/L S
2-
SO4
2-
: 0.8 mmol/L H 2S-S: 16 mmol/L
(SO4
2-
-S: 26 mg/L) (H 2S-S: 456 mg/L)
H 2S: 107 mg/L S
2-
(3 mmol)
ST: 546 mg/L S
2-
HS
-
: 413 mg/L S
2-
(13mmol)
H 2S-S: 120 mg/L
H 2S-S: 3.7 mmol/L
Gaseous H2S
Over 99.9% removal of
gaseous H 2S
(complete conversion)
Gaseous effluent less
than 0.01% of the total
sulfur
Aqueous H
2
S stripping at pH=6.0
(90% total sulfide removal based on
the Stripping test)
H 2S-S: 468 mg/L
H 2S-S: 14.5 mmol/L
E ffluent: 360 mg/L
SO4
2-
: 75 mg/L H 2S: 11 mg/L
SO4
2-
: 0.8 mmol/L HS
-
: 41 mg/L
(SO4
2-
-S: 26 mg/L) ST: 78 mg/L
H
2
S Stripping by
nitrogen gas
(90% Conversion)
70% ST
82%
ST
18% S T
12% ST

Figure 5-41: Mass Balance for FBR with Acetate and Effluent H
2
S Stripping

154
In the case of the FBR using lactate, the operating conditions were a pH of
7.0 and a temperature of 30°C. The estimation of ionization constant at the FBR
salinity and temperature are explained below.

The conditional ionization constant is based on Equation 5-21:
] [
] ][ [
'
2
1
S H
HS H
K
− +
= (5-21)
The dissociation constant for zero ionic strength pK
1
at T = 30°C (303.15K) is
estimated as follows:

pK
1
= 32.55 + 1519.44/T – 15.672 logT + 0.02722T (5-22)

= 32.55 + 1519.44/303.15 – 15.672 log303.15 + 0.02722×303.15

= 6.9214

Ionic strength can be calculated from Equation 5-23:
I = TDS × 2.5 × 10
-5
(5-23)

The TDS in the FBR with lactate is 15,000 mg/L (for lactate at 2000 mg/L
sulfate a mixed system was used that decreased the TDS)
I(lactate) = 15000 × 2.5 × 10
-5
= 0.4
The Debye-Huckel parameter “A“ can be estimated as follows:
A = 0.7083 – 2.277 × 10
-3
T

+ 5.399 × 10
-6
T
2
(5-24)

155
= 0.7083 – 2.277 × 10
-3
× 303.15 + 5.399 × 10
-6
× 303.15
2

= 0.5142

The ion activity coefficient parameter is then defined by Equation 5-25 as follows:








−
+
= I
I
I
A pf
m
3 . 0
1
(5-25)
1375 . 0 4 . 0 3 . 0
4 . 0 1
4 . 0
5142 . 0
,
=








× −
+
=
Lactate m
pf
Therefore, the conditional ionization constant for lactate is estimated as follows:
K′
1
= 10
-pK1+2pfm
(5-26)
[H
+
] = 10
-pH+pfm
(5-27)
so that
K′
1
= 10
(-6.9214+2×0.1375)
= 2.26 × 10
-7

[H
+
] = 10
(-7.5+0.1375)
= 4.34 × 10
-8

and

] [
'
1
] [
1
2
+
+
=
H
K
S
S H
T
(5-28)
The concentrations of aqueous forms of H
2
S in the FBR using lactate at 30
o
C
and pH of 7.0 are as follows:
Total dissolved sulfide concentration and its dissociated forms are as shown
below:
Since 456 mg/L S
2-
= 456 × 10
-3
/ (32.07) = 0.014 M (S
2-
)

156
003 . 0
10 32 . 4
10 64 . 1
1
014 . 0
] [
8
7
2
=
×
×
+
=
−
−
S H M = 96 mg/L as S
2-

and
[HS
-
] = 456-96 = 360 mg/L as S
2-

The influent and effluent sulfate concentrations of the sample obtained from
sulfide analysis under steady-state condition for the FBR using acetate are as
follows:
Influent SO
4
2-
concentration: 2040 mg/L
Effluent SO
4
2-
concentration: 160 mg/L
SO
4
2 -
removal: 2040-160= 1880 mg/L SO
4
2 -

628
07 . 96
07 . 32 1880
=
×
mg S
2-
/L

The concentration of the H
2
S in the gas phase was measured by forming a
precipitate of zinc sulfide due to the reaction between hydrogen sulfide and zinc
acetate and back titration of the solution. The H
2
S concentration in the gas phase was
170 mg/L as S
2-
. The sulfur mass balance between the gas and liquid phase is
presented in Table 5-17.

157
Table 5-17: The Sulfur Mass Balance between Liquid and Gas Phase in the FBR
with Lactate
Influent to FBR Effluent from FBR
Unit SO
4
2 -
SO
4
2 -
H
2
S HS
-
H
2
S (gas)
mg/L 2040 160 96 360 170
mmol/L 21.23 1.7 3 11 5.3
mg/L (S
2 -
) 681 54 96 360 170

The fate of 99.85% of the sulfur in the system was theoretically accounted for
during this analysis. The observed error (0.15%) could be attributed to minor errors
in measurement of sulfur species. In the laboratory scale FBR studies with lactate, a
second reactor is used to convert dissolved H
2
S in the aqueous phase into elemental
sulfur while using nitrate as electron acceptor. In the pilot-scale system, the use of a
H
2
S sparging column with nitrogen gas was recommended to effectively transfer H
2
S
into the gas phase and use the gas to produce elemental sulfur and hydrogen biofuel.
The mass balances for both systems are presented in Figures 5-42 and 5-43. The H
2
S
sparging data were used to estimate the H
2
S in the FBR with a sparging column.
The standard for discharge of the sulfate is 250 mg/L S
2-
, the concentrations
in the FBR effluents are 75 and 160 mg/L S
2-
for the acetate and lactate FBRs,
respectively, which is less than the MCL.

158

67%
ST
C/S ratio: 2.0, pH: 7.0,
Temperature: 30
o
C
Lactate
FBR
(92% SO4
2-
removal)
Biofilter
ST: 170 mg/L
25% ST remained as
elemental sulfur
Influent: 360 ml/hr
SO4
2-
: 2040 mg/L
SO4
2-
: 21 mmol/L
ST: 681 mg/L
SO 4
2-
: 160 mg/L Total H 2S: 456 mg/L S
2-
SO 4
2-
: 1.7 mmol/L H 2S-S: 14 mmol/L
(SO 4
2-
-S: 54 mg/L) H 2S: 96 mg/L S
2-
(3 mmol)
S T: 510 mg/L S
2-
HS
-
: 360 mg/L S
2-
(11mmol)
H 2S-S: 170 mg/L
H 2S-S: 5.3 mmol/L
Gaseous H 2S
Gaseous effluent less
than 0.01% of the total
sulfur
E ffluent: 360 mg/L
SO4
2-
: 160 mg/L H 2S: 0.2 mg/L
SO4
2-
: 1.7 mmol/L HS
-
: 0.8 mg/L
(SO4
2-
-S: 54 mg/L) ST: 55 mg/L
H
2
S removing
reactor
(99.8% conv ersion to
elemental sulfur)
75%
S T
25% S T
8% ST
S: 455 mg/L
S: 14 mmol/L
ST: 455 mg/L
Over 99.9% remov al of
gaseous H2S
(complete conversion)

Figure 5-42: Mass Balance for the FBR with Lactate with Effluent H
2
S Removal from the Bioreactor

159

C/S ratio: 2.0, pH: 7.0,
Temperature: 30
o
C
Biofilter
S
T
: 580 mg/L
85% S
T
remained as
elemental sulfur
Aqueous H
2
S stripping at pH=6.0
(90% total sulfide removal based on
the Stripping test)
H
2
S-S: 410 mg/L
H
2
S-S: 13 mmol/L
H
2
S Stripping by
nitrogen gas
(90% Conversion)
60% S
T
Lactate
FBR
(92% SO
4
2-
removal)
Influent: 360 ml/hr
SO
4
2-
: 2040 mg/L
SO
4
2-
: 21 mmol/L
S
T
: 681 mg/L
SO
4
2-
: 160 mg/L Total H
2
S: 456 mg/L S
2-
SO
4
2-
: 1.7 mmol/L H
2
S-S: 14 mmol/L
(SO
4
2-
-S: 54 mg/L) H
2
S: 96 mg/L S
2-
(3 mmol)
S
T:
510 mg/L S
2-
HS
-
: 360 mg/L S
2-
(11mmol)
H
2
S-S: 170 mg/L
H
2
S-S: 5.3 mmol/L
Gaseous H
2
S
Over 99.9% removal of
gaseous H
2
S
(complete conversion)
Gaseous effluent less
than 0.01% of the total
sulfur
Effluent: 360 mg/L
SO
4
2-
: 160 mg/L H
2
S: 10 mg/L
SO
4
2-
: 1.7 mmol/L HS
-
: 36 mg/L
(SO
4
2-
-S: 54 mg/L) S
T
: 100 mg/L
75%
S
T
25% S
T
15% S
T

Figure 5-43: Mass Balance for the FBR using Lactate with Effluent H
2
S Stripping

160
5.7.3 Mass Balance in the Anaerobic Biofilter
The sulfur mass balance was performed for the anaerobic biofiltration of
hydrogen sulfide in the FBR using lactate and acetate. The FBR studies showed a
steady state, sulfate removal efficiency of 96% for an influent sulfate concentration
of 2000 mg/L under optimal conditions. The optimal conditions were the following,
C/S=1.0, pH=7.5, and temperature=30°C for acetate and C/S=2.0, pH=7.0, and
temperature=30°C for lactate. The total dissolved sulfide concentration of 520 mg/L
was generated at the FBR study with acetate at a steady state removal of 96 %. The
sulfur mass balance studies for the FBR system using acetate revealed that 20% of
the total dissolved sulfide was produced in the form of aqueous H
2
S at a pH of 7.5,
accounting for 78% of the total sulfide. Consequently, about 79.5% of the total
dissolved sulfide existed as HS
-
, corresponding to 45% of the total sulfide. After
nitrogen stripping, 90% of the dissolved hydrogen sulfide was transferred into the
gas phase, which accounts for 70% of the total sulfide. Thus, the laboratory-scale
FBR system with influent flow rate of 360 mL/hr generated about 590 mg/hr (S) of
gaseous H
2
S. The biofilter unit was capable of maintaining more than 99.9% of H
2
S
removal efficiency, and the H
2
S levels in the effluent gas stream from the biofilter
were below the analytical detection limits. Therefore, it can be stated that 88% of the
total sulfur (S: 588 mg/L) remained in the biofilter in the form of elemental sulfur
(visible in the biofilter bed as deposits), an important factor from the standpoint of
both sulfur recovery and process economics.

161
In the FBR study using lactate at a steady-state sulfate removal of 96%, the
total dissolved sulfide generation was 456 mg/L. The sulfur mass balance studies for
this FBR system revealed that 21% of the total dissolved sulfide was produced in the
form of aqueous H
2
S at a pH of 7.5, accounting for 14% of the total sulfide.
Consequently, about 79% of the total dissolved sulfide existed as HS
-
, corresponding
to 53% of the total sulfide. After stripping with nitrogen, 90% of the dissolved
hydrogen sulfide was transferred into the gas phase, accounting for 60% of the total
sulfide. Thus, the laboratory-scale FBR system with influent flow rate of 360 mL/hr
generated about 580 mg/hr (S) of gaseous H
2
S. The biofilter was capable of
achieving more than 99.9% of H
2
S removal efficiency, so that H
2
S levels in the
biofilter effluent were below the analytical detection limits. Therefore, it can be
stated that 85% of the total sulfur (S: 580 mg/L) remained in the biofilter in the form
of elemental sulfur, visible in the reactor bed as deposits.

162
5.8 Managing the Produced H
2
S and Production of Hydrogen Fuel
To remove the hydrogen sulfide from the effluent wastewater, one of the
following methods could be used: sparging, membrane contactor process, oxidation
with air, biological reduction, or gas removal contactors.
Sparging the hydrogen sulfide out of the system by using nitrogen gas was
tested in this study, and the results were presented in Section 5.5. As discussed
previously, the H
2
S sparging proved to be very effective. Another potential method
of H
2
S gas removal would entail the use of membrane contactors (Polypore
Company, 2007). These contactors would remove the undissolved gas by using a
vacuum. Application of vacuum in the gas removal line would reduce the partial
pressure of the gas in contact with water, so that the dissolved gas would be
progressively transferred from liquid to gas phase.
Higher degrees of H
2
S removal might be obtained by adjusting the solution
pH to 6.0, as discussed in Section 5.5. Although no report is currently available on
the application of membrane contactors for hydrogen sulfide removal, the
mechanism and design of the system as presented in Figure 5-44 and discussed by
the manufacture appears to be potentially viable.

163

Figure 5-44: Liqui-Cel Membrane Contactor System
(Ref.: Polypore Company, 2007)

Once H
2
S is transferred from the liquid phase to the gas phase, it may be
effectively used to yield other products. Several studies have been performed
successfully to dissociate H
2
S into hydrogen gas and elemental sulfur. Harkness et
al. (1990) successfully dissociated H
2
S by using microwave plasma. They also
reported that the energy recovered from hydrogen gas production was more than the
energy needed by the plasma for the dissociation. These researchers further stated
that the H
2
S dissociation was possible even in the presence of water, CO
2
, and
methane.

164
Borton and Rogers (1990) reported that the solar dissociation of H
2
S can
serve as a source of renewable energy. Traus et al. (1993) employed a rotating high-
pressure-glow discharge for H
2
S dissociation, wherein concentric electrodes were
used in an axial magnetic field to remove the sulfur produced in the discharge line.
Krasheninov et al. (1986) reported the use of radio frequency (RF) discharge
from a plasma-generating gas to dissociate the H
2
S with nearly 100% efficiency.
Traus and Suhr (1992) used a high-temperature (130-560°C) ozonizer to convert H
2
S
into elemental sulfur and hydrogen. Dong et al. (1997) used a microwave plasma
reactor to dissociate H
2
S under atmospheric pressure.Using an anaerobic FBR in
combination with any H
2
S dissociation methods could effectively transfer sulfate
into two valuable products, elemental sulfur and hydrogen fuel. This method can be
used in any industry that produces sulfate-rich wastewaters, including reverse
osmosis (RO) brines from various desalination systems.
Anaerobic biofilters were used in this study, and they managed to effectively
remove the H
2
S from the off-gas by oxidizing sulfide to elemental sulfur. In the
biofilter, a mixed group of anaerobic bacteria, possibly green sulfur bacteria, were
used to utilize hydrogen sulfide as the energy source. These phototrophic bacteria
anaerobically oxidize H
2
S, according to Reaction 2-45, to elemental sulfur. This
sulfur can be recovered and sold for economic purposes.

165
5.9 Scanning Electron Microscopy of the Fluidized Bed Reactors GAC
Sample preparation had three stages: treatment, dehydration, and sputter
coating, as explained in Section 4.2.7 (Pirbazari, et al., 1990). Figures 5-45 to 5-48
presents typical scanning electron microscopy (SEM) of the GAC particles from
randomly withdrawn FBR system.

Figure 5-45: SEM micrograph demonstrating morphologically different SRBs in the
GAC biofilm

Figure 5-45 presents a typical SEM micrograph from GAC particles taken
from the middle of the acetate FBR column. Two morphologically different SRBs
are observed in the micrograph, i.e., vibroids as well as rod shape. The average

166
lengths of the SRBs are about 2.1 μm for the vibroids and 3.6 μm for the rod-shape
strain. The micrograph also shows the presence of microbial biopolymers.

Figure 5-46: SEM micrograph demonstration the GAC biofilm thickness

Figure 5-46 presents another typical micrograph demonstrating the depth of
biofilm on carbon particle.

167

Figure 5-47: A typical SEM micrograph showing the heterogeneous nature of
biofilm

Figure 5-47 presents another SEM micrograph demonstrating the non-
uniform and heterogeneous nature of biofilm. As can be observed in the micrograph,
portions of the surface are covered with dense biomass and secreted biopolymers.

5.10 Managing the BOD By-products in the FBR Effluent
The biological oxygen demand (BOD) discharge permits should follow one
of the following standards. The effluent would comply with the technology-based
standards for publicly owned treatment works, implying that a POTW at a minimum

168
should provide secondary treated wastewater with established limits for BOD of 30
mg/L as a 30-day average and 45 mg/L as a weekly average. Some California
permits have also included a daily maximum of 50 mg/L. However, if the POTW is
discharging to a stream that provides little or no dilution (as a standard, less than
20:1), more stringent restrictions should be applied. These restrictions will protect
the beneficial uses of receiving water for municipal, recreational and agricultural use.
These limitations could be equivalent to tertiary treated wastewater levels having
BOD effluent limitations of 10 mg/L as a monthly average, 15 mg/L as a weekly
average, and 20 mg/L as a daily maximum(California Water Resource Board,
personal communication, 2007).
The second concern for BOD relates to protection of the COLD (Cold Fresh
Water Habitat-COLD) is the uses of waters that support cold water ecosystems
including, but not limited to, preservation or enhancement of aquatic habitats,
vegetation, fish or wildlife, including invertebrates. If certain receiving waters have
the COLD beneficial use, then the dissolved oxygen (DO) level of 7 mg/L in the
receiving water can drive the BOD limitations to more stringent levels, according to
the Streeter Phelps equation, the equation for calculating the oxygen deficit (Yen,
1998).
The monetary penalty for BOD discharge is $3000 for every effluent limit
violation, meaning that if a discharge has a limit for 20 mg/L BOD as a daily
maximum, and monitoring shows a 25 or 50 mg/L BOD, then that constitutes a

169
violation and there would be a $3000 penalty (California Water Resource Board,
personal communication, 2007).
A primary goal of this research is to remove the sulfate from the effluent, so
that the wastewater can be used for methane production in the next step. The organic
by-product can be treated in a methanogenic bioreactor to produce methane fuel. The
effluent BOD in the lactate FBR after sulfate removal reached steady-state
concentration of 930 mg/L. However the chemical oxygen demand (COD) can
represent BOD in cases where the organic constituents manifest an extremely high
degree of biodegradability. The theoretical or stochiometric amount of COD based
on Equation 5-29 for residual lactate is 925 mg/L (based on lactate concentration of
716 mg/L in the FBR effluent aqueous stream).
2CH
3
CHOHCOOH + 15/2 O
2
→ 6CO
2
+9 H
2
O (5-29)
The effluent BOD in the FBR with acetate was not measured, but it can be
correlated to theoretical COD. Based on the acetate level in the effluent (292 mg/L
on day 125) and theoretical calculations of COD according to equation 5-30, the
COD in the effluent will be about 234 mg/L.
CH
3
COOH + 3/2 O
2
→ 2CO
2
+2 H
2
O (5-30)
For these electron donors, the amount of BOD and COD is almost identical.
This amount of BOD can be lowered to the standard level of 30 mg/L in a
methanogenic process, as it will be discussed later in Figures 5-48 to 5-51. Finally,
the methane produced in the process can be used as fuel.

170
Chapter 6

SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

6.1 Summary and Conclusions
6.1.1 CMBR Studies
Thermodynamic evaluations of several electron donors including acetate,
lactate, hydrogen, molasses, ethanol, acetic acid, and formic acid were performed
to choose the most promising.

In-depth analyses of the reactions and associated thermodynamics pertaining
to biological sulfate reduction were performed to identify the best electron
donors. Acetate, lactate, hydrogen gas, molasses, ethanol, acetic acid, and formic
acid were the electron donors that have been studied.

CMBR biokinetic studies for acetate as the electron donor, investigated the
effect of various environmental parameters such as temperature, pH, and carbon-
to-sulfur (C/S) ratio on the desulfurization of the wastewater. The results
indicated that the optimum temperature, C/S ratio, and pH are 30
o
C, 1.0, and 7.5,
respectively.

CMBR biokinetic studies using lactate as electron donor demonstrated that
the optimum temperature, C/S ratio, and pH were 30
o
C, 2.0 and 7.0, respectively.

171
Therefore, a working pH of 7.0, C/S of 2.0, and temperature of 30
o
C were chosen
for the FBR system with lactate.

The CMBR studies with molasses and citric acid did not show significant
bacterial growth; therefore, they were not used in the FBR studies.

CMBR with ethanol had good sulfate reduction rates but production of
copious amounts of acetate as reaction by-product discouraged the use of this
electron donor in the FBR studies.

Preliminary batch biokinetic studies using acetate and lactate as electron
donors revealed that sulfate removals of more than 96% were obtainable.

The biokinetic parameters for optimal process conditions were estimated
from CMBR studies for acetate and lactate, including the Monod maximum
substrate utilization rate, the yield coefficient, Y, and specific consumption rate.
These parameters were used to choose the best electron donor from a reaction
kinetic standpoint.

172
6.1.2 Anaerobic FBR Studies

Laboratory-scale FBR-GAC studies with acetate as electron donor yielded
encouraging results. Sulfate removal efficiencies as high as 96% were observed
at sulfate concentration of 2000 mg/L.

The most significant parameter affecting the sulfate reduction rates were the
amount of active biomass supported by the GAC in the column. The amount of
attached biomass in FBR with lactate at the end point steady-state condition were
13 (mg biomass/g dried GAC) and that for FBR with acetate were 12.5 (mg
biomass/ g dried GAC).

Despite the operational problems, the FBR studies with hydrogen gas as the
electron donor showed a viable technology for sulfate reduction. It is important
to note that this system did not yield any undesirable by-product, except H
2
S.
Nonetheless, the H
2
S produced in the system can be dissociated into hydrogen
gas and elemental sulfur (Section 5.8). The hydrogen gas can then be used as the
electron donor in the FBR system.

FBR studies with acetate as the electron donor were performed in two phases:
i) low sulfate ranges from 700 to 1100 mg/L sulfate; and ii) high sulfate ranges,
from 1100 to 2000 mg/L sulfate. Sulfate removals as high as 96% were observed.

173
FBR studies with lactate as the electron donor were conducted in two
different systems. In the first system the influent sulfate concentration started at
700 mg/L and was gradually increased to 2000 mg/L. No H
2
S sequestration was
deemed necessary. In the second system the influent sulfate concentration was
diluted with a portion of the column effluent to maintain concentration level
between 1200-1300 mg/L. Sulfate removals as high as 96% were obtained in
both systems.

FBR systems with acetate and lactate as electron donors resulted in H
2
S
concentrations as high as 600 mg/L S
2-
. Nonetheless, hydrogen sulfide did not
cause system inhibition and consequently there was no need for its sequestration.

Gas stripping tests were performed on dissolved H
2
S in the effluent solution
stream from acetate FBR. In these studies, nitrogen gas was used to sparge the
H
2
S from the liquid to gas phase. These sparging tests with nitrogen gas revealed
that at a pH of 6, and relatively low flow rates H
2
S could be removed from the
system with more than 90% efficiency. It is important to note that using air
(instead of nitrogen) could reoxidize H
2
S into sulfate.

Specially-designed anaerobic biofilters were used to convert H
2
S in the FBR
off-gas into elemental sulfur. Some bacteria species responsible for this
conversion were possibly photolithotrophic green and purple sulfur bacteria.

174
Economical analysis of the FBR indicated that acetate could be the best
electron donor for sulfate-rich wastewaters.

6.2 Recommendations
Conduct further biological sulfate reduction studies on a pilot-scale system to
assess the efficiency and cost- effectiveness of acetate as the electron donor.

Investigate higher concentrations of sulfate in the FBR systems to determine
the H
2
S toxicity levels, and the need for H
2
S sequestration.

Conduct experimental studies to investigate methods for H
2
S dissociation
into the hydrogen gas and elemental sulfur and to assess the hydrogen fuel
production rates and purity as well as the elemental sulfur production rate.

Test the potential of the membrane contactors (section 5-8) for removal of
H
2
S from the FBR effluent liquid, stream.

Conduct FBR studies to improve and optimize the hydrogen delivery system.

Investigate other potential electron donors for biological sulfate reduction of
sulfate-rich wastewaters.

175

Study the possibility of using the effluent from the FBR system to dilute
seawater in hydrogen fuel production.

176
REFERENCES

Annachhatre, A. P., and Suktrakoolvait, S. 2001 Biological Sulfate Reduction Using
Molasses as a Carbon Source. Water Environmental Research, 73 (1), 118–126.

Badziong, W., and Thauer, R. K. 1978 Growth Yields and Growth Rates of
Desulfovibrio vulgaris (Marburg) Growing on Hydrogen Plus Sulfate and Hydrogen
Plus Thiosulfate as the Sole Energy Sources. Archives of Microbiology, 117 (2),
209–214.

Basu, R., Clausen, C., and Gaddy, J. L. 2006 Biological Conversion of Hydrogen
Sulfide into Elemental Sulfur. Environmental Progress, 15 (4), 234-238.

Bayoumy, M. E., Bewtra, j. k., Hamdy, I. A., and Biswas, N. 1999 Removal of
Heavy Metals and COD by SRB in UAFF Reactors. Journal of Environmental
Engineering, 125 (6), 532-538.

Bhattacharya, S. K., Uberoi, V., and Dronamaraju, M., M., 1996 Interaction between
Acetate Fed Sulfate Reducers and Methanogens. Water Science and Technology, 30
(10), 2239-2246.

Bohn, H. 1992 Consider Biofiltration for Decontaminating Gases. Chemical
Engineering Progress, 88, 35–40.

Borton, D. N., and Rogers, W. E. 1990 Solar Dissociation of Hydrogen Sulfide: A
First Step in Producing a Renewable Fuel. Proceedings of the Intersociety Energy
Conversion Engineering Conference, 4, 315–319.

Brinkhoff, T., Muyzer, G., Wirsen, O. C., and Kueven, J. 1999 Thiomicrospira
kuenenii Sp. Nov. and Thiomicrospira frisia Sp. Nov., two Mesophilic Obligately
Chemolithoautotrophic Sulfur-Oxidizing Bacteria Isolated from an Intertidal Mud
Flat. International Journal of Systematic Bacteriology, 49: 385–392.

177
Cadenhead, P., and Sublette, K. L. 1990 Oxidation of Hydrogen Sulfide by
Thiobacilli. Biotechnology and Bioengineering, 35, 1150-1154.
Cha, J. M., W. S. Cha, and J. H. Lee. 1999 Removal of Organo-Sulfur Odour
Compounds by Thiobacillus novellas SRM, Sulfur-Oxidizing Microorganisms.
Process Biochemistry, 34 (6–7), 659–665.

Chitwood, D. E., and Devinny J. S. 1999 Evaluation of a Two-Stage Biofilter for
Treatment of POTW Waste Air. Environmental Progress, 18 (3), 212–221.

Cho, K. S., Hirai, M., and Shoda, M. 1991 Degradation Characteristics of Hydrogen
Sulfide, Methanethiol, Dimethyl Sulfide and Dimethyl Disulfide by Thiobacillus
thioparus DW44 Isolated from Peat Biofilter. Journal of Fermentation and
Bioengineering 71 (6), 384–389

Choi, E. R., and Jay, M. 1991 Competition and Inhibition of Sulfate Reducers and
Methane Producers in Anaerobic Treatment. Water Science and Technology, 23 (7–
9), 1259–1264.

Chung, Y. C., Huang, C., and Tseng, C. P. 1996 Operation Optimization of
Thiobacillus thioparus CH11 Biofilter for Hydrogen Sulfide Removal. Journal of
Biotechnology, 52, 31–38.

Chung, Y. C., Huang, C., and Tseng, C. P. 2001 Biological Elimination of H
2
S and
NH
3
from Waste Gases by Biofilter Packed with Immobilized Heterotrophic
Bacteria. Chemosphere, 43, 1043–1050.

Colleran, E., Finnegan, S., and O’Keeffe, R. B. 1994 Anaerobic Digestion of High
Sulfate Content Wastewater from the Industrial Production of Citric Acid. Water
Science and Technology, 30 (12), 263–273.

Colleran, E., Finnegan, S., and Lens, P. 1995 Anaerobic Treatment of Sulphate-
Containing Waste Streams. Antonie van Leeuwenhoek, 67, 29–46.

178
Cooney, M. J., Roschi, E., Marison, I. W., Comninellis, C., and Von Stockar, U.
1996 Physiologic Studies with the Sulfate-Reducing Bacterium Desulfovibrio
desulfuricans: Evaluation for Use in a Biofuel Cell. Enzyme and Microbial
Technology, 18 (5), 358–365.

Cox, H. H. J., and Deshusses, A. M. 2002 Biotrickling Filters for Air Pollution
Control. The Encyclopedia of Environmental Microbiology, ed. G. Britton 2,782–
2,795, New York, J. Wiley & Sons.

Dong, Y., Wang, H., and Yu, J. 1997 Hydrogen Production by H
2
S Microwave
Plasma Dissociation. Taiyangneng Xuebao/Acta Energiae Solaris Sinica 18 (2), 142–
145.

Duan, H., Koea, L. C. C., and Yan, R. 2006 Biological Treatment of H
2
S Using
Pellet Activated Carbon as a Carrier of Microorganisms in a Biofilter. Water
Research, 40, 2629–2636.

El Bayoumy, M., Bewtra, J. K., Hamdy, I. A., and Biswas, N. 1999 Removal of
Heavy Metals and COD by SRB in UAFF Reactor. Journal of Environmental
Engineering, 125 (6), 532–539.

Elias, A., Barona, A., Arreguy, A., Rios, J., Aranguiz, I, and Penas, J. 2002
Evaluation of a Packing Material for the Biodegradation of H
2
S and Product
Analysis. Process Biochemistry, 37, 813–820.

EPA. 2003 Nickel Plating: Industry Practices Control Technology and
Environmental Management. National Risk Management Research Laboratory
Office of Research and Development U.S. Environmental Protection Agency
Cincinnati, Ohio.

Friedrich, C. 1998 Physiology and Genetics of Sulfur-oxidizing Bacteria. Advances
in Microbial Physiology, 39, 233–289.

Friedrich, C. G., Rother, D., Bardschewsky, F., Quentmeier, A., and Fischer, J. 2001
Oxidation of Reduced Inorganic Sulfur Compounds by Bacteria: Emergence of a
Common Mechanism. Applied and Environmental Microbiology, July, 2873–2882.

179

FTN Associates, Ltd. 2006 TMDLs for Turbidity, Sediment, TSS, Chloride, Sulfate,
and TDS for Subsegments 100309, 100602, and 100603 in the Red River Basin,
Louisiana. Prepared for USEPA, Region 6, Water Quality Protection Division,
Dallas, Texas.

Galiana-Aleixandre, M. V., Iborra-Clar, A., Bes-Pifi, A., Mendoza-Roca, J. A.,
Cuartas-Uribe, B., and Iborra-Clar, M. I. 2005 Nanofiltration for Sulfate Removal
and Water Reuse of the Pickling and Tanning Processes in a Tannery. Desalination,
179, 307–313.

Garrett, R. H., and Grisham, C. M. 2005 Biochemistry, Third Edition, McGraw-Hill.

Genchow, E., Hegemann, W., and Maschke, C. 1996 Biological Sulfate Removal
from Tannery Wastewater in a Two-Stage Anaerobic Treatment. Water Research, 30
(9), 2072–2078.

Glombitza, F. 2001 Treatment of Acid Lignite Mine Flooding Water by Means of
Microbial Sulfate Reduction. Waste Management, 21, 197–203.

Goorissen, H. P. 2002 Thermophilic Methanol Utilization by Sulfate Reducing
Bacteria. Dissertation, University of Groningen.

Harkness, J. B. L., Gorski, A. J., and Daniels, E. J. 1990 Hydrogen Sulfide Waste
Treatment by Microwave Plasma Dissociation. Proceedings of the 25th Intersociety
Energy Conversion Engineering Conference, 6, 197–202.

Heijnen, J. J. 1988 Review on the Application of Anaerobic Fluidized Bed Reactors
in Wastewater treatment. Chemical Engineering, (London), 41 (B37).

Iza, J. E. Colleran, Paris, J. M., and Wu, W. M. 1991 International Workshop on
Anaerobic Treatment Technology for Municipal and Industrial Wastewaters:
Summary Paper. Water Science Technology, 24 (8), 1–16.

180
Janssen, A. J. H. Lettinga, G., and De Keizer, A. 1999 Removal of Hydrogen
Sulphide from Wastewater and Waste Gases by Biological Conversion to Elemental
Sulphur—Colloidal and Interfacial Aspects of Biologically Produced Sulphur
Particles. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 151
(1), 389–397.

Kabdasli, K., Tūnay O., and Orhon D. 1995 Sulfate Removal from Indigo Dyeing
Textile Wastewaters. Water Science Technology, 32 (12), 21–27.
Kalyuzhnyi, S., Fedorovich, V., Lens, P., Hulshoff Pol, L., and Lettinga, G. 1998
Mathematical Modelling as a Tool to Study Population Dynamics between Sulfate-
Reducing and Methanogenic Bacteria. Biodegradation, 9, 187–199.

Karhadkar, P. P., Audic, J. M., Faup, G. M., and Khanna, P. 1987 Sulfide and Sulfate
Inhibition of Methanogenesis. Water Research, 21, 1061.

Karnovsky, M. J. 1965 A Formaldehyde-Glutaraldehyde Fixative of High
Osmolarity for Use in Electron Microscopy. Journal of Cell Biology, 27,137A.

Kettuen, R. H., and Rintala, J. A. 1995 Sequential Anaerobic–Aerobic Treatment of
Sulfur Rich Phenolic Leachates. Journal of Chemical Technology and
Biotechnology, 62, 177–184.

Kelly, P. D., and Wood P. A. 2000 Confirmation of Thiobacillus denitrificans as a
Species of the Genus Thiobacillus, in the Subclass of the Proteobacteria, with Strain
NCIMB 9548 as the Type Strain. International Journal of Systematic and
Evolutionary Microbiology. 50, 547–550.

Khanal, S. K., and Huang, J. C. 2003 Anaerobic Treatment of High Sulfate
Wastewater with Oxygenation to Control Sulfide Toxicity. Water Science and
Technology, 47 (12), 183–189.

Kim, H., Kim, J. Y., Chung, S. J., and Xie, Q. 2002 Long-Term Operation of a
Biofilter for Simultaneous Removal of H
2
S and NH
3
. Air & Waste Management
Association, 52, 1389–1398.

181
Kleerebezem, R., and Mendez, R. 2002 Autotrophic Denitrification for Combined
Hydrogen Sulfide Removal from Biogas and Post-Denitrification. Water Science and
Technology, 45 (10), 349–356.

Kohl, A. L., and Nielsen, R. B. 1997 Gas Purification, Fifth Edition, Gulf Publishing
Company.

Kolmert, A., and Johnson, D. B. 2001 Remediation of Acidic Waste Waters Using
Iimmobilised, Acidophilic Sulfate-Reducing Bacteria. Journal of Chemical
Technology and Biotechnology, 76 (8), 836–843.

Koster, I. W., Rinzema, A., De Vegt, A. L., and Lettinga, G. 1986 Sulfide Inhibition
of the Methanogenic Activity of Granular Sludge of Various H-levels. Water
Research (G. B.), 12, 1561.

Krasheninkov, E.G., Rusanov, V. D., Sanyuk, S. V., and Fridman, A. A. 1986
Dissociation of Hydrogen Sulfide in an RF Discharge. Soviet Physics—Technical
Physics, 31(6), 645–648.

Kroiss, H., and Wabnegg, F. 1983 Testing Methods to Characterize Anaerobic
Sludge and Anaerobic Removal of Substrates. Poster presented at 3
rd
Int. Conf. of
Anaerobic Digestion, Boston, MA.

Larry Walker Associates, 2006 Calleguas Creek Watershed Boron, Chloride, TDS,
and Sulfate TMDL. First stakeholder draft, technical report submitted to Los Angeles
Regional Water Quality Control Board and the United States Environmental
Protection Agency.

Lawrence, A. W., McCay, P. L., and Guerin, F. J. A. 1966 The Effects of Sulfides on
Anaerobic Treatment. Air Water Pollution International Journal, 10, 207.

Lee, E. Y., Lee N. Y., Cho K., and Ryu, H. W. 2006 Removal of Hydrogen Sulfide
by Sulfate-Resistant Acidithiobacillus thiooxidans AZ11. Journal of Bioscience and
Bioengineering, 101 (4), 309–314.

182

Lens, P. N. L., Visser, A., Janssen, A. J. H., Hulshoff, Pol, L. W., and Lettinga, G.
1998 Biotechnical Treatment of Sulfate-rich Wastewaters. Critical Reviews in
Environmental Science and Technology, 28 (1), 41–88.

Li, H., Lueking, D. R., Mihelcic, J. R., and Peterson, K. 2005 Biogeochemical
Analysis of Hydrogen Sulfide Removal by a Lava-Rock Packed Biofilter. Water
Environmental Research, 77 (2), 179–186.

Maier, R. M., Pepper, I. L., and Gerba, C. P. 2000 Environmental Microbiology.
Academic Press.

Malhautier, L., Gracian, C., Roux, C. J., Fanlo, L. J. and Le Cloirec, P. 2003
Biological Treatment Process of Air Loaded with an Ammonia and Hydrogen
Sulfide Mixture. Chemosphere, 50, 145–153.

McCartney, D. M. 1991 Effects of Sulfate and Sulfide on Methanogenic and Sulfate
Reducing Activity during Degradation of Simple Organics: Role of Propionate and
Acclimation. Ph.D. thesis, University of Manitoba.

McCartney, D. M., and Oleszkiewicz, J. A. 1991 Sulfide Inhibition of Anaerobic
Degradation of Lactate and Acetate. Water Research, 25, 203.

McCartney, D. M., and Oleszkiewicz, J. A. 1993 Competition between Methanogens
and Sulfate Reducers: Effect of COD: Sulfate Ratio and Acclimation. Water
Environment Research, 65 (5), 655–664.

McCarty, P. L. 1964 Anaerobic Waste Treatment Fundamental, Part Three: Toxic
Material and Their Control. Public Work, 95 (11).

Metcalf and Eddy Inc., 2003 Wastewater Engineering Treatment and Reuse.
McGraw-Hill Professional, Fourth Edition.

Michiko, M. N., and Zuber, P. 2004 Strict and Facultative Anaerobes: Medical and
Environmental Aspects. CRC Press.

183

Mizuno, O., Li, Y. Y., and Noike, T. 1994 Effects of Sulfate Concentration and
Sludge Retention Time on the Interaction between Methane Production and Sulfate
Reduction for Butyrate. Water Science and Technology, 30 (8), 45–54.

Moosa, M. and Harrison, S. T. L. 2002 A Kinetic Study on the Anaerobic Reduction
of Sulfate. Part I: Effect of Sulfate Concentration. Chemical Engineering Science,
57, 2773–2780.

Morgan-Segastume, M. J., Noyola, A., Raveh, S., and Ergas, J. S. 2003 Changes in
Physical Properties of a Compost Biofilter Treating Hydrogen Sulfide. Journal of Air
& Waste Management Association, 53, 1011–1021.

Muthumbi, W., Boon, N., Boterdaele, R., DeVreese, I., Top, E. M., and Verstraete,
W., 2001 Microbial Sulfate Reduction with Acetate: Process Performance and
Composition of the Bacterial Communities in the Reactor at Different Salinity
Levels. Applied Microbiology and Biotechnology, 55 (6), 787–793.

Nagpal, S., Chuichulcherm, S., and Peeva, L. 2000 Microbial Sulfate Reduction in a
Liquid-Solid Fluidized Bed Reactor. Biotechnology and Bioengineering, 70 (4),
370–380.

Nagpal, S., Chuichulcherm, S., Livingston, A., and Peeva, L. 2000 Ethanol
Utilization by Sulfate-Reducing Bacteria: An Experimental and Modeling Study.
Biotechnology and Bioengineering, 70 (5), 533–543.

Nedwell, D. B., and Reynolds, P. J. 1996 Treatment of Landfill Leachate by
Methanogenic, and Sulfate- Reducing Digestion. Water Research, 30, 21–28.

Ng, Y. L., Yan, R., Chen, X. G., Geng, A. L., Goulg, W. D., Liang, D. T., and Koe,
L. C. C. 2004 Use of Activated Carbon as a Support Medium for H
2
S Biofiltration
and Effect of Bacterial Immobilization on Available Pore Surface. Applied Microbial
Biotechnology, 66 (3), 259–265.

184
Oude Elferink, S. J. W. H., Visser, A., Hulshoff Pol, L. W., and Stams, A. J. M. 1994
Sulfate Reduction in Methanogenic Bioreactors. FEMS Microbiol. Rev., 15, 119–
136.

Parkin, G. F., Lynch, H. A., Kui, W. C., Van Keuren, E. L., and Bhattacharya, S. K.
1990 Interaction between Sulfate Reducers and Methanogens for Acetate and
Proponate. Research Journal of the Water Pollution Control Federation, 62, 780.

Perry. J., Staley, J., and Lory, S. 2002 Microbial Life. Sinauer Associates.

Pirbazari, M., Voice, T. C., and Weber, W. J. Jr. 1990 Evaluation of Biofilm
Development on Various Natural and Synthetic Media. Hazardous Wastes and
Hazardous Materials, 7 (3), 239–250.

Pirbazari, M., Ravindran, V., Jung, J., Samee, M., and Vahdati-Nikzad, A. 2006
Biological Sulfate Reduction for Recovering Reverse Osmosis Concentrate. Final
Project Report, submitted to the Metropolitan Water District of Southern California
and U.S. Environmental Protection Agency under the auspices of the Desalination
Research and Innovation Partnership (DRIP) II.

Polypore Company, 2007 Liqui-Cel Membrane Contactor Product Information,
www.liqui-cel.com.

Postgate, J. R. 1984 The Sulfate-Reducing Bacteria, Cambridge University Press,
Second Edition.

Prescott, M. L., Harley P. J., and Klein A.D. 2003 Microbiology, 5
th
Edition. New
York, McGraw-Hill Companies.

Robinson, J. A., Tiedje, J. M. 1984 Competition between Sulfate-Reducing and
Methanogenic Bacteria for H
2
Under Resting and Growing Conditions. Archives of
Microbiology, 137 (1), 26–32.

Ricklefs, R. E., and Miller G. L. 2000 Ecology W. H. Freeman and Company.

185
Rittmann, B. E., and McCarty, P. L. 2001 Environmental Biotechnology: Principals
and Applications. New York, McGraw-Hill.

Rittmann, B. E. 2004 Comparative Performance of Biofilm Reactor Types.
Biotechnology and Bioengineering, 24 (6), 1341–1370.

Robinson, J. A., Tiedje, J. M. 1984 Competition between Sulfate-Reducing and
Methanogenic Bacteria for H
2
under Resting and Growing Conditions. Archives of
Microbiology, 137 (1), 26–32.

Rodgers, M. and Zhan, X. M. 2005 Moving-Medium Biofilm Reactors. Reviews in
Environmental Science and Biotechnology, 2 (2-4), 213–224.

Schieder, D., Quicker, P., Schneider, R., Winter, H., Prechtl, S., and Faulstich, M.
2003 Microbiological Removal of Hydrogen Sulfide from Biogas by Means of a
Separate Biofilter System: Experience with Technical Operation. Water Science and
Technology, 48 (4), 209–212.

Shareefdeen, Z., Herner, B., and Wilson, B. 2002 Biofiltration of Nuisance Sulfur
Gaseous Odors from a Meat Rendering Plant. Journal of Chemical Technology and
Biotechnology, 77, 1296–1299.

Shareefdeen, Z., Herner, B., Webb, D., and Wilson, S. 2003 Hydrogen Sulfide
Removal in Synthetic Media Biofilters. Environmental Progress, 22 (3), 207–213.

Shin, H., S., Oh, S. E., and Lee, C. Y., 1997 Influence of Sulfur Compounds and
Heavy Metals on the Methanization of Tannery Wastewater. Water Science and
Technology, 35 (8), 239–245.

Song, Y., Piak, B., Shin, H., and La, S. 1998 Influence of Electron Donor, and Toxic
Materials on the Activity of Sulfate Reducing Bacteria for the Treatment of
Electroplating Wastewater. Water Science and Technology, 38 (4–5), 187–194.

186
Sorokin, Y. D., Tourova, P. T., Kolganova, V. T., Sjollema, A. K., and Kuenen, G. J.
2002 Thioalkalispira microaerophila Gen. Nov., Sp. Nov., a Novel Lithoautotrophic,
Sulfuroxidizing Bacterium from a Soda Lake. International Journal of Systematic
and Evolutionary Microbiology, 52: 2175–2182.

Speece, R. E. 1996 Anaerobic Biotechnology for Industrial Wastewaters. Archae
Press.

Stams, A. J. M., Oude Elferink, S. J. W. H., and Westermann, P. 2003 Metabolic
Interactions Between Methanogenic Consortia and Anaerobic Respiring Bacteria.
Adv. Biochem. Eng. Biotechnol., 81, 151–203.

Stams, A. J. M., Plugge, C. M., deBok, F. A. M., van Houten, B. H. G. W., Lens, P.,
Dijkman, H., and Weijma, J. 2005 Metabolic Interaction in Methanogencan Sulfate-
Reducing Bioreactors. Water Environment Research, 52 (1–2), 13–20.

Steed, V. S., Suidan, M. T., Gupta, M., Miyahara, T., Acheson, C. M., and Sayles,
G. D. 2000 Development of a Sulfate-Reducing Biological Process to Remove
Heavy Metals from Acid Mine Drainage. Water Environment Research, 72 (5), 530–
535.

Steudel, R., 1989 On the Nature of the Elemental Sulfur (S
0
) Reduced by Sulfur-
Oxidizing bacteria- A model for S
0
Globules. In Autotrophic Bacteria, Science
Technology Publishers, 289–303.

Syed, M., Soreanu, G., Falletta, P., and Béland M. 2006 Removal of Hydrogen
Sulfide from Gas Streams Using Biological Processes—A Review. Canadian
Biosystems Engineering, 48, 2.1–2.14.

Takano, B., Koshida, M., Fujiwara, Y., Sugimori, K., and Takayangi, S. 1997
Influence of Sulfur-Oxidizing Bacteria on the Budget of Sulphate in Yugama Crater
Lake, Kusatsu-Shirane Volcano, Japan. Biogeochemistry, 38: 227–253.

187
TNRCC 2000 TMDLs for Total Dissolved Solids and Sulfate, in E. V. Spence
Reservoir. Prepared by Strategic Assessment Division, TNRCC Colorado River
Municipal Water District, and Distributed by Texas Natural Resource Conservation
Commission.

Thauer, R. K., Jungermann, K. and Decker, K. 1977 Energy Conservation in
Chemotrophic Anaerobic Bacteria. Bacteriol. Rev., 41, 100–180.

Thauer, R. K., Zinkhan, D. M., and Spormann, A. M. 1989 Biochemistry of Acetate
Catabolism in Anaerobic Chemotrophic Bacteria. Annual Review of Microbiology,
43, 43–67.

Tichy, R., Grotenhuis, J. T. C., Bos, P., and Lens, P. 1996 Solid State Reduced
Sulfur Compounds: Environmental Aspects and Bioremediation. Critical Revue
Environmental Science and Technology, 28 (1), 1–40.

Traus, I., and Suhr, H. 1992 Hydrogen Sulfide Dissociation in Ozonizer Discharges
and Operation of Ozonizers at Elevated Temperatures. Plasma Chemistry and
Plasma Processing, 12 (3), 275–285.

Traus, I., Suhr, H., Harry, J. E., and Evans, D. R. 1993 Application of a Rotating
High-Pressure Glow Discharge for the Dissociation of Hydrogen Sulfide. Plasma
Chemistry and Plasma Processing, 13 (1), 77–91.

Tursman, J. F., and Cork, D. J. 1989 Influence of Sulfate and Sulfate-Reducing
Bacteria on Anaerobic Digestion Technology. Advances in Biotechnological
Processes, 12, 273–285.

Van Houten, R. T., Hulshoff Pol, L. W., and Lettinga, G. 1994 Biological Sulfate
Reduction Using Gas-Lift Reactors Fed with Hydrogen, and Carbon Dioxide as
Energy and Carbon Source. Biotechnology and Bioengineering, 44 (5), 586–594.

Vetter, r. D. 1985 Elemental Sulfur in the Gills of Three Species of Clams
Containing Chemoautotrophic Symbiotic Bacteria: A Possible Inorganic Energy
Storage Compound. Marine Biology, 88, 33–42.

188

Vlasceanu, L., Popa R., and Kinkle K. B. 1997 Characterization of Thiobacillus
thioparus LV43 and Its Distribution in a Chemoautotrophically Based Groundwater
Ecosystem. Applied and Environmental Microbiology, 63(8): 3123–3127.

California State Water Resources Control Board, 2005 State of California S.B.
TMDL Guidance: A process for Addressing Impaired Waters in California.

Wani, A. H., Lau, A. K., and Branion, M. R. 1999 Biofiltration Control of Pulping
Odors-Hydrogen Sulfide: Performance, Microkinetics and Coexistance Effects of
Organo-Sulfur Species. Journal of Chemical Technology and Biotechnology, 74, 9–
16.

Webster, T. S., Devinny, J. S.,

Torres, E. M., and Basrai, S. S. 1997 Microbial
Ecosystems in Compost and Granular Activated Carbon Biofilters. Biotechnology
and Bioengineering, 53 (3), 296–303.

Weijma J., Hushoff Pol, L. W., Stams, A. J., and Lettinga, G. 2000 Performance of a
Thermophilic Sulfate and Sulfite Reducing High Rate Anaerobic Reactor Fed with
Methanol. Biodegradation, 11 (6), 429–439.

Widdle, F., and Pfenning, N. 1981 Studies on Dissimilatory Sulfate Reducing
Bacteria that Decompose Fatty acids. 1. Isolation of New Sulfate Reducing Bacteria
Enriched with Acetate from Saline Environments—Description of Desulfabacter
postgatei Gen-Nov, Sp-Nov. Archives of Microbiology, 129 (5), 395–400.

Widdle, F., and Pfenning, N. 1982 Studies on Dissimilatory Sulfate Reducing
Bacteria that Decompose Fatty acids: 2. Incomplete Oxidation of Propionate by
Desulfubulbus propionicus. Archives of Microbiology, 131, 360–365.

Widdle, F. 1988. Microbiology and Ecology of Sulfate- and Sulfur-Reducing
Bacteria. In A. J. B. Zebnder (ed.), Biology of Anaerobic Microorganisms. New
York, John Wiley and Sons, Inc., pp. 469–585.

189
Williams, M., Evangelista, R., and Cohen, Y. 2002 Non-thermal Process for
Recovering Reverse Osmosis Concentrate: Process Chemistry and Kinetics.
Proceedings, 2002 AWWA Water Quality Technology Conference, Seattle, WA
Yann, A. Le Gouellec, Elimelech, M. 2002 Calcium Sulfate (gypsum) Scaling in
Nanofiltration of Agricultural Drainage Water. Journal of Membrane Science, 205,
279–291.

Yen, T. F. 1998 Environmental Chemistry: Essentials of Chemistry for Engineering
Practice, Volume 4B. Prentice Hall, 1
st
Edition.

Yoda, M, Kitagawa, M., and Miyaji, Y. 1987 Long Term Competition between
Sulfate-reducing, and Methane-producing Bacteria for Acetate in Anaerobic Biofilm.
Water Research, 21 (12), 1547–1556.

Zitomer, D. H., Shrout, J. D. 2000 High Sulfate, High Chemical Oxygen Demand
Wastewater Treatment Using Aerated Methanogenic Fluidized Beds. Water
Environment Research, 72 (1), 90–97.

190

APPENDIXES

191
APPENDIX A

Recommendations to Improve the FBR System

1. Several operational problems were encountered in the FBR system used in
this study. The liquid influent tube from the feed tank and the recirculation line both
entered the reactor from the bottom of the FBR column, and the influent passed a
stainless mesh screen that supported the column packing. This mesh screen was
prone to severe clogging, which resulted in pressure loss. The cause of the clogging
was excess biomass and some GAC particles that had been crushed in the
recirculation pump. To solve this problem, a solid-liquid separator should be
installed in the recirculation loop (Figure A1).

Figure A1: Gas-Liquid Separator Installation

q
Q
Gas /liquid
separator
Pump

Check
Valve

Fluidized Bed Reactor

192
2. A check valve should be installed in the discharge pie of the recirculation
pump to prevent backflow.

3. Ball valves should be installed at the column entrance to prevent water
discharge when cleaning the influent and recirculation lines.

4. Degassing of the Recirculation Pump: During the cleaning process, the
water level may fall below the recirculation exit line and cause air to enter into the
pump. The air must be withdrawn by using a syringe. Even hydrogen sulfide gas
could trap inside the pump, which must be removed before further use.

5. Re-fluidization of the System: The system needs to be stopped for regular
maintenance. This would include cleaning the pumps, and checking valves,
recirculation line, screen mesh, etc. Stopping the operation usually causes the GAC
particles to stick together. After re-start up, a gentle increase in the recirculation flow
can return the system into fluidization. If this alone does not help, gentle mixing
might be used to provide separation of the GAC particles. This action must be done
very gently to reduce the separation of the biomass from the GAC particles.

193

APPENDIX B

Recommendations for the Successful Operation of the FBR with
Hydrogen Gas as Electron Donor

Laboratory experiments with H
2
and CO
2
revealed several operational issues
that must be considered for successful laboratory-scale studies.

1. Hydrogen Gas Introduction into the System
Hydrogen gas has very low water solubility (1.82% at 20
o
C); therefore, it
must be introduced into the system in ultra-fine bubbles to increase the mass transfer.
In this study, ultra-fine diffusers were used to provide bubbles within a 40-μm size
range. However, these diffusers are prone to clogging, and therefore should not be
installed directly inside the FBR column.

2. Introducing Carbon Dioxide Gas into the System
Carbon dioxide has a high aqueous solubility, and therefore it can easily be
introduced into the column through the influent line.

3. Hydrogen Gas Measurement

194
Because of the low solubility of hydrogen in water, the soluble portion and
the uptake rate by bacteria should be measured with great caution and precision.

4. pH Adjustment
Hydrogen and carbon dioxide by utilizing SRBs manifest a high degree of
sensitivity to solution pH, and therefore pH needs to be monitored continuously.

5. Gas Metering Pumps
An accurate measurement of the gas flow rates is of paramount importance.
Therefore, efforts should be made to select the appropriate flow meters and to adapt
effective flow-controlling strategies.

195

APPENDIX C

(CMBR Studies Data with Acetate and Lactate as Electron Donors)

196

Table C1: CMBR Data for Acetate as the Electron Donor
(Different pHs)

C/S=1
pH=8 pH=7.5 pH=8 pH=7.5
Time
(hr)
Sulfate
(mg/L)
Sulfate
(mg/L)
Acetate
(mg/L)
Acetate
(mg/L)
0 687.89 662.75 594.35 592.26
3 673.95 661.45 580.79 565.64
9 644.87 664.67 529.52 548.98
15 632.95 669.84 ------ -----
21 622.97 659.04 506.72 516.34
27 656.53 646.36 526.12 502.62
33 666.56 652.74 516.52 506.21
39 654.96 627.12 492.64 482.60
45 625.02 612.50 478.33 470.81
51 651.01 634.58 480.24 486.55
57 652.74 620.36 488.56 471.78
63 636.57 568.60 450.59 436.32
69 636.20 572.52 473.21 431.18
75 620.78 550.43 457.84 413.08
81 603.00 531.76 440.78 406.42
87 589.69 500.46 438.87 381.27
93 563.02 468.11 400.16 342.89
99 582.95 448.01 397.78 323.45
105 531.57 419.24 394.27 311.87
111 514.39 414.40 375.61 259.51
117 496.14 379.12 354.56 229.91
123 487.31 369.77 325.42 215.65

197
Table C1: CMBR Data for Acetate as the Electron Donor (Cont.)
(Different pHs)

C/S=1
pH=8 pH=7.5 pH=8 pH=7.5
Time
(hr)
Sulfate
(mg/L)
Sulfate
(mg/L)
Acetate
(mg/L)
Acetate
(mg/L)
129 475.11 327.34 309.21 193.35
135 467.22 292.21 248.51 163.74
141 439.10 282.87 217.48 149.48
147 420.19 268.92 190.96 135.57
153 414.60 233.79 198.68 92.39
159 372.58 247.38 156.90 80.82
165 350.25 233.67 154.26 77.49
171 268.92 241.28 135.57 34.95
177 341.50 179.35 113.76 23.98
183 330.41 161.03 71.66 25.95
189 283.27 162.29 49.13 3.13
195 304.12 174.97 44.39 0.00
201 289.91 38.88
207 283.63 18.14
213 250.66 0.00

198

Table C1: CMBR Data for Acetate as the Electron Donor (Cont.)
(Different pHs)

pH=6 pH=7 pH=7 pH=6
Time (hr) SO
4
(mg/L) SO
4
(mg/L)
Acetate
(mg/L)
Acetate
(mg/L)
0 702.57 690.22 507.07 514.73
21 683.80 672.50 385.54 413.80
28 644.87 648.71 346.05 358.13
35 614.82 620.21 305.86 325.81
49 528.66 549.16 197.13 217.28
56 520.06 495.09 152.57 182.80
63 454.70 396.38 125.20 132.29
77 368.04 386.97 41.01 55.13
84 332.27 396.09 0.00 0.00
91 351.36 427.87 0.00 0.00
105 348.72 406.10 0.00
112 346.26 424.71 3.27
119 358.98 0.00

199

Table C2: CMBR Data for Acetate as the Electron Donor (Different Temperatures)

C/S=1 T=35 T=30 T=20 T=25
Time
(hr)
Sulfate
(mg/L)
Sulfate
(mg/L)
Sulfate
(mg/L)

Time (hr) Sulfate
0 724.73 690.22 695 0 693.14
21 725.6 672.5 711.20 6 696.16
28 654.17 648.71 715.33 20 676.83
35 662.06 620.21 706.67 24 660.68
49 546.81 549.16 700.25 30 673.45
56 495.89 495.09 726.22 42 646.84
63 462.83 396.38 684.95 52.5 618.16
77 357.82 386.97 663.42 72.5 570.90
84 323.04 396.09 644.10 78.5 542.41
91 282.16 427.87 582.64 91.5 502.94
105 262.67 426.1 535.87 96 486.55
112 214.81 424.71 524.00 102 461.56
119 196.45 474.46 115.5 416.68
140 175.26 452.33 120 406.19
147 154.16 439.67 126 382.54
161 136.62 439.20 139.5 345.44
168 134.71 144 325.30
175 135.16 150 309.11
189 120.98 163.5 273.04
196 123.36 167.5 260.62
173.5 251.39
190.5 204.10
196.5 172.68

200
Table C2: CMBR Data for Acetate as the Electron Donor (Cont.)
(Different Temperatures)

C/S=1 T=35 T=30 T=20 T=25
Time
(hr)
Sulfate
(mg/L)
Sulfate
(mg/L)
Time
(hr)
Sulfate
(mg/L)
Time
(hr) Sulfate
21 725.6 672.5 77 663.42 42 618.16
28 654.17 648.71 84 644.10 52 570.90
35 662.06 620.21 91 582.64 72 542.41
49 546.81 549.16 105 535.87 78 502.94
56 495.89 495.09 112 524.00 91 486.55
63 462.83 396.38 119 474.46 96 461.56
77 357.82 386.97 133 452.33 102 416.68

Table C2: CMBR Data for Acetate as the Electron Donor (Cont.)
(Different Temperatures)

T=35 T=20 T=25 T=25
Time
(hr)
Acetate
(mg/L)
Acetate
(mg/L)
Time
(hr) Acetate
0 653 624.64 0 570.96
21 647.31 509.47 6 575.10
28 568.94 488.55 20 564.16
35 468.59 450.98 24 557.04
49 433.11 373.22 30 556.91
56 392.64 340.19 42 532.08
63 315.32 316.77 52.5 513.62
77 279.47 237.64 72.5 490.24

201
Table C2: CMBR Data for Acetate as the Electron Donor (Cont.)
( Different Temperatures)

T=35 T=20 T=25 T=25
Time
(hr)
Acetate
(mg/L)
Acetate
(mg/L)
Time
(hr) Acetate
84 256.81 206.91 78.5 453.09
91 175.45 203.46 91.5 426.27
105 156.5 97.56 96 410.86
112 175.48 72.14 102 394.78
119 126.84 59.06 115.5 363.56
133 116.38 0 120 358.23
140 102.49 126 337.93
147 87.61 139.5 311.82
161 75.76 144 300.44
168 61.01 150 281.46
175 50.09 163.5 234.67
189 39.35 167.5 223.60
196 173.5 201.04
190.5 145.22
196.5 153.86
209 128.71
212.5 118.51
217.5 103.09
220 95.20
225.5 85.33
230.5 65.10
243.5 47.55
253.5 0.00

202

Table C3: CMBR Data for Acetate as the Electron Donor (Different pHs)
pH=7 C/S= 0.8 pH=7 C/S= 1.0
Time (hr) Sulfate Acetate Time (hr) Sulfate Acetate
0 721.64 471.92 0 690.22 507.07
1 644.99 438.25 21 672.50 385.54
7 630.55 406.83 28 648.71 346.05
13 612.88 350.24 35 620.21 305.86
19 611.26 350.33 49 549.16 197.13
25 608.44 335.22 56 152.57
31 607.51 315.95 63 495.09 125.20
37 594.56 302.88 77 396.38 41.01
43 578.63 290.69 84 386.97 0.00
49 583.93 250.00 91 396.09 0.00
55 561.66 230.70 105 427.87 4.19
61 549.05 249.48 112 426.10 0.35
67 525.75 212.01 119 424.71 0
73 522.47 194.27
79 512.70 145.99
87 491.17 129.49
93 474.31 104.30

203
Table C3: CMBR Data for Acetate as the Electron Donor (Cont.)
(Different pHs)

pH=7 C/S= 1.2 C/S= 1.2 pH=7 C/S= 1.4 C/S= 1.4
Time (hr) Sulfate Acetate Time (hr) Sulfate Acetate
0 703.89 742.34 0 778.83 934.43
2 702.51 726.77 21 748.07 800.38
16 668.54 603.43 28 700.53 711.98
20 646.07 581.14 35 639.24 647.84
26 624.54 541.53 49 511.15 497.20
38 525.45 434.81 56 462.85 452.38
44 492.69 388.09 63 417.32 407.87
50 444.09 340.26 77 329.13 275.88
62 330.43 270.20 84 288.11 241.10
70 281.10 216.85 91 213.78 203.59
74 237.49 193.27 105 85.85 85.64
92 57.07 82.88 112 48.32 50.86
98 98.95 66.16 119 32.58 58.65
110 38.24 0.00 133 15.50 27.08
116 42.91 0.00 140 15.75 21.13
124 37.47 0.00 147 14.82 15.81
161 12.89 13.65
168 11.79 20.34

204

Table C4: CMBR Data for Lactate as the Electron Donor
(Different pHs)

C/S=1 pH=6.0 C/S=1 pH=6.5
Time
(hr)
Lactate
acid Sulfate
Time
(hr)
Lactate
acid Sulfate
0 334.06 660.46 0 305.40 701.76
3 282.96 631.86 12 264.59 697.37
11 202.75 543.68 24 260.76 682.37
25 169.38 522.42 36 181.37 616.77
37 136.13 484.01 48 121.63 579.64
49 128.85 478.83 60 100.72 565.05
61 130.32 478.99 72 96.66 547.44
72 115.17 479.71 84 115.96 568.84
C/S=1 pH=7.0 C/S=1 pH=7.5
Time
(hr)
Lactate
acid Sulfate
Time
(hr)
Lactate
acid Sulfate
0 328.78 672.08 0 339.33 692.18
3 272.55 630.78 12 304.62 694.11
11 170.09 513.97 24 307.56 661.20
25 93.77 438.58 36 220.04 563.21
37 117.68 452.06 48 168.75 502.41
49 117.43 451.06 60 122.54 470.33
61 122.59 459.55 72 119.59 454.19
73 124.58 504.06 84 123.41 476.20
110 124.83 483.13

205

Table C4: CMBR Data for Lactate as the Electron Donor (Cont.)
(Different pHs)

C/S=1.0 pH=7.0 C/S=2.0 pH=7.0
Time
(hr)
Lactate
acid Sulfate
Time
(hr) Lactate Sulfate
0 328.78 672.08 0.00 986.46 701.80
3 272.55 630.78 11.00 981.02 701.09
11 170.09 513.97 23.00 874.69 666.29
25 93.77 438.58 35.00 650.80 547.18
37 117.68 452.06 45.00 521.82 475.16
49 117.43 451.06 60.00 293.81 227.50
61 122.59 459.55 71.00 295.06 246.36
83.00 480.71 27.18
95.00 453.81 28.68
107.00 432.88 30.49

206
Table C4: CMBR Data for Lactate as the Electron Donor (Cont.)
(Different pHs)

C/S=2.5 pH=7.0 C/S=3.0 pH=7.0
Time
(hr) Lactate Sulfate
Time
(hr) Lactate Sulfate
0 1404.50 650.36 0 1471.05 689.26
11 1505.36 700.33 11 1452.56 681.18
23 1308.65 677.48 23 1106.16 673.29
35 1132.59 591.09 35 1102.26 602.86
45 1076.08 525.73 45 1056.85 565.28
60 807.59 271.77 60 852.48 390.23
71 584.14 231.06 71 730.42 356.24
83 577.46 134.81 83 334.96 103.06
C/S=2.5 pH=7.0 C/S=3.0 pH=7.0
Time
(hr) Lactate Sulfate
Time
(hr) Lactate Sulfate
95 413.00 76.14 95 334.55 99.69
107 414.18 78.13 107 331.99 98.81
119 442.87 26.62 119 430.56 80.50
131 413.60 32.05 131 341.99 15.74

207
Table C5: CMBR Data for Lactate as the Electron Donor
(Different Temperatures)

T=25 C/S= 2 T=30 C/S=2 T=35 C/S=2
Time
(hr) Lactate Sulfate
Time
(hr) Lactate Sulfate
Time
(hr) Lactate Sulfate
0 1250.63 688.13 0.00 986.46 701.80 0 1022.67 689.97
11 1248.38 698.89 11.00 981.02 701.09 23 871.71 571.90
23 980.17 664.58 23.00 874.69 666.29 35 647.74 392.90
35 891.94 623.35 35.00 650.80 547.18 47 383.26 139.80
45 885.24 567.46 45.00 521.82 475.16 59 374.80 105.82
60 673.58 405.60 60.00 293.81 227.50 71 364.78 117.28
71 514.17 280.55 71.00 295.06 246.36
83 297.81 272.31 83.00 480.71 27.18
95 285.94 242.39 95.00 453.81 28.68
107 338.52 110.79 107.00 432.88 30.49
119 341.99 93.23
131 346.40 98.01

208

APPENDIX D

(FBR Studies Data with Hydrogen Gas, Acetate and Lactate as Electron Donors)

209
Table D1: Sulfate Data in FBR with Hydrogen Gas as Electron Donor

Day Influent Effluent Removal (%) pH
0 847.67 652.66 23.01 6.42
1 847.67 532.14 37.22 6.42
2 898.66 258.03 71.29 7.50
3 925.33 692.65 25.15 7.80
7 957.39 692.65 27.65 6.00
9 908.74 864.61 4.86 6.00
13 945.88 469.25 50.39 6.50
15 920.31 603.63 34.41 7.30
17 907.39 510.24 43.77 6.70
19 915.49 553.30 39.56 6.80
20 921.19 519.21 43.64 7.40
22 921.19 565.35 38.63 6.60
24 916.04 241.11 73.68 6.80
29 901.71 404.64 55.13 6.60
30 909.16 349.75 61.53 7.60
31 910.63 39.33 95.68 6.60
37 905.66 232.19 74.36 6.62
38 897.80 179.94 79.96 6.72
39 916.79 51.61 94.37 6.80
40 883.77 54.66 93.82 6.85
41 883.77 55.25 93.75 6.82
44 1008.45 372.74 63.04 6.78
45 1008.45 345.22 65.77 6.80
49 1008.39 436.87 56.68 6.79
50 993.67 376.89 62.07 6.78
53 1013.19 354.20 65.04 6.89
54 1013.19 383.11 62.19 6.89
57 1000.10 407.55 59.25 6.78
59 1000.10 367.58 63.25 6.81
60 1000.10 552.68 44.74 6.79
62 1020.99 632.70 38.03 6.75
63 1020.99 609.56 40.30 6.76

210
Table D1: Sulfate Data in FBR with Hydrogen Gas as Electron Donor (Con.)

Day Influent Effluent Removal (%) pH
66 1036.72 523.92 49.46 6.82
68 1011.56 562.76 44.37 6.85
70 1010.95 411.22 59.32 6.82
71 1001.49 509.64 49.11 6.88
72 1001.49 413.29 58.73 6.87
73 1001.49 375.71 62.49 7.02
75 1001.49 596.60 40.43 7.02
76 1066.46 646.38 39.39 7.02
77 1062.75 652.74 38.58 7.10
78 1062.75 600.86 43.46 7.10
79 1062.75 620.27 41.64 7.10
80 1067.31 650.48 39.05 7.10
84 1062.75 890.97 16.16 7.10
85 1073.93 738.86 31.20 7.10
86 1051.29 626.90 40.37 7.10
87 1051.29 488.82 53.50 7.02
88 941.97 361.75 61.60 7.02
89 941.97 294.31 68.76 7.02
90 941.97 176.98 81.21 7.02
91 941.97 133.39 85.84 7.02
92 941.97 112.10 88.10 6.98
93 941.97 75.28 92.01 6.98
94 987.34 133.75 86.45 6.98
95 987.34 238.37 75.86 11.00
97 987.34 599.48 39.28 11.00
98 987.34 845.65 14.35 7.02
99 955.07 853.93 10.59 7.02
100 955.07 905.77 5.16 7.02
101 992.29 873.68 11.95 7.02
102 992.29 836.69 15.68 7.02
104 992.29 755.61 23.85 7.02
105 992.29 660.17 33.47 6.80
107 1019.98 607.67 40.42 7.25

211
Table D1: Sulfate Data in FBR with Hydrogen Gas as Electron Donor (Con.)

Day Influent Effluent Removal (%) pH
109 1019.98 596.73 41.50 7.20
110 1019.98 489.42 52.02 7.20
111 1019.98 397.41 61.04 7.20
112 1019.98 357.56 64.94 6.80
113 1019.98 345.72 66.11 6.80
114 1019.98 543.42 46.72 6.61
115 1019.98 714.25 29.97 6.40
116 1019.98 782.04 23.33 6.39
117 1019.98 772.25 24.29 6.40
119 1055.59 565.31 46.45 6.80
120 1055.59 466.71 55.79 7.22
121 1055.59 422.95 59.93 7.22
124 1064.02 301.81 71.64 7.22
125 1064.02 526.50 50.52 7.22
126 1053.35 530.31 49.66 7.13
128 1053.35 460.26 56.31 7.33
129 1053.35 393.20 62.67 7.11
130 1053.35 393.01 62.69 7.18
131 1053.35 675.55 35.87 6.40
132 1034.58 868.63 16.04 6.40
133 1034.58 763.00 26.25 6.40
134 1034.58 803.63 22.32 6.63
135 1034.58 804.77 22.21 6.60
136
1034.23
813.75 21.32 6.80
137
1034.23
528.95 48.86 6.76
138
1034.23
519.50 49.77 6.70
139
1034.23
666.92 35.52 6.60
140
1034.23
767.13 25.83 7.00
141
1034.23
803.48 22.31 7.00
142
1034.23
570.46 44.84 7.00
143 1078.36 555.93 48.45 7.00
145 1078.36 440.96 59.11 7.00
146 1078.36 381.36 64.64 7.00

212
Table D1: Sulfate Data in FBR with Hydrogen Gas as Electron Donor (Con.)

Day Influent Effluent Removal (%) pH
147 1078.36 309.98 71.26 7.00
148 1078.36 265.19 75.41 7.00
149 1082.62 263.38 75.67 7.00
150 1082.62 270.71 74.99 7.00
151 1082.62 301.72 72.13 7.00
153 1082.62 286.44 73.54 7.00
154 1082.62 367.61 66.04 7.00
155 1049.16 495.48 52.77 7.00
156 1049.16 609.71 41.89 7.00
160 1049.16 650.72 37.98 7.00
161 1082.62 680.38 37.15 7.00
162 1082.62 703.93 34.98 7.00
163 1082.62 759.16 29.88 7.00
164 1082.62 768.07 29.05 7.00
165 1451.68 744.18 48.74 7.00
167 1451.68 761.43 47.55 7.00
168 1515.40 559.40 63.09 7.00
169 1515.40 549.24 63.76 7.00
170 1515.40 541.10 64.29 7.00

213
Table D2: Hydrogen and Carbon Dioxide Flow Data in FBR with Hydrogen Gas as
Electron Donor
Flow meter Flow meter ml/min ml/min
Day
Q
CO2
Q
H2
Corrected Q CO2 Corrected Q H2 % Q
CO2
% Q
H2

0 4.00 18.00
6.20
130.00 12.30 91.60
1 4.00 18.00
6.20
130.00 12.30 91.60
2 4.00 18.00
6.20
130.00 12.30 91.60
3 20.00 10.00 31.00 72.20 61.40 85.00
7 20.00 10.00 31.00 72.20 61.40 85.00
9 20.00 10.00 31.00 72.20 61.40 85.00
13 20.00 10.00 31.00 72.20 61.40 85.00
15 20.00 10.00 31.00 72.20 61.40 85.00
17 20.00 10.00 31.00 72.20 61.40 85.00
19 20.00 10.00 31.00 72.20 61.40 85.00
20 20.00 10.00 31.00 72.20 61.40 85.00
22 20.00 10.00 31.00 72.20 61.40 85.00
24 20.00 10.00 31.00 72.20 61.40 85.00
29 20.00 10.00 31.00 72.20 61.40 85.00
30 20.00 10.00 31.00 13.70 61.40 85.00
31 15.00 10.00 13.70 48.88 28.03 71.97
37 7.00 6.00 7.35 34.76 21.15 78.85
38 6.00 6.50 8.88 44.76 19.83 80.17
39 6.00 6.50 8.88 44.76 19.83 80.17
40 6.00 6.50 8.88 44.76 19.83 80.17
41 6.00 6.50 8.88 44.76 19.83 80.17
44 9.50 7.00 9.94 50.74 19.59 80.41
45 9.50 7.00 9.94 50.74 19.59 80.41
49 9.50 7.00 9.94 50.74 19.59 80.41
50 8.00 7.00 9.92 50.74 19.56 80.44
53 7.00 7.00 9.86 50.74 19.42 80.58
54 7.00 7.00 9.86 50.74 19.42 80.58
57 7.00 7.00 9.86 50.74 19.42 80.58
59 7.00 7.00 9.86 50.74 19.42 80.58
60 7.00 7.00 9.86 50.74 19.42 80.58
62 7.00 7.00 9.86 50.74 19.42 80.58

214
Table D2: Hydrogen and Carbon Dioxide Flow Data in FBR with Hydrogen Gas as
Electron Donor (Cont.)

Flow meter Flow meter ml/min ml/min
Day Q
CO2
Q
H2
Corrected Q CO2 Corrected Q H2 % Q
CO2
% Q
H2

63 7.00 7.00 9.86 50.74 19.42 80.58
66 11.00 11.00 10.25 50.06 20.47 79.53
68 11.00 11.00 10.25 50.06 20.47 79.53
70 11.00 11.00 10.25 50.06 20.47 79.53
71 7.00 7.00 10.87 50.57 21.49 78.51
72 7.00 7.00 10.87 50.57 21.49 78.51
73 7.00 7.00 10.87 50.57 21.49 78.51
75 7.00 7.00 10.87 50.57 21.49 78.51
76 7.00 7.00 10.87 50.57 21.49 78.51
77 7.00 7.00 10.87 50.57 21.49 78.51
78 7.00 7.00 10.87 50.57 21.49 78.51
79 7.00 7.00 10.87 50.57 21.49 78.51
80 7.00 7.00 10.87 50.57 21.49 78.51
84 7.00 7.00 10.87 50.57 21.49 78.51
85 5.00 5.00 7.40 34.40 21.51 78.49
86 5.00 5.00 7.40 34.40 21.51 78.49
87 5.00 5.00 7.40 34.40 21.51 78.49
88 5.00 5.00 7.40 34.40 21.51 78.49
89 5.00 5.00 7.40 34.40 21.51 78.49
90 5.00 5.00 7.40 34.40 21.51 78.49
91 5.00 5.00 7.40 34.40 21.51 78.49
92 5.00 5.00 7.40 34.40 21.51 78.49
93 5.00 5.00 7.40 34.40 21.51 78.49
94 5.00 5.00 7.40 34.40 21.51 78.49
95 5.00 5.00 7.40 34.40 21.51 78.49
97 5.00 5.00 7.40 34.40 21.51 78.49
98 0.00 5.00
7.40
34.40 0.00 100.00
99 0.00 5.00
7.40
34.40 0.00 100.00
100 5.00 5.00 7.40 34.40 21.51 78.49
101 5.00 5.00 7.40 34.40 21.51 78.49
102 5.00 5.00 7.40 34.40 21.51 78.49
104 5.00 5.00 7.40 34.40 21.51 78.49

215
Table D2: Hydrogen and Carbon Dioxide Flow Data in FBR with Hydrogen Gas as
Electron Donor (Cont.)
Flow meter Flow meter ml/min ml/min
Day Q
CO2
Q
H2
Corrected Q CO2 Corrected Q H2 % Q
CO2
% Q
H2

105 5.00 5.00 7.40 34.40 21.51 78.49
107 5.00 5.00 7.40 34.40 21.51 78.49
109 5.00 5.00 7.40 34.40 21.51 78.49
110 5.00 5.00 7.40 34.40 21.51 78.49
111 5.00 5.00 7.40 34.40 21.51 78.49
112 5.00 5.00 7.40 34.40 21.51 78.49
113 5.00 5.00 7.40 34.40 21.51 78.49
114 5.00 5.00 7.40 34.40 21.51 78.49
115 5.00 5.00 7.40 34.40 21.51 78.49
116 5.00 5.00 7.40 34.40 21.51 78.49
117 5.00 5.00 7.40 34.40 21.51 78.49
119 5.00 5.00 7.40 34.40 21.51 78.49
120 5.00 5.00 7.40 34.40 21.51 78.49
121 5.00 5.00 7.40 34.40 21.51 78.49
124 5.00 5.00 7.40 34.40 21.51 78.49
125 5.00 5.00 7.40 34.40 21.51 78.49
126 5.00 5.00 7.40 34.40 21.51 78.49
128 5.00 5.00 7.40 34.40 21.51 78.49
129 5.00 5.00 7.40 34.40 21.51 78.49
130 5.00 5.00 7.40 34.40 21.51 78.49
131 5.00 5.00 7.40 34.40 21.51 78.49
132 5.00 5.00 7.40 34.40 21.51 78.49
133 5.00 5.00 7.40 34.40 21.51 78.49
134 5.00 5.00 7.40 34.40 21.51 78.49
135 5.00 5.00 7.40 34.40 21.51 78.49
136 5.00 5.00 7.40 34.40 21.51 78.49
137 5.00 5.00 7.40 34.40 21.51 78.49
138 5.00 5.00 7.40 34.40 21.51 78.49
139 5.00 5.00 7.40 34.40 21.51 78.49
140 5.00 5.00 7.40 34.40 21.51 78.49
141 5.00 5.00 7.40 34.40 21.51 78.49
142 5.00 5.00 7.40 34.40 21.51 78.49

216
Table D2: Hydrogen and Carbon Dioxide Flow Data in FBR with Hydrogen Gas as
Electron Donor (Cont.)
Flow meter Flow meter ml/min ml/min
Day Q
CO2
Q
H2
Corrected Q CO2 Corrected Q H2 % Q
CO2
% Q
H2

143 5.00 5.00 7.40 34.40 21.51 78.49
145 5.00 5.00 7.40 34.40 21.51 78.49
146 5.00 5.00 7.40 34.40 21.51 78.49
147 5.00 5.00 7.40 34.40 21.51 78.49
148 5.00 5.00 7.40 34.40 21.51 78.49
149 5.00 5.00 7.40 34.40 21.51 78.49
150 5.00 5.00 7.40 34.40 21.51 78.49
151 5.00 5.00 7.40 34.40 21.51 78.49
153 5.00 5.00 7.40 34.40 21.51 78.49
154 5.00 5.00 7.40 34.40 21.51 78.49
155 5.00 5.00 7.40 34.40 21.51 78.49
156 5.00 5.00 7.40 34.40 21.51 78.49
160 5.00 5.00 7.40 34.40 21.51 78.49
161 5.00 5.00 7.40 34.40 21.51 78.49
162 5.00 5.00 7.40 34.40 21.51 78.49
163 5.00 5.00 7.40 34.40 21.51 78.49
164 5.00 5.00 7.40 34.40 21.51 78.49
165 5.00 5.00 7.40 34.40 21.51 78.49
167 5.00 5.00 7.40 34.40 21.51 78.49
168 5.00 5.00 7.40 34.40 21.51 78.49
169 5.00 5.00 7.40 34.40 21.51 78.49
170 5.00 5.00 7.40 34.40 21.51 78.49

217

Table D3: Hydrogen Sulfide Production Data in FBR with Hydrogen as Electron
Donor
Time (Days) H
2
S mg S
-2
/L Time (Days) H
2
S mg S
-2
/L
20 84 115 92
24 128 116 92
28 108 119 108
39 166 120 152
43 152 125 120
50 112 126 144
59 136 128 184
62 78.8 129 244
65 76 130 172
73 68 131 120
86 48 132 100
88 160 133 90
89 176 134 88
91 182 135 80
92 188 136 80
95 192 137 140
99 72 138 120
101 80 139 100
105 116 140 112
106 120 141 120
107 120 142 140
111 160 143 152
114 136 144 160

218
Table D4: Sulfate Data in FBR with Acetate as Electron Donor

Day Influent Effluent Removal pH
1
846.37
740.59 12.50 7.44
2
846.37
676.73 20.04 7.44
4
846.37
661.57 21.83 7.44
6
846.37
649.52 23.26 7.44
9
846.37
576.40 31.90 7.44
10 962.03 544.87 43.36 7.44
12 962.03 521.29 45.81 7.44
14 962.03 563.94 41.38 7.44
16 962.03 506.90 47.31 7.44
18 1050.49 464.23 55.81 7.44
20 1065.52 333.14 68.73 7.49
23 1065.52 329.11 69.11 7.53
26 1065.52 311.70 70.75 7.48
28 1133.22 249.40 77.99 7.53
30 1133.22 229.47 79.75 7.48
33 1133.22 226.13 80.05 7.42
35 1135.57 245.12 78.41 7.48
37 1135.57 251.12 77.89 7.52
39 1135.57 253.83 77.65 7.50
41 1135.57 260.15 77.09 7.48
49 1135.57
320.57
71.77 6.50
51 1135.57 309.49 72.75 7.63
55 1137.66 207.60 81.75 7.68
58 1137.66 113.76 90.00 7.53
59 1148.57 102.11 91.11 7.54
61 1148.57 184.07 83.97 7.53
62 1148.57 138.62 87.93 7.53
63 1148.57 73.14 93.63 7.53
65 1148.57 62.70 94.54 7.48
67 1134.69 59.99 94.71 7.53
69 1134.69 56.84 94.99 7.53
70 1134.69 46.62 95.89 7.56

219
Table D4: Sulfate Data in FBR with Acetate as Electron Donor (Cont.)

Day Influent Effluent Removal pH
72 1189.47 88.66 92.55 7.53
73 1189.47 81.54 93.14 7.53
74 1189.47 78.90 93.37 7.53
75 1189.47 73.24 93.84 7.53
76 1189.47 64.49 94.58 7.53
77 1189.47 61.22 94.85 7.53
79 1259.04 74.34 94.10 7.53
80 1259.04 60.30 95.21 7.53
81 1259.04 49.98 96.03 7.53
82 1271.10 40.90 96.78 7.53
83 1271.10 53.27 95.81 7.53
85 1271.10 47.17 96.29 7.53
86 1271.10 34.05 97.32 7.53
87 1271.10 26.29 97.93 7.52
88 1461.57 45.18 96.91 7.52
89 1461.57 63.87 95.63 7.52
90 1487.57 45.75 96.92 7.49
91 1487.57 106.61 92.83 7.49
92 1487.57 88.77 94.03 7.49
93 1487.57 75.89 94.90 7.48
94 1487.57 67.72 95.45 7.48
95 1487.57 65.30 95.61 7.47
96 1487.57 70.24 95.28 7.47
97 1487.57 33.39 97.76 7.44-43
98
1591.69
144.11 90.95 7.44-44
99
1591.69
116.65 92.67 7.40
100
1591.69
107.26 93.26 7.49
101
1591.69
86.64 94.56 7.49
102
1591.69
73.79 95.36 7.49
103
1591.69
69.39 95.64 7.48
104
1591.69
47.30 97.03 7.47
105
1644.96
78.88 95.20 7.44
106
1644.96
55.32 96.64 7.44

220
Table D4: Sulfate Data in FBR with Acetate as Electron Donor (Cont.)

Day Influent Effluent Removal pH
107 1704.29 121.72 92.86 7.50
108 1704.29 81.93 95.19 7.50
109 1704.29 36.90 97.83 7.51
110 1804.13 124.77 93.08 7.51
111 1804.13 99.30 94.50 7.50
112 1804.13 88.08 95.12 7.51
113 1804.13 92.33 94.88 7.51
114 1804.13 89.91 95.02 7.50
115 1804.13 69.93 96.12 7.51
116 1804.13 71.18 96.05 7.50
117 1804.13 82.24 95.44 7.49
118 1877.42 118.54 93.69 7.48
119 1877.42 85.93 95.42 7.48
120 1877.42 97.21 94.82 7.47
121 1877.42 95.42 94.92 7.46
122 1877.42 101.05 94.62 7.45
123 1877.42 71.98 96.17 7.44
124 1877.42 91.03 95.15 7.44
125 1979.73 74.87 96.22 7.50
126 1979.73 41.27 97.92 7.50
129 1979.73 66.84 96.62 7.80
130 1979.73 71.74 96.38 8-7.5

221
Table D5: Acetate Utilization Data in FBR with Acetate as Electron Donor

Day Influent Effluent Removal
1
617.7482
564.40 8.64
2
617.7482
508.88 17.62
4
617.7482
477.48 22.71
6
617.7482
342.33 44.58
9
617.7482
214.82 65.22
10
617.7482
109.68 82.24
12
617.7482
160.59 74.00
14 810.62 141.65 82.53
16 885.97 180.32 79.65
18 878.90 162.35 81.53
20 844.34 182.56 78.38
23 844.34 135.88 83.91
26 844.34 111.55 86.79
28 777.97 122.86 84.21
30 777.97 114.3 85.31
33 777.97 86.35 88.90
35 777.97 108.01 86.12
37 830.03 118.1 85.77
39 830.03 110.38 86.70
41 830.03 93.17 88.78
49 830.03 191.59 76.92
51 865.43 186.71 78.43
55 833.62 112.82 86.47
58 833.62 69.02 91.72
59 958.08 54.91 94.27
61 958.08 46.54 95.14
62 966.99 85.96 91.11
63 966.99 79.44 91.78
65 966.99 81.76 91.54
67 966.99 100.76 89.58
69 971.46 78.49 91.92
70 971.46 86.9 91.05

222
Table D5: Acetate Utilization Data in FBR with Acetate as
Electron Donor (Cont.)

Day Influent Effluent Removal
72 915.88 71.36 92.21
73 915.88 58.26 93.64
74 915.88 53.23 94.19
75 915.88 51.52 94.37
76 915.88 55.35 93.96
77 915.88 46.89 94.88
79 930.82 152.33 83.63
80 930.82 153.67 83.49
81 930.82 129.4 86.10
82 1099.97 143.28 86.97
83 1099.97 142.44 87.05
85 1099.97 210.12 80.90
86 1099.97 207.42 81.14
87 1099.97 184.71 83.21
88 1229.54 90.91 92.61
89 1229.54 145.35 88.18
90 1229.54 125.05 89.83
91 1229.54 105.38 91.43
92 1229.54 125.55 89.79
93 1229.54 124.75 89.85
94 1229.54 125.39 89.80
95 1229.54 116.67 90.51
96 1229.54 226.66 81.57
97 1251.37 194.15 84.49
98 1251.37 180.36 85.59
99 1360.9 162.86 88.03
100 1360.9 157.95 88.39
101 1360.9 190.44 86.01
102 1360.9 178.84 86.86
103 1360.9 176.68 87.02
104 1360.9 168.4 87.63
105 1360.9 143.97 89.42
106 1360.9 198.71 85.40

223
Table D5: Acetate Utilization Data in FBR with Acetate as
Electron Donor (Cont.)

Day Influent Effluent Removal
107 1407.88 243.47 82.71
108 1407.88 158.33 88.75
109 1407.88 148.3 89.47
110 1407.88 213.73 84.82
111 1554.56 237.35 84.73
112 1554.56 232.31 85.06
113 1554.56 298.2 80.82
114 1554.56 254.76 83.61
115 1554.56 254.77 83.61
116 1554.56 287.13 81.53
117 1623.2 292.57 81.98
118 1623.2 235.81 85.47
119 1623.2 213.78 86.83
120 1623.2 274.07 83.12
121 1623.2 254.98 84.29
122 1623.2 249.27 84.64
123 1623.2 273.01 83.18
124 1623.2 264.05 83.73
125 1763.77 292.48 83.42
126 1763.77 183.79 89.58
129 1763.77 210.97 88.04
130 1763.77 219.86 87.53

224
Table D6: Sulfate Data in FBR with Lactate as Electron Donor

Day Influent Mixed concentration Effluent Removal
1 762.18 762.18 720.22 5.51
2 762.18 762.18 748.17 1.84
4 762.18 762.18 738.40 3.12
5 762.18 762.18 466.87 38.75
6 762.18 762.18 453.63 40.48
7 762.18 762.18 438.51 42.47
8 762.18 762.18 353.62 53.60
10 762.18 762.18 458.53 39.84
11 762.18 762.18 398.09 47.77
12 762.18 762.18 358.84 52.92
13 802.15 802.15 398.83 50.28
14 802.15 802.15 396.41 50.58
15 802.15 802.15 403.84 49.66
16 802.15 802.15 381.03 52.50
17 802.15 802.15 379.29 52.72
18 802.15 802.15 345.61 56.91
19 802.15 802.15 342.42 57.31
20 802.15 802.15 309.44 61.42
21 802.15 802.15 289.58 63.90
22 802.15 802.15 249.67 68.88
23 802.15 802.15 241.04 69.95
24 802.15 802.15 222.36 72.28
25 802.15 802.15 218.70 72.74
26 802.15 802.15 200.12 75.05
27 995.28 995.28 285.58 71.31
29 995.28 995.28 302.17 69.64
30 995.28 995.28 286.52 71.21
31 995.28 995.28 275.87 72.28
32 995.28 995.28 259.67 73.91
33 995.28 995.28 237.76 76.11
34 1179.93 1179.93 226.64 80.79

225
Table D6: Sulfate Data in FBR with Lactate as Electron Donor (Cont.)

Day Influent Mixed concentration Effluent Removal
35 1179.93 1179.93 242.83 79.42
36 1179.93 1179.93 292.81 75.18
37 1179.93 1179.93 271.82 76.96
38 1179.93 1179.93 257.50 78.18
39 1179.93 1179.93 244.30 79.30
40 1179.93 1179.93 203.32 82.77
41 1179.93 1179.93 191.91 83.74
42 1179.93 1179.93 83.49 92.92
43 1383.31 1263.35 76.44 93.95
44 1383.31 1263.35 146.68 88.39
46 1383.31 1263.35 207.59 83.57
47 1383.31 1263.35 224.26 82.25
48 1383.31 1263.35 219.47 82.63
49 1383.31 1263.35 229.70 81.82
50 1383.31 1263.35 209.69 83.40
51 1471.97 1319.85 356.16 73.02
52 1471.97 1319.85 232.89 82.35
53 1471.97 1319.85 156.91 88.11
54 1471.97 1319.85 156.86 88.12
55 1471.97 1319.85 106.59 91.92
56 1471.97 1319.85 159.25 87.93
57 1471.97 1319.85 125.00 90.53
58 1707.29 1271.85 146.40 88.49
59 1707.29 1271.85 76.16 94.01
60 1707.29 1271.85 32.66 97.43
61 1707.29 1271.85 34.08 97.32
62 1707.29 1271.85 34.64 97.28
63 1707.29 1271.85 40.26 96.83
64 2042.62 1370.30 138.59 89.89
65 2042.62 1370.30 108.00 92.12
66 2042.62 1370.30 297.84 78.26
67 2042.62 1370.30 181.71 86.74
68 2042.62 1370.30 286.77 79.07
69 2042.62 1370.30 299.31 78.16
70 2042.62 1370.30 278.46 79.68

226
Table D6: Sulfate Data in FBR with Lactate as Electron Donor (Cont.)

Day Influent Mixed concentration Effluent Removal
71 2042.62 1370.30 264.07 80.73
72 2042.62 1370.30 160.10 88.32
73 2042.62 1370.30 59.14 95.68
74 2042.62 1370.30 29.36 97.86
75 2042.62 1370.30 24.19 98.23
76 2373.79 1370.30 26.63 98.06
77 2373.79 1370.30 35.51 97.41
78 2373.79 1370.30 55.15 95.98
79 2373.79 1370.30 54.50 96.02

Table D7: Lactate Utilization Data in FBR with Lactate as Electron Donor

Time (Days) Influent Effluent Removal
1 202.01 165.11 18.27
2 202.01 116.47 42.35
4 202.01 42.35 79.03
5 1013.95 377.54 62.76
6 1013.95 392.99 61.24
7 1013.95 374.88 63.03
8 1013.95 384.65 62.06
10 1013.95 375.08 63.01
11 1013.95 366.37 63.87
12 1013.95 364.07 64.09
13 1218.18 347.88 71.44
14 1218.18 339.29 72.15
15 1218.18 335.39 72.47
16 1368.03 345.43 74.75
17 1368.03 345.44 74.75
18 1368.03 335.31 75.49

227
Table D7: Lactate Utilization Data in FBR with Lactate as Electron Donor (Cont.)

Time (Days) Influent Effluent Removal
19 1368.03 354.50 74.09
20 1368.03 391.64 71.37
21 1368.03 390.78 71.43
22 1368.03 381.41 72.12
23 1368.03 429.16 68.63
24 1368.03 469.28 65.70
25 1368.03 464.00 65.70
26 1368.03 443.43 66.08
27 1568.85 520.85 71.74
29 1568.85 481.91 66.80
30 1568.85 465.29 69.28
31 1568.85 470.42 70.34
32 1568.85 482.79 70.02
33 1568.85 436.77 69.23
34 1789.53 491.88 75.59
35 1789.53 569.03 72.51
36 1789.53 565.11 68.20
37 1789.53 579.22 68.42
38 1789.53 593.10 67.63
39 1789.53 581.92 66.86
40 1789.53 581.92 67.48
41 1789.53 569.68 67.48
42 1789.53 532.23 68.17
43 1789.53 1135.57 70.26
44 1916.27 938.96 40.74
45 1916.27 645.24 51.00
46 1916.27 619.34 66.33
47 1916.27 631.84 67.68
48 1916.27 633.55 67.03
49 1916.27 623.11 66.94
50 2158.27 688.31 71.13
51 2158.27 707.18 68.11
52 2158.27 672.98 67.23
53 2158.27 720.85 68.82
54 2158.27 676.80 66.60
55 2158.27 757.05 68.64

228
Table D7: Lactate Utilization Data in FBR with Lactate as Electron Donor (Cont.)

Time (Days) Influent Effluent Removal
56 2158.27 758.35 64.92
57 2158.27 758.07 64.86
58 2158.27 741.40 64.88
59 2158.27 715.11 65.65
60 2033.52 746.86 64.83
61 2033.52 787.08 63.27
62 2033.52 794.99 61.29
63 2033.52 889.23 60.91
64 2033.52 730.50 56.27
65 1976.525 778.125 63.04
66 1976.525 760.58 60.63
67 1976.525 771.73 61.52
68 1976.525 747.19 60.96
69 1976.525 764.75 62.20
70 1976.525 729.74 61.31
71 1976.525 753.42 63.08
72 1843.217 798.26 59.12
73 1843.217 800.01 56.69
74 1843.217 845.57 56.60
75 1843.217 761.64 54.13
76 1843.217 761.64 58.68
77 2128.69 764.31 64.22
78 2128.69 716.41 64.09
79 2128.69 708.33 66.34

229
Table D8: Hydrogen Sulfide Production Data in FBR with Lactate as Electron Donor

Time (days) H
2
S (mg/L S
2-
) Time (days) H
2
S (mg/L S
2-
)
13 200 50 400
20 220 52 416
22 240 54 420
25 280 56 414
27 260 58 420
30 280 59 424
33 268 61 420
35 296 63 432
37 324 65 408
38 344 66 400
40 340 69 380
42 372 70 444
44 388 71 456
46 358 73 456
48 376 75 400
49 380 77 348

230
Table D9: Hydrogen Sulfide Production Data in FBR with Acetate as Electron
Donor

Time (days) H
2
S (mg/L S
2-
) Time (days) H
2
S (mg/L S
2-
)
18 280 79 428
20 320 81 420
23 332 83 412
26 352 85 420
28 364 86 424
30 380 88 440
33 382 90 448
35 380 92 452
37 380 94 440
39 380 96 420
41 392 97 460
47 340 99 452
49 352 100 480
50 380 102 496
54 384 104 500
57 452 106 504
58 460 108 516
60 484 110 548
62 440 114 552
64 392 116 552
66 396 118 552
68 394 120 552

231

Table D9: Hydrogen Production Data in FBR with Acetate as Electron Donor (Cont.)

Time (days) H
2
S (mg/L S
2-
) Time (days) H
2
S (mg/L S
2-
)
69 412 122 608
71 408 123 608
72 408 124 432
74 412 125 520
76 408 127 404

232
APPENDIX E

H
2
S Stripping Data

Table E1: H
2
S Stripping Data at Different pHs

Experimental conditions
Stripping gas : N
2

Gas flowrate (G) : 0, 1, 3, 5, and 7 L/min
Liquid Flowrate (L) : 6
mL/min

Liquid pH : 6, 6.5, 7, and 7.5

Gas flowrate G L Stripping ratio
(L/min) (L/m
2
-sec) (L/m
2
-sec) (R, G/L)
0 0 0.0316 0
1 5.27 0.0316 64.27
3 15.80 0.0316 192.80
5 26.33 0.0316 321.33
7 36.86 0.0316 449.86

pH = 6
Gas flowrate
Stripping
ratio
Sulfide Conc. Removal rate
(L/min) (R, G/L) (mg S
2-
/L) (%)
Influent 240
0 0 100 58.33
1 64.27 13 94.58
3 192.80 13 94.58
5 321.33 10 95.83
7 449.86 14 94.17

233
Table E1: H
2
S Stripping Data at Different pHs (Cont.)

pH = 6.5
Gas
flowrate
Stripping
ratio
Sulfide Conc. Removal rate
(L/min) (R, G/L) (mg S
2-
/L) (%)
Influent 242
0 0 111 54.13
1 64.27 35.2 85.45
3 192.80 19.2 92.07
5 321.33 14.8 93.88
7 449.86 13.2 94.55

pH = 7
Gas
flowrate
Stripping
ratio
Sulfide Conc. Removal rate
(L/min) (R, G/L) (mg S
2-
/L) (%)
Influent 245
0 0 190 22.45
1 64.27 63 74.29
3 192.80 39 84.08
5 321.33 27 88.98
7 449.86 18 92.65

234
Table E1: H
2
S Stripping Data at Different pHs (Cont.)

pH = 7.5
Gas
flowrate
Stripping
ratio
Sulfide Conc. Removal rate
(L/min) (R, G/L) (mg S
2-
/L) (%)
Influent 235, 218
0 0 215 8.51
1 64.27 105 55.32
3 192.80 43 80.28
5 321.33 32.4 85.14
7 449.86 27.2 87.52

Table E2: H
2
S Stripping Data at Different Liquid Flow Rates

G= 5 ml/min
pH=6.5
Liquid Flow Rate
L (ml/min) H
2
S Removal (%)
5 85.51
6 85.37
10 84.82
15 83.57
20 79.30
25 75.98

235
Table E2: H
2
S Stripping Data at Different Liquid Flow Rates (Cont.)

pH=6.5 L=6 ml/min
G= 5 ml/min
Height
Removal
(%)
0 0
5 68.05
10 82.54
15 85.30
20 85.85
25 86.40

Abstract (if available)

Abstract High-sulfate wastewaters are a major problem in industry because they increase the total dissolved solid content and interfere with methanogenesis, resulting in a decrease in the production of methane, which is a valuable fuel. A large variety of industries, including pulp and paper production, molasses fermentation, seafood processing, potato-starch factories, and tanneries, that produce wastewaters with a high sulfate concentration, have major problems in discharging their wastewaters. The reason for this is that the discharge of industrial wastes into water bodies is governed by National Pollutant Discharge Elimination System (NPDES) program, which limits the amount of pollutants, especially chemical oxygen demand (COD), received by the surface waters. Unfortunately, high sulfate content in the wastewater limits the usage of anaerobic methanogenesis for COD reduction.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Vahdati Nikzad, Atosa (author)

Core Title Biological sulfate reduction in sulfate rich industrial wastewaters by anaerobic fluidized-bed reactors: effect of electron donors

School Viterbi School of Engineering

Degree Doctor of Philosophy

Degree Program Civil Engineering (Environmental Engineering)

Publication Date 10/08/2009

Defense Date 06/29/2007

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biological treatment,electron donor,energy production,fluidized bed reactors,hydrogen sulfide control,industrial wastewaters,OAI-PMH Harvest,sulfate reduction

Language English

Advisor Pirbazari, Massoud (committee chair), Shing, Katherine S. (committee member), Yen, Teh Fu (committee member)

Creator Email vahdati@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m850

Unique identifier UC1157493

Identifier etd-VahdatiNikzad-20071008 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-583602 (legacy record id),usctheses-m850 (legacy record id)

Legacy Identifier etd-VahdatiNikzad-20071008.pdf

Dmrecord 583602

Document Type Dissertation

Rights Vahdati Nikzad, Atosa

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu