Biological hydrogen production by anaerobic fermentation

137 Pages·2015·3.83 MB·English

Iowa State University Capstones, Theses and
Retrospective Theses and Dissertations
Dissertations
2006
Biological hydrogen production by anaerobic
fermentation
Wen-Hsing Chen
Iowa State University
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Chen, Wen-Hsing, "Biological hydrogen production by anaerobic fermentation " (2006).Retrospective Theses and Dissertations. 1863.
https://lib.dr.iastate.edu/rtd/1863
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Biological hydrogen production by anaerobic fermentation

by

Wen-Hsing Chen

A thesis submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY

Major: Civil Engineering (Environmental Engineering)

Program of Study Committee:
Shihwu Sung (Major Professor)
Kenneth J. Koehler
Say Kee Ong
Timothy Ellis
Dennis Bazylinski

Iowa State University
Ames, Iowa
2006

Copyright © Wen-Hsing Chen, 2006. All rights reserved.
UMI Number: 3243819
UMI Microform3243819
Copyright2007 by ProQuest Information and Learning Company.
All rights reserved. This microform edition is protected against
unauthorized copying under Title 17, United States Code.
ProQuest Information and Learning Company
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ii
TABLE OF CONTENTS
LIST OF FIGURES...........................................................................................................iv
LIST OF TABLES.............................................................................................................vi
ABSTRACT......................................................................................................................vii
CHAPTER 1. INTRODUCTION.......................................................................................1
1.1 Research Background................................................................................................1
1.2 Research Goals..........................................................................................................3
1.3 References.................................................................................................................5
CHAPTER 2. LITERATURE REVIEW............................................................................8
2.1 Hydrogen Production................................................................................................8
2.1.1 Water electrolysis...............................................................................................8
2.1.2 Thermal-chemical hydrogen production............................................................9
2.1.3 Biological hydrogen production......................................................................10
2.2 Dark Fermentation..................................................................................................11
2.2.1 Fundamentals of dark fermentation.................................................................11
2.3 Environmental Factors Affecting Hydrogen Production........................................14
2.3.1 Inocula and start-up..........................................................................................14
2.3.2 Substrate...........................................................................................................15
2.3.3 pH.....................................................................................................................16
2.3.4 Hydraulic retention time..................................................................................17
2.3.5 Product inhibition.............................................................................................18
2.4 Fluorescence in situ Hybridization..........................................................................19
2.4.1 Hybridization reaction kinetics........................................................................20
2.4.2 Technical aspects of FISH...............................................................................21
2.4.3 FISH application on wastewater treatment......................................................23
2.5 References...............................................................................................................25
CHAPTER 3. KINETIC STUDY OF BIOLOGICAL HYDROGEN PRODUCTION BY
ANAEROBIC FERMENTATION............................................................40
3.1 Abstract...................................................................................................................40
3.2 Introduction.............................................................................................................41
3.3 Materials and Methods............................................................................................44
3.3.1 Seed microorganisms.......................................................................................44
3.3.2 Hydrogen production experiments...................................................................44
3.3.3 Analysis............................................................................................................45
3.3.4 Data analysis....................................................................................................45
3.4 Results and Discussion............................................................................................46
3.4.1 Kinetic analysis of hydrogen production.........................................................46
3.4.2 Variations of biomass, pH and carbohydrate at different substrate
concentrations..................................................................................................48
3.4.3 Growth kinetics of hydrogen-producing bacteria for three different
substrates..........................................................................................................49
3.5 Conclusions.............................................................................................................51
3.6 References...............................................................................................................52
iii
CHAPTER 4. BIOLOGICAL HYDROGEN PRODUCTION IN ANAEROBIC
SEQUENCING BATCH REACTOR........................................................68
4.1 Abstract...................................................................................................................68
4.2 Introduction.............................................................................................................69
4.3 Materials and Methods............................................................................................71
4.3.1 Seed sludge and substrate preparation.............................................................71
4.3.2 Reactor operation.............................................................................................72
4.3.3 Analysis............................................................................................................73
4.4 Results and Discussion............................................................................................74
4.4.1 Effect of HRT..................................................................................................74
4.4.2 Effect of pH......................................................................................................77
4.4.3 Effect of substrate concentration.....................................................................81
4.4.4 Effect of cyclic duration...................................................................................85
4.5 Conclusions.............................................................................................................87
4.6 References...............................................................................................................88
CHAPTER 5. DIAGNOSIS OF HYDROGEN FERMENTATION BY
FLUORESCENCE IN SITU HYBRIDIZATION...................................103
5.1 Introduction...........................................................................................................103
5.2 Materials and Methods..........................................................................................105
5.2.1 Reactor operation...........................................................................................105
5.2.2 FISH...............................................................................................................105
5.2.3 Microscopy and quantificantion analysis.......................................................106
5.3 Results and Discussion..........................................................................................107
5.3.1 Hybridization performance from different 16S rDNA probes.......................107
5.3.2 Quantification of bacteria community...........................................................109
5.4 Conclusions...........................................................................................................110
5.5 References.............................................................................................................110
CHAPTER 6. CONCLUSIONS......................................................................................120
6.1 Closing Statements................................................................................................120
6.2 Recommendations for Future Work......................................................................123
ACKNOWLEDGEMENTS.............................................................................................125
iv
LIST OF FIGURES

Figure 2.1 Catabolic pathway of Clostridia in hydrogen fermentation (Jones and Woods,
1989; Mitchell, 2001).....................................................................................39
Figure 3.1 Cumulative hydrogen production with time: (a) sucrose, (b) NFDM, and (c)
food waste......................................................................................................61
Figure 3.2 Hydrogen yield at different S/X ratios: (a) sucrose, (b) NFDM, and (c) food
waste..............................................................................................................62
Figure 3.3 Specific hydrogen production rate at different S/X ratios: (a) sucrose; (b)
NFDM; and (c) food waste............................................................................63
Figure 3.4 Metabolism of hydrogen-producing bacteria at different sucrose
concentrations: (a) mixed liquor volatile suspended solid concentration, (b)
final pH, (c) removed carbohydrate concentration, and (d) carbohydrate
removal efficiency..........................................................................................64
Figure 3.5 Metabolism of hydrogen-producing bacteria at different NFDM
concentrations: (a) mixed liquor volatile suspended solid concentration, (b)
final pH, (c) removed carbohydrate concentration, and (d) carbohydrate
removal efficiency..........................................................................................65
Figure 3.6 Metabolism of hydrogen-producing bacteria at different food waste
concentrations: (a) mixed liquor volatile suspended solid concentration; (b)
final pH; (c) removed carbohydrate concentration; and (d) carbohydrate
removal efficiency..........................................................................................66
Figure 3.7 Effect of substrate concentration on the hydrogen production rate from
different substrate...........................................................................................67
Figure 4.1 Performance of ASBR operated at HRT of 16 h with pH controlled of 6.7:
(a) Gas production rate; (b) gas content; (c) acid concentration; and (d)
solvent concentration.....................................................................................98
Figure 4.2 Performance of hydrogen fermentation at different pHs: (a) hydrogen yield;
(b) hydrogen conversion efficiency; (c) hydrogenic activity; (d) carbohydrate
removal efficiency..........................................................................................99
Figure 4.3 Variation of soluble microbial product concentration and MLVSS
concentration at different pHs: (a) soluble microbial product concentration;
(b) HBu/HAc ratio; and (c) MLVSS concentration.....................................100
Figure 4.4 (a) MLVSS and effluent VSS concentrations; (b) SRT; and (c) F/M ratio at
different sucrose concentration....................................................................101
Figure 4.5 Effect of batch feeding on substrate concentration at different cyclic duration
in ASBR (Sung and Dague, 1995)...............................................................102
v
Figure 5.1 Hydrogen-producing sludge hybridized with the EUB338 probe and stained
with DAPI. Scale bars. 5 µm......................................................................116
Figure 5.2 Clostridium pasteurianum hybridized with the ARC915 probe and stained
with DAPI. Scale bars, 5 µm......................................................................117
Figure 5.3 Escherichia coli hybridized with the ARC915 probe and stained with DAPI.
Scale bars, 5 µm...........................................................................................118
Figure 5.4 (a) Hydrogen-producing sludge hybridized with the CLOST I probe labeled
by Oregon-green, and stained with DAPI; (b) hydrogen-producing sludge
hybridized with the CLOST I probe labeled by Texas-red, and stained with
DAPI; and (c) Clostridium pasteurianum hybridized with the CLOST I probe
labeled by Texas-red, and stained with DAPI. Scale bars, 5 µm................119
vi
LIST OF TABLES

Table 1.1 Life time of fossil fuels with the current consumption rate (Lodhi, 1997)......7
Table 2.1 Summary of hydrogen production from thermo-chemical reactions (Sørensen,
2005)..............................................................................................................37
Table 2.2 Comparison of biological hydrogen production processes (Nath and Das,
2004)..............................................................................................................38
Table 3.1 Characteristics of NFDM and food waste......................................................57
Table 3.2 Components of the food waste (on wet weight basis) (Li et al., 2003).........58
Table 3.3 Estimated parameters of Gompertz equation for hydrogen production.........59
Table 3.4 Summary of growth kinetics of hydrogen-producing bacteria......................60
Table 4.1 Summary of experimental design..................................................................94
Table 4.2 Results of hydrogen production at different HRT.........................................95
Table 4.3 Hydrogen production and soluble microbial products in ASBR at each
influent sucrose concentration.......................................................................96
Table 4.4 Cyclic effect on hydrogen production and soluble microbial products in
ASBR.............................................................................................................97
Table 5.1 16S rDNA oligonucleotide probes used for hybridization...........................114
Table 5.2 Summary of the percentage of EUB338 to DAPI at different operating
conditions.....................................................................................................115
vii
ABSTRACT

Considering the energy security and the global environment, there is a pressing
need to develop non-polluting and renewable energy sources. Alternatively, hydrogen is
a clean energy carrier, producing water as its only by-product when it burns. Anaerobic
bioconversion of organic wastes to hydrogen gas is an attractive option that not only
stabilizes the waste/wastewater, but also generates a benign renewable energy carrier.
The purposes of this study were to determine the kinetics of hydrogen production using
different characteristics of substrates and to evaluate hydrogen production potential from
different operating conditions in continuous operation.
The growth kinetics of hydrogen-producing bacteria using three different
substrates including sucrose, non-fat dry milk (NFDM), and food waste were investigated
through a series of batch experiments. The results demonstrated that hydrogen
production potential and hydrogen production rate increased with an increasing substrate
concentration. The maximum hydrogen yields from sucrose, NFDM, and food waste
were 234, 119, and 101 mL/g COD, respectively. The low pH (pH < 4) inhibited
hydrogen production and resulted in lower carbohydrate fermentation at high substrate
concentrations. The Michaelis-Menten equation was employed to model the hydrogen
production rate at different substrate concentrations. The equation gave a good
approximation of the maximum hydrogen production rate and the half saturation constant
(K ) with correlation coefficient (R2) over 0.85. The values of half saturation constant
S
(K ) for sucrose, NFDM, and food waste were 1.4, 6.6, and 8.7 g COD/L, respectively.
S
Based on the Ks values, the substrate affinity of the enriched hydrogen-producing culture
viii
was found to depend on the carbohydrate content of the substrate. The substrate
containing high carbohydrates showed a lower K value. The maximum hydrogen
S
production rate was governed by the complexity of carbohydrates in the substrate.
Biological hydrogen production from sucrose-rich substrate was investigated in an
anaerobic sequential batch reactor (ASBR). The goal of this study was to investigate the
effect of different hydraulic retention times (HRT) (8, 12, 16, 24, and 48 h), pHs (4.9, 5.5,
6.1, and 6.7), substrate concentrations (15, 25, and 35 g COD/L), and cyclic durations (4,
6, and 8 h) on biological hydrogen production. The maximum hydrogen yield of 2.53
mol H /mol sucrose consumed and the maximum hydrogenic activity of 538 mL H /g
2 2
VSS-d were obtained at HRT of 16h, pH 4.9, sucrose concentration of 25 g COD/L, and
feeding cycle of 4 h. Methane was detected in the biogas when solids retention time
(SRT) exceeded 100 h at pH of 6.7. Based on the low ethanol concentration of nearly
300 mg/L, the metabolic pathway shift to solvent fermentation was not observed at pH of
4.9. The ratios of butyrate (HBu) to acetate (HAc) decreased from 1.25 to 0.54 mol/mol,
when the sucrose concentration was increased from 15 to 35 g COD/L. This suggests that
the metabolic pathway of acetate fermentation was predominant at higher sucrose
concentrations. Hydrogen production was found to improve at a shorter feeding cycle of
4 h.
Fluorescent in situ hybridization (FISH) was applied for identifying and
quantifying the specific microbial populations in the study. Most bacteria successfully
identified by an EUB338 probe were counted and the percentages of 16S rDNA of
EUB338 to DAPI at different reactor operating conditions were determined. Due to the
false positive hybridization results, the ARC915 probe was found unsuitable for