Biohydrogen production from renewable biomass is expected to replace the current fossil fuels and combat the anthropogenic CO2 emissions. Dark fermentation could be the future industrial process due to its higher production rates than light-dependent fermentation process. This study aimed to enhance the biohydrogen production from an industrial feedstock (beverage wastewater), focusing on process improvements in suspended and immobilized cells operational system. In order to select the efficient hydrogen-producers for beverage wastewater (BWW), initially sucrose was chosen as a sole carbon source to enrich efficient hydrogen-producers from two different compost seeds. Among these two seeds enriched culture from compost of food waste (C1) provides the stable and enhanced hydrogen production. Peak hydrogen yield (HY) of 3.76 mol/mol sucrose, hydrogen production rate (HPR) of 1.15 L/L-d were achieved under the conditions of (temperature 37 °C, pH 7.0 and substrate concentration of 10g/L). Once the most efficient enriched mixed culture was chosen, further optimization of pH, temperature and substrate concentration from BWW was carried out and resulted in an optimized level of pH 5.5, temperature 37 °C and a substrate concentration of 20 g/L. Under these conditions, the optimal HPR and HY were observed as 2.16 L/L-d and 1.30 mol/mol hexose utilized. Ratwosky kinetic model was used to evaluate the effect of temperature and provided the optimal temperature values of 38.5. While, Monod model was used to predict the substrate concentration effects and it showed the Rm and Ks values as 3.57 L/L-d and 8.29 g/L, respectively.
CSTR was seeded with selective enriched mixed culture and operated under the suitable conditions obtained from batch experiments (pH 5.5-6.5, temperature 37 °C, substrate concentration 20 g/L). During continuous operation at hydraulic retention time (HRT) 8 h, propionic acid accumulation was appeared in the reactor with a concentration of 2.36 g/L leading to a drop in hydrogen production rate (HPR) from 10 to 8.5 L/L-d. To overcome the HPr inhibition, a temperature shift (from 37 °C) to 45 °C for 8 h was applied. This significantly improved the inhibited HPR and HY to 13.6 L/L-d. and 1.68 mol/mol hexose, respectively, with a simultaneous reduction in the HPr concentration to 0.7 g/L. Microbial community analysis based on PCR-DGGE after temperature shift revealed the absence of Selenomonas lacticifex and Bifidobacterium catenulatum (involved in HPr formation), and presence of hydrogen producing bacteria namely Clostridium butyricum, Clostridium perfringenes, Clostridium acetobutylicum, and Ethanoligenens harbinense. Temperature shift strategy could overcome the HPr inhibition and significantly improve the hydrogen fermentation of an industrial wastewater.
In order to improve the efficiency of hydrogen production from BWW with suspended cells operation, CSTR was further operated with different HRTs (6, 4, 3, 2 and 1.5 h) to find out optimal hydrogen production rate and operational stability of the bioreactor. The peak HPR 37.5 L/L-d and energy production rate 441 KJ/L-d were observed at 1.5 h HRT, while the maximum HY of 1.59 mol/mol hexose was observed at 6 h HRT. Butyrate-type fermentative pathway was observed at all tested HRTs. Lactate concentration increased with decreasing in HRT, which significantly affects the HY. At optimal HRT 1.5 h with peak HPR 37.5 L/L-d, the four species (C. butyricum, C. tyrobutyricum, C. perfringenes and K. oxytoca), were observed.
Hydrogen production efficiency from BWW was further studied with immobilized cell operation. A novel material (hybrid immobilization material, HY-IM) was developed by the combination of calcium-alginate, activated carbon (prepared from de-oiled jatropha waste), wako gel (silica gel) and chitosan for immobilization of hydrogen-producing bacteria. The Scanning electron microscopy (SEM) images of the HY-IM showed bacterial cells entrapment. The hydrogen production performance of the reactor with HY-IM (1.12 ±0.04 mol H2/mol hexose utilized) was comparable to suspended cells reactor (1.07±0.01 mol H2/mol hexose utilized), and demonstrated repeated usage of same HY-IM in the reactor with high stability under ten repeated batch operations.
The hydrogen production from beverage industry wastewater (20 g/L hexose equivalent) using an immobilized cell reactor was studied at various HRT (8-1.5 h) in a mode of continuous operation. Maximum hydrogen production rate (HPR) of 55 L/L-d was obtained at HRT 1.5 h (an organic loading of 320 g/L-d). This HPR value is much higher than those of other industrial wastewaters employed in fermentative hydrogen production. The cell biomass concentration peaked at 3 h HRT with a volatile suspended solids (VSS) concentration value of 6.31 g/L (with presence of self-flocculating Selenomonas sp.) but dropped to 3.54 gVSS/L at 1.5 h HRT. With the shortening of HRT, lactate concentration increased, but butyrate concentration did not vary significantly and remained as the dominant metabolite at all HRTs. The Clostridium species (C. butyricum, C. tyrobutyricum, C. celerecrescens, C. pasteurianum and C. acetobutylicum) were present as seen from PCR-DGGE analyses.
Comprehensive discussion provided the information about advantages and disadvantages of using suspended and immobilized cell systems on improving the hydrogen production performances in terms of yield and production rate. Additionally, the energy production rate (EPR) also compared to show the overall efficiency of the process. The maximum EPR (641.05 KJ/L-d) and HPR (55.4 L/L-d) obtained from the immobilized cells were 47 % higher than the suspended cells (441.52 KJ/L-d) (37.5 L/L-d). Based on these results, it is concluded that, immobilized cell operation is suitable for efficient and stable hydrogen production than suspended cell systems from beverage wastewater. Overall this study demonstrated that beverage wastewater is a high energy yielding feedstock capable of producing both hydrogen and ethanol

Abstract….. i
Preface and Acknowledgement v
Contents…….. vii
List of Tables x
List of Publications xiv
Chapter 1 Introduction 1
1.1 Motivation and purpose 1
1.2 Outline of this thesis 4
Chapter 2 Literature review 6
2.1 Introduction 6
2.2 Biohydrogen production methods 7
2.3 Dark fermentative hydrogen production 8
2.4 Strategies for improving hydrogen fermentation in continuous operation 12
2.4.1 Start-up period reduction 12
2.4.2 pH 14
2.4.3 Hydraulic retention time (HRT) 15
2.4.4 Organic loading rate (OLR) 16
2.4.5 Temperature 17
2.4.6 Immobilization 28
2.4.7 Bioaugmentation 30
2.4.8 Gas Sparging 31
2.5 Wastewater feedstock in continuous hydrogen production 32
Chapter 3 Biohydrogen production potential of selectively enriched mixed cultures 38
3.1 Introduction 38
3.2 Materials and Methods 39
3.2.1 Enrichment of hydrogen producing mixed cultures 39
3.2.2 Batch fermentation 40
3.2.3 Effects of pH, temperature and substrate concentration 40
3.2.4 Analytical procedures 41
3.2.5 Kinetic models 42
3.2.6 Microbial community analysis 43
3.3 Results and Discussions 45
3.3.1 Stable hydrogen production by enriched mixed cultures 45
3.3.2 Selection of the efficient mixed culture for hydrogen production 46
3.3.3 Microbial community 50
3.3.4 Effects of pH, temperature and substrate concentration on the fermentation of BWW 52
3.4 Summary 63
Chapter 4 Hydrogen production by suspended cell systems 65
4.1 Introduction 65
4.2 Materials and Methods 67
4.2.1 Microbial source and beverage wastewater 67
4.2.2 Experimental setup for continuous operation 67
4.3 Results and Discussions 69
4.3.1 Hydrogen production from BWW at HRT 8 h 69
4.3.2 Effects of HRT (6 to 1.5 h) on continuous hydrogen production 87
4.3.3 Significance of the study 103
4.4 Summary 105
Chapter 5 Hydrogen production by immobilized cells system 106
5.1 Introduction 106
5.2 Materials and Methods 108
5.2.1 Immobilization of enriched hydrogen producing microbes 108
5.2.2 Batch biohydrogen fermentation 109
5.2.3 Continuous operation of immobilized cell bioreactor (ICB) 109
5.2.4 Scanning Electron Microscopy 110
5.3 Results and Discussion 111
5.3.1 Characterization of activated carbon and immobilized beads 111
5.3.2 Batch hydrogen fermentation in suspended and immobilized cell systems 112
5.3.3 Repeated batch operation of hydrogen production in immobilized cell system 113
5.3.4 Continuous operation of immobilized cell systems 114
5.3.5 Significance of the study 130
5.4 Summary 132
Chapter 6 Comprehensive discussion of the suspended and immobilized cells operation 134
6.1 Introduction 134
6.2 Comparison of hydrogen production between suspended and immobilized cells operation 137
6.3 Feasibility analysis of hydrogen production from BWW for electricity generation and CO2 emission reduction 142
Chapter 7 Conclusions and Recommendations 146
7.1 Conclusions 146
7.2 Recommendations 148
Bibliography 150

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