The production of biohydrogen by a novel strain Clostridium sp. YM1 in dark fermentation process

In view of increasing attempts for the production of renewable energy, the production of biohydrogen energy by a new mesophilic bacterium Clostridium sp. YM1 was performed for the first time in the dark fermentation. Experimental results showed that the fermentative hydrogen was successfully produced by Clostridium sp. YM1 with the highest cumulative hydrogen volume of 3821 ml/L with a hydrogen yield of 1.7 mol H₂/mol glucose consumed. Similar results revealed that optimum incubation temperature and pH value of culture medium were 37 °C and 6.5, respectively. The study of hydrogen production from glucose and xylose revealed that this strain was able to generate higher hydrogen from glucose compared to that from xylose. The profile of volatile fatty acids produced showed that hydrogen generation by Clostridium sp. YM1 was butyrate-type fermentation. Moreover, the findings of this study indicated that an increase in head space of fermentation culture positively enhanced hydrogen production.     

Fermentative hydrogen production by a new alkaliphilic Clostridium sp. (strain PROH2) isolated from a shallow submarine hydrothermal chimney in Prony Bay,New Caledonia

The hydrogen-producing strain PROH2 pertaining to the genus Clostridium was successfully isolated from a shallow submarine hydrothermal chimney (Prony Bay, New Caledonia) driven by serpentinization processes. Cell biomass and hydrogen production performances during fermentation by strain PROH2 were studied in a series of batch experiments under various conditions of pH, temperature, NaCl and glucose concentrations. The highest hydrogen yield, 2.71 mol H₂/mol glucose, was observed at initial pH 9.5, 37 °C, and glucose concentration 2 g/L, and was comparable to that reported for neutrophilic clostridial species. Hydrogen production by strain PROH2 reached the maximum production rate (0.55 mM-H₂/h) at the late exponential phase. Yeast extract was required for growth of strain PROH2 and improved significantly its hydrogen production performances. The isolate could utilize various energy sources including cellobiose, galactose, glucose, maltose, sucrose and trehalose to produce hydrogen. The pattern of end-products of metabolism was also affected by the type of energy sources and culture conditions used. These results indicate that Clostridium sp. strain PROH2 is a good candidate for producing hydrogen under alkaline and mesothermic conditions.     

The effect of pH on the production of biohydrogen by clostridia: Thermodynamic and metabolic considerations

This study evaluates the effect of pH (4-7) on fermentative biohydrogen production by utilizing three isolated Clostridium species. Fermentative batch experiments show that the maximum hydrogen yield for Clostridium butyricum CGS2 (1.77 mmol/mmol glucose) is achieved at pH 6, whereas a high hydrogen production with Clostridium beijerinckii L9 (1.72 mmol/mmol glucose) and Clostridium tyrobutyricum FYa102 (1.83 mmol/mmol glucose) could be achieved under uncontrolled pH conditions (initial pH of 6.4-6.6 and final pH of 4-4.2). Low hydrogen yields (0-0.6 mmol/mmol glucose) observed at pH 4 are due likely to inhibitory effects on the microbial growth, although a low pH can be thermodynamically favorable for hydrogen production. The low hydrogen yields (0.12-0.64 mmol/mmol glucose) observed at pH 7 are attributed not only to thermodynamically unfavorable, but also metabolically unfavorable for hydrogen production. The relatively high levels of lactate, propionate, or formate observed at pH 7 reflect presumably the high enzymatic activities responsible for their production, together with the low hydrogenase activity, resulting in a low hydrogen production. A correlation analysis of the data from present and previous studies on biohydrogen production with pure Clostridium cultures and mixed microflora indicates a close relation between the hydrogen yield (YH₂) and the (YH₂)/(2(YHAc+YHBu)) ratio, with the observed correlation coefficient (0.787) higher than that (0.175) between YH₂ and the molar ratio of butyrate to acetate (B/A). Based on the (YH₂)/(2(YHAc+YHBu)) ratios observed at different pHs, a control of pH at 5.5-6.8 would seem to be an effective means to enhance the fermentative biohydrogen production.     

Co-digestion of cassava starch wastewater with buffalo dung for bio-hydrogen production

Batch and continuous modes for bio-hydrogen production by co-digesting cassava starch wastewater with buffalo dung were investigated. Response surface methodology with central composite design was used to optimize the bio-hydrogen production conditions. A hydrogen production potential of 1787 mL H₂/L was achieved under optimal conditions of 2.84 g/L of NaHCO₃, an initial pH of 6.77 and a total chemical oxygen demand (tCOD)/total nitrogen ratio of 42.36. A continuous stirred tank reactor was operated under the optimum conditions from batch mode to investigate the effects of hydraulic retention time (HRT) of 72, 60 and 48 h on hydrogen production. The highest hydrogen content, hydrogen production rate and hydrogen yield of 33%, 839 mL H₂/L.d and 16.90 mL H₂/g-COD_added, respectively, were achieved at a HRT of 60 h. The predominant hydrogen producer under the optimal conditions in batch mode was Clostridium sp. while Clostridium sp., Megasphaera sp. and Chloroflexi sp. were observed in the continuous hydrogen production mode at an optimal HRT.     

Isolation and characterization of a new hydrogen-producing strain Bacillus sp. FS2011

A new fermentative hydrogen-producing strain FS2011 was isolated from an effluent of bio-hydrogen production reactor, and identified as Bacillus amyloliquefaciens on the basis of 16S rDNA gene sequence. The strain could utilize various carbon and nitrogen sources to produce hydrogen in a broad range of initial pH (5.29–7.38). Phosphate buffer concentration and fermentation temperature significantly affected hydrogen production and cell growth. The maximum hydrogen yield of 2.26 mol/mol was observed at glucose concentration of 10 g/l, beef extract concentration of 2 g/l, initial pH 6.98, phosphate buffer of 20 mmol/l, and 35 °C, indicating FS2011 was a high-efficiency hydrogen-producing bacterium.     

Effect of Green synthesized silver oxide nanoparticle on biological hydrogen production

Investigation of the effect of different nanoparticles on dark fermentation is very popular nowadays. Among the nanoparticle production methods, the use of nanoparticles produced by the green synthesis method, which is more environmentally friendly, is important for a sustainable environment. This study investigated the effect of green synthesized silver oxide nanoparticles on bio-hydrogen yield. Nanoparticle synthesis was carried out by using Chlorella sp. microalgae as a reducing agent. SEM, EDX and UV–Visible spectrum analyzes were performed for the characterization of the synthesized nanoparticles. The synthesized silver nanoparticles have a uniform structure and an average particle size of 85 nm. Hydrogen production performance by Clostridium sp. was evaluated using different ratios of produced nanoparticles (100–600 μg/L). The addition of 400 μg/L nanoparticles increased the production of dark fermentative bio-hydrogen by 17% compared to the control group. The highest hydrogen yield was 2.44 mol H₂/mol glucose.     

Effect of initial pH,nutrients and temperature on hydrogen production from palm oil mill effluent using thermotolerant consortia and corresponding microbial communities

Thermotolerant consortia were obtained by heat-shock treatment on seed sludge from palm oil mill. Effect of the initial pH (4.5–6.5) on fermentative hydrogen production palm oil mill effluent (POME) showed the optimum pH at 6.0, with the maximum hydrogen production potential of 702.52 mL/L-POME, production rate of 74.54 mL/L/h. Nutrients optimization was investigated by response surface methodology with central composite design (CCD). The optimum nutrients contained 0.25 g urea/L, 0.02 g Na₂HPO₄/L and 0.36 g FeSO₄·7H₂O/L, giving the predicted value of hydrogen production of 1075 mL/L-POME. Validation experiment revealed the actual hydrogen production of 968 mL/L-POME. Studies on the effect of temperature (25–55 °C) revealed that the maximum hydrogen production potential (985.3 mL/L-POME), hydrogen production rate (75.99 mL/L/h) and hydrogen yield (27.09 mL/g COD) were achieved at 55, 45 and 37 °C, respectively. Corresponding microbial community determined by the DGGE profile demonstrated that Clostridium spp. was the dominant species. Clostridium paraputrificum was the only dominant bacterium presented in all temperatures tested, indicating that the strain was thermotolerant.     

Hydrogen production by Escherichia coli ΔhycA ΔlacI using cheese whey as substrate

This study reports a fermentative hydrogen production by Escherichia coli using cheese whey as substrate. To improve the biohydrogen production, an E. coli ΔhycA ΔlacI strain (WDHL) was constructed. The absence of hycA and lacI genes had a positive effect on the biohydrogen production. The strain produced 22% more biohydrogen in a shorter time than the wild-type (WT) strain. A Box-Behnken experimental design was used to optimize pH, temperature and substrate concentration. The optimal initial conditions for biohydrogen production by WDHL strain were pH 7.5, 37 °C and 20 g/L of cheese whey. The specific production rate was improved from 3.29 mL H₂/optical density at 600 nm (OD_600nm) unit-h produced by WDHL under non-optimal conditions to 5.88 mL H₂/OD_600nm unit-h under optimal conditions. Using optimal initial conditions, galactose can be metabolized by WDHL strain. The maximum yield obtained was 2.74 mol H₂/mol lactose consumed, which is comparable with the yield reached in other hydrogen production processes with Clostridium sp. or mixed cultures.     

Optimization of fermentative hydrogen production process by response surface methodology

The effect of temperature, initial pH and glucose concentration on fermentative hydrogen production by mixed cultures was investigated in batch tests, and the optimization of fermentative hydrogen production process was conducted by response surface methodology with a central composite design. Experimental results showed that temperatures, initial pH and glucose concentrations had impact on fermentative hydrogen production individually and interactively. The maximum hydrogen yield of 289.8 mL/g glucose was estimated at the temperature of 38.6 °C, the initial pH of 7.2 and the glucose concentration of 23.9 g/L. The maximum hydrogen production rate of 28.2 mL/h was estimated at the temperature of 37.8 °C, the initial pH of 7.2 and the glucose concentration of 27.6 g/L. The maximum substrate degradation efficiency of 96.9% was estimated at the temperature of 39.3 °C, the initial pH of 7.0 and the glucose concentration of 26.8 g/L. Response surface methodology was a better method to optimize the fermentative hydrogen production process. Modified logistic model could describe the progress of cumulative hydrogen production in the batch tests of this study successfully.     

Evaluating biohydrogen production by Clostridium hydrogenum sp. nov. strain CUEA01 isolated from mangrove sediments in Thailand

The hydrogen (H₂) fermentative Clostridium hydrogenum sp. nov. strain CUEA01 was isolated from a mangrove sediment in Thailand. Genome sequencing and analysis revealed a genome size of 5,501,482 bp that encoded for 3,292 predicted protein coding genes with annotated functional assignments and many genes associated with carbon utilization and H₂ evolution. The H₂ production performance was evaluated in batch fermentation, and revealed that this strain can grow and produce H₂ at a broad range of temperatures (15–40 °C), pH (4–10), and initial glucose concentrations (5–60 g/L). The maximum H₂ yield (3.11 mol_H2/mol_glucose) was obtained at 37 °C, pH 8, and an initial glucose concentration of 10 g/L. Furthermore, this strain could utilize various carbon sources, including xylose, xylan, starch, mannose, glycerol, and avicel cellulose, amongst others. Additionally, CUEA01 was compatible with agro-industrial wastes and could achieve a maximum CHP of 4639 mL/L and 4024 mL/L from sugarcane molasses and cassava pulp, respectively. This demonstrates that CUEA01 has a potential for H₂ fermentation from complex organic wastes since it can secrete enzyme cocktails that consolidate the fermentation process.     

Effects of pretreatment of natural bacterial source and raw material on fermentative biohydrogen production

Effects of pretreatment of natural bacterial source and raw material on biohydrogen production in fermentative biohydrogen production process were investigated systematically. Biohydrogen production from stale corn slurry was found to be feasible and effective by A1 (water soak) pretreated compost and B1 (gelatinization) pretreated stale corn. The results were further confirmed by the verification test with working volume of 8 L, in which the maximum hydrogen yield of 262 mL H₂/g-substrate, hydrogen production rate of 39 mL/g h⁻¹ and the corresponding hydrogen content of 50% was observed at fixed substrate concentration of 10 g/L, working pH 5.0-5.5 and 36 ± 1 °C. The effluent was mostly composed of acetate and butyrate. Subsequently, two new hydrogen producing strains were isolated from the effluent sludge in the running bioreactor, and they were preliminarily identified as Clostridium and Enterobacter, respectively, according to the routine screening examinations.     

Isolation and characterization of a novel strain Clostridium butyricum INET1 for fermentative hydrogen production

A novel hydrogen-producing strain was isolated from gamma irradiated digested sludge and identified as Clostridium butyricum INET1. The fermentative hydrogen production performance of the newly isolated C. butyricum INET1 was characterized. Various carbon sources, including glucose, xylose, sucrose, lactose, starch and glycerol were used as substrate for hydrogen production. The operational conditions, including temperature, initial pH, substrate concentration and inoculation proportion were evaluated for their effects on hydrogen production, and the optimal condition was determined to be 35 °C, initial pH 7.0, 10 g/L glucose and 10% inoculation ratio. Cumulative hydrogen production of 218 mL/100 mL and hydrogen yield of 2.07 mol H₂/mol hexose was obtained. The results showed that C. butyricum INET1 is capable of utilizing different substrates (glucose, xylose, sucrose, lactose, starch and glycerol) for efficient hydrogen production, which is a potential candidate for fermentative hydrogen production.     

Batch fermentative hydrogen production utilising sago (Metroxylon sp.) starch processing effluent by enriched sago sludge consortia

Sago starch processing effluent (SSPE) is an ideal bio-resource that can be utilised as a substrate for fermentative reactions due to its relatively high organic content. Annually in Malaysia, about 2.5 million tonnes of effluent are generated from the processing of sago starch. In this study, the potential use of SSPE as a substrate for fermentative hydrogen production was confirmed under all the experimental conditions studied. The maximum hydrogen production and volumetric hydrogen production rate were 575 mL H₂/L SSPE and 57.54 mL H₂/hr.L SSPE, respectively, from cultures with an initial pH of 7 and substrate concentration of 11 g soluble carbohydrate/L SSPE. The final soluble metabolites were comprised mainly of acetate (24–43%), butyrate (4–20%), propionate (1–7%) and ethanol (44–66%), suggesting an acetic acid-ethanol type fermentation pathway.     

Prevention of substrate and product inhibitions by using a dilution strategy during dark fermentative hydrogen production from molasses

Substrate and product inhibitions have a significant effect on dark fermentative hydrogen gas production. Particularly, rapid formation of volatile fatty acids leads to fast pH decreases shifting the metabolic pathway. Therefore, controlling volatile fatty acid accumulation has great importance in maintaining effective hydrogen production. In this context, a dilution strategy was applied to regulate volatile fatty acids levels within the desired concentration range. A three-factor Box-Behnken statistical experiment design was established to assess the effects of dilution time, dilution percentage and initial COD concentration on hydrogen formation yield and rate. Highest hydrogen yield (7.7 mL H₂/mL_reactor) and rate (21. 47 mL H₂/h or 9.38 mmol/L_reactor.h) were achieved when 85 gCOD/L containing fermentation media was diluted with a percentage of 130 of the initial working volume at the 3rd hour of the fermentation period. Moreover, this strategy enabled to start fermentation with 55 g glucose/L.     

Fermentative hydrogen production from macro-algae Laminaria japonica using anaerobic mixed bacteria

In this study, one macro-alga (Laminaria japonica) was used for fermentative hydrogen production by anaerobic mixed bacteria. The saccharification efficiency and hydrogen production by L. japonica with four different pretreatment methods, including heat, acid, alkaline and ultrasonic treatment, were investigated. The results showed that the saccharification efficiency from L. japonica that was pretreated with acid was the highest among the four methods. The saccharification efficiency for the total reducing sugars in the acid-pretreated L. japonica was 350.54 ± 19.89 mg/g (mean ± S.E.). The cumulative hydrogen production was 66.68 ± 5.68 mL/g from the heat-pretreated L. japonica, whereas that of L. japonica that was subjected to acid, alkaline, and ultrasonic pretreatment and the control was 43.65 ± 6.87 mL/g, 15.00 ± 3.89 mL/g, 23.56 ± 4.56 mL/g and 10.00 ± 1.21 mL/g, respectively. In addition, the effects of substrate concentration and initial pH on hydrogen production from heat-pretreated L. japonica were also analyzed. The results showed that the maximum hydrogen production was 83.45 ± 6.96 mL/g with a hydrogen concentration of approximately 28.4% from heat-pretreated L. japonica when the initial pH and substrate concentration were determined to be 6.0 and 2%, respectively. Heat pretreatment was the most effective method for increasing fermentative hydrogen production when L. japonica was used as the only substrate.     

Comparison of metabolic pathway for hydrogen production in wild-type and mutant Clostridium tyrobutyricum strain based on metabolic flux analysis

There has been a great interest in fermentative hydrogen production during recent decades. However, the low H₂ yield associated with fermentative hydrogen production process continues to hinder its industrial application. It is delectable that a maximum 3.9 mol H₂ per mol glucose was obtained in fed-batch fermentation mode with a butyric acid over-producing Clostridium tyrobutyricum mutant, which to our knowledge is the highest H₂ yield ever got in the fermentation process with Clostridium sp. This study aimed to better understand the change of flux profile within the whole metabolic network and to conduct the metabolic flux analysis of fermentative hydrogen production. For the first time, we constructed a metabolic flux model for the anaerobic glucose metabolism of C. tyrobutyricum ATCC 25755, and revealed the internal mechanism responsible for the redistribution of the carbon flux in the mutant strain in comparison with the wide-type. The MFA methodology was used to study the fractional flux response to variations in operational pH, and revealed that pH was a significant operational parameter effecting on the fermentative hydrogen production process. Furthermore, the presence of NADH-ferredoxin oxidoreductase activity in this anaerobe was demonstrated. By measuring the activities of related enzymes in the biosynthesis pathway of hydrogen, we thus concluded that the increased specific activities of both NFOR and hydrogen-catalyzing enzyme (hydrogenase) would be attributed to the hydrogen over-producing.     

Improvement of hydrogen production with thermophilic mixed culture from rice spent wash of distillery industry

Biohydrogen production processes were investigated using thermophilic bacterial consortia enriched from sludge of the anaerobic digester. A multiple parameter optimization viz. temperature, pH and substrate concentration was performed for maximization of hydrogen production. Heat shock pre-treatment followed by BES (2-bromo ethane sulfonate) treatment was done for the enrichment of hydrogen producing bacteria. Box–Behnken design and response surface methodology were adopted to investigate the mutual interaction among the process parameters. Experimental optimization of process parameters (60 °C, pH 6.5 and 10 g/L) gave the maximum hydrogen production and yield of 3985 mL/L and 2.7 mol/mol glucose respectively in the batch system which is higher than the reported value on UASB. These experimental parameters found concurrent with the values obtained from the theoretical model i.e. 58.4 °C, pH 6.6, 10.8 g/L and yield of 2.71 mol/mol glucose. At optimized conditions, maximum hydrogen production rate (R_m) of 850 mL/h, gas production potential (P) of 4551 mL/L and lag time (λ) of 1.98 h were determined using modified Gompertz equation. Using the optimum conditions, hydrogen production from rice spent wash was conducted in which hydrogen yield of 464 mL/g carbohydrate and hydrogen production rate of 168 mL/L h were obtained. PCR-DGGE profile showed that the thermophilic mixed culture was predominated with species closely affiliated to Thermoanaerobacterium sp.     

Application of desirability function based on neural network for optimizing biohydrogen production process

A fractional factorial design was carried out to investigate the effects of temperature, initial pH and glucose concentration on fermentative hydrogen production by mixed cultures in batch tests and then the experimental data of substrate degradation efficiency, hydrogen yield and average hydrogen production rate were described by a neural network, based on which the simultaneous optimization of the three responses was performed by the method of desirability function. The analysis showed that the neural network could successfully describe the effects of temperature, initial pH and glucose concentration on the substrate degradation efficiency, hydrogen yield and average hydrogen production rate of this study. The maximum substrate degradation efficiency of 95.3%, hydrogen yield of 305.3 mL/g glucose and average hydrogen production rate of 23.9 mL/h were all obtained at the optimal temperature of 39.0 °C, initial pH of 7.0 and glucose concentration of 24.6 g/L identified by the method of desirability function based on a neural network. In sum, the method of desirability function based on a neural network was a useful tool to optimize several responses for fermentative hydrogen production processes simultaneously.     

Optimization of fermentative hydrogen production by Enterococcus faecium INET2 using response surface methodology

A newly isolated strain Enterococcus faecium INET2 was used as inoculum for biohydrogen production through dark fermentation. The individual and interactive effect of initial pH, operation temperature, glucose concentration and inoculation amount on the accumulation of hydrogen during fermentation was examined by a Box–Behnken Design (BBD), and hydrogen production process was analyzed at the optimal condition. A significant interactive effect between glucose concentration and pH was observed, the optimal condition was initial pH 7.1, operation temperature 34.8 °C, glucose concentration 11.3 g/L and inoculation amount 10.4%. Hydrogen yield, maximum hydrogen production rate and hydrogen production potential were determined to be 1.29 mol H₂/mol glucose, 86.7 L H₂/L/h and 1.35 L H₂/L. Metabolites analysis showed that E. faecium INET2 followed the pyruvate: formate lyase (Pfl) pathway in first 16 h, followed by the acetate-type fermentation and then shifted to butyrate-type fermentation. Maximum hydrogen production rate was accompanied with a quick formation of acetic acid.     

Thermophilic dark fermentation of untreated rice straw using mixed cultures for hydrogen production

Biohydrogen production from untreated rice straw using different heat-treated sludge, initial cultivation pH, substrate concentration and particle size was evaluated at 55 °C. The peak hydrogen production yield of 24.8 mL/g TS was obtained with rice straw concentration 90 g TS/L, particle size <0.297 mm and heat-treated sludge S1 at pH 6.5 and 55 °C in batch test. Hydrogen production using sludge S1 resulted from acetate-type fermentation and was pH dependent. The maximum hydrogen production (P), production rate (R_m) and lag (λ) were 733 mL, 18 mL/h and 45 h respectively. Repeated-batch operation showed decreasing trend in hydrogen production probably due to overloading of substrate and its non-utilization. PCR-DGGE showed both hydrolytic and fermentative bacteria (Clostridium pasteurianum, Clostridium stercorarium and Thermoanaerobacterium saccharolyticum) in the repeated-batch reactor, which perhaps in association led to the microbial hydrolysis and fermentation of raw rice straw avoiding the pretreatment step.