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.     

Biohydrogen production from wastewater by Clostridium beijerinckii: Effect of pH and substrate concentration

An investigation of biological hydrogen production from glucose by Clostridium beijerinckii was conducted in a synthetic wastewater solution. A study examining the effect of initial pH (range 5.7–6.5) and substrate loading (range 1–3 g COD/L) on the specific conversion and hydrogen production rate has shown interaction behaviour between the two independent variables. Highest conversion of 10.3 mL H₂/(g COD/L) was achieved at pH of 6.1 and glucose concentration of 3 g COD/L, whereas the highest production rate of 71 mL H₂/(h L) was measured at pH 6.3 and substrate loading of 2.5 g COD/L. In general, there appears to be a strong trend of increasing hydrogen production rate with an increase in both substrate concentration and pH. Butyrate (14–63%), formate (10–45%) and ethanol (16–40%) were the main soluble products with other volatile fatty acids and alcohols present in smaller quantities.     

Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3

A hydrogen producing strain newly isolated from anaerobic sludge in an anaerobic bioreactor, was identified as Clostridium beijerinckii Fanp3 by 16S rDNA gene sequence analysis and detection by BioMerieux Vitek. The strain could utilize various carbon and nitrogen sources to produce hydrogen, which indicates that it has the potential of converting renewable wastes into hydrogen. In batch cultivations, the optimal initial pH of the culture medium was between 6.47 and 6.98. Using 0.15 M phosphate as buffer could alleviate the medium acidification and improve the overall performance of C. beijerinckii Fanp3 in hydrogen production. Culture temperature of 35 °C was established to be the most favorable for maximum rate of hydrogen production. The distribution of soluble metabolic products (SMP) was also greatly affected by temperature. Considering glucose as a substrate, the activation energy (E_a) for hydrogen production was calculated as 81.01 kcal/mol and 21.4% of substrate energy was recovered in the form of hydrogen. The maximal hydrogen yield and the hydrogen production rate were obtained as 2.52 mol/mol-glucose and 39.0 ml/g-glucose h⁻¹, respectively. These results indicate that C. beijerinckii Fanp3 is an ideal candidate for the fermentative hydrogen production.     

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.     

Comparative study of biohydrogen production by four dark fermentative bacteria

Dark fermentation is a promising biological method for hydrogen production because of its high production rate in the absence of light source and variety of the substrates. In this study, hydrogen production potential of four dark fermentative bacteria (Clostridium butyricum, Clostridium pasteurianum, Clostridium beijerinckii, and Enterobacter aerogenes) using glucose as substrate was investigated under anaerobic conditions. Batch experiments were conducted to study the effects of initial glucose concentration on hydrogen yield, hydrogen production rate and concentration of volatile fatty acids (VFA) in the effluents. Among the four different fermentative bacteria, C. butyricum showed great performance at 10 g/L of glucose with hydrogen production rate of 18.29 mL-H₂/L-medium/hand specific hydrogen production rate of 3.90 mL-H₂/g-biomass/h. In addition, it was found that the distribution of volatile fatty acids was different among the fermentative bacteria. C. butyricum and C. pasteurianum had higher ratio of acetate to butyrate compared to the other two species, which favored hydrogen generation.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum TERI S7 from oil reservoir flow pipeline

Thermophilic dark fermentative hydrogen producing bacterial strain, TERI S7, isolated from an oil reservoir flow pipeline located in Mumbai, India, showed 98% identity with Thermoanaerobacterium thermosaccharolyticum by 16S rRNA gene analysis. It produced 1450–1900 ml/L hydrogen under both acidic and alkaline conditions; at a temperature range of 45–60 °C. The maximum hydrogen yield was 2.5 ± 0.2 mol H₂/mol glucose, 2.2 ± 0.2 mol H₂/mol xylose and 5.2 ± 0.2 mol H₂/mol sucrose, when the respective sugars were used as carbon source. The cumulative hydrogen production, hydrogen production rate and specific hydrogen production rate by the strain TERI S7 with sucrose as carbon source was found to be 1704 ± 105 ml/L, 71 ± 6 ml/L/h and 142 ± 13 ml/g/h respectively. Major soluble metabolites produced during fermentation were acetic acid and butyric acid. The strain TERI S7 was also observed to produce hydrogen continuously up to 48 h at pH 3.9.     

Biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent

A hydrogen producer was successfully isolated from anaerobic digested palm oil mill effluent (POME) sludge. The strain, designated as Clostridium butyricum EB6, efficiently produced hydrogen concurrently with cell growth. A controlled study was done on a synthetic medium at an initial pH value of 6.0 with 10 g/L glucose with the maximum hydrogen production at 948 mL H₂/L-medium and the volumetric hydrogen production rate at 172 mL H₂/L-medium/h. The supplementation of yeast extract was shown to have a significant effect with a maximum hydrogen production of 992 mL H₂/L-medium at 4 g/L of yeast extract added. The effect of pH on hydrogen production from POME was investigated. Experimental results showed that the optimum hydrogen production ability occurred at pH 5.5. The maximum hydrogen production and maximum volumetric hydrogen production rate were at 3195 mL H₂/L-medium and 1034 mL H₂/L-medium/h, respectively. The hydrogen content in the biogas produced was in the range of 60–70%.     

Characteristics of fermentative hydrogen production with Klebsiella pneumoniae ECU-15 isolated from anaerobic sewage sludge

Klebsiella pneumoniae ECU-15 (EU360791), which was isolated from anaerobic sewage sludge, was investigated in this paper for its characteristics of fermentative hydrogen production. It was found that the anaerobic condition favored hydrogen production than that of the micro-aerobic condition. Culture temperature and pH of 37 °C and 6.0 were the most favorable for the hydrogen production. The strain could grow in several kinds of monosaccharide and disaccharide, as well as the complicated corn stalk hydrolysate, with the best results exhibited in glucose. The maximum hydrogen production rate and yield of 482 ml/l/h and 2.07 mol/mol glucose were obtained at initial glucose concentration of 30 g/L and 5 g/L, respectively. Fermentation results in the diluent corn stalk hydrolysate showed that cell growth was not inhibited. However, the hydrogen production of 0.65 V/V was relatively lower than that of the glucose (1.11 V/V), which was mainly due to the interaction between xylose and glucose.     

Hydrogen production using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

Fermentative hydrogen production was carried out using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). This work investigates the effects of initial substrate concentration, initial medium pH, and temperature. The hydrogen yield was about 3.1 mol (mol glucose)⁻¹ when starting with an initial glucose concentration of 10 gl⁻¹ and initial a pH of 6.0 ± 0.2 at a temperature of 37 °C. The volume of hydrogen produced decreased when higher initial glucose concentrations were applied. The most suitable conditions for hydrogen production in a batch reactor were observed at initial pH 6.0 ± 0.2 and 37 °C.     

Fermentative hydrogen production from starch using natural mixed cultures

Batch and continuous tests were conducted to evaluate fermentative hydrogen production from starch (at a concentration of chemical oxygen demand (COD) 20 g/L) at 35 °C by a natural mixed culture of paper mill wastewater treatment sludge. The optimal initial cultivation pH (tested range 5–7) and substrate concentration (tested range 5–60-gCOD/L) were evaluated by batch reactors while the effects of hydraulic retention time (HRT) on hydrogen production, as expressed by hydrogen yield (HY) and hydrogen production rate (HPR), were evaluated by continuous tests. The experimental results indicate that the initial cultivation pH markedly affected HY, maximum HPR, liquid fermentation product concentration and distribution, butyrate/acetate concentration ratio and metabolic pathway. The optimal initial cultivation pH was 5.5 with peak values of HY 1.1 mol-H₂/mol-hexose maximum HPR 10.4 mmol-H₂/L/h and butyrate concentration 7700 mg-COD/L. In continuous hydrogen fermentation, the optimal HRT was 4 h with peak HY of 1.5 mol-H₂/mol-hexose, peak HPR of 450 mmol-H₂/L/d and lowest butyrate concentration of 3000 mg-COD/L. The HPR obtained was 280% higher than reported values. A shift in dominant hydrogen-producing microbial population along with HRT variation was observed with Clostridium butyricum, C. pasteurianum, Klebshilla pneumoniae, Streptococcus sp., and Pseudomonas sp. being present at efficient hydrogen production at the HRTs of 4–6 h. Strategies based on the experimental results for optimal hydrogen production from starch are proposed.     

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.     

Effect of temperature on fermentative hydrogen production by mixed cultures

Effect of temperatures ranging from 20 °C to 55 °C on fermentative hydrogen production by mixed cultures was investigated in batch tests. The experimental results showed that, at initial pH 7.0, during the fermentative hydrogen production using glucose as substrate, the substrate degradation efficiency and hydrogen production potential increased with increasing temperatures from 20 °C to 40 °C. The maximal substrate degradation efficiency was 98.1%, the maximal hydrogen production potential was 269.9 mL, the maximal hydrogen yield was 275.1 mL/g glucose and the shortest lag time was 7.0 h. The temperature for fermentative hydrogen production by mixed cultures was optimized to be 40 °C. The expanded Ratkowsky models could be used to describe the effect of temperatures on the hydrogen production potential, maximum hydrogen production rate and the lag time during fermentative hydrogen production.     

High biohydrogen yielding Clostridium sp. DMHC-10 isolated from sludge of distillery waste treatment plant

A mesophilic high hydrogen producing strain DMHC-10 was isolated from a lab scale anaerobic reactor being operated on distillery wastewater for hydrogen production. DMHC-10 was identified as Clostridium sp. on the basis of 16S rRNA gene sequencing. Various medium components (carbon and nitrogen sources) and environmental factors (initial pH, temperature of incubation) were optimized for hydrogen production by Clostridium sp. DMHC-10. The strain, in late exponential growth phase, showed maximum hydrogen production (3.35 mol-H₂ mol⁻¹ glucose utilized) at 37 °C, pH 5.0 in a medium supplemented with organic nitrogen source. Butyric acid to acetic acid ratio was ca. 2.3. Hydrogen production declined when organic nitrogen was replaced with inorganic nitrogen.     

Biological hydrogen production of the genus Clostridium: Metabolic study and mathematical model simulation

The biochemical hydrogen potential (BHP) tests were conducted to investigate the metabolism of glucose fermentation and hydrogen production performance of four Clostridial species, including C. acetobutylicum M121, C. butyricum ATCC19398, C. tyrobutyricum FYa102, and C. beijerinckii L9. Batch experiments showed that all the tested strains fermented glucose, reduced medium pH from 7.2 to a value between 4.6 and 5.0, and produced butyrate (0.37–0.67 mmol/mmol-glucose) and acetate (0.34–0.42 mmol/mmol-glucose) as primary soluble metabolites. Meanwhile, a significant amount of hydrogen gas was produced accompanied with glucose degradation and acid production. Among the strains examined, C. beijerinckii L9 had the highest hydrogen production yield of 2.81 mmol/mmol-glucose. A kinetic model was developed to evaluate the metabolism of glucose fermentation of those Clostridium species in the batch cultures. The model, in general, was able to accurately describe the profile of glucose degradation as well as production of biomass, butyrate, acetate, ethanol, and hydrogen observed in the batch tests. In the glucose re-feeding experiments, the C. tyrobutyricum FYa102 and C. beijerinckii L9 isolates fermented additional glucose during re-feeding tests, producing a substantial amount of hydrogen. In contrast, C. butyricum ATCC19398 was unable to produce more hydrogen despite additional supply of glucose, presumably due to the metabolic shift from acetate/butyrate to lactate/ethanol production.     

Direct hydrogen production from cellulosic waste materials with a single-step dark fermentation process

Biohydrogen production from cellulosic waste materials using dark fermentation is a promising technology for producing renewable energy. The purpose of this study was to evaluate residual cellulosic materials generated from local sources for their H₂ production potential without any pretreatment. Clostridium thermocellum ATCC 27405, a cellulolytic, thermophilic bacterium that has been shown to be capable of H₂ production on both cellobiose and α-cellulose substrates, was used in simultaneous batch fermentation experiments with dried distillers grain (DDGs), barley hulls (BH) and fusarium head blight contaminated barley hulls (CBH) as the carbon source. Overall, the dried distillers grain produced the highest concentration of hydrogen gas at 1.27 mmol H₂/glucose equivalent utilized. CBH and BH produced 1.18 and 1.24 mmol H₂/glucose equivalent utilized, respectively. Overall, this study indicates that hydrogen derived from a variety of cellulosic waste biomass sources is a possible candidate for the development of sustainable energy.     

Optimization of hydrogen production by hyperthermophilic eubacteria, Thermotoga maritima and Thermotoga neapolitana in batch fermentation

The batch fermentations of two hyperthermophilic eubacteria Thermotoga maritima strain DSM 3109 and Thermotoga neapolitana strain DSM 4359 were carried out to optimize the hydrogen production. The simple and economical culture medium using cheap salts with strong buffering capacity was designed based on T. maritima basal medium (TMB). Both strains cultivated under strictly anaerobic conditions showed the best growth at temperature of 75–80 °C and pH of 6.5–7.0. The maximum cell growth of 3.14 g DCW/L and hydrogen production of 342 mL H₂ gas/L were obtained, respectively, in the modified TB medium containing 7.5 g/L of glucose and 4 g/L of yeast extract. Hydrogen accumulation in the headspace was more than 30% of the gaseous phase. Cells were also cultivated in cellulose-containing medium to test the feasibility of hydrogen production.     

High yield simultaneous hydrogen and ethanol production under extreme-thermophilic (70 °C) mixed culture environment

The effect of pH and medium composition on extreme-thermophilic (70 °C) dark fermentative simultaneous hydrogen and ethanol production (process performance and microbial ecology) was investigated. Hydrogen and ethanol yields were optimized with respect to glucose, peptone, FeSO₄, NaHCO₃, yeast extract, trace mineral salts, vitamins, and phosphate buffer concentrations as well as initial pH as independent variables. A combination of low levels of both glucose (≤2 g/L) and vitamin solutions (≤1 mL/L) and high levels of initial pH (≥7), mineral salts solution (≥5 mL/L) and FeSO₄ (≥100 mg/L) stimulated the hydrogen production, while high level of glucose (≥5 g/L) and low levels of both initial pH (≤5.5) and mineral salts solution (≤1 mL/L) enhanced the ethanol production. High yield of simultaneous hydrogen and ethanol production (1.58 mol H₂/mol glucose combined with an ethanol yield of 0.90 mol ethanol/mol glucose) was achieved under extreme-thermophilic mixed culture environment. Results obtained showed that the shift of the metabolic pathways favouring either hydrogen or ethanol production was affected by the change in cultivation conditions (pH and medium composition). The mixed culture in this study demonstrated flexible ability for simultaneous hydrogen and ethanol production, depending on pH and nutrients formulation. The microorganisms involved could be regarded as simultaneous hydrogen/ethanol producers, as hydrogen and ethanol fermentation under all conditions was carried out by a group of extreme-thermophilic bacterial species related to Thermoanaerobacter, Thermoanaerobacterium and Caldanaerobacter.     

Fermentative hydrogen production in recombinant Escherichia coli harboring a [FeFe]-hydrogenase gene isolated from Clostridium butyricum

The [FeFe]-hydrogenase (hydA) from Clostridium butyricum TERI BH05-2 strain was isolated to elucidate its molecular characterization. A 1953 bp DNA fragment encompassing the ORF and the putative promoter region of hydA gene was PCR amplified and subcloned into pGEM^®-T-Easy cloning vector (pGEM^®-T-hydA). The hydA DNA sequence revealed the presence of a 1725 bp length ORF (including the stop codon) encoding 574 amino acids with a predicted isoelectric point and molecular mass of 6.8 and 63097.67 Da, respectively. The hydA ORF was PCR amplified from pGEM^®-T-hydA and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-5X-hydA) and transformed into Escherichia coli BL-21. The recombinant E. coli BL-21 was investigated for fermentative hydrogen production under anaerobic condition from glucose. Heterologous expression of the Clostridium butyricum hydA resulted in 1.9 fold increase in hydrogen productivity as compared to that from the wild type strain, C. butyricum TERI BH05-2. The hydrogen yield of the recombinant strain was 3.2 mol H₂/mol glucose, 1.68 fold higher than the wild type parent strain.     

Impact of regulated pH on proto scale hydrogen production from xylose by an alkaline tolerant novel bacterial strain, Enterobacter cloacae DT-1

A hydrogen producing facultative anaerobic alkaline tolerant novel bacterial strain was isolated from crude oil contaminated soil and identified as Enterobacter cloacae DT-1 based on 16S rRNA gene sequence analysis. DT-1 strain could utilize various carbon sources; glycerol, CMCellulose, glucose and xylose, which demonstrates that DT-1 has potential for hydrogen generation from renewable wastes. Batch fermentative studies were carried out for optimization of pH and Fe²⁺ concentration. DT-1 could generate hydrogen at wide range of pH (5–10) at 37 °C. Optimum pH was; 8, at which maximum hydrogen was obtained from glucose (32 mmol/L), when used as substrate in BSH medium containing 5 mg/L Fe²⁺ ion. Decrease in hydrogen partial pressure by lowering the total pressure in the fermenter head space, enhanced the hydrogen production performance of DT-1 from 32 mmol H₂/L to 42 mmol H₂/L from glucose and from 19 mmol H₂/L to 33 mmol H₂/L from xylose. Hydrogen yield efficiency (HY) of DT-1 from glucose and xylose was 1.4 mol H₂/mol glucose and 2.2 mol H₂/mol xylose, respectively. Scale up of batch fermentative hydrogen production in proto scale (20 L working volume) at regulated pH, enhanced the HY efficiency of DT-1 from 2.2 to 2.8 mol H₂/mol xylose (1.27 fold increase in HY from laboratory scale). 84% of maximum theoretical possible HY efficiency from xylose was achieved by DT-1. Acetate and ethanol were the major metabolites generated during hydrogen production.     

Fermentative hydrogen production by the newly isolated Enterobacter asburiae SNU-1

A new fermentative hydrogen-producing bacterium was isolated from a domestic landfill and identified as Enterobacter asburiae using 16S rRNA gene sequencing and DNA–DNA hybridization methods. The isolated bacterium, designated as Enterobacter asburiae SNU-1, is a new species that has never been examined as a potential hydrogen-producing bacterium. This study examined the hydrogen-producing ability of Enterobacter asburiae SNU-1. During fermentation, the hydrogen was mainly produced in the stationary phase. The hydrogen yield based on the formate consumption was 0.43 mol hydrogen/mol formate. This strain was able to produce hydrogen over a wide range of pH (4–7.5), with the optimum pH being pH 7. The level of hydrogen production was also affected by the initial glucose concentration, and the optimum value was found to be 25 g glucose/l. The maximum and overall hydrogen productivities were 398 and 174 ml/l/hr, respectively, at pH 7 with an initial glucose concentration of 25 g/l. This strain could produce hydrogen from glucose and many other carbon sources such as fructose, sucrose, and sorbitol.