Effect of carbon sources on the photobiological production of hydrogen using Rhodobacter sphaeroides RV

Rhodobacter sphaeroides RV was employed to produce hydrogen for the photo-fermentation of sole (acetate, propionate, butyrate, lactate, malate, succinate, ethanol, glucose, citrate and sodium carbonate) and compound carbon sources (malate and succinate, lactate and succinate). The concentrations of sole carbon sources on hydrogen production were investigated in batch assays at 0.8 g/L sodium glutamate and the maximum hydrogen yield was 424 mmol H₂/mol-substrate obtained at 0.8 g/L sodium propionate. The maximum hydrogen yield reached 794 mmol H₂/mol-substrate for 2.02 g lactate and 2.0 g succinate as the compound carbon source. The results showed hydrogen production for the compound carbon source was better than the sole carbon source.     

Photofermentative hydrogen production from volatile fatty acids present in dark fermentation effluents

In the present study, the growth and hydrogen production of Rhodobacter sphaeroides O.U. 001, was investigated in media containing five different volatile fatty acids (VFA) individually (malate, acetate, propionate, butyrate and lactate) and in media containing mixtures of these acids that reflect the composition of dark fermentation effluents. The highest hydrogen production rate was obtained in malate (24 ml_hydrogen/l_reactor h) and the highest biomass concentration was obtained in acetate containing media (1.65 g/l). The substrate conversion efficiencies for different volatile fatty acids were found to vary between 14 and 50%. The malate and butyrate consumption rates were first order with consumption rate constants of 0.026 h⁻¹ and 0.015 h⁻¹, respectively. In the case of substrate mixtures, it was observed that the bacteria consumed acetate first, followed by propionate and then butyrate. It was also found that the consumption rate of the main substrate significantly increased when the minor substrates were depleted.     

Hydrogen production and end-product synthesis patterns by Clostridium termitidis strain CT1112 in batch fermentation cultures with cellobiose or α-cellulose

Hydrogen (H₂) production and end-product synthesis were characterized in a novel, mesophilic, cellulolytic, anaerobic bacterium, Clostridium termitidis strain CT1112, isolated from the gut of the termite, Nasutitermes lujae. Growth curves, pH patterns, protein content, organic acid synthesis, and H₂ production were determined. When grown on 2 g l⁻¹ cellobiose and 2 g l⁻¹ α-cellulose, C. termitidis displayed a cell generation time of 6.5 h and 18.9 h, respectively. The major end-products synthesized on cellobiose included acetate, hydrogen, CO₂, lactate, formate and ethanol, where as on cellulose, the major end-products included hydrogen, acetate, CO₂ and ethanol. The concentrations of acetate were greater than ethanol, formate and lactate on both cellobiose and α-cellulose throughout the entire growth phase. Maximum yields of acetate, ethanol, hydrogen and formate on cellobiose were 5.9, 3.7, 4.6 and 4.2 mmol l⁻¹ culture, respectively, where as on cellulose, the yields were 7.2, 3.1, 7.7 and 2.9 mmol l⁻¹ culture, respectively. Hydrogen and ethanol production rates were slightly higher in C. termitidis cultured on cellobiose when compared to α-cellulose. Although, the generation time on α-cellulose was longer than on cellobiose, H₂ production was favored corresponding to acetate synthesis, thereby restricting the carbon flowing to ethanol. During log phase, H₂, CO₂ and ethanol were produced at specific rates of 4.28, 5.32, and 2.99 mmol h⁻¹ g dry weight⁻¹ of cells on cellobiose and 2.79, 2.59, and 1.1 mmol h⁻¹ g dry weight⁻¹ of cells on α-cellulose, respectively.     

Production of biohydrogen from sugars and lignocellulosic biomass using Thermoanaerobacter GHL15

The effect of culture parameters on hydrogen production using strain GHL₁₅ in batch culture was investigated. The strain belongs to the genus Thermoanaerobacter with 98.9% similarity to Thermoanaerobacter yonseiensis and 98.5% to Thermoanaerobacter keratinophilus with a temperature optimum of 65–70 °C and a pH optimum of 6–7. The strain metabolizes various pentoses, hexoses, and disaccharides to acetate, ethanol, hydrogen, and carbon dioxide. However substrate inhibition was observed above 10 mM glucose concentration. Maximum hydrogen yields on glucose were 3.1 mol H₂ mol⁻¹ glucose at very low partial pressure of hydrogen. Hydrogen production from various lignocellulosic biomass hydrolysates was investigated in batch culture. Various pretreatment methods were examined including acid, base, and enzymatic (Celluclast^® and Novozyme 188) hydrolysis. Maximum hydrogen production (5.8–6.0 mmol H₂ g⁻¹ dw) was observed from Whatman paper (cellulose) hydrolysates although less hydrogen was produced by hydrolysates from other examined lignocellulosic materials (maximally 4.83 mmol H₂ g⁻¹ dw of grass hydrolysate). The hydrogen yields from all lignocellulosic hydrolysates were improved by acid and alkaline pretreatments, with maximum yields on grass, 7.6 mmol H₂ g⁻¹ dw.     

Effect of pH on glucose and starch fermentation in batch and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production rates and yields were investigated at 30 °C in a 2.3 L bioreactor operated in batch and sequenced-batch mode using glucose and starch as substrates. In order to study the precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 mol H₂ mol⁻¹ glucose) was measured when the pH was maintained at 5.2. The acetate and butyrate yields were 0.35 mol acetate mol⁻¹ glucose and 0.6 mol butyrate mol⁻¹ glucose. For starch a maximum yield of 2.0 mol H₂ mol⁻¹ hexose, and a maximum production rate of 15 mol H₂ mol⁻¹ hexose h⁻¹ were obtained at pH 5.6 when the acetate and butyrate yields were 0.47 mol acetate mol⁻¹ hexose and 0.67 mol butyrate mol⁻¹ hexose.     

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.     

Evaluation of hydrogen production by Rhodobacter sphaeroides O.U.001 and its hupSL deficient mutant using acetate and malate as carbon sources

Rhodobacter sphaeroides O.U.001 is one of the candidates for photobiological hydrogen production among purple non-sulfur bacteria. Hydrogen is produced by Mo-nitrogenase from organic acids such as malate or lactate. A hupSL in frame deletion mutant strain was constructed without using any antibiotic resistance gene. The hydrogen production potential of the R. sphaeroides O.U.001 and its newly constructed hupSL deleted mutant strain in acetate media was evaluated and compared with malate containing media. The hupSL⁻R. sphaeroides produced 2.42 l H₂/l culture and 0.25 l H₂/l culture in 15 mM malate and 30 mM acetate containing media, respectively, as compared to the wild type cells which evolved 1.97 l H₂/l culture and 0.21 l H₂/l culture in malate and acetate containing media, correspondingly. According to the results, hupSL⁻R. sphaeroides is a better hydrogen producer but acetate alone does not seem to be an efficient carbon source for photoheterotrophic H₂ production by R. sphaeroides.     

The buffer composition impacts the hydrogen production and the microbial community composition in non-axenic cultures

Hydrogen obtained from biomass via dark fermentation is considered a sustainable and clean energy carrier. Batch fermentations with cheese whey powder were performed to assess total hydrogen production (H_max), volumetric hydrogen production rate (VHPR), maximum lactose consumption (S_max), maximum lactose consumption rate (R_max,S), hydrogen molar yield (HMY) and the bacterial species present using two mineral media formulation (A, B). The highest VHPR was 304.8 cm³ dm⁻³ h⁻¹ and the HMY was 1.8 mol mol⁻¹. Medium B yielded around twice the VHPR than the attained with medium A, but HMY only had a slight increment with the use of medium B. The values reached for S_max (17.3 g dm⁻³), H_max (4.863 dm³) and R_max,S (2.7 g dm⁻³ h⁻¹) were also enhanced with medium B. Results suggest that butyrate levels and lower pH are the reasons for diminished hydrogen production with medium A. The microbial communities were analyzed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Only one band was observed in the experiments with medium A, the sequence retrieved from this band presented a closest relative match to the sequence from Citrobacter freundii JCM (100% identity); whereas for medium B, three bands were detected. Sequences from these bands presented high homology to sequences from Clostridium perfringens W11 (95% identity), uncultured Lachnospiraceae bacterium clone MS146A1 E12 (100% identity) and Enterobacter cloacae GH1 (100% identity). From the results obtained it is clear that the formulation of culture media had a strong effect on hydrogen production, kinetics and also on the microbial diversity.     

The effects of seed sludge and hydraulic retention time on the production of hydrogen from a cassava processing wastewater and glucose mixture in an anaerobic fluidized bed reactor

The potential for co-fermentation of a cassava processing wastewater and glucose mixture was studied in anaerobic fluidized bed reactors. The effects of different hydraulic retention times (HRTs) (10–2 h) and varying sources of inoculum are reported. The sludge from a UASB reactor that had been used to treat poultry slaughterhouse wastewater (SP) resulted in the highest yields of hydrogen (HY) and ethanol (EtOHY) of 1.0 mmol H₂ g⁻¹ COD (10 h) and 3.0 mmol EtOH g⁻¹ COD (6 h). The sludge from a UASB reactor used for the treatment of swine wastewater (SW) resulted in a maximum HY of 0.65 mmol H₂ g⁻¹ COD (6 h) and EtOHY of 2.1 mmol g⁻¹ COD (10 and 8 h). Methane was produced with a maximum production of 9.68 L CH₄ d⁻¹ L⁻¹. Based on phylogenetic analysis of 16S rRNA, bacteria and methanogenic archaea similar to Lactobacillus and Methanobacterium, respectively, were identified.     

Sugarcane vinasse as substrate for fermentative hydrogen production: The effects of temperature and substrate concentration

The present study aimed to evaluate the hydrogen production of a microbial consortium using different concentrations of sugarcane vinasse (2–12 g COD L⁻¹) at 37 °C and 55 °C. In mesophilic tests, the increase in vinasse concentration did not significantly impact the hydrogen yield (HY) (from 1.72 to 2.23 mmol H₂ g⁻¹ COD_influent) but had a positive effect on the hydrogen production potential (P) and hydrogen production rate (R_m). On the other hand, the increase in the substrate concentration caused a drop in HY from 2.31 to 0.44 mmol H₂ g⁻¹ COD_influent in the tests performed at 55 °C with vinasse concentrations from 2 to 12 g COD L⁻¹. The mesophilic community was composed of different species within the Clostridium genus, and the thermophilic community was dominated by organisms affiliated with the Thermoanaerobacter genus. Not all isolates affiliated with the Clostridium genus contributed to a high HY, as the homoacetogenic pathway can occur.     

Enhanced hydrogen production performance of Rubrivivax gelatinosus M002 using mixed carbon sources

A new isolated photosynthetic bacterium, Rubrivivax gelatinosus M002, can produce hydrogen with glucose or lactate as sole carbon source, and grow on butyrate and acetate without hydrogen evolution. Experiments on studying its hydrogen production performance from glucose mixed with acetate, butyrate or lactate were carried out. The results showed that the hydrogen yield increased significantly and the pH value of the photo-fermentations could retain around 7 in these mixed carbon sources cultures. A hydrogen yield of 9.9 mol H₂/mol-glucose was observed when 20 mM acetate and 15 mM glucose was co-fed as substrate. The maximum hydrogen production rate was 44 mL/(L·h), which was 37.5% higher than the highest rate obtained with glucose as sole carbon source. The results suggest an alternative way for high-yield hydrogen production with mixed carbon source in one-step process instead of two-step fermentation process.     

Hydrogen production from soft-drink wastewater in an upflow anaerobic packed-bed reactor

The production of hydrogen from soft-drink wastewater in two upflow anaerobic packed-bed reactors was evaluated. The results show that soft-drink wastewater is a good source for hydrogen generation. Data from both reactors indicate that the reactor without medium containing macro- and micronutrients (R2) provided a higher hydrogen yield (3.5 mol H₂ mol⁻¹ of sucrose) as compared to the reactor (R1) with a nutrient-containing medium (3.3 mol H₂ mol⁻¹ of sucrose). Reactor R2 continuously produced hydrogen, whereas reactor R1 exhibited a short period of production and produced lower amounts of hydrogen. Better hydrogen production rates and percentages of biogas were also observed for reactor R2, which produced 0.4 L h⁻¹ L⁻¹ and 15.8% of H₂, compared to reactor R1, which produced 0.2 L h⁻¹ L⁻¹ and 2.6% of H₂. The difference in performance between the reactors was likely due to changes in the metabolic pathway for hydrogen production and decreases in bed porosity as a result of excessive biomass growth in reactor R1. Molecular biological analyses of samples from reactors R1 and R2 indicated the presence of several microorganisms, including Clostridium (91% similarity), Enterobacter (93% similarity) and Klebsiella (97% similarity).     

Sulfate-reducing bacteria as new microorganisms for biological hydrogen production

Sulfate-reducing bacteria (SRB) have an extremely high hydrogenase activity and in natural habitats where sulfate is limited, produce hydrogen fermentatively. However, the production of hydrogen by these microorganisms has been poorly explored. In this study we investigated the potential of SRB for H₂ production using the model organism Desulfovibrio vulgaris Hildenborough. Among the three substrates tested (lactate, formate and ethanol), the highest H₂ production was observed from formate, with 320 mL L⁻¹_medium of H₂ being produced, while 21 and 5 mL L⁻¹_medium were produced from lactate and ethanol, respectively. By optimizing reaction conditions such as initial pH, metal cofactors, substrate concentration and cell load, a production of 560 mL L⁻¹_medium of H₂ was obtained in an anaerobic stirred tank reactor (ASTR). In addition, a high specific hydrogen production rate (4.2 L g⁻¹_dcw d⁻¹; 7 mmol g⁻¹_dcw h⁻¹) and 100% efficiency of substrate conversion were achieved. These results demonstrate for the first time the potential of sulfate reducing bacteria for H₂ production from formate.     

Isolation of Clostridium perfringens strain W11 and optimization of its biohydrogen production by genetic modification

Clostridium perfringens strain W11, which we previously identified as the major hydrogen producer in a hydrogen-producing microbial flora, was isolated in this study. The hydrogen yield from sucrose of this strain was 1.53 mol H₂/mol hexose. To exclude potential safety problems, the plc gene, encoding an alpha toxin protein, was permanently knocked out using the Targetron gene knockout system, creating strain W12. Strains W11 and W12 both produced lactate, acetate, and butyrate during hydrogen production. Furthermore, yields of these metabolites and hydrogen were near-identical by the two strains. When the ldh gene encoding lactate dehydrogenase in strain W12 was deleted, the hydrogen yield and acetate and butyrate concentrations in the resulting mutant, W13, increased by 51%, 26%, and 57%, respectively. Lactate production by strain W13 decreased almost to zero. The growth rates of the wild-type strain W11 and its mutant derivatives were similar.     

Developing a thermophilic hydrogen-producing co-culture for efficient utilization of mixed sugars

Previous studies on the extreme thermophile Caldicellulosiruptor saccharolyticus revealed that the organism produces high yields of hydrogen on glucose and xylose, the major components of lignocellulosic hydrolysates. Preliminary experiments on mixed sugar substrates, however, indicated that xylose was preferred over glucose. The sugar preference of some other extreme thermophiles, including Caldicellulosiruptor owensensis, Caldicellulosiruptor kristjanssonii and newly enriched, thermophilic compost sludge microflora, was investigated in an attempt to find complementary organisms to C. saccharolyticus for rapid and efficient utilization of lignocellulosic sugars. The behavior of C. owensensis and C. kristjanssonii appeared to be similar to that of C. saccharolyticus, either in pure cultures or in co-cultures with the latter. Co-culturing C. saccharolyticus with the enriched compost microflora resulted in fast, simultaneous consumption of both glucose and xylose in the medium with a relatively high specific hydrogen production rate, 40 mmol (gCDW)⁻¹ h⁻¹, and high volumetric productivity, 22.5 mmol l⁻¹ h⁻¹.     

Biohydrogen production by Anabaena sp. PCC 7120 wild-type and mutants under different conditions: Light, nickel, propane, carbon dioxide and nitrogen

Increasing awareness of environmental problems caused by the current use of fossil fuel-based energy, has led to the search for alternatives. Hydrogen is a good alternative and the cyanobacterium Anabaena sp. PCC 7120 is naturally able to produce molecular hydrogen, photosynthetically from water and light. However, this H₂ is rapidly consumed by the uptake hydrogenase.This study evaluated the hydrogen production of Anabaena sp. PCC 7120 wild-type and mutants: hupL⁻ (deficient in the uptake hydrogenase), hoxH⁻ (deficient in the bidirectional hydrogenase) and hupL⁻/hoxH⁻ (deficient in both hydrogenases) on several experimental conditions, such as gas atmosphere (argon and propane with or without N₂ and/or CO₂ addition), light intensity (54 and 152 ??Em⁻²s⁻¹), light regime (continuous and light/dark cycles 16 h/8 h) and nickel concentrations in the culture medium.In every assay, the hupL⁻ and hupL⁻/hoxH⁻ mutants stood out over wild-type cells and the hoxH⁻ mutant. Nevertheless, the hupL⁻ mutant showed the best hydrogen production except in an argon atmosphere under 16 h light/8 h dark cycles at 54 ??Em⁻²s⁻¹ in the light period, with 1 ??M of NiCl₂ supplementation in the culture medium, and under a propane atmosphere.In all strains, higher light intensity leads to higher hydrogen production and if there is a daily 1% of CO₂ addition in the gas atmosphere, hydrogen production could increase 5.8 times, related to the great increase in heterocysts differentiation (5 times more, approximately), whereas nickel supplementation in the culture medium was not shown to increase hydrogen production. The daily incorporation of 1% of CO₂ plus 1% of N₂ did not affect positively hydrogen production rate.     

Hydrogen evolution catalyzed by viable and non-viable cells on biocathodes

The presence of microorganisms on cathodes has been shown to enhance the hydrogen evolution reaction (HER), but a requirement for viable cells has not been sufficiently examined. HER was examined using live or killed biocathodes of exoelectrogenic (Geobacter sulfurreducens) and non-exoelectrogenic (Escherichia coli) bacteria, and a hydrogenotrophic methanogen (Methanosarcina barkeri). Electrodes at a set potential of −0.6 V (versus a standard hydrogen electrode) containing G. sulfurreducens biofilms or killed controls produced hydrogen at a similar rates (118 ± 15 nmold⁻¹ mL⁻¹) over 5 months. Electrodes containing cell extracts produced hydrogen at approximately half this rate (56 ± 6 nmold⁻¹ mL⁻¹). Biocathodes fed lactate produced only 14 ± 2 nmol/d-mL. Electrodes inoculated with M. barkeri produced hydrogen at a rate (120 ± 18 nmold⁻¹ mL⁻¹) similar to the G. sulfurreducens, but no methane was recovered after the initial inoculation cycle. Non-exoelectrogenic E. coli cells and extracts produced hydrogen at a slower rate (13 ± 1 and 4 ± 1 nmold⁻¹ mL⁻¹, over 3 cycles). Electrodes exposed to viable cells that were examined after 5 months had increased levels of in nitrogen, sulfur, iron, nickel, cobalt, and peptides (possibly remnants of hydrogenases and other oxidoreductases) relative to uninoculated controls, and no intact cells. These results show that enhanced HER can result from cell debris and that living cells are not required.     

Optimization of thermophilic fermentative hydrogen production by the newly isolated Caloranaerobacter azorensis H53214 from deep-sea hydrothermal vent environment

A unique thermophilic fermentative hydrogen-producing strain H53214 was isolated from a deep-sea hydrothermal vent environment, and identified as Caloranaerobacter azorensis based on bacterial 16S rRNA gene analysis. The optimum culture condition for hydrogen production by the bacterium, designated C. azorensis H53214, was investigated by the response surface methodology (RSM). Eight variables including the concentration of NaCl, glucose, yeast, tryptone, FeSO₄ and MgSO₄, initial pH and incubation temperature were screened based on the Plackett–Burman design. The results showed that initial pH, tryptone and yeast were significant variables, which were further optimized using the steepest ascent method and Box–Behnken design. The optimal culture conditions for hydrogen production were an initial pH of 7.7, 8.3 g L⁻¹ tryptone and 7.9 g L⁻¹ yeast. Under these conditions, the maximum cumulative hydrogen volume, hydrogen yield and maximum H₂ production rate were 1.58 L H₂ L⁻¹ medium, 1.46 mol H₂ mol⁻¹ glucose and 25.7 mmol H₂ g⁻¹ cell dry weight (CDW) h⁻¹, respectively. By comparison analysis, strain H53214 was superior to the most thermophilic hydrogen producers because of the high hydrogen production rate. In addition, the isolation of C. azorensis H53214 indicated the deep-sea hydrothermal environment might be a potential source for fermentative hydrogen-producing thermophiles.     

Bioethanol production from sweet potato by co-immobilization of saccharolytic molds and Saccharomyces cerevisiae

To investigate the bioethanol production from sweet potato, the saccharification and fermentation conditions of co-immobilization of saccharolytic molds (Aspergillus oryzae and Monascus purpureus) with Saccharomyces cerevisiae were analyzed. The immobilized yeast cells showed that at 10% glucose YPD (yeast extract peptone dextrose) the maximum fermentation rate was 80.23%. Viability of yeasts cells were 95.70% at a final ethanol concentration of 6%. Immobilization enhanced the ethanol tolerance of yeast cells. In co-immobilization of S. cerevisiae with A. oryzae or M. purpureus, the optimal hardening time of gel beads was between 15 and 60 min. Bioethanol production was 3.05-3.17% (v v⁻¹) and the Y_E/s (yield of ethanol production/starch consumption) was 0.31-0.37 at pH 4, 30 °C and 150 rpm during 13 days fermentation period. Co-immobilization of S. cerevisiae with a mixed cultures of A. oryzae and M. purpureus at a ratio of 2:1, the bioethanol production was 3.84% (v v⁻¹), and the Y_E/s was 0.39 for a 11 days incubation. However a ratio of A. oryzae and M. purpureus at 1:2 resulted a bioethanol production rate of 4.08% (v v⁻¹), and a Y_E/s of 0.41 after 9 days of fermentation.     

Overcoming propionic acid inhibition of hydrogen fermentation by temperature shift strategy

A novel temperature shift strategy has been proposed to overcome an inhibition on hydrogen fermentation of beverage industry wastewater (BW) due to the accumulation of propionic acid (HPr) during continuous reactor operation. The continuous performance at constant pH 5.5, temperature 37 °C and hydraulic retention time (HRT) 8 h with BW concentration of 20 g/L_{hexose-equivalent} in a stirred tank reactor (2 L) showed an accumulation of HPr to 2.36 g/L leading to a drop in hydrogen production rate (HPR) from 10 to 8.5 L L⁻¹ d⁻¹. 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⁻¹ d⁻¹ and 1.68 mol-H₂ mol⁻¹ 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 non-dominance of Selenomonas lacticifex and Bifidobacterium catenulatum (involved in HPr formation), and dominance of hydrogen producing bacteria namely Clostridium butyricum, Clostridium perfringenes, Clostridium acetobutylicum, and Ethanoligenens harbinense. This study demonstrated that temperature shift strategy could overcome the HPr inhibition and significantly improve the hydrogen fermentation of an industrial wastewater.