Phytosynthesized iron oxide nanoparticles and ferrous iron on fermentative hydrogen production using Enterobacter cloacae: Evaluation and comparison of the effects

The effects of FeSO₄ and synthesized iron oxide nanoparticles (0–250 mg/L) on fermentative hydrogen production from glucose and sucrose, using Enterobacter cloacae were investigated, to find out the enhancement of efficiency. The maximum hydrogen yields of 1.7 ± 0.017 mol H₂/mol glucose and 5.19 ± 0.12 mol H₂/mol sucrose were obtained with 25 mg/L of ferrous iron supplementation. In comparison, the maximum hydrogen yields of 2.07 ± 0.07 mol H₂/mol glucose and 5.44 ± 0.27 mol H₂/mol sucrose were achieved with 125 mg/L and 200 mg/L of iron oxide nanoparticles, respectively. These results indicate that the enhancement of hydrogen production on the supplementation of iron oxide nanoparticles was found to be considerably higher than that of ferrous iron supplementation. The activity of E. cloacae in a glucose and sucrose fed systems was increased by the addition of iron oxide nanoparticles, but the metabolic pathway was not changed. The results revealed that the glucose and sucrose fed systems conformed to the acetate/butyrate fermentation type.     

Enhancement of biohydrogen production from sustainable orange peel wastes using Enterobacter species isolated from domestic wastewater

Five facultative anaerobic bacterial isolates were recovered from domestic wastewater. These isolates were identified based on the 16S rRNA as Enterobacter aerogenes (one isolate), Enterobacter cloacae (two isolates), and Cronobacter sakazakii (three isolates). These isolates were examined for their potential to evolve hydrogen on a glucose medium. The most potent hydrogen‐producing isolates, E aerogenes (KY549389) and E cloacae (KY524293), were examined for their capacity to generate hydrogen, acetone, butanol, and ethanol using orange peel (OP) hydrolysate. OP powder was pretreated with n‐hexane to remove the toxicity of d ‐limonene. Different concentrations (4%, 6%, and 8% w/v) of limonene‐free OP were subjected to the boiling water (temperature of 100°C) or acid (HCl) treatments. The maximum fermentative H₂ production of 1700 and 1620 mL/L was obtained from 6% OP hydrolysate extracted with boiling water using facultative anaerobic E aerogenes (KY549389) and E cloacae (KY524293), respectively. Hydrogen production efficiency was 0.99 and 1.19 mol H₂/mol glucose for E aerogenes and E cloacae, respectively. The total fermentative acetone, butanol, and ethanol (ABE) generated by E aerogenes and E cloacae were 0.78 and 0.38 g/L including acetone (0.05 and 0.04 g/L), butanol (0.011 and 0.013 g/L), and ethanol (0.71 and 0.32 g/L), respectively. The maximum ABE productivity was 0.01 and 0.005 g/L/h generated at 60 g/L OP hydrolysate by E aerogenes and E cloacae, respectively. These strains were positive for nitrogen fixation (nitrogenase) capability estimated by the acetylene reduction assay. Application of OP hydrolysate without the addition of any nutritional components or reducing agent is considered an eco‐friendly, economical, and commercial substrate for desired biofuel production.     

Hydrogen production performance by Enterobacter cloacae KBH3 isolated from termite guts

Two out of six bacterial isolates obtained from the guts of Globitermes sp. termites were identified as hydrogen-producing bacteria. One isolate, Enterobacter cloacae KBH3, was characterised using the BIOLOG identification system and 16S rRNA gene analysis. In a batch fermentation study to evaluate its growth in defined medium, E. cloacae KBH3 produced 154 ml H₂ per litre medium with approximately 50% hydrogen content. The carbon utilisation results suggest that E. cloacae KBH3 have the potential to be a good hydrogen producer. This strain is also able to produce hydrogen within a wide range of temperatures (28–40 °C) and pH (4.5–8). In several fermentation runs, the pH of the culture dropped from 6.5 to 5.36 within the first 3 h, which was mostly due to the biosynthesis of formate. An increase of cumulative hydrogen production was recorded as well as a decrease in the concentration of formate, indicating the importance of the formate pathway for hydrogen production. The highest rate of hydrogen production of 180.74 ml H₂/l/h was achieved when lactate and acetate were at their highest concentrations. Most of the hydrogen gas was produced during the exponential growth phase, and the biogas continued to be produced during the stationary phase. The specific growth rate was calculated to be 0.224 per hour while the hydrogen yield was 1.8 mol of hydrogen per mol of glucose. At the end of the batch study, the highest cumulative hydrogen production was 2404 ml H₂ per litre of fermentation medium.     

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.     

Comparative study on the production of biohydrogen from rice mill wastewater

This study presents the production of biohydrogen from rice mill wastewater. The acid hydrolysis and enzymatic hydrolysis operating conditions were optimized, for better reducing sugar production. The effect of pH and fermentation time on biohydrogen production from acid and enzymatic hydrolyzed rice mill wastewater was investigated, using Enterobacter aerogenes and Citrobacter ferundii. The enzymatic hydrolysis produced the maximum reducing sugar (15.8 g/L) compared to acid hydrolysis (14.2 g/L). The growth data obtained for E. aerogenes and C. ferundii, fitted well with the Logistic equation. The hydrogen yields of 1.74 mol H₂/mol reducing sugar, and 1.40 mol H₂/mol reducing sugar, were obtained from the hydrolyzate obtained from enzymatic and acid hydrolysis, respectively. The maximum hydrogen yield was obtained from E. aerogenes compared to C. ferundii, and the optimum pH for better hydrogen production was found to be in the range from 6.5 to 7.0. The chemical oxygen demand (COD) reduction obtained was around 71.8% after 60 h of fermentation.     

Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor

The production of hydrogen from glucose by using Enterobacter aerogenes ATCC 13048 (E. aerogenes) in an immobilized cell reactor (ICR) was investigated. The effect of several factors, such as the glucose concentration, feed flow rate, and fermentation time were examined. The highest amount of hydrogen (9.44 mmol H₂/g glucose) was obtained at a glucose concentration of 8 g/L, flow rate of 0.5 mL/min, retention time of 24 h and at a temperature of 30 °C. Meanwhile, the highest amount of carbon dioxide (1.68 mmol CO₂/g glucose) was obtained at a glucose concentration of 10 g/L, flow rate of 0.7 mL/min, hydraulic retention time of 24 h and at a temperature of 30 °C. The hydrogen and carbon dioxide production were affected by glucose concentration, hydraulic retention time (HRT) and fermentation time. This study showed that the ICR was a very efficient method for the production of hydrogen and carbon dioxide gases.     

Enzymatic saccharification and fermentation of paper and pulp industry effluent for biohydrogen production

Paper and pulp industry effluent was enzymatically hydrolysed using crude cellulase enzyme (0.8–2.2FPU/ml) obtained from Trichoderma reesei and from the hydrolysate biohydrogen was produced using Enterobacter aerogenes. The influence of temperature and incubation time on enzyme production was studied. The optimum temperature for the growth of T. reesei was found to be around 29 °C. The enzyme activity of 2.5 FPU/ml was found to produce about 22 g/l of total sugars consisting mainly of glucose, xylose and arabinose. Relevant kinetic parameters with respect to sugars production were estimated using two fraction model. The enzymatic hydrolysate was used for the biohydrogen production using E. aerogenes. The growth data obtained for E. aerogenes were fitted well with Monod and Logistic equations. The maximum hydrogen yield of 2.03 mol H₂/mol sugar and specific hydrogen production rate of 225 mmol of H₂/g cell/h were obtained with an initial concentration of 22 g/l of total sugars. The colour and COD of effluent was also decreased significantly during the production of hydrogen. The results showed that the paper and pulp industry effluent can be used as a substrate for biohydrogen production.     

Fermentative hydrogen production by two novel strains of Enterobacter aerogenes HGN-2 and HT 34 isolated from sea buried crude oil pipelines

Present study investigated fermentative hydrogen production of two novel isolates of Enterobacter aerogenes HGN-2 and HT 34 isolated from oil water mixtures. The two isolates were identified as novel strains of E. aerogenes based on 16S rRNA gene. The batch fermentations of two strains from glucose and xylose were carried out using economical culture medium under various conditions such as temperature, initial pH, NaCl, Ni⁺/Fe⁺⁺, substrate concentrations for enhanced fermentation process. Both the strains favoured wide range of pH (6.5–8.0) at 37 °C for optimum production (2.20–2.23 mol H₂/mol-glucose), which occurred through acetate/butyrate pathway. At 55 °C, both strains favoured 6.0–6.5 and acetate type fermentation was predominant in HT 34. Hydrogen production by HT 34 from xylose was highly pH dependant and optimum production was at pH 6.5 (circa 1.98 mol-H₂/mol-xylose) through acetate pathway. The efficiency of the strain HGN-2 at pH 6.5 was 1.92–1.94 mol-H₂/mol-xylose, and displayed both acetate and butyrate pathways. At 55 °C, very low hydrogen production was detected (less than 0.5 m mol/mol-xylose).     

Biological hydrogen production by extremely thermophilic novel bacterium Thermoanaerobacter mathranii A3N isolated from oil producing well

Hydrogen producing novel bacterial strain was isolated from formation water from oil producing well. It was identified as Thermoanaerobacter mathranii A3N by 16S rRNA gene sequencing. Hydrogen production by novel strain was pH and substrate dependent and favored pH 8.0 for starch, pH 7.5 for xylose and sucrose, pH 8.0–9.0 for glucose fermentation at 70 °C. The highest H₂ yield was 2.64 ± 0.40 mol H₂ mol glucose at 10 g/L, 5.36 ± 0.41 mol H₂ mol – sucrose at 10 g/L, 17.91 ± 0.16 mmol H₂ g – starch at 5 g/L and 2.09 ± 0.21 mol H₂ mol xylose at 5 g/L. The maximum specific hydrogen production rates 6.29 (starch), 9.34 (sucrose), 5.76 (xylose) and 4.89 (glucose) mmol/g cell/h. Acetate-type fermentation pathway (approximately 97%) was found to be dominant in strain A3N, whereas butyrate formation was found in sucrose and xylose fermentation. Lactate production increased with high xylose concentrations above 10 g/L.     

Biohydrogen production from various feedstocks by Bacillus firmus NMBL-03

In present work our objective was to find out the potential substrates for fermentative hydrogen production using microbial culture of Bacillus firmus NMBL-03 isolated from municipal sludge. A wide variety of substrates (glucose, xylose, arabinose, lactose, sucrose, and starch) and carbohydrate rich waste products (bagasse hydrolysate, molasses, potato peel and cyanobacterial mass) have been used for dark fermentative hydrogen production. All studies were done at optimized physico-chemical conditions. Among all substrates glucose, arabinose, lactose, starch, molasses and bagasse hydrolysate were found to be the favorable substrates for hydrogen production. The highest VHPR i.e. 177.5 ± 7.07 ml/L.h (7.95 ± 0.29 mmol H₂/L.h) and maximum H₂ production (22.58 ± 2.65 mmol H₂/L) was achieved using starch as the substrate. The maximum yield 1.29 ± 0.11 mol H₂/mol reducing sugar was obtained from bagasse hydrolysate as substrate. Butyrate and acetate were detected as the end product in all the cases while lactate was also detected from glucose and cyanobacterial hydrolysate. Since considerable amount of H₂ also evolved when cyanobacterial mass was used, therefore this microbe can be exploited for hydrogen production through a three stage integrated system. Residual carbohydrate containing biomass left after cyanobacterial H₂ production can be utilized in dark fermentative H₂ production. Spent media obtained after dark fermentative H₂ production contain considerable amount of volatile fatty acids that can be potential substrate for photo fermentative H₂ production.     

Inhibitory effects of acetate and ethanol on biohydrogen production of Ethanoligenens harbinese B49

Inhibitory effects of acetate and ethanol on biohydrogen production from glucose by Ethanoligenens harbinese B49 were investigated in this study. In batch test, sodium acetate (0, 10, 20, 50, 100 and 150 mmol/l) and ethanol (0, 20, 40, 80, 100 and 200 mmol/l) were added respectively. Their inhibitory effects on glucose degradation, cell growth, distribution of liquid products and hydrogen production were discussed. Compared with ethanol, acetate exhibited more significant inhibition on growth and hydrogen producing performance of E. harbinese B49. The inhibitory effects of acetate and ethanol were compared and analyzed on the basis of a noncompetitive product inhibition model. For acetate addition, the maximum specific hydrogen production rate r_max = 722 ml/gVSS/h, inhibition constant K_C = 55 mmol/l and the exponent of inhibition n = 0.6 were estimated, whereas the maximum hydrogen yield r_max = 2.2 mol H₂/mol glucose, K_C = 57 mmol/l and n = 0.8 were calculated from kinetic analysis. For ethanol addition, the maximum specific rate r_max = 729 ml/g VSS/h, K_C = 139 mmol/l and n = 0.8 were estimated, whereas the maximum hydrogen yield r_max = 2.2 mol H₂/mol glucose, K_C = 153 mmol/l and n = 0.9 were calculated. In addition, deducing from dose-response curves, the C_I,50 values of ethanol and acetate were 154 and 62 mmol/l, respectively. Acetate has a strong inhibitory effect on hydrogen production with ethanol-type fermentation. Thus, hydrogen production can be improved by optimizing the fermentation strategy through removing the acetate as soon as it was produced.     

Biohydrogen production by immobilized Enterobacter aerogenes on functionalized multi-walled carbon nanotube

Hydrogen production by immobilized Enterobacter aerogenes on functionalized multi-walled carbon nanotube (MWCNT-COOH) in repeated batch mode was studied. Fourier transform infrared (FTIR) spectroscopy and field emission scanning electron microscopy (FESEM) were employed to confirm immobilization of E. aerogenes successfully. The effect of MWCNT-COOH concentrations (0.2, 0.6, and 1.2 mg/mL) on hydrogen production was investigated. The present study showed that immobilized E. aerogenes on 1.2 mg/mL MWCNT-COOH resulted in higher hydrogen yield (2.2 moL/mol glucose), hydrogen production rate (2.72 L/L.h), and glucose degradation efficiency (96.20%) and shorter the lag phase (1 h) compared to the free E. aerogenes. Modified Gompertz and Logistic models were employed to predict the cumulative hydrogen production successfully.     

Bioreactor for glycerol conversion into H2 by bacterium Enterobacter aerogenes

Glycerol was used as a substrate for H₂ production by bacterium Enterobacter aerogenes in the test tubes and bioreactor. A BioFlo/CelliGen 115 bioreactor (10 L working volume) was utilized to conduct the experiments for conversion of glycerol into H₂ by E. aerogenes cells. The highest H₂ production rate was observed under 2% glycerol in the culture medium. The glycerol uptake efficiency by bacteria in the bioreactor was found to be 65% during the 6 day period, matching glycerol uptake efficiency observed in the test tubes experiment (65%).Hydrogen production from glycerol (2% glycerol, v/v) by E. aerogenes in the bioreactor and test tubes was measured over the 6 days, showing the maximal H₂ rate at 650 mL g⁻¹ dry weight h⁻¹. The yield of H₂ production from glycerol at 0.89 mol/mol in the bioreactor was high, corresponding to the theoretical yield of 1 mol of H₂ per 1 mol of glycerol.     

Hydrogen production from the monomeric sugars hydrolyzed from hemicellulose by Enterobacter aerogenes

Relatively large percentages of xylose with glucose, arabinose, mannose, galactose and rhamnose constitute the hydrolysis products of hemicellulose. In this paper, hydrogen production performance of facultative anaerobe (Enterobacter aerogenes) has been investigated from these different monomeric sugars except glucose. It was shown that the stereoisomers of mannose and galactose were more effective for hydrogen production than those of xylose and arabinose. The substrate of 5 g/l xylose resulted in a relative high level of hydrogen yield (73.8 mmol/l), hydrogen production efficiency (2.2 mol/mol) and a maximum hydrogen production rate (249 ml/l/h). The hydrogen yield, hydrogen production efficiency and the maximum hydrogen production rate reached 104 mmol/l, 2.35 mol/mol and 290 ml/l/h, respectively, on a substrate of 10 g/l galactose. The hydrogen yields and the maximum hydrogen production rates increased with an increase of mannose concentrations and reached 119 mmol/l and 518 ml/l/h on the culture of 25 g/l mannose. However, rhamnose was a relative poor carbon resource for E. aerogenes to produce hydrogen, from which the hydrogen yield and hydrogen production efficiency were about one half of that from the mannose substrate. E. aerogenes was found to be a promising strain for hydrogen production from hydrolysis products of hemicellulose.     

Optimization of photo-hydrogen production based on cheese whey spent medium

Cheese whey wastewater diluted to 10 g lactose/L was initially subjected to dark-fermentation by Enterobacter aerogenes MTCC 2822, and the VFAs-rich spent medium (acetic acid 1900 mg/L, butyric acid 537 mg/L, and traces of propionic acid) was subjected to photo-fermentation through enrichment by Ni²⁺ (0–8 μmol/L), Fe²⁺ (0–100 μmol/L) or Mg²⁺ (0–15 mmol/L) in batch mode by Rhodopseudomonas BHU 01 strain. The maximum cumulative H₂ production (144 ml) and yield (58 mmol) was obtained at 4 μmol Ni²⁺/L. Likewise, Fe²⁺ (60 μmol/L) resulted in maximum cumulative H₂ production (139 ml) and yield (56 mmol). Nevertheless, 6 mmol of Mg²⁺ did not significantly affect H₂ production (110 ml) or yield (44 mmol); the latter value in close proximity with the control (37 mmol). The concomitant reduction in COD was maximum (15.61%) for 4 μmol Ni²⁺/L, followed by 15.33% for 60 μmol Fe²⁺/L, and the least for 6 mmol Mg²⁺/L (14.5%). The observations suggest the role of Fe²⁺ and Ni²⁺ in regulation of nitrogenase and hydrogenase, while that of Mg²⁺ mainly in the biosynthesis of photopigment bacteriochlorophyll (Bchl).     

Enhancement in hydrogen production by co-cultures of Bacillus and Enterobacter

Defined co-cultures of hydrogen (H₂) producers belonging to Citrobacter, Enterobacter, Klebsiella and Bacillus were used for enhancing the efficiency of biological H₂ production. Out of 11 co-cultures consisting of 2–4 strains, two co-cultures composed of Bacillus cereus EGU43, Enterobacter cloacae HPC123, and Klebsiella sp. HPC793 resulted in H₂ yield up to 3.0 mol mol⁻¹ of glucose. Up-scaling of the reactor by 16-fold resulted in a corresponding increase in H₂ production with an actual evolution of 7.44 L of H₂. It constituted 58.2% of the total biogas. Continuous culture evolution of H₂ by co-cultures (B. cereus EGU43 and E. cloacae HPC123) immobilized on ligno-cellulosic materials resulted in 6.4-fold improvement in H₂ yield compared to free floating bacteria. This synergistic influence of B. cereus and E. cloacae can offer a better strategy for H₂ production than undefined or mixed cultures.     

Optimization of selected salts concentration for improved biohydrogen production from biodiesel-based glycerol using Enterobacter aerogenes

Enterobacter aerogenes have a known ability to convert glycerol (GL) in a fermentative process to yield hydrogen and ethanol as the main by-products. The concentration of some media constituents was optimized to maximize biohydrogen yield and rate of production. E. aerogenes were cultured in aerobic conditions, and then transferred into anaerobic conditions before being cultured in a minimum mineral synthetic media (MMSM) containing 15 g/L GL. The concentration of selected salts were optimized in the following ranges: 0–300 mg/L MgSO₄, 0–14 g/L Na₂EDTA, 0–10 mg/L CaCL₂, 0–10 g/L Na₂HPO₄, and 0–9.7 g/L KH₂PO₄. The results of the full factorial design indicated that the production of biohydrogen required a minimal concentration of 3.5 mg/L EDTA, 200 mg/L MgSO₄.7H₂O and no CaCl₂.2H₂O. A significant interaction between EDTA and MgSO₄ was also observed. Results from the phosphate salts optimization showed that Na₂HPO₄ gave better results than KH₂PO₄. The optimal conditions determined using pure glycerol (commercial grade glycerol), were successfully applied to the fermentation of crude glycerol from biodiesel production. The results indicated promising yields of 0.79 and 0.84 mol/mol of glycerol for bioethanol and biohydrogen, respectively, and this at a faster rate than reported previously for E. aerogenes.     

Dark fermentative hydrogen production by defined mixed microbial cultures immobilized on ligno-cellulosic waste materials

Mixed microbial cultures (MMCs) based on 11 isolates belonging to Bacillus spp. (Firmicutes), Bordetella avium, Enterobacter aerogenes and Proteus mirabilis (Proteobacteria) were employed to produce hydrogen (H₂) under dark fermentative conditions. Under daily fed culture conditions (hydraulic retention time of 2 days), MMC6 and MMC4, immobilized on ligno-cellulosic wastes – banana leaves and coconut coir evolved 300–330 mL H₂/day. Here, H₂ constituted 58–62% of the total biogas evolved. It amounted to a H₂ yield of 1.54–1.65 mol/mol glucose utilized over a period of 60 days of fermentation. The involvement of various Bacillus spp. – Bacillus sp., Bacillus cereus, Bacillus megaterium, Bacillus pumilus and Bacillus thuringiensis as components of the defined MMCs for H₂ production has been reported here for the first time.     

Biohydrogen production from beet molasses by sequential dark and photofermentation

Biological hydrogen production using renewable resources is a promising possibility to generate hydrogen in a sustainable way. In this study, a sequential dark and photofermentation has been employed for biohydrogen production using sugar beet molasses as a feedstock. An extreme thermophile Caldicellulosiruptor saccharolyticus was used for the dark fermentation, and several photosynthetic bacteria (Rhodobacter capsulatus wild type, R. capsulatus hup⁻ mutant, and Rhodopseudomonas palustris) were used for the photofermentation. C. saccharolyticus was grown in a pH-controlled bioreactor, in batch mode, on molasses with an initial sucrose concentration of 15 g/L. The influence of additions of NH₄⁺ and yeast extract on sucrose consumption and hydrogen production was determined. The highest hydrogen yield (4.2 mol of H₂/mol sucrose) and maximum volumetric productivity (7.1 mmol H₂/L_c.h) were obtained in the absence of NH₄⁺. The effluent of the dark fermentation containing no NH₄⁺ was fed to a photobioreactor, and hydrogen production was monitored under continuous illumination, in batch mode. Productivity and yield were improved by dilution of the dark fermentor effluent (DFE) and the additions of buffer, iron-citrate and sodium molybdate. The highest hydrogen yield (58% of the theoretical hydrogen yield of the consumed organic acids) and productivity (1.37 mmol H₂/L_c.h) were attained using the hup⁻ mutant of R. capsulatus. The overall hydrogen yield from sucrose increased from the maximum of 4.2 mol H₂/mol sucrose in dark fermentation to 13.7 mol H₂/mol sucrose (corresponding to 57% of the theoretical yield of 24 mol of H₂/mole of sucrose) by sequential dark and photofermentation.     

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.