Technological advances in hydrogen production by Enterobacter bacteria upon substrate,luminosity and anaerobic conditions

The enhancement of hydrogen production by Enterobacter aerogenes and Enterobacter cloacae from fermentation of carbon sources such as glucose and lactose (from cheese whey permeate) was investigated. Also, the influence of the luminosity (2200 lux) and anaerobic condition (nitrogen and argon gases) were evaluated. The assays were carried out in 50 mL reactors during 108 h. To E. aerogenes/nitrogen/luminosity condition and using glucose as substrate, H₂ production (73.8 mmol/L.d) was higher than using lactose (15.5 mmol/L.d). In the dark fermentation, hydrogen yields were 1.60 mol H₂/mol glucose and 1.36 molH₂/mol lactose. When using E. cloacae, the light fermentation using nitrogen gas resulted in 77 mmol H₂/L.d and 1.62 mol H₂/mol glucose. In addition, for E. cloacae, hydrogen yields using argon gas and luminosity provided 2.39 mol/mol glucose and 2.53 mol/mol lactose. In general, butyric and acetic acid fermentation were observed and favored the target-product (H₂).     

Effects of substrate loading and co-substrates on thermophilic anaerobic conversion of microcrystalline cellulose and microbial communities revealed using high-throughput sequencing

Batch tests were conducted to investigate the effect of co-substrates, including glucose, xylose and starch, on thermophilic anaerobic conversion of microcrystalline cellulose using mixed culture enriched from anaerobic digestion sludge (ADS). Up to 30.9% of cellulose was utilized with xylose as co-substrate. When using glucose as co-substrate, cellulose conversion rate reached the maximum of 0.048 g/l/h at cellulose loading of 5.0 g/l. Illumina high-throughput sequencing of the 16S rRNA gene revealed that the thermophilic consortium exclusively consisted of Clostridium (more than 70% of all sequences). Growth of Thermoanaerobacterium over Clostridium would inhibit cellulose conversion capacity of the consortium. But the growth of Thermoanaerobacterium could be repressed by pH higher than pH 6.0. Co-substrates caused noticeable variation of bacterial community structure. Predominance of Thermoanaerobacterium over Clostridium was observed when monosugars (glucose and xylose) were used as co-substrate without pH control. Starch was ineffective as co-substrate because it competed with cellulose for Clostridium.     

Improvement of hydrogen production under decreased partial pressure by newly isolated alkaline tolerant anaerobe, Clostridium butyricum TM-9A: Optimization of process parameters

A mesophilic alkaline tolerant fermentative microbe was isolated from estuarine sediment samples and designated as Clostridium butyricum TM-9A, based on 16S rRNA gene sequence. Batch experiments were conducted for investigation of TM-9A strain for its growth and hydrogen productivity from glucose, in an iron containing basal solution supplemented with yeast extract as organic nitrogen source. Hydrogen production started to evolve when cell growth entered exponential phase and reached maximum production rate at late exponential phase. Maximum hydrogen production was observed at 37 °C, initial pH of 8.0 in the presence of 1% glucose. Optimization of process parameters resulted in increase in hydrogen yield from 1.64 to 2.67 mol of H₂/mol glucose. Molar yield of H₂ increased further from 2.67 to 3.1 mol of H₂/mol of glucose with the decrease in hydrogen partial pressure, obtained by lowering the total pressure in the head space of the batch reactor. Acetate and butyrate were the measure volatile fatty acids generated during hydrogen fermentation. TM-9A strain produced hydrogen efficiently from a range of pentose and hexose sugars including di-, tri and poly-saccharides like; xylose, ribose, glucose, rhamnose, galactose, fructose, mannose, sucrose, arabinose, raffinose, cellulose, cellobiose and starch.     

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.     

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.     

Hydrogen production by mixed bacteria through dark and photo fermentation

Mixed bacteria were used to improve hydrogen yield from cassava starch in combination of dark and photo fermentation. In dark fermentation, mixed anaerobic bacteria (mainly Clostridium species) were used to produce hydrogen from cassava starch. Substrate concentration, fermentation temperature and pH were optimized as 10.4 g/l, 31 °C and 6.3 by response surface methodology (RSM). The maximum hydrogen yield and production rate in dark fermentation were 351 ml H₂/g starch (2.53 mol H₂/mol hexose) and 334.8 ml H₂/l/h, respectively. In photo fermentation, immobilized mixed photosynthetic bacteria (PSB, mainly Rhodopseudomonaspalustris species) were used to produce hydrogen from soluble metabolite products (SMP, mainly acetate and butyrate) of dark fermentation. The maximum hydrogen yield in photo fermentation was 489 ml H₂/g starch (3.54 mol H₂/mol hexose). The total hydrogen yield was significantly increased from 402 to 840 ml H₂/g starch (from 2.91 to 6.07 mol H₂/mol hexose) by mixed bacteria and cell immobilization in combination of dark and photo fermentation.     

Enhancement of fermentative hydrogen production from green algal biomass of Thermotoga neapolitana by various pretreatment methods

Biomass of the green algae has been recently an attractive feedstock source for bio-fuel production because the algal carbohydrates can be derived from atmospheric CO₂ and their harvesting methods are simple. We utilized the accumulated starch in the green alga Chlamydomonas reinhardtii as the sole substrate for fermentative hydrogen (H₂) production by the hyperthermophilic eubacterium Thermotoga neapolitana. Because of possessing amylase activity, the bacterium could directly ferment H₂ from algal starch with H₂ yield of 1.8–2.2 mol H₂/mol glucose and the total accumulated H₂ level from 43 to 49% (v/v) of the gas headspace in the closed culture bottle depending on various algal cell-wall disruption methods concluding sonication or methanol exposure. Attempting to enhance the H₂ production, two pretreatment methods using the heat-HCl treatment and enzymatic hydrolysis were applied on algal biomass before using it as substrate for H₂ fermentation. Cultivation with starch pretreated by 1.5% HCl at 121 °C for 20 min showed the total accumulative H₂ yield of 58% (v/v). In other approach, enzymatic digestion of starch by thermostable α-amylase (Termamyl) applied in the SHF process significantly enhanced the H₂ productivity of the bacterium to 64% (v/v) of total accumulated H₂ level and a H₂ yield of 2.5 mol H₂/mol glucose. Our results demonstrated that direct H₂ fermentation from algal biomass is more desirably potential because one bacterial cultivation step was required that meets the cost-savings, environmental friendly and simplicity of H₂ production.     

Behavior of acidogenesis during biohydrogen production with formate and glucose as carbon source: Substrate associated dehydrogenase expression

Experiments were designed to enumerate variation in biohydrogen (H₂) production pattern with formate and glucose as carbon source under acidogenic mixed microenvironment. High H₂ production was observed with glucose (180 ml) when compared to formate (152 ml). The process was validated with modified Gompertz model (R² = 0.98). Substrate degradation also showed higher removal of glucose (ξ_COD, 82%) compared to formate (ξ_COD, 53%). Nevertheless, specific H₂ yield of formate (6.6 mol H₂/kg COD_R) obtained was comparatively higher than glucose (5 mol H₂/kg COD_R). Variation in H₂ production was manifested by change in fatty acid composition and substrate degradation pattern. Acetate was obtained as a major metabolic intermediate from the degradation of glucose and a shift in the biochemical pathway towards formation of butyrate occurred after maximum substrate degradation. The role of substrate-dependent dehydrogenase activity was deciphered during H₂ production and was evidenced with bio-electrochemical analysis.     

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.     

Co-fermentation of sewage sludge and algae and Fe2+ addition for enhancing hydrogen production

Co-fermentation of sewage sludge and algae was performed for enhancing the hydrogen production, and the effect of Fe²⁺ on co-fermentation process was examined. Results showed that both co-fermentation process and Fe²⁺ addition promoted hydrogen production. Highest hydrogen production of 28 mL/100 mL (14.8 mL H₂/g VS_added) was obtained from the co-fermentation group with 600 mg/L Fe²⁺ addition, which was 2.15 times, 2.00 times and 1.87 times of mono-fermentation of sludge, mono-fermentation of algae, and the co-fermentation group without Fe²⁺ addition. Both volatile solids and protein degradation were stimulated by co-fermentation process. Microbial analysis showed that co-fermentation groups with Fe²⁺ addition enriched Clostridium sensu stricto 13, Clostridium tertium and Terrisporobacter, which were positively correlated with cumulative hydrogen production. This study suggested that the co-fermentation of sludge and algae in the presence of Fe²⁺ could significantly improve the hydrogen production by stimulating the hydrogen-producing metabolism.     

Pentoses,hexoses and glycerin as substrates for biohydrogen production: An approach for Brazilian biofuel integration

Biofuels production in Brazil is a traditional activity, and it has becoming more important each year. Within this context, biohydrogen production could exploit residual streams from first generation ethanol (ethanol 1G), second generation ethanol (ethanol 2G) and biodiesel production. Therefore hexoses, pentoses and glycerin were tested as substrates for hydrogen production. Firstly, the effects of different inoculum pretreatments (acid, alkaline and heat) on bacterial communities' performance were evaluated through the levels of Clostridium hydrogenase expression. The heat pretreated inoculum provided the highest yield of H₂ (4.62 mol H₂/mol sucrose) and also the highest level of hydrogenase expression, 64 times higher when compared with untreated inoculum after 72 h. Then C5 and C6 sugars and also glycerin were tested for H₂ production (35 °C and pH 5.5), which resulted in promising yields of H₂: sucrose (4.24 mol H₂/mol sucrose), glucose (2.19 mol H₂/mol glucose), fructose (2.09 mol H₂/mol fructose), xylose (1.88 mol H₂/mol xylose) and glycerin (0.80 mol H₂/mol glycerin).     

Characteristics of the process of biohydrogen production from simple and complex substrates with different biopolymer composition

The work investigated the characteristics of the dark fermentation (DF) process of a number of simple (starch, sunflower oil, peptone, both separately and mixed) and complex (dog food, pig feed, sewage sludge) substrates using a mixed culture of microorganisms, with a controlled pH (5.5), at 55 °C. Peptone and sunflower oil were characterized by the lowest production of H₂, namely 5.0 and 2.3 ml H₂/g COD, respectively. The specific hydrogen yield from starch was 1.55 mol H₂/mol hexose. The addition of peptone and sunflower oil to starch reduced the specific yield of hydrogen from starch by 23%. A large difference in hydrogen production was observed during DF of complex substrates. The specific hydrogen yield from dog food was 46.5 ml H₂/g COD or 143.4 ml H₂/g carbohydrates; from pig feed – 32.1 ml H₂/g COD or 91.6 ml H₂/g carbohydrates; and from sewage sludge – 9.3 ml H₂/g COD or 98.0 ml H₂/g carbohydrates. Possible relationships between the biopolymer composition of substrates and characteristics of the DF process were analyzed using Spearman's rank correlation coefficients. The concentration of carbohydrates, as well as the ratio of carbohydrates/proteins and carbohydrates/fats, were the main factors influencing the high specific yield of H₂, its content in biogas, as well as the ratio of H₂/soluble metabolites. The concentration of proteins had a statistically significant positive effect on the accumulation of acetate and succinate, and carbohydrates - on the accumulation of caproate.     

Simultaneous biohydrogen (H2) and bioplastic (poly-β-hydroxybutyrate-PHB) productions under dark,photo, and subsequent dark and photo fermentation utilizing various wastes

The present study is focused on bio hydrogen (H₂) and bioplastic (i.e., poly-β-hydroxybutyrate; PHB) productions utilizing various wastes under dark fermentation, photo fermentation and subsequent dark-photo fermentation. Potential bio H₂ and PHB producing microbes were enriched and isolated. The effects of substrate (rice husk hydrolysate, rice straw hydrolysate, dairy industry wastewater, and rice mill wastewater) concentration (10–100%) and pH (5.5–8.0) were examined in the batch mode under the dark and photo fermentation conditions. Using 100% rice straw hydrolysate at pH 7, the maximum bio H₂ (1.53 ± 0.04 mol H₂/mol glucose) and PHB (9.8 ± 0.14 g/L) were produced under dark fermentation condition by Bacillus cereus. In the subsequent dark-photo fermentation, the highest amounts of bio H₂ and PHB were recorded utilizing 100% rice straw hydrolysate (1.82 ± 0.01 mol H₂/mol glucose and 19.15 ± 0.25 g/L PHB) at a pH of 7.0 using Bacillus cereus (KR809374) and Rhodopseudomonas rutila. The subsequent dark-photo fermentative bio H₂ and PHB productions obtained using renewable biomass (i.e., rice husk hydrolysate and rice straw hydrolysate) can be considered with respect to the sustainable management of global energy sources and environmental issues.     

Thermophilic fermentative hydrogen production from various carbon sources by anaerobic mixed cultures

In the present work, various carbon sources, xylose, glucose, galactose, sucrose, cellobiose, and starch were tested for thermophilic (60 °C) fermentative hydrogen production (FHP) by using the anaerobic mixed culture. An inoculum was obtained from a continuously-stirred tank reactor (CSTR) operated at pH 5.5 and HRT 12 h, and fed with tofu processing waste. The dominant species in the CSTR were found to be Thermoanaerobacterium thermosaccharolyticum and Clostridium thermosaccharolyticum, which are well known thermophilic H₂-producers in anaerobic-state, and have the ability to utilize a wide range of carbohydrates. When initial pH was adjusted to 6.8 ± 0.1 but not controlled during fermentation, vigorous pH drop began within 5 h, and finally reached 4.0–4.5 in all carbon sources. Although over 90% of substrate removal was achieved for all carbon sources except cellobiose (71.7%), the fermentation performances were profoundly different with each other. Glucose, galactose, and sucrose exhibited relatively higher H₂ yields whereas lower H₂ yields were observed for xylose, cellobiose, and starch. On the other hand, when pH was controlled (pH ≥ 5.5), the fermentation performance was enhanced in all carbon sources but to a different extent. A substantial increase in H₂ production was observed for cellobiose, a 1.9-fold increase of H₂ yield along with a substrate removal increase to 93.8%, but a negligible increase for xylose. H₂ production capabilities of all carbon sources tested were as follows: sucrose > galactose > glucose > cellobiose > starch > xylose. The maximum H₂ yield of 3.17 mol H₂/mol hexose_added achieved from sucrose is equivalent to a 26.5% conversion of energy content in sucrose to H₂. Acetic and butyric acids were the main liquid-state metabolites of all carbon sources while lactic acid was detected only in cellobiose, starch and xylose exhibiting relatively lower H₂ yields.     

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.     

Co-fermentation of carbohydrates and proteins for biohydrogen production: Statistical optimization using Response Surface Methodology

In this study, the co-fermentation of carbohydrates and proteins at different ratios (C1–C5) was explored. The rates of particulate carbohydrates degradation in the co-substrate mixtures, not only increased with starch concentrations, but negatively impacted the degradation rates of the particulate proteins. Particulate proteins also negatively impacted particulate carbohydrate degradation rates, albeit to a lesser extent. Generally, there was a synergistic impact on hydrogen production and the optimum ratio that required no pH control occurred at C4 (80% carbohydrates + 20% proteins) with a hydrogen yield of 350 mL H₂/gCOD_added which was 38% higher than the expected, and the fermentation followed the acetate-ethanol pathway. Response Surface Methodology (RSM) was used to optimize the responses to the co-fermentation process at C4. By fitting 20 experimental data points, the responses adequately fitted second-order polynomial models. At the optimized VFA and ammonia concentrations of 580 mg/L and 40 mg/L, respectively, the biohydrogen production process would be feasible without pH control at a carbohydrate-to-protein COD ratio of 4:1.     

Fermentative hydrogen production by new marine Clostridium amygdalinum strain C9 isolated from offshore crude oil pipeline

The present study investigated hydrogen production potential of novel marine Clostridium amygdalinum strain C9 isolated from oil water mixtures. Batch fermentations were carried out to determine the optimal conditions for the maximum hydrogen production on xylan, xylose, arabinose and starch. Maximum hydrogen production was pH and substrate dependant. The strain C9 favored optimum pH 7.5 (40 mmol H₂/g xylan) from xylan, pH 7.5–8.5 from xylose (2.2–2.5 mol H₂/mol xylose), pH 8.5 from arabinose (1.78 mol H₂/mol arabinose) and pH 7.5 from starch (390 ml H₂/g starch). But the strain C9 exhibited mixed type fermentation was exhibited during xylose fermentation. NaCl is required for the growth and hydrogen production. Distribution of volatile fatty acids was initial pH dependant and substrate dependant. Optimum NaCl requirement for maximum hydrogen production is substrate dependant (10 g NaCl/L for xylose and arabinose, and 7.5 g NaCl/L for xylan and starch).     

Effect of the type and concentration of cellulose and temperature on metabolite formation by a fermentative thermophilic consortium

In this study, we hypothesized that anaerobic biodegradation of cellulose is influenced by cellulose type and concentration, temperature, and their interactions. Cellulose biodegradation by an anaerobic consortium was tested in thermophilic batch experiments that combined cellulase action, hydrolysis, and fermentation. Initially, the main constituents in the inocula were Thermoanaerobacter, Clostridium, and Acetivibrio spp. Four types of cellulose and a range of concentrations were used as feedstock with pathways involving hydrolysis and glycolysis to produce H₂, CO₂, acetate, and ethanol. Long fibrous cellulose, two types of microcrystalline cellulose, and filter paper squares were tested at several concentrations between 2 and 20 g/l as substrates. The yields ranged between 0.1 and 2.9 mmol H₂ and 0.7–2.6 mmol ethanol per g cellulose. The rates ranged between 0.01 and 0.2 mmol H₂, 0.03–0.2 mmol CO₂, and 0.01–0.05 mmol ethanol per g cellulose·h. Statistical analyses indicated that the rates and yields of metabolite production were influenced by two-way interactions between the temperature, type, and concentration of cellulose. The results suggest that two-way interactions between experimental variables may impact the outcomes in cellulose bioconversion studies.     

Dark fermentative hydrogen production from xylose by a hot spring enrichment culture

Dark fermentative hydrogen production by a hot spring culture was studied from different sugars in batch assays and from xylose in continuous stirred tank reactor (CSTR) with on-line pH control. Batch assays yielded hydrogen in following order: xylose > arabinose > ribose > glucose. The highest hydrogen yield in batch assays was 0.71 mol H₂/mol xylose. In CSTR the highest H₂ yield and production rate at 45 °C were 1.97 mol H₂/mol xylose and 7.3 mmol H₂/h/L, respectively, and at 37 °C, 1.18 mol H₂/mol xylose and 1.7 mmol H₂/h/L, respectively. At 45 °C, microbial community consisted of only two bacterial strains affiliated to Clostridium acetobutulyticum and Citrobacter freundii, whereas at 37 °C six Clostridial species were detected. In summary hydrogen yield by hot spring culture was higher with pentoses than hexoses. The highest H₂ production rate and yield and thus, the most efficient hydrogen producing bacteria were obtained at suboptimal temperature of 45 °C for both mesophiles and thermophiles.     

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.