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

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

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.     

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 by Ethanoligenens harbinense B49: Nutrient optimization

A new hydrogen-producing bacterial strain Ethanoligenens harbinense B49 was examined for its capability of H₂ production with glucose as sole carbon source. The H₂ production was significantly affected by the concentration of the yeast powder and phosphate in the synthetic medium. The optimized concentration of yeast powder was 0.3–0.5 g/L and the maximum hydrogen yield was obtained at the concentration of phosphate about 100–150 mmol/L. The dynamics of hydrogen production showed that rapid evolution of hydrogen appeared to start after the middle-phase of exponential growth (about 8 h). The maximum H₂ yield and specific hydrogen production rate were estimated to be 2.26 mol H₂/mol glucose and 27.74 mmol H₂/g cell, respectively, when 10 g/L of glucose was present in the medium. The possible pathway of hydrogen production by Ethanoligenens sp. B49 during glucose fermentation was oxidative decarboxylation of pyruvate and the NADH pathway.     

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 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.     

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.     

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.     

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.     

The kinetic characterization of photofermentative bacterium Rhodopseudomonas faecalis RLD-53 and its application for enhancing continuous hydrogen production

In this work, the kinetic characterization of hydrogen production by the photofermentative bacteria Rhodopseudomonas faecalis RLD-53 was investigated at different growth phase. During entire fermentation, 89.30% of total biomass was accumulated in exponential growth phase, while hydrogen yield was only 1.82 mol H₂/mol acetate at the expense of 51.25% substrate. In the stationary phase, biomass synthesis was minimal (7.51%), and 38.17% of the substrate was directly converted into hydrogen. As a result, hydrogen (59.19%) was mainly produced in stationary phase with highest hydrogen yield of 3.67 mol H₂/mol acetate. Consequently, bacteria in stationary phase were most effective for hydrogen production. Based on these findings, a novel membrane photobioreactor was developed to retain bacteria during stationary phase in reactor through membrane separation. Maximum rate (32.82 ml/l/h) and yield (3.27 mol H₂/mol acetate) of hydrogen production were achieved using membrane photobioreactor under the continuous operation. Therefore, using bacteria in stationary phase as hydrogen producer can offer considerable benefits for enhancing photo-hydrogen production.     

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.     

Statistical optimization of fermentative hydrogen production from xylose by newly isolated Enterobacter sp. CN1

Statistical experimental designs were applied for the optimization of medium constituents for hydrogen production from xylose by newly isolated Enterobacter sp. CN1. Using Plackett–Burman design, xylose, FeSO₄ and peptone were identified as significant variables which highly influenced hydrogen production. The path of steepest ascent was undertaken to approach the optimal region of the three significant factors. These variables were subsequently optimized using Box–Behnken design of response surface methodology (RSM). The optimum conditions were found to be xylose 16.15 g/L, FeSO₄ 250.17 mg/L, peptone 2.54 g/L. Hydrogen production at these optimum conditions was 1149.9 ± 65 ml H₂/L medium. Under different carbon sources condition, the cumulative hydrogen volume were 1217 ml H₂/L xylose medium, 1102 ml H₂/L glucose medium and 977 ml H₂/L sucrose medium; the maximum hydrogen yield were 2.0 ± 0.05 mol H₂/mol xylose, 0.64 mol H₂/mol glucose. Fermentative hydrogen production from xylose by Enterobacter sp. CN1 was superior to glucose and sucrose.     

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.     

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

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

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.     

Hydrogen production and metabolic flux analysis of metabolically engineered Escherichia coli strains

Escherichia coli can produce H₂ from glucose via formate hydrogen lyase (FHL). In order to improve the H₂ production rate and yield, metabolically engineered E. coli strains, which included pathway alterations in their H₂ production and central carbon metabolism, were developed and characterized by batch experiments and metabolic flux analysis. Deletion of hycA, a negative regulator for FHL, resulted in twofold increase of FHL activity. Deletion of two uptake hydrogenases (1 (hya) and hydrogenase 2 (hyb)) increased H₂ production yield from 1.20 mol/mol glucose to 1.48 mol/mol glucose. Deletion of lactate dehydrogenase (ldhA) and fumarate reductase (frdAB) further improved the H₂ yield; 1.80 mol/mol glucose under high H₂ pressure or 2.11 mol/mol glucose under reduced H₂ pressure. Several batch experiments at varying concentrations of glucose (2.5–10 g/L) and yeast extract (0.3 or 3.0 g/L) were conducted for the strain containing all these genetic alternations, and their carbon and energy balances were analyzed. The metabolic flux analysis revealed that deletion of ldhA and frdABdirected most of the carbons from glucose to the glycolytic pathway leading to H₂ production by FHL, not to the pentose phosphate pathway.     

Biohydrogen production from xylose by Thermoanaerobacterium thermosaccharolyticum KKU19 isolated from hot spring sediment

A thermophilic hydrogen producer was isolated from hot spring sediment and identified as Thermoanaerobacterium thermosaccharolyticum KKU19 by biochemical tests and 16S rRNA gene sequence analysis. The strain KKU19 showed the ability to utilize various kinds of carbon sources. Xylose was the preferred carbon source while peptone was the preferred organic nitrogen source. The optimum conditions for hydrogen production and cell growth on xylose were an initial pH of 6.50, temperature of 60 °C, a carbon to nitrogen ratio of 20:1, and a xylose concentration of 10.00 g/L. This resulted in a maximum cumulative hydrogen production, hydrogen production rate and hydrogen yield of 3020 ± 210 mL H₂/L, 3.95 ± 0.20 mmol H₂/L h and 2.09 ± 0.02 mol H₂/mol xylose consumed, respectively. Acetic and butyric acids were the main soluble metabolite products suggesting acetate and butyrate type 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.