Improvement of hydrogen production by transposon-mutagenized strain of Pantoea agglomerans BH18

Pantoea agglomerans BH18, isolated from mangrove sludge, could produce hydrogen under marine culture condition. To improve the hydrogen-producing capacity of this strain, we constructed a stable transposon-mutagenized library of P. agglomerans BH18. A Tn7-based transposon was randomly inserted into genomic DNA of P. agglomerans BH18. Mutants were identified by kanamycin resistance and amplification of the inserted transposon sequences. A transposon mutant, named as strain TB212, was screened for the highest hydrogen production ability. The total volume of hydrogen gas evolved by this mutant strain TB212 was 60% higher than that of the wild type. The mutant strain TB212 was able to produce hydrogen over a wide range of initial pH from 5.0 to 10.0, with an optimum initial pH of 7.0, and hydrogen production was 2.52 ± 0.02 mol H₂/mol glucose (mean ± S.E.) under marine culture condition. The mutant strain TB212 could produce hydrogen at the salt concentration from 3 to 6%. It was concluded that the transposon-mutagenized library may be a useful tool for investigation of high efficiency hydrogen-producing bacteria.     

Hydrogen production from glucose by a mutant strain of Rhodovulum sulfidophilum P5 in single-stage photofermentation

Single-stage hydrogen production from glucose was investigated using the marine photosynthetic bacterium Rhodovulum sulfidophilum TH-79, a Tn7 transposon mutant of strain P5. The mutation in strain TH-79 did not affect its cell growth in glucose medium compared with the parent strain. TH-79 displayed improved photoheterotrophic hydrogen production performance when the medium contained glucose or galactose as the sole carbon source. The mutant produced about 7.07 mol H₂/mol glucose, which is similar to the yields of more complicated integration systems. A one-stage photofermentation system using a seawater culture medium appears to be a promising alternative to the integration of dark- and photofermentation systems.     

Hydrogen production by a marine photosynthetic bacterium, Rhodovulum sulfidophilum P5, isolated from a shrimp pond

A new hydrogen-producing photosynthetic bacterium, designated as Rhodovulum sulfidophilum P5, was enriched and isolated from the sludge of a marine shrimp cultivation farm. During fermentation, hydrogen was mainly produced in the late exponential and stationary phases. The optimum culture conditions of strain P5 for hydrogen production were NaCl concentration of 20 g/L, initial pH of 8, temperature of 30 °C, and light intensity of 100 μmol photons/m² s. The maximum hydrogen yield and rate were 2.56 ± 0.18 mol/mol acetate and 19.4 ± 1.6 mL/L h, respectively. Under optimum culture conditions, the hydrogen conversion efficiencies of P5 from acetate, propionate, and butyrate were (64.62 ± 5.05)%, (17.95 ± 0.72)%, and (41.83 ± 2.68)%, respectively. Taken together, these results suggest that this strain has a high salt tolerance and the potential to be used for biohydrogen production and biological treatment of marine organic wastewater.     

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.     

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.     

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.     

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

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

Enhanced hydrogen production from xylose and bamboo stalk hydrolysate by overexpression of xylulokinase and xylose isomerase in Klebsiella oxytoca HP1

Biofuels production from lignocellulose hydrolysates by microbe fermentation has merited attention because of the mild reaction conditions involved and the clean nature of the process. In this work, xylulokinase (XK) and xylose isomerase (XI) were overexpressed in Klebsiella oxytoca HP1 to enhance hydrogen production by the fermentation of xylose. The recombinant strains exhibited higher enzyme activity of XI or XK compared with the wild strain. Hydrogen production from pure xylose, xylose/glucose mixtures and bamboo stalk hydrolysate was significantly enhanced with the overexpression of XI and XK in K. oxytoca HP1 in terms of total hydrogen yield (THY), hydrogen yield per mole substrate (HYPM) and hydrogen production rate (HPR). The HYPM of K. oxytoca HP1/xylB and K. oxytoca HP1/xylA reached 1.93 ± 0.05 and 2.46 ± 0.05 mol H₂/mol xylose, respectively in pure xylose, while the value for the wild strain was 1.68 ± 0.04 mol H₂/mol xylose. The xylose consumption rate (XCR) for the recombinant strain was significantly higher than that for the wild strain, particularly in the early stage of fermentation. Relative to the wild type, hydrogen yield (HY) from 1 g of preprocessed bamboo powder of HP1/xylB and HP1/xylA increased by 33.04 and 41.31%, respectively. It was concluded that overexpression of XK or XI was able to promote hydrogen production from xylose and xylose/glucose mixtures by simultaneously increasing the utilization efficiency of xylose and weakening the inhibitory effect of glucose on xylose use. In addition, the results indicated that overexpression technology was an effective way to further increase hydrogen production from lignocellulosic hydrolysates.     

Isolation and characterization of a new hydrogen-producing strain Bacillus sp. FS2011

A new fermentative hydrogen-producing strain FS2011 was isolated from an effluent of bio-hydrogen production reactor, and identified as Bacillus amyloliquefaciens on the basis of 16S rDNA gene sequence. The strain could utilize various carbon and nitrogen sources to produce hydrogen in a broad range of initial pH (5.29–7.38). Phosphate buffer concentration and fermentation temperature significantly affected hydrogen production and cell growth. The maximum hydrogen yield of 2.26 mol/mol was observed at glucose concentration of 10 g/l, beef extract concentration of 2 g/l, initial pH 6.98, phosphate buffer of 20 mmol/l, and 35 °C, indicating FS2011 was a high-efficiency hydrogen-producing bacterium.     

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.     

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

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

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.     

Effect of pH on continuous biohydrogen production from liquid swine manure with glucose supplement using an anaerobic sequencing batch reactor

pH is considered as one of the most important factors governing the hydrogen fermentation process. In this project, five pH levels, ranging from 4.4 to 5.6 at 0.3 increments, were tested to evaluate the pH effect on hydrogen production from swine manure supplemented with glucose in an anaerobic sequencing batch reactor system with 16 h of hydraulic retention time (HRT). The optimal hydrogen yield (1.50 mol H₂/mol glucose) was achieved at pH 5.0 when the maximum production rate of 2.25 L/d/L was obtained. Continuous hydrogen production was achieved for over 3 weeks for pH 5.0, 4.7, and 4.4, with no significant methane produced. However, as pH increased to 5.3 and 5.6, methane production was observed in the biogas with concurrent reductions in hydrogen production, indicating that methanogens could become increasingly activated for pH 5.3 or higher. Acetate, propionate, butyrate, valerate, and ethanol were the main aqueous products whose distribution was significantly affected by pH as well.     

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).     

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.     

Isolation and evaluation of a high H2-producing lab isolate from cow dung

Hydrogen producing bacterial strain was isolated from Indian cow dung and identified of the bacterial family Enterobacteriaceae. This lab isolate was differentiated from Citrobacter Y-19 at molecular level by using RAPD, PCR based technique, and OPO-03₄₆₀ and OPO-17₈₀₀ RAPD marker for this specific strain (lab isolate) was identified. Fermentative studies were investigated for important parameters, starting with pH of the culture, temperature, inoculum age and inoculum volume, initial substrate concentration and different substrates. Among different substrates, dextrose and sucrose were the preferred substrates for hydrogen production. The optimal starting pH of the culture was found to be 5.0. The H₂ production increased with increase in temperature up to 30 °C. The maximum value of H₂ production was recorded when inoculum volume was 12.5% of the culture broth and inoculum age was 14 h. Under batch fermentation conditions, the maximum hydrogen production rate and yield were 355.2 ml l⁻¹ h⁻¹ and 2.1 mol/mol glucose (conversion 35%), respectively. These results indicate that this lab isolate is an ideal hydrogen producer.     

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.     

Effect of substrate concentration on fermentative hydrogen production from sweet sorghum extract

The aim of the present study was to assess the influence of substrate concentration on the fermentative hydrogen production from sweet sorghum extract, in a continuous stirred tank bioreactor. The reactor was operated at a Hydraulic Retention Time (HRT) of 12 h and carbohydrate concentrations ranging from 9.89 to 20.99 g/L, in glucose equivalents. The maximum hydrogen production rate and yield were obtained at the concentration of 17.50 g carbohydrates/L and were 2.93 ± 0.09 L H₂/L reactor/d and 0.74 ± 0.02 mol H₂/mol glucose consumed, corresponding to 8.81 ± 0.02 L H₂/kg sweet sorghum, respectively. The main metabolic product at all steady states was butyric acid, while ethanol production was high at high substrate concentrations. The experiments showed that hydrogen productivity depends significantly on the initial carbohydrate concentration, which also influences the distribution of the metabolic products.     

Hydrogen production from butyrate by a marine mixed phototrophic bacterial consort

A newly enriched marine phototrophic bacterial consort was studied for its capability of hydrogen production in batch cultivations using butyrate as the sole carbon source. Analyses of denaturing gradient gel electrophoresis (DGGE) profiles showed that the mixed bacterial consort consisted mainly of Ectothiorhodospira, Sporolactobacillus, and Rhodovulum. Important parameters investigated include temperature, light intensity, initial pH, and butyrate concentration. The pH of the culture medium significantly increased as fermentation proceeded. Optimal cell growth was observed at temperature of 25–35 °C, light intensity of 80–120 μmol photons/m² s, initial pH of 8, butyrate concentration of 20–40 mmol/l. Optimal conditions for hydrogen production were 30 °C, light intensity of 80 μmol photons/m² s, initial pH 8. The increase of butyrate concentration (10–50 mmol/l) resulted in higher hydrogen production, but the yield of hydrogen production (mol H₂/mol butyrate) gradually decreased with increasing butyrate concentration. The maximal hydrogen yield and hydrogen production rate were estimated to be 2.52 ± 0.12 mol H₂/mol butyrate and 19.40 ± 2.32 ml/l h, respectively. These results indicate that optimization of the culture conditions resulted in a simultaneous increase in biohydrogen production and cell growth.     

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.