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.     

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.     

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.     

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.     

Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 °C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0–2.3 mol H₂/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans.     

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).     

Dark fermentative hydrogen production with crude glycerol from biodiesel industry using indigenous hydrogen-producing bacteria

Glycerol is an inevitable by-product from biodiesel synthesis process and could be a promising feedstock for fermentative hydrogen production. In this study, the feasibility of using crude glycerol from biodiesel industry for biohydrogen production was evaluated using seven isolated hydrogen-producing bacterial strains (Clostridium butyricum, Clostridium pasteurianum, and Klebsiella sp.). Among the strains examined, C. pasteurianum CH4 exhibited the best biohydrogen-producing performance under the optimal conditions of: temperature, 35 °C; initial pH, 7.0; agitation rate, 200 rpm; glycerol concentration, 10 g/l. When using pure glycerol as carbon source for continuous hydrogen fermentation, the average H₂ production rate and H₂ yield were 103.1 ± 8.1 ml/h/l and 0.50 ± 0.02 mol H₂/mol glycerol, respectively. In contrast, when using crude glycerol as the carbon source, the H₂ production rate and H₂ yield was improved to 166.0 ± 8.7 ml/h/l and 0.77 ± 0.05 mol H₂/mol glycerol, respectively. This work demonstrated the high potential of using biodiesel by-product, glycerol, for cost-effective biohydrogen 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.     

Characteristics of hydrogen production by an aciduric transposon-mutagenized strain of Pantoea agglomerans BH18

Based on the generation of Pantoea agglomerans BH18 library using Tn7 transposon system, mutant screens were conducted for improvement the ability of hydrogen production of this strain. These mutants were used to test for ability of hydrogen production in the initial pH of 5.0. In contrast to wild type strain BH18, a transposon mutant, named as strain TB108, was screened for high hydrogen-producing capability and acid tolerance in the initial pH of 5.0. The factors required for hydrogen production of the aciduric transposon-mutagenized strain TB108 were determined. The mutant strain TB108 similar as wild type strain BH18 was able to produce hydrogen over a wide range of salt concentration from 0.4% to 6%. Under the marine conditions with the initial pH of 5.0 and glucose concentration of 10 g/L, the total hydrogen production of the mutant TB108 was (1.36 ± 0.04) mol H₂/mol glucose (mean ± S.E.), increasing by 55% compared with wild type. In addition, the mutant strain TB108 could produce hydrogen using many carbon sources such as fructose, glucose, sucrose, sorbitol and so on. This result demonstrated that the mutant strain with high acid tolerance is beneficial for improvement of hydrogen production.     

Sequential dark–photo fermentation and autotrophic microalgal growth for high-yield and CO2-free biohydrogen production

Dark fermentation, photo fermentation, and autotrophic microalgae cultivation were integrated to establish a high-yield and CO₂-free biohydrogen production system by using different feedstock. Among the four carbon sources examined, sucrose was the most effective for the sequential dark (with Clostridium butyricum CGS5) and photo (with Rhodopseudomonas palutris WP3-5) fermentation process. The sequential dark–photo fermentation was stably operated for nearly 80 days, giving a maximum H₂ yield of 11.61 mol H₂/mol sucrose and a H₂ production rate of 673.93 ml/h/l. The biogas produced from the sequential dark–photo fermentation (containing ca. 40.0% CO₂) was directly fed into a microalga culture (Chlorella vulgaris C–C) cultivated at 30 °C under 60 μmol/m²/s illumination. The CO₂ produced from the fermentation processes was completely consumed during the autotrophic growth of C. vulgaris C–C, resulting in a microalgal biomass concentration of 1999 mg/l composed mainly of 48.0% protein, 23.0% carbohydrate and 12.3% lipid.     

Enhancing the performance of dark fermentative hydrogen production using a reduced pressure fermentation strategy

The partial pressure of hydrogen is an extremely important factor for hydrogen generation. This study investigated the effect of reduced pressure (via vacuum) on hydrogen production in a CSTR reactor. The results show that the reduced pressure condition is more effective in enhancing H₂ production at lower HRT (e.g., 8–4 h) than at higher HRT (e.g., 12 h). The optimal hydrogen yield and overall hydrogen production efficiency occurred at a HRT of 6 h with a value of 4.50 mol H₂/mol sucrose and 56.2%, respectively. Meanwhile, at HRT 6 h the hydrogen production rate was 0.937 mol/L/d. In addition, the HPR could be further improved to 1.196 mol/L/d when the HRT was shortened to 4 h, obtaining a 37–271% increase in HPR when compared with that described in the relevant reports. For all experiments, butyrate and acetate were the two primary soluble metabolites, accounting for 85–99% of total soluble microbial products. Predominant production of acetate and butyrate demonstrates the efficient H₂ fermentation with reduced pressure processes.     

Statistical optimization of biohydrogen production from sucrose by a co-culture of Clostridium acidisoli and Rhodobacter sphaeroides

Statistically based experimental designs were applied to optimize the fermentation process parameters for hydrogen (H₂) production by co-culture of Clostridium acidisoli and Rhodobacter sphaeroides with sucrose as substrate. An initial screening using the Plackett–Burman design identified three factors that significantly influenced H₂ yield: sucrose concentration, initial pH, and inoculum ratio. These factors were considered to have simultaneous and interdependent effects. A central composite design and response surface analysis were adopted to further investigate the mutual interactions among the factors and to identify the values that maximized H₂ production. The optimal substrate concentration, initial pH, and inoculum ratio of C. acidisoli to R. sphaeroides were 11.43 g/L sucrose, 7.13, and 0.83, respectively. Using these optimal culture conditions, substrate conversion efficiency was determined as 10.16 mol H₂/mol sucrose (5.08 mol H₂/mol hexose), which was near the expected value of 10.70 mol H₂/mol sucrose (5.35 mol H₂/mol hexose).     

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.     

Enhanced photo-hydrogen production of Rhodopseudomonas faecalis RLD-53 by EDTA addition

This study investigated the effect of EDTA concentration in medium on the growth, hydrogen production and nitrogenase activity of Rhodopseudomonas faecalis RLD-53 by batch cultures. Experimental results indicated that bacterial growth and hydrogen production were strongly inhibited with EDTA concentration increasing to 0.6–0.7 g/L. However, the lag time of hydrogen production and the trends of biomass at EDTA concentration of 0–0.5 g/L were similar. The maximum cumulative hydrogen volume of 3325 ml H₂/L culture, hydrogen production rate of 27.6 ml H₂/L/h and hydrogen yield of 2.97 mol H₂/mol acetate were obtained when EDTA concentration was at 0.3 g/L, and the maximum OD₆₆₀ attained 3.83. And the nitrogenase activity also reached a maximum value of 1331.9 μl-C₂H₄/h/mg dry weight in the medium containing Fe²⁺ and EDTA. These results showed that a proper concentration of EDTA can promote the availability of iron, thereby further enhancing the activity of nitrogenase and photo-hydrogen production.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum KKU-ED1: Culture conditions optimization using xylan as the substrate

Thermophilic hydrogen production from xylan by Thermoanaerobacterium thermosaccharolyticum KKU-ED1 isolated from elephant dung was investigated using batch fermentation. The optimum conditions for hydrogen production from xylan by the strain KKU-ED1 were an initial pH of 7.0, temperature of 55 °C and xylan concentration of 15 g/L. Under the optimum conditions, the hydrogen yield (HY), hydrogen production rate (HPR) and xylanase activity were 120.05 ± 15.07 mL H₂/g xylan, 11.53 ± 0.19 mL H₂/L h and 0.41 units/mL, respectively. The optimum conditions were then used to produce hydrogen from 62.5 g/L sugarcane bagasse (SCB) (equivalent to 15 g/L xylan) in which the HY and HPR of 1.39 ± 0.10 mL H₂/g SCB (5.77 ± 0.41 mL H₂/g xylan) and 0.66 ± 0.04 mL H₂/L h, respectively, were achieved. In comparison to the other strains, the HY of the strain KKU-ED1 (120.05 ± 15.07 mL H₂/g xylan) was close to that of Clostridium sp. strain X53 (125.40 mL H₂/g xylan) and Clostridium butyricum CGS5 (90.70 mL H₂/g xylan hydrolysate).     

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 production of hydrogen and soluble metabolites from crude glycerol of biodiesel plant by the newly isolated thermotolerant Klebsiella pneumoniae TR17

A thermotolerant fermentative hydrogen-producing strain was isolated from crude glycerol contaminated soil and identified as Klebsiella pneumoniae on the basis of the 16S rRNA gene analysis as well as physiological and biochemical characteristics. The selected strain, designated as K. pneumoniae TR17, gave good hydrogen production from crude glycerol. Culture conditions influencing the hydrogen production were investigated. The strain produced hydrogen within a wide range of temperature (30–50 °C), initial pH (4.0–9.0) and crude glycerol concentration (20–100 g/L) with yeast extract as a favorable nitrogen source. In batch cultivation, the optimal conditions for hydrogen production were: cultivation temperature at 40 °C, initial pH at 8.0, 20 g/L crude glycerol and 2 g/L yeast extract. This resulted in the maximum cumulative hydrogen production of 27.7 mmol H₂/L and hydrogen yield of 0.25 mol H₂/mol glycerol. In addition, the main soluble metabolites were 1,3-propanediol, 2,3-butanediol and ethanol corresponding to the production of 3.52, 2.06 and 3.95 g/L, respectively.     

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.     

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.