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.     

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.     

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

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

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.     

Biohydrogen production from wastewater by Clostridium beijerinckii: Effect of pH and substrate concentration

An investigation of biological hydrogen production from glucose by Clostridium beijerinckii was conducted in a synthetic wastewater solution. A study examining the effect of initial pH (range 5.7–6.5) and substrate loading (range 1–3 g COD/L) on the specific conversion and hydrogen production rate has shown interaction behaviour between the two independent variables. Highest conversion of 10.3 mL H₂/(g COD/L) was achieved at pH of 6.1 and glucose concentration of 3 g COD/L, whereas the highest production rate of 71 mL H₂/(h L) was measured at pH 6.3 and substrate loading of 2.5 g COD/L. In general, there appears to be a strong trend of increasing hydrogen production rate with an increase in both substrate concentration and pH. Butyrate (14–63%), formate (10–45%) and ethanol (16–40%) were the main soluble products with other volatile fatty acids and alcohols present in smaller quantities.     

Selection and isolation of hydrogen-producing fermentative bacteria with high yield and rate and its bioaugmentation process

One of the most effective approaches is to obtain bacteria with high hydrogen-producing capacity and introduce into hydrogen production process in order to perform utmost efficiency. It was proved by analysis of 16S rDNA sequence that it, R3, possibly belongs to a novel genus. The hydrogen production varies depending on glucose concentration and pH respectively. It was found that the optimal glucose concentration is 12.0 g/l and the optimal initial pH is about 5.5. Glucose degradation rate of R3 conforms to Michaelis–Menten formula. There are three stages to actualize bioaugmentation which are cultivated hydrogen-producing fermentative bacteria with high yield and rate, actualize maximum bearing capacity and high physiological activity of hydrogen-producing bacterium added in the system for improving a special performance in the mixed system. It is very important for bioaugmentation application to the biological hydrogen-producing system in order to further enhance hydrogen-producing capacity of the system and stability of the system operation.     

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.     

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.     

Acidophilic biohydrogen production from rice slurry

Batch experiment results showed that hydrogen production from rice slurry was found most effective at pH 4.5, 37 °C treating a slurry containing 5.5 g-carbohydrate/L. An anaerobic digester sludge was used as seed after a 100 °C heat treatment for 30 min. After a 36 h acclimation period, the sludge had a maximum specific hydrogen production rate of 2.1 L/(g-VSS d) and a hydrogen yield of 346 mL/g-carbohydrate, corresponding to 62.6% of stoichiometric yield. The effluent was composed mostly of acetate (28.3–43.0%) and butyrate (51.4–70.9%). Based on the 16S rDNA analysis, the 28 clones developed from this acidophilic hydrogen-producing sludge may be classified into nine OTUs, all of which are affiliated with the genus Clostridium. Phylogenetic analysis shows that eight OTUs (96.4% of population) form a distinct group with Clostridium sp. 44a-T5zd. Results indicate the acidophilic hydrogen-producing bacteria found in this study are unknown, and warrant further studies.     

Control strategies for hydrogen production through co-culture of Ethanoligenens harbinense B49 and immobilized Rhodopseudomonas faecalis RLD-53

This study evaluated hydrogen production by co-culture of Ethanoligenens harbinense B49 and immobilized Rhodopseudomonas faecalis RLD-53 with different control strategies. To enhance cooperation of dark and photo-fermentation bacteria during hydrogen production process, the glucose concentration, phosphate buffer concentration and initial pH were controlled at 6 g/l, 50 mmol/l and 7.5, respectively. The maximum yield and rate of hydrogen production were 3.10 mol H₂/mol glucose and 17.2 mmol H₂/l/h, respectively. Ethanol from E. harbinense B49 in acetate medium can enhance hydrogen production by R. faecalis RLD-53 except the ratio of ethanol to acetate (R_E/A) among 0.8 to 1.0. Control of the proper phosphate buffer concentration (50 mmol/l) not only increased acetic acid production by E. harbinense B49, but also maintained stable pH of co-culture system. Therefore, the results showed that co-culture of E. harbinense B49 and immobilized R. faecalis RLD-53 was a promising way of converting glucose into hydrogen.     

Characteristics of fermentative hydrogen production with Klebsiella pneumoniae ECU-15 isolated from anaerobic sewage sludge

Klebsiella pneumoniae ECU-15 (EU360791), which was isolated from anaerobic sewage sludge, was investigated in this paper for its characteristics of fermentative hydrogen production. It was found that the anaerobic condition favored hydrogen production than that of the micro-aerobic condition. Culture temperature and pH of 37 °C and 6.0 were the most favorable for the hydrogen production. The strain could grow in several kinds of monosaccharide and disaccharide, as well as the complicated corn stalk hydrolysate, with the best results exhibited in glucose. The maximum hydrogen production rate and yield of 482 ml/l/h and 2.07 mol/mol glucose were obtained at initial glucose concentration of 30 g/L and 5 g/L, respectively. Fermentation results in the diluent corn stalk hydrolysate showed that cell growth was not inhibited. However, the hydrogen production of 0.65 V/V was relatively lower than that of the glucose (1.11 V/V), which was mainly due to the interaction between xylose and glucose.     

The effect of Ni,Fe and Mg concentration on photo-hydrogen production by Rhodopseudomonas faecalis RLD-53

In the present study, the effect of Ni²⁺ (0–10 μmol/l), Fe²⁺ (0–200 μmol/l) and Mg²⁺ (0–15 mmol/l) concentration on photo-hydrogen production from acetate was investigated by batch culture. Results showed that under a proper concentration range, Ni²⁺ was able to enhance the hydrogen production rate and the hydrogen yield; Fe²⁺ was able to increase the hydrogen yield, and hydrogen production rate was enhanced only when the culturing time was 24–72 h. Ni²⁺ and Fe²⁺ at a higher concentration inhibited cell growth. When Ni²⁺ and Fe²⁺ concentrations were 4 μmol/l and 80 μmol/l, respectively, maximal hydrogen yield of 2.87 and 2.78 mol H₂/mol acetate was obtained when batch culturing at 35 °C with initial pH 7.0. Mg²⁺ did not significantly affect hydrogen production and hydrogen yield which maintained at about 2.45 mol H₂/mol acetate, but it was favorable to cell growth.     

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

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

Biological hydrogen production of the genus Clostridium: Metabolic study and mathematical model simulation

The biochemical hydrogen potential (BHP) tests were conducted to investigate the metabolism of glucose fermentation and hydrogen production performance of four Clostridial species, including C. acetobutylicum M121, C. butyricum ATCC19398, C. tyrobutyricum FYa102, and C. beijerinckii L9. Batch experiments showed that all the tested strains fermented glucose, reduced medium pH from 7.2 to a value between 4.6 and 5.0, and produced butyrate (0.37–0.67 mmol/mmol-glucose) and acetate (0.34–0.42 mmol/mmol-glucose) as primary soluble metabolites. Meanwhile, a significant amount of hydrogen gas was produced accompanied with glucose degradation and acid production. Among the strains examined, C. beijerinckii L9 had the highest hydrogen production yield of 2.81 mmol/mmol-glucose. A kinetic model was developed to evaluate the metabolism of glucose fermentation of those Clostridium species in the batch cultures. The model, in general, was able to accurately describe the profile of glucose degradation as well as production of biomass, butyrate, acetate, ethanol, and hydrogen observed in the batch tests. In the glucose re-feeding experiments, the C. tyrobutyricum FYa102 and C. beijerinckii L9 isolates fermented additional glucose during re-feeding tests, producing a substantial amount of hydrogen. In contrast, C. butyricum ATCC19398 was unable to produce more hydrogen despite additional supply of glucose, presumably due to the metabolic shift from acetate/butyrate to lactate/ethanol production.     

Biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent

A hydrogen producer was successfully isolated from anaerobic digested palm oil mill effluent (POME) sludge. The strain, designated as Clostridium butyricum EB6, efficiently produced hydrogen concurrently with cell growth. A controlled study was done on a synthetic medium at an initial pH value of 6.0 with 10 g/L glucose with the maximum hydrogen production at 948 mL H₂/L-medium and the volumetric hydrogen production rate at 172 mL H₂/L-medium/h. The supplementation of yeast extract was shown to have a significant effect with a maximum hydrogen production of 992 mL H₂/L-medium at 4 g/L of yeast extract added. The effect of pH on hydrogen production from POME was investigated. Experimental results showed that the optimum hydrogen production ability occurred at pH 5.5. The maximum hydrogen production and maximum volumetric hydrogen production rate were at 3195 mL H₂/L-medium and 1034 mL H₂/L-medium/h, respectively. The hydrogen content in the biogas produced was in the range of 60–70%.     

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.     

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 thermophilic fermentative hydrogen production by the newly isolated Caloranaerobacter azorensis H53214 from deep-sea hydrothermal vent environment

A unique thermophilic fermentative hydrogen-producing strain H53214 was isolated from a deep-sea hydrothermal vent environment, and identified as Caloranaerobacter azorensis based on bacterial 16S rRNA gene analysis. The optimum culture condition for hydrogen production by the bacterium, designated C. azorensis H53214, was investigated by the response surface methodology (RSM). Eight variables including the concentration of NaCl, glucose, yeast, tryptone, FeSO₄ and MgSO₄, initial pH and incubation temperature were screened based on the Plackett–Burman design. The results showed that initial pH, tryptone and yeast were significant variables, which were further optimized using the steepest ascent method and Box–Behnken design. The optimal culture conditions for hydrogen production were an initial pH of 7.7, 8.3 g L⁻¹ tryptone and 7.9 g L⁻¹ yeast. Under these conditions, the maximum cumulative hydrogen volume, hydrogen yield and maximum H₂ production rate were 1.58 L H₂ L⁻¹ medium, 1.46 mol H₂ mol⁻¹ glucose and 25.7 mmol H₂ g⁻¹ cell dry weight (CDW) h⁻¹, respectively. By comparison analysis, strain H53214 was superior to the most thermophilic hydrogen producers because of the high hydrogen production rate. In addition, the isolation of C. azorensis H53214 indicated the deep-sea hydrothermal environment might be a potential source for fermentative hydrogen-producing thermophiles.     

Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species

In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 °C and pH 7.5, giving a yield of 0.46–0.53 g reducing sugar/g starch. Five H₂-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H₂ from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H₂ production rate of 116 and 118 ml/g VSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H₂ yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H₂/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H₂ production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H₂ production rate increased from 250 to 534 ml/g  VSS/h, whereas the H₂ yield decreased from 2.03 to 1.50  mol H₂/mol glucose. While operating at 2 h HRT, the volumetric H₂ production rate reached a high level of 1.5 l/h/l.