Fermentative hydrogen production from indigenous mesophilic strain Bacillus anthracis PUNAJAN 1 newly isolated from palm oil mill effluent

In the present study, a new mesophilic bacterial strain, identified as Bacillus anthracis strain PUNAJAN 1 was isolated from palm oil mill effluent (POME) sludge, and tested for its hydrogen production ability. Effect of physico-chemical factors such as temperature, initial pH, nitrogen source and carbon sources were investigated in order to determine the optimal conditions for hydrogen production. The maximum hydrogen yield of 2.42 mol H₂/mol mannose was obtained at 35 °C and initial pH of 6.5. Yeast and mannose were used as the main carbon and nitrogen sources respectively in the course of the hydrogen production. Apart from synthetic substrate, specific hydrogen production potentials of the strain using POME was calculated and found to be 236 ml H₂/g chemical oxygen demand (COD). The findings of this study demonstrate that the indigenous strain PUNAJAN 1 could be a potential candidate for hydrogen using POME as substrate.     

Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3

A hydrogen producing strain newly isolated from anaerobic sludge in an anaerobic bioreactor, was identified as Clostridium beijerinckii Fanp3 by 16S rDNA gene sequence analysis and detection by BioMerieux Vitek. The strain could utilize various carbon and nitrogen sources to produce hydrogen, which indicates that it has the potential of converting renewable wastes into hydrogen. In batch cultivations, the optimal initial pH of the culture medium was between 6.47 and 6.98. Using 0.15 M phosphate as buffer could alleviate the medium acidification and improve the overall performance of C. beijerinckii Fanp3 in hydrogen production. Culture temperature of 35 °C was established to be the most favorable for maximum rate of hydrogen production. The distribution of soluble metabolic products (SMP) was also greatly affected by temperature. Considering glucose as a substrate, the activation energy (E_a) for hydrogen production was calculated as 81.01 kcal/mol and 21.4% of substrate energy was recovered in the form of hydrogen. The maximal hydrogen yield and the hydrogen production rate were obtained as 2.52 mol/mol-glucose and 39.0 ml/g-glucose h⁻¹, respectively. These results indicate that C. beijerinckii Fanp3 is an ideal candidate for the fermentative hydrogen production.     

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.     

Evaluating biohydrogen production by Clostridium hydrogenum sp. nov. strain CUEA01 isolated from mangrove sediments in Thailand

The hydrogen (H₂) fermentative Clostridium hydrogenum sp. nov. strain CUEA01 was isolated from a mangrove sediment in Thailand. Genome sequencing and analysis revealed a genome size of 5,501,482 bp that encoded for 3,292 predicted protein coding genes with annotated functional assignments and many genes associated with carbon utilization and H₂ evolution. The H₂ production performance was evaluated in batch fermentation, and revealed that this strain can grow and produce H₂ at a broad range of temperatures (15–40 °C), pH (4–10), and initial glucose concentrations (5–60 g/L). The maximum H₂ yield (3.11 mol_H2/mol_glucose) was obtained at 37 °C, pH 8, and an initial glucose concentration of 10 g/L. Furthermore, this strain could utilize various carbon sources, including xylose, xylan, starch, mannose, glycerol, and avicel cellulose, amongst others. Additionally, CUEA01 was compatible with agro-industrial wastes and could achieve a maximum CHP of 4639 mL/L and 4024 mL/L from sugarcane molasses and cassava pulp, respectively. This demonstrates that CUEA01 has a potential for H₂ fermentation from complex organic wastes since it can secrete enzyme cocktails that consolidate the fermentation process.     

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).     

Fermentative hydrogen production from different sugars by Citrobacter sp. CMC-1 in batch culture

In this study, production of hydrogen (H₂) from glucose, xylose, galactose, mannose, arabinose and rhamnose by a strain isolated from activated sludge was investigated. The strain, named as Citrobacter sp. CMC-1, was enriched in cellobiose amended minimal media. Based on 16S rRNA sequence, the CMC-1 strain is a close relative of Citrobacter amalonaticus strain SA01 (99%). Optimal cultivation parameters for H₂ production and growth such as pH and temperature were investigated. H₂ yields from glucose at optimal conditions (pH 6.0 and 34 °C) were 1.82 ± 0.02 mol-H₂/mol-glucose. Strain CMC-1 fermented galactose, mannose, xylose, arabinose and rhamnose. After 48 h incubation, the strain CMC-1 completely fermented all sugars tested, except arabinose. Increase in fermentation period lowered residual formate level in the media and improved H₂ production for galactose, mannose and xylose (1.68 ± 0.24, 1.93 ± 0.14 and 1.63 ± 0.07 mol-H₂/mol-substrate respectively).     

Fermentative hydrogen production from cassava stillage by mixed anaerobic microflora: Effects of temperature and pH

Fermentative hydrogen production from cassava stillage was conducted to investigate the influences of temperature (37 °C, 60 °C, 70 °C) and initial pH (4–10) in batch experiments. Although the seed sludge was mesophilic anaerobic sludge, maximum hydrogen yield (53.8 ml H₂/gVS) was obtained under thermophilic condition (60 °C), 53.5% and 198% higher than the values under mesophilic (37 °C) and extreme-thermophilic (70 °C) conditions respectively. The difference was mainly due to the different VFA and ethanol distributions. Higher hydrogen production corresponded with higher ratios of butyrate/acetate and butyrate/propionate. Similar hydrogen yields of 66.3 and 67.8 ml H₂/gVS were obtained at initial pH 5 and 6 respectively under thermophilic condition. The total amount of VFA and ethanol increased from 3536 to 7899 mg/l with the increase of initial pH from 4 to 10. Initial pH 6 was considered as the optimal pH due to its 19% higher total VFA and ethanol concentration than that of pH 5. Homoacetogenesis and methonogenesis were very dependent on the initial pH and temperature even when the inoculum was heat-pretreated. Moreover, a difference between measured and theoretical hydrogen was observed in this study, which could be attributed to homoacetogenesis, methanogenesis and the degradation of protein.     

Fermentative biohydrogen production by a new chemoheterotrophic bacterium Citrobacter sp. Y19

A newly isolated Citrobacter sp. Y19 for CO-dependent H₂ production was studied for its capability of fermentative H₂ production in batch cultivation. When glucose was used as carbon source, the pH of the culture medium significantly decreased as fermentation proceeded and H₂ production was seriously inhibited. The use of fortified phosphate at 60–180 mM alleviated this inhibition. By increasing culture temperatures (25–36°C), faster cell growth and higher initial H₂ production rates were observed but final H₂ production and yield were almost constant irrespective of temperature. Optimal specific H₂ production activity was observed at 36°C and pH 6–7. The increase of glucose concentration (1–20 g/l) in the culture medium resulted in higher H₂ production, but the yield of H₂ production (mol H₂/mol glucose) gradually decreased with increasing glucose concentration. Carbon mass balance showed that, in addition to cell mass, ethanol, acetate and CO₂ were the major fermentation products and comprised more than 70% of the carbon consumed. The maximal H₂ yield and H₂ production rate were estimated to be 2.49 molH₂/mol glucose and 32.3 mmolH₂/gcellh, respectively. The overall performance of Y19 in fermentative H₂ production is quite similar to that of most H₂-producing bacteria previously studied, especially to that of Rhodopseudomonas palustris P4, and this indicates that the attempt to find an outstanding bacterial strain for fermentative H₂ production might be very difficult if not impossible.     

Effect of cornstalk hydrolysis on photo-fermentative hydrogen production by R. capsulatus

Cornstalk is a typical cellulose material, which can be used by photo-fermentative H₂ production after pretreatment. However, the pretreatment methods have different influence on photo fermentation. In this study, 25.0 g cornstalk was pretreated by HCl/NaOH/cellusase. The hydrolysis rates increased from 45.51% by ddH₂O-treatment to 60.79% by diluted HCl-treatment and 51.6% by NaOH-treatment. The corresponding reducing sugar yields were 0.13 g/g, 0.42 g/g and 0.01 g/g, respectively. Enzymatic treatment enhanced the corresponding cornstalk hydrolysis rates to 50.81%, 67.60% and 64.10% with reducing sugar yields of 0.22 g/g, 0.62 g/g and 0.26 g/g. The sorts and concentrations of carbon source for H₂ production vary among different hydrolysates. Photo-fermentative H₂ production of strain R. capsulatus JL1 and mutant JL1601 (cheR2^-) with hydrolysates were investigated. The maximum H₂ yield of 123.8 ± 14.2 mL/g by strain JL1 was obtained from alkali-enzyme pretreated cornstalk, while the H₂ yield of 224.9 ± 5.2 mL/g by mutant JL1601 (cheR2^-) was obtained with acid-enzyme hydrolysate as the substrates. Meanwhile, the alkali pretreated cornstalk was the worst for photo-fermentation of both strain JL1 and mutant JL1601 (cheR2^-). Nevertheless, the highest substrate conversion efficiencies for both strains were obtained from ddH₂O-pretreated hydrolysate. Two-step pretreated hydrolysates were more beneficial to H₂ production for mutant JL1601 (cheR2^-) but not for strain JL1.     

Optimization of conditions for hydrogen production from complex dairy wastewater by anaerobic sludge using desirability function approach

Present study deals with the multiple-response optimization for biohydrogen production using anaerobic sludge and outstanding approach to overcome the drawbacks of conventional response surface methodology (RSM). Dairy wastewater was used as source in batch fermentation was followed for this study. Response surface methodology (RSM), based on a three level, four variable Box–Behnken design, was employed to obtain the best possible combination of substrate concentration, pH, COD/N ratio and COD/P ratio for maximum H₂ yield (HY) and specific hydrogen production rate (SHPR). Experimental data were evaluated by applying RSM integrating a desirability function approach. The optimum H₂ yield and SHPR conditions were: substrate concentration 15.3 g COD/L, pH 5.5, COD/N ratio 100.5 and COD/P ratio 120 with maximum overall desirability D of 0.94. The confirmation experiment under these optimal condition showed a HY and SHPR of 13.54 mmol H₂/g COD and 29.91 mmol H₂/g-VSS.d, respectively. This was only 0.22% and 0.20%, respectively, different from the predicted values, suggesting that the desirability function approach with RSM was a useful technique to get the maximum H₂ yield and SHPR simultaneously.     

Fermentative hydrogen production by two novel strains of Enterobacter aerogenes HGN-2 and HT 34 isolated from sea buried crude oil pipelines

Present study investigated fermentative hydrogen production of two novel isolates of Enterobacter aerogenes HGN-2 and HT 34 isolated from oil water mixtures. The two isolates were identified as novel strains of E. aerogenes based on 16S rRNA gene. The batch fermentations of two strains from glucose and xylose were carried out using economical culture medium under various conditions such as temperature, initial pH, NaCl, Ni⁺/Fe⁺⁺, substrate concentrations for enhanced fermentation process. Both the strains favoured wide range of pH (6.5–8.0) at 37 °C for optimum production (2.20–2.23 mol H₂/mol-glucose), which occurred through acetate/butyrate pathway. At 55 °C, both strains favoured 6.0–6.5 and acetate type fermentation was predominant in HT 34. Hydrogen production by HT 34 from xylose was highly pH dependant and optimum production was at pH 6.5 (circa 1.98 mol-H₂/mol-xylose) through acetate pathway. The efficiency of the strain HGN-2 at pH 6.5 was 1.92–1.94 mol-H₂/mol-xylose, and displayed both acetate and butyrate pathways. At 55 °C, very low hydrogen production was detected (less than 0.5 m mol/mol-xylose).     

Impact of cultural conditions on photoproduction of hydrogen by Allochromatium sp. GSKRLMBKU-01 isolated from marine water of Visakhapatnam

In the present study, photoproduction of hydrogen by Allochromatium sp. strain GSKRLMBKU-01 isolated from marine water was measured under different cultural conditions. Hydrogen production was measured by using a Gas chromatography using argon gas as a carrier. Among different carbon and nitrogen sources used, succinate induced maximum hydrogen (5.68 ± 0.27 mL) production by immobilized cells, while free cells recorded 4.24 ± 0.30 mL of hydrogen under anaerobic light conditions. Immobilization of cells resulted in increased production of hydrogen. Ammonium chloride promoted more amounts of hydrogen production (2.68 ± 0.29 mL) by free cells, whereas glycine enhanced the hydrogen production upto 4.82 ± 0.36 mL in immobilized cells. In formic acid and urea, less hydrogen production was observed in both free and immobilized cells compared to other sources. Cumulative hydrogen production by the bacterium was recorded with the progress in incubation period. Incubation period of 192 h, pH of 7.0 and temperature at 30 °C were found to be optimum for the maximum hydrogen production. Significance of the above results was discussed in light of existing literature.     

Start-up study of biohydrogen production from palm oil mill effluent in a lab-scale up-flow anaerobic sludge blanket fixed-film reactor

A start-up study of lab-scale up-flow anaerobic sludge blanket fixed-film reactor (UASFF) was conducted to produce biohydrogen from palm oil mill effluent (POME). The reactor was fed with POME at different hydraulic retention time (HRT) and organic loading rate (OLR) to obtain the optimum fermentation time for maximum hydrogen yield (HY). The results showed the HY, volumetric hydrogen production rate (VHPR), and COD removal of 0.5–1.1 L H₂/g COD_consumed, 1.98–4.1 L H₂ L^?1 day^?1, and 33.4–38.5%, respectively. The characteristic study on POME particles was analyzed by particle size distribution (PSD), Scanning electron microscopy (SEM), and Energy-dispersive X-ray spectroscopy (EDX). The microbial Shannon and Simpson diversity indices and Principal Component Analysis assessed the alpha and beta diversity, respectively. The results indicated the change of bacterial community diversity over the operation, in which Clostridium sensu stricto 1 and Lactobacillus species were contributed to hydrogen fermentation.     

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.     

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.     

Efficient hydrogen production from lignocellulosic feedstocks by a newly isolated thermophlic Thermoanaerobacterium sp. strain F6

Consolidated bioprocessing (CBP) is a promising approach for hydrogen production from lignocellulose owing to its lower cost and higher efficiency. In this study, the newly isolated theromphilic Thermoanaerobacterium sp. strain F6 exhibited the capability of direct utilization of various hemicellulosic and cellulosic materials for hydrogen production, including xylan, Avicel and filter paper etc. Especially, the maximum cumulative hydrogen production reached 370.7 mmoL/L from 60 g/L of xylan. In addition, natural lignocellulosic materials, such as corn cob and sugarcane bagasse without any hydrolytic pretreatment could also be directly utilized as the sole carbon source for hydrogen production. 1822.6 and 826.3 mL H₂/L of hydrogen were produced from corn cob and sugarcane bagasse, respectively. The high hydrogen production from cellulosic and hemicellulosic materials were both benefit from its efficient secretion of hydrolytic enzymes. Thus, Thermoanaerobacterium sp. strain F6 is a potential candidate for effective conversion of lignocellulose to hydrogen through CBP.     

Evaluation of hydrogen production from corn cob with the mesophilic bacterium Clostridium hydrogeniproducens HR-1

Corn cob is a promising hydrogen fermentation substrate, not only because of its abundant and low cost, but also because of its high cellulose and hemicellulose content. However, little information is available on the use of corn cob as a feedstock for hydrogen production. In this study, corn cob was hydrolyzed by cellulase after acid steam-explosion, alkali soaking, or steam-explosion pretreatment. The liquid products of pretreatment and the enzymatic hydrolysates were then used as carbon sources for hydrogen production by Clostridium hydrogeniproducens HR-1. Pretreatment followed by enzymatic hydrolysis yielded 720, 670, and 530 mg reducing sugars/g corn cob, and the hydrogen yield from corn cob reached 119, 100, and 83 ml H₂/g corn cob, which is 55.9%, 46.7%, and 38.8% of the theoretical hydrogen yield from corn cob using C. hydrogeniproducens HR-1, respectively.     

Effect of culture conditions on the kinetics of hydrogen production by photosynthetic bacteria in batch culture

Biochemical kinetic characteristics of photo-fermentative hydrogen production were experimentally and numerically investigated to optimize the photo-fermentation hydrogen-producing process in this work. It is found that a maximum specific growth rate of 0.26 h⁻¹ was achieved under the optimal conditions of illumination intensity 6000 lux, 30 °C culture temperature and pH 7.0 of culture medium. These experimental results also led to an empirical formula of the maximum specific microbial growth rate (μ_max) as a function of illumination intensity, pH and temperature. With the empirical formula, the modified Monod equation along with the kinetic equations for biomass growth, glucose consumption and hydrogen production is then developed to simulate the photofermentation hydrogen-producing process. The modeling results are in good agreements with the experimental data, indicating that the developed kinetic models are able to objectively describe the characteristics of hydrogen production by PSB under different culture conditions.     

Photofermentative production of hydrogen from organic acids by the purple sulfur bacterium Thiocapsa roseopersicina

A mutant strain of the anaerobic purple sulfur bacterium Thiocapsa roseopersicina, containing only nitrogenase as a functionally active enzyme for H₂ generation was utilized to study the production of H₂ from organic acids (acetate, pyruvate and succinate). Two types of potential substrates for H₂ production, thiosulfate and salts of various organic acids, were compared under photoheterotrophic growth conditions. Thiosulfate proved to be the preferred electron donor for T. roseopersicina; the consumption of organic acids became pronounced only following depletion of the thiosulfate supply. The system is suitable for the generation of H₂ from effluents of heterotrophic dark fermentation processes or waste streams rich in inorganic reduced sulfur compounds and/or simple organic acids.