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.     

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.     

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

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

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).     

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.     

Simultaneous hydrogen and ethanol production from a mixture of glucose and xylose using extreme thermophiles II: Effect of hydraulic retention time

In this study, biofuels (hydrogen and ethanol) fermentation from glucose and xylose by extreme thermophiles in an Up-flow Anaerobic Sludge Bed (UASB) reactor was successfully demonstrated. Autoclaved methanogenic granules were used as carriers for the extreme thermophiles. High yields of hydrogen and ethanol were achieved at various HRTs from 24 h to 6 h. The highest hydrogen production rate of 121 ± 23 mL/(L h) and highest ethanol production rate of 6.7 ± 1.2 mmol/(L h) were observed at HRT = 12 h. The highest simultaneous hydrogen and ethanol yields were 0.58 ± 0.11 mol H₂/(mol hexose) and 0.72 ± 0.13 mol ethanol/(mol hexose), reaching a total energy yield of 1151 kJ/mol hexose. The substrate conversion efficiency was maintained over 90% at three HRTs (24, 18, and 12 h).     

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).     

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.     

Direct fermentation of Laminaria japonica for biohydrogen production by anaerobic mixed cultures

A few studies have been made on fermentative hydrogen production from marine algae, despite of their advantages compared with other biomass substrates. In this study, fermentative hydrogen production from Laminaria japonica (one brown algae species) was investigated under mesophilic condition (35 ± 1 °C) without any pretreatment method. A feasibility test was first conducted through a series of batch cultivations, and 0.92 mol H₂/mol hexose_added, or 71.4 ml H₂/g TS of hydrogen yield was achieved at a substrate concentration of 20 g COD/L (based on carbohydrate), initial pH of 7.5, and cultivation pH of 5.5. Continuous operation for a period of 80 days was then carried out using anaerobic sequencing batch reactor (ASBR) with a hydraulic retention time (HRT) of 6 days. After operation for approximately 30 days, a stable hydrogen yield of 0.79 ± 0.03 mol H₂/mol hexose_added was obtained. To optimize bioenergy recovery from L. japonica, an up-flow anaerobic sludge blanket reactor (UASBr) was applied to treat hydrogen fermentation effluent (HFE) for methane production. A maximum methane yield of 309 ± 12 ml CH₄/g COD was achieved during the 90 days operation period, where the organic loading rate (OLR) was 3.5 g COD/L/d.     

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.     

Dark fermentative hydrogen production from xylose by a hot spring enrichment culture

Dark fermentative hydrogen production by a hot spring culture was studied from different sugars in batch assays and from xylose in continuous stirred tank reactor (CSTR) with on-line pH control. Batch assays yielded hydrogen in following order: xylose > arabinose > ribose > glucose. The highest hydrogen yield in batch assays was 0.71 mol H₂/mol xylose. In CSTR the highest H₂ yield and production rate at 45 °C were 1.97 mol H₂/mol xylose and 7.3 mmol H₂/h/L, respectively, and at 37 °C, 1.18 mol H₂/mol xylose and 1.7 mmol H₂/h/L, respectively. At 45 °C, microbial community consisted of only two bacterial strains affiliated to Clostridium acetobutulyticum and Citrobacter freundii, whereas at 37 °C six Clostridial species were detected. In summary hydrogen yield by hot spring culture was higher with pentoses than hexoses. The highest H₂ production rate and yield and thus, the most efficient hydrogen producing bacteria were obtained at suboptimal temperature of 45 °C for both mesophiles and thermophiles.     

Exogenous anthrahydroquinone-2,6-disulfonate specifically increases xylose utilization during mixed sugar fermentation by Clostridium beijerinckii NCIMB 8052

The influence of a reduced extracellular electron shuttle (anthrahydroquinone-2,6-disulfonate, AH₂QDS) on substrate utilization and H₂ production was investigated at different glucose:xylose ratios using the fermentative culture Clostridium beijerinckii NCIMB 8052. Adding 250 μM AH₂QDS increased the total substrate utilization by 23–66%, and specifically xylose utilization by 20–54% at glucose:xylose ratios of 1:1, 1:3 and 1:9. Adding 250 μM AH₂QDS also increased the substrate utilization by 40–88% at all tested glucose:xylose ratios (which ranged from 1:9 to 9:1). Increasing AH₂QDS concentrations from 250 μM to 2 mM increased xylose utilization (>99% xylose consumed) as well as cumulative hydrogen production during mixed sugar fermentation. The extent of glucose utilization was consistent amongst all tests, which was expected. However, hydroquinone amendment specifically increased the extent of xylose utilized irrespective of the starting glucose:xylose ratio. To the best of our knowledge this is the first report of 100% xylose utilization by C. beijerinckii NCIMB 8052 grown under mixed sugar conditions. These data demonstrate that the substrate utilization, particularly xylose utilization, can be manipulated by amending extracellular redox active compounds. This will improve pentose utilization during fermentation of hydrolyzates that result from pre-treatment of lignocellulosic materials with wild type (non-GMO) cultures.     

Influence of linoleic acid,pH and HRT on anaerobic microbial populations and metabolic shifts in ASBRs during dark hydrogen fermentation of lignocellulosic sugars

In this study, controlling an anaerobic microbial community to increase the hydrogen (H₂) yield during the degradation of lignocelluosic sugars was accomplished by adding linoleic acid (LA) at low pH and reducing the hydraulic retention time (HRT) of an anaerobic sequencing batch reactor (ASBR). At pH 5.5 and a 1.7 d HRT, the maximum H₂ yield for LA treated cultures fed glucose or xylose reached 2.89 ± 0.18 mol mol⁻¹ and 1.94 ± 0.17 mol mol⁻¹, respectively. The major soluble metabolites at pH 5.5 with a 1.7 day HRT differed between the control and LA treated cultures. A metabolic shift toward H₂ production resulted in increased hydrogenase activity in both the xylose (13%) and glucose (34%) fed LA treated cultures relative to the controls. In addition, the Clostridia population and the H₂ yield were elevated in cultures treated with LA. A flux balance analysis for the LA treated cultures showed a reduction in homoacetogenic activity which was associated with reducing the Bacteriodes levels from 12% to 5% in the glucose fed cultures and 16% to 10% in the xylose fed cultures. Strategies for controlling the homoacetogens and optimal hydrogen production from glucose and xylose are proposed.     

Anthrahydroquinone-2,6-disulfonate increases the rate of hydrogen production during Clostridium beijerinckii fermentation with glucose,xylose, and cellobiose

Fermentative hydrogen production is considered a reasonable alternative for generating H₂ as an energy carrier for electricity production using hydrogen fuel cells. The kinetics of hydrogen production from glucose, xylose and cellobiose were investigated using pure culture Clostridium beijerinckii NCIMB 8052. Adding anthrahydroquinone-2,6-disulfonate (AH₂QDS) at concentrations ranging from 100 μM to 500 μM increased the hydrogen production rates from 0.80 to 1.35 mmol/L-hr to 1.20–2.70 mmol/L-hr with glucose, xylose, or cellobiose as the primary substrates. AH₂QDS amendment also increased the substrate utilization rate and biomass growth rate by at least two times. These findings suggest that adding hydroquinone reducing equivalents influence cellular metabolism with hydrogen production rate, substrate utilization rate, and growth rate being simultaneously affected. Resting cell suspensions were conducted to investigate the influence of AH₂QDS on the hydrogen production rate from glyceraldehyde 3-phosphate, which is a shared intermediate in both glycolysis and pentose phosphate pathway. Data demonstrated that hydrogen production rate increased by 1.5 times when glyceraldehyde 3-phosphate was the sole carbon source, suggesting that the hydroquinone may alter reactions starting with or after glyceraldehyde 3-phosphate in central metabolism. These data demonstrate that adding hydroquinones increased overall metabolic activity of C. beijerinckii. This will eventually increase the efficiency of industrial scale production once appropriate hydroquinone equivalents are identified that work well in large-scale operations, since fermentation rate is one of the two critical factors (production rate and yield) influencing efficiency and cost.     

Characterization on hydrogen production performance of a newly isolated Clostridium beijerinckii YA001 using xylose

A mesophilic bacterium was isolated from cow manure, and identified as Clostridium beijerinckii YA001 based on 16S rRNA gene sequence calculation using MEGA 5.0 and biochemical tests. This strain showed the ability to digest a wide range of carbon and nitrogen sources for hydrogen production, and it showed high hydrogen production performance using xylose as substrate. The optimum parameters for bio-hydrogen production in batch tests were pH 8.0, 1% substrate concentration, 40 °C and yeast extract as nitrogen source. The maximum hydrogen yield and the hydrogen production rate were obtained at 2.31 mol/mol xylose and 311.3 mL H₂/(Lh), respectively. These results indicate that C. beijerinckii YA001 is an ideal candidate for fermentative 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 from Enterobacter cloacae IIT-BT 08 using distillery effluent

Distillery effluent poses severe environmental pollution problem mainly due to its high organic content. During alcohol fermentation, most of the essential macro- and micro-nutrients get utilized. Therefore, supplementation of these nutrients becomes imperative for the improvement of biohydrogen production. In the present study, starch based distillery effluent was used for dark fermentative hydrogen production using Enterobacter cloacae IIT-BT 08. Hence, this study was undertaken to evaluate the effect of supplementation of yeast extract, malt extract, Fe⁺⁺, Cu⁺⁺ and Mg⁺⁺ on biohydrogen production. The interaction among supplements and their mutual effect on the hydrogen production was studied using five factor–five level central composite design). Optimum hydrogen yield of 7.4 mol H₂/kg COD_reduced was predicted by the model, which showed an excellent correlation with experimental hydrogen yield of 7.38 ± 0.24 mol H₂/kg COD_reduced. An average hydrogen production rate of 80 mL/L h was achieved after supplementation, having 2.2 times higher hydrogen yield as compared to non-supplemented distillery effluent.     

Simultaneous hydrogen and ethanol production from a mixture of glucose and xylose using extreme thermophiles I: Effect of substrate and pH

The simultaneous hydrogen and ethanol production from glucose and xylose was investigated. The effect of carbon sources on hydrogen and ethanol production was examined in batches. When the substrate concentration was increased from 1 g/L to 7 g/L, the hydrogen yield decreased from 0.74 mol/mol to 0.15 mol/mol and from 0.67 mol/mol to 0.07 mol/mol for glucose and xylose. The highest ethanol yield of 1.19 mol/(mol·glucose) was obtained at 5 g/L glucose and 6 g/L xylose concentrations. For the co-fermentation of glucose and xylose, the highest ethanol yield 1.54 mol/(mol·hexose) was obtained at 2.5 g/L glucose to 2.5 g/L xylose (1:1). However, the hydrogen yield was not significantly affected by the glucose to xylose ratio. Continuous co-fermentation of glucose and xylose by extreme thermophiles was successfully demonstrated using an upflow anaerobic reactor. The hydrogen production rate, the ethanol concentration, and the substrate degradation efficiency increased along with pH. The optimal pH for the continuous mode was determined to be in the range of 5.8–6.6.     

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 acid and enzymatic oat straw hydrolysates in an anaerobic sequencing batch reactor: Performance and microbial population analysis

Feasibility of hydrogen production from acid and enzymatic oat straw hydrolysates was evaluated in an anaerobic sequencing batch reactor at 35 °C and constant substrate concentration (5 g chemical oxygen demand/L). In a first experiment, hydrogen production was replaced by methane production. Selective pressures applied in a second experiment successfully prevented methane production. During this experiment, initial feeding with glucose/xylose, as model substrates, promoted biomass granulation. Also, the highest hydrogen molar yield (HMY, 2 mol H₂/mol sugar consumed) and hydrogen production rate (HPR, 278 mL H₂/L-h) were obtained with these model substrates. Gradual substitution of glucose/xylose by acid hydrolysate led to disaggregation of granules and lower HPR and HMY. When the model substrates were completely substituted by enzymatic hydrolysate, the HMY and HPR were 0.81 mol H₂/mol sugar consumed and 29.6 mL H₂/L-h, respectively. Molecular analysis revealed a low bacterial diversity in the stages with high hydrogen production and vice versa. Furthermore, Clostridium pasteurianum was identified as the most abundant species in stages with a high hydrogen production. Despite that feasibility of hydrogen production from hydrolysates was demonstrated, lower performance from hydrolysates than from model substrates was obtained.