Fermentative hydrogen production by a new mesophilic bacterium Clostridium sp. 6A-5 isolated from the sludge of a sugar mill

The fermentative hydrogen production capability of the newly isolated Clostridium sp. 6A-5 bacterium was studied in a batch cultivation experiment. Various culture conditions (temperature, initial pH, and glucose concentration) were evaluated for their effects on cell growth and hydrogen production (including the yield and rate) of Clostridium sp. 6A-5. Optimal cell growth was observed at 40 °C, initial pH 7.5–8, and glucose concentration 16–26 g/L. The optimal hydrogen yield was obtained at 43 °C, initial pH 8, and glucose concentration 10–16 g/L. Hydrogen began to evolve when cell growth entered the mid-exponential phase and reached the maximum production rate at the late exponential and stationary phases. The maximum hydrogen yield, and rate were 2727 mL/L, and 269.3 mL H₂/L h, respectively. These results indicate that Clostridium sp. 6A-5 is a good candidate for mesophilic fermentative hydrogen production.     

Hydrogen production from xylose by moderate thermophilic mixed cultures using granules and biofilm up-flow anaerobic reactors

Continuous H₂ production from xylose by granules and biofilm up-flow anaerobic reactor using moderate thermophilic mixed cultures was investigated. The maximum H₂ yield of 251 mL H₂/g-xylose with H₂production rate of 15.1 L H₂/L⋅d was obtained from granules reactor operating at the organic loading rate (OLR) of 60 g-xylose/L⋅d and hydraulic retention time (HRT) of 4 h. Meanwhile the highest H₂ production rate of 13.3 L H₂/L⋅d with an H₂ yield of 221 mL H₂/g–xylose was achieved from the biofilm reactor. Both reactors were dominated by Thermoanaerobacterium species with acetate and butyrate as main fermentation products. The microbial community of the biofilm reactor was composed of Thermoanaerobacterium species, while granules reactor was composed of Clostridium sp., Thermoanaerobacterium sp. and Caloramator sp. The granular reactor was more microbial diversity and more balance between economic efficiency in term of the hydrogen production rate and technical efficiency in term of hydrogen yield.     

Non-sterile bio-hydrogen fermentation from food waste in a continuous stirred tank reactor (CSTR): Performance and population analysis

Bio-hydrogen production from food waste by anaerobic mixed cultures was conducted in a continuous stirred tank reactor (CSTR). The hydraulic retention time (HRT) was optimized in order to maximize hydrogen yield (HY) and hydrogen production rate (HPR). The maximum hydrogen content (38.6%), HPR (379 mL H₂/L. d) and HY (261 mL H₂/g-VS_added) were achieved at the optimum HRT of 60 h. The major soluble metabolite products were butyric and acetic acids which indicated a butyrate-acetate type fermentation. Operation of CSTR at HRT 60 h could select hydrogen producing bacteria and eliminate lactic acid bacteria and acetogenic bacteria. The microbial community analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) revealed that the predominant hydrogen producer was Clostridium sp.     

Effects of substrate concentration and hydraulic retention time on hydrogen production from common reed by enriched mixed culture in continuous anaerobic bioreactor

This study aims to investigate the effect of substrate concentration and hydraulic retention time (HRT) on hydrogen production in a continuous anaerobic bioreactor from unhydrolyzed common reed (Phragmites australis) an invasive wetland and perennial grass. The bioreactor has capacity of 1 L and working volume of 600 mL. It was operated at pH 5.5, temperature at 37 °C, hydraulic retention time (HRT) 12 h, and variation of substrate concentration from 40, 50, and 60 g COD/L, respectively. Afterward, the HRT was then varied from 12, 8, to 4 h for checking the optimal biohydrogen production. Each condition was run until reach steady state on hydrogen production rate (HPR) which based on hydrogen percentage and daily volume. The results were obtained the peak of substrate concentration was at the 50 g COD/L with HRT 12 h, average HPR and H₂ concentration were 28.71 mL/L/h and 36.29%, respectively. The hydrogen yield was achieved at 106.23 mL H₂/g CODre. The substrate concentration was controlled at 50 g COD/L for the optimal HRT experiments. It was found that the maximum of average HPR and H₂ concentration were 43.28 mL/L/h and 36.96%, respectively peak at HRT 8 h with the corresponding hydrogen yield of 144.35 mL H₂/g CODre. Finally, this study successful produce hydrogen from unhydrolyzed common reed by enriched mixed culture in continuous anaerobic bioreactor.     

Exploring optimal conditions for thermophilic fermentative hydrogen production from cassava stillage

This study investigated the effects of seed sludges, alkalinity and HRT on the thermophilic fermentative hydrogen production from cassava stillage. Five different kinds of sludges were used as inocula without any pretreatment. Though batch experiments showed that mesophilic anaerobic sludge was the best inoculum, the hydrogen yields with different seed sludges were quite similar in continuous experiments in the range of 82.9–92.3 ml H₂/gVS without significant differences which could be attributed to the establishment of Uncultured Thermoanaerobacteriaceae bacterium-dominant microbial communities in all reactors. It is indicated that results obtained from batch experiments are not consistent with those from continuous experiments and all the tested seed sludges are good sources for continuous thermophilic hydrogen production from cassava stillage. The influent alkalinity of 6 g NaHCO₃/L and HRT 24 h were optimal for hydrogen production with hydrogen yield of 76 ml H₂/gVS and hydrogen production rate of 3215 ml H₂/L/d. Butyrate was the predominant metabolite in all experiments. With the increase in alkalinity of more than 6 g/L, the concentration of VFA/ethanol increased while hydrogen yield decreased due to the higher concentration of acetate and propionate. The decrease in HRT resulted in the higher hydrogen production rate but lower hydrogen yield. Variation of hydrogen yields were quite correlated with butyrate/acetate (B/A) ratio with different influent alkalinities, however, butyrate was important parameter to justify the hydrogen yields with various HRTs.     

Enhanced bio-hydrogen production by immobilized Clostridium sp. T2 on a new biological carrier

Biological mycelia pellets, which are formed spontaneously in the process of Aspergillus niger Y3 fermentation, were explored as carrier for immobilization of Clostridium sp. T2 to improve hydrogen production. Batch fermentation tests showed that optimal dosage and size of mycelia pellets for hydrogen production were 0.350 g 150 ml⁻¹ medium and 1.5 mm. Under these conditions, hydrogen production with immobilized cells on mycelia pellets was further investigated in continuous stirred-tank reactor (CSTR) with hydraulic retention time (HRT) ranging from 12 to 8 h. It obtained that the maximum hydrogen production rate reached 2.76 mmol H₂ L⁻¹ h⁻¹ at 10 h HRT, which was 40.8% higher than the carrier-free process, but slightly lower than the counterpart immobilized in sodium alginate with the value of 3.15 mmol H₂ L⁻¹ h⁻¹. SEM observation showed that abundant cells were closely adhered to mycelia pellets. The present results indicate the potential of using mycelia pellets as biological carrier for enhancing hydrogen production.     

Effect of Green synthesized silver oxide nanoparticle on biological hydrogen production

Investigation of the effect of different nanoparticles on dark fermentation is very popular nowadays. Among the nanoparticle production methods, the use of nanoparticles produced by the green synthesis method, which is more environmentally friendly, is important for a sustainable environment. This study investigated the effect of green synthesized silver oxide nanoparticles on bio-hydrogen yield. Nanoparticle synthesis was carried out by using Chlorella sp. microalgae as a reducing agent. SEM, EDX and UV–Visible spectrum analyzes were performed for the characterization of the synthesized nanoparticles. The synthesized silver nanoparticles have a uniform structure and an average particle size of 85 nm. Hydrogen production performance by Clostridium sp. was evaluated using different ratios of produced nanoparticles (100–600 μg/L). The addition of 400 μg/L nanoparticles increased the production of dark fermentative bio-hydrogen by 17% compared to the control group. The highest hydrogen yield was 2.44 mol H₂/mol glucose.     

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 by Escherichia coli ΔhycA ΔlacI using cheese whey as substrate

This study reports a fermentative hydrogen production by Escherichia coli using cheese whey as substrate. To improve the biohydrogen production, an E. coli ΔhycA ΔlacI strain (WDHL) was constructed. The absence of hycA and lacI genes had a positive effect on the biohydrogen production. The strain produced 22% more biohydrogen in a shorter time than the wild-type (WT) strain. A Box-Behnken experimental design was used to optimize pH, temperature and substrate concentration. The optimal initial conditions for biohydrogen production by WDHL strain were pH 7.5, 37 °C and 20 g/L of cheese whey. The specific production rate was improved from 3.29 mL H₂/optical density at 600 nm (OD_600nm) unit-h produced by WDHL under non-optimal conditions to 5.88 mL H₂/OD_600nm unit-h under optimal conditions. Using optimal initial conditions, galactose can be metabolized by WDHL strain. The maximum yield obtained was 2.74 mol H₂/mol lactose consumed, which is comparable with the yield reached in other hydrogen production processes with Clostridium sp. or mixed cultures.     

Optimization of key factors affecting hydrogen production from food waste by anaerobic mixed cultures

Key factors (inoculums concentration, substrate concentration and citrate buffer concentration) affecting hydrogen yield (HY) and specific hydrogen production rate (SHPR) from food waste in batch fermentation by anaerobic mixed cultures were optimized using Response Surface Methodology with Central Composite Design. The experiments were conducted in 120 ml serum bottles with a working volume of 70 mL. Under the optimal condition of 2.30 g-VSS/L of inoculums concentration, 2.54 g-VS/L of substrate concentration, and 0.11 M of citrate buffer concentration, the predicted maximum HY and SHPR of 104.79 mL H₂/g-VS_added and 16.90 mL H₂/g-VSS.h, respectively, were obtained. Concentrations of inoculums, substrate and citrate buffer all had an individual effect on HY and SHPR (P < 0.05). The substrate concentration and citrate buffer concentration had the greatest interactive effect on SHPR (P = 0.0075) while their effects on HY (P = 0.0131) were profound. These results were reproduced in confirmation experiments under optimal conditions and generated an HY of 104.58 mL H₂/g-VS_added and an SHPR of 16.86 mL H₂/g-VSS.h. This was only 0.20% and 0.24%, respectively, different from the predicted values. Microbial community analysis by PCR-DGGE indicated that Clostridium was the pre-dominant hydrogen producer at the optimum and worst conditions. The presence of Lactobacillus sp. and Enterococcus sp. might be responsible for the low HY and SHPR at the worst condition.     

Simultaneous saccharification and fermentation of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung

The objective of this study was to optimize the culture conditions for simultaneous saccharification and fermentation (SSF) of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung under thermophilic temperature. Carboxymethyl cellulose (CMC) was used as the model substrate. The investigated parameters included initial pH, temperature and substrate concentration. The experimental results showed that maximum hydrogen yield (HY) and hydrogen production rate (HPR) of 7.22 ± 0.62 mmol H₂/g CMC_added and 73.4 ± 3.8 mL H₂/L h, respectively, were achieved at an initial pH of 7.0, temperature of 55 °C and CMC concentration of 0.25 g/L. The optimum conditions were then used to produce hydrogen from the cellulose fraction of sugarcane bagasse (SCB) at a concentration of 0.40 g/L (equivalent to 0.25 g/L cellulose) in which an HY of 7.10 ± 3.22 mmol H₂/g cellulose_added. The pre-dominant hydrogen producers analyzed by polymerase chain reaction-denaturing gel gradient electrophoresis (PCR-DGGE) were Thermoanaerobacterium thermosaccharolyticum and Clostridium sp. The lower HY obtained when the cellulose fraction of SCB was used as the substrate might be due to the presence of lignin in the SCB as well as the presence of Lactobacillus parabuchneri and Lactobacillus rhamnosus in the hydrogen fermentation broth.     

Application of immobilized upflow anaerobic sludge blanket reactor using Clostridium LS2 for enhanced biohydrogen production and treatment efficiency of palm oil mill effluent

Polyethylene glycol (PEG) gel was used to immobilize hydrogen producing Clostridium LS2 bacteria for hydrogen production in an upflow anaerobic sludge blanket (UASB) reactor. The UASB reactor with a PEG-immobilized cell packing ratio of 10% weight to volume ratio (w/v) was optimal for dark hydrogen production. The performance of the UASB reactor fed with palm oil mill effluent (POME) as a carbon source was examined under various hydraulic retention time (HRT) and POME concentration. The best volumetric hydrogen production rate of 365 mL H₂/L/h (or 16.2 mmol/L/h) with a hydrogen yield of 0.38 L H₂/g COD_added was obtained at POME concentration of 30 g COD/L and HRT of 16 h. The average hydrogen content of biogas and COD reduction were 68% and 65%, respectively. The primary soluble metabolites were butyric acid and acetic acid with smaller quantities of other volatile fatty acid and alcohols formed during hydrogen fermentation. More importantly, the feasibility of PEG-immobilized cell UASB reactor for the enhancement of the dark-hydrogen production and treatment of wastewater is demonstrated.     

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).     

Biological hydrogen production from sweet sorghum syrup by mixed cultures using an anaerobic sequencing batch reactor (ASBR)

Continuous biological hydrogen production from sweet sorghum syrup by mixed cultures was investigated by using anaerobic sequencing batch reactor (ASBR). The ASBR was conducted based on the optimum condition obtained from batch experiment i.e. 25 g/L of total sugar concentration, 1.45 g/L of FeSO₄ and pH of 5.0. Feasibility of continuous hydrogen fermentation in ASBR operation at room temperature (30 ± 3 °C) with different hydraulic retention time (HRT) of 96, 48, 24 and 12 hr and cycle periods consisting of filling (20 min), settling (20 min), and decanting (20 min) phases was analyzed. Results showed that hydrogen content decreased with a reduction in HRT i.e. from 42.93% (96 hr HRT) to 21.06% (12 hr HRT). Decrease in HRT resulted in a decrease of solvents produced which was from 10.77 to 2.67 mg/L for acetone and 78.25 mg/L to zero for butanol at HRT of 96 hr-12 hr, respectively. HRT of 24 hr was the optimum condition for ASBR operation indicated by the maximum hydrogen yield of 0.68 mol H₂/mol hexose. The microbial determination in DGGE analysis indicated that the well-known hydrogen producers Clostridia species were dominant in the reacting step. The presence of Sporolactobacillus sp. which could excrete the bacteriocins causing the adverse effect on hydrogen-producing bacteria might responsible for the low hydrogen content obtained.     

Fermentative hydrogen production from starch using natural mixed cultures

Batch and continuous tests were conducted to evaluate fermentative hydrogen production from starch (at a concentration of chemical oxygen demand (COD) 20 g/L) at 35 °C by a natural mixed culture of paper mill wastewater treatment sludge. The optimal initial cultivation pH (tested range 5–7) and substrate concentration (tested range 5–60-gCOD/L) were evaluated by batch reactors while the effects of hydraulic retention time (HRT) on hydrogen production, as expressed by hydrogen yield (HY) and hydrogen production rate (HPR), were evaluated by continuous tests. The experimental results indicate that the initial cultivation pH markedly affected HY, maximum HPR, liquid fermentation product concentration and distribution, butyrate/acetate concentration ratio and metabolic pathway. The optimal initial cultivation pH was 5.5 with peak values of HY 1.1 mol-H₂/mol-hexose maximum HPR 10.4 mmol-H₂/L/h and butyrate concentration 7700 mg-COD/L. In continuous hydrogen fermentation, the optimal HRT was 4 h with peak HY of 1.5 mol-H₂/mol-hexose, peak HPR of 450 mmol-H₂/L/d and lowest butyrate concentration of 3000 mg-COD/L. The HPR obtained was 280% higher than reported values. A shift in dominant hydrogen-producing microbial population along with HRT variation was observed with Clostridium butyricum, C. pasteurianum, Klebshilla pneumoniae, Streptococcus sp., and Pseudomonas sp. being present at efficient hydrogen production at the HRTs of 4–6 h. Strategies based on the experimental results for optimal hydrogen production from starch are proposed.     

Biohydrogen production from organic solid waste in a sequencing batch reactor: An optimization of the hydraulic and solids retention time

Hydrogen production from organic solids waste was evaluated using a sequencing batch reactor (SBR) under mesophilic conditions, to investigate the effect of the hydraulic retention time (HRT) and solids retention time (SRT) on hydrogen production. The examined HRT and SRT values were from 4.6 to 27 h and 17–102 h, respectively. The results showed high hydrogen production rates (1.86 LH₂/L·d) and a yield of 127.26 mLH₂/gCOD_removed for an SRT of 60 h and an HRT of 16 h. The highest chemical oxygen demand (COD) removal (38.6 ± 6.9%) was also obtained under those conditions. The highest substrate hydrolysis percentage (73.0 ± 11.4%) was obtained at an HRT of 16 h and an SRT of 102 h. A short SRT of 20 h affected hydrogen production, which decreased up to 90%. With an SRT of 20 h and an HRT of 16 h, acetic acid-like fatty acids were mainly obtained. In experiments with a long SRT (60 h), the obtained fatty acid was butyrate. The conversion efficiencies for converting particulate material into fatty acids were 51–47% using a long SRT; a short HRT resulted in percentages of 37–40%. A 3D surface analysis was performed using the maximum hydrogen yield conditions as the central point, showing that the optimal hydrogen production can be obtained with an HRT of 16 h and an SRT of 55 h. Microbial analysis showed the predominance of the Olsenella genus at an HRT< 8 h and the presence of Clostridium at an HRT of 16 h. The HRT is the main parameter leading the community composition in the process.     

Magnetite nanoparticles enhanced glucose anaerobic fermentation for bio-hydrogen production using an expanded granular sludge bed (EGSB) reactor

The feasibility and efficiency of magnetite nanoparticles (Fe₃O₄NPs) enhanced bio-hydrogen production from glucose anaerobic fermentation were evaluated in this study. The results demonstrated that the maximum hydrogen yield (HY) of 12.97 mL H₂/g-VSS was obtained with 50 mg/L and 40–60 nm of Fe₃O₄NPs in batch experiments. Moreover, the optimum dosage of Fe₃O₄NPs produced hydrogen production (HP) of 4.95 L H₂/d in an expanded granular sludge bed (EGSB) reactor. Fe₃O₄NPs involved could promote ethanol and acetic acid accumulation. Fe²⁺ as by-product of iron corrosion could effectively promote the activity of key coenzymes and soluble microbial products (SMPs). Importantly, Fe₃O₄NPs addition resulted in the formation of electronic conductor chains to enhance the electron transport efficiency in the granular sludge. Microbial community analysis revealed that the relative abundance of butyrate-hydrogen-producing bacteria (Clostridium) decreased from 40.55% to 11.45%, while the relative abundance of ethanol-hydrogen-producing bacteria (Acetanaerobacterium and Ethanoligenens) increased from 19.62% to 35.35% with Fe₃O₄NPs involved, confirming that the fermentation type was transformed from butyrate-type to ethanol-type, which finally facilitated more hydrogen production.     

Experimental verification of various methods for biological hydrogen production

The aim of the work was to compare two different biological methods for hydrogen production: fermentative and photosynthetic based upon the modality of batch cultures. For testing of fermentative bio-hydrogen production four mixed cultures representing anaerobic microorganisms (dominant strain Clostridium) were selected. The kinetic parameters on the intensity of bio-hydrogen production were established. The efficiency coefficient of transformation ranged from 1.65 mol H₂/mol glucose in the pectin culture up to 2.45 in the mixed culture. The bio-hydrogen concentration never exceeded 30%. The carbon dioxide was produced in a ratio of CO₂ to H₂ (0.5–0.67)/1. The testing of green algae proved that the most effective was the algae species Scenedesmus. High bio-hydrogen purity was analytically verified. The fermentative method of H₂ production is more efficient; it does not need light, has a longer efficiency of one charge and enables effective use of different biological wastes.     

Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 in a UASB reactor and bacterial quantification under non-sterile conditions

Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 was investigated in an up-flow anaerobic sludge blanket (UASB) reactor. The reactor was operated under non-sterile conditions at 40^○C and initial pH 8.0 at different hydraulic retention times (HRTs) (2–12 h) and glycerol concentrations (10–30 g/L). Decreasing the HRT led to an increase in hydrogen production rate (HPR) and hydrogen yield (HY). The highest HPR of 242.15 mmol H₂/L/d and HY of 44.27 mmol H₂/g glycerol consumed were achieved at 4 h HRT and glycerol concentrations of 30 and 10 g/L, respectively. The main soluble metabolite was 1,3-propanediol, which implies that Klebsiella sp. was dominant among other microorganisms. Fluorescence in situ hybridization (FISH) revealed that the microbial community was dominated by Klebsiella sp. with 56.96, 59.45, and 63.47% of total DAPI binding cells, at glycerol concentrations of 10, 20, and 30 g/L, respectively.     

Discontinuous biomass recycling as a successful strategy to enhance continuous hydrogen production at high organic loading rates

A novel strategy to discontinuously increase the biomass concentration in a continuous stirred-tank reactor was evaluated to enhance the performance of dark fermentation. Different concentrations of biomass were evaluated at organic loading rates (OLR) ranging from 90 to 160 g lactose/L-d with a hydraulic retention time (HRT) of 6 h. The study revealed that the discontinuous increase of biomass enhanced the hydrogen (H₂) production rates and carboxylic acids concentrations by 19–25% and 8–23%, respectively. In particular, a maximum H₂ production rate of 30.8 L H₂/L-d with carboxylic acids concentration of 20 g/L was reached at an OLR of 138 g lactose/L-d with a biomass concentration of 15 g volatile suspended solids/L. The analysis of microbial communities showed the co-dominance of Clostridium and lactic acid bacteria. Overall, the discontinuous increase of biomass was an effective strategy to improve the performance of suspended-biomass reactors operated at high OLR and low HRT.