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.     

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

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

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.     

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.     

Enhancement of fermentative hydrogen production from green algal biomass of Thermotoga neapolitana by various pretreatment methods

Biomass of the green algae has been recently an attractive feedstock source for bio-fuel production because the algal carbohydrates can be derived from atmospheric CO₂ and their harvesting methods are simple. We utilized the accumulated starch in the green alga Chlamydomonas reinhardtii as the sole substrate for fermentative hydrogen (H₂) production by the hyperthermophilic eubacterium Thermotoga neapolitana. Because of possessing amylase activity, the bacterium could directly ferment H₂ from algal starch with H₂ yield of 1.8–2.2 mol H₂/mol glucose and the total accumulated H₂ level from 43 to 49% (v/v) of the gas headspace in the closed culture bottle depending on various algal cell-wall disruption methods concluding sonication or methanol exposure. Attempting to enhance the H₂ production, two pretreatment methods using the heat-HCl treatment and enzymatic hydrolysis were applied on algal biomass before using it as substrate for H₂ fermentation. Cultivation with starch pretreated by 1.5% HCl at 121 °C for 20 min showed the total accumulative H₂ yield of 58% (v/v). In other approach, enzymatic digestion of starch by thermostable α-amylase (Termamyl) applied in the SHF process significantly enhanced the H₂ productivity of the bacterium to 64% (v/v) of total accumulated H₂ level and a H₂ yield of 2.5 mol H₂/mol glucose. Our results demonstrated that direct H₂ fermentation from algal biomass is more desirably potential because one bacterial cultivation step was required that meets the cost-savings, environmental friendly and simplicity of H₂ production.     

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).     

Comparison of mesophilic and thermophilic anaerobic hydrogen production by hot spring enrichment culture

Anaerobic hydrogen producing mesophilic and thermophilic cultures were enriched and studied from an intermediate temperature (45 °C) hot spring sample. H₂ production yields at 37 °C and 55 °C were highest at the initial pH of 6.5 and 7.5, respectively. Optimum glucose, iron and nickel concentrations were 9 g/l, 25 mg/l and 25 mg/l both at 37 °C and 55 °C, respectively. The highest H₂ yields at 37 °C and 55 °C were 1.8 and 1.0 mol H₂/mol glucose, respectively, with the optimal pH, glucose concentration and iron addition. Hydrogen production from glucose at 55 °C and 37 °C was associated with ethanol- and acetate–butyrate type fermentations, respectively. Bacterial composition was analyzed by 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). Clostridium species dominated at both temperatures and the microbial diversity decreased with increasing temperature. At 55 °C, Clostridium ramosum was the dominant organism.     

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

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

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.     

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

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

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.     

Anaerobic H2 production at elevated temperature (60 °C) by enriched mixed consortia from mesophilic sources

Two different enriched mixed consortia from mesophilic sources were used for H₂ production from glucose at 60 °C. The variables were initial pH, nitrogen source, iron and sulfate. pH had a crucial effect and iron was slightly positive for the biohydrogen production performance of the mixed culture. On the other hand, H₂ production decreased with the increasing of ammonia, peptone and sulfate concentrations. Metabolic pathways of mixed culture were affected in different ways depending on the differences in microbial community. The PCR-DGGE and sequencing based microbial community analysis revealed that two enriched mixed cultures had different microbial diversity and both culture were dominated mainly by Thermoanaerobacterium species.     

Sugarcane vinasse as substrate for fermentative hydrogen production: The effects of temperature and substrate concentration

The present study aimed to evaluate the hydrogen production of a microbial consortium using different concentrations of sugarcane vinasse (2–12 g COD L⁻¹) at 37 °C and 55 °C. In mesophilic tests, the increase in vinasse concentration did not significantly impact the hydrogen yield (HY) (from 1.72 to 2.23 mmol H₂ g⁻¹ COD_influent) but had a positive effect on the hydrogen production potential (P) and hydrogen production rate (R_m). On the other hand, the increase in the substrate concentration caused a drop in HY from 2.31 to 0.44 mmol H₂ g⁻¹ COD_influent in the tests performed at 55 °C with vinasse concentrations from 2 to 12 g COD L⁻¹. The mesophilic community was composed of different species within the Clostridium genus, and the thermophilic community was dominated by organisms affiliated with the Thermoanaerobacter genus. Not all isolates affiliated with the Clostridium genus contributed to a high HY, as the homoacetogenic pathway can occur.     

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.     

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.     

Fermentative hydrogen production by Clostridium butyricum CGS5 using carbohydrate-rich microalgal biomass as feedstock

In this work, a carbohydrate-rich microalga, Chlorella vulgaris ESP6, was grown photoautotrophically to fix the CO₂. The resulting microalgal biomass was hydrolyzed by acid or alkaline/enzymatic treatment and was then used for biohydrogen production with Clostridium butyricum CGS5. The C. vulgaris biomass could be effectively hydrolyzed by acid pretreatment while similar hydrolysis efficiency was achieved by combination of alkaline pretreatment and enzymatic hydrolysis. The biomass of C. vulgaris ESP6 containing a carbohydrate content of 57% (dry weight basis) was efficiently hydrolyzed by acid treatment with 1.5% HCl, giving a reducing sugars (RS) yield of nearly 100%. C. butyricum CGS5 could utilize RS from C. vulgaris ESP6 biomass to produce hydrogen without any additional organic carbon sources. The optimal conditions for hydrogen production were 37 °C and a microalgal hydrolysate loading of 9 g RS/L with pH-controlled at 5.5. Under the optimal conditions, the cumulative H₂ production, H₂ production rate, and H₂ yield were 1476 ml/L, 246 ml/L/h, and 1.15 mol/mol RS, respectively. The results demonstrate that the C. vulgaris biomass has the potential to serve as effective feedstock for dark fermentative H₂ production.     

Biohydrogen production from pentose-rich oil palm empty fruit bunch molasses: A first trial

Oil palm empty fruit bunch (OPEFB) was pretreated by local plantation industry to increase the accessibility towards its fermentable sugars. This pretreatment process led to the formation of a dark sugar-rich molasses byproduct. The total carbohydrate content of the molasses was 9.7 g/L with 4.3 g/L xylose (C₅H₁₀O₅). This pentose-rich molasses was fed as substrate for biohydrogen production using locally isolated Clostridium butyricum KBH1. The effect of initial pH and substrate concentration on the yield and productivity of hydrogen production were investigated in this study. The best result for the fermentation performed in 70 mL working volume was obtained at the initial reaction condition of pH 9, 150 rpm, 37 °C and 5.9 g/L total carbohydrate. The maximum hydrogen yield was 1.24 mol H₂/mol pentose and the highest productivity rate achieved was 0.91 mmol H₂/L/h. The optimal pH at pH 9 was slightly unusual due to the presence of inhibitors, mainly furfural. The furfural content decreased proportionally as pH was increased. The optimal experiment condition was repeated and continued in fermentation volume of 200 mL. The maximum hydrogen yield found for this run was 1.21 mol H₂/mol pentose while the maximum productivity was 1.1 mmol H₂/L/h. The major soluble metabolites in the fermentation were n-butyric acid and acetic acid.     

Comparative study on the production of biohydrogen from rice mill wastewater

This study presents the production of biohydrogen from rice mill wastewater. The acid hydrolysis and enzymatic hydrolysis operating conditions were optimized, for better reducing sugar production. The effect of pH and fermentation time on biohydrogen production from acid and enzymatic hydrolyzed rice mill wastewater was investigated, using Enterobacter aerogenes and Citrobacter ferundii. The enzymatic hydrolysis produced the maximum reducing sugar (15.8 g/L) compared to acid hydrolysis (14.2 g/L). The growth data obtained for E. aerogenes and C. ferundii, fitted well with the Logistic equation. The hydrogen yields of 1.74 mol H₂/mol reducing sugar, and 1.40 mol H₂/mol reducing sugar, were obtained from the hydrolyzate obtained from enzymatic and acid hydrolysis, respectively. The maximum hydrogen yield was obtained from E. aerogenes compared to C. ferundii, and the optimum pH for better hydrogen production was found to be in the range from 6.5 to 7.0. The chemical oxygen demand (COD) reduction obtained was around 71.8% after 60 h of fermentation.     

Cellulosic hydrogen production and microbial community characterization in hyper-thermophilic continuous bioreactor

A continuous stirred tank reactor was used for the dark hydrogen fermentation of cellulose by mixed microflora at hyper-thermophilic temperature (70 ± 1 °C) for 240 days. A total of twenty six batch experiments were conducted to investigate the effect of temperature on the activity of cellulosic-hydrogen producing bacteria. The results show that the system reached a steady state condition after 90 days. A stable hydrogen yield of 7.07 ± 0.23 mmol H₂/g cellulose was maintained for 150 days with acetate, butyrate, ethanol and propionate as main soluble byproducts. Analysis of 16S rRNA sequences showed that the cellulolytic bacteria were close to Thermoanaerobacterium genus. The cellulosic-hydrogen producing bacteria were able to utilize the cellulose or glucose within a wide range of fermentation temperatures (45–80 °C) to produce hydrogen. The activation energy for cellulose and glucose were estimated at 133.2 and 117.7 kJ/mol, respectively.     

Continuous thermophilic hydrogen production and microbial community analysis from anaerobic digestion of diluted sugar cane stillage

The aim of this study was to promote biohydrogen production in an thermophilic anaerobic fluidized bed reactor (AFBR) at 55 °C using a mixture of sugar cane stillage and glucose at approximately 5000–5300 mg COD L⁻¹. During a reduction in the hydraulic retention time (HRT) from 8, 6, 4, 2 and 1 h, H₂ yields of 5.73 mmol g COD_added⁻¹ were achieved (at HRT of 4 h, with organic loading rate of 52.7 kg COD m⁻³ d⁻¹). The maximum volumetric H₂ production of 0.78 L H₂ h⁻¹ L⁻¹ was achieved using stillage as carbon source. In all operational phases, the H₂ average content in the biogas was between 31.4 and 52.0%. Butyric fermentation was the predominant metabolic pathway. The microbial community in accordance with the DGGE bands profile was found similarity coefficient between 91 and 95% without significant changes in bacterial populations after co-substrate removal. Bacteria like Thermoanaerobacterium sp. and Clostridium sp. were identified.