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

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

Biogenic hydrogen and methane production from reed canary grass

The composition, biodegradability, abundance, availability and cost determine the amenability of carbonaceous substrate for fermentative hydrogen and methane production systems. The aim of the present work was to determine suitability of lignocellulosic material, reed canary grass (RCG) (Phalaris arundinacea L.), for hydrogen and methane production at 35 °C by utilizing solid RCG and acid hydrolyzed soluble RCG. Synthetic cellulose was used as control substrate. Acid hydrolysis released 61.7 mg g⁻¹ (dw) and 115 mg g⁻¹ (dw) of reducing sugars from synthetic cellulose and chopped RCG, respectively. More hydrogen was produced from acid hydrolyzed RCG than from solid RCG, the highest yield being 1.25 mmol H₂ per g (dw) RCG. Methane production from solid RCG resulted in the highest yield of 8.26 mmol CH₄ per g (dw) RCG. In summary hydrogen and methane was produced from RCG, and acid hydrolysis was required for hydrogen, but not for methane production.     

The effects of seed sludge and hydraulic retention time on the production of hydrogen from a cassava processing wastewater and glucose mixture in an anaerobic fluidized bed reactor

The potential for co-fermentation of a cassava processing wastewater and glucose mixture was studied in anaerobic fluidized bed reactors. The effects of different hydraulic retention times (HRTs) (10–2 h) and varying sources of inoculum are reported. The sludge from a UASB reactor that had been used to treat poultry slaughterhouse wastewater (SP) resulted in the highest yields of hydrogen (HY) and ethanol (EtOHY) of 1.0 mmol H₂ g⁻¹ COD (10 h) and 3.0 mmol EtOH g⁻¹ COD (6 h). The sludge from a UASB reactor used for the treatment of swine wastewater (SW) resulted in a maximum HY of 0.65 mmol H₂ g⁻¹ COD (6 h) and EtOHY of 2.1 mmol g⁻¹ COD (10 and 8 h). Methane was produced with a maximum production of 9.68 L CH₄ d⁻¹ L⁻¹. Based on phylogenetic analysis of 16S rRNA, bacteria and methanogenic archaea similar to Lactobacillus and Methanobacterium, respectively, were identified.     

Developing a thermophilic hydrogen-producing co-culture for efficient utilization of mixed sugars

Previous studies on the extreme thermophile Caldicellulosiruptor saccharolyticus revealed that the organism produces high yields of hydrogen on glucose and xylose, the major components of lignocellulosic hydrolysates. Preliminary experiments on mixed sugar substrates, however, indicated that xylose was preferred over glucose. The sugar preference of some other extreme thermophiles, including Caldicellulosiruptor owensensis, Caldicellulosiruptor kristjanssonii and newly enriched, thermophilic compost sludge microflora, was investigated in an attempt to find complementary organisms to C. saccharolyticus for rapid and efficient utilization of lignocellulosic sugars. The behavior of C. owensensis and C. kristjanssonii appeared to be similar to that of C. saccharolyticus, either in pure cultures or in co-cultures with the latter. Co-culturing C. saccharolyticus with the enriched compost microflora resulted in fast, simultaneous consumption of both glucose and xylose in the medium with a relatively high specific hydrogen production rate, 40 mmol (gCDW)⁻¹ h⁻¹, and high volumetric productivity, 22.5 mmol l⁻¹ h⁻¹.     

Different ratios of carbon sources in the fermentation of cheese whey and glucose as substrates for hydrogen and ethanol production in continuous reactors

The fermentation of glucose, cheese whey and the mixture of glucose and cheese whey were evaluated in this study from two inocula sources (sludge from a UASB reactor for swine wastewater treatment and poultry slaughterhouse) for hydrogen production in continuous anaerobic fluidized bed reactors (AFBR). For all fermentations, a hydraulic retention time (HRT) of 6 h and a substrate concentration of 5 g COD L⁻¹ were used. In glucose fermentation, the maximum hydrogen yield (HY) was 1.37 mmol H₂ g⁻¹ COD. The co-fermentation of the cheese whey and glucose mixture was favorable for the concomitant production of hydrogen and ethanol, with yields of up to 1.7 mmol H₂ g⁻¹ COD and 3.45 mol EtOH g⁻¹ COD in AFBR2. The utilization of cheese whey as a sole substrate resulted in an HY of 1.9 mmol H₂ g⁻¹ COD. Throughout the study, ethanol fermentation was evident.     

Effect of pH on glucose and starch fermentation in batch and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production rates and yields were investigated at 30 °C in a 2.3 L bioreactor operated in batch and sequenced-batch mode using glucose and starch as substrates. In order to study the precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 mol H₂ mol⁻¹ glucose) was measured when the pH was maintained at 5.2. The acetate and butyrate yields were 0.35 mol acetate mol⁻¹ glucose and 0.6 mol butyrate mol⁻¹ glucose. For starch a maximum yield of 2.0 mol H₂ mol⁻¹ hexose, and a maximum production rate of 15 mol H₂ mol⁻¹ hexose h⁻¹ were obtained at pH 5.6 when the acetate and butyrate yields were 0.47 mol acetate mol⁻¹ hexose and 0.67 mol butyrate mol⁻¹ hexose.     

Hydrogen production and end-product synthesis patterns by Clostridium termitidis strain CT1112 in batch fermentation cultures with cellobiose or α-cellulose

Hydrogen (H₂) production and end-product synthesis were characterized in a novel, mesophilic, cellulolytic, anaerobic bacterium, Clostridium termitidis strain CT1112, isolated from the gut of the termite, Nasutitermes lujae. Growth curves, pH patterns, protein content, organic acid synthesis, and H₂ production were determined. When grown on 2 g l⁻¹ cellobiose and 2 g l⁻¹ α-cellulose, C. termitidis displayed a cell generation time of 6.5 h and 18.9 h, respectively. The major end-products synthesized on cellobiose included acetate, hydrogen, CO₂, lactate, formate and ethanol, where as on cellulose, the major end-products included hydrogen, acetate, CO₂ and ethanol. The concentrations of acetate were greater than ethanol, formate and lactate on both cellobiose and α-cellulose throughout the entire growth phase. Maximum yields of acetate, ethanol, hydrogen and formate on cellobiose were 5.9, 3.7, 4.6 and 4.2 mmol l⁻¹ culture, respectively, where as on cellulose, the yields were 7.2, 3.1, 7.7 and 2.9 mmol l⁻¹ culture, respectively. Hydrogen and ethanol production rates were slightly higher in C. termitidis cultured on cellobiose when compared to α-cellulose. Although, the generation time on α-cellulose was longer than on cellobiose, H₂ production was favored corresponding to acetate synthesis, thereby restricting the carbon flowing to ethanol. During log phase, H₂, CO₂ and ethanol were produced at specific rates of 4.28, 5.32, and 2.99 mmol h⁻¹ g dry weight⁻¹ of cells on cellobiose and 2.79, 2.59, and 1.1 mmol h⁻¹ g dry weight⁻¹ of cells on α-cellulose, respectively.     

Light fermentation of dark fermentation effluent for bio-hydrogen production by different Rhodobacter species at different initial volatile fatty acid (VFA) concentrations

Three different Rhodobacter sphaeroides (RS) strains (RS–NRRL, RS–DSMZ and RS–RV) and their combinations were used for light fermentation of dark fermentation effluent of ground wheat containing volatile fatty acids (VFA). In terms of cumulative hydrogen formation, RS–NRRL performed better than the other two strains producing 48 ml H₂ in 180 h. However, RS–RV resulted in the highest hydrogen yield of 250 ml H₂ g⁻¹ TVFA. Specific hydrogen production rate (SHPR) with the RS–NRRL was also better in comparison to the others (13.8 ml H₂ g⁻¹ biomass h⁻¹). When combinations of those three strains were used, RS–RV + RS–DSMZ resulted in the highest cumulative hydrogen formation (90 ml H₂ in 330 h). However, hydrogen yield (693 ml H₂ g⁻¹ TVFA) and SHPR (12.1 ml H₂ g⁻¹ biomass h⁻¹) were higher with the combination of the three different strains. On the basis of Gompertz equation coefficients mixed culture of the three different strains gave the highest cumulative hydrogen and formation rate probably due to synergistic interaction among the strains. The effects of initial TVFA and NH₄–N concentrations on hydrogen formation were investigated for the mixed culture of the three strains. The optimum TVFA and NH₄–N concentrations maximizing the hydrogen formation were determined as 2350 and 47 mg L⁻¹, respectively.     

Evaluation of continuous biohydrogen production from enzymatically treated cornstalk hydrolysate

Enzymatically treated cornstalk hydrolysate was tested as substrate for H₂ production by Thermoanaerobacterium thermosaccharolyticum W16 in a continuous stirred tank reactor. The performance of strain W16 to ferment the main components of hydrolysate, mixture of glucose and xylose, in continuous culture was conducted at first, and then T. thermosaccharolyticum W16 was evaluated to ferment fully enzymatically hydrolysed cornstalk to produce H₂ in continuous operation mode. At the dilution rate of 0.020 h⁻¹, the H₂ yield and production rate reached a maximum of 1.9 mol H₂ mol⁻¹ sugars and 8.4 mmol H₂ L⁻¹ h⁻¹, respectively, accompanied with the maximum glucose and xylose utilizations of 86.3% and 77.6%. Continuous H₂ production from enzymatically treated cornstalk hydrolysate in this research provides a new direction for economic, efficient, and harmless H₂ production.     

Hydrogen production and consumption of organic acids by a phototrophic microbial consortium

Alternative fuel sources have been extensively studied. Hydrogen gas has gained attention because its combustion releases only water, and it can be produced by microorganisms using organic acids as substrates. The aim of this study was to enrich a microbial consortium of photosynthetic purple non-sulfur bacteria from an Upflow Anaerobic Sludge Blanket reactor (UASB) using malate as carbon source. After the enrichment phase, other carbon sources were tested, such as acetate (30 mmol l⁻¹), butyrate (17 mmol l⁻¹), citrate (11 mmol l⁻¹), lactate (23 mmol l⁻¹) and malate (14.5 mmol l⁻¹). The reactors were incubated at 30 °C under constant illumination by 3 fluorescent lamps (81 μmol m⁻² s⁻¹). The cumulative hydrogen production was 7.8, 9.0, 7.9, 5.6 and 13.9 mmol H₂ l⁻¹ culture for acetate, butyrate, citrate, lactate and malate, respectively. The maximum hydrogen yield was 0.6, 1.4, 0.7, 0.5 and 0.9 mmol H₂ mmol⁻¹ substrate for acetate, butyrate, citrate, lactate and malate, respectively. The consumption of substrates was 43% for acetate, 37% for butyrate, 100% for citrate, 49% for lactate and 100% for malate. Approximately 26% of the clones obtained from the Phototrophic Hydrogen-Producing Bacterial Consortium (PHPBC) were similar to Rhodobacter, Rhodospirillum and Rhodopseudomonas, which have been widely cited in studies of photobiological hydrogen production. Clones similar to the genus Sulfurospirillum (29% of the total) were also found in the microbial consortium.     

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.     

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.     

Enhanced hydrogen production from xylose and bamboo stalk hydrolysate by overexpression of xylulokinase and xylose isomerase in Klebsiella oxytoca HP1

Biofuels production from lignocellulose hydrolysates by microbe fermentation has merited attention because of the mild reaction conditions involved and the clean nature of the process. In this work, xylulokinase (XK) and xylose isomerase (XI) were overexpressed in Klebsiella oxytoca HP1 to enhance hydrogen production by the fermentation of xylose. The recombinant strains exhibited higher enzyme activity of XI or XK compared with the wild strain. Hydrogen production from pure xylose, xylose/glucose mixtures and bamboo stalk hydrolysate was significantly enhanced with the overexpression of XI and XK in K. oxytoca HP1 in terms of total hydrogen yield (THY), hydrogen yield per mole substrate (HYPM) and hydrogen production rate (HPR). The HYPM of K. oxytoca HP1/xylB and K. oxytoca HP1/xylA reached 1.93 ± 0.05 and 2.46 ± 0.05 mol H₂/mol xylose, respectively in pure xylose, while the value for the wild strain was 1.68 ± 0.04 mol H₂/mol xylose. The xylose consumption rate (XCR) for the recombinant strain was significantly higher than that for the wild strain, particularly in the early stage of fermentation. Relative to the wild type, hydrogen yield (HY) from 1 g of preprocessed bamboo powder of HP1/xylB and HP1/xylA increased by 33.04 and 41.31%, respectively. It was concluded that overexpression of XK or XI was able to promote hydrogen production from xylose and xylose/glucose mixtures by simultaneously increasing the utilization efficiency of xylose and weakening the inhibitory effect of glucose on xylose use. In addition, the results indicated that overexpression technology was an effective way to further increase hydrogen production from lignocellulosic hydrolysates.     

Biological hydrogen production from corn stover by moderately thermophile Thermoanaerobacterium thermosaccharolyticum W16

This study addressed the utilization of an agro-waste, corn stover, as a renewable lignocellulosic feedstock for the fermentative H₂ production by the moderate thermophile Thermoanaerobacterium thermosaccharolyticum W16. The corn stover was first hydrolyzed by cellulase with supplementation of xylanase after delignification with 2% NaOH. It produced reducing sugar at a yield of 11.2 g L⁻¹ glucose, 3.4 g L⁻¹ xylose and 0.5 g L⁻¹ arabinose under the optimum condition of cellulase dosage 25 U g⁻¹ substrate with supplement xylanase 30 U g⁻¹ substrate. The hydrolyzed corn stover was sequentially introduced to fermentation by strain W16, where, the cell density and the maximum H₂ production rate was comparable to that on simulated medium, which has the same concentration of reducing sugars with hydrolysate. The present results suggest a promising combined hydrogen production process from corn stover with enzymatic hydrolysis stage and fermentation stage using W16.     

Microbial culture selection for bio-hydrogen production from waste ground wheat by dark fermentation

Hydrogen formation performances of different anaerobic bacteria were investigated in batch dark fermentation of waste wheat powder solution (WPS). Serum bottles containing wheat powder were inoculated with pure cultures of Clostridium acetobutylicum (CAB), Clostridium butyricum (CB), Enterobacter aerogenes (EA), heat-treated anaerobic sludge (ANS) and a mixture of those cultures (MIX). Cumulative hydrogen formation (CHF), hydrogen yield (HY) and specific hydrogen production rate (SHPR) were determined for every culture. The heat-treated anaerobic sludge was found to be the most effective culture with a cumulative hydrogen formation of 560 ml, hydrogen yield of 223 ml H₂ g⁻¹ starch and a specific hydrogen production rate of 32.1 ml H₂ g⁻¹ h⁻¹.     

Sulfate-reducing bacteria as new microorganisms for biological hydrogen production

Sulfate-reducing bacteria (SRB) have an extremely high hydrogenase activity and in natural habitats where sulfate is limited, produce hydrogen fermentatively. However, the production of hydrogen by these microorganisms has been poorly explored. In this study we investigated the potential of SRB for H₂ production using the model organism Desulfovibrio vulgaris Hildenborough. Among the three substrates tested (lactate, formate and ethanol), the highest H₂ production was observed from formate, with 320 mL L⁻¹_medium of H₂ being produced, while 21 and 5 mL L⁻¹_medium were produced from lactate and ethanol, respectively. By optimizing reaction conditions such as initial pH, metal cofactors, substrate concentration and cell load, a production of 560 mL L⁻¹_medium of H₂ was obtained in an anaerobic stirred tank reactor (ASTR). In addition, a high specific hydrogen production rate (4.2 L g⁻¹_dcw d⁻¹; 7 mmol g⁻¹_dcw h⁻¹) and 100% efficiency of substrate conversion were achieved. These results demonstrate for the first time the potential of sulfate reducing bacteria for H₂ production from formate.     

Improved hydrogen production of the downstream bioreactor by coupling single chamber microbial fuel cells between series-connected photosynthetic biohydrogen reactors

To obtain additional hydrogen recovery from the downstream photosynthetic biohydrogen reactor (PBR), a system (PBR1–MFCs–PBR2) that combined PBRs with three single chamber microbial fuel cells (MFCs) was proposed in this study. The results revealed that the PBR2 in PBR1–MFCs–PBR2 showed a hydrogen production rate of 0.44 ± 0.22 mmol L h⁻¹, which was 15 and 4 times higher than those obtained by direct connecting the two PBRs (PBR1–PBR2) and pH regulated system (PBR1–pH regulation–PBR2), respectively. In addition, the PBR1–MFCs–PBR2 exhibited the highest glucose utilization (η_g) of 97.6 ± 2.1 %, while lower η_g values of 75.6 ± 2.2% and 70.1 ± 1.2% was obtained for PBR1–PBR2 and PBR1–pH regulation–PBR2, respectively. These improvements were due to the removal of inhibitory byproduct and H⁺ from the PBR1 effluent by the MFCs.     

Mesophilic biohydrogen production by Clostridium butyricum CWBI1009 in trickling biofilter reactor

This study investigates the mesophilic biohydrogen production from glucose using a strictly anaerobic strain, Clostridium butyricum CWBI1009, immobilized in a trickling bed sequenced batch reactor (TBSBR) packed with a Lantec HD Q-PAC^® packing material (132 ft²/ft³ specific surface). The reactor was operated for 62 days. The main parameters measured here were hydrogen composition, hydrogen production rate and soluble metabolic products. pH, temperature, recirculation flow rate and inlet glucose concentration at 10 g/L were the controlled parameters. The maximum specific hydrogen production rate and the hydrogen yield found from this study were 146 mmol H₂/L.d and 1.67 mol H₂/mol glucose. The maximum hydrogen composition was 83%. Following a thermal treatment, the culture was active without adding fresh inoculum in the subsequent feeding and both the hydrogen yield and the hydrogen production rate were improved. For all sequences, the soluble metabolites were dominated by the presence of butyric and acetic acids compared to other volatile fatty acids. The results from the standard biohydrogen production (BHP) test which was conducted using samples from TBSBR as inoculum confirmed that the culture generated more biogas and hydrogen compared to the pure strain of C. butyricum CWBI1009. The effect of biofilm activity was studied by completely removing (100%) the mixed liquid and by adding fresh medium with glucose. For three subsequent sequences, similar results were recorded as in the previous sequences with 40% removal of spent medium. The TBSBR biofilm density varied from top to bottom in the packing bed and the highest biofilm density was found at the bottom plates. Moreover, no clogging was evidenced in this packing material, which is characterized by a relatively high specific surface area. Following a PCA test, contaminants of the Bacillus genus were isolated and a standard BHP test was conducted, resulting in no hydrogen production.     

Biohydrogen production from cellobiose in phenol and cresol-containing medium using Clostridium sp. R1

Cellobiose fermentation in batch test using an isolated strain, Clostridium sp. R1, was investigated. The Clostridium sp. R1 achieved a maximum hydrogen yield of 3.5 mol H₂ mol⁻¹ cellobiose at pH 6 and 30 °C, higher than most yields reported in literature. This strain can generate hydrogen from a number of carbohydrates, including galactose, glucose, mannose, maltose, sucrose, and starch. This strain can also convert cellobiose to hydrogen in the presence of toxic phenol or cresol. The inhibition effects of phenolic compounds on strain R1 activity followed phenol > p-cresol > o-cresol > m-cresol. Co-culturing with another strain, Clostridium butyricum, can co-degrade some of the phenol as substrates. The new isolated strain can yield hydrogen from phenol-containing wastewaters.     

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.