Dark fermentative biohydrogen production by mesophilic bacterial consortia isolated from riverbed sediments

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated for hydrogen production. A series of batch experiments were conducted to study the effect of pH, substrate concentration and temperature on hydrogen production from a selected bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized all the five sugars to produce fermentative hydrogen. Glucose was the most preferred carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid were the major soluble metabolites detected. Investigation on optimization of pH, temperature, and substrate concentration revealed that TERI BH05 produced maximum hydrogen at 37 °C, pH 6 with 8 g/L of glucose supplementation and maximum yield of hydrogen production observed was 2.0–2.3 mol H₂/mol glucose. Characterization of TERI BH05 revealed the presence of two different bacterial strains showing maximum homology to Clostridium butyricum and Clostridium bifermentans.     

Co-culture of Clostridium beijerinckii L9, Clostridium butyricum M1 and Bacillus thermoamylovorans B5 for converting yeast waste into hydrogen

In order to enhance anaerobic hydrogen production from yeast waste, a series of 120-mL batch co-cultures of Clostridium beijerinckii L9, Clostridium butyricum M1, and Bacillus thermoamylovorans B5 under mesophilic conditions were established according to full factorial design (FFD) and mixture design (MD). The experimental results were subjected to multivariate and response surface analyses to determine the relationships between bacteria converting yeast waste into hydrogen. The results indicated clearly that C. beijerinckii L9 and C. butyricum M1 had significant potential to convert yeast waste into hydrogen. There was no significant hydrogen generation when B. thermoamylovorancs B5 alone was cultured with yeast waste. However, B. thermoamylovorancs B5 could significantly shorten the co-culture’s hydrogen-producing lag phase. Response surface analyses demonstrate that B. thermoamylovorancs B5 can stimulate the specific hydrogen production rate of C. beijerinckii L9 and C. butyricum M1, greater in the case of the former than of the latter. An ultimate hydrogen yield of 46 mL H₂/g COD added yeast waste was obtained with an optimal volumetric ratio C. beijerinckii L9: C. butyricum M1: B. thermoamylovoranc B5 of 8.9:4.8:10.3. Highly reproducible co-culture results confirm that FFD and MD, via response surface analysis, are applicable to assess the roles of the individual microorganisms in the defined co-culture.     

Biological hydrogen production from phenol-containing wastewater using Clostridium butyricum

Toxicity renders certain industrial effluents unfit for recovering its bioenergy content. An enriched single strain, Clostridium butyricum, was herein applied to fermentatively produce hydrogen from glucose in the presence of 200–1500 mg L⁻¹ of phenol. The enriched C. butyricum yielded hydrogen at approximately 1.4 mol H₂ mol⁻¹ glucose in the presence of 200–400 mg L⁻¹ phenol. Significant inhibition of cell metabolism was noted at phenol concentration >1000 mg L⁻¹. During glucose fermentation, phenol dosed at 200–400 mg L⁻¹ was partly co-degraded. Ethanol and acetate were the primary metabolites, whose yields increased with increasing phenol concentration. The present results revealed the potential to harvest hydrogen from a toxic (phenol-containing) wastewater.     

Characterization and overexpression of a [FeFe]-hydrogenase gene of a novel hydrogen-producing bacterium Ethanoligenens harbinense

Ethanoligenens, a novel ethanologenic and hydrogen-producing genus, has capability of hydrogen production at low pH. A [FeFe]-hydrogenase gene with [4Fe-4S] and [2Fe-2S] clusters from Ethanoligenens harbinense YUAN-3 was cloned and overexpressed in a non-hydrogen-producing Escherichia coli BL-21. This hydA gene consisted of an open reading frame of 1743 bp encoding 580 amino acids with an estimated molecular weight of 63 188.1 Da. Six characteristic sequence signatures were present within the H-cluster domain of [FeFe]-H₂ases, and three of them were described previously. The overexpressed and purified hydrogenases from recombinant cells showed catalytic activity in vitro and in vivo.     

Clostridium strain co-cultures for biohydrogen production enhancement from condensed molasses fermentation solubles

An anaerobic continuous-flow hydrogen fermentor was operated at a hydraulic retention time of 8 h using condensed molasses fermentation solubles (CMS) substrate of 40 g-COD/L. Serum bottles were used for seed micro-flora cultivation and batch hydrogen fermentation tests (CMS substrate concentrations of 10–160 g-COD/L). Three hydrogen-producing bacterial strains Clostridium sporosphaeroides F52, Clostridium tyrobutyricum F4 and Clostridium pasteurianum F40 were isolated from the seed fermentor and used as the seeding microbes in single and mixed-culture cultivations for determining their hydrogen productivity. These strains possessed specific hydrogenase genes that could be detected from CMS-fed hydrogen fermentors and were major hydrogen producers. C. pasteurianum F40 was the dominant strain with a high hydrogen production rate while C. sporosphaeroides F52 may play a main role in degrading carbohydrate and glutamate. These strains could be co-cultivated as a symbiotic mixed-culture process to enhance hydrogen productivity. C. pasteurianum F40 or C. tyrobutyricum F4 co-culture with the glutamate-utilizing strain C. sporosphaeroides F52 efficiently enhanced hydrogen production by 12–220% depending on the substrate CMS concentrations.     

Fermentative hydrogen production by Clostridium butyricum and Escherichia coli in pure and cocultures

The effect of coculture of Clostridium butyricum and Escherichia coli on hydrogen production was investigated. C. butyricum and E. coli were grown separately and together as batch cultures. Gas production, growth, volatile fatty acid production and glucose degradation were monitored. Whilst C. butyricum alone produced 2.09 mol-H₂/mol-glucose the coculture produced 1.65 mol-H₂/mol-glucose. However, the coculture utilized glucose more efficiently in the batch culture, i.e., it was able to produce more H₂ (5.85 mmol H₂) in the same cultivation setting than C. butyricum (4.62 mmol H₂), before the growth limiting pH was reached.     

Harvesting biohydrogen from toxic wastewater using isolated strain

Toxicity prevents the bioenergy content of certain industrial effluents from being recovered. An enriched Clostridium butyricum strain was employed to produce hydrogen by fermentation from cellobiose in the presence of phenol at 200–1500 mgl⁻¹. The enriched Cl. butyricum yielded the most hydrogen at 2.1 mol H₂ mol⁻¹ cellobiose with 600 mgl⁻¹ phenol. Butyrate was the main metabolite. Cell metabolism was substantially inhibited at a phenol concentration of 1500 mgl⁻¹. Part of the phenol was co-degraded during the test, helping to eliminate the toxicity of wastewater. Both the pyruvate oxidative decarboxylation pathway and the NADH pathway contributed to biohydrogen production. Phenol toxicity more strongly inhibits soluble hydrogenase than it does membrane-bound hydrogenase. Although the NADH pathway dominated at low phenol concentration, increasing the phenol concentration shifted the biohydrogen pathway toward decarboxylation.     

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.     

Optimization of biohydrogen production by Clostridium butyricum EB6 from palm oil mill effluent using response surface methodology

Clostridium butyricum EB6 successfully produced hydrogen gas from palm oil mill effluent (POME). In this study, central composite design and response surface methodology were applied to determine the optimum conditions for hydrogen production (P_c) and maximum hydrogen production rate (R_max) from POME. Experimental results showed that the pH, temperature and chemical oxygen demand (COD) of POME affected both the hydrogen production and production rate, both individually and interactively. The optimum conditions for hydrogen production (P_c) were pH 5.69, 36 °C, and 92 g COD/l; with an estimated P_c value of 306 ml H₂/g carbohydrate. The optimum conditions for maximum hydrogen production rate (R_max) were pH 6.52, 41 °C and 60 g COD/l; with an estimated R_max value of 914 ml H₂/h. An overlay study was performed to obtain an overall model optimization. The optimized conditions for the overall model were pH 6.05, 36 °C and 94 g COD/l. The hydrogen content in the biogas produced ranged from 60% to 75%.     

Effects of pH, glucose and iron sulfate concentration on the yield of biohydrogen by Clostridium butyricum EB6

A local bacterial isolate from palm oil mill effluent (POME) sludge, identified as Clostridium butyricum EB6, was used for biohydrogen production. Optimization of biohydrogen production was performed via statistical analysis, namely response surface methodology (RSM), with respect to pH, glucose and iron concentration. The results show that pH, glucose concentration and iron concentration significantly influenced the biohydrogen gas production individually, interactively and quadratically (P < 0.05). The center composite design (CCD) results indicated that pH 5.6, 15.7 g/L glucose and 0.39 g/L FeSO₄ were the optimal conditions for biohydrogen production, yielding 2.2 mol H₂/mol glucose. In confirmation of the experimental model, t-test results showed that curve fitted to the experimental data had a high confidence level, at 95% with t = 2.225. Based on the results of this study, optimization of the culture conditions for C. butyricum EB6 significantly increased the production of biohydrogen.     

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.     

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.     

Isolation and characterization of high hydrogen-producing strain Clostridium beijerinckii PS-3 from fermented oil palm sap

Felled oil palm trunk (OPT) (25 years old) is an abundant biomass in Southern Thailand. The OPT composition was 31.28–42.85% cellulose, 19.73–25.56% hemicellulose, 10.74–18.47% lignin, 1.63–2.25% protein, 1.60–1.83% fat, 1.12–1.35% ash and trace amount of minerals (0.01–0.40%). Oil palm sap extracted from OPT was found to contain 15.72 g/L glucose, 2.25 g/L xylose, and 0.086 g/L arabinose. A total of twenty samples from hot springs (45–75 °C and pH 6.5–8.4), oil palm sap and palm oil mill effluent were enriched for isolation of hydrogen-producing bacteria. The highest hydrogen-producing strain was isolated from oil palm sap and identified as Clostridium beijerinckii PS-3 using biochemical test and 16S rRNA gene analysis. Among various carbon sources tested, glucose, xylose, starch and cellulose were the preferred substrates for hydrogen production. The strain PS-3 could produce the maximum hydrogen yield of 140.9 ml H₂/g total sugar and the cumulative hydrogen production of 1973  ml/L-oil palm sap. Therefore, C. beijerinckii PS-3 is a potential candidate for fermentative hydrogen production from mixed sugars of the oil palm sap.     

Application of Plackett–Burman experimental design to optimize biohydrogen fermentation by E. coli (XL1-BLUE)

Escherichia coli is attractive for biotechnological hydrogen production. Compared to other biohydrogen producing bacteria e.g. Clostridium species, E. coli is able to tolerate oxygen, fast growing and well-characterized in physiological and biochemical terms. According to the well known metabolic pathways of E. coli, the hydrogen production from different substrates is dependent on the membrane-boundary formate-hydrogen lyase (FHL) enzyme complex. The efficiency and economic success of hydrogen fermentation are influenced by the applied operational conditions. In this work the optimal conditions (composition of broth, inoculum size, stirring speed) for biohydogen fermentation using E. coli (XL1-BLUE) were investigated by experimental design. We found that among the several variables only formate compound plays a key role in hydrogen formation and the optimal conditions for biohydrogen production were identified as follows: 30 mM formate, 5 g/l yeast extract, 10 g/l tryptone, 3.33 g/l NaCl, 0.05 g dry cell weight/l initial cell density and 220 rpm stirring rate, where productivity and yield were 426 ml H₂ l⁻¹ d⁻¹ and 0.41 mol H₂/mol formate, respectively.     

Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16

Lignocellulosic biomass, if properly hydrolyzed, can be an ideal feedstock for fermentative hydrogen production. This work considered the pretreatment of corn stover (CS) using a dilute acid hydrolysis process and studied its fermentability for hydrogen production by the strain Thermoanaerobacterium thermosaccharolyticum W16. The effects of sulfuric acid concentration and reaction time in the hydrolysis stage of the process were determined based on a 2² central composite experimental design with respect to maximum hydrogen productivity. The optimal hydrolysis conditions to yield the maximum quantity of hydrogen by W16 were 1.69% sulfuric acid and 117 min reaction time. At these conditions, the hydrogen yield was shown to be 3305 ml H₂ L⁻¹ medium, which corresponds to 2.24 mol H₂ mol⁻¹ sugar. The present results indicate the potential of using T. thermosaccharolyticum W16 for high-yield conversion of CS hemicellulose into bio-H₂ integrated with acid hydrolysis.     

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.     

Homologous overexpression of [FeFe] hydrogenase in Enterobacter cloacae IIT-BT 08 to enhance hydrogen gas production from cheese whey

The present study investigated the influence of increase in intracellular [FeFe] hydrogenase levels, in Enterobacter cloacae IIT-BT 08, on the formation of molecular hydrogen. The hydA gene from E. cloacae IIT-BT 08 was successfully amplified and cloned downstream of a tac promoter in an Escherichiacoli-Enterobacter reconstructed pGEX-Kan shuttle vector and introduced into E. cloacae. Finally E. cloacae strain carrying multiple copies of pGEX-Kan-hydA vector was developed. Homologous overexpression of the [FeFe] hydrogenase gene increased the hydrogenase activity by1.3-fold as compared to the wild type. SDS-PAGE confirmed the successful expression of the GST-tagged hydA protein. The hydrogen yield and rate of production in recombinant strain were found to be 1.2-fold and 1.6-fold higher, respectively, compared to the wild type strain. This was found to be concomitant with the shift in the metabolic pathway. In addition, feasibility of using cheese whey as a substrate for biohydrogen production and the effect of its supplementation with yeast extract as nitrogen source was studied for both the wild type and the recombinant strain. It was found that supplementation with 0.3% (w/v) yeast extract enhanced hydrogen production from whey. Further, the yield and rate of hydrogen production from the recombinant was found to be more promising as compared to the wild type.     

Bio-hydrogen production from thin stillage using conventional and acclimatized anaerobic digester sludge

To assess the viability of biohydrogen production from thin stillage, a comparative evaluation of anaerobic digester sludge (ADS) and acclimatized anaerobic digester sludge (AADS) for biohydrogen production over a wide range of S⁰/X⁰ ratio (0.5-8 gCOD/gVSS) was performed. A maximum hydrogen yield of 19.5 L H₂/L thin stillage was achieved for the AADS while tests with ADS achieved a maximum yield of only 7.5 L H₂/L thin stillage. The optimum range of S⁰/X⁰ ratio for hydrogen production was found to be 1 to 2 gCOD/gVSS using conventional ADS and 3 to 6 gCOD/gVSS using AADS. The biomass specific hydrogen production rate for the AADS was 3.5 times higher than rate for the ADS throughout the range of S⁰/X⁰ ratio examined in this study. The DGGE profiles of the 16S rDNA gene fragments confirmed the superior performance of the AADS over the ADS, showing that the widely known hydrogen producers Clostridium acetobutyricum, Klebsiella pneumonia, Clostridium butyricum and Clostridium pasteurianum were the predominant species.     

Quantitative analysis of microorganism composition in a pilot-scale fermentative biohydrogen production system

Five specific real-time polymerase chain reaction primers targeting the 16S rRNA gene of Clostridium spp., Klebsiella spp., Streptococcus spp., Pseudomonas spp., and Bifidobacterium spp., and two primer sets targeting the hydrogenase genes of hydrogen-producing Clostridium pasteurianum and Clostridium butyricum were designed and tested in the present study to quantify the microorganisms in fermentative biohydrogen production systems. The former primers revealed the composition of all coexisting microorganisms, whereas the latter ones provided information on which clostridia were responsible for the biohydrogen production in various operational conditions. When sucrose was selected as the feeding substrate, the biogas production and hydrogen production rate (HPR) of the system increased as the percentage of Clostridium spp. (especially C. pasteurianum) increased. The cell count of C. pasteurianum increased up to 90% of the total cell population when the system approached its maximum hydrogen production. C. butyricum was identified as the main hydrogen-producing clostridium in the condensed molasses soluble wastewater feeding system, but there was no significant correlation between system HPR and C. butyricum cell count. At the same time, other microorganisms, such as Bifidobacterium spp. and Klebsiella spp., were the predominant ones throughout the whole operation and possibly caused the unsatisfied biohydrogen production. The composition of microorganisms is the principal factor affecting biohydrogen production. Aside from the well-known hydrogen-producing Clostridium spp., several other microorganisms not only coexist but can also significantly affect system performance. The monitoring method established in the present study provides a fast quantification procedure to help operators understand how the system works and therefore quickly respond in operations.