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%.     

Phototrophic hydrogen production from glucose by pure and co-cultures of Clostridium butyricum and Rhodobacter sphaeroides

Phototrophic hydrogen production from glucose by pure and co-cultures of Clostridium butyricum and Rhodobacter sphaeroides was studied in batch experiments. Results showed that in all batches hydrogen was produced after a lag phase of about 10 h; pure culture of R. sphaeroides produced hydrogen at rates substantially lower than C. butyricum. In co-culture systems, R. sphaeroides even with cell populations 5.9 times higher still could not compete with C. butyricum for glucose. In co-culture systems, R. sphaeroides syntrophically interacted with C. butyricum, using the acetate and butyrate produced by the latter as substrate for hydrogen production. Hydrogen production was ceased in all batches when the pH was lowered to the level of pH 6.5, resulting from the accumulation of fatty acids. It was also demonstrated in this study that fluorescence in situ hybridization (FISH) was an effective means for the quantification of the relative abundance of individual bacteria in a co-culture system.     

Fermentative hydrogen production in recombinant Escherichia coli harboring a [FeFe]-hydrogenase gene isolated from Clostridium butyricum

The [FeFe]-hydrogenase (hydA) from Clostridium butyricum TERI BH05-2 strain was isolated to elucidate its molecular characterization. A 1953 bp DNA fragment encompassing the ORF and the putative promoter region of hydA gene was PCR amplified and subcloned into pGEM^®-T-Easy cloning vector (pGEM^®-T-hydA). The hydA DNA sequence revealed the presence of a 1725 bp length ORF (including the stop codon) encoding 574 amino acids with a predicted isoelectric point and molecular mass of 6.8 and 63097.67 Da, respectively. The hydA ORF was PCR amplified from pGEM^®-T-hydA and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-5X-hydA) and transformed into Escherichia coli BL-21. The recombinant E. coli BL-21 was investigated for fermentative hydrogen production under anaerobic condition from glucose. Heterologous expression of the Clostridium butyricum hydA resulted in 1.9 fold increase in hydrogen productivity as compared to that from the wild type strain, C. butyricum TERI BH05-2. The hydrogen yield of the recombinant strain was 3.2 mol H₂/mol glucose, 1.68 fold higher than the wild type parent strain.     

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.     

Optimization of conditions for hydrogen production from complex dairy wastewater by anaerobic sludge using desirability function approach

Present study deals with the multiple-response optimization for biohydrogen production using anaerobic sludge and outstanding approach to overcome the drawbacks of conventional response surface methodology (RSM). Dairy wastewater was used as source in batch fermentation was followed for this study. Response surface methodology (RSM), based on a three level, four variable Box–Behnken design, was employed to obtain the best possible combination of substrate concentration, pH, COD/N ratio and COD/P ratio for maximum H₂ yield (HY) and specific hydrogen production rate (SHPR). Experimental data were evaluated by applying RSM integrating a desirability function approach. The optimum H₂ yield and SHPR conditions were: substrate concentration 15.3 g COD/L, pH 5.5, COD/N ratio 100.5 and COD/P ratio 120 with maximum overall desirability D of 0.94. The confirmation experiment under these optimal condition showed a HY and SHPR of 13.54 mmol H₂/g COD and 29.91 mmol H₂/g-VSS.d, respectively. This was only 0.22% and 0.20%, respectively, different from the predicted values, suggesting that the desirability function approach with RSM was a useful technique to get the maximum H₂ yield and SHPR simultaneously.     

Value analysis for commercialization of fermentative hydrogen production from biomass

In addition to producing hydrogen gas, biohydrogen production is also used to process wastewater. Therefore, this study specifically conducted value analyses of two different scenarios of fermentative hydrogen production from a biomass system: to increase the value of a wastewater treatment system and to specifically carry out hydrogen production. The analytical results showed that fermentative hydrogen production from a biomass system would increase the value of a wastewater treatment system and make its commercialization more feasible. In contrast, fermentative hydrogen production from a biomass system designed specifically for producing hydrogen gas would have a lower system value, which indicated that it is not yet ready for commercialization. The main obstacle to be overcome in promoting biohydrogen production technology and system application is the lack of sales channels for the system's products such as hydrogen gas and electricity. Thus, in order to realize its commercialization, this paper suggests that governments provide investment subsidies for the use of biohydrogen production technology and establish a buy-back tariff system for fuel cells.     

Effect of arabinose concentration on dark fermentation hydrogen production using different mixed cultures

Dark fermentation hydrogen production from arabinose at concentrations ranging between 0 and 100 g/L was examined in batch assays for three different mixed anaerobic cultures, two suspended sludges (S1, S2) obtained from two different sludge digesters and one granular sludge (G) obtained from a brewery wastewater treatment plant. After elimination of the methanogenic activity by heat treatment, all mixed cultures produced hydrogen, and optimal hydrogen rates and yields were generally observed for concentrations between 10 and 40 g/L of substrate. Higher concentrations of arabinose up to 100 g/L inhibited hydrogen production, although the effect was different from inoculum to inoculum. It was evident that the granular biomass was less affected by increased initial arabinose concentrations when calculating the rate of decrease in hydrogen yields versus arabinose concentrations, compared against the two suspended sludges.     

Predictive functional profiling of microbial communities in fermentative hydrogen production system using PICRUSt

PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) is based on 16S rRNA sequencing data, which can analyze the microbial functions in fermentation system. In this study, PICRUSt was adopted to figure out the direct evidence of functions of the microbial community in biohydrogen production system. PICRUSt analysis demonstrated that metabolic flux shifted from acid-producing pathway to hydrogen-producing pathway, and the hydrogen-consuming homoacetogenic pathway was eliminated by ionizing radiation pretreatment at 5 kGy. KEGG (Kyoto Encyclopedia of Genes and Genomes) based functional genes analysis showed enriched energy metabolism and diminished homoaceto gene, which enhanced the hydrogen production. However, the diminished carbohydrate metabolism indicated that the pretreatment reduced the activity of microbial consortia to degrade various substrates. This study suggested that PICRUSt is an effective approach to analyze the functional profiling of microbial community in fermentative hydrogen production system.     

Effect of Ni2+ concentration on fermentative hydrogen production using waste activated sludge as substrate

The influence of Ni²⁺ concentration on biohydrogen production was investigated using waste activated sludge as substrate. The degradation of substrate, accumulation of volatile fatty acids (VFAs) and distribution of microbial community were analyzed to provide information for influencing mechanisms of Ni²⁺ addition. The experimental results demonstrated that the efficiency of hydrogen fermentation from waste activated sludge could be significantly improved. The optimal Ni²⁺ concentration was 5 mg/L, and under this concentration, the cumulative hydrogen production was 1.29 times of the control group. The degradation of soluble chemical oxygen demand (SCOD) increased from 25.21% to 27.69% when the added Ni²⁺ concentration was 5 mg/L. The analysis of microbial community distribution revealed that Ni²⁺ decreased the microbial diversity, and provided more suitable condition for the microbial growth and activity of hydrogen-producers. Citrobacter was the dominant hydrogen-producers in the control group, they changed into Enterococcus when 5 mg/L Ni²⁺ was added. Besides, the proportion of Clostridium_sensu_stricto_1, which is regarded as the primary hydrogen-producing bacteria under numerous operating conditions, was also significantly increased in the presence of Ni²⁺.     

Mesophilic fermentative hydrogen production from sago starch-processing wastewater using enriched mixed cultures

Biohydrogen (bioH₂) production from starch-containing wastewater is an energy intensive process as it involves thermophilic temperatures for hydrolysis prior to dark fermentation. Here we report a low energy consumption bioH₂ production process with sago starch powder and wastewater at 30 °C using enriched anaerobic mixed cultures. The effect of various inoculum pretreatment methods like heat (80 °C, 2 h), acid (pH 4, 2.5 N HCl, 24 h) and chemical (0.2 g L⁻¹ bromoethanesulphonic acid, 24 h) on bioH₂ production from starch powder (1% w/v) showed highest yield (323.4 mL g⁻¹ starch) in heat-treatment and peak production rate (144.5 mL L⁻¹ h⁻¹) in acid-treatment. Acetate (1.07 g L⁻¹) and butyrate (1.21 g L⁻¹) were major soluble metabolites of heat-treatment. Heat-treated inoculum was used to develop mixed cultures on sago starch (1% w/v) in minimal medium with 0.1% peptone-yeast extract (PY) at initial pH 7 and 30 °C. The effect of sago starch concentration, pH, inoculum size and nutrients (PY and Fe ions) on batch bioH₂ production showed 0.5% substrate, pH 7, 10% inoculum size and 0.1% PY as the best H₂ yielding conditions. Peak H₂ yield and production rate were 412.6 mL g⁻¹ starch and 78.6 mL L⁻¹ h⁻¹, respectively at the optimal conditions. Batch experiment results using sago-processing wastewater under similar conditions showed bioH₂ yield of 126.5 mL g⁻¹ COD and 456 mL g⁻¹ starch. The net energy was calculated to be +2.97 kJ g⁻¹ COD and +0.57 kJ g⁻¹ COD for sago starch powder and wastewater, respectively. Finally, the estimated net energy value of +2.85 × 10¹³ kJ from worldwide sago-processing wastewater production indicates that this wastewater can serve as a promising feedstock for bioH₂ production with low energy input.     

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.     

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.     

Enhancing biohydrogen production from sugar industry wastewater using metal oxide/graphene nanocomposite catalysts in microbial electrolysis cell

Biohydrogen production through Microbial Electrolysis Cell (MEC) has drifted towards the development of suitable cost-effective cathode catalysts. In this study, two graphene hybrid metal oxide nanocomposites were used as catalysts to investigate hydrogen production in the MEC operated with sugar industry wastewater as substrate against phosphate buffer catholyte. Electrochemical characterizations exposed the better performance of NiO.rGO coated cathode which showed lesser overpotential at 600 mV and overall lowest resistance in the Nyquist plots than Ni-foam and Co₃O₄.rGO cathodes. The experimental results showed that at an applied voltage 1.0 V, NiO.rGO nanocomposite had exhibited maximum hydrogen production rate of 4.38 ± 0.11 mmol/L/D, Coloumbic efficiency of 65.6% and Cathodic hydrogen recovery of 20.8% respectively. The MEC performance in terms of biohydrogen production was 1.19 and 2.68 times higher than Co₃O₄.rGO and uncoated Ni-Foam. Hence, economical hybrid nanocomposite catalysts were demonstrated in MEC using industrial effluent for energy and environment sustainability.     

Feasible pretreatment of textile wastewater for dark fermentative hydrogen production

In this study, the yield of hydrogen production was investigated under different feedstock pretreatment conditions. The feedstock for dark fermentative hydrogen production was textile wastewater which was obtained from the de-sizing process in a textile factory, located in northern Taiwan. The wastewater was pretreated with activated carbon, cation exchange resin or was not pretreated before being fed into the batch bottles. Biohydrogen production was carried out in a batch reactor with the sludge of mixed-culture using the feedstock from the pretreated wastewater. The sludge was obtained from the Taichung municipal wastewater treatment plant. The yield of hydrogen production using the two pretreatment methods or non – treatment were compared.     

Emerging technologies for hydrogen production from wastewater

Wastewater treatment is essential to shield the environment. The production of H₂ is substantial for prospering its applications in diversified sectors; hence the study of wastewater treatment for H₂ production is accomplished. Various technologies have been developed and studied considering the potential of wastewater to generate hydrogen-rich gas. These technologies have different mechanisms, diversified setups, and processes. Perhaps these technologies are proven to be exceptional exposures for hydrogen production. Fortunately, a valuable contribution to the environment and the H₂ economy is that some technological processes have been promoted to synthesize H₂ from lab scale to pilot scale. Contemplating such comprehensive exposure to H₂ synthesis from wastewater, the critical information of eight emerging technologies, including their mechanism and reaction parameters influencing the process, pros, cons, and future developmental scopes, are described in this review by classifying them into three different classes, namely light-dependent technologies, light-independent technologies, and other technologies.     

An integrated system development including PEM fuel cell/biogas purification during acidogenic biohydrogen production from dairy wastewater

Biohydrogen production from dairy wastewater with subsequent biogas purification by hollow fiber membrane module was investigated in this study. The purified and not purified (raw) biohydrogen were used as fuel in polymer electrolyte membrane (PEM) fuel cell. Furthermore, the effect of CO₂ on the performance of PEM fuel cell was evaluated considering cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and polarization curves. The maximum H₂ production rate was 0.015 mmol H₂/mol glucose and the biohydrogen concentration in biogas was ranged 33%–60% (v/v). CO₂/H₂ selectivity decreased with increasing pressure and maximum selectivity was obtained as 4.4 at feed pressure of 1.5 bar. The electrochemical active surface (EASA) areas were decreased with increasing CO₂ ratio. The maximum power densities were 0.2, 0.08 and 0.045 W cm⁻² for 100%, 80% and 60% (v/v) H₂, respectively. The results indicated that integrated PEM fuel cell/biogas purification system can be used as a potential clean energy sources during acidogenic biohydrogen production from dairy wastewater.     

Effect of the nitrogen source on the hydrogen production metabolism and hydrogenases of Clostridium butyricum CWBI1009

Investigations were carried out to determine the effect of various concentrations of organic and ammonium nitrogen sources on fermentative hydrogen production by the strain Clostridium butyricum CWBI1009. The results indicated that the H₂-producing metabolism of the strain is favoured within the range (0.56–0.062 g_N/L) of peptone and (NH₄)₂SO₄. Optimal overall performance (i.e. 1.43 ± 0.08 mol H₂/mol glucose and 1.08 ± 0.03 mL_H2/h, respectively) was achieved with 0.062 g_N/L of casein peptone. The study of the amino acid uptake and the gene expression pattern for four [FeFe]-hydrogenases and the nitrogenase showed that nitrogen was in excess in all the experiments with a nitrogen concentration above 0.062 g_N/L and, at that optimal concentration, the expression of the HydB2 gene would be responsible for the much higher H₂ yield.     

Effect of system optimizing conditions on biohydrogen production from herbal wastewater by slaughterhouse sludge

The study evaluates the biohydrogen production from herbal wastewater as the substrate by the enriched mixed slaughterhouse sludge as the seed source. In the following experiments, batch-fermentations are carried out with the optimum substrate concentrations, fermentation pH and fermentation temperature to observe the effects of H₂ production, hydrogen yield and other fermentation end products at different conditions. The hydrogen production is increased as substrate concentration increased up to 8 g COD/L WW, but drastically decreased at 10 g COD/L WW. When the pH of fermentation is controlled to 6.5, a maximum amount of hydrogen yield could be obtained. The hydrogen production is maximum at 50 °C (930 ± 30 mL/L WW) compared to 30 °C (436 ± 16 mL/L WW). Acid-forming pathway with butyric acid as a major metabolite dominated the metabolic flow during the hydrogen production. The experimental results indicated that effective hydrogen production from the herbal wastewater could be obtained by thermophilic acidogenesis at proper operational conditions.