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.     

Characteristics of cured and wild strains of Enterobacter cloacae IIT-BT 08 for the improvement of biohydrogen production

Enterobacter cloacae IIT-BT 08 was found to harbor multiple endogenous plasmids. The plasmids were successfully cured by the combined action of SDS and mitomycin C. The cured strain exhibited an altered pattern of antibiotic and metal resistance. The effect of plasmid curing on biohydrogen production was determined. LP model showed that hydrogen was a growth associated product for both the wild type and the cured strain. However, modified Gompertz equation showed that the productivity of the cured strain was comparable to the wild type. Further, comparative kinetic parameter analysis showed that the maximum specific growth rate (μ_max) and saturation constant (K_s) were 1.26 and 2.2 times higher respectively, for the cured strain as compared to the wild type. Similarly, maintenance coefficient (m) was determined to be function of fermentation time and was lower for the cured strain. This was related to the decrease in plasmid load in the cured strain.     

High biohydrogen yielding Clostridium sp. DMHC-10 isolated from sludge of distillery waste treatment plant

A mesophilic high hydrogen producing strain DMHC-10 was isolated from a lab scale anaerobic reactor being operated on distillery wastewater for hydrogen production. DMHC-10 was identified as Clostridium sp. on the basis of 16S rRNA gene sequencing. Various medium components (carbon and nitrogen sources) and environmental factors (initial pH, temperature of incubation) were optimized for hydrogen production by Clostridium sp. DMHC-10. The strain, in late exponential growth phase, showed maximum hydrogen production (3.35 mol-H₂ mol⁻¹ glucose utilized) at 37 °C, pH 5.0 in a medium supplemented with organic nitrogen source. Butyric acid to acetic acid ratio was ca. 2.3. Hydrogen production declined when organic nitrogen was replaced with inorganic nitrogen.     

Biohydrogen production: Current perspectives and the way forward

Global research is moving forward in developing biological production of hydrogen (biohydrogen) as a renewable energy source to alleviate stresses due to carbon dioxide emissions and depleting fossil fuels resource. Biohydrogen has the potential to replace current hydrogen production technologies relying heavily on fossil fuels through electricity generation. While biohydrogen research is still immature, extensive work on laboratory- and pilot-scale systems with promising prospects has been reported. This work presents a review of advances in biohydrogen production focusing on production pathways, microbiology, as well as bioreactor configuration and operation. Challenges and prospects of biohydrogen production are also outlined.     

Pentoses,hexoses and glycerin as substrates for biohydrogen production: An approach for Brazilian biofuel integration

Biofuels production in Brazil is a traditional activity, and it has becoming more important each year. Within this context, biohydrogen production could exploit residual streams from first generation ethanol (ethanol 1G), second generation ethanol (ethanol 2G) and biodiesel production. Therefore hexoses, pentoses and glycerin were tested as substrates for hydrogen production. Firstly, the effects of different inoculum pretreatments (acid, alkaline and heat) on bacterial communities' performance were evaluated through the levels of Clostridium hydrogenase expression. The heat pretreated inoculum provided the highest yield of H₂ (4.62 mol H₂/mol sucrose) and also the highest level of hydrogenase expression, 64 times higher when compared with untreated inoculum after 72 h. Then C5 and C6 sugars and also glycerin were tested for H₂ production (35 °C and pH 5.5), which resulted in promising yields of H₂: sucrose (4.24 mol H₂/mol sucrose), glucose (2.19 mol H₂/mol glucose), fructose (2.09 mol H₂/mol fructose), xylose (1.88 mol H₂/mol xylose) and glycerin (0.80 mol H₂/mol glycerin).     

Comparative study of biohydrogen production by four dark fermentative bacteria

Dark fermentation is a promising biological method for hydrogen production because of its high production rate in the absence of light source and variety of the substrates. In this study, hydrogen production potential of four dark fermentative bacteria (Clostridium butyricum, Clostridium pasteurianum, Clostridium beijerinckii, and Enterobacter aerogenes) using glucose as substrate was investigated under anaerobic conditions. Batch experiments were conducted to study the effects of initial glucose concentration on hydrogen yield, hydrogen production rate and concentration of volatile fatty acids (VFA) in the effluents. Among the four different fermentative bacteria, C. butyricum showed great performance at 10 g/L of glucose with hydrogen production rate of 18.29 mL-H₂/L-medium/hand specific hydrogen production rate of 3.90 mL-H₂/g-biomass/h. In addition, it was found that the distribution of volatile fatty acids was different among the fermentative bacteria. C. butyricum and C. pasteurianum had higher ratio of acetate to butyrate compared to the other two species, which favored hydrogen generation.     

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.     

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.     

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.     

A pilot-scale high-rate biohydrogen production system with mixed microflora

A pilot-scale high-rate dark fermentative hydrogen production plant has been established in the campus of Feng Chia University to develop biohydrogen production pilot-plant technology. This pilot-plant system is composed of two feedstock storage tanks (0.75 m³ each), a nutrient storage tank (0.75 m³), a mixing tank (0.6 m³), an agitated granular sludge bed fermentor (working volume 0.4 m³), a gas-liquid-solid separator (0.4 m³) and a control panel. The seed mixed microflora was obtained from a lab-scale agitated granular sludge bed bioreactor. This pilot-scale fermentor was operated for 67 days at 35 °C, an organic loading rate (OLR) of 40-240 kg COD/m³/d, and the influent sucrose concentration of 20 and 40 kg COD/m³. Both biogas and hydrogen production rates increased with increasing OLR. However, the biomass concentration (volatile suspended solids, VSS) only increased with an increasing OLR at an OLR range of 40-120 kg COD/m³/d, whereas it decreased when OLR was too high (i.e., 240 kg COD/m³/d). The biogas consisted mainly of H₂ and CO₂ with a H₂ content range of 23.2-37.8%. At an OLR of 240 kg COD/m³/d, the hydrogen content in biogas reached its maximum value of 37% with a hydrogen production rate (HPR) of 15.59 m³/m³/d and a hydrogen yield of 1.04 mol H₂/mol sucrose. This HPR value is much higher than 5.26 m³/m³/d (fermented molasses substrate) and 1.56 m³/m³/d (glucose substrate) reported by other pilot-scale systems. Moreover, HPR was also greatly affected by pH. At an optimal pH of 5.5, the bacterial community became simple, while the efficient hydrogen producer Clostridium pasteurianum was dominant. The factors of energy output compared with the energy input (E_f) ranged from 13.65 to 28.68 on biohydrogen, which is higher than the E_f value on corn ethanol, biodiesel and sugarcane ethanol but in the similar range of cellulosic ethanol.     

Whey waste as potential feedstock for biohydrogen production

In-house isolate Clostridium sp. IODB-O3 was exploited for biohydrogen production using cheese whey waste in batch fermentation. Analysis of cheese whey shows, it is enriched with lactose, lactic acid and protein components which were observed most favourable for biohydrogen production. Biohydrogen yield by IODB-O3 was compared with the cultures naturally occurring in waste solely or in combinations, and found that Clostridium sp. IODB-O3 was the best producer. The maximum biohydrogen yield obtained was 6.35 ± 0.2 mol-H₂/mol-lactose. The cumulative H₂ production (ml/L), 3330 ± 50, H₂ production rate (ml/L/h), 139 ± 5, and specific H₂ production (ml/g/h), 694 ± 10 were obtained. Clostridium sp. IODB-O3 exhibited better H₂ yield from cheese whey than the reported values in literature. Importantly, the enhancement of biohydrogen yield was observed possibly due to absence of inhibitory compounds, presence of essential nutrients, protein and lactic acid fractions which supported better cell growth than that of the lactose and glucose media. Carbon balance was carried out for the process which provided more insights in IODB-O3 metabolic pathway for biohydrogen production. This study may help for effective utilization of whey wastes for economic large scale biohydrogen production.     

Mathematical modelling for biohydrogen production by Clostridium beijerinckii

Hydrogen is an energy source that can be produced by Clostridium sporogenes microorganism. In the present work, modeling of dark fermentation using Clostridium beijerinckii and dextrose as substrate was performed to evaluate how the gases and liquid by-products affect the biological process. A mathematical model was developed according to ADM1. The developed model takes into account biochemical reactions, physicochemical equilibrium as well as mass transfer processes during dark fermentation. Findings revealed that Clostridium beijerinckii reached a yield as high as 3.58 mol of H₂/mol of dextrose and generates by-products in the aqueous phase that may either be used as raw materials in a chemical process. Clostridium beijerinckii is very sensitive to acid media (pH < 5.0) and shows a low rate of biohydrogen production (even the absence of metabolic activity) at pH lower than 4.5. The developed model is able to predict (R² > 0.95) dextrose consumption profile, cumulative biohydrogen production and the maximum concentrations of liquid by-products.     

Techno-economic evaluation of biohydrogen production from wastewater and agricultural waste

The world is facing serious climate change caused in part by human consumption of fossil fuel. Therefore, developing a clean and environmentally friendly energy resource is necessary given the depletion of fossil fuels, the preservation of the earth's ecosystem and self-preservation of human life. Biological hydrogen production, using dark fermentation is being developed as a promising alternative and renewable energy source, using biomass feedstock. In this study, beverage wastewater and agricultural waste were examined as substrates for dark fermentation to produce clean biohydrogen energy.     

Optimization of culture conditions for biohydrogen production from sago wastewater by Enterobacter aerogenes using Response Surface Methodology

Sago wastewater (SWW) causes pollution to the environment due to its high organic content. Annually, about 2.5 million tons of SWW is produced in Malaysia. In this study, the potential of SWW as a substrate for biohydrogen production by Enterobacter aerogenes (E. aerogenes) was evaluated. Response Surface Methodology (RSM) was employed to find the optimum conditions. From preliminary optimization, it was found that the most significant factors were yeast extract, temperature, and inoculum size. According to Face Centered Central Composite Design (FCCCD), the maximum hydrogen concentration and yield were 630.67 μmol/L and 7.42 mmol H₂/mol glucose, respectively, which is obtained from the sample supplemented with 4.8 g/L yeast extract concentration, 5% inoculum, and incubated at the temperature of 31 °C. Cumulative hydrogen production curve fitted by the modified Gompertz equation suggested that H_max, R_max, and λ from this study were 15.10 mL, 2.18 mL/h, and 9.84 h, respectively.     

Fermentative biohydrogen production: Evaluation of net energy gain

Most dark fermentation (DF) studies had resorted to above-ambient temperatures to maximize hydrogen yield, without due consideration of the net energy gain. In this study, literature data on fermentative hydrogen production from glucose, sucrose, and organic wastes were compiled to evaluate the benefit of higher fermentation temperatures in terms of net energy gain. This evaluation showed that the improvement in hydrogen yield at higher temperatures is not justified as the net energy gain not only declined with increase of temperature, but also was mostly negative when the fermentation temperature exceeded 25 °C. To maximize the net energy gain of DF, the following two options for recovering additional energy from the end products and to determine the optimal fermentation temperature were evaluated: methane production via anaerobic digestion (AD); and direct electricity production via microbial fuel cells (MFC). Based on net energy gain, it is concluded that DF has to be operated at near-ambient temperatures for the net energy gain to be positive; and DF + MFC can result in higher net energy gain at any temperature than DF or DF + AD.     

The prospects of brewery waste application in biohydrogen production by photofermentation of Rhodobacter sphaeroides

Selection of the source for biohydrogen (H₂) production is essential, since it affects bacterial metabolism. Pure organic substrates give fast H₂ generation with high yields, but they increase the production cost. Using industrial wastes as a source provides inexpensive energy generation with simultaneous waste utilization. H₂ production by photofermentation of Rhodobacter sphaeroides is monitored during cultivation on brewery waste. Maximum specific growth rate is observed for 5–10% waste containing media. H₂ production by cells, grown on waste, is detected at 48–96 h of growth; it is comparable or higher than that of control medium with expensive carbon and nitrogen sources. N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity of R. sphaeroides membrane vesicles from growth on waste containing media is 1.6–1.7-fold higher compared to control, correlating with enhanced H₂ production. Growth medium containing optimal amount of wastes may be a successful alternative to expensive media for high H₂ yield in R. sphaeroides during photofermentation.     

Continuous biohydrogen production by a degenerated strain of Clostridium acetobutylicum ATCC 824

Degenerated strains of Clostridium acetobutylicum lack the ability to produce solvents and to sporulate, allowing the continuous production of hydrogen and organic acids. A degenerated strain of Clostridium acetobutylicum was obtained through successive batch cultures. Its kinetic characterization showed a similar specific growth rate than the wild type (0.25 h^?1), a higher butyric acid production of 6.8 g·L^?1 and no solvents production. A steady state was reached in a continuous culture at a dilution rate of 0.1 h^?1, with a constant hydrogen production of 507 mL·h^?1, corresponding to a volumetric rate of 6.10 L·L^?1 d^?1, and a yield of 2.39 mol of H₂ per mole of glucose which represents 60% of the theoretical maximum yield. These results suggest that the degeneration is an interesting alternative for hydrogen production with this strain, obtaining a high hydrogen production in a continuous culture with cells in a permanent acidogenic state.     

Enzymatic saccharification and fermentation of paper and pulp industry effluent for biohydrogen production

Paper and pulp industry effluent was enzymatically hydrolysed using crude cellulase enzyme (0.8–2.2FPU/ml) obtained from Trichoderma reesei and from the hydrolysate biohydrogen was produced using Enterobacter aerogenes. The influence of temperature and incubation time on enzyme production was studied. The optimum temperature for the growth of T. reesei was found to be around 29 °C. The enzyme activity of 2.5 FPU/ml was found to produce about 22 g/l of total sugars consisting mainly of glucose, xylose and arabinose. Relevant kinetic parameters with respect to sugars production were estimated using two fraction model. The enzymatic hydrolysate was used for the biohydrogen production using E. aerogenes. The growth data obtained for E. aerogenes were fitted well with Monod and Logistic equations. The maximum hydrogen yield of 2.03 mol H₂/mol sugar and specific hydrogen production rate of 225 mmol of H₂/g cell/h were obtained with an initial concentration of 22 g/l of total sugars. The colour and COD of effluent was also decreased significantly during the production of hydrogen. The results showed that the paper and pulp industry effluent can be used as a substrate for biohydrogen production.