Biohydrogen production from Enterobacter cloacae IIT-BT 08 using distillery effluent

Distillery effluent poses severe environmental pollution problem mainly due to its high organic content. During alcohol fermentation, most of the essential macro- and micro-nutrients get utilized. Therefore, supplementation of these nutrients becomes imperative for the improvement of biohydrogen production. In the present study, starch based distillery effluent was used for dark fermentative hydrogen production using Enterobacter cloacae IIT-BT 08. Hence, this study was undertaken to evaluate the effect of supplementation of yeast extract, malt extract, Fe⁺⁺, Cu⁺⁺ and Mg⁺⁺ on biohydrogen production. The interaction among supplements and their mutual effect on the hydrogen production was studied using five factor–five level central composite design). Optimum hydrogen yield of 7.4 mol H₂/kg COD_reduced was predicted by the model, which showed an excellent correlation with experimental hydrogen yield of 7.38 ± 0.24 mol H₂/kg COD_reduced. An average hydrogen production rate of 80 mL/L h was achieved after supplementation, having 2.2 times higher hydrogen yield as compared to non-supplemented distillery effluent.     

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.     

Advances in biohydrogen production processes: An approach towards commercialization

Biological H₂ production has an edge over its chemical counterpart mainly because it is environmentally benign. Despite having simpler technology, higher evolution rate of H₂ and the wide spectrum of substrate utilization, the major deterrent of anaerobic dark fermentation process stems from its lower achievable yields. Theoretically, the maximum H₂ yield is 4 mol H₂/mol glucose when glucose is completely metabolized to acetate or acetone in the anaerobic process. But it is somewhat difficult to achieve the complete degradation of glucose to carbon dioxide and H₂ through anaerobic dark fermentation. Moreover, this yield appears too low to be economically viable as an alternative to the existing chemical or electrochemical processes of hydrogen generation. Intensive research studies have already been carried out on the advancement of these processes, such as the development of genetically modified microorganism, improvement of the reactor designs, use of different solid matrices for the immobilization of whole cells, development of two-stage processes, and higher H₂ production rates. Maximum H₂ yield is found to be 5.1 mol H₂/mol glucose. However, major bottlenecks for the commercialization of these processes are lower H₂ yield and rate of H₂ production. Competent microbial cultures are required to handle waste materials efficiently, which are usually complex in nature. This will serve dual purposes: clean energy generation and bioremediation. Scale-up studies on fermentative H₂ production processes have been done successfully. Pilot plant trials of the photo-fermentation processes require more attention. Use of cheaper raw materials and efficient biological H₂ production processes will surely make them more competitive with the conventional H₂ generation processes in near future.     

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.     

Phytosynthesized iron oxide nanoparticles and ferrous iron on fermentative hydrogen production using Enterobacter cloacae: Evaluation and comparison of the effects

The effects of FeSO₄ and synthesized iron oxide nanoparticles (0–250 mg/L) on fermentative hydrogen production from glucose and sucrose, using Enterobacter cloacae were investigated, to find out the enhancement of efficiency. The maximum hydrogen yields of 1.7 ± 0.017 mol H₂/mol glucose and 5.19 ± 0.12 mol H₂/mol sucrose were obtained with 25 mg/L of ferrous iron supplementation. In comparison, the maximum hydrogen yields of 2.07 ± 0.07 mol H₂/mol glucose and 5.44 ± 0.27 mol H₂/mol sucrose were achieved with 125 mg/L and 200 mg/L of iron oxide nanoparticles, respectively. These results indicate that the enhancement of hydrogen production on the supplementation of iron oxide nanoparticles was found to be considerably higher than that of ferrous iron supplementation. The activity of E. cloacae in a glucose and sucrose fed systems was increased by the addition of iron oxide nanoparticles, but the metabolic pathway was not changed. The results revealed that the glucose and sucrose fed systems conformed to the acetate/butyrate fermentation type.     

Impact of regulated pH on proto scale hydrogen production from xylose by an alkaline tolerant novel bacterial strain, Enterobacter cloacae DT-1

A hydrogen producing facultative anaerobic alkaline tolerant novel bacterial strain was isolated from crude oil contaminated soil and identified as Enterobacter cloacae DT-1 based on 16S rRNA gene sequence analysis. DT-1 strain could utilize various carbon sources; glycerol, CMCellulose, glucose and xylose, which demonstrates that DT-1 has potential for hydrogen generation from renewable wastes. Batch fermentative studies were carried out for optimization of pH and Fe²⁺ concentration. DT-1 could generate hydrogen at wide range of pH (5–10) at 37 °C. Optimum pH was; 8, at which maximum hydrogen was obtained from glucose (32 mmol/L), when used as substrate in BSH medium containing 5 mg/L Fe²⁺ ion. Decrease in hydrogen partial pressure by lowering the total pressure in the fermenter head space, enhanced the hydrogen production performance of DT-1 from 32 mmol H₂/L to 42 mmol H₂/L from glucose and from 19 mmol H₂/L to 33 mmol H₂/L from xylose. Hydrogen yield efficiency (HY) of DT-1 from glucose and xylose was 1.4 mol H₂/mol glucose and 2.2 mol H₂/mol xylose, respectively. Scale up of batch fermentative hydrogen production in proto scale (20 L working volume) at regulated pH, enhanced the HY efficiency of DT-1 from 2.2 to 2.8 mol H₂/mol xylose (1.27 fold increase in HY from laboratory scale). 84% of maximum theoretical possible HY efficiency from xylose was achieved by DT-1. Acetate and ethanol were the major metabolites generated during hydrogen production.     

Bio-hydrogen production from glycerol by immobilized Enterobacter aerogenes ATCC 13048 on heat-treated UASB granules as affected by organic loading rate

Bio-hydrogen production from glycerol by immobilized Enterobacter aerogenes ATCC 13048 on heat-treated upflow anaerobic sludge blanket (UASB) granules was examined in a UASB reactor. The organic loading rate (OLR) was optimized in order to maximize the hydrogen production rate (HPR). The maximum hydrogen content (37.1% and 24.2%) and HPR (9 and 6.2 mmol H₂/L h) were achieved at the optimum OLR of 50 g/L d using pure and waste glycerol as the substrate, respectively. The major soluble metabolite products (SMPs) were ethanol, 1,3-propanediol (1,3-PD), formic acid, and acetic acid. The microbial community and microbial structure, analyzed by fluorescent in situ hybridization (FISH) and scanning electron microscopy (SEM), revealed that the predominant hydrogen producers were E. aerogenes ATCC 13048 and firmicutes bacteria including Clostridium, Bacillus, and Dialister sp.     

Microbial fuel cell of Enterobacter cloacae: Effect of anodic pH microenvironment on current, power density, internal resistance and electrochemical losses

The aim of this study was to evaluate the electrical generating characteristics of a double-chamber microbial fuel cell with pure culture of Enterobacter cloacae as a function of pH variation of its microenvironment. The performance was analyzed over 5 batch cycles (around 16-21 days) with community wastewater by adjusting the pH between 6.5 and 9.5. Operations under pH 6.5 (0.40 mA) and 7.4 (0.42 mA) showed highly effective performance with respect to maximum current generation and maximum power density corresponding to pH 8.5 (0.38 mA) and 9.5 (0.27 mA). This better performance could be attributed to the low internal resistance under the low pH microenvironment. Short experiments conducted for 60 min with different external resistances to calculate maximum current and internal resistance were remarkably shown to increase current and decrease internal resistance with respect to pH 6.5 and 7.4. Maximum power density obtained from the polarization curve was observed to follow the same behavior as current generation with a maximum of 0.0042 mW/cm² for pH 7.4. COD removal efficiencies increased as a function of pH, and maximum amounted to pH 9.5 respectively, due to long operating time. Coulombic efficiency attained different trend with a maximum of 3.4% at pH 6.5. Low pH of 6.5 and 7.4 were associated with dominant electrochemical activity, which was proved by cyclic voltammetry. These results demonstrate the importance of pH environment in the biocurrent generation with wastewater containing E. cloacae.     

Bioreactor for glycerol conversion into H2 by bacterium Enterobacter aerogenes

Glycerol was used as a substrate for H₂ production by bacterium Enterobacter aerogenes in the test tubes and bioreactor. A BioFlo/CelliGen 115 bioreactor (10 L working volume) was utilized to conduct the experiments for conversion of glycerol into H₂ by E. aerogenes cells. The highest H₂ production rate was observed under 2% glycerol in the culture medium. The glycerol uptake efficiency by bacteria in the bioreactor was found to be 65% during the 6 day period, matching glycerol uptake efficiency observed in the test tubes experiment (65%).Hydrogen production from glycerol (2% glycerol, v/v) by E. aerogenes in the bioreactor and test tubes was measured over the 6 days, showing the maximal H₂ rate at 650 mL g⁻¹ dry weight h⁻¹. The yield of H₂ production from glycerol at 0.89 mol/mol in the bioreactor was high, corresponding to the theoretical yield of 1 mol of H₂ per 1 mol of glycerol.     

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.     

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.     

Simultaneous production of hydrogen and ethanol from waste glycerol by Enterobacter aerogenes KKU-S1

Factors affecting simultaneous hydrogen and ethanol production from waste glycerol by a newly isolated bacterium Enterobacter aerogenes KKU-S1 were investigated employing response surface methodology (RSM) with central composite design (CCD). The Plackett-Burman design was first used to screen the factors influencing simultaneous hydrogen and ethanol production, i.e., initial pH, temperature, amount of vitamin solution, yeast extract (YE) concentration and glycerol concentration. Results indicated that initial pH, temperature, YE concentration, and glycerol concentration had a statistically significant effect (p ≤ 0.05) on hydrogen production rate (HPR) and ethanol production. The significant factors were further optimized using CCD. Optimum conditions for simultaneously maximizing HPR and ethanol production were YE concentration of 1.00 g/L, glycerol concentration of 37 g/L, initial pH of 8.14, and temperature of 37 °C in which a maximum HPR and ethanol production of 0.24 mmol H₂/L h and 120 mmol/L were achieved.     

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.     

Fermentative hydrogen production by the newly isolated Enterobacter asburiae SNU-1

A new fermentative hydrogen-producing bacterium was isolated from a domestic landfill and identified as Enterobacter asburiae using 16S rRNA gene sequencing and DNA–DNA hybridization methods. The isolated bacterium, designated as Enterobacter asburiae SNU-1, is a new species that has never been examined as a potential hydrogen-producing bacterium. This study examined the hydrogen-producing ability of Enterobacter asburiae SNU-1. During fermentation, the hydrogen was mainly produced in the stationary phase. The hydrogen yield based on the formate consumption was 0.43 mol hydrogen/mol formate. This strain was able to produce hydrogen over a wide range of pH (4–7.5), with the optimum pH being pH 7. The level of hydrogen production was also affected by the initial glucose concentration, and the optimum value was found to be 25 g glucose/l. The maximum and overall hydrogen productivities were 398 and 174 ml/l/hr, respectively, at pH 7 with an initial glucose concentration of 25 g/l. This strain could produce hydrogen from glucose and many other carbon sources such as fructose, sucrose, and sorbitol.     

Regulation of hydrogen production by Enterobacter aerogenes by external NADH and NAD

Experiments involving the addition of external nicotinamide adenine dinucleotide, reduced form (NADH) or nicotinamide adenine dinucleotide (NAD⁺) have been designed to examine how the hydrogen in Enterobacter aerogenes is liberated by NADH or NAD⁺. The addition of external NADH or NAD⁺ was found to regulate hydrogen production by E. aerogenes in resting cells, batch cultures, and chemostat cultures. Particularly in chemostat cultivation, with the external addition of NADH, hydrogen production via the NADH pathway was decreased, while that via the formate pathway was increased; in the end, the overall hydrogen p was decreased. The addition of NAD⁺, on the other hand, gave the opposite results. The membrane-bound hydrogenase was found to play a central role in regulating hydrogen production. The occurrence of NADH oxidation (NAD⁺ reduction) on the cell membrane resulted in an electron flow across the membrane; this changed the oxidation state and metabolic pattern of the cells, which eventually affected the hydrogen evolution.     

Enhancement in hydrogen production by co-cultures of Bacillus and Enterobacter

Defined co-cultures of hydrogen (H₂) producers belonging to Citrobacter, Enterobacter, Klebsiella and Bacillus were used for enhancing the efficiency of biological H₂ production. Out of 11 co-cultures consisting of 2–4 strains, two co-cultures composed of Bacillus cereus EGU43, Enterobacter cloacae HPC123, and Klebsiella sp. HPC793 resulted in H₂ yield up to 3.0 mol mol⁻¹ of glucose. Up-scaling of the reactor by 16-fold resulted in a corresponding increase in H₂ production with an actual evolution of 7.44 L of H₂. It constituted 58.2% of the total biogas. Continuous culture evolution of H₂ by co-cultures (B. cereus EGU43 and E. cloacae HPC123) immobilized on ligno-cellulosic materials resulted in 6.4-fold improvement in H₂ yield compared to free floating bacteria. This synergistic influence of B. cereus and E. cloacae can offer a better strategy for H₂ production than undefined or mixed cultures.     

Hydrogen production by Escherichia coli ΔhycA ΔlacI using cheese whey as substrate

This study reports a fermentative hydrogen production by Escherichia coli using cheese whey as substrate. To improve the biohydrogen production, an E. coli ΔhycA ΔlacI strain (WDHL) was constructed. The absence of hycA and lacI genes had a positive effect on the biohydrogen production. The strain produced 22% more biohydrogen in a shorter time than the wild-type (WT) strain. A Box-Behnken experimental design was used to optimize pH, temperature and substrate concentration. The optimal initial conditions for biohydrogen production by WDHL strain were pH 7.5, 37 °C and 20 g/L of cheese whey. The specific production rate was improved from 3.29 mL H₂/optical density at 600 nm (OD_600nm) unit-h produced by WDHL under non-optimal conditions to 5.88 mL H₂/OD_600nm unit-h under optimal conditions. Using optimal initial conditions, galactose can be metabolized by WDHL strain. The maximum yield obtained was 2.74 mol H₂/mol lactose consumed, which is comparable with the yield reached in other hydrogen production processes with Clostridium sp. or mixed cultures.     

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.     

Bio-hydrogen production enhancement by co-cultivating Rhodopseudomonas palustris WP3-5 and Anabaena sp. CH3

Photobiological H₂ production is a promising method for renewable energy development. An innovative system that co-cultivating Rhodopseudomonas palustris WP3-5 and Anabaena sp. CH3 was carried out to estimate the effect of co-cultivation on H₂ production enhancement. H₂ production prolongation and enhancement were observed due to the light and metabolic compatibility of these two strains. Co-culture system served by acetate and fructose as carbon source can accumulate H₂ in 140.8 mL, almost double than the sum of individuals. Moreover, the enhancement of H₂ production was significantly affected by the mixed ratio of two strains. The mixed ratio (WP3-5:CH3) of 1:2 showed a highest H₂ production rate in 44.8 mL-H₂/L-culture/h, and both 2:1 and 1:2 exhibited a relatively high substrate conversion efficiency during the latest period of cultivation, whereas the mixed ratio of 1:1 and 3:1 only revealed a prolongation in H₂ production due to metabolic compatibility of two strains.