Impairment of NADH dehydrogenase for increased hydrogen production and its effect on metabolic flux redistribution in wild strain and mutants of Enterobacter aerogenes

An NADH dehydrogenase encoded by the nuo cluster was isolated and impaired by knocking out the nuoB gene in Enterobacter aerogenes to examine its effect on hydrogen production. Three nuoB-deleted mutant strains were constructed from the wild-type strain E. aerogenes IAM1183 and two recombinant strains, IAM1183-A (ΔhycA) and IAM1183-O (ΔhybO), from which the hycA and hybO genes had already been deleted previously, respectively. Compared with the performance of the wild-type strain, the overall hydrogen production of the mutants IAM1183-B (ΔnuoB), IAM1183-AB (ΔhycA/ΔnuoB) and IAM1183-BO (ΔhybO/ΔnuoB) was increased by 49.2%, 54.0%, and 52.4% in batch culture, respectively. The hydrogen yields from glucose by the three mutants IAM1183-B, IAM1183-AB, IAM1183-BO were 1.38, 1.49, and 1.39 mol H₂/mol glucose, respectively, while it was 1.16 mol H₂/mol glucose in the wild-type strain. Metabolic flux analysis indicated that all three mutants exhibited reduced fluxes to lactate production, and enhanced fluxes toward the generation of hydrogen, acetate, ethanol, succinate and 2,3-butanediol. Both the formate pathway and the NADH pathway contributed to increased hydrogen production in the mutant strains. The assay of 4 NADH-mediated enzyme activities (H₂ase, LDH, ADH and BDDH) was in accordance with the redistributions of the metabolic fluxes in the mutant strains.     

Cloning and knockout of formate hydrogen lyase and H2-uptake hydrogenase genes in Enterobacter aerogenes for enhanced hydrogen production

A 5431-bp DNA fragment partially encoding the formate hydrogen lyase (FHL) gene cluster hycABCDE was isolated and identified from Enterobacter aerogenes IAM1183 chromosomal DNA. All the five putative gene products showed a high degree of homology to the reported bacterial FHL proteins. The gene hycA, encoding the FHL repressor protein, and hybO, encoding the small subunit of the uptake hydrogenase, were targeted for genetic knockout for improving the hydrogen production. The pYM-Red recombination system was adopted to form insertional mutations in the E. aerogenes genome, thereby creating mutant strains of IAM1183-A (△hycA), IAM1183-O (△hybO), and IAM1183-AO (△hycA/△hybO double knockout). The hydrogen production experiments with these mutants showed that the maximum specific hydrogen productivities of IAM1183-A, IAM1183-O, and IAM1183-AO were 2879.466 ± 38.59, 2747.203 ± 13.25 and 3372.019 ± 4.39 (ml h⁻¹ g⁻¹dry cell weight), respectively, higher than that of the wild strain (2321.861 ± 15.34 ml h⁻¹ g⁻¹dry cell weight). The total H₂ yields by the three mutants IAM1183-A, IAM1183-O and IAM1183-AO were 0.73, 0.78, and 0.83 mol-H₂/mol glucose, respectively, while the wild-type IAM1183 was only 0.65 mol-H₂/mol glucose. The metabolites of the mutants including acetate, ethanol, 2,3-butanediol and succinate were all increased compared with that of the wild type, implying the changed metabolic flux by the mutation. In the fermentor cultivation with IAM1183△hycA/△hybO, the total hydrogen volume after 16 h cultivation reached 4.4 L, while that for the wild type was only 2.9 L.     

Perturbation of formate pathway for hydrogen production by expressions of formate hydrogen lyase and its transcriptional activator in wild Enterobacter aerogenes and its mutants

To examine perturbation effects of formate pathway on hydrogen productivity in Enterobacter aerogenes (Ea), formate dehydrogenase FDH-H gene (fdhF) and formate hydrogen lyase activator protein FHLA gene (fhlA) originated from Escherichia coli, were overexpressed in the wild strain Ea, its hycA-deleted mutant (A) by knockout the formate hydrogen lyase repressor and hybO-deleted mutant (O) by knockout of the uptake hydrogenase, respectively. Overexpression of fdhF and fhlA promoted cell growth and volumetric hydrogen production rates of all the strains, and the hydrogen production per gram cell dry weight (CDW) for Ea, A and O was increased by 38.5%, 21.8% and 5.25%, respectively. The fdhF and fhlA overexpression improved the hydrogen yield per mol glucose of strains Ea and A, but declined that of strain O. The increase of hydrogen yield of the strain Ea with fdhF and fhlA expression was mainly attributed to the increase of formate pathway, while for the mutant A, the improved hydrogen yield with fdhF and fhlA expression was mainly due to the increase of NADH pathway. Analysis of the metabolites and ratio of ethanol-to-acetate showed that the cellular redox state balance and energy level were also changed for these strains by fdhF and fhlA expression. These findings demonstrated that the hydrogen production was not only dependent on the hydrogenase genes, but was also affected by the regulation of the whole metabolism. Therefore, fdhF and fhlA expression in different strains of E. aerogenes could exhibit different perturbation effects on the metabolism and the hydrogen productivity.     

Hydrogen production of Enterobacter aerogenes altered by extracellular and intracellular redox states

Enterobacter aerogenes HU-101, tested for its hydrogen production in batch cultures on various substrates, produced the highest amount of hydrogen when the substrate was glycerol. The yield of hydrogen is a function of the degree to which the substrates are reduced. To examine the effect of intracellular redox state on hydrogen yield, glucose-limiting chemostat cultures were carried out at various pH using strain HU-101 and its mutant AY-2. For both strains, the molar yield and the production rate of hydrogen and the hydrogenase activity in the cell-free extract were optimal at the culture pH of 6.3. The highest NADH/NAD ratio in both strains was also observed at pH 6.3, at which the ratio in AY-2 was more than two-fold that of HU-101. Furthermore, NAD(P)H-dependent hydrogen formation was observed in the cell-free extract of AY-2, and hydrogenase activity was found not in the cytoplasmic but in the cell membrane fraction, suggesting that a high intracellular redox state, that is a high NADH/NAD ratio, would accelerate hydrogen production by driving membrane-bound NAD(P)H-dependent hydrogenase.     

Effects of increasing the NAD(H) pool on hydrogen production and metabolic flux distribution in Enterobacter aerogenes mutants

The effects of combining two strategies, recycling NAD and improving the availability of NADH, on hydrogen production in Enterobacter aerogenes were investigated. The NAD synthetase encoded by nadE gene was homologously overexpressed in AB91002-O, which had been obtained previously, to increase the intracellular concentration of the NAD(H/⁺) pool. This overexpression was duplicated in mutant strains in which the phosphoenolpyruvate carboxylase (PEPC) gene (ppc) and hybO gene were knocked out, yielding AB91102-OP (ΔhybO/Δppc), AB91102-ON (ΔhybO/nadE), and AB91102-OP/N (ΔhybO/Δppc/nadE). Chemostat experiments showed that the total NAD(H) pool size in AB91102-ON increased 2-fold compared with the control strain AB91102-OC, but the NADH/NAD⁺ ratio decreased by 24%. Metabolic analysis of batch experiments indicated that a larger NAD(H/⁺) pool and inactivation of PEPC led to a significant shift in metabolic patterns, whereas a smaller NADH/NAD⁺ ratio improved glucose uptake. Thus, compared with the control strain, the hydrogen yields per glucose of the mutant strains AB91102-OP, AB91102-ON, and AB91102-OP/N were enhanced by 36.2%, 66.0%, and 149%, respectively, and the total volumes of hydrogen production increased by 27%, 165%, and 301%, respectively. The maximum hydrogen production of 5.1 L/L was achieved by AB91102-OP/N, suggesting that the double modification strategy exhibits markedly positive synergistic effects on hydrogen production.     

Isolation of Clostridium perfringens strain W11 and optimization of its biohydrogen production by genetic modification

Clostridium perfringens strain W11, which we previously identified as the major hydrogen producer in a hydrogen-producing microbial flora, was isolated in this study. The hydrogen yield from sucrose of this strain was 1.53 mol H₂/mol hexose. To exclude potential safety problems, the plc gene, encoding an alpha toxin protein, was permanently knocked out using the Targetron gene knockout system, creating strain W12. Strains W11 and W12 both produced lactate, acetate, and butyrate during hydrogen production. Furthermore, yields of these metabolites and hydrogen were near-identical by the two strains. When the ldh gene encoding lactate dehydrogenase in strain W12 was deleted, the hydrogen yield and acetate and butyrate concentrations in the resulting mutant, W13, increased by 51%, 26%, and 57%, respectively. Lactate production by strain W13 decreased almost to zero. The growth rates of the wild-type strain W11 and its mutant derivatives were similar.     

Effect of biohydrogen by overexpressing small RNA RyhB combined with ldh impairment in novel Klebsiella sp. FSoil 024

Biotechnological hydrogen production is considered as an environmentally sustainable alternative to petrochemical sources or electrolysis. Here, disruption of lactate dehydrogenase (LDH) and metabolic regulation via the small RNA RyhB were adopted to improve hydrogen production in the novel Klebsiella sp. strain FSoil 024. The hydrogen production of FSoil 024-L (Δldh) and FSoil 024-L/R (Δldh/RyhB) from glucose in a 5-L fermenter respectively increased by 40 and 50% compared to the wild type. When glycerol was adopted as a more favorable substrate, FSoil 024-L generated 3.3 L/L of hydrogen after 52 h of fermentation, implying its great potential for the utilization of crude glycerol to produce hydrogen. Overexpression of RyhB downregulated formate biosynthesis in FSoil 024, thereby redirecting NADH toward the hydrogen production pathway. This finding provides new insights into the role of cellular reducing power in hydrogen metabolism and establishes a rationale for improving hydrogen production.     

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.     

An in silico re-design of the metabolism in Thermotoga maritima for increased biohydrogen production

Microbial hydrogen production is currently hampered by lack of efficiency. We examine how hydrogen production in the hyperthermophilic bacterium Thermotoga maritima can be increased in silico. An updated genome-scale metabolic model of T. maritima was used to i) describe in detail the H₂ metabolism in this bacterium, ii) identify suitable carbon sources for enhancing H₂ production, and iii) to design knockout strains, which increased the in silico hydrogen production up to 20%. A novel synthetic oxidative module was further designed, which connects the cellular NADPH and ferredoxin pools by inserting into the model a NADPH-ferredoxin reductase. We then combined this in silico knock-in strain with a knockout strain design, resulting in an in silico production strain with a predicted 125% increase in hydrogen yield. The in silico strains designs presented here may serve as blueprints for future metabolic engineering efforts of T. maritima.     

Biohydrogen production by immobilized Enterobacter aerogenes on functionalized multi-walled carbon nanotube

Hydrogen production by immobilized Enterobacter aerogenes on functionalized multi-walled carbon nanotube (MWCNT-COOH) in repeated batch mode was studied. Fourier transform infrared (FTIR) spectroscopy and field emission scanning electron microscopy (FESEM) were employed to confirm immobilization of E. aerogenes successfully. The effect of MWCNT-COOH concentrations (0.2, 0.6, and 1.2 mg/mL) on hydrogen production was investigated. The present study showed that immobilized E. aerogenes on 1.2 mg/mL MWCNT-COOH resulted in higher hydrogen yield (2.2 moL/mol glucose), hydrogen production rate (2.72 L/L.h), and glucose degradation efficiency (96.20%) and shorter the lag phase (1 h) compared to the free E. aerogenes. Modified Gompertz and Logistic models were employed to predict the cumulative hydrogen production successfully.     

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.     

Influence of sulfuric acid concentration on biohydrogen production from rice mill wastewater using pure and coculture of Enterobacter aerogenes and Citrobacter freundii

We report a concise study on the influence of sulfuric acid concentration used in the hydrolysis of rice mill wastewater on biohydrogen production using pure as well as co-culture of Enterobacter aerogenes and Citrobacter freundii. A higher total reducing sugar release of 14.2 g/L was obtained with 1.5% sulfuric acid (v/v). The effect of pH on cumulative biohydrogen production from 1.5% acid hydrolyzed rice mill wastewater was investigated using pure and coculture of Enterobacter aerogenes and Citrobacter ferundii. The fermentative hydrogen production using mixed culture showed a maximum hydrogen yield of 1.61 mol/mol sugar. In addition, about 70% reduction in chemical oxygen demand (COD) and higher volatile fatty acids (VFA) production were observed. Modified Logistic, Gompertz and Richards models were used to fit cumulative hydrogen production data whereas Logistic equation was used fit growth data. Richards model showed best fit and Logistic equation gave least error in predicting maximum hydrogen producing potential.     

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.     

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.     

Hydrogen production from sludge of poultry slaughterhouse wastewater treatment plant pretreated with microwave

This study aims to produce hydrogen from sludge of poultry slaughterhouse wastewater treatment plant (5% total solid) by anaerobic batch fermentation with Enterobactor aerogenes or mixed cultures from hot spring sediment as the inoculums. Sludge was heated in microwave at 850 W for 3 min. Results indicated that a soluble chemical oxygen demand (sCOD) of pretreated sludge was higher than that of raw sludge. Pretreated sludge inoculated with E. aerogenes and supplemented with the Endo nutrient had a higher hydrogen yield (12.77 mL H₂/g tCOD) than the raw sludge (0.18 mL H₂/g tCOD). When considered the hydrogen yield, the optimum initial pH for hydrogen production from microwave pretreated sludge was 5.5 giving the maximum value of 12.77 mL H₂/g tCOD. However, when considered the hydrogen production rate (R_m), the optimum pH for hydrogen production would be 9.0 with the maximum R_m of 22.80 mL H₂/L sludge·h.     

Hydrogen production from the monomeric sugars hydrolyzed from hemicellulose by Enterobacter aerogenes

Relatively large percentages of xylose with glucose, arabinose, mannose, galactose and rhamnose constitute the hydrolysis products of hemicellulose. In this paper, hydrogen production performance of facultative anaerobe (Enterobacter aerogenes) has been investigated from these different monomeric sugars except glucose. It was shown that the stereoisomers of mannose and galactose were more effective for hydrogen production than those of xylose and arabinose. The substrate of 5 g/l xylose resulted in a relative high level of hydrogen yield (73.8 mmol/l), hydrogen production efficiency (2.2 mol/mol) and a maximum hydrogen production rate (249 ml/l/h). The hydrogen yield, hydrogen production efficiency and the maximum hydrogen production rate reached 104 mmol/l, 2.35 mol/mol and 290 ml/l/h, respectively, on a substrate of 10 g/l galactose. The hydrogen yields and the maximum hydrogen production rates increased with an increase of mannose concentrations and reached 119 mmol/l and 518 ml/l/h on the culture of 25 g/l mannose. However, rhamnose was a relative poor carbon resource for E. aerogenes to produce hydrogen, from which the hydrogen yield and hydrogen production efficiency were about one half of that from the mannose substrate. E. aerogenes was found to be a promising strain for hydrogen production from hydrolysis products of hemicellulose.     

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.     

Biohydrogen production by Ethanoligenens harbinense B49: Nutrient optimization

A new hydrogen-producing bacterial strain Ethanoligenens harbinense B49 was examined for its capability of H₂ production with glucose as sole carbon source. The H₂ production was significantly affected by the concentration of the yeast powder and phosphate in the synthetic medium. The optimized concentration of yeast powder was 0.3–0.5 g/L and the maximum hydrogen yield was obtained at the concentration of phosphate about 100–150 mmol/L. The dynamics of hydrogen production showed that rapid evolution of hydrogen appeared to start after the middle-phase of exponential growth (about 8 h). The maximum H₂ yield and specific hydrogen production rate were estimated to be 2.26 mol H₂/mol glucose and 27.74 mmol H₂/g cell, respectively, when 10 g/L of glucose was present in the medium. The possible pathway of hydrogen production by Ethanoligenens sp. B49 during glucose fermentation was oxidative decarboxylation of pyruvate and the NADH pathway.     

The effect of nutrient limitation on hydrogen production by batch cultures of Escherichia coli

In order to understand some limiting factors in microbial hydrogen fermentation we have examined hydrogen production by different strains of Escherichia coli grown in batch cultures under different limiting nutrient regimes. The effect of mutations in uptake hydrogenases, in lactate dehydrogenase (ldhA), and fhlA, coding for the regulator of formate hydrogen lyase (fhl) component synthesis, were studied. Each mutation contributed to a modest increase in hydrogen evolution and the effects were synergistic. Various elements were used as limiting nutrient. In batch experiments, limitation for sulfate was without great effect. There was some affect of limiting phosphate with yields approaching 1 mol per mol of glucose. However, strains showed the highest yield of hydrogen per glucose (∼2$\sim 2$) when cultured at limiting concentrations of either ammonia or glucose.     

Improved bio-hydrogen production by overexpression of glucose-6-phosphate dehydrogenase and FeFe hydrogenase in Clostridium acetobutylicum

Clostridium acetobutylicum is an attractive industrial microorganism for biochemical production, but there have been few attempts for bio-hydrogen production based on metabolic engineering. In this study, metabolically engineered C. acetobutylicum carrying glucose-6-phosphate dehydrogenase (zwf) and FeFe hydrogenase (hydA) were constructed as recombinant strains CA-zwf(pIMP-zwf) and CA-hydA(pMTL-hydA), respectively, to improve hydrogen productivity. The results showed that the engineered strains produced 1.15 and 1.39-fold higher hydrogen yield, respectively, than the wild type. Furthermore, when pH and glucose concentration were optimized for the CA-hydA strain, enhanced hydrogen productivity of 25.8% was achieved in 7 L jar scale fermentation. This result provides an insight into the future direction for metabolic engineering of C. acetobutylicum for improved hydrogen production.