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.     

Regulation of the NADH supply by nuoE deletion and pncB overexpression to enhance hydrogen production in Enterobacter aerogenes

In this study, seven mutants from E. aerogenes IAM1183 wildtype were constructed via different strategies including deletion of lactate dehydrogenase, disruption of NADH dehydrogenase gene nuoE, overexpression of pncB and a combination of both to regulate of the NADH supply to enhance hydrogen production. Compared with the parental strain, the hydrogen yields of the strains IAM1183-E, IAM1183-L and IAM1183-EL increased by 23.3, 81.7 and 97.9%, respectively. When the pncB gene was overexpressed, the hydrogen yield of IAM1183/P, IAM1183-E/P, IAM1183-L/P and IAM1183-EL/P increased by 39.0, 6.5, 5.9, and 5.1% compared with the respective original knockout strains. Among them, the total hydrogen yield of strain IAM1183-EL/P with highest production efficiency was 58% higher than IAM1183. Further metabolite analysis indicated that the knockout of nuoE and ldhA, combined with the overexpression of pncB, resulted in a redistribution of the metabolic fluxes in E. aerogenes, which led to an improvement of the hydrogen yield.     

Bio-hydrogen production from acid hydrolyzed wheat starch by photo-fermentation using different Rhodobacter sp

Hydrogen gas production from sugar solution derived from acid hydrolysis of ground wheat starch by photo-fermentation was investigated. Three different pure strains of Rhodobacter sphaeroides (RV, NRLL and DSZM) were used in batch experiments to select the most suitable strain. The ground wheat was hydrolyzed in acid solution at pH = 3 and 90 °C in an autoclave for 15 min. The resulting sugar solution was used for hydrogen production by photo-fermentation after neutralization and nutrient addition. R. sphaeroides RV resulted in the highest cumulative hydrogen gas formation (178 ml), hydrogen yield (1.23 mol H₂ mol⁻¹ glucose) and specific hydrogen production rate (46 ml H₂ g⁻¹ biomass h⁻¹) at 5 g l⁻¹ initial total sugar concentration among the other pure cultures. Effects of initial sugar concentration on photo-fermentation performance were investigated by varying sugar concentration between 2.2 and 13 g l⁻¹ using the pure culture of R. sphaeroides RV. Cumulative hydrogen volume increased from 30 to 232 ml when total sugar concentration was increased from 2.2 to 8.5 g l⁻¹. Further increases in initial sugar concentration resulted in decreases in cumulative hydrogen formation. The highest hydrogen formation rate (3.69 ml h⁻¹) and yield (1.23 mol H₂ mol⁻¹ glucose) were obtained at a sugar concentration of 5 g l⁻¹.     

Investigation of the combined effects of acetate and photobioreactor illuminated fraction in the induction of anoxia for hydrogen production by Chlamydomonas reinhardtii

In the context of hydrogen production by microalgae, the growth of Chlamydomonas reinhardtii was characterized under autotrophic and mixotrophic conditions in a fully controlled photobioreactor (PBR). The combined effect of light transfer conditions, as represented by the illuminated fraction γ, with acetate consumption was observed upon establishment of anoxia. Anoxia was reached in batch cultures when γ was close to 1 (almost fully illuminated culture) in mixotrophic conditions while a value of γ ≈ 0.46 in autotrophic conditions was not sufficient. Based on these results, continuous hydrogen production was established in a cylindrical PBR operated in luminostat with constant illumination and in mixotrophic conditions. Maximum hydrogen gas production was equal to 1.4 ± 0.1 ml_H2 l⁻¹ h⁻¹ for photon flux density of 110 μmol m⁻² s⁻¹ and reactor illuminated fraction of γ = 0.5. Carbon mass balance was realized, emphasizing the necessity to work in strictly autotrophic conditions for hydrogen production with no concomitant CO₂ release.     

Microstructure and hydrogenation thermokinetics of ZrTi0.2V1.8 alloy

The microstructures and phase composition of the pseudobinary ZrTi_0.2V_1.8 alloy were examined by scan electron microscope (SEM) and X-ray diffraction (XRD). Before hydrogenation, the hypoeutectic structure accompanied with ZrV₂ + (ZrV₂ + Zr) spherical-like texture has been observed in ZrTi_0.2V_1.8 and the dominant phase could be ascribed to the C15 Laves phase. Hydrogen absorption pressure–composition isotherms (P–C isotherms) and hydriding kinetics of ZrTi_0.2V_1.8 were investigated by pressure reduction method using Sievert apparatus from 673 to 823 K. At hydrogen concentration 0.65 (H/A), the relative partial molar enthalpy and entropy calculated by Van’t Hoff equation are −60 ± 1 kJ mol⁻¹ and −119 ± 1 J mol⁻¹ K⁻¹, respectively. In addition, two stages in the hydrogen absorption reaction between 673 and 823 K could be attributed to the different hydrogen absorption mechanisms including redistribution of the hydrogen atoms in the hydride phase and the diffusion of hydrogen in the β-phase. The activation energy E_a of the alloy is ∼3.6 kJ mol⁻¹ for the first absorption stage and ∼61.9 kJ mol⁻¹ for the second one.     

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.     

High hydrogen production from glycerol or glucose by electrohydrogenesis using microbial electrolysis cells

The use of glycerol for hydrogen gas production was examined via electrohydrogenesis using microbial electrolysis cells (MECs). A hydrogen yield of 3.9 mol-H₂/mol was obtained using glycerol, which is higher than that possible by fermentation, at relatively high rates of 2.0 ± 0.4 m³/m³ d (E_ap = 0.9 V). Under the same conditions, hydrogen was produced from glucose at a yield of 7.2 mol-H₂/mol and a rate of 1.9 ± 0.3 m³/m³ d. Glycerol was completely removed within 6 h, with 56% of the electrons in intermediates (primarily 1,3-propanediol), with the balance converted to current, intracellular storage products or biomass. Glucose was removed within 5 h, but intermediates (mainly propionate) accounted for only 19% of the electrons. Hydrogen was also produced using the glycerol byproduct of biodiesel fuel production at a rate of 0.41 ± 0.1 m³/m³ d. These results demonstrate that electrohydrogenesis is an effective method for producing hydrogen from either pure glycerol or glycerol byproducts of biodiesel fuel production.     

High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana

Efficient conversion of glycerol waste from biodiesel manufacturing processes into biohydrogen by the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 was investigated. Biohydrogen production by T. neapolitana was examined using the batch cultivation mode in culture medium containing pure glycerol or glycerol waste as the sole substrate. Pre-treated glycerol waste showed higher hydrogen (H₂) production than untreated waste. Nitrogen (N₂) sparging and pH control were successfully implemented to maintain the culture pH and to reduce H₂ partial pressure in the headspace for optimal growth rate and to enhance hydrogen production from the glycerol waste. It was found that hydrogen production increased from 1.24 ± 0.06 to 1.98 ± 0.1 mol-H₂ mol⁻¹ glycerol_consumed by optimising N₂ sparging and pH control. We observed that in medium containing 0.05 M HEPES, with three cycles of N₂ sparging, the H₂ yield increased to 2.73 ± 0.14 mol-H₂ mol⁻¹ glycerol_consumed, which was 2.22-fold higher than the non-N₂ sparged H₂ yield (1.23 ± 0.06 mol-H₂ mol⁻¹ glycerol_consumed).     

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.     

The effect of irradiance growing on hydrogen photoevolution and on the kinetic growth in Rhodopseudomonas palustris, strain 42OL

The relationship between hydrogen generation and the age of culture was investigated under fed-batch growth conditions. The specific growth rate (μ_e) was determined during the log phase of the growth curve and the μ_eMax was 0.02643 h⁻¹. Boltzmann's sigmoidal regression model was used to determine the specific rate of hydrogen evolution (μH): the maximum was 0.04440 h⁻¹. At low irradiance (36–75 W m⁻²), an inverse relationship was found between μH and I; after increasing the irradiance further, μH reached a plateau (0.00916 h⁻¹). The maximum reactor yield of cumulative hydrogen (4.5 l) was obtained at an irradiance of 320 W m⁻², but the highest hydrogen evolution rate (17.217 ml h⁻¹) was achieved at 500 W m⁻². The light conversion efficiency reached its maximum (6.91%) at the lowest irradiance investigated (36 W m⁻²); when the irradiance increased further, it decreased progressively down to 0.36%.     

Manipulating the hydrogen production from acetate in a microbial electrolysis cell-microbial fuel cell-coupled system

A biological hydrogen-producing system is configured through coupling an electricity-assisting microbial fuel cell (MFC) with a hydrogen-producing microbial electrolysis cell (MEC). The advantage of this biocatalyzed system is the in-situ utilization of the electric energy generated by an MFC for hydrogen production in an MEC without external power supply. In this study, it is demonstrated that the hydrogen production in such an MEC-MFC-coupled system can be manipulated through adjusting the power input on the MEC. The power input of the MEC is regulated by applying different loading resistors connected into the circuit in series. When the loading resistance changes from 10 Ω to 10 kΩ, the circuit current and volumetric hydrogen production rate varies in a range of 78 ± 12 to 9 ± 0 mA m⁻² and 2.9 ± 0.2 to 0.2 ± 0.0 mL L⁻¹ d⁻¹, respectively. The hydrogen recovery (RH₂), Coulombic efficiency (CE), and hydrogen yield (YH₂) decrease with the increase in loading resistance. Thereafter, in order to add power supply for hydrogen production in the MEC, additional one or two MFCs are introduced into this coupled system. When the MFCs are connected in series, the hydrogen production is significantly enhanced. In comparison, the parallel connection slightly reduces the hydrogen production. Connecting several MFCs in series is able to effectively increase power supply for hydrogen production, and has a potential to be used as a strategy to enhance hydrogen production in the MEC-MFC-coupled system from wastes.     

Hydrogen production using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

Fermentative hydrogen production was carried out using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). This work investigates the effects of initial substrate concentration, initial medium pH, and temperature. The hydrogen yield was about 3.1 mol (mol glucose)⁻¹ when starting with an initial glucose concentration of 10 gl⁻¹ and initial a pH of 6.0 ± 0.2 at a temperature of 37 °C. The volume of hydrogen produced decreased when higher initial glucose concentrations were applied. The most suitable conditions for hydrogen production in a batch reactor were observed at initial pH 6.0 ± 0.2 and 37 °C.     

Evaluation of hydrogen production by Rhodobacter sphaeroides O.U.001 and its hupSL deficient mutant using acetate and malate as carbon sources

Rhodobacter sphaeroides O.U.001 is one of the candidates for photobiological hydrogen production among purple non-sulfur bacteria. Hydrogen is produced by Mo-nitrogenase from organic acids such as malate or lactate. A hupSL in frame deletion mutant strain was constructed without using any antibiotic resistance gene. The hydrogen production potential of the R. sphaeroides O.U.001 and its newly constructed hupSL deleted mutant strain in acetate media was evaluated and compared with malate containing media. The hupSL⁻R. sphaeroides produced 2.42 l H₂/l culture and 0.25 l H₂/l culture in 15 mM malate and 30 mM acetate containing media, respectively, as compared to the wild type cells which evolved 1.97 l H₂/l culture and 0.21 l H₂/l culture in malate and acetate containing media, correspondingly. According to the results, hupSL⁻R. sphaeroides is a better hydrogen producer but acetate alone does not seem to be an efficient carbon source for photoheterotrophic H₂ production by R. sphaeroides.     

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.     

Biological hydrogen production from corn stover by moderately thermophile Thermoanaerobacterium thermosaccharolyticum W16

This study addressed the utilization of an agro-waste, corn stover, as a renewable lignocellulosic feedstock for the fermentative H₂ production by the moderate thermophile Thermoanaerobacterium thermosaccharolyticum W16. The corn stover was first hydrolyzed by cellulase with supplementation of xylanase after delignification with 2% NaOH. It produced reducing sugar at a yield of 11.2 g L⁻¹ glucose, 3.4 g L⁻¹ xylose and 0.5 g L⁻¹ arabinose under the optimum condition of cellulase dosage 25 U g⁻¹ substrate with supplement xylanase 30 U g⁻¹ substrate. The hydrolyzed corn stover was sequentially introduced to fermentation by strain W16, where, the cell density and the maximum H₂ production rate was comparable to that on simulated medium, which has the same concentration of reducing sugars with hydrolysate. The present results suggest a promising combined hydrogen production process from corn stover with enzymatic hydrolysis stage and fermentation stage using W16.     

Some physical aspects of hydrogen behaviour in the H-Storage bcc alloys Ti35VxCr65−x, Ti40VxMn50−xCr10 and TixCr97.5−xMo2.5

Hydrogen absorption/desorption has been investigated in the three series of solid solution bcc alloys Ti₃₅V_xCr_65−x (x = 18,22), Ti₄₀V_xMn_50−xCr₁₀ (x = 32,36) and Ti_xCr_97.5−xMo_2.5 (x = 43,46). It has been found that the H absorption at pressures smaller than 1 bar can only occur after elimination of the oxide films by heating the alloys to temperatures higher than 600 K. Hydrogen desorption from pre-loaded materials (n_H = H/Me ≤ 0.27) takes place on heating at much lower temperatures in the Ti₄₀V_xMn_50−xCr₁₀ and Ti₃₅V_xCr_65−x than in the Ti_xCr_97.5−xMo_2.5 alloys. The H diffusion parameters W and D_o deduced from high temperature (>450 K) absorption experiments are as follows: W = 0.318 ± 0.005 eV, D_o = (4 ± 1)×10⁻⁷ m²/s for Ti₄₀V_xMn_50−xCr₁₀; W = 0.32 ± 0.02 eV, D_o = (3 ± 2)×10⁻⁷ m²/s for Ti₃₅V_xCr_65−x; W = 0.79 ± 0.06 eV, D_o = (4 ± 2)×10⁻⁸ m²/s for Ti_xCr_97.5−xMo_2.5. The higher value of the activation energy for H diffusion in Mo containing alloys is most likely due to remarkable attractive interactions between H and Mo atoms.     

Bio-hydrogen production by photo-fermentation of dark fermentation effluent with intermittent feeding and effluent removal

Pure culture of Rhodobacter sphaeroides (NRRL- B1727) was used for continuous photo-fermentation of volatile fatty acids (VFA) present in the dark fermentation effluent of ground wheat starch. The feed contained 1950 ± 50 mg L⁻¹ total VFA with some nutrient supplementation. Hydraulic residence time (HRT) was varied between 24 and 120 hours. The highest steady-state daily hydrogen production (55 ml d⁻¹) and hydrogen yield (185 ml H₂ g⁻¹ VFA) were obtained at HRT = 72 hours (3 days). Biomass concentration increased with increasing HRT. Volumetric and specific hydrogen formation rates were also maximum at HRT = 72 h. High extent of TVFA fermentation at HRT = 72 h resulted in high hydrogen gas production.     

Enhanced hydrogen production by means of sulfur-deprived Chlamydomonas reinhardtii cultures grown in pretreated olive mill wastewater

Under sulfur-deprived conditions, the metabolism of Chlamydomonas reinhardtii switches to the photoproduction of hydrogen. This process is sustained by both photosystem II-driven water splitting and by the fermentation of stored carbohydrates. We investigated the possibility of using diluted pretreated olive mill wastewaters (OMW), which contain organic acids and sugars, as a substrate on which to grow Chlamydomonas, in order to obtain suitable biomass to produce hydrogen. The cells grown on a mixture of pretreated OMW and TAP (tris-acetate-phosphate) (50% dilution) were found to be richer in carbohydrates and exhibited a greater production of hydrogen (150 ml H₂ l⁻¹ culture), compared to the control cells (100 ml H₂ l⁻¹ culture). In these cultures, the hydrogen production process was characterized by a shorter aerobic phase and a longer hydrogen-production period. The results offer a useful perspective for the utilization of olive mill wastewaters, which constitute an environmental problem, particularly in Mediterranean areas, and for increasing the output for hydrogen production with Chlamydomonas.     

Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3

A hydrogen producing strain newly isolated from anaerobic sludge in an anaerobic bioreactor, was identified as Clostridium beijerinckii Fanp3 by 16S rDNA gene sequence analysis and detection by BioMerieux Vitek. The strain could utilize various carbon and nitrogen sources to produce hydrogen, which indicates that it has the potential of converting renewable wastes into hydrogen. In batch cultivations, the optimal initial pH of the culture medium was between 6.47 and 6.98. Using 0.15 M phosphate as buffer could alleviate the medium acidification and improve the overall performance of C. beijerinckii Fanp3 in hydrogen production. Culture temperature of 35 °C was established to be the most favorable for maximum rate of hydrogen production. The distribution of soluble metabolic products (SMP) was also greatly affected by temperature. Considering glucose as a substrate, the activation energy (E_a) for hydrogen production was calculated as 81.01 kcal/mol and 21.4% of substrate energy was recovered in the form of hydrogen. The maximal hydrogen yield and the hydrogen production rate were obtained as 2.52 mol/mol-glucose and 39.0 ml/g-glucose h⁻¹, respectively. These results indicate that C. beijerinckii Fanp3 is an ideal candidate for the fermentative hydrogen production.