Molecular hydrogen production by nitrogenase of Rhodobacter sphaeroides and by Fe-only hydrogenase of Rhodospirillum rubrum

The genes coding for two PII-like proteins, GlnB and GlnK, which play key roles in repressing the nitrogenase expression in the presence of ammonium ion, were interrupted from the chromosome of Rhodobacter sphaeroides. The glnB–glnK mutant exhibits the less ammonium ion-mediated repression for nitrogenase compared with its parental strain, which results in more H₂ accumulation by the mutant under the conditions. Rhodospirillum rubrum produces H₂ by both nitrogenase and hydrogenase. R. rubrum containing the recombinant pRK415 with an insert of hydC coding for its own Fe-only hydrogenase showed twofold higher accumulation of H₂ in the presence of pyruvate under photoheterotrophic conditions, which was not observed in the absence of pyruvate. The same was true with R. rubrum containing the recombinant pRK415 cloned with hydA coding for Fe-only hydrogenase of Clostridium acetobutylicum. Thus, Fe-only hydrogenase requires pyruvate as an electron donor for the production of H₂.     

Molecular characterization and homologous overexpression of [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1

The H₂-evoving [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1 was isolated to elucidate molecular characterization and modular structure of the hydrogenase. Then, homologous overexpression of the hydrogenase gene was for the first time performed to enhance hydrogen production. The hydA open reading frame (ORF) was 1734-bp, encodes 577 amino acids with a predicted molecular mass of 63,970 Da, and presents 80% and 75% identity at the amino acid level with the [FeFe]-hydrogenase genes of Clostridium kluyveri DSM 555 and Clostridium acetobutylicum ATCC 824, respectively. One histidine residue and 19 cysteine residues, known to fasten one [2Fe–2S] cluster, three [4Fe–4S] clusters and one H-cluster, were conserved in hydA of C. tyrobutyricum.     

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.     

Prospecting hydrogen production of Escherichia coli by metabolic network modeling

Genome-scale model was applied to analyze the anaerobic metabolism of Escherichia coli. Three different methods were used to find deletions affecting fermentative hydrogen production: flux balance analysis (FBA), algorithm for blocking competing pathways (ABCP), and manual selection. Based on these methods, 81 E. coli mutants possessing one gene deletion were selected and cultivated in batch experiments. Experimental results of H₂ and biomass production were compared against the results of FBA. Several gene deletions enhancing H₂ production were found. Correctness of gene essentiality predictions of FBA for the selected genes was 78% and 77% in glucose and galactose media, respectively. 33% of the mutations that were predicted by FBA to increase H₂ production had a positive effect in experiments. Batch cultivation is a simple and straightforward experimental way to screen improvements in H₂ production. However, the ability of FBA to predict the H₂ production rate cannot be evaluated by batch experiments. Metabolic network models provide a method for gaining broader understanding of the complicated metabolic system of a cell and can aid in prospecting suitable gene deletions for enhancing H₂ production.     

Sustained hydrogen production from formate using immobilized recombinant Escherichia coli SH5

Biohydrogen is an ideal energy carrier for mobile chemical fuel cells, but its use is often limited by unavailability of sustained H₂ production system(s). Here, we developed a compact system for H₂ production from formate based on immobilized cells of recombinant Escherichia coli SH5. Three different matrices were tested as immobilization medium, among which agar showed the best performance in mechanical stability and permeability of substrate(s) and/or gaseous products (H₂ and CO₂). To explore and optimize the H₂ production capability of the immobilized cells, the conditions for cell immobilization including cell loading and agar concentration as well as the factors affecting H₂ production rate such as temperature, pH, and substrate concentration were studied in detail. A maximum volumetric production rate of 2.4 L H₂ L⁻¹ h⁻¹ was obtained when the immobilized cells were incubated with 350 mM sodium formate at pH 6.5 and 37 °C. Periodic supplementation of 200 mM formate with 20 mM glucose at pH 6.5 maintained the high H₂ production rate for a prolonged period of 10 h. We believe that our process can be developed for sustained H₂ production and is applicable to the operation of fuel cells in small-scale.     

Hydrogen producing activity by Escherichia coli hydrogenase 4 (hyf) depends on glucose concentration

Escherichia coli produces molecular hydrogen (H₂) during glucose fermentation. This production of H₂ occurs via multiple and reversible membrane-associated hydrogenases (Hyd). Dependence of H₂ producing rate (VH₂)$\left({\text{V}}_{{\text{H}}_2}\right)$ by Hyd-4 (hyf) on glucose concentration was studied at different pHs. During growth on 0.2% glucose at pH 7.5 in JRG3615 (hyfA-B) and JRG3621 (hyfB-R  ) mutants (VH₂)$\left({\text{V}}_{{\text{H}}_2}\right)$ was decreased ∼6.7 and ∼5 fold, respectively, compared to wild type. Only in JRG3621 mutant at pH 6.5 and 5.5 (VH₂)$\left({\text{V}}_{{\text{H}}_2}\right)$ was severely decreased ∼7.8 and ∼3.8 fold, respectively. But when cells were grown on 0.8% glucose no difference between wild type and mutants was detected at any of the tested pHs. The results indicate Hyd-4 H₂ producing activity inhibition by high concentration of glucose mainly at pH 7.5. This is of significance to regulate Hyd activity and H₂ production by E. coli during fermentation.     

Ferredoxin has a pivotal role in the biosynthesis of the hydrogen-oxidizing hydrogenases in Escherichia coli

Iron–sulphur clusters (FeS) are essential cofactors in the small, electron-transfer subunits of [NiFe]-hydrogenases (Hyd). In this study we analyzed the in vivo role of ferredoxin in the biosynthesis of three of the Hyd in Escherichia coli. Our results reveal that a fdx mutant, which is unable to synthesize ferredoxin, lacks the activity of both hydrogen-oxidizing enzymes Hyd-1 and Hyd-2. In the case of Hyd-2 this was due to the absence of the FeS cluster-containing small subunit. In the case of Hyd-1, stability of the catalytic subunit was also impaired. Partial activity of the hydrogen-evolving Hyd-3 enzyme, as well as that of both respiratory formate dehydrogenases was retained in the fdx mutant. Analysis of lacZ fusions demonstrated that the fdx mutation had a limited effect on expression of the operon encoding Hyd-1. Rather, these data suggest that ferredoxin has a role in the maturation or assembly of the hydrogen-oxidizing [NiFe]-hydrogenases.     

Influence of Escherichia coli hydrogenases on hydrogen fermentation from glycerol

Since the actual role of Escherichia coli hydrogenases on fermentation from glycerol has not been clear, we evaluated the effect of inactivation of each E. coli hydrogenase on cell growth, hydrogen production, organic acids production, and ethanol production. Inactivation of hydrogenase 2 and hydrogenase 3 reduced cell growth, hydrogen and succinate production as well as glycerol utilization while acetate increased. Inactivation of hydrogenase 2 in minimal medium at pH 7.5 impaired hydrogen production, but no significant effect occurred at pH 6.5 or in complex medium. Inactivation of hydrogenase 3 impaired hydrogen production in minimal and rich medium, pH 6.5 and pH 7.5 accumulating formate in all conditions. Therefore during fermentation from glycerol, hydrogenase 3 is the main hydrogenase with hydrogen synthesis activity through the formate hydrogen lyase complex. Hydrogenase 2 seems mainly required for optimum glycerol metabolism rather than hydrogen synthesis. There were no significant impacts by inactivating hydrogenase 1 and hydrogenase 4.     

Escherichia coli hydrogenase activity and H2 production under glycerol fermentation at a low pH

Hydrogenase (Hyd) activity and H₂ production by Escherichia coli were studied at a low pH. H₂ production at pH 5.5 under glycerol fermentation was shown to be ∼1.5-fold higher than that at pH 6.5 or above but less than that under glucose fermentation. It was inhibited by N,N′-dicyclohexylcarbodiimide: H₂ production inhibition was increased with decreasing pH and almost maximal inhibition was observed at pH 5.5. The data on H₂ production by single and double mutants with defects in different Hyd-enzymes and in fhlA gene suggest that under glycerol fermentation at a low pH, Hyd-1, Hyd-2 and Hyd-4 were operating in a reversed, non-H₂ producing mode. Moreover, a role of fhlA gene in Hyd-3 and Hyd-4 activity in H₂ production is proposed under glucose fermentation at a low pH.     

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.     

Bacterial hydrogen production in recombinant Escherichia coli harboring a HupSL hydrogenase isolated from Rhodobacter sphaeroides under anaerobic dark culture

In this study, recombinant plasmid was constructed to analyze the effect of hydrogen production on the expression HupSL hydrogenase isolated from Rhodobacter sphaeroides in Escherichia coli. Although most of recombinant HupSL hydrogenase was produced as inclusion bodies the solubility of the protein increased significantly when the expression temperature shifted from 37 °C to 30 °C. Hydrogen production by expression of HupSL hydrogenase from recombinant E. coli increased 20.9-fold compared to control E. coli and 218-fold compared to wild type R. sphaeroides under anaerobic dark condition. The results demonstrate that HupSL hydrogenase, consisting of small and large subunits of hydrogenase isolated from R. sphaeroides, increases hydrogen production in recombinant E. coli. In addition conditions for enhancing the activity of HupSL hydrogenase in E. coli were suggested and were used to increase bacterial hydrogen production.     

Hydrogen production by Escherichia coli depends on glucose concentration and its combination with glycerol at different pHs

Escherichia coli produces molecular hydrogen (H₂) during glucose or mixed carbon (glucose and glycerol) fermentation. Dependence of H₂ production rate (VH₂)$\left(V_{{\text{H}}_2}\right)$ on glucose at different pHs was studied in a concentration dependent manner. During growth of wild-type on glucose, increasing glucose concentration from 0.05% to 0.2% resulted in the marked inhibition of VH₂$V_{{\text{H}}_2}$. Inhibitory effect of glucose was shown at pH 7.5 and 6.5 but not pH 5.5. However, glycerol added in the growth medium with 0.1% glucose significantly increased VH₂$V_{{\text{H}}_2}$ but different effects at different pHs were established upon glucose or glycerol assays. The results indicate that H₂ production is inhibited by glucose in a concentration dependent manner during glucose fermentation but glucose in combination with glycerol might enhance H₂ production during mixed carbon fermentation.     

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

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

Expression of the [FeFe] hydrogenase in the chloroplast of Chlamydomonas reinhardtii

Biological hydrogen generation from phototrophic organisms is a promising source of renewable fuel. The nuclear-expressed [FeFe] hydrogenase from Chlamydomonas reinhardtii has an extremely high turnover rate, and so has been a target of intense research. Here, we demonstrate that a codon-optimized native hydrogenase can be successfully expressed in the chloroplast. We also demonstrate a curiously strong negative selective pressure resulting from unregulated hydrogenase expression in this location, and discuss management of its expression with a vitamin-controlled gene repression system. To the best of our knowledge, this represents the first example of a nuclear-expressed, chloroplast-localized metalloprotein being synthesized in situ. Control of this process opens up several bioengineering possibilities for the production of biohydrogen.     

Hydrogen production by Escherichia coli without nitrogen sparging and subsequent use of the waste culture for fast mass scale one-pot green synthesis of silver nanoparticles

In view of the transition to hydrogen as a major energy carrier in the future, new routes for bringing down the cost of biological hydrogen production need to be explored. The current study was devoted to optimizing the dark fermentation by Escherichia coli HD701 for hydrogen production from an acid-hydrolyzed potato starch residue stream without nitrogen sparging to reduce the cost. To further increase the economic feasibility of hydrogen production by E. coli, this study explores the use of the waste culture after hydrogen production in mass scale one-pot green synthesis of silver nanoparticles.     

Fermentative hydrogen yields from different sugars by batch cultures of metabolically engineered Escherichia coli DJT135

Future sustainable production of biofuels will depend upon the ability to use complex substrates present in biomass if the use of simple sugars derived from food crops is to be avoided. Therefore, organisms capable of using a variety of fermentable carbon sources must be found or developed for processes that could produce hydrogen via fermentation. Here we have examined the ability of a metabolically engineered strain of Escherichia coli, DJT135, to produce hydrogen from glucose as well as various other carbon sources, including pentoses. The effects of pH, temperature and carbon source were investigated in batch experiments. Maximal hydrogen production from glucose was obtained at an initial pH of 6.5 and temperature of 35 °C. Kinetic growth studies showed that the μmax was 0.0495 h⁻¹ with a Ks of 0.0274 g L⁻¹ when glucose was the sole carbon source in M9 (1X) minimal medium. Among the many sugar and sugar derivatives tested, hydrogen yields were highest with fructose, sorbitol and d-glucose; 1.27, 1.46 and 1.51 mol H₂ mol⁻¹ substrate respectively.     

Escherichia coli hydrogenase 4 (hyf) and hydrogenase 2 (hyb) contribution in H2 production during mixed carbon (glucose and glycerol) fermentation at pH 7.5 and pH 5.5

Molecular hydrogen (H₂) production by Escherichia coli was studied during mixed carbon sources (glucose and glycerol) fermentation at pH 7.5 and pH 5.5. H₂ production rate (V_H2) by bacterial cells grown on mixed carbon was assayed with either adding glucose (glucose assay) or glycerol (glycerol assay) and compared with the cells grown on sole carbon (glucose or glycerol only) and appropriately assayed. Wild type cells grown on mixed carbon, in the assays with adding glucose, produced H₂ at pH 7.5 with the same level as in the cells grown on glucose only. At pH 7.5 V_H2 in fhlA single and fhlA hyfG double mutants decreased ∼6.5 and ∼7.9 fold, respectively. In wild type cells grown on mixed carbon V_H2 at pH 5.5 was lowered ∼2 fold, compared to the cells grown on glucose only. But in hyfG and hybC single mutants V_H2 was decreased ∼2 and ∼1.6 fold, respectively. However, at pH 7.5, in the assays with glycerol, V_H2 was low, when compared to the cells grown on glycerol only. At pH 5.5 in the assays with glycerol V_H2 was absent. Moreover, V_H2 in wild type cells was inhibited by 0.3 mM N,N^′-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F₀F₁-ATPase, in a pH dependent manner. At pH 7.5 in wild type cells V_H2 was decreased ∼3 fold but at pH 5.5 the inhibition was ∼1.7 fold. At both pHs in fhlA mutant V_H2 was totally inhibited by DCCD. Taken together, the results obtained indicate that at pH 7.5, in the presence of glucose, glycerol can also be fermented. They point out that Hyd-4 mainly and Hyd-2 to some extent contribute in H₂ production by E. coli during mixed carbon fermentation at pH 5.5 whereas Hyd-1 is only responsible for H₂ oxidation.     

Characterization and overexpression of a [FeFe]-hydrogenase gene of a novel hydrogen-producing bacterium Ethanoligenens harbinense

Ethanoligenens, a novel ethanologenic and hydrogen-producing genus, has capability of hydrogen production at low pH. A [FeFe]-hydrogenase gene with [4Fe-4S] and [2Fe-2S] clusters from Ethanoligenens harbinense YUAN-3 was cloned and overexpressed in a non-hydrogen-producing Escherichia coli BL-21. This hydA gene consisted of an open reading frame of 1743 bp encoding 580 amino acids with an estimated molecular weight of 63 188.1 Da. Six characteristic sequence signatures were present within the H-cluster domain of [FeFe]-H₂ases, and three of them were described previously. The overexpressed and purified hydrogenases from recombinant cells showed catalytic activity in vitro and in vivo.