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.     

Occurrence and characterisation of the hydrogen-evolving enzyme in Frankia sp.

An increase in hydrogen evolution from the hydrogen-evolving enzyme in the actinomycete Frankia was recorded in the presence of nickel. Immunogold localisation analysis of the intracellular distribution of hydrogenase proteins indicated that they were evenly distributed in the membranes and cytosol of both hyphae and vesicles. In addition, molecular characterisation of the hydrogen-evolving enzyme at the proteomic level, using two-dimensional gel electrophoresis combined with mass spectrometry, confirmed that the Frankia hydrogen-evolving enzyme is similar to the cyanobacterial bidirectional hydrogenase of Anabena siamensis.     

Engineered clostridial [FeFe]-hydrogenase shows improved O2 tolerance in Chlamydomonas reinhardtii

Hydrogenase intolerance to oxygen remains a critical hurdle on the road to photosynthetic hydrogen production for sustainable energy demands. Although the engineering of the intrinsic oxygen tolerance mechanism of hydrogenase using mutagenesis is an ambitious approach, recent in-vitro studies reported a novel and improved synthetic [FeFe]-Hydrogenase variants. To corroborate these findings in-vivo, we expressed either an engineered variant or its cognate wild type enzyme in the chloroplast genome of Chlamydomonas reinhardtii. We characterized their activity using a customized photosynthetic hydrogen production in-vivo assay to test whether the improved variant could maintain a greater fraction of its activity following oxygen exposure. We found that the mutated variant exhibited a superior oxygen tolerance while persevering its photosynthetic performance in terms of hydrogen production yield. Importantly, we show for the first time that this approach can potentially address the inherent O₂ sensitivity of [FeFe]-Hydrogenases for photosynthetic hydrogen production.     

Three-dimensional carbon nanotube-polypyrrole-[NiFe] hydrogenase electrodes for the efficient electrocatalytic oxidation of H2

Hydrogenase electrodes for hydrogen oxidation are elaborated by an innovative immobilization strategy of [NiFe] hydrogenases from Desulfovibrio fructosovorans on highly porous single-walled (SWCNT) and multi-walled (MWCNT) carbon nanotube electrodes.The bioelectrode fabrication involved the adsorption of hydrogenase and amphiphilic pyrrole monomer functionalized by a methylviologen moiety on the nanotube deposits.The electropolymerization of the adsorbed monomer then leads to the enzyme entrapment in polypyrrole film surrounding the nanotubes. In addition, the redox polypyrrole achieves an efficient electrical wiring of hydrogenase on SWCNT and MWCNT electrodes via a mediated electron transfer. The latter configuration showed improved performances in catalytic responses (up to 0.30 ± 0.01 mA cm⁻²) at stationary electrodes due to the more appropriate wettability of MWCNTs. This led to a better coating of the nanostructured surface and thus, to an enhanced mediated electron transfer between the enzyme and the nanotubes.     

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.     

Dark hydrogen production in nitrogen atmosphere – An approach for sustainability by marine cyanobacterium Leptolyngbya valderiana BDU 20041

Biological hydrogen production is an ideal system for three main reasons i) forms a renewable energy source, ii) gives clean fuel and iii) serves as a good supplement to oil reserves. The major challenges faced in biological hydrogen production are the presence of uptake hydrogenase and lack of sustainability in the cyanobacterial hydrogen production system. Three different marine cyanobacterial species viz. Leptolyngbya valderiana BDU 20041, Dichothrix baueriana BDU 40481 and Nostoc calcicola BDU 40302 were studied for their potential use in hydrogen production. Among these, L. valderiana BDU 20041, was found to produce hydrogen even in 100% nitrogen atmosphere which was 85% of the hydrogen produced in argon atmosphere. This is the first report of such a high rate of production of hydrogen in a nitrogen atmosphere by a cyanobacterium, which makes it possible to develop sustained hydrogen production systems. L. valderiana BDU 20041, a dark hydrogen producer uses the reductant essentially supplied by the respiratory pathway for hydrogen production. Using inhibitors, this organism was found to produce hydrogen due to the activities of both nitrogenase and bidirectional hydrogenase, while it had no ‘uptake’ hydrogenase activity. The other two organisms though had low levels of bidirectional hydrogenase, possessed considerable ‘uptake’ hydrogenase activity and hence could not release much hydrogen either in argon or nitrogen atmosphere.     

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.     

Introducing pyruvate oxidase into the chloroplast of Chlamydomonas reinhardtii increases oxygen consumption and promotes hydrogen production

In an anaerobic environment, the unicellular green algae Chlamydomonas reinhardtii can produce hydrogen (H₂) using hydrogenase. The activity of hydrogenase is inhibited at the presence of molecular oxygen, forming a major barrier for large scale production of hydrogen in autotrophic organisms. In this study, we engineered a novel pathway to consume oxygen and correspondingly promote hydrogen production in Chlamydomonas reinhardtii. The pyruvate oxidase from Escherichia coli and catalase from Synechococcus elongatus PCC 7942 were cloned and integrated into the chloroplast of Chlamydomonas reinhardtii. These two foreign genes are driven by a HSP70A/RBCS2 promoter, a heat shock inducing promoter. After continuous heat shock treatments, the foreign genes showed high expression levels, while the growth rate of transgenic algal cells was slightly inhibited compared to the wild type. Under low light, transgenic algal cells consumed more oxygen than wild type. This resulted in lower oxygen content in sealed culture conditions, especially under low light condition, and dramatically increased hydrogen production. These results demonstrate that pyruvate oxidase expressed in Chlamydomonas reinhardtii increases oxygen consumption and has potential for improving photosynthetic hydrogen production in Chlamydomonas reinhardtii.     

Interplay between hydrogen production and photosynthesis in a green alga expressing an active photosystem I-hydrogenase chimera

We have previously created and expressed a chimeric polypeptide joining the PsaC subunit of Photosystem I (PSI) to the HydA2 hydrogenase of Chlamydomonas reinhardtii and demonstrated that it assembles into the PSI complex and feeds electrons directly to the hydrogenase domain, allowing for prolonged photobiological hydrogen production. Here we describe a new PSI-hydrogenase chimera using HydA1, the more abundant and physiologically active endogenous hydrogenase of this alga. When the PsaC-HydA1 polypeptide was expressed in a C. reinhardtii strain lacking endogenous hydrogenases, it was assembled into active PSI-HydA1 complexes that were accumulated at a level ～75% that of PSI, which is ～5 times higher than the PSI-HydA2 chimera. Hydrogen production by the chimera could be restored after complete inactivation by oxygen without requiring new synthesis of PSI or the PsaC-HydA1 polypeptide, demonstrating that the complex could be repaired in vivo. The PSI-HydA1 chimera reduces ferredoxin in vivo to such an extent that it can drive the Calvin-Benson-Bassham cycle, leading to high O₂ production rates, and eventually resulting in inactivation of the hydrogenase; use of media that drastically diminished CO₂ fixation and an O₂-scavenging material allowed H₂ production for at least 4 days.     

Direct electron transfer to hydrogenase for catalytic hydrogen production using a single-walled carbon nanotube forest

The production of hydrogen through the direct electron transfer from electrode to hydrogenase was successfully performed using hydrogenase combined with a single-walled carbon nanotube forest (SWNT-forest). The SWNT-forest has a unique structure comprising dense and vertically aligned SWNTs with millimeter-scale height. The vertically aligned SWNTs became rearranged into a wall-like architecture after immersing in water. Hydrogenase was spontaneously incorporated within the SWNT-forest and confined on the sidewall of the rearranged architecture. The SWNT-forest was a very stable protein carrier in aqueous solution, and an SWNT-forest integrated with hydrogenase could easily be constructed. With a hydrogenase assembled SWNT-forest we were able to electrochemically produce hydrogen with an electron transfer efficiency exceeding 30% without the use of any chemical mediator. Furthermore, a reverse oxidative reaction of hydrogen was also successfully performed using the same device. Thus, the SWNT-forest can work as an effective and direct electron mediator for the oxidation/reduction reaction of hydrogenase. The usefulness of the SWNT-forest as a beneficial material for electrochemistry was demonstrated, underlining the potential of protein assembled SWNT-forests in fabricating highly efficient biofuel cell devices.     

Stimulatory effect of ascorbate,the alternative electron donor of photosystem II,on the hydrogen production of sulphur-deprived Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is capable of photoproducing molecular hydrogen following sulphur deprivation, which results in anaerobiosis and a suppression of oxygen evolution and thus an alleviation of the inhibitory effect of oxygen on the hydrogenase. At the same time it transiently maintains a limited supply of electrons arising from photosystem II (PSII) to the hydrogenase (Melis and Happe Plant Physiol 2001; 127:740–748). In this work, using fast chl a fluorescence and P700 measurements, we show that ascorbate (Asc), a naturally occurring PSII alternative electron donor, is capable of donating electrons to PSII in heat-treated and sulphur-deprived cells and this can be significantly accelerated by supplementing the culture with 10 mM Asc. It also enhances, about three-fold, the photoproduction of hydrogen in cells subjected to sulphur deprivation as shown by gas chromatography. Similar stimulation was obtained in the presence of diphenylcarbazide (DPC), an artificial PSII electron donor. Asc and DPC also facilitated the anaerobiosis of cells, probably via super reducing the oxygen evolving complex while feeding electrons to PSII reaction centres and the linear electron transport chain, and ultimately to the hydrogenase – as shown by the significant DCMU-sensitivity of the light-induced Asc- and DPC-dependent re-reduction of P700⁺ and hydrogen evolution.     

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.     

Comparison of metabolic pathway for hydrogen production in wild-type and mutant Clostridium tyrobutyricum strain based on metabolic flux analysis

There has been a great interest in fermentative hydrogen production during recent decades. However, the low H₂ yield associated with fermentative hydrogen production process continues to hinder its industrial application. It is delectable that a maximum 3.9 mol H₂ per mol glucose was obtained in fed-batch fermentation mode with a butyric acid over-producing Clostridium tyrobutyricum mutant, which to our knowledge is the highest H₂ yield ever got in the fermentation process with Clostridium sp. This study aimed to better understand the change of flux profile within the whole metabolic network and to conduct the metabolic flux analysis of fermentative hydrogen production. For the first time, we constructed a metabolic flux model for the anaerobic glucose metabolism of C. tyrobutyricum ATCC 25755, and revealed the internal mechanism responsible for the redistribution of the carbon flux in the mutant strain in comparison with the wide-type. The MFA methodology was used to study the fractional flux response to variations in operational pH, and revealed that pH was a significant operational parameter effecting on the fermentative hydrogen production process. Furthermore, the presence of NADH-ferredoxin oxidoreductase activity in this anaerobe was demonstrated. By measuring the activities of related enzymes in the biosynthesis pathway of hydrogen, we thus concluded that the increased specific activities of both NFOR and hydrogen-catalyzing enzyme (hydrogenase) would be attributed to the hydrogen over-producing.