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.     

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 different types of [FeFe]-hydrogenase genes in bacteria isolated from a population of a bio-hydrogen pilot-scale plant

[FeFe]-hydrogenases are the enzymes responsible for high yield H₂ production during dark fermentation in bio-hydrogen production plants. The culturable bacterial population present in a pilot-scale plant efficiently producing H₂ from waste materials was isolated, classified and identified by means of 16S rDNA gene analysis. The culturable part of the mixed population consists of nine bacterial species that include non-hydrogen producers (Lactobacillus, Enterococcus and Staphylococcus) and several Clostridium that are directly responsible for H₂ production.     

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.     

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.     

Bioproduction of hydrogen by simultaneous saccharification and fermentation of cassava starch with 2-deoxyglucose-resistant mutant strains of Clostridium tyrobutyricum

Laboratory mutagenesis of microorganisms offers the possibility of relating acquired mutations to improve the quality of microbial cultures. In the present study, a mutant strain, Clostridium tyrobutyricum ATCC 25755 DG-8, with significantly elevated α-amylase activity as well as resistant to the non-metabolizable and toxic glucose analog 2-deoxyglucose (2-DG) was obtained by implanting the low-energy nitrogen ion beam. DG-8 was further developed to produce hydrogen by simultaneous saccharification and fermentation (SSF) directly form cassava starch in batch fermentation mode, which to our knowledge is at the first attempt in genus Clostridium. Our results demonstrated that the increased activity of α-amylase would be attributed to the hydrogen over-producing. Higher hydrogen yield (3.2 mol/mol glucose) was achieved with the volumetric productivity of 0.41 L/h/L when the initial total sugar concentration of cassava starch rise up to 100 g/L. The present work will help to decrease the cost of hydrogen fermentation process and stimulate its industrial application in the near future.     

A polyene-based polymer functionalized with a model of [FeFe]-hydrogenase and film electrodes assembled from the polymer via spin-coating

A diiron hexacarbonyl complex possessing an alkynyl group as a model complex of the diiron sub-unit of [FeFe]-hydrogenase was polymerized under the catalysis of WCl₆-SnPh₄. The polymer (Poly-{Fe₂}) functionalized with {Fe₂(CO)₆} units which is dominated by cis-form was fully characterised using FTIR, NMR, SEM, TEM, and TGA techniques. Through modifying the monomer, the properties, for example, solubility, of the resultant polymer could be tuned and much larger molecule weight, which was estimated as 7.93 × 10⁵ g mol⁻¹ using static light scattering technique was achieved without compromising its solubility. Spin-coating the functionalized polymer onto the surface of vitreous carbon electrode with or without multi-wall carbon nanotubes (MWCNTs) produced film electrodes which show electrochemical responses. Adding MWCNTs into the film enhances significantly the electrochemical response probably via not only improving the conductivity of the film, but also the increase in its effective surface area after being doped with MWCNTs.     

Purification and characterization of a highly thermostable, oxygen-resistant, respiratory [NiFe]-hydrogenase from a marine, aerobic hydrogen-oxidizing bacterium Hydrogenovibrio marinus

The membrane-bound [NiFe]-hydrogenase from Hydrogenovibrio marinus (HmMBH) was purified homogeneously under anaerobic conditions. Its molecular weight was estimated as 110 kDa, consisting of a heterodimeric structure of 66 kDa and 37 kDa subunits. The purified enzyme exhibited high activity in a wide temperature range: 185 U/mg at 30 °C and 615 U/mg at 85 °C (the optimum temperature). The K_m and k_cat/K_m values for H₂ were, respectively, 12 μM and 8.58 × 10⁷ M⁻¹ s⁻¹. The optimum reaction pH was 7.8, but its stability was particularly high at pH 4.0-7.0. Results show that HmMBH was remarkably thermostable and oxygen-resistant: its half-life was 75 h at 80 °C under H₂, and more than 72 h at 4 °C under air. The air-oxidized HmMBH for 72 h showed only weak EPR signals of Ni-B, suggesting a structural feature in which the active center is not easily oxidized.     

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.     

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.     

Hydrogen production based on the heterologous expression of NAD+-reducing [NiFe]-hydrogenase from Cupriavidus necator in different genetic backgrounds of Escherichia coli strains

This study explored the genetic engineering of Escherichia coli for hydrogen (H₂) production. In E. coli W3110, the introduction of NAD⁺-reducing [NiFe]-hydrogenase from Cupriavidus necator, combined with the inactivation of three endogenous [NiFe]-hydrogenases, exhibited not only H₂ production but also H₂ uptake based on exogenous hydrogenase. Although the H₂ production ability was much lower than the H₂ uptake ability, inactivation of the ethanol, lactate, and succinate production pathways resulted in a marked increase in H₂ production, demonstrating the bidirectional hydrogenase function in vivo depending on NADH/NAD⁺. Unexpectedly, H₂ production was completely repressed under conditions for high expression of exogenous hydrogenase. Furthermore, the introduction of the heterologous enzyme markedly repressed the endogenous H₂ production ability of E. coli W3110 but not the HST02. These in vivo behaviors largely correlated with in vitro hydrogenase activity suggested complicated interactions between the native and nonnative functional expression of [NiFe]-hydrogenases.     

Heterologous functionality and roles of conserved cysteine motifs of the [NiFe]-hydrogenase accessory protein,HupK/HoxV

Numerous auxiliary proteins participate in the complex posttranslational modification process of [NiFe]-hydrogenases. In Thiocapsa roseopersicina, the HupK protein is important for the formation of active membrane-bound hydrogenases. The HupK proteins of various origins have moderate similarity to each other and to the large subunits of [NiFe]-hydrogenases. Site directed mutagenesis experiments were performed to disclose the role of the highly conserved cysteines in HupK. Cys54 was shown to be indispensable for the proper function of HupK and recreation of a large subunit like cysteine profile had negative effect on the activity of HupSL hydrogenase. Although, the results of the mutagenesis study slightly differed from that obtained for Ralstonia eutropha HoxV, it was clearly demonstrated that HupK from T. roseopersicina and HoxV from R. eutropha can substitute each other. It was also demonstrated that HoxV could be involved in the maturation of both Hup and Hyn hydrogenases in T. roseopersicina.     

Maturation and processing of the recombinant [FeFe] hydrogenase from Desulfovibrio vulgaris Hildenborough (DvH) in Escherichia coli

The need of an efficient and well-characterized heterologous expression system of [FeFe]-hydrogenase for the production of O₂-resistant mutants prompted us to explore the use of Escherichia coli as a possible expression system. O₂-resistant hydrogenase mutants could be instrumental when coupling oxygenic photosynthesis with hydrogen bio-production. In general, expression of Desulfovibrio vulgaris Hildenborough active enzyme in E. coli was very modest indicating that the co-expression of the HydE, HydF and HydG maturases with hydrogenase structural genes in this bacterium is not optimal. A 28-fold increase in activity was obtained when these proteins were co-expressed with the Iron–Sulfur Cluster operon, indicating that one of the problems with over-expression is the correct insertion of FeS clusters. However, the measured activity is still about 4000-fold lower than the one measured in the native hydrogenase indicating that additional, so far unidentified factors may be necessary for optimal heterologous expression of [FeFe]-hydrogenase.     

Artificial water-soluble systems inspired by [FeFe]-hydrogenases for electro- and photocatalytic hydrogen production

[FeFe]-hydrogenases efficiently catalyze the hydrogen evolution reactions (HERs) at rates of up to 10⁴ s⁻¹ with low overpotentials in aqueous media. Although the small-molecule diiron mimetics of the active site of [FeFe]-hydrogenases have been studied for years, most of the synthetic models mediate the catalysis in organic solvents, seriously limiting the application of bioinspired catalytic systems in large-scale H₂ production. Herein, we systematically present the state-of-the-art artificial water-soluble systems inspired by [FeFe]-hydrogenases for potentially electro- and photocatalytic HERs utilizing either electrical or solar energy inputs. The engineering motifs and catalytic properties of these water-soluble mimetic systems have been surveyed and discussed. We hope the present review will shed light on some helpful aspects for designing artificial assembling catalysts for HERs in aqueous milieu and provide mechanistic insights into a broad array of natural oxidoreductases.     

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.     

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.     

Effect of pH on glucose and starch fermentation in batch and sequenced-batch mode with a recently isolated strain of hydrogen-producing Clostridium butyricum CWBI1009

This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production rates and yields were investigated at 30 °C in a 2.3 L bioreactor operated in batch and sequenced-batch mode using glucose and starch as substrates. In order to study the precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 mol H₂ mol⁻¹ glucose) was measured when the pH was maintained at 5.2. The acetate and butyrate yields were 0.35 mol acetate mol⁻¹ glucose and 0.6 mol butyrate mol⁻¹ glucose. For starch a maximum yield of 2.0 mol H₂ mol⁻¹ hexose, and a maximum production rate of 15 mol H₂ mol⁻¹ hexose h⁻¹ were obtained at pH 5.6 when the acetate and butyrate yields were 0.47 mol acetate mol⁻¹ hexose and 0.67 mol butyrate mol⁻¹ hexose.