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.     

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.     

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.     

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.     

Full length obtains of hydA and phylogenetic analysis of bio-hydrogen production new species of Clostridium based on efficient hydA degenerate primers

In fermentative bio-hydrogen production, bacteria from genus Clostridium are considered to be efficient candidates. Studies on bacteria isolation and functional gene analysis of Clostridium make contribution to the H₂ production efficiency improvement. In this study, six degenerate primers were designed based on [FeFe]-hydrogenase (hydA) gene sequences from representative biohydrogen producers of Clostridium, and employed to obtain the hydA gene open reading frames (ORFs) of C. guangxiense and C. neuense. Their phylogenetic relationships were also evaluated with hydA gene partial sequences. The ORFs of hydA gene from strains C. guangxiense ZGM211^T and C. neuense G1^T were obtained with the same length of 1719 bp and theoretical pI 8.09, but different stop codons of TAA and TAG, and Mw of 63.03 and 63.13 kDa, respectively. The phylogenetic tree constructed with hydA partial gene sequences was very similar to that of full-length 16S rRNA gene. It more accurately reflected the phylogenetic relationship of C. guangxiense, C. neuense and related species, and suggested that the hydA gene could be an assistant or reference to 16S rRNA gene in the phylogenetic relationship investigation of bio-hydrogen producers within genus Clostridium.     

Simultaneous saccharification and fermentation of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung

The objective of this study was to optimize the culture conditions for simultaneous saccharification and fermentation (SSF) of cellulose for bio-hydrogen production by anaerobic mixed cultures in elephant dung under thermophilic temperature. Carboxymethyl cellulose (CMC) was used as the model substrate. The investigated parameters included initial pH, temperature and substrate concentration. The experimental results showed that maximum hydrogen yield (HY) and hydrogen production rate (HPR) of 7.22 ± 0.62 mmol H₂/g CMC_added and 73.4 ± 3.8 mL H₂/L h, respectively, were achieved at an initial pH of 7.0, temperature of 55 °C and CMC concentration of 0.25 g/L. The optimum conditions were then used to produce hydrogen from the cellulose fraction of sugarcane bagasse (SCB) at a concentration of 0.40 g/L (equivalent to 0.25 g/L cellulose) in which an HY of 7.10 ± 3.22 mmol H₂/g cellulose_added. The pre-dominant hydrogen producers analyzed by polymerase chain reaction-denaturing gel gradient electrophoresis (PCR-DGGE) were Thermoanaerobacterium thermosaccharolyticum and Clostridium sp. The lower HY obtained when the cellulose fraction of SCB was used as the substrate might be due to the presence of lignin in the SCB as well as the presence of Lactobacillus parabuchneri and Lactobacillus rhamnosus in the hydrogen fermentation broth.     

Evaluation of bio-hydrogen production using rice straw hydrolysate extracted by acid and alkali hydrolysis

This study was conducted to investigate the properties of hydrolysates obtained from acid and alkali hydrolysis and to evaluate the feasibility of employing them for bio-hydrogen production. High sugar concentrations of 16.8 g/L and 13.3 g/L were present in 0.5% and 1.0% H₂SO₄ hydrolysates, respectively. However, H₂SO₄ hydrolysis resulted in large amounts of short-chain fatty acids (SCFAs) and furan derivatives, which were removed by detoxification. In bio-hydrogen production, 1.0% H₂SO₄ hydrolysate showed a 55.6 mL of highest hydrogen production and 1.14 mol-H₂/mol-hexose equivalent_added of hydrogen yield. In control and 1.0% NaOH hydrolysate, 29.7 mL and 36.9 mL of hydrogen were produced, respectively. Interestingly, relatively high acetate and butyrate production resulted in lactate reduction. Also, NH₄OH hydrolysate produced less than 10 mL of hydrogen. Thus, these results indicate that hydrogen production and metabolite distribution can vary depending on the sugars and by-product composition in the hydrolysate.     

Comparison of bio-hydrogen production from hydrolyzed wheat starch by mesophilic and thermophilic dark fermentation

Hydrogen gas production potentials of acid-hydrolyzed and boiled ground wheat were compared in batch dark fermentations under mesophilic (37 °C) and thermophilic (55 °C) conditions. Heat-treated anaerobic sludge was used as the inoculum and the hydrolyzed ground wheat was supplemented by other nutrients. The highest cumulative hydrogen gas production (752 ml) was obtained from the acid-hydrolyzed ground wheat starch at 55 °C and the lowest (112 ml) was with the boiled wheat starch within 10 days. The highest rate of hydrogen gas formation (7.42 ml H₂ h⁻¹) was obtained with the acid-hydrolyzed and the lowest (1.12 ml H₂ h⁻¹) with the boiled wheat at 55 °C. The highest hydrogen gas yield (333 ml H₂ g⁻¹ total sugar or 2.40 mol H₂ mol⁻¹ glucose) and final total volatile fatty acid (TVFA) concentration (10.08 g L⁻¹) were also obtained with the acid-hydrolyzed wheat under thermophilic conditions (55 °C). Dark fermentation of acid-hydrolyzed ground wheat under thermophilic conditions (55 °C) was proven to be more beneficial as compared to mesophilic or thermophilic fermentation of boiled (partially hydrolyzed) wheat starch.     

Effects of pretreatment method of natural bacteria source on microbial community and bio-hydrogen production by dark fermentation

The effects of pretreatment method of cow dung compost, which was employed as natural hydrogen bacteria source, on the microbial community, population distribution of microbes and hydrogen production potential were investigated in the batch tests. The maximum hydrogen yield of 290.8 mL/L-culture appeared in the pretreated method A (infrared drying) by dark fermentation. The pretreated method of compost significantly affected microbial succession, population distribution of microbes. Both Clostridium sp. and Enterobacter sp. were found to be two species of preponderant hydrogen-producing bacteria, the next best was Bacteroides sp. and Veillonella sp., the last was Lactobacillus sp. and Streptococcus sp., which were also essential. The results showed that the mutualism and symbiosis relations of the mixed bacteria played a critical role in hydrogen fermentation process.     

Effects of the substrate and cell concentration on bio-hydrogen production from ground wheat by combined dark and photo-fermentation

Effects of the substrate and cell concentration on bio-hydrogen production from ground wheat solution were investigated in combined dark-light fermentations. The ratio of the dark to light bacteria concentration (D/L) was kept constant at 1/10 while the wheat powder (WP) concentration was changed between 2.5 and 20 g L⁻¹ with a total cell concentration of 0.41 g L⁻¹ in the first set of experiments. Cell concentration was changed between 0.5 and 5 g L⁻¹ in the second set of experiments while the wheat powder concentration was constant at 5 g L⁻¹ with a D/L ratio of 1/7. The highest cumulative hydrogen (135 ml) and formation rate (3.44 ml H₂ h⁻¹) were obtained with the 20 g L⁻¹ wheat powder concentration. However, the highest yield (63.9 ml g⁻¹ starch) was obtained with the 2.5 g L⁻¹ wheat powder. In variable cell concentration experiments, the highest cumulative hydrogen (118 ml) and yield (156.8 ml H₂ g⁻¹ starch) were obtained with 1.1 g L⁻¹ cell concentration yielding an optimal biomass/substrate ratio of 0.22 g cells/g WP.     

Dark fermentation of acid hydrolyzed ground wheat starch for bio-hydrogen production by periodic feeding and effluent removal

Dark fermentation of acid hydrolyzed ground wheat starch for bio-hydrogen production by periodic feeding and effluent removal was investigated at different feeding intervals. Ground wheat was acid hydrolyzed at pH = 3 and T = 121 °C for 30 min using an autoclave. The resulting sugar solution was subjected to dark fermentation with periodic feeding and effluent removal. The feed solution contained 9 ± 0.5 g L⁻¹ total sugar supplemented with some nutrients. Depending on the feeding intervals hydraulic residence time (HRT) was varied between 6 and 60 h. Steady-state daily hydrogen production increased with decreasing HRT. The highest daily hydrogen production (305 ml d⁻¹) and volumetric hydrogen production rate (1220 ml H₂ L⁻¹ d⁻¹) were obtained at HRT of 6 h. Hydrogen yield (130 ml H₂ g⁻¹ total sugar) reached the highest level at HRT = 24 h. Effluent total sugar concentration decreased, biomass concentration and yield increased with increasing HRT indicating more effective sugar fermentation at high HRTs. Dark fermentation end product profile shifted from acetic to butyric acid with increasing HRT. High acetic/butyric acid ratio obtained at low HRTs resulted in high hydrogen yields.     

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.     

Effect of organic loading rate and solids retention time on microbial population during bio-hydrogen production by dark fermentation in large lab-scale

The experimental investigation aimed at the study of the microbial population during the continuous operation of a complete mixed reactor in large lab-scale (30 L) by variation of the Organic Loading Rate (OLR) and Sludge Retention Time (SRT) ranging from 10 g sucrose/(L?d) to 30 g/(L?d) and from 12 h to 48 h respectively. H₂ yield reached to 1.72 mol H₂/mol hexose for HRT = 1.6 d and OLR = 20 g sucrose/(L?d). In each phase the dominant microbial genera were identified by sequencing after a Polymerase Chain Reaction (PCR) with universal primers for the domains of Archaea and Eubacteria and specific for Clostridium species and genetic material isolation by Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The phylogenetic analyses showed that hydrogen producing Clostridium species could be affiliated in all experimental phases. Other dominant genera were affiliated mainly to Ethanoligenes harbinense and uncultured Prevotella and Selonomonas species. Bio-hydrogen production was associated to a mixed butyric/ethanol type fermentation facilitated mainly by Clostridium tyrobutyricum and E. harbinense in the presence of lactate as intermediate metabolic product.     

Consolidated bioprocessing of hydrogen production from agave biomass by Clostridium acetobutylicum and bovine ruminal fluid

The biological production of H₂ represents a renewable and eco-friendly energy alternative compared to fossil fuels. However, its production from lignocellulose involves the use of expensive enzymatic complexes. In the present work, the production of H₂ from pretreated agave biomass was evaluated by means of a Consolidated Bioprocess (CBP). This strategy was carried through the interaction of cellulose-degrading microorganisms obtained from bovine ruminal fluid (BRF) capable of enhancing H₂ production by Clostridium acetobutylicum. The results obtained show the capacity of BRF to hydrolyze the acid pretreated agave, improving the production of H₂ in the experiments where the inoculum of Clostridium was greater. According to the results, production of H₂ is significantly affected by the increase of the solids loading, obtaining a maximum H₂ production at a 10% of solids loading, pH 5.5 and 35 °C, representing a yield of 150 L of H₂ per Kg of biomass in 264 h.     

Bio-hydrogen production by mixed culture of photo- and dark-fermentation bacteria

Clostridium butyricum and Rhodopseudomonas faecalis RLD-53 were employed to produce hydrogen in mixed culture with glucose as sole substrate. Due to the great difference on growth rate and acid-resistant capacity between photo-fermentative bacteria and dark-fermentative bacteria, directly mixed culture of the two kinds of bacteria in different ratio was studied in this work. Hydrogen yield, volatile acids, pH and biomass in different periods were evaluated. Acetic acid and butyric acid produced by C. butyricum were dominant terminal fermentation products, and they were effective substrates for photo-fermentative bacteria. The cooperation was formed in a way like food chain. But compared to the production rate of volatile acids produced by C. butyricum, the utilization rate by photo-fermentative bacteria was far slower. The results demonstrated that the growth of photo-fermentative bacteria was limited when pH decreased sharply. The best ratio of C. butyricum to R. faecalis RLD-53 was 1:600. The maximum yield of hydrogen reached 122.4 ml-H₂/vessel and hydrogen production rate was 0.5 ml-H₂/ml-culture/day.     

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.     

Effects of light source,intensity and lighting regime on bio-hydrogen production from ground wheat starch by combined dark and photo-fermentations

Combined dark and photo-fermentation of ground wheat starch was carried out by using different light sources, intensities and lighting regime. A mixture of heat treated anaerobic sludge and Rhodobacter sphaeroides-RV with a certain light/dark bacteria ratio was used in batch experiments. Tungsten, fluorescent, infrared (IR), tungsten + infrared, halogen lamps were used as light sources with a light intensity of 270 Wm⁻² along with sunlight. Halogen lamp was found to be the most suitable light source yielding the highest cumulative hydrogen formation (178 ml) and yield (218 ml g⁻¹ starch). Combined fermentations were performed at different light intensities (1–10 klux) using the halogen lamp in the second set of experiments. The optimum light intensity was found to be 10 klux (approx. 352 Wm⁻²) resulting in the highest cumulative hydrogen (111 ml) and hydrogen yield (139 ml H₂ g⁻¹ starch). Hydrogen formation was limited by the availability of light at low light intensities below 10 klux. Durations of dark/light cycles were changed to determine the most suitable lighting regime. Hydrogen gas formation increased with increasing cycle time and continuous lighting resulted in the highest cumulative hydrogen formation and hydrogen yield.     

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.     

Cell degeneration and performance decline of immobilized Clostridium acetobutylicum on bagasse during hydrogen and butanol production by repeated cycle fermentation

The cell degeneration and the fermentation performance decline during repeated cycle fermentation with immobilized Clostridium acetobutylicum on bagasse for hydrogen and butanol production was studied. The cell degeneration has been characterized in abnormality of a long-chain morphology through 7 cycles of repeated fermentation. The fermentation performance decline has been indicated by decrease of glucose consumption rate from 0.82 g/L/h to 0.22 g/L/h, reduction of hydrogen production and productivity from 6 L/L to 2.5 L/L and 170 mL/L/h to 40 mL/L/h, as well as the decrease of butanol production and butanol productivity from 6.5 g/L to 1.0 g/L and 0.18 g/L/h to 0.02 g/L/h, respectively. The ratio of hydrogen production and butanol production was in the range of 6–10 and 17–20 during the earlier three and later four cycles, respectively. The carbon flow directed to ethanol was higher during the later period of fermentation.