Specific Activity Of Hydrogenase Research Articles

Ferric oxide/date seed activated carbon nanocomposites mediated dark fermentation of date fruit wastes for enriched biohydrogen production

Biohydrogen production from biomass waste, not only addresses the energy demand in a renewable manner but also resolves the safe disposal issues associated with these biowastes. Also, scalable and low-cost techniques to enhance biohydrogen production have gained more attraction and are highly explored. In this research work, date-palm fruit wastes have been studied for their biohydrogen production potential using Enterobacter aerogenes by dark fermentation. Hydrogen yield and productivity were improved through the addition of iron oxide nanoparticles (Fe3O4 NPs) and its date seed activated carbon nanocomposites (Fe3O4/DSAC) to the fermentation media. Studies on discrete inclusions of these NPs showed that the appropriate dosage of NPs promoted, while higher dosages repressed the hydrogen production performance. Optimal dosage and fermentation time was observed as 150 mg/L and 24 h for both the additives. Fe3O4/DSAC nanocomposites showed better hydrogen production enhancement than Fe3O4 NPs. Maximum hydrogen yield of 238.7 mL/g was obtained for the 150 mg/L nanocomposites, which was 65.7% higher than that of the standalone Fe3O4 NPs and three folds higher than the yield of the control run without any NPs inclusion (78.4 mL/g). Metabolites analysis showed that the hydrogen evolution followed the ethanol-acetate pathway. Formation levels of longer chain propionate and butyrate co-metabolites were significantly low in the presence of Fe3O4/DSAC than Fe3O4. The carbon support in the nanocomposites acted as an adsorbent-buffer, which favored the medium pH in-addition to the stimulatory effects of Fe3O4 NPs. Cell growth and specific hydrogenase activity analysis were also performed to supplement the hydrogen production results. Gompertz and modified Logistic kinetic models were employed for kinetic modeling of experimental hydrogen production values. The Fe3O4/DSAC nanocomposites exhibited significant application potential for the production of biohydrogen from date fruit wastes.

Absolute quantification of selected photosynthetic electron transfer proteins in Chlamydomonas reinhardtii in the presence and absence of oxygen.

The absolute amount of plastocyanin (PC), ferredoxin-NADP+-oxidoreductase (FNR), hydrogenase (HYDA1), and ferredoxin 5 (FDX5) were quantified in aerobic and anaerobic Chlamydomonas reinhardtii whole cells using purified (recombinant) proteins as internal standards in a mass spectrometric approach. Quantified protein amounts were related to the estimated amount of PSI. The ratios of PC to FNR to HYDA1 to FDX5 in aerobic cells were determined to be 1.4:1.2:0.003:0. In anaerobic cells, the ratios changed to 1.1:1.3:0.019:0.027 (PC:FNR:HYDA1:FDX5). Employing sodium dithionite and methyl viologen as electron donors, the specific activity of hydrogenase in whole cells was calculated to be 382 ± 96.5 μmolH2min-1mg-1. Importantly, these data reveal an about 70-fold lower abundance of HYDA1 compared to FNR. Despite this great disproportion between both proteins, which might further enhance the competition for electrons, the alga is capable of hydrogen production under anaerobic conditions, thus pointing to an efficient channeling mechanism of electrons from FDX1 to the HYDA1.

Glycerol and mixture of carbon sources conversion to hydrogen by Clostridium beijerinckii DSM791 and effects of various heavy metals on hydrogenase activity

Hydrogen is a carbon-neutral energy feedstock which is produced during fermentation of various carbon sources. The genomes of clostridia encode mainly [Fe-Fe]-hydrogenases. Clostridium beijerinckii DSM791 performed anaerobic fermentation of glycerol in batch culture at pH 7.5 and pH 5.5 and produced H2. At pH 7.5, the glycerol consumption rate was 3.7 g/g cell mass/h, which was higher than that at pH 5.5. H2 production reached 5 mmol/h/g cell mass at pH 7.5. The specific hydrogenase activity was ∼1.4 fold higher if cells were grown on glycerol compared to cells grown on glucose. Single (Fe2+, Fe3+, Ni2+) or mixed supply of metals (Fe2+ and Ni2+) increased the specific hydrogenase activity by ∼50%. These results suggest that C. beijerinckii DSM791 could be used as a potential H2 producer. It may help to further enhance H2 production using different industrial or agricultural wastes where glycerol and other carbon sources are present.

Photoautotrophic hydrogen production by eukaryotic microalgae under aerobic conditions

Eukaryotic algae and cyanobacteria produce hydrogen under anaerobic and limited aerobic conditions. Here we show that novel microalgal strains (Chlorella vulgaris YSL01 and YSL16) upregulate the expression of the hydrogenase gene (HYDA) and simultaneously produce hydrogen through photosynthesis, using CO2 as the sole source of carbon under aerobic conditions with continuous illumination. We employ dissolved oxygen regimes that represent natural aquatic conditions for microalgae. The experimental expression of HYDA and the specific activity of hydrogenase demonstrate that C. vulgaris YSL01 and YSL16 enzymatically produce hydrogen, even under atmospheric conditions, which was previously considered infeasible. Photoautotrophic H2 production has important implications for assessing ecological and algae-based photolysis.

Characterization of hydrogenase from purple sulfur bacterium Thiocapsa roseopersicina and its prolonged in vitro hydrogen evolution

Hydrogenase from a purple sulfur bacterium, Thiocapsa roseopersicina NCIB 8347, was purified, characterized and investigated for in vitro hydrogen ( H 2 ) production. The cell concentration and the specific hydrogenase activity were increased when T. roseopersicina NCIB 8347 was photo-autotropically cultivated with the addition of nutrients every 24 h. Crude extracts of the cells were prepared by sonication and then subjected sequentially to ultra-centrifugation, ammonium sulfate fractionation and heat-treatment to remove the impurities. Four-step separation of chromatographic procedures utilizing Q-Sepharose, Phenyl Superose, Mono-Q and Superdex resulted in a 9.56-fold purification with a 0.25% yield of crude cell extract. The optimum pH and temperature for the H 2 evolution of the purified enzyme was 7.0 and 70 °C, respectively, and those for optimum cell growth were 7.0 and 25 °C, respectively. The maximum H 2 -evolution activity was exhibited at 70 °C, and when the reaction temperature was decreased to 50 °C, the manifest activity fell to 44%. Hydrogenase activities were initially increased in the course of the 50–70 °C heat-treatment, whereas the remaining activities after 5-h heat-treatment at 50 and 70 °C were 110% and 60% of the initial ones, respectively. The purified hydrogenase, at 50 °C and pH 7.0, evolved H 2 gas for approximately 10 days under in vitro conditions, using sodium dithionite and methyl viologen as the electron donor and carrier, respectively.

Development of a Genetic System for the Chemolithoautotrophic Bacterium Thiobacillus denitrificans

Thiobacillus denitrificans is a widespread, chemolithoautotrophic bacterium with an unusual and environmentally relevant metabolic repertoire, which includes its ability to couple denitrification to sulfur compound oxidation; to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV); and to oxidize mineral electron donors. Recent analysis of its genome sequence also revealed the presence of genes encoding two [NiFe]hydrogenases, whose role in metabolism is unclear, as the sequenced strain does not appear to be able to grow on hydrogen as a sole electron donor under denitrifying conditions. In this study, we report the development of a genetic system for T. denitrificans, with which insertion mutations can be introduced by homologous recombination and complemented in trans. The antibiotic sensitivity of T. denitrificans was characterized, and a procedure for transformation with foreign DNA by electroporation was established. Insertion mutations were generated by in vitro transposition, the mutated genes were amplified by the PCR, and the amplicons were introduced into T. denitrificans by electroporation. The IncP plasmid pRR10 was found to be a useful vector for complementation. The effectiveness of the genetic system was demonstrated with the hynL gene, which encodes the large subunit of a [NiFe]hydrogenase. Interruption of hynL in a hynL::kan mutant resulted in a 75% decrease in specific hydrogenase activity relative to the wild type, whereas complementation of the hynL mutation resulted in activity that was 50% greater than that of the wild type. The availability of a genetic system in T. denitrificans will facilitate our understanding of the genetics and biochemistry underlying its unusual metabolism.

Fermentation of biomass‐generated synthesis gas: Effects of nitric oxide

The production of renewable fuels, such as ethanol, has been steadily increasing owing to the need for a reduced dependency on fossil fuels. It was demonstrated previously that biomass-generated synthesis gas (biomass-syngas) can be converted to ethanol and acetic acid using a microbial catalyst. The biomass-syngas (primarily CO, CO(2), H(2), and N(2)) was generated in a fluidized-bed gasifier and used as a substrate for Clostridium carboxidivorans P7(T). Results showed that the cells stopped consuming H(2) when exposed to biomass-syngas, thus indicating that there was an inhibition of the hydrogenase enzyme due to some biomass-syngas contaminant. It was hypothesized that nitric oxide (NO) detected in the biomass-syngas could be the possible cause of this inhibition. The specific activity of hydrogenase was monitored with time under varying concentrations of H(2) and NO. Results indicated that NO (at gas concentrations above 40 ppm) was a non-competitive inhibitor of hydrogenase activity, although the loss of hydrogenase activity was reversible. In addition, NO also affected the cell growth and increased the amount of ethanol produced. A kinetic model of hydrogenase activity with inhibition by NO was demonstrated with results suggesting there are multiple binding sites of NO on the hydrogenase enzyme. Since other syngas-fermenting organisms utilize the same metabolic pathways, this study estimates that NO < 40 ppm can be tolerated by cells in a syngas-fermentation system without compromising the hydrogenase activity, cell growth, and product distribution.

Continuous culture of Methanococcus jannaschii, an extremely thermophilic methanogen

Methanococcus jannaschii, an extremely thermophilic methanogen isolated from a deep-sea hydrothermal vent, was grown at 80 degrees C in continuous culture on a mineral salts medium gassed with H(2) and CO(2) at three different flow rates. The maximum specific growth rate was 0.56 h(-1), and the maximum specific methane productivity was 0.32 (mol g(-1) h(-1)). Uncoupling of growth and methane production was evidenced by an increase in teh non-growth-associated rate of methane formation, beta, with increasing gaseous input. The specific hydrogenase activity exhibited growth-assiciated behaviour at low growth rates, but showed no dependence on growth at higher growth rates. The growth dependence of hydrogenase activity is consistent with the pressure dependence of hydrogenase activity measured in previous experiments. In contrast, the specific protease activity was independent of the growth rate over the entire range of dilution rates studied. (c) 1994 John Wiley & Sons, Inc.

Autotrophic growth of nitrogen-fixingAzospirillum species and partial characterization of hydrogenase from strain CC

Out of 15 strains ofAzospirillum spp. isolated from the roots of different plants, only 4 (CY, M, CC, and AM) were able to grow autotrophically with H2 and CO2. All of them showed H2 uptake in the presence of oxygen or methylene blue and ribulose-1,5-bisphosphate carboxylase activity. Among the four strains, strain CC isolated from the roots ofCenchrus cilliaris showed maximum H2+O2 uptake (32.5 μl/min. mg protein) as well as H2 uptake in the presence of methylene blue (41.4 μl/min·mg protein) and also the maximum activity of ribulose-1,5-bisphosphate carboxylase (17 units [U]/g protein). The doubling time of this strain under autotrophic growth conditions and at low oxygen concentration (2.5%, vol/vol) was 10 h. At the same O2 concentration the maximal rates of H2+O2 uptake were reached. The distribution of hydrogenase activity among soluble and particulate protein fractions revealed that the hydrogenase ofAzospirillum strain CC is a membrane-bound enzyme. It showed cross-reaction with antibodies raised against the membrane-bound hydrogenase ofAlcaligenes eutrophus. The hydrogenase in intact cells and crude extracts reacted with methylene blue, phenazine methosulfate, and ferricyanide, but not with NAD or FMN. The specific hydrogenase activity, with methylene blue as an acceptor, was 5.71 U/mg protein in crude extract at 9.38 U/mg protein in the membrane suspension. Hydrogen evolution from reduced viologen dyes could not be demonstrated. The hydrogenase is oxygen sensitive and can be optimally stabilized by addition of dithionite to H2-gased samples.

PRODUCTION OF ACETIC ACID FROM HYDROGEN AND CARBON DIOXIDE BY CLOSTRIDIUM SPECIES ATCC 29797

Abstract Clostridium sp. ATCC 29797 converts H2 and CO2 to acetic acid in stationary phase in almost 100% yield. The resting cells are stable for up to 300 h and produce acetate to a final product concentration of 221 mM. The specific productivity of acetate during the stationary phase appears limited by the specific activity of hydrogenase. A high pressure fermentor was constructed to investigate the use of pressure, as a general strategy, for increasing the rates of microbial syngas conversions. Fermentations were conducted at pressures up to 1000 psig using various mixtures of H2 and CO2. The use of high pressure proved to be an effective method for eliminating gas transfer resistances and increasing the volumetric productivity. Hydrostatic pressure had little effect on the specific productivity of acetate, however, high partial pressures of hydrogen caused the specific productivity and total acetate accumulated to decrease. High pressures of hydrogen also caused a decrease in the specific activity of ...

Effect of enzyme concentration on apparent specific activity of hydrogenase

The effect of enzyme concentration on the H 2-uptake and H 2-evolving activities of the reversible hydrogenase from Thiocapsa roseopersicina was examined. In the activity range assayed by a spectrophotometric technique the apparent H 2-uptake specific activity varied greatly with hydrogenase concentration. Study of H 2-evolving activity measured by the H 2 electrode method and compared with a gas chromatographic assay also indicated that specific activity was highly dependent on enzyme concentration. The results indicate that the widely applied hydrogenase assays give systematically erroneous specific activity values. These assays should be used only for relative measurements and the hydrogenase concentration in the reaction mixture should be kept constant. To make the data from various laboratories comparable the assay parameters should be standardized.

Autotrophic growth of strains ofRhizobium and properties of isolated hydrogenase

Four strains ofRhizobium (R. trifolii RCL10,R. japonicum S19 and SB16, andRhizobium sp. NEA4) were demonstrated to grow lithoautotrophically with molecular hydrogen as sole electron donor and with ammonium or with N2 as N source. All of them showed ribulose-1,5-bisphosphate carboxylase activity and hydrogenase (H2-uptake) activity with methylene blue and oxygen as electron acceptors. ForR. japonicum SB 16, a doubling time under autotrophic conditions of 30 h and a specific hydrogenase activity (methylene blue reduction) in crude extracts of 1.4 U/mg protein were calculated.Rhizobium hydrogenase is a membrane-bound enzyme. It is mainly detectable in particulate cell fractions, it cross-reacts with the antibodies of the membrane-bound hydrogenase ofAlcaligenes eutrophus, and is unable to reduce NAD. The isolated hydrogenase is a relatively oxygen-sensitive enzyme with a half-life of three days when stored at 4°C under air.

Physiological characterization ofPseudomonas pseudoflava GA3

Pseudomonas pseudoflava GA3, an aerobic hydrogen-oxidizing bacterium, was characterized physiologically. The doubling time of autotrophically growing cells was 2.5 h for cells growing heterotrophically on sucrose it was 1.5 h; and for cells growing heterotrophically on fructose, succinate, lactate, glutamate, treahalose, glucose, and pyruvate it was about 1.6 h. Glucose and fructose were degraded via the Entner-Doudoroff pathway. 6-Phosphogluconate dehydrogenases (NAD or NADP-dependent) were not detected. Hydrogen did not suppress or inhibit the utilization of organic substrates. The specific hydrogenase activity was influenced by the growth conditions. An oxygen concentration of 20% (vol/vol) in the gas atmosphere provided conditions for continued exponential growth but diminished the specific hydrogenase activity. Cells contained a single membrane-bound hydrogenase; intact membranes were able to reduce menadion, ubiquinone Q10, methylene blue, phenazine methosulfate, and dichlorophenolindophenol. The hydrogenase was solubilized from the membrane fraction by treatment with 0.2% Triton X-100 and 10% sucrose, resulting in a yield of about 60% of total activity.

The activities of hydrogenase and enoate reductase in two Clostridium species, their interrelationship and dependence on growth conditions.

The enzyme activities of Clostridium La 1 and Clostridium kluyveri involved in the stereospecific hydrogenation of α,β-unsaturated carbonyl compounds with hydrogen gas were measured. In C. La 1 the specific activities of hydrogenase and enoate reductase depended heavily on the growth phase and the composition of the medium. During growth in batch cultures on 70 mM crotonate the specific activity of hydrogenase increased and then dropped to about 10% of its maximum value, whereas the activity of enoate reductase reached its maximum in cells of the stationary phase. Under certain conditions during growth the activity ratio hydrogenase: enoate reductase changed from 120 to 1. Thus, the rate limiting enzyme for the hydrogenation can be either the hydrogenase or the enoate reductase, depending on the growth conditions of the cells.

The regulation of hydrogenase formation as a differentiating character of strains of Paracoccus denitrificans.

Paracoccus denitrificans strains Stanier 381 (DSM 65), Morris (DSM 413), and Vogt 11 (DSM 415) and eleven newly isolated strains were compared with respect to the localization of hydrogenase and its regulation. In all strains hydrogenase was found to be membrane-bound and not able to reduce pyridine nucleotides. The enzyme was inducible in strain 381 and was found only in cells grown with hydrogen as the sole hydrogen donor; in cells grown under mixotrophic or heterotrophic conditions the hydrogenase activity was zero. In all other strains hydrogenase was constitutive and was present in cells grown under autotrophic, mixotrophic and heterotrophic conditions. Under the latter conditions the specific hydrogenase activity was even higher than under mixotrophic conditions.

Hydrogen Evolution from Alfalfa and Clover Nodules and Hydrogen Uptake by Free-Living Rhizobium meliloti

A series of Rhizobium meliloti and Rhizobium trifolii strains were used as inocula for alfalfa and clover, respectively, grown under bacteriologically controlled conditions. Replicate samples of nodules formed by each strain were assayed for rates of H(2) evolution in air, rates of H(2) evolution under Ar and O(2), and rates of C(2)H(2) reduction. Nodules formed by all strains of R. meliloti and R. trifolii on their respective hosts lost at least 17% of the electron flow through nitrogenase as evolved H(2). The mean loss from alfalfa nodules formed by 19 R. meliloti strains was 25%, and the mean loss from clover nodules formed by seven R. trifolii strains was 35%. R. meliloti and R. trifolii strains also were cultured under conditions that were previously established for derepression of hydrogenase synthesis. Only strains 102F65 and 102F51 of R. meliloti showed measurable activity under free-living conditions. Bacteroids from nodules formed by the two strains showing hydrogenase activity under free-living conditions also oxidized H(2) at low rates. The specific activity of hydrogenase in bacteroids formed by either strain 102F65 or strain 102F51 of R. meliloti was less than 0.1% of the specific activity of the hydrogenase system in bacteroids formed by H(2) uptake-positive Rhizobium japonicum USDA 110, which has been investigated previously. R. meliloti and R. trifolii strains tested possessed insufficient hydrogenase to recycle a substantial proportion of the H(2) evolved from the nitrogenase reaction in nodules of their hosts. Additional research is needed, therefore, to develop strains of R. meliloti and R. trifolii that possess an adequate H(2)-recycling system.

One carbon metabolism in methanogenic bacteria. Cellular characterization and growth of Methanosarcina barkeri.

Two strains of Methanosarcina (M. Barkeri strain MS, isolated from sewage sludge, and strain UBS, isolated from lake sediments) were found to have similar cellular properties and to have DNA base compositions of 44 mol percent guanosine plus cytosine. Strain MS was selected for further studies of its one-carbon metabolism. M. barkeri grew autotrophically via H2 oxidation/CO2 reduction. The optimum temperature for growth and methanogenesis was 37°C. H2 oxidation proceeded via an F420-dependent NADP+-linked hydrogenase. A maximum specific activity of hydrogenase in cell-free extracts, using methyl viologen as electron acceptor, was 6.0 μmol min · mg protein at 37°C and the optimum pH (9.0). M. barkeri also fermented methanol andmethylamine as sole energy sources for growth. Cell yields during growth on H2/CO2 and on methanol were 6.4 and 7.2 mg cell dry weight per mmol CH4 formed, respectively. During mixotrophic growth on H2/CO2 plus methanol, most methane was derived from methanol rather than from CO2. Similar activities of hydrogenase were observed in cell-free extracts from H2/CO2-grown and methanol-grown cells. Methanol oxidation apparently proceeded via carrierbound intermediates, as no methylotrophy-type of methanol dehydrogenase activity was observed in cell-free extracts. During growth on methanol/CO2, up to 48% of the cell carbon was derived from methanol indicating that equivalent amounts of cell carbon were derived from CO2 and from an organic intermediate more reduced than CO2. Cell-free extracts lacked activity for key cell carbon synthesis enzymes of the Calvin cycle, serine path, or hexulose path.

A new coryneform hydrogen bacterium: Corynebacterium autotrophicum strain 7 C

1. Corynebacterium autotrophicum strain 7 C was isolated from an enrichment culture designed for propane oxidizing bacteria. The cells are Grampositive, immotile, short, irregularly formed rods. The colonies are yellow-pigmented and slimy. The yellow pigmentation is due to carotenoids. 2. Growth occurs either autotrophically in mineral medium under an atmosphere of 70% H2+20% O2+10% CO2 or heterotrophically with fructose or many organic acids as substrates. 3. The hexoses and gluconate are degraded via the Entner-Doudoroff pathway. 6-Phosphogluconate dehydrogenase is not detectable. 4. A NAD reducing hydrogenase has not been detected; the hydrogenase is localized in the particle fraction of the crude extract and reduces methylene blue. The specific activity of hydrogenase in the crude extract of autotrophically grown cells is 2400 μl H2/mg protein · hr. During growth on fructose the enzyme is constitutively formed (1200 μl H2/mg protein · hr). 5. The utilization of fructose was suppressed by hydrogen. The inhibitory effect was significant, when either fully adapted or autotrophically grown cells were exposed to a hydrogen containing atmosphere.