Aerobic Hydrogen-oxidizing Bacterium Research Articles

Enrichment of hydrogen-oxidizing bacteria using a hybrid biological-inorganic system.

Hybrid biological-inorganic (HBI) systems comprising inorganic water-splitting catalysts and aerobic hydrogen-oxidizing bacteria (HOB) have previously been used for CO2 conversion. In order to identify new biocatalysts for CO2 conversion, the present study used an HBI system to enrich HOB directly from environmental samples. Three sediment samples (from a brackish water pond, a beach, and a tide pool) and two activated sludge samples (from two separate sewage plants) were inoculated into HBI systems using a cobalt phosphorus (Co-P) alloy and cobalt phosphate (CoPi) as inorganic catalysts with a fixed voltage of 2.0V. The gas composition of the reactor headspaces and electric current were monitored. An aliquot of the reactor medium was transferred to a new reactor when significant consumption of H2 and CO2 was detected. This process was repeated twice (with three reactors in operation for each sample) to enrich HOB. Increased biomass concomitant with increased H2 and CO2 consumption was observed in the third reactor, indicating enrichment of HOB. 16S rRNA gene amplicon sequencing demonstrated enrichment of sequences related to HOB (including bacteria from Mycobacterium, Hydrogenophaga, and Xanthobacter genera) over successive sub-cultures. Finally, four different HOB belonging to the Mycobacterium, Hydrogenophaga, Xanthobacter, and Acidovorax genera were isolated from reactor media, representing potential candidates as HBI system biocatalysts.

Aerobic hydrogen-oxidizing bacteria in soil: from cells to ecosystems

Aerobic hydrogen-oxidizing bacteria in soil: from cells to ecosystems

Aerobic Hydrogen-oxidizing Bacteria and Utilization for Carbon Recycle and Reduction

Aerobic Hydrogen-oxidizing Bacteria and Utilization for Carbon Recycle and Reduction

Complete genome sequence of Hydrogenobacter thermophilus type strain (TK-6T)

Hydrogenobacter thermophilus Kawasumi et al. 1984 is the type species of the genus Hydrogenobacter. H. thermophilus was the first obligate autotrophic organism reported among aerobic hydrogen-oxidizing bacteria. Strain TK-6T is of interest because of the unusually efficient hydrogen-oxidizing ability of this strain, which results in a faster generation time compared to other autotrophs. It is also able to grow anaerobically using nitrate as an electron acceptor when molecular hydrogen is used as the energy source, and able to aerobically fix CO2 via the reductive tricarboxylic acid cycle. This is the fifth completed genome sequence in the family Aquificaceae, and the second genome sequence determined from a strain derived from the original isolate. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,742,932 bp long genome with its 1,899 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

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 H 2 were, respectively, 12 μM and 8.58 × 10 7 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 H 2, 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.

Complete Genome Sequence of the Thermophilic, Obligately Chemolithoautotrophic Hydrogen-Oxidizing Bacterium Hydrogenobacter thermophilus TK-6

Hydrogenobacter thermophilus is a thermophilic, obligately chemolithoautotrophic and aerobic hydrogen-oxidizing bacterium. It is unique in its ability to fix carbon dioxide via the reductive tricarboxylic acid cycle under aerobic conditions. It utilizes molecular hydrogen, elemental sulfur, or thiosulfate as the sole energy source. Here, we report the complete genome sequence of H. thermophilus TK-6.

Detection in soil of aerobic hydrogen-oxidizing bacteria related to Alcaligenes eutrophus by PCR and hybridization assays targeting the gene of the membrane-bound (NiFe) hydrogenase

The presence and abundance of the aerobic, chemolithoautotrophic Alcaligenes eutrophus, and related hydrogen-oxidizing bacteria, in soil samples were determined by using a hydrogenase gene probe and two different PCR primer sets that were derived from the alignment of (NiFe) hydrogenase gene sequences available in the EMBL data bank. The specificity of both the gene probe and the primer sets was tested on the genomic DNAs of 19 hydrogenase-containing, or hydrogenase-lacking, bacteria. The hydrogenase gene probe detected hydrogenase-containing Knallgas bacteria, N2-fixing bacteria, and enterobacteria, belonging to the alpha, beta, and gamma subclasses of the Proteobacteria. Hydrogenase-containing sulfate reducers (delta subclass) were not detected. One primer set was degenerated and resulted in the amplification of gene fragments of Knallgas bacteria and N2-fixing bacteria. The other primer set was specifically designed for the gene of the small subunit of the membrane-bound hydrogenase of A. eutrophus. In addition to the target organism, only Variovorax (Alcaligenes) paradoxus gave a gene product of the expected length. A. eutrophus-related bacteria were detected in soil by using two different strategies. The first strategy required cultivation and isolation of soil bacteria on complex media. Colonies were isolated from six different soils and tested by colony hybridization with the hydrogenase gene probe. Fifteen of the positive colonies were chosen for further characterization with the PCR primers. Two of the isolates gave amplification products with the A. eutrophus-specific PCR primers, which were sequenced and found to be 89–90% identical on a nucleotide level to the hydrogenase gene of A. eutrophus H16. The second strategy avoided a cultivation step. Total DNA was isolated from the rhizosphere of rice plants and tested in PCR amplification experiments with the A. eutrophus-specific PCR primers. Two different types of hydrogenase fragments were cloned and partially sequenced. The gene fragment sequences showed 73 and 75% identity to the A. eutrophus hydrogenase sequence. In quantitative PCR amplification assays, the number (about 2.6×107 g−1 fresh roots) of these two species of hydrogenase-containing bacteria was determined in the rice rhizosphere and was found to be similar to the number (about 7.4×106 g−1 fresh roots) of Knallgas bacteria that were determined by enumeration on selective media. Thus, it was apparently possible to detect and enumerate A. eutrophus-related bacteria in soil samples by molecular techniques.

還元的ＴＣＡサイクル 好熱好気性・絶対独立栄養性水素細菌Ｈｙｄｒｏｇｅｎｏｂａｃｔｅｒ ｔｈｅｒｍｏｐｈｉｌｕｓ ＴＫ‐６株のＣＯ２・エネルギー代謝を中心として

還元的ＴＣＡサイクル 好熱好気性・絶対独立栄養性水素細菌Ｈｙｄｒｏｇｅｎｏｂａｃｔｅｒ ｔｈｅｒｍｏｐｈｉｌｕｓ ＴＫ‐６株のＣＯ２・エネルギー代謝を中心として

Detection in soil of aerobic hydrogen-oxidizing bacteria related to Alcaligenes eutrophus by PCR and hybridization assays targeting the gene of the membrane-bound (NiFe) hydrogenase

The presence and abundance of the aerobic, chemolithoautotrophic Alcaligenes eutrophus, and related hydrogen-oxidizing bacteria, in soil samples were determined by using a hydrogenase gene probe and two different PCR primer sets that were derived from the alignment of (NiFe) hydrogenase gene sequences available in the EMBL data bank. The specificity of both the gene probe and the primer sets was tested on the genomic DNAs of 19 hydrogenase-containing, or hydrogenase-lacking, bacteria. The hydrogenase gene probe detected hydrogenase-containing Knallgas bacteria, N 2-fixing bacteria, and enterobacteria, belonging to the alpha, beta, and gamma subclasses of the Proteobacteria. Hydrogenase-containing sulfate reducers (delta subclass) were not detected. One primer set was degenerated and resulted in the amplification of gene fragments of Knallgas bacteria and N 2-fixing bacteria. The other primer set was specifically designed for the gene of the small subunit of the membrane-bound hydrogenase of A. eutrophus. In addition to the target organism, only Variovorax ( Alcaligenes) paradoxus gave a gene product of the expected length. A. eutrophus-related bacteria were detected in soil by using two different strategies. The first strategy required cultivation and isolation of soil bacteria on complex media. Colonies were isolated from six different soils and tested by colony hybridization with the hydrogenase gene probe. Fifteen of the positive colonies were chosen for further characterization with the PCR primers. Two of the isolates gave amplification products with the A. eutrophus-specific PCR primers, which were sequenced and found to be 89–90% identical on a nucleotide level to the hydrogenase gene of A. eutrophus H16. The second strategy avoided a cultivation step. Total DNA was isolated from the rhizosphere of rice plants and tested in PCR amplification experiments with the A. eutrophus-specific PCR primers. Two different types of hydrogenase fragments were cloned and partially sequenced. The gene fragment sequences showed 73 and 75% identity to the A. eutrophus hydrogenase sequence. In quantitative PCR amplification assays, the number (about 2.6×10 7 g −1 fresh roots) of these two species of hydrogenase-containing bacteria was determined in the rice rhizosphere and was found to be similar to the number (about 7.4×10 6 g −1 fresh roots) of Knallgas bacteria that were determined by enumeration on selective media. Thus, it was apparently possible to detect and enumerate A. eutrophus-related bacteria in soil samples by molecular techniques.

Electron Transport System in Pseudomonas hydrogenothermophila Strain TH-1, a Thermophilic Hydrogen-oxidizing Bacterium.

The electron transport system in Pseudomonas hydrogenothermophila strain TH-1, a thermophilic, aerobic hydrogen-oxidizing bacterium, was studied using a membrane fraction, an ubiquinone-depleted membrane fraction (Q(-) MF), and an ubiqunone-reconstituted membrane fraction (Q(+) MF). From spectrophotometric and TLC analyses, cytochromes b and c, ubiquinone-8 (Q8), and cytochrome o were detected as respiratory components in the membrane fraction of P. hydrogenothermophila. Spectrophotometric analyses also showed that reduction of all the components in the membrane fraction depends on a membrane-bound hydrogenase reaction. In Q(-) MF, reduction of cytochromes b and c depending on membrane-bound hydrogenase reaction could not be observed. However, the reduction was restored in Q(+) MF. From these results, a possible electron transport system in P. hydrogenothermophila strain TH-1 is proposed.

High-Level Nickel Resistance in Alcaligenes xylosoxydans 31A and Alcaligenes eutrophus KTO2

Two new nickel-resistant strains of Alcaligenes species were selected from a large number (about 400) of strains isolated from ecosystems polluted by heavy metals and were studied on the physiological and molecular level. Alcaligenes xylosoxydans 31A is a heterotrophic bacterium, and Alcaligenes eutrophus KTO2 is an autotrophic aerobic hydrogen-oxidizing bacterium. Both strains carry-among other plasmids-a megaplasmid determining resistance to 20 to 50 mM NiCl(2) and 20 mM CoCl(2) (when growing in defined Tris-buffered media). Megaplasmids pTOM8, pTOM9 from strain 31A, and pGOE2 from strain KTO2 confer nickel resistance to the same degree to transconjugants of all strains of A. eutrophus tested but were not transferred to Escherichia coli. However, DNA fragments carrying the nickel resistance genes, cloned into broad-hostrange vector pVDZ'2, confer resistance to A. eutrophus derivatives as well as E. coli. The DNA fragments of both bacteria, TBA8, TBA9, and GBA (14.5-kb BamHI fragments), appear to be identical. They share equal size, restriction maps, and strong DNA homology but are largely different from fragment HKI of nickel-cobalt resistance plasmid pMOL28 of A. eutrophus CH34.

Methionaquinone is a Direct Natural Electron Acceptor for the Membrane-bound Hydrogenase in Hydrogenobacter thermophilus Strain TK-6.

The hydrogenase reaction in Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, aerobic hydrogen-oxidizing bacterium, was studied in the membrane fraction, methionaquinone-depleted membrane, and purified membrane-bound hydrogenase. Both b and c type cytochromes were involved in the hydrogen oxidation. Methionaquinone mediated an electron transport between membrane-bound hydrogenase and cytochrome b560. Methionaquinone was reduced directly by purified hydrogenase. From these results, we conclude that methionaquinone is a direct natural electron acceptor for the membrane-bound hydrogenase in the strain.

Growth characteristics and high cell-density cultivation of a marine obligately chemolithoautotrophic hydrogen-oxidizing bacterium Hydrogenovibrio marinus strain MH-110 under a continuous gas-flow system

A marine obligately chemolithoautotrophic aerobic hydrogen-oxidizing bacterium Hydrogenovibrio marinus strain MH-110 was cultivated in a 2- l jar fermentor with continuous supply of a mixed gas of H 2/O 2/CO 2. Fast growth began rapidly without a lag period, even in an atmosphere of 40% oxygen. Apparent specific growth rate of the initial stage of growth was 0.60 – 0.67 h −1 irrespective of any oxygen tension tested. Under an atmosphere containing hydrogen, the growth was promoted by the addition of tetrathionate to the medium. A dense culture was achieved by increasing the tension of oxygen and adding ferrous and magnesium ions before these factors became growth limiting. Under this culture condition, exponential growth continued until the cell density reached 14 g/ l dry weight. The final cell concentration reached approximately 26 g/ l dry weight after 30-h cultivation.

Hydrogenobacter thermophilus: its unusual physiological properties and phylogenic position in the microbial world

Hydrogenobacter thermophilus (IAM12695) is an obligately autotrophic, thermophilic and aerobic hydrogen-oxidizing bacterium. Chemotaxonomic studies revealed various unusual features of this microorganism. H. thermophilus operates a reductive TCA cycle, which is the only confirmed example of the operation of a non-Calvin type carbon dioxide fixation pathway among aerobic organisms. Isolation of strains similar to Hydrogenobacter from different areas suggests a possible role of these microorganisms as primary producers of organic compounds from carbon dioxide in geothermal and/or aquatic environments.

Hydrogenobacter thermophilus: its unusual physiological properties and phylogenic position in the microbial world

Hydrogenobacter thermophilus (IAM12695) is an obligately autotrophic, thermophilic and aerobic hydrogen-oxidizing bacterium. Chemotaxonomic studies revealed various unusual features of this microorganism. H. thermophilus operates a reductive TCA cycle, which is the only confirmed example of the operation of a non-Calvin type carbon dioxide fixation pathway among aerobic organisms. Isolation of strains similar to Hydrogenobacter from different areas suggests a possible role of these microorganisms as primary producers of organic compounds from carbon dioxide in geothermal and/or aquatic environments.

Nickel resistance of Alcaligenes denitrificans strain 4a-2 is chromosomally coded

A newly isolated aerobic hydrogen-oxidizing bacterium, Alcaligenes denitrificans strain 4a-2, differs from related autotrophic bacteria by containing only a single cytoplasmic, NAD-reducing hydrogenase, and by its high resistance to nickel ions, i.e. tolerance to 20 mM NiCl2. Strain 4a-2 harbors a single plasmid of about 250 kb. On helper-assisted mating of 4a-2 with Alcaligenes eutrophus strains H16,G29, and M85 nickelresistant transconjugants were selected; these did not contain the donor plasmid, however. All three transconjugants tolerated 3 to 10 mM NiCl2. The resistance was constitutively expressed. DNA/DNA hybridization showed homology with EcoRI-digested DNA of the wild type 4a-2 and transconjugants using a DNA probe containing nickel resistance genes of pMOL28. This indicated that the 4a-2 nickel resistance genes are located on the chromosome.

A new isolate of Hydrogenobacter, an obligately chemolithoautotrophic, thermophilic, halophilic and aerobic hydrogen-oxidizing bacterium from seaside saline hot spring

An obligately chemolithoautotrophic and aerobic hydrogen-oxidizing bacterium was isolated from a seaside saline hot spring in Izu Peninsula, Japan. The isolate was a Gram-negative, non-motile, non-spore-forming rod cell measuring 0.3 to 0.5 by 1.0 to 2.5 μm. The optimal temperature for growth was around 70°C, and no growth was observed at 40°C or 80°C. Elemental sulfur or thiosulfate could be an alternative to molecular hydrogen as the sole energy source. The DNA base composition of the isolate was 46.0 mol% G+C. 2-Methylthio-3-VI,VII-tetrahydromultiprenyl7-1,4-naphthoquinone (methionaquinone) was the major component of the quinone system. C18:0, C18:1 and C20:1 were the major components of the cellular fatty acids. These properties clearly indicate that the isolate belongs to genus Hydrogenobacter, but differed from H. thermophilus in some respects. Specifically, the isolate was a halophile which grew optimally at around 0.3–0.5 M NaCl, while H. thermophilus could not grow at such NaCl concentration levels. A new species name H. halophilus is proposed for this new halophilic isolate.

Isolation of an obligately chemolithoautotrophic, halophilic and aerobic hydrogen-oxidizing bacterium from marine environment

An obligately chemolithoautotrophic and aerobic hydrogen-oxidizing bacterium was isolated from seawater of the Shonan Coast, Kanagawa Pref., Japan. The isolate was a Gram-negative, comma-shaped rod cell measuring 0.2 to 0.5 by 1 to 2 μm. The cells occurred singly and were motile by a polar flagellum. The deoxyribonucleic acid base composition of the isolate was 44.1 mol% guanine plus cytosine. The optimal temperature for autotrophic growth on H2−O2−CO2 was around 37°C, and no growth was observed at 5° C or 45° C. The optimal pH for growth was around 6.5. NaCl was required for growth with an optimum of 0.5 M. Elemental sulfur, thiosulfate or tetrathionate was utilized, as well as molecular hydrogen, as the sole energy source. No heterotrophic growth was observed on organic media tested. To our knowledge, this is the first report of the isolation of a marine, aerobic hydrogen-oxidizing bacterium, and of an obligately chemolithoautotrophic, mesophilic and aerobic hydrogen-oxidizing bacterium.

The CO2 assimilation via the reductive tricarboxylic acid cycle in an obligately autotrophic, aerobic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus

The incorporation of 14CO2 by the cell suspensions of an extremely thermophilic, aerobic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus was studied. After short time incubation of the cell suspensions with 14CO2, the radiactivity was initially present in aspartate, glutamate, succinate, phosphorylated compounds, citrate, malate and fumarate. All of these compounds except phosphorylated compounds were related to the members of the tricarboxylic acid cycle. The proportion of labelled aspartate onglutamate in total radioactivity on each chromatogram decreased with incubation time, while the percentage of the radioactivity incorporated in phosphorylated compounds increased with time up to 10 s. These indicated that aspartate and glutamate is derived from primary products of CO2 fixation.

Hydrogen evolution by strictly aerobic hydrogen bacteria under anaerobic conditions

When strains and mutants of the strictly aerobic hydrogen-oxidizing bacterium Alcaligenes eutrophus are grown heterotrophically on gluconate or fructose and are subsequently exposed to anaerobic conditions in the presence of the organic substrates, molecular hydrogen is evolved. Hydrogen evolution started immediately after the suspension was flushed with nitrogen, reached maximum rates of 70 to 100 mumol of H2 per h per g of protein, and continued with slowly decreasing rates for at least 18 h. The addition of oxygen to an H2-evolving culture, as well as the addition of nitrate to cells (which had formed the dissimilatory nitrate reductase system during the preceding growth), caused immediate cessation of hydrogen evolution. Formate is not the source of H2 evolution. The rates of H2 evolution with formate as the substrate were lower than those with gluconate. The formate hydrogenlyase system was not detectable in intact cells or crude cell extracts. Rather the cytoplasmic, NAD-reducing hydrogenase is involved by catalyzing the release of excessive reducing equivalents under anaerobic conditions in the absence of suitable electron acceptors. This conclusion is based on the following experimental results. H2 is formed only by cells which had synthesized the hydrogenases during growth. Mutants lacking the membrane-bound hydrogenase were still able to evolve H2. Mutants lacking the NAD-reducing or both hydrogenases were unable to evolve H2.