Theoretical hypothesis in a direct electron transfer between non-interacting Fe-S proteins within an artificial fusion.

Reduction of CO2 to formate utilizing formate dehydrogenases (FDHs) has been attempted biologically and electrochemically. However, the conversion efficiency is very low due to the low energy potential of electron donors and/or electron competition with other electron acceptors. To overcome such a low conversion efficiency, I focused on a direct electron transfer between two unrelated redox enzymes for the efficient reduction of CO2 and utilized the quantum mechanical magnetic properties of the [Fe-S] ([iron-sulfur]) cluster to develop a novel electron path. Using this electron path, we connected non-interacting carbon monoxide dehydrogenase and FDH, constructing a synthetic carbon monoxide:formate oxidoreductase as a single functional enzyme complex in the previous study. Here, a theoretical hypothesis that can explain the direct electron transfer phenomenon based on the magnetic properties of the [Fe-S] cluster is proposed.

Genomic potential and physiological characteristics of C1 metabolism in novel acetogenic bacteria.

Acetogenic bacteria can utilize C1 compounds, such as carbon monoxide (CO), formate, and methanol, via the Wood-Ljungdahl pathway (WLP) to produce biofuels and biochemicals. Two novel acetogenic bacteria of the family Eubacteriaceae ES2 and ES3 were isolated from Eulsukdo, a delta island in South Korea. We conducted whole genome sequencing of the ES strains and comparative genome analysis on the core clusters of WLP with Acetobacterium woodii DSM1030T and Eubacterium limosum ATCC8486T. The methyl-branch cluster included a formate transporter and duplicates or triplicates copies of the fhs gene, which encodes formyl-tetrahydrofolate synthetase. The formate dehydrogenase cluster did not include the hydrogenase gene, which might be replaced by a functional complex with a separate electron bifurcating hydrogenase (HytABCDE). Additionally, duplicated copies of the acsB gene, encoding acetyl-CoA synthase, are located within or close to the carbonyl-branch cluster. The serum bottle culture showed that ES strains can utilize a diverse range of C1 compounds, including CO, formate, and methanol, as well as CO2. Notably, ES2 exhibited remarkable resistance to high concentrations of C1 substrates, such as 100% CO (200 kPa), 700 mM formate, and 500 mM methanol. Moreover, ES2 demonstrated remarkable growth rates under 50% CO (0.45 h-1) and 200 mM formate (0.34 h-1). These growth rates are comparable to or surpassing those previously reported in other acetogenic bacteria. Our study introduces novel acetogenic ES strains and describes their genetic and physiological characteristics, which can be utilized in C1-based biomanufacturing.

Thermococcus argininiproducens sp. nov., an arginine biosynthesis archaeal species isolated from the Central Indian Ocean ridge.

A strictly anaerobic hyperthermophilic archaeon, designated strain IOH2T, was isolated from a deep-sea hydrothermal vent (Onnuri vent field) area on the Central Indian Ocean Ridge. Strain IOH2T showed high 16S rRNA gene sequence similarity to Thermococcus sibiricus MM 739T (99.42 %), Thermococcus alcaliphilus DSM 10322T (99.28 %), Thermococcus aegaeus P5T (99.21 %), Thermococcus litoralis DSM 5473T (99.13 %), 'Thermococcus bergensis' T7324T (99.13 %), Thermococcus aggregans TYT (98.92 %) and Thermococcus prieurii Bio-pl-0405IT2T (98.01 %), with all other strains showing lower than 98 % similarity. The average nucleotide identity and in silico DNA-DNA hybridization values were highest between strain IOH2T and T. sibiricus MM 739T (79.33 and 15.00 %, respectively); these values are much lower than the species delineation cut-offs. Cells of strain IOH2T were coccoid, 1.0-1.2 µm in diameter and had no flagella. Growth ranges were 60-85 °C (optimum at 80 °C), pH 4.5-8.5 (optimum at pH 6.3) and 2.0-6.0 % (optimum at 4.0 %) NaCl. Growth of strain IOH2T was enhanced by starch, glucose, maltodextrin and pyruvate as a carbon source, and elemental sulphur as an electron acceptor. Through genome analysis of strain IOH2T, arginine biosynthesis related genes were predicted, and growth of strain IOH2T without arginine was confirmed. The genome of strain IOH2T was assembled as a circular chromosome of 1 946 249 bp and predicted 2096 genes. The DNA G+C content was 39.44 mol%. Based on the results of physiological and phylogenetic analyses, Thermococcus argininiproducens sp. nov. is proposed with type strain IOH2T (=MCCC 4K00089T=KCTC 25190T).

Laboratory Evaluation of ESGFIBER in Asphalt Paving Mixture.

The global desire to improve the performance of road pavements and move towards a sustainable transportation system has immensely encouraged the usage of fibers in asphalt paving materials. In this study, glass fibers trademarked as ESGFIBER produced by the ESG Industry company Limited from Daejeon, Korea were added in dense-graded asphalt mix. The purpose of this study was to evaluate effects that fibers have on volumetric properties, mechanical properties, and long-term performance of asphalt concrete mixes. ESGFIBER were mixed together with aggregates and asphalt binder in asphalt mix and five different asphalt mixes with different dosage of fibers were evaluated in this study. The Marshall mix design method was used for designing all asphalt mixes, and laboratory tests indirect tensile strength test, deformation strength test and Hamburg wheel tracking test were conducted to evaluate moisture susceptibility, fatigue cracking behavior and rutting resistance of asphalt concrete mixes. The results showed that when ESGFIBER were added in asphalt mix moisture susceptibility, fatigue cracking and rutting resistance were both improved. The usage of ESGFIBER in asphalt concrete mixes can be very beneficial since the mechanical and long-term performance were improved upon the addition of fibers.

Bioconversion of CO to formate by artificially designed carbon monoxide:formate oxidoreductase in hyperthermophilic archaea.

Ferredoxin-dependent metabolic engineering of electron transfer circuits has been developed to enhance redox efficiency in the field of synthetic biology, e.g., for hydrogen production and for reduction of flavoproteins or NAD(P)+. Here, we present the bioconversion of carbon monoxide (CO) gas to formate via a synthetic CO:formate oxidoreductase (CFOR), designed as an enzyme complex for direct electron transfer between non-interacting CO dehydrogenase and formate dehydrogenase using an electron-transferring Fe-S fusion protein. The CFOR-introduced Thermococcus onnurineus mutant strains showed CO-dependent formate production in vivo and in vitro. The maximum formate production rate from purified CFOR complex and specific formate productivity from the bioreactor were 2.2 ± 0.2 µmol/mg/min and 73.1 ± 29.0 mmol/g-cells/h, respectively. The CO-dependent CO2 reduction/formate production activity of synthetic CFOR was confirmed, indicating that direct electron transfer between two unrelated dehydrogenases was feasible via mediation of the FeS-FeS fusion protein.

Thermococcus indicus sp. nov., a Fe(III)-reducing hyperthermophilic archaeon isolated from the Onnuri Vent Field of the Central Indian Ocean ridge.

A strictly anaerobic, dissimilatory Fe(III)-reducing hyperthermophilic archaeon, designated as strain IOH1T, was isolated from a new deep-sea hydrothermal vent (Onnuri Vent Field) area in the Central Indian Ocean ridge. Strain IOH1T showed > 99% 16S rRNA gene sequence similarity with Thermococcus celericrescens TS2T (99.4%) and T. siculi DSM 12349T (99.2%). Additional three species T. barossii SHCK-94T (99.0%), T. celer Vu13T (98.8%), and T. piezophilus (98.6%) showed > 98.6% of 16S rRNA gene sequence similarity, however, the maximum OrthoANI value is 89.8% for the genome of T. celericrescens TS2T. Strain IOH1T cells are coccoid, 1.2-1.8 µm in diameter, and motile by flagella. Growth was at 70-82°C (optimum 80°C), pH 5.4-8.0 (optimum pH 6.0) with 2-4% (optimum 3%) NaCl. Growth of strain IOH1T was enhanced by starch, pyruvate, D(+)-maltose and maltodextrin as a carbon sources, and elemental sulfur as an electron acceptor; clearly different from those of related species T. celecrescens DSM 17994T and T. siculi DSM 12349T. Strain IOH1T, T. celercrescence DSM 17994T, and T. siculi DSM 12349T reduced soluble Fe(III)-citrate present in the medium, whereas the amount of total cellular proteins increased with the concomitant accumulation of Fe(II). We determined a circular chromosome of 2,234 kb with an extra-chromosomal archaeal plasmid, pTI1, of 7.7 kb and predicted 2,425 genes. The DNA G + C content was 54.9 mol%. Based on physiological properties, phylogenetic, and genome analysis, we proposed that strain IOH1T (= KCTC 15844T = JCM 39077T) is assigned to a new species in the genus Thermococcus and named Thermococcus indicus sp. nov.

Paradesulfovibrio onnuriensis gen. nov., sp. nov., a chemolithoautotrophic sulfate-reducing bacterium isolated from the Onnuri vent field of the Indian Ocean and reclassification of Desulfovibrio senegalensis as Paradesulfovibrio senegalensis comb. nov.

An anaerobic, rod-shaped, mesophilic, chemolithoautotrophic, sulfate-reducing bacterial strain IOR2T was isolated from a newly found deep-sea hydrothermal vent (OVF, Onnuri Vent Field) area in the central Indian Ocean ridge (11°24'88″ S 66°25'42″ E, 2021 m water depth). The 16S rRNA gene sequence analysis revealed that the strain IOR2T was most closely related to Desulfovibrio senegalensis BLaC1T (96.7%). However, it showed low similarity with the members of the family Desulfovibrionaceae, such as Desulfovibrio tunisiensis RB22T (94.0%), D. brasiliensis LVform1T (93.9%), D. halophilus DSM 5663T (93.7%), and Pseudodesulfovibrio aespoeensis Aspo-2T (93.2%). The strain IOR2T could grow at 23-42°C (optimum 37°C), pH 5.0-8.0 (optimum pH 7.0) and with 0.5-6.5% (optimum 3.0%) NaCl. The strain could use lactate, pyruvate, H2, and glycerol as electron donors and sulfate, thiosulfate, and sulfite as electron acceptors. The major fatty acids of the strain IOR2T were iso-C15:0, iso-C17:0, ante-iso-C15:0, and summed feature 9 (C16:0 methyl/iso-C17:1ω9c). Both the strains IOR2T and BLaC1T could grow with CO2 and H2 as the sole sources of carbon and energy, respectively. Genomic evidence for the Wood-Ljungdahl pathway in both the strains reflects chemolithoautotrophic growth. The DNA G + C content of the strain IOR2T and BLaC1T was 58.1-60.5 mol%. Based on the results of the phylogenetic and physiologic studies, Paradesulfovibrio onnuriensis gen. nov., sp. nov. with the type strain IOR2T (= KCTC 15845T = MCCC 1K04559T) was proposed to be a member of the family Desulfovibrionaceae. We have also proposed the reclassification of D. senegalensis as Paradesulfovibrio senegalensis comb. nov.

Direct Electron Transfer between the frhAGB-Encoded Hydrogenase and Thioredoxin Reductase in the Nonmethanogenic Archaeon Thermococcus onnurineus NA1.

To date, NAD(P)H, ferredoxin, and coenzyme F420 have been identified as electron donors for thioredoxin reductase (TrxR). In this study, we present a novel electron source for TrxR. In the hyperthermophilic archaeon Thermococcus onnurineus NA1, the frhAGB-encoded hydrogenase, a homolog of the F420-reducing hydrogenase of methanogens, was demonstrated to interact with TrxR in coimmunoprecipitation experiments and in vitro pulldown assays. Electrons derived from H2 oxidation by the frhAGB-encoded hydrogenase were transferred to TrxR and reduced Pdo, a redox partner of TrxR. Interaction and electron transfer were observed between TrxR and the heterodimeric hydrogenase complex (FrhAG) as well as the heterotrimeric complex (FrhAGB). Hydrogen-dependent reduction of TrxR was 7-fold less efficient than when NADPH was the electron donor. This study not only presents a different type of electron donor for TrxR but also reveals new functionality of the frhAGB-encoded hydrogenase utilizing a protein as an electron acceptor.IMPORTANCE This study has importance in that TrxR can use H2 as an electron donor with the aid of the frhAGB-encoded hydrogenase as well as NAD(P)H in T. onnurineus NA1. Further studies are needed to explore the physiological significance of this protein. This study also has importance as a significant step toward understanding the functionality of the frhAGB-encoded hydrogenase in a nonmethanogen; the hydrogenase can transfer electrons derived from oxidation of H2 to a protein target by direct contact without the involvement of an electron carrier, which is distinct from the mechanism of its homologs, F420-reducing hydrogenases of methanogens.

A rapid and sensitive enzymatic assay for 2,3-butanediol.

In this study, we developed a rapid and sensitive enzymatic assay for 2,3-butanediol (2,3-BDO) detection. The concentration of 2,3-BDO was determined by measuring the reduction of NADP+ using Clostridium ljungdahlii 2,3-butanediol dehydrogenase (CL-Bdh). The enzymatic assay could detect as low as 0.01 mM of 2,3-BDO, while the high-performance liquid chromatography (HPLC) method required a much higher concentration than 0.15 mM. Gratifyingly, the developed method was 15 times more sensitive than the HPLC method. When the enzymatic assay was applied to high-throughput screening, the enzymatic assay detected 14 positive samples out of 23 tested, as compared to 8 by the HPLC method. These results suggest that the enzymatic assay is an effective screening method for the detection of 2,3-BDO-producing microbes in a microtiter plate-based format.

A simple biosynthetic pathway for 2,3-butanediol production in Thermococcus onnurineus NA1.

The biosynthetic pathway of 2,3-butanediol (2,3-BDO) production from pyruvate under anaerobic conditions includes three enzymes: acetolactate synthase (ALS), acetolactate decarboxylase (ALDC), and acetoin reductase (AR). Recently, in anaerobic hyperthermophilic Pyrococcus furiosus, it has been reported that acetoin, a precursor of 2,3-BDO, is produced from pyruvate by ALS through a temperature-dependent metabolic switch. In this study, we first attempted to produce 2,3-BDO from Thermococcus onnurineus NA1 using a simple biosynthetic pathway by two enzymes (ALS and AR) at a high temperature. Two heterologous genes, acetolactate synthase (alsS) from Pyrococcus sp. NA2 and alcohol dehydrogenase (adh) from T. guaymacensis, were introduced and expressed in T. onnurineus NA1. The mutant strain produced approximately 3.3 mM 2,3-BDO at 80 °C. An acetyl-CoA synthetase IIIα (TON_1001) was further deleted to enhance 2,3-BDO production, and the mutant strain showed a 25% increase in the specific production of 2,3-BDO. Furthermore, when carbon monoxide (CO) gas was added as a reductant, specific production of 2,3-BDO increased by 45%. These results suggest a new biosynthetic pathway for 2,3-BDO and demonstrate the possibility of T. onnurineus NA1 as a platform strain for 2,3-BDO production at high temperatures.

Redox regulation of SurR by protein disulfide oxidoreductase in Thermococcus onnurineus NA1.

Protein disulfide oxidoreductases are redox enzymes that catalyze thiol-disulfide exchange reactions. These enzymes include thioredoxins, glutaredoxins, protein disulfide isomerases, disulfide bond formation A (DsbA) proteins, and Pyrococcus furiosus protein disulfide oxidoreductase (PfPDO) homologues. In the genome of a hyperthermophilic archaeon, Thermococcus onnurineus NA1, the genes encoding one PfPDO homologue (TON_0319, Pdo) and three more thioredoxin- or glutaredoxin-like proteins (TON_0470, TON_0472, TON_0834) were identified. All except TON_0470 were recombinantly expressed and purified. Three purified proteins were reduced by a thioredoxin reductase (TrxR), indicating that each protein can form redox complex with TrxR. SurR, a transcription factor involved in the sulfur response, was tested for a protein target of a TrxR-redoxin system and only Pdo was identified to be capable of catalyzing the reduction of SurR. Electromobility shift assay demonstrated that SurR reduced by the TrxR-Pdo system could bind to the DNA probe with the SurR-binding motif, GTTttgAAC. In this study, we present the TrxR-Pdo couple as a redox-regulator for SurR in T. onnurineus NA1.

Duodenal Loop Obstruction as an Unusual Cause of Acute Pancreatitis: A Case Series.

Duodenal loop obstruction is an unusual cause of acute pancreatitis. Increased intraluminal pressure hinders pancreatic flow, causing dilatation of the pancreatic duct and inducing acute pancreatitis. We experienced three cases of acute pancreatitis that resulted from duodenal loop obstruction after (1) an esophagectomy with gastric pull-up procedure for esophageal cancer, (2) a gastrectomy with Billroth I reconstruction for gastric cancer, and (3) a gastrojejunostomy for abdominal trauma. An abdominal CT scan revealed a distended duodenal loop, dilated pancreatic duct, and inflamed pancreas with fluid collection. Acute pancreatitis with duodenal loop obstruction was diagnosed by abdominal pain, elevated serum amylase/lipase, and abdominal CT findings. Immediate decompression with a nasogastric tube was performed, and all patients showed improvement within one week after admission. Each patient was followed up for more than two years without recurrence. Our findings suggest the usefulness of nasogastric tube decompression as the first line of treatment for acute pancreatitis related to duodenal loop obstruction.

Na+ transport by the A1AO-ATP synthase purified from Thermococcus onnurineus and reconstituted into liposomes.

The ATP synthase of many archaea has the conserved sodium ion binding motif in its rotor subunit, implying that these A1AO-ATP synthases use Na(+) as coupling ion. However, this has never been experimentally verified with a purified system. To experimentally address the nature of the coupling ion, we have purified the A1AO-ATP synthase from T. onnurineus. It contains nine subunits that are functionally coupled. The enzyme hydrolyzed ATP, CTP, GTP, UTP, and ITP with nearly identical activities of around 40 units/mg of protein and was active over a wide pH range with maximal activity at pH 7. Noteworthy was the temperature profile. ATP hydrolysis was maximal at 80 °C and still retained an activity of 2.5 units/mg of protein at 45 °C. The high activity of the enzyme at 45 °C opened, for the first time, a way to directly measure ion transport in an A1AO-ATP synthase. Therefore, the enzyme was reconstituted into liposomes generated from Escherichia coli lipids. These proteoliposomes were still active at 45 °C and coupled ATP hydrolysis to primary and electrogenic Na(+) transport. This is the first proof of Na(+) transport by an A1AO-ATP synthase and these findings are discussed in light of the distribution of the sodium ion binding motif in archaea and the role of Na(+) in the bioenergetics of archaea.

One-carbon substrate-based biohydrogen production: microbes, mechanism, and productivity.

Among four basic mechanisms for biological hydrogen (H2) production, dark fermentation has been considered to show the highest hydrogen evolution rate (HER). H2 production from one-carbon (C1) compounds such as formate and carbon monoxide (CO) is promising because formate is an efficient H2 carrier, and the utilization of CO-containing syngas or industrial waste gas may render the industrial biohydrogen production process cost-effective. A variety of microbes with the formate hydrogen lyase (FHL) system have been identified from phylogenetically diverse groups of archaea and bacteria, and numerous efforts have been undertaken to improve the HER for formate through strain optimization and bioprocess development. CO-dependent H2 production has been investigated to enhance the H2 productivity of various carboxydotrophs via an increase in CO gas-liquid mass transfer rates and the construction of genetically modified strains. Hydrogenogenic CO-conversion has been applied to syngas and by-product gas of the steel-mill process, and this low-cost feedstock has shown to be promising in the production of biomass and H2. Here, we focus on recent advances in the isolation of novel phylogenetic groups utilizing formate or CO, the remarkable genetic engineering that enhances H2 productivity, and the practical implementation of H2 production from C1 substrates.

Characterization of the frhAGB-encoding hydrogenase from a non-methanogenic hyperthermophilic archaeon.

The F420-reducing hydrogenase has been known as a key enzyme in methanogenesis. Its homologs have been identified in non-methanogenic hyperthermophilic archaea, including Thermococcus onnurineus NA1, but neither physiological function nor biochemical properties have been reported to date. The enzyme of T. onnurineus NA1 was distinguished from those of other methanogens and the members of the family Desulfurobacteriaceae with respect to the phylogenetic distribution of the α and ß subunits, organization of frhAGB genes and conservation of F420-coordinating residues. RT-qPCR and Western blot analyses revealed frhA gene is not silent but is expressed in T. onnurineus NA1 grown in the presence of sulfur, carbon monoxide, or formate. The trimeric enzyme complex was purified to homogeneity via affinity chromatography from T. onnurineus NA1 and exhibited catalytic activity toward the electron acceptors such as viologens and flavins but not the deazaflavin coenzyme F420. This is the first biochemical study on the function of the frhAGB-encoding enzyme from a non-methanogenic archaea.

A novel CO-responsive transcriptional regulator and enhanced H2 production by an engineered Thermococcus onnurineus NA1 strain.

Genome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism in Thermococcus onnurineus NA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes of Thermococcus species and "Candidatus Korarchaeum cryptofilum" OPF8. In-frame deletion of either corQ or corR caused a severe impairment in CO-dependent growth and H2 production. When corQ and corR deletion mutants were complemented by introducing the corQR genes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integrated corQR (ΔCorR/corQR(↑)) compared with those in the wild-type strain. In addition, the ΔCorR/corQR(↑) strain exhibited a much higher H2 production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2 production rate (191.9 mmol liter(-1) h(-1)) and the specific H2 production rate (249.6 mmol g(-1) h(-1)) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that the corQR genes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2 production.

Energy conservation by oxidation of formate to carbon dioxide and hydrogen via a sodium ion current in a hyperthermophilic archaeon.

Thermococcus onnurineus NA1 is known to grow by the anaerobic oxidation of formate to CO2 and H2, a reaction that operates near thermodynamic equilibrium. Here we demonstrate that this reaction is coupled to ATP synthesis by a transmembrane ion current. Formate oxidation leads to H(+) translocation across the cytoplasmic membrane that then drives Na(+) translocation. The ion-translocating electron transfer system is rather simple, consisting of only a formate dehydrogenase module, a membrane-bound hydrogenase module, and a multisubunit Na(+)/H(+) antiporter module. The electrochemical Na(+) gradient established then drives ATP synthesis. These data give a mechanistic explanation for chemiosmotic energy conservation coupled to formate oxidation to CO2 and H2. Because it is discussed that the membrane-bound hydrogenase with the Na(+)/H(+) antiporter module are ancestors of complex I of mitochondrial and bacterial electron transport these data also shed light on the evolution of ion transport in complex I-like electron transport chains.

CO-dependent H2 production by genetically engineered Thermococcus onnurineus NA1.

Hydrogenogenic CO oxidation (CO + H(2)O → CO(2) + H(2)) has the potential for H(2) production as a clean renewable fuel. Thermococcus onnurineus NA1, which grows on CO and produces H(2), has a unique gene cluster encoding the carbon monoxide dehydrogenase (CODH) and the hydrogenase. The gene cluster was identified as essential for carboxydotrophic hydrogenogenic metabolism by gene disruption and transcriptional analysis. To develop a strain producing high levels of H(2), the gene cluster was placed under the control of a strong promoter. The resulting mutant, MC01, showed 30-fold-higher transcription of the mRNA encoding CODH, hydrogenase, and Na(+)/H(+) antiporter and a 1.8-fold-higher specific activity for CO-dependent H(2) production than did the wild-type strain. The H(2) production potential of the MC01 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The H(2) production rate of the engineered strain was severalfold higher than those of any other CO-dependent H(2)-producing prokaryotes studied to date. The engineered strain also possessed high activity for the bioconversion of industrial waste gases created as a by-product during steel production. This work represents the first demonstration of H(2) production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe.

Thermodynamics of formate-oxidizing metabolism and implications for H2 production.

Formate-dependent proton reduction to H(2) (HCOO(-) + H(2)O → HCO(3)(-) + H(2)) has been reported for hyperthermophilic Thermococcus strains. In this study, a hyperthermophilic archaeon, Thermococcus onnurineus strain NA1, yielded H(2) accumulation to a partial pressure of 1 × 10(5) to 7 × 10(5) Pa until the values of Gibbs free energy change (ΔG) reached near thermodynamic equilibrium (-1 to -3 kJ mol(-1)). The bioenergetic requirement for the metabolism to conserve energy was demonstrated by ΔG values as small as -5 kJ mol(-1), which are less than the biological minimum energy quantum, -20 kJ mol(-1), as calculated by Schink (B. Schink, Microbiol. Mol. Biol. Rev. 61:262-280, 1997). Considering formate as a possible H(2) storage material, the H(2) production potential of the strain was assessed. The volumetric H(2) production rate increased linearly with increasing cell density, leading to 2,820 mmol liter(-1) h(-1) at an optical density at 600 nm (OD(600)) of 18.6, and resulted in the high specific H(2) production rates of 404 ± 6 mmol g(-1) h(-1). The H(2) productivity indicates the great potential of T. onnurineus strain NA1 for practical application in comparison with H(2)-producing microbes. Our result demonstrates that T. onnurineus strain NA1 has a highly efficient metabolic system to thrive on formate in hydrothermal systems.