Biohydrogen production of obligate anaerobic archaeon Thermococcus onnurineus NA1 under oxic conditions via overexpression of frhAGB-encoding hydrogenase genes

Background: The production of biohydrogen (H ₂ ) as a promising future fuel in anaerobic hyperthermophiles has attracted great attention because H ₂ formation is more thermodynamically feasible at elevated temperatures and fewer undesired side products are produced. However, these microbes require anoxic culture conditions for growth and H ₂ production, thereby necessitating costly and time-consuming physical or chemical methods to remove molecular oxygen (O ₂ ). Therefore, the development of an O ₂ -tolerant strain would be useful for industrial applications. Results: In this study, we found that the overexpression of frhAGB-encoding hydrogenase genes in Thermococcus onnurineus NA1, an obligate anaerobic archaeon and robust H ₂ producer, enhanced O ₂ tolerance. When the recombinant FO strain was exposed to levels of O ₂ up to 20% in the headspace of a sealed bottle, it showed significant growth. Whole transcriptome analysis of the FO strain revealed that several genes involved in the stress response such as chaperonin β subunit, universal stress protein, peroxiredoxin, and alkyl hydroperoxide reductase subunit C, were significantly up-regulated. The O ₂ tolerance of the FO strain enabled it to grow on formate and produce H ₂ under oxic conditions, where prior O ₂ -removing steps were omitted, such as the addition of reducing agent Na ₂ S, autoclaving, and inert gas purging. Conclusions: Via the overexpression of frhAGB genes, the obligate anaerobic archaeon T. onnurineus NA1 gained the ability to overcome the inhibitory effect of O ₂ . This O ₂ -tolerant property of the strain may provide another advantage to this hyperthermophilic archaeon as a platform for biofuel H ₂ production.