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

Background: The production of biohydrogen (H2 ) as a promising future fuel in anaerobic hyperthermophiles has attracted great attention because H2 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 H2 production, thereby necessitating costly and time-consuming physical or chemical methods to remove molecular oxygen (O2 ). Therefore, the development of an O2 -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 H2 producer, enhanced O2 tolerance. When the recombinant FO strain was exposed to levels of O2 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 O2 tolerance of the FO strain enabled it to grow on formate and produce H2 under oxic conditions, where prior O2 -removing steps were omitted, such as the addition of reducing agent Na2 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 O2 . This O2 -tolerant property of the strain may provide another advantage to this hyperthermophilic archaeon as a platform for biofuel H2 production.