Enhanced hydrogen production by a laboratory-evolved archaeon in formate

Abstract

The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been reported to grow on formate coupled with H2 production and ATP generation (HCOO- + H2O→HCO3- + H2, ΔG80oC = -2.6 kJ/mol). In this study, we applied adaptive evolution toimprove H2 productivity of the strain. The strain was grown in a medium containing formate and then transferred to a fresh medium every 15 hours. Through serial transfers, physiological changes were monitored and gradual increases in cell density, H2 production and formate consumption were observed. After 156 transfers, the evolved strain showed 1.7-, 1.9- and 1.9-fold higher values in cell density, H2 production and formate consumption in comparison with those of the parental strain. In order to obtain an integrative understanding of the genetic and phenotypic changes during evolution, genome sequencing was performed and 11 mutation sites, including 2 insertions, 2 deletions and 7 substitutions either at the intergenic (2 sites) or coding regions (9 sites), were observed. When we introduced each reverse mutation in the evolved strain, two revertants for TON_1561 and TON_1573 encoding F420-reducing hydrogenase β subunit and formate transporter,respectively exhibited significant decreases in the cell density and hydrogen production. On the other hand, the mutations in TON_1561 or TON_1573 were introduced to the wild-type strain and only TON_1573 A52T mutant displayed increases ofmprove H2 productivity of the strain. The strain was grown in a medium containing formate and then transferred to a fresh medium every 15 hours. Through serial transfers, physiological changes were monitored and gradual increases in cell density, H2 production and formate consumption were observed. After 156 transfers, the evolved strain showed 1.7-, 1.9- and 1.9-fold higher values in cell density, H2 production and formate consumption in comparison with those of the parental strain. In order to obtain an integrative understanding of the genetic and phenotypic changes during evolution, genome sequencing was performed and 11 mutation sites, including 2 insertions, 2 deletions and 7 substitutions either at the intergenic (2 sites) or coding regions (9 sites), were observed. When we introduced each reverse mutation in the evolved strain, two revertants for TON_1561 and TON_1573 encoding F420-reducing hydrogenase β subunit and formate transporter,respectively exhibited significant decreases in the cell density and hydrogen production. On the other hand, the mutations in TON_1561 or TON_1573 were introduced to the wild-type strain and only TON_1573 A52T mutant displayed increases of