ABSTRACT
The hyperthermophilic archaeon Thermococcus kodakarensis can utilize sugars or pyruvate for growth. In the absence of elemental sulfur, the electrons via oxidation of these substrates are accepted by protons, generating molecular hydrogen (H2). The hydrogenase responsible for this reaction is a membrane-bound [NiFe]-hydrogenase (Mbh). In this study, we have examined several possibilities to increase the protein levels of Mbh in T. kodakarensis by genetic engineering. Highest levels of intracellular Mbh levels were achieved when the promoter of the entire mbh operon (TK2080-TK2093) was exchanged to a strong constitutive promoter from the glutamate dehydrogenase gene (TK1431) (strain MHG1). When MHG1 was cultivated under continuous culture conditions using pyruvate-based medium, a nearly 25% higher specific hydrogen production rate (SHPR) of 35.3 mmol H2 g-dcw(-1) h(-1) was observed at a dilution rate of 0.31 h(-1). We also combined mbh overexpression using an even stronger constitutive promoter from the cell surface glycoprotein gene (TK0895) with disruption of the genes encoding the cytosolic hydrogenase (Hyh) and an alanine aminotransferase (AlaAT), both of which are involved in hydrogen consumption (strain MAH1). At a dilution rate of 0.30 h(-1), the SHPR was 36.2 mmol H2 g-dcw(-1) h(-1), corresponding to a 28% increase compared to that of the host T. kodakarensis strain. Increasing the dilution rate to 0.83 h(-1) or 1.07 h(-1) resulted in a SHPR of 120 mmol H2 g-dcw(-1) h(-1), which is one of the highest production rates observed in microbial fermentation.
ABSTRACT
The hydrogen (H2) production potential of the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 was evaluated at 85 degrees C. In batch cultivation using a complex medium supplemented with elemental sulfur (S0), evolution of H2S and CO2 was observed in the gas phase. When S0 was omitted and pyruvate or starch was added in the medium, the cells produced H2 at high levels instead of H2S. As the level of H2 appeared to correlate with the specific growth rate, analysis in continuous cultures was performed to develop a continuous H2 production system. In a steady-state condition at a dilution rate of 0.2 h-1, a continuous H2 production rate (per gram dry weight, gdw) of 24.9 and 14.0 mmol gdw-1 h-1 was observed in media supplemented with pyruvate and starch, respectively. In both cultivations, a high accumulation of acetate and alanine was found as metabolites. When the dilution rates were elevated in the medium with pyruvate, steady-state growth was observed up to 0.8 h-1, and a maximum H2 production rate of 59.6 mmol gdw-1 h-1 was obtained. Based on the experimental results along with data of the entire genome sequence, the metabolic pathway of the strain relating to starch and pyruvate degradation is discussed.
