A medium optimization strategy for improvement of growth and methane production by Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum, strain Hveragerdi, has been cultivated in a completely defined mineral salts medium, under strictly anaerobic conditions with CO₂ and H₂ as sole carbon and energy source, respectively. During optimization of the medium an iron limitation was identified that could not be overcome by the simple addition of iron—or iron complexed with nitrilotriacetate (NTA)—to the medium, due to the formation of insoluble FeS complexes. In order to define a medium optimization strategy, and to avoid laborious empirical optimization procedures, a theoretical model has been developed in order to describe the solubility of iron and other mineral species in the medium as a function of the concentration of sulfur and NTA. This model may be applied for the optimization of any medium component. With this information sulfide has been replaced by a combination of cysteine and thiosulphate in conjunction with a non-toxic reducing agent (titanium (III) citrate). Using this defined medium precipitation was avoided and an iron limitation was overcome resulting in a 5-fold improvement of the final biomass concentration from 2–3 g l^?1 to 11.2 g l^?1 together with a 2-fold increase (from 45 to almost 100%) in the conversion of CO₂ and H₂ to CH₄, even at gas flow rates as high as 6 l min^?1.