Enhanced biomass and desulfurization by recombinant Rhodococcus qingshengii IGTS8 in bioreactor cultures

Rhodococcus qingshengii IGTS8 is a model strain well-known for its ability to remove sulfur from dibenzothiophene (DBT) and its derivatives via the 4S pathway, a process that catalyzes the conversion of DBT to 2-hydroxybiphenyl (2-HBP) and sulfite without losses in the energy content of the fuel. The desulfurization ability of the wild-type (wt) strain is limited due to repression of the dsz operon in the presence of bioavailable sulfur sources, including methionine and in some cases, sulfates, whereas genetic deletion of the reverse transsulfuration pathway enzymes, Cbs and MetB, alleviates this effect. Herein, we examined the growth and biodesulfurization activity of two genetically engineered R. qingshengii IGTS8 strains, Δcbs and ΔmetB, when grown in a stirred tank bioreactor in the presence of their preferred sulfur sources, sulfate and methionine, respectively. The Δcbs strain demonstrated the highest biomass concentration, but the lowest desulfurization activity, whereas ΔmetB, when grown in a repressive sulfur source, exhibited desulfurization activity comparable to that of the wt grown under non-repressive conditions. Investigation of the influence of ethanol and methionine supply for ΔmetB strain, highlighted carbon, and not sulfur concentration, as the critical factor for more efficient growth and desulfurization activity. The highest rates in batch mode were achieved in the presence of 165 mM ethanol and 2 mM methionine. Finally, fed-batch cultures of ΔmetB improved desulfurization efficiency up to 99.3% compared to batch mode, upon optimization of ethanol feeding rates. Thus, we identify carbon source as an important factor for process upscale, while dynamic control of nutrient supply may contribute to balancing biomass production and enzymatic activity. KEY POINTS: • Recombinant R. qingshengii IGTS8 cultivated under sulfur-repression in bioreactors. • Carbon supply limits growth and desulfurization activity in batch cultures. • Fed-batch ethanol control boosts longevity, biomass, and desulfurization activity.