Improved glycerol utilization by a triacylglycerol-producing Rhodococcus opacus strain for renewable fuels

Background: Glycerol generated during renewable fuel production processes is potentially an attractive substrate for the production of value-added materials by fermentation. An engineered strain MITXM-61 of the oleaginous bacterium Rhodococcus opacus produces large amounts of intracellular triacylglycerols (TAGs) for lipid-based biofuels on high concentrations of glucose and xylose. However, on glycerol medium, MITXM-61 does not produce TAGs and grows poorly. The aim of the present work was to construct a TAG-producing Rhodococcus opacus strain capable of high-cell-density cultivation at high glycerol concentrations. Results: An adaptive evolution strategy was applied to improve the conversion of glycerol to TAGs in Rhodococcus opacus MITXM-61. An evolved strain, MITGM-173, grown on a defined medium with 16 g L-1 glycerol, produced 2.3 g L-1 of TAGs, corresponding to 40.4% of the cell dry weight (CDW) and 0.144 g g(-1) of TAG yield per glycerol consumed. MITGM-173 was able to grow on high concentrations (greater than 150 g L-1) of glycerol. Cultivated in a medium containing an initial concentration of 20 g L-1 glycerol, 40 g L-1 glucose, and 40 g L-1 xylose, MITGM-173 was capable of simultaneously consuming the mixed substrates and yielding 13.6 g L-1 of TAGs, representing 51.2% of the CDM. In addition, when 20 g L-1 glycerol was pulse-loaded into the culture with 40 g L-1 glucose and 40 g L-1 xylose at the stationary growth phase, MITGM-173 produced 14.3 g L-1 of TAGs corresponding to 51.1% of the CDW although residual glycerol in the culture was observed. The addition of 20 g L-1 glycerol in the glucose/xylose mix resulted in a TAG yield per glycerol consumed of 0.170 g g(-1) on the initial addition and 0.279 g g(-1) on the pulse addition of glycerol. Conclusion: We have generated a TAG-producing Rhodococcus opacus MITGM-173 strain that shows significantly improved glycerol utilization in comparison to the parental strain. The present study demonstrates that the evolved Rhodococcus opacus strain shows significant promise for developing a cost-effective bioprocess to generate advanced renewable fuels from mixed sugar feedstocks supplemented with glycerol.