Efficient bioconversion of crude glycerol from biodiesel to optically pure D-lactate by metabolically engineered Escherichia coli

Biodiesel has attracted considerable attention as one of the best choices among alternative and renewable fuels. Large quantities of crude glycerol are produced as a main co-product with increasing biodiesel production. Currently, the problem of waste glycerol utilization needs to be crucially addressed, not only for environmental protection but also for the economy of the biodiesel industry. In this paper, the use of crude glycerol for the production of D-lactate by engineered Escherichia coli was investigated. Engineered E. coli B0013-070 with a homolactic pathway for D-lactate synthesis by elimination of byproduct pathways (ethanol, succinate, formate and acetate) could convert 20 g L−1 of crude glycerol to 13.6 g L−1 of D-lactate with a yield of 0.67 g g−1 glycerol. Overexpression of D-lactate dehydrogenase by a low-copy vector in E. coli B0013-070 resulted in the increased production and yield of D-lactate, in which 14.5 g L−1 of D-lactate was produced with a yield of 0.72 g g−1 glycerol from crude glycerol. The effect of temperature on D-lactate fermentation by the engineered strain E. coli B0013-070-pTHldhA was also investigated, and 34 °C and 40 °C were found to be the optimal temperatures for cell growth and lactate production, respectively. The engineered strain B0013-070-pTHldhA produced 100.3 g L−1 of D-lactate with 99.97% optical purity from 531.5 g of crude glycerol with an overall productivity of 2.78 g L−1 h−1 and a yield of 75.4 g per 100 g glycerol (0.77 mol mol−1) using two phase fermentation combined with a temperature shifting strategy in a 7 L bioreactor. In summary, this paper shows that crude glycerol could be directly converted to D-lactate without any prior purification.