Homoethanol Production from Glycerol and Gluconate Using Recombinant Klebsiella oxytoca Strains

Gluconic acid, an oxidized cellulose degradation product, could be produced from cellulosic biomass. Glycerol is an inexpensive and renewable resource for fuels and chemicals production and is available as a byproduct of biodiesel production. Gluconate is a more oxidized substrate than glucose, whereas glycerol is a more reduced substrate than glucose. Although the production of homoethanol from glucose can be achieved, the conversion of gluconate to ethanol is accompanied by the production of oxidized byproduct such as acetate, and reduced byproducts such as 1,3-propanediol are produced, along with ethanol, when glycerol is used as the carbon source. When gluconate and glycerol are used as the sole carbon source by Klebsiella oxytoca BW21, the ethanol yield is about 62 to 64%. Coutilization of both gluconate and glycerol in batch fermentation increased the yield of ethanol to about 78.7% and decreased by-product accumulation (such as acetate and 1,3-propanediol) substantially. Decreasing by-product formation by deleting the pta, frd, ldh, pflA and pduC genes in strain BW21 increased the ethanol yield to 89.3% in the batch fermentation of a glycerol-gluconate mixture. These deletions produced the strain K. oxytoca WT26. However, the utilization rate of glycerol was significantly slower than that of gluconate in batch fermentation. In addition, substantial amounts of glycerol remain unutilized after gluconate was depleted in batch fermentation. Continuous fed-batch fermentation was used to solve the utilization rate mismatch problem for gluconate and glycerol. An ethanol yield of 97.2% was achieved in continuous fed-batch fermentation of these two substrates, and glycerol was completely used at the end of the fermentation. IMPORTANCE Gluconate is a biomass-derived degradation product, and glycerol can be obtained as a biodiesel byproduct. Compared to glucose, using them as the sole substrate is accompanied by the production of by-products. Our study shows that through pathway engineering and adoption of a fed-batch culture system, high-yield homoethanol production that usually can be achieved by using glucose as the substrate is achievable using gluconate and glycerol as cosubstrates. The same strategy is expected to be able to achieve homofermentative production of other products, such as lactate and 2,3-butanediol, which can be typically achieved using glucose as the substrate and inexpensive biodiesel-derived glycerol and biomass-derived gluconate as the cosubstrates.