ABSTRACT
BACKGROUND: Simultaneous saccharification and fermentation (SSF) of pre-treated lignocellulosics to biofuels and other platform chemicals has long been a promising alternative to separate hydrolysis and fermentation processes. However, the disparity between the optimum conditions (temperature, pH) for fermentation and enzyme hydrolysis leads to execution of the SSF process at sub-optimal conditions, which can affect the rate of hydrolysis and cellulose conversion. The fermentation conditions could be synchronized with hydrolysis optima by carrying out the SSF at a higher temperature, but this would require a thermo-tolerant organism. Economically viable production of platform chemicals from lignocellulosic biomass (LCB) has long been stymied because of the significantly higher cost of hydrolytic enzymes. The major objective of this work is to develop an SSF strategy for D-lactic acid (D-LA) production by a thermo-tolerant organism, in which the enzyme loading could significantly be reduced without compromising on the overall conversion. RESULTS: A thermo-tolerant strain of Lactobacillus bulgaricus was developed by adaptive laboratory evolution (ALE) which enabled the SSF to be performed at 45 °C with reduced enzyme usage. Despite the reduction of enzyme loading from 15 Filter Paper Unit/gLCB (FPU/gLCB) to 5 FPU/gLCB, we could still achieve ~ 8% higher cellulose to D-LA conversion in batch SSF, in comparison to the conversion by separate enzymatic hydrolysis and fermentation processes at 45 °C and pH 5.5. Extending the batch SSF to SSF with pulse-feeding of 5% pre-treated biomass and 5 FPU/gLCB, at 12-h intervals (36th-96th h), resulted in a titer of 108 g/L D-LA and 60% conversion of cellulose to D-LA. This is one among the highest reported D-LA titers achieved from LCB. CONCLUSIONS: We have demonstrated that the SSF strategy, in conjunction with evolutionary engineering, could drastically reduce enzyme requirement and be the way forward for economical production of platform chemicals from lignocellulosics. We have shown that fed-batch SSF processes, designed with multiple pulse-feedings of the pre-treated biomass and enzyme, can be an effective way of enhancing the product concentrations.
ABSTRACT
D-Lactic acid (D-LA) is an enantiomer of lactic acid, which has a niche application in synthesis of poly-lactic acid based (PLA) polymer owing to its contribution to the thermo-stability of stereo-complex PLA polymer. Utilization of renewable substrates such as whey permeate is pivotal to economically viable production of D-LA. In present work, we have demonstrated D-LA production from whey permeate by Lactobacillus delbrueckii and engineered Lactococcus lactis. We observed that lactose fermentation by a monoculture of L. delbrueckii yields D-LA and galactose as major products. The highest yield of D-LA obtained was 0.48 g g-1 when initial lactose concentration was 30 g L-1. Initial lactose concentration beyond 20 g L-1 resulted in accumulation of glucose and galactose, and hence, reduced the stoichiometric yield of D-LA. L. lactis naturally produces L-lactic acid (L-LA), so a mutant strain of L. lactis (L. lactis Δldh ΔldhB ΔldhX) was used to prevent L-LA production and engineer it for D-LA production. Heterologous over-expression of D-lactate dehydrogenase (ldhA) in the recombinant strain L. lactis TSG1 resulted in 0.67 g g-1 and 0.44 g g-1 of D-LA yield from lactose and galactose, respectively. Co-expression of galactose permease (galP) and α-phosphoglucomutase (pgmA) with ldhA in the recombinant strain L. lactis TSG3 achieved a D-LA yield of 0.92 g g-1 from galactose. A co-culture batch process of L. delbrueckii and L. lactis TSG3 achieved an enhanced stoichiometric yield of 0.90 g g-1 and ~45 g L-1D-LA from whey permeate (lactose). This is the highest reported yield of D-LA from lactose substrate, and the titres can be improved further by a suitably designed fed-batch co-culture process.