Evolutionary engineering of Lactobacillus bulgaricus reduces enzyme usage and enhances conversion of lignocellulosics to D-lactic acid by simultaneous saccharification and fermentation

J Vishnu Prasad,Tridweep K Sahoo,Guhan Jayaraman,S Naveen

doi:10.1186/s13068-020-01812-x

Abstract

BackgroundSimultaneous 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.ResultsA 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.ConclusionsWe 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.

Highlights

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
Batch enzymatic hydrolysis The effect of enzyme loading on batch cellulose hydrolysis was studied at the optimal condition of temperature 50 °C and pH 4.5 [34,35,36]
Though there is no significant difference in the extent of cellulose hydrolysis between 37 and 45 °C in the later stages of batch enzymatic hydrolysis irrespective of enzyme loading (Additional file 1: Fig S4), we could see a significant increase of ~ 20% in the cellulose to glucose and D-lactic acid (D-Lactic acid (LA)) conversions by increasing the operating temperature of SSF from 37 to 45 °C at an enzyme loading of 5 Filter Paper Unit (FPU)/gLCB (Table 2)

Summary

Introduction

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. 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. 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. Valorization of LCB to bulk chemicals like D-LA requires a sequence of separate processes such as pretreatment of LCB, enzymatic hydrolysis of cellulose and fermentation of the hydrolysate. Physical and temporal separation of the enzymatic hydrolysis and fermentation processes makes the whole process time-intensive and results in low productivity [1]

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Conclusion