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

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.

Rapid Enabling of Gluconobacter oxydans Resistance to High D-Sorbitol Concentration and High Temperature by Microdroplet-Aided Adaptive Evolution

“…However, due to the difficulty in genetic modification, future studies need to verify these mutation sites for enhancing specific phenotypes rather than simply modifying the membrane components or other known processes. High-temperature fermentation has great advantages in industrial production, especially for reducing cooling costs and risk of contaminations, and simultaneous saccharification and fermentation of pretreated lignocellulosic for biofuel production (Zhang et al, 2016;Vishnu Prasad et al, 2020). However, most of the strains produced in the industry have poor heat resistance.…”

Simultaneous saccharification and lactic acid fermentation of the cellulosic fraction of municipal solid waste using Bacillus smithii

“…Although the lactic acid titres reported here do not compare with established platform producers, such as B. coagulans and Lactobacillus sp., (Aulitto et al 2019;Prasad et al 2020) this study serves to demonstrate that, when cellulosic SSF is being considered, other thermophilic Bacillus spp. and Parageobacillus spp.…”

Fermentative Lactic Acid Production From Lignocellulosic Feedstocks: From Source to Purified Product

“…Transformation of L. gasseri with plasmids carrying additional genes for L-or D-lactate dehydrogenases didn't affect the ratio of produced stereoisomers, but inactivation of the endogenous genes created strains which yielded 0.96 g/g glucose of either L-or D-lactate. Constructed strains efficiently fermented wheat straw hydrolysate and produced 0.37-0.42 g of lactate/g wheat straw (Žunar et al, 2020). Prasad et al (2020) described the application of an evolutionary engineered thermo-tolerant strain of Lactobacillus bulgaricus strain in D-lactic acid production from rice straw. The engineered strain was capable to work at 45 °C and thus be used in an SSF process enzyme significantly reducing the enzyme loading.…”

“…SHF and batch and fed-bach SSF mode of fermentation were carried out. In batch SSF experiments 11.8-32.4 g/l D-lactic acid was produced, depending on the enzyme and total solid loading, while in fed-batch SSF the final obtained lactic acid concentration was 108.6 g/L (Prasad et al, 2020). Tian et al (2021) developed through the CRISPR-Cas9 geneediting platform a high optical purity L-lactic acid producing strains.…”