Enhancement of lignocellulosic degradation in high-moisture alfalfa via anaerobic bioprocess of engineered Lactococcus lactis with the function of secreting cellulase

Abstract

Background: Butyric fermentation and a substantial loss of dry matter (DM) often occur in alfalfa silage during the rainy season, which is not conducive to subsequent biofuel production. Currently, there have been negative effects on the combination of cellulases and lactic acid bacteria (LAB) on processing high-moisture alfalfa silage; however, transgenically engineered LAB strains that secrete cellulase have been proposed as an alternative approach to avoid the above problem. The objective of the present study was to construct engineered Lactococcus lactis strains with high-efficiency secretory-expressing cellulase genes from Trichoderma reesei and to investigate the effects of the combination of transgenically engineered L. lactis strains HT1/pMG36e-usp45-bgl1, HT1/pMG36e-usp45-cbh2, and HT1/pMG36e-usp45-egl3 (HT2) on fermentation quality, structural carbohydrate degradability and nonstructural carbohydrate fermentation kinetics of high-moisture alfalfa silage treated without additive as a negative control (Control), or/and with cellulase (EN), wild-type L. lactis subsp. lactis MG1363 (HT1) and the combination of HT1 and EN (HT1 + EN) as positive additive controls. Results: Engineered L. lactis strains were successfully constructed and efficiently secreted endoglucanase (1118 mU/mL), cellobiohydrolase (222 mU/mL), and β-glucosidase (131 mU/mL) and had high filter paper activity (236 mU/mL). Ensiling experiments verified that HT2 obtained the highest fermentation quality score (83.6) and most efficiently processed high-moisture alfalfa silage, demonstrated by a low pH (4.49) and ammonia-N content (106 g/kg nitrogen) and a high lactic acid content (67.1 g/kg DM) and without butyric acid. Change curves of structural carbohydrates revealed that HT2 degraded more lignocelluloses, demonstrated by the lowest contents of neutral detergent fibre, acid detergent fibre, cellulose and hemicellulose after ensiling for 60 days. Kinetic analysis showed that the most residual water-soluble carbohydrates, glucose, fructose and xylose generated by lignocellulose degradation were produced by HT2, followed by HT1 + EN. The HT2-treated silages had the highest DM recovery, had the fewest Clostridia spores, emitted a fragrance and were not sticky. Conclusion: HT2 improved the conversion of lignocellulose to sugars and processed high-moisture alfalfa silage efficiently. This is a novel strategy that can be used to enhance lignocellulosic degradation in high-moisture alfalfa via a bioprocess with transgenically engineered L. lactis strains, which could enhance the development of alfalfa as a biomass feedstock and promote second-generation biofuel development in the rainy season.