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- usp 45- bgl 1, HT1 / pMG36e- usp 45- cbh 2, and HT1 / pMG36e- usp 45- egl 3 (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 ...

Low-Carbohydrate Tolerant LAB Strains Identified from Rumen Fluid: Investigation of Probiotic Activity and Legume Silage Fermentation

“…Fermentation highly depends on the presence of natural microbiota, the chemical composition of the forage crops, mainly WSC, and the presence of nitrogenous components [ 51 ]. Recently, Liu et al (2019) [ 52 ] studied alfalfa silage and reported that LAB-inoculated silage has distinct shapes, colors, smells, and textures after 60 days of fermentation. The control silage (without inoculum) produced an unpleasant odor and seemed black and sticky.…”

Clarifying Key Cellulase Component Cooperated With Lactic Acid Bacteria for Alfalfa Lignocellulose Degradation to Improve Lactic Acid Production

“…The engineered strains of MG1363 containing bgl1, cbh2, and egl3 gene of T. reesei were constructed in our lab by Liu et al (2019). After culturing in GM17 broth medium at 30°C for 30 h, engineered strains containing bgl1, cbh2, and egl3 gene were mixed at 1:1:1, 2:1:1, 1:2:1, 1:1:2, 1:1:0, 1:0:1, and 0:1:1 and were named HT2, HT3, HT4, HT5, E1C1, E1B1, and C1B1 separately.…”

“…According to the method reported by Liu et al (2019), the LAB, aerobic bacteria, and clostridia in alfalfa materials were counted after harvesting from the eld for 2 h. The BC of alfalfa materials was measured by the method reported by Liu et al (2018). The DM, NDF, ADF, ADL, and WSC in alfalfa materials were measured by the same method reported by Li et al (2019).…”

“…However, some previous studies have described negative effects of the combined treatment of cellulase and LAB on ensiling alfalfa, such as ineffectiveness on decreasing DM loss, neutral detergent ber (NDF) and acid detergent ber (ADF), and increasing LA content compared with the control (Kozelov et al 2007;Lynch et al 2014). The proposed explanation is that some cellulases cannot degrade lignocellulose of alfalfa (Lynch et al 2014), some lignocellulose degradation products of cellulase (high-molecularweight oligosaccharides) cannot be fermented by LAB (Stokes and Chen 1994;Tarraran and Mazzoli 2018), or the timeliness of cellulase and LAB was different (Liu et al 2019). However, the bottleneck of lignocellulose degradation was that little known about which cellulase component played synergistic roles with LAB in fermenting alfalfa lignocellulose into lactic acid.…”

“…lactis MG1363 (MG1363) strains separately containing the bgl1, cbh2, and egl3 gene of Trichoderma reesei. The combination proportion of engineered MG1363 strains (HT2) containing bgl1, cbh2, and egl3 genes at 1:1:1 could secrete cellulase (236 mU/ml FPase activity) (Liu et al 2019). We hypnotized that optimizing the combinations of engineered MG1363 strains could clarify key cellulase component playing synergistic roles with LAB in fermenting alfalfa lignocellulose to improve LA production.…”