Abstract
BackgroundButyric 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.ResultsEngineered 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.ConclusionHT2 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.
Highlights
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
The lactic acid content in the control silage increased to 41.1 g/kg DM during 18 days of ensiling but decreased to 16.3 g/kg DM at the final ensiling time (60 days), while the contents of acetic acid and ammonia-N in the control silage individually increased to 42.5 g/kg DM and 246 g/kg N, respectively, during 60 days of ensiling
Silage treated without additives; DM, dry matter; Combination of HT1 and EN (EN), cellulase; HT1, wild-type L. lactis subsp. lactis MG1363; HT1 + EN, combination of HT1 and EN; HT2, combination of transgenically engineered L. lactis strains HT1/pMG36e-usp45-bgl1, HT1/pMG36e-usp45-cbh2, and HT1/pMG36e-usp45-egl3; N, nitrogen; Ratio of lactic acid to acetic acid (LA/AA), ratio of lactic acid to acetic acid; Standard error of the means (SEM), standard error of the means
Summary
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. Alfalfa contains low contents of dry matter (DM) and water-soluble carbohydrates (WSC), especially in the rainy season, and has a high buffer capacity that often causes increased butyric acid and substantial DM loss [5]. The few nutrients in poor-quality alfalfa silage are useless for feeding animals and decrease biofuel production due to insufficient soluble nonstructural carbohydrates, which deliver energy sources to yeast for producing bioethanol [6]. In the rainy season, maximally preserving high-moisture alfalfa via the ensiling bioprocess is an interesting and potential strategy for subsequent biofuel production
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
