Abstract
Using gas chromatography mass spectrometry and the PacBio single molecule with real-time sequencing technology (SMRT), we analyzed the detailed metabolomic profiles and microbial community dynamics involved in ensiled Medicago sativa (alfalfa) inoculated without or with the homofermenter Lactobacillus plantarum or heterofermenter Lactobacillus buchneri. Our results revealed that 280 substances and 102 different metabolites were present in ensiled alfalfa. Inoculation of L. buchneri led to remarkable up-accumulation in concentrations of 4-aminobutyric acid, some free amino acids, and polyols in ensiled alfalfa, whereas considerable down-accumulation in cadaverine and succinic acid were observed in L. plantarum-inoculated silages. Completely different microbial flora and their successions during ensiling were observed in the control and two types of inoculant-treated silages. Inoculation of the L. plantarum or L. buchneri alters the microbial composition dynamics of the ensiled forage in very different manners. Our study demonstrates that metabolomic profiling analysis provides a deep insight in metabolites in silage. Moreover, the PacBio SMRT method revealed the microbial composition and its succession during the ensiling process at the species level. This provides information regarding the microbial processes underlying silage formation and may contribute to target-based regulation methods to achieve high-quality silage production.
Highlights
Ensiling is a traditional method of green fodder conservation involving lactic acid fermentation by epiphytic lactic acid bacteria (LAB) under anaerobic conditions
Metagenomic analysis by 454 high-throughput sequencing provided more comprehensive insight into the composition of the whole microbial community involved in grass ensiling[5], this technique is restricted to genus-level identification because only a partial sequence of the 16S rRNA
Inoculants are commonly used to rapidly decrease silage pH and ensure the fermentation quality of ensiled alfalfa[15,17]. Fermentation parameters, such as pH and lactic and acetic acid concentrations of the ensiled alfalfa, suggest that high quality-silages were prepared in our mini-silos
Summary
Ensiling is a traditional method of green fodder conservation involving lactic acid fermentation by epiphytic lactic acid bacteria (LAB) under anaerobic conditions. The SMRT sequencing platform is applicable for profiling ensiling communities Metabolites in silage, such as organic acids (lactic acid, acetic acid, propionic acid, butyric acid), ethanol, and 1,2-propanediol, are conventionally detected to evaluate the fermentation quality of ensiled forage. Previous studies showed that LAB can improve silage aerobic stability by producing different metabolites, such as acetic acid and 1,2-propanediol7,8, 3-phenyllactic acid and 3-hydroxydecanoic acid[9], and a phenolic-related antibiotic[10]. These studies suggested that many other metabolites are present in ensiled forage in addition to the conventional metabolites commonly detected when assessing silage quality. We comprehensively analyzed the metabolomic profiles and bacterial community dynamics using the PacBio SMRT method in ensiled alfalfa, using the homofermenter L. plantarum and heterofermenter L. buchneri for modulation
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