Dynamics Variation Of Microbial Diversity And Functional Geneomics During Alfalfa Silage Fermentation Process

Silage production has a long history.Nowadays,silage ensiling has drawn much attention because it is easy to make and preserve;it is good for animal digestion and absorption.Also,it can be used as alternative feedstuffs for livestocks in the season of lacking fresh forage.The silage process is accompanied by the proliferation and metabolism of a large number of microorganisms,which play an important role in the quality of silage.In this study,Lactobacillus plantarum P-8 was used as alfalfa silage starter.A total of 50 silage samples were made according to the actual production process of silage.The dynamic changes of viable counts of microorganisms in silage were detected by traditional culture methods.The results showed that the viable counts of lactic acid bacteria grew rapidly during silage fermentation,while the viable counts of harmful microorganisms such as clostridium,coliform bacteria,yeasts and molds decreased significantly.During the silage fermentation process,the contents of lactic acid,acetic acid(LAB),phenyllactic acid and gamma-aminobutyric acid increased in alfalfa silage,while the content of neutral detergent fiber decreased.Protein degradation was inhibited,and the quality and safety of alfalfa silage were improved.Microbial biodiversity of silage was analyzed by the PacBio single molecule real-time sequencing technology combined with propidium monoazide(PMA)technology.The results showed that 544 species belonging to 278 genus of bacteria and 95 species belonging to 82 genus of fungal were identified in silage samples.L.plantarum became the dominant species after 7 days of ensiling.As the ensiling process continuted,a core flora dominated by L.plantarum along with Weissella jogaejeotgali,Weissella paramesenteroides,Lactobacillus pentosus,Lactobacillus hammesii,Lactobacillus paralimentarius,Lactobacillus brevis and Pediococcus pentosaceus was gradually formed,which inhibited the growth of other bacteria and fungi such as Weissella,Pantoea,Erwinia,Pseudomonas,Staphylococcus,Volutella,Cryptococcus,Leucosporidium and Fusarium in silage.The relationship between physicochemical index of silage and microbial flora was analyzed by the Spearman correlation analysis.The beneficial effect of LAB on silage quality was elaborated at the species level.Metagenome sequencing and functional genome(based on Illumina HiSeq 4000 platform)results showed that L.plantarum was positively correlated with the functional genes which produced metabolites of lactic acid,phenyllactic acid and acetic acid,while negatively correlated with genes related to butyric acid produced.L.plantarum and L.brevis were positively correlated with gamma-aminobutyric acid.These results indicate that L.plantarum was an important factor affecting the whole network relationship.At the gene level,the contents of COG functional categories E(amino acid transport and metabolism),L(replication,recombination and repair),G(carbohydrate transport and metabolism),J(translation,ribosome structure and biosynthesis),H(coenzyme transport and metabolism),P(inorganic particle transport and metabolism),T(signal),transduction mechanism,F(nucleotide transport and metabolism),M(cell wall/membrane/envelope biosynthesis)were relatively high during the silage fermentation.The results of carbohydrate active enzyme analysis showed that the carbohydrate diversity and the content of glycoside hydrolase and polysaccharide lyase significantly increased after the ensiling.The changes of metabolic pathways at different stages of silage fermentation were analyzed based on KEGG database.The results showed that glutamate metabolism,pyruvate metabolism,fructose and mannose metabolism,glycolysis,and cell cycle(cell growth and death)pathways were active during 7 days of silage,which suggested that microorganisms may compete with each other.Compared with metabolic pathways between 7th and 14h day,the metabolic pathways were more active in the 14th days.Compared with metabolic pathways between 14th and 28h day,all the metabolic pathways were more active in the 14th days,which proved the silage entered a stable period at day 28,and microbial metabolism slowed down.In conclusion,by combining PacBio single molecule real-time sequencing technology with metagenome sequencing on the Illumina HiSeq 4000 platform,we could monitor changes in the microbiota as well as its functional genes accurately during the silage ensiling process,which provides new ideas for the research and development of silage starter.