Evaluating the fermentation characteristics, bacterial community, and predicted functional profiles of native grass ensiled with different additives

Abstract

Bioaugmentation of native grass ensiling with Lactobacillus plantarum or Lactobacillus buchneri or Pediococcus pentosaceus on the ensiling performance and bacterial community was investigated after 30 days of the fermentation process. The native grass was inoculated with distilled water, Lactobacillus plantarum, Lactobacillus plantarum, and Lactobacillus buchneri, and Lactobacillus plantarum, Lactobacillus buchneri, and Pediococcus pentosaceus as the CON treatment, T1 treatment, T2 treatment, and T3 treatment, respectively. The addition of lactic acid bacteria was added at a total of 1 × 10⁶ colony-forming unit/g of fresh weight. As expected, the markedly (p < 0.05) lower water-soluble carbohydrate content was tested in the T2 and T3 treatments compared to the CON and T1 treatments. Compared to the CON and T1 treatment, significantly (p < 0.05) higher crude protein content, and lower acid detergent fiber and neutral detergent fiber contents were found in the T2 and T3 treatments. Compared to the CON treatment, the pH significantly (p < 0.05) decreased in the lactic acid bacteria (LAB) inoculated silage, and the lowest pH was measured in the T3 treatment. Similarly, significantly higher lactic acid and acetic acid contents were also found in the T3 treatment compared to those in other treatments. After 30 days of ensiling, the Shannon and Chao1 indexes in silages decreased compared to that in the fresh materials (FMs). The principal coordinate analysis indicated that both FM and silage were distinctly separated in each treatment with no interactions on the confidence ellipse (R = 0.8933, p = 0.001). At the phylum level, the dominant phylum was shifted from Proteobacteria to Firmicutes after the fermentation process. Interestingly, Weissella dominated the fermentation in the CON treatment and Lactobacillus dominated the fermentation in all inoculated LAB silages at the genus level. Results of functional prediction analyses showed that the metabolism of amino acid, cofactors, and vitamins, and membrane transport was reduced, while the metabolism of nucleotide and majority carbohydrates was increased after ensiling. The complex LAB (Lactobacillus plantarum, Lactobacillus buchneri, and Pediococcus pentosaceus) exhibited the potential possibility to decrease pH and enhance the relative abundance of LAB in response to obtaining high-quality silage by the synergistic effects. These results suggested that the complex LAB could improve the ensiling performance of native grass silage, and lay a theoretical basis for inoculant application in native grass.

Keywords: bacterial community; fermentation quality; lactic acid bacteria; native grass; silage.

Figure 1

Bacterial community diversities and dissimilarities of native grass silage. FM, fresh material; CON, Samples without inoculants; T1, native grass inoculated with Lactobacillus plantarum; T2, native grass inoculated with Lactobacillus plantarum and Lactobacillus buchneri; T3, native grass inoculated with Lactobacillus plantarum, Lactobacillus buchneri, and Pediococcus pentosaceus. (A) Venn diagram representing the unique and common OTUs. (B) The bacterial community dissimilarities in different treatments were calculated by Bray–Curtis with coordinates calculated by principal coordinates analysis. (C,D) Relative abundances of native grass silage bacterial phylum and genus across different treatments. (E) Differences of bacterial taxa in native grass silage with different treatments.

Figure 2

Correlation analysis between genera (top 10 significant genera) and ensiling performance of native grass silage inoculated without or with lactic acid bacteria. *Significant correlation at p < 0.05 level, **Significant correlation at p < 0.01 level. DM, dry matter; WSC, water-soluble carbohydrates; CP, crude protein; ADF, acid detergent fiber; NDF, neutral detergent fiber; NH₃-N, ammonia nitrogen; LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid.

Figure 3

Dynamics of bacterial functional profiles in different treatments analyzed by PICRUSt (n = 3). (A) Level 1 metabolic pathway. (B) Level 2 KEGG ortholog functional predictions of the relative abundances with significant differences in the top 20 metabolic functions. (C) Level 3 KEGG ortholog functional predictions of the relative abundances of the top 30 metabolic functions. FM, fresh materials; CON, Samples without inoculants; FM, fresh material; T1, native grass inoculated with Lactobacillus plantarum; T2, native grass inoculated with Lactobacillus plantarum and Lactobacillus buchneri; T3, native grass inoculated with Lactobacillus plantarum, Lactobacillus buchneri, and Pediococcus pentosaceus.de. * Indicates significant at p < 0.05, ** indicates significant at p < 0.01 and *** indicates significant at p < 0.001.