Time of Day for Harvest Affects the Fermentation Parameters, Bacterial Community, and Metabolic Characteristics of Sorghum-Sudangrass Hybrid Silage

Abstract

To characterize the effects of time of day for harvest on the fermentation parameters, bacterial community, and metabolic characteristics of sorghum-sudangrass hybrid (SSG) silage, SSG (vegetative stage) harvested at 7:00 (AM), 12:00 (M), and 17:00 (PM) on three sunny days were ensiled for 1, 3, 7, 14, 30, and 60 days. Compared to AM silage, M and PM silages were characterized by delayed fermentation, unnormal lower final pH, and lower acetic acid production. In addition, PM silage contained higher residual water-soluble carbohydrates than other silages. After 60 days of ensiling, AM silage was dominated by Lactobacillus, whereas the bacterial communities of M and PM silages were complex and mainly composed of bacteria such as Delftia, Methylobacterium-Methylorubrum, Enhydrobacter, Acinetobacter, and Bacillus. The harvest time affected a wide range of metabolic pathways including "Metabolism" and "Cellular Processes" and "Organismal Systems" in SSG silage. Particularly, at the late stage of ensiling M silage exhibited highest relative abundances of amino acid metabolisms including "glycine, serine, and threonine metabolism," "phenylalanine metabolism," and lowest relative abundances of "lysine biosynthesis." These results suggest that the time of day for harvest could affect the fermentation parameters, bacterial community, and metabolic characteristics of SSG silage. Better SSG silage characteristics could be achieved through morning harvest. IMPORTANCE Ensiling is a common way for preserving green forages worldwide. Silage fermentation quality can vary greatly depending on the chemical and microbial characteristics of forage crop being ensiled. It is well documented that forages exhibit considerable variations in chemical composition and epiphytic microbiota during daylight. However, the effects of the time of day for harvest on silage fermentation is less investigated. Our results demonstrate that the time of day for harvest could affect the fermentation parameters, bacterial community, and metabolic characteristics of SSG hybrid silage. Harvesting SSG late in the day delayed fermentation process, lowered acetic acid production and final pH, and increased the residual water-soluble carbohydrates content in silage. Moreover, the delayed harvest time increased the relative abundances of bacteria such as Delftia, Methylobacterium-Methylorubrum, Acinetobacter, Enhydrobacter, and Bacillus, and amino acid metabolisms at the late stage of SSG ensiling. This study highlights the importance of diurnal changes in forage to fermentation characteristics, providing a strategy to improve silage quality through optimizing the harvest time.

Keywords: bacterial community; fermentation parameters; metabolic characteristics; sorghum-sudangrass hybrid; time of day for harvest.

FIG 1

Bacterial diversity during SSG ensiling. AM, 7:00; M, 12:00; PM, 17:00. FM. fresh material. Arabic numbers indicate the days of ensiling. (A) Alpha diversity estimators for SSG silage harvested at various times within a day. (B) Principal coordinates analysis (PCoA) of bacterial communities on OTU level.

FIG 2

The structures and comparison of bacterial community of SSG silage harvested at various times. The bacterial community structures on phylum (A), genus (B), and species (C) level in fresh and ensiled SSG. Phyla and genera detected at less than 1.0% of total sequence reads are not included. Only species ranked in top 15 were presented. AM, 7:00; M, 12:00; PM, 17:00. Statistical comparison of the relative abundance of bacterial communities (top 10) on genus level in raw material (D) and silage (E). Error bars denote the standard error of means. Different lowercase letters (a–c) above each bar denote significant differences among harvest times according to Tukey’s test (P < 0.05).

FIG 3

Interaction networks of top 20 genera during SSG ensiling. A connection stands for a significant (P < 0.05) and strong (Spearman’s |P| >0.6) correlation. Size of each node is proportional to the relative abundance, and the nodes are colored by phylum. The color of the edges corresponds to a positive (red) or negative (blue) relationship. AM, 7:00; M, 12:00; PM, 17:00. (A) AM silage on day (d) 1. (B) M silage on d 1. (C) PM silage on d 1. (D) AM silage on d 3. (E) M silage on d 3. (F) PM silage on d 3. (G) AM silage on d 60. (H) M silage on d 60. (I) PM silage on d 60.

FIG 4

Correlation analysis of the top 15 genera with fermentation characteristics. (A) Canonical correspondence analysis (CCA) of the correlations between fermentation characteristics and bacterial community. The canonical axes are labeled with the percentage of total variance explained (%). Arrow lengths indicate the variance explained by fermentation characteristics. SSG silages harvested at various times ensiled for different days are presented as individual data points. Arabic numbers indicate days of ensiling. (B) Spearman’s correlation heatmap of the correlations between fermentation characteristics and bacterial community composition. The corresponding value of the middle heatmap is the Spearman correlation coefficient r, which ranges between −1 and 1, r < 0 indicates a negative correlation (blue), r > 0 indicates a positive correlation (red), and *, P < 0.05, **, P < 0.01, and ***, P < 0.001, respectively. WSC, water-soluble carbohydrates; LA, lactic acid; LA/AA, lactic/acetic acid ratio; DM, dry matter; CP, crude protein; ETH, ethanol; AA, acetic acid; PA, propionic acid.

FIG 5

The changes of KEGG metabolic pathways on the first (A), second (B), and carbohydrate and amino acid pathways (C) on the third level obtained with Tax4Fun in raw material and silage. AM, 7:00; M, 12:00; PM, 17:00. Arabic numbers indicate days of ensiling. Error bars denote the standard error of means. Different lowercase letters (a–c) above each bar denote significant differences among harvest times according to Tukey’s test (P < 0.05).