The gas production, ruminal fermentation parameters, and microbiota in response to Clostridium butyricum supplementation on in vitro varying with media pH levels

Abstract

The aim of this study was to investigate the gas production (GP), dry matter disappearance (DMD), fermentation parameters, and rumen microbiota in response to Clostridium butyricum (CB) supplementation in batch culture using a high forage substrate. The doses of CB were supplemented at 0 (Control), 0.5 × 10⁶, 1 × 10⁶, and 2 × 10⁶ CFU/bottle, respectively, at either media pH 6.0 or pH 6.6. The 16S rRNA gene sequencing was used to detect the microbiota of fermentation culture in control and 1 × 10⁶ CFU/bottle after 24 h of incubation. The results showed that the GP (p < 0.001), DMD (p = 0.008), total volatile fatty acid (VFA) concentration (p < 0.001), acetate to propionate ratio (p < 0.001), and NH₃-N concentration (p < 0.001) were greater at media pH 6.6 than pH 6.0. Furthermore, the linearly increased DMD (pH 6.0, p = 0.002; pH 6.6, p < 0.001) and quadratically increased butyrate proportion (pH 6.0, p = 0.076; pH 6.6, p < 0.053) and NH₃-N concentration (pH 6.0, p = 0.003; pH 6.6, p = 0.014) were observed with increasing doses of CB. The Alpha diversity indexes of OTU number and Chao1 were higher (p = 0.045) at media pH 6.6 than pH 6.0, but they were not affected by CB supplementation. The PCoA analysis (unweighted uniFrac) demonstrated that the clustering of the bacterial microbiota of control and CB were distinctly separated from each other at media pH 6.0. At the phylum level, the abundance of Bacteroidota (p < 0.001) decreased, whereas that of Firmicutes (p = 0.026) increased when the media pH was elevated from 6.0 to 6.6. Supplementation of CB increased relative abundances of Rikenellaceae_RC9_gut_group (p = 0.002), Christensenellaceae_R-7_group (p < 0.001), and NK4A214_group (p = 0.002) at genus level. Interactions between media pH and CB addition were observed for bacteria at both phylum and genus levels. These results indicated that increasing the media pH level and CB supplementation increased in vitro rumen digestibility, and altered the ruminal fermentation pattern (by media pH) and microbiota.

Keywords: Clostridium butyricum; batch culture; media pH level; microbiota; rumen fermentation.

Figure 1

Principal coordinates analysis (PCoA) based on unweighted uniFrac distance matrices of microbiota in ruminal fermentation fluid supplemented with CB at low or high media pH after 24 h of in vitro incubation. CONH, control at media pH 6.6; CONL, control at media pH 6.0; CBH, CB supplemented at 1 × 10⁶ CFU/bottle at media pH 6.6; and CBL, CB supplemented at 1 × 10⁶ CFU/bottle at media pH 6.0.

Figure 2

Analysis of OTUs exclusiveness of microbiota in ruminal fermentation fluid supplemented with or without CB at low or high media pH after 24 h of in vitro incubation. CONH, control at media pH 6.6; CONL, control at media pH 6.0; CBH, CB supplemented at 1 × 10⁶ CFU/bottle at media pH 6.6; and CBL, CB supplemented at 1 × 10⁶ CFU/bottle at media pH 6.0.