AUTHOR=Jiao Peixin , Wang Ziwei , Wang Xin , Zuo Yanan , Yang Yuqing , Hu Guanghui , Lu Changming , Xie Xiaolai , Wang Li , Yang Wenzhu 

TITLE=Effect of Clostridium butyricum Supplementation on in vitro Rumen Fermentation and Microbiota With High Grain Substrate Varying With Media pH Levels

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.912042

DOI=10.3389/fmicb.2022.912042

ISSN=1664-302X

ABSTRACT=<p><italic>Clostridium butyricum</italic> (<italic>C. butyricum</italic>) can survive at low pH, and it has been widely used as an alternative to antibiotics for the improvement of feed efficiency and animal health in monogastrics. A recent study suggested that the improved ruminal fermentation with supplementing <italic>C. butyricum</italic> is may be associated with increasing the abundance of rumen microbiota in Holstein heifers, as ruminal pH plays a key role in rumen microbiota and the probiotics are often active in a dose-dependent manner. The objective of this study was to determine the effects of increasing the doses of <italic>C. butyricum</italic> on gas production (GP) kinetics, dry matter disappearance (DMD), fermentation characteristics, and rumen microbiota using a high grain substrate in batch culture varying with media pH levels. The doses of <italic>C. butyricum</italic> were supplemented at 0 (control), 0.5 × 10<sup>6</sup>, 1 × 10<sup>6</sup>, and 2 × 10<sup>6</sup> CFU/bottle, respectively, at either media pH 6.0 or pH 6.6. The fermentation microbiota at 0 and 1 × 10<sup>6</sup> CFU/bottle were determined using the 16S rRNA high throughput sequencing technology. Overall, the GP, DMD, total volatile fatty acid (VFA) concentration, and the ratio of acetate:propionate were higher (<italic>P</italic> &lt;0.01) at media pH 6.6 than at pH 6.0. However, there was interaction between pH × dose of C. butyricum for rate constant of GP (<italic>P</italic> = 0.01), average GP rate (<italic>P</italic> = 0.07), and volume of GP (<italic>P</italic> = 0.06); with the increase in <italic>C. butyricum</italic> supplementation, the GP kinetics were not changed at media pH 6.0, but the volume (<italic>P</italic> = 0.02), rate of GP (<italic>P</italic> = 0.01), and average GP rate (<italic>P</italic> = 0.01) were quadratically changed at media pH 6.6. The DMD was not affected by increasing the supplementation of C. butyricum. The molar proportions of propionate (<italic>P</italic> &lt;0.09), butyrate (<italic>P</italic> &lt;0.06), and NH<sub>3</sub>-N concentration (<italic>P</italic> = 0.02) were quadratically changed with increasing supplementation of <italic>C. butyricum</italic> regardless of media pH levels. The interactions between media pH level and dose of <italic>C. butyricum</italic> supplementation were noticed for alpha diversity indexes of Shannon (<italic>P</italic> = 0.02) and Evenness (<italic>P</italic> = 0.04). The alpha diversity indexes increased (<italic>P</italic> &lt;0.05) except for Chao1 with supplementation of <italic>C. butyricum</italic>. The unweighted uniFrac analysis showed that the group of control at media pH 6.0 and control at media pH 6.6, and supplementation of <italic>C. butyricum</italic> and control at media pH 6.0 clustered separately from each other. At the phylum level, relative abundance (RA) of <italic>Bacteroidota</italic> was lower (<italic>P</italic> &lt;0.01) and <italic>Firmicutes</italic> was higher (<italic>P</italic> &lt;0.01) at media pH 6.6 than pH 6.0. Moreover, RA of <italic>Proteobacteria</italic> decreased (<italic>P</italic> &lt;0.05) with supplemented <italic>C. butyricum</italic> at either media pH 6.6 or pH 6.0. At media pH 6.6, RA of <italic>Rikenellaceae_RC9_gut_group</italic> and <italic>Prevotella</italic> were decreased, and CAG-352 was increased (at genus level) compared to pH 6.0. Supplementation of <italic>C. butyricum</italic> decreased RA of <italic>Rikenellaceae_RC9_gut_group</italic> and increased <italic>CAG-352</italic> at media pH 6.0. It could hence be concluded that manipulating media pH level and supplementation of <italic>C. butyricum</italic> effectively modulated <italic>in vitro</italic> rumen fermentation characteristics and microbiota but in a dose depending manner of <italic>C. butyricum</italic> addition.</p>