# Effect of active and heat-killed Clostridium butyricum on in vitro gas production, ruminal fermentation parameters, and microbiota at varying media pH levels

## TL;DR

This study examines how active and heat-killed Clostridium butyricum affect rumen fermentation and gas production under different pH conditions.

## Contribution

The study reveals how pH levels modulate the effects of active and heat-killed Clostridium butyricum on rumen fermentation and microbial communities.

## Key findings

- Lower media pH (5.8) inhibited gas production, dry matter disappearance, and microbial diversity.
- Supplementing ICB increased gas volume and butyrate proportion at pH 6.5.
- Both active and inactive Clostridium butyricum had dose-dependent effects on fermentation parameters.

## Abstract

The objective of this study was to evaluate the effects of inactive (heat-killed) Clostridium butyricum (ICB) on gas production kinetics, fermentation parameters, and microbiota with varying media pH levels in batch culture.

The in vitro experiment was designed as a completely randomized factorial arrangement, with 2 media pH levels (5.8 and 6.5)×2 Clostridium butyricum (CB) products (active and inactive)×4 dosages of CB. Two lactating dairy cows with ruminal fistulas, fed a diet comprising 40% forage and 60% concentrate, served as donors for rumen inoculum. Following 24 h of incubation, the gas production, dry matter disappearance (DMD), volatile fatty acid (VFA), ammonia nitrogen (NH3-N) and microbial profile were measured to determine the effect of treatment on fermentation.

The gas volume (GV), DMD, total VFA concentration, NH3-N concentration, acetate concentration and microbial alpha diversity were inhibited when the media pH decreased from 6.5 to 5.8. Increasing the supplemental doses of ICB linearly increased the GV, DMD (trend) and butyrate proportion at media pH 6.5. Moreover, the increasing supplemental dose of active Clostridium butyricum (ACB) linearly increased GV, butyrate proportion and NH3-N concentration (trend) regardless of media pH, and linearly increased DMD, total VFA concentration and A:P ratio at media pH 6.5. Supplementing ICB decreased the relative abundance of Actinobacteria and Butyrivibrio in the fermentation fluid.

Increasing media pH promotes rumen fermentation and alter bacterial community. Although both ACB and ICB have the potential to stimulate rumen fermentation in a dose-dependent manner, their effects change depending on media pH levels. Furthermore, both ACB and ICB rarely altered the rumen bacterial community.