# The effect of Methylococcus capsulatus in mono- or co-culture with Methanobrevibacter smithii or with mixed rumen fluid on bacterial growth and methane gas production

## TL;DR

This study shows that adding Methylococcus capsulatus to rumen systems can significantly reduce methane emissions, though it may increase nitrous oxide emissions.

## Contribution

The study introduces a novel biological approach using Methylococcus capsulatus to mitigate methane emissions in rumen fermentation.

## Key findings

- Methylococcus capsulatus significantly reduced methane production when co-cultured with Methanobrevibacter smithii.
- Methylococcus capsulatus addition increased nitrous oxide emissions in both grain- and forage-based diets.
- Continuous use of Methylococcus capsulatus in rumen systems led to a significant reduction in methane flux over time.

## Abstract

Providing an alternate electron sink to methane (CH4) is a crucial step in reducing enteric CH4 emissions. Comparing the effects of CH4-utilizing methanotroph bacteria (Methylococcus capsulatus; MC) and its combination with pure strains of rumen bacteria or mixed rumen fluid can offer biological insights into methanogenesis pathways and CH4 consumption. The primary objectives of this study were to investigate the impact of inoculating with M. capsulatus on the growth rates of rumen bacteria, including methanogens and mixed rumen fluid, as well as fermentation rates, ruminal gas production, CH4 emissions, and other environmental-impacting gases (N2O, H2S). Three experiments were carried out using in vitro ANKUM gas production systems (Exp. 1 and 2) and continuous recirculating flux chamber systems (Exp. 3). In Exp. 1, four strains of rumen bacteria—Streptococcus bovis [SB], Ruminococcus flavefaciens [RF], Methanobrevibacter smithii [MS], and M. capsulatus [MC]—were used to determine the effect of CH4-utilizing bacteria (e.g., MC) on specific growth rate, volatile fatty acid (VFA) production, and ruminal CH4 emissions in a combination with these bacterial strains. Results from Experiment 1 showed that MS produced the most CH4 among the strains. When cocultured with MC, no CH4 was detected, indicating that MC could utilize most of the CH4 produced in coculture with MS and other bacterial strains. There was little difference in total and cumulative gas production with varying MC doses (Exp. 2). However, in the presence of MC, CH4 production (percentage or g DM) decreased significantly (P < 0.01) as MC addition increased. Conversely, substrates containing both grain- and forage- based diets with MC increased N2O emissions per gram of DM (µg/g DM) or total N2O production (ppm), with treatment and basal diet interactions (P < 0. 01). In Exp. 3, using a continuous recirculating flux chamber system, CH4 flux significantly reduced (P < 0.001) over time in both basal diets with MC inoculum. However, fermentation rates varied between treatments and diets. These findings demonstrate that adding MC inoculum to in vitro rumen fermentation chambers significantly reduces CH4 emissions compared to controls.

Our findings showed that Methanobrevibacter smithii increased nitrous oxide (N2O) emissions per gram of DM (µg/g DM) or total N2O during 24 h (ppm), with a significant interaction between treatment and basal diet (P < 01; Table 4). The rumen fermentation rate, as measured by total volatile fatty acids (VFA), was similar across treatment groups. Using a continuous recirculating flux chamber system, CH4 production over time in both forage- and grain-based diets with the M. capsulatus inoculum to in vitro rumen fermentation chambers significantly reduces CH4 was detected, suggesting that M. capsulatus emissions in ruminant animals or the agricultural compost industry. Such a mitigation strategy could influence nutrient metabolism and CH4 dynamics, underscoring the importance of dietary approaches in optimizing rumen function and productivity in ruminant animals.