Effect of Pine Derived Biochar on Ruminal Methanogen Communities in Beef Steers

Abstract Biochar is a carbon-rich charcoal-like material produced from the anaerobic heating of biomass. Historically used to enhance soil health and sequester carbon, biochar has recently been investigated for its potential to reduce methane emissions in cattle. However, there has not yet been a metagenomic analysis of the impact of biochar on rumen microbial populations, including methanogens. Further, an efficacious dietary inclusion rate of biochar has not been established in cattle. Therefore, the objective of this study was to evaluate an increasing dietary inclusion of pinewood-based biochar on microbial community composition in rumen (solid and liquid fractions) and fecal samples in beef steers. Four ruminally cannulated steers were utilized in a 4 × 4 Latin square design with four 14-d periods, each consisting of 8-d for treatment adaptation, 5-d for measures of intake and digestibility, and 1-d for measures of rumen fermentation and collection of rumen and fecal samples for microbial analysis. Steers consumed ad libitum forage and were supplemented cottonseed meal (125 mg N/kg BW) once daily. One of four biochar treatments was provided as a percentage of previous 3-d forage intake: 0% (CON), 0.8% (LO), 1.6% (MED) and 2.4% (HI). Rumen and fecal samples were analyzed using the 16S rRNA gene through PCR-amplification of the V1-V3 regions from microbial genomic DNA, followed by Illumina MiSeq 2X300 sequencing. Sequence data were processed using QIIME 2 to generate a curated set of amplicon sequence variants (ASV) which represent unique bacterial sequences. ASVs were then analyzed using rank-transformed ANOVA and treatment effects were analyzed using orthogonal polynomial contrasts. F-Bt-0001 was consistently the most abundant ASV across rumen and fecal samples and was closely related to Methanobrevibacter ruminantium (98.9% identity), which is a methanogen identified in cattle supplemented biochar in previous literature. Increasing dietary biochar linearly increased the relative abundance of ASV F-Bt-0001 (P ≤ 0.01) in the rumen solid fraction, from 5.33% in CON to 11.4% in HI, but there was no effect of treatment on F-Bt-0001 in the liquid fraction (P = 0.51). In fecal samples, ASV F-Bt-0001 did not respond linearly or quadratically to biochar inclusion. These data suggest that biochar promotes enrichment of Methanobrevibacter-like hydrogenotrophic methanogens, likely through alterations in the rumen environment as this effect does not translate to feces. We suggest further research directly measuring methane flux in cattle supplemented biochar to investigate how these metagenomic findings translate to actual gas production.