Effects of cotton straw biochar on methane emissions using rumen simulations

The 16S rRNA gene sequencing analysis was performed to investigate the microbial community structure and populations in the rumen fluid. Total DNA was extracted from the fermentation broth using the CTAB and bead-beating method. The concentration and purity of the extracted DNA were measured using a NanoDrop spectrophotometer. Real-time PCR was employed to quantify the DNA copy numbers of various microbial species and groups, using specific primers targeting the V3-V4 region of the 16S rDNA for bacteria and the V4-V5 region of the 16S rRNA gene for archaea. The primers used are listed in Table 3. The sequencing data were processed through a series of steps, including data filtering, merging, and clustering into operational taxonomic units (OTUs) at a 97% sequence similarity threshold. Species annotation was achieved using the usearch -sintax command against the SILVA (16S) database with a confidence threshold of 0.8. The analysis revealed that the addition of cotton straw biochar did not significantly affect the total bacterial DNA copy number or the total methanogen DNA copy number in the rumen fluid. The bacterial OTU numbers were 2,073, 2,099, and 2,136 for the 0%, 3%, and 6% treatment groups, respectively, while the archaeal OTU numbers were 30, 34, and 35 for the corresponding groups. No significant differences were observed in the alpha diversity or beta diversity of bacteria and archaea among the treatment groups. These results indicate that the cotton straw biochar supplementation did not have a substantial impact on the overall microbial diversity or the total DNA copy numbers of bacteria and methanogens in the rumen fluid under the conditions of this experiment.