Biochar supplementation affects the microbiome of recycled manure solids for cow bedding: A metagenomic analysis

The widespread use of recycled manure solids (RMS) as cow bedding material is not without risks, because cattle manure may act as a vehicle for pathogenic and antimicrobial-resistant bacteria dissemination. Thus, our aim was to evaluate RMS supplemented with a pine biochar produced in Portugal as a new cow bedding material, because the use of biochar has been shown to have the potential to mitigate the effect of relevant bacterial species when added to animal manure microbiota. Our experimental setup consisted on fresh RMS samples that were collected on a commercial dairy farm and placed in naturally-ventilated containers for a total of 4 groups: (1) nonsupplemented RMS, (2) RMS supplemented with 2.5% (wt/wt) of biochar, (3) RMS supplemented with 5% (wt/wt) of biochar, and (4) RMS supplemented with 10% (wt/wt) of biochar. Sampling was performed at 4 different incubation times (0, 5, 15, and 30 d) and in 2 distinct seasons: April through May (humid season) and June through July (dry season). The resulting 32 samples were subjected to DNA extraction and their microbiome profile determined through complete 16S rDNA gene sequencing using Nanopore next-generation sequencing. We observed that biochar supplementation clearly altered the microbiome of RMS, which was reflected in changes in populations' diversity and the relative abundance of relevant pathogenic bacteria. In particular, we found that long-term storage (30 d) was more beneficial than shortterm storage, an effect that was more evident for samples supplemented with 2.5% or 5% biochar. In both seasons, those concentrations of biochar led to a decrease in the levels of several mastitis-causing agents (Enterobacteriaceae, streptococci, enterococci, and staphylococci). In addition, we also observed a reduction in the levels of Salmonella spp. and gram-positive bacilli in the biocharsupplemented samples. Unexpectedly, however, those same conditions yielded an increase in the abundance of Brucella spp., a group that includes important infectious agents, highlighting the need for a deeper evaluation of the effect of biochar supplementation of RMS to ensure the future safe and sustainable use of this environmentally-friendly resource in animal production.