Enhancing the conventional culture: The evaluation of several culture media and growth conditions improve the isolation of ruminal bacteria

Abstract

The rumen microbiota is crucial in cattle digestion, but its low cultivability makes it difficult to study its ecological function and biotechnological potential. To improve the recovery of ruminal microorganisms, this study combined the evaluation of several cultivation parameters with metabarcoding analysis. The parameters tested were culture media (CAN, ER, GC, GOOD, KNT, KYO, NYO, TRB), rumen fluid dilution (10-2, 10-6, and 10-12), and incubation time (3 and 7 days). The bacterial populations were determined through Illumina sequencing of 16S rRNA from three biological replicates of each treatment. Results showed that the culture media did not recover all rumen populations and altered the dominant phyla's relative abundance. In the rumen, Bacteroidetes and Firmicutes had a relative abundance of 75 % and 15 %, respectively, but in the culture media, it was 15 % and 60 %. The PCoA analysis showed that dilution had a significant impact on population composition, with 10-2 and 10-6 populations clustering together while 10-12 populations were more distant. Incubation time had no impact on populations diversity. After comparing PCoA distances, two media (CAN, KNT) were selected based on their ability to recover similar populations to the rumen. 64 cultures were obtained and 54 were identified through 16s rRNA gene sequencing. The metataxonomic analysis showed that CAN media had reproducibility over time, while KNT showed enrichment of different taxa due to the use of rumen fluid as a substrate.