Assessment of Biolog EcoplateTM method for functional metabolic diversity of the pig fecal microbiota

In the last decades, gut microbiota and its role in mammals host development and health has been increasingly investigated. Metabolites produced by gut microbiota can affect intestinal homeostasis, immune system maturity and activation, and in turn, they can influence health and growth performance of livestock. Therefore, a better understanding of the functional metabolic capability of the gastrointestinal microbiota has come to be appreciated by scientific community. In this study, Biolog EcoPlates® technology was used for studying metabolic potential and activity of the microbial community in pig fecal samples, evaluating the interference of different storage conditions and cell concentrations. Furthermore, the specific length of time that a sample will retain its detectable and invariate metabolic activity in a given storage condition was also investigated. Two assays were carried out and aimed to evaluate differences in the metabolic activities between fresh and snap frozen fecal samples at different dilutions (approximately 9 x 105 cells/g, 9 x 104 and 9 x 103 cells/g) and at different times of presenvation at -80°C: 15 hours (T0) and 15 (T1), 45 (T2) and 150 (T3) days after sample collection. Furthermore the V3-V4 hypervariable regions of the 16S rRNA gene were also analysed in order to describe the biodiversity of community composition and predicted functionality. The results highlighted that snap freezing of pig fecal samples preserved the metabolic activity of the microbial community when compared to fresh feces (P > 0.1). Sample storage at -80 °C did not significantly affect metabolism of the microbial community, whose activity remained stable throughout the study period (P > 0.1). The carbon source utilization by pig fecal microbiota was significantly affected by bacterial cell density (P < 0.05). Indeed, a cell concentration of 9 x 104 and 9 x 105 cells/g allows to detect the lofty metabolic activity of microbial community. Overall, after 96 hours of incubation of Ecoplates, carbohydrates were the highest carbon source category, while amines were the lowest. The results evidenced that functional metabolic activity of the pig fecal microbial community can be preserved without significant variation until 150 days of storage at -80°C. Biolog EcoPlates® technology is a rapid and useful method to explore the metabolic capabilities of the microbial community in animal samples, thereby contributing to explain the health status and the physiology of the host animals.