An inoculating fluid

We used the AN MicroPlate (Biolog) containing 95 carbon sources to determine metabolic activity of F. necrophorum. The assay was carried out according to the manufacturer’s instructions. Briefly, F. necrophorum KG34 (GenBank accession no. SRX5402669) was grown on Wilkins-Chalgren agar medium (Sigma-Aldrich) and suspended in 14 mL of AN inoculating fluid (Biolog) to make bacterial suspension with a transmittance level of about 65%. A 100 μL of bacterial suspension was quickly plated into each well of the AN MicroPlate and incubated at 37 °C in the GasPak EZ anaerobic pouch system (BD). Utilization of the carbon sources by F. necrophorum was indicated by tetrazolium violet forming a blue color, which was measured at 3, 6, 9, 12, 18, and 24 h post inoculation at OD590 using SmartSpec 3000 spectrophotometer (Bio-Rad).

The metabolic repertoire of KH365_2^T, KH569_7, and B. uniformis ATCC 8492 ^T was studied by performing a series of assays using Biolog AN MicroPlates™ (Biolog, Hayward, CA), following the protocol provided by the manufacturer with minor modifications. The strains were grown on CM agar under anaerobic conditions at 37 °C for 5 days to obtain colonies of sufficient size. Individual colonies were picked and inoculated in AN Inoculating Fluid (Biolog, Hayward, CA) such that the turbidity (600 nm) of 0.05–0.1 was achieved. The Inoculating Fluid was then pipetted into Biolog AN MicroPlates™ (100 μl per well), and the initial turbidity (590 nm) was measured using a Spark® microplate reader (TECAN, Männedorf, Switzerland). The plates were then incubated in anaerobic jars at 37 °C for 5 days, after which the final turbidity (590 nm) was measured. The difference between the final and initial turbidity_, ΔT (590 nm), was used to determine the extent to which each of the metabolites were utilized by the strains. The metabolites differentially utilized by the strains were identified by testing whether the difference in ΔT for each metabolite was significantly different between each pair of strains (KH365_2^T vs. B. uniformis ATCC 8492 T, KH569_7 vs. B. uniformis ATCC 8492 T, and KH365_2^T vs. KH569_7) using the Kruskal–Wallis test.