Bug agar

The metabolic abilities of R7 and BCP1 strains were also assayed on 41 organic/xenobiotic compounds added as the only carbon and energy source, such as aliphatic hydrocarbons, polycyclic aromatic hydrocarbons, aromatic and compounds belonging to the BTEX group, and naphthenic acids. Strains were grown at 30°C on BUG agar (Biolog), and then, each strain was picked with a sterile cotton swab from the agar surface and suspended in 1 mL of sterile mineral medium. Each strain suspension was added to 15 mL of mineral medium without carbon source until cell density of 79% transmittance was reached on a Biolog turbidimeter. 1% dye G (vol/vol) was added to each suspension before inoculation, while the substrate was supplied to the inoculated wells with a minimum of two wells of distance. The plates were incubated at 30°C in an OmniLog reader and were monitored automatically every 15 min. Readings were recorded for 72 h and the data were analyzed using OminoLog PM software (release OM_PM_109M). Each strain was analyzed at least in duplicate and the results were checked for consistency. The growth was also evaluated using OD₅₉₀ after 72 hours.

The phenotype analysis for different pH was carried out by using a new tool, Phenotype MicroArrays (PMs). The 2,3-butanediol over-producer [2,3-B (++)] and non-producer [2,3-B (-)] were assayed on PM (Biolog) lane A1 to A12 of microplates PM10, testing different pH range from 3.5 to 10. PM technology uses the irreversible reduction of tetrazolium violet to formazan as a reporter of active metabolism. All procedures were performed as indicated by the manufacturer and previous study (Zhang and Biswas, 2009 (link)). Strains were grown at 30°C on BUG agar (Biolog), and then, each strain was picked with a sterile cotton swab from the agar surface and suspended in 15 ml of inoculation fluid (IF-0; Biolog) until a cell density of 85% transmittance was reached on a Biolog turbidimeter. In order to inoculate microplates PM10, 1% tetrazolium violet (vol/vol) was added to the suspension and the mixture was inoculated (100 μl per well). The photo was taken at 24 h after bacterial inoculation.

The Biolog MicroStation System and GEN III microplate (Biolog Inc., Hayward, CA, United States) is an automated microbial identification system based on aerobic metabolic activities. The GEN III plate contains 95 different carbon substrates based on interpreting patterns of sole carbon substrate utilization indicated by color development in a 96-well microtiter plate. By analyzing the similarity of the metabolic fingerprints between test strains and standard strains in the kinetic database by Biolog software, the strains are identified. In this study, the strains M13, M15, and M17 were first cultured on BUG agar (provided by Biolog) and inoculated into a GEN III plate. After being cultured at 30°C for 24 h, the plate was read by a Biolog MicroStation reader to generate strain identification (Wozniak et al., 2019 (link)).