Turbidimeter

Strain Pt-3 was cultured on commercial BUG medium (Biolog, Hayward, CA) at 28 °C for 24 h. The fresh bacteria clone was inoculated into solution A (Biolog, Hayward, CA) and adjusted to percent transmittance 95% through Biolog Turbidimeter (Biolog, Hayward, CA). The bacteria suspensions were mixed well and distributed into Microplate (Biolog, Hayward, CA) with 100 μL in each hole. The plates were cultured at 28 °C for 12 h. The biochemical results of Pt-3 were recorded through Biolog Gen III system (Biolog, Hayward, CA) [24 ].

Isolate XC-1908 was cultured in BHI solid medium, and a single colony was inoculated in Biolog universal growth agar (BUG, Thinkfar, Wuhan, China) identification plates; the plates were incubated at 28 °C for 16–24 h, and a colony of suitable size was resuspended in IF-A inoculation fluid provided in the Biolog Bacterial Identification Kit (Biolog, Hayward, CA, USA). The turbidity of the solution was measured using the Biolog turbidimeter and adjusted (between 92% T and 98% T; as per Biolog). The inoculation fluid with the resuspended bacterial colony was added to a GEN III plate (100 μL per well) using a pipette, and the plate was incubated in the Biolog system, which recorded the absorbance of each well at different time-points, and identified the isolate.

A high-throughput analysis for microbial growth phenotypes using a colorimetric reaction, Phenotype MicroArrays (Biolog Inc., Hayward, CA, USA), was performed in accordance with the manufacturer’s protocol. Briefly, several colonies of E. coli grown on LB agar were transferred in 10 mL of a mixture of Biolog IF-0a medium (Biolog) and sterilized water into a sterile capped test tube. The suspension was mixed gently, and the turbidity was adjusted to achieve the appropriate transmittance using the Biolog turbidimeter (Biolog). The cell suspension was diluted with the IF-0a Plus dye mix, as mentioned in the manufacturer’s protocol. One hundred microliters of the mixture suspension was inoculated into phenotypic microarray (PM) plates 1 to 3 and 9 to 20 and incubated for 72 h at 37°C. The absorbance of each well was taken every 15 min. The OmniLog software (Biolog) was used to view and edit data, to compare data lists, and to generate reports.

Biolog PM analysis (Biolog Inc., Hayward, CA, USA) was employed to examine the metabolic profile of Metschnikowia sp. strain UM 1034. Ten MicroPlate panels (PM1 to PM10) including carbon, nitrogen, phosphorus, sulphur, nutrient supplements, peptide nitrogen, osmolytes and pH sources were used in this study. Metschnikowia sp. strain UM 1034 was grown on SDA at 25°C for 24 hours. The colonies on SDA were picked and suspended into 15 mL of FF inoculum media (Biolog Inc., Hayward, CA, USA). The cell suspension was adjusted to 62% transmittance at 590 nm using a turbidimeter (Biolog Inc., Hayward, CA, USA). The adjusted cell suspension was inoculated to PM1-10 inoculating fluids (Biolog Inc., Hayward, CA, USA) as indicated in S5 Table. The resultant inoculating fluid (100 μL) was then transferred into each well of PM1-10 microplate and incubated at 25°C for 60 hours in the Omnilog machine (Biolog Inc., Hayward, CA, USA). Negative control measurement was obtained from a well containing water. Data for each assay was reported as an average value from duplicate runs. Heat maps were constructed using Omnilog values ≥ 200,000 recorded over 60 hours.

Biolog Phenotype Microarrays were performed as previously described^{36 (link)}. In short, PAO1 and PAO1 ΔmexZ strains were streaked on LB agar plates and incubated at 37 °C. Cells were swabbed from the plates and suspended in IF-0 GN Base (inoculation fluid) at a density corresponding to 42% transmittance in the Biolog turbidimeter. The cell suspensions were diluted in IF-0 minimal medium containing Biolog redox dye mixture D (tetrazolium), and aliquots were added to the two different carbon-source plates; PM1 and PM2A. The plates were incubated at 37 °C in an OmniLog plate reader (Biolog) for 48 h, and growth/respiration was measured kinetically by determining the colorimetric reduction of the tetrazolium dye. Export of OmniLog data was performed using OmniLog OL_FM/Kin 1.20.02 software (Biolog). The average area beneath each kinetic curve was used for analysis. Total catabolic function was calculated as previously described^{37 (link)}.

D. shibae Dshi-5 and Dshi-6 strains were tested for their ability to utilize 95 different carbon sources using the Biolog PM01 microplate (34 (link)). Biolog experiments were performed with freshly grown D. shibae cells that were resuspended in 10 mL inoculating fluid IF-0 to achieve 85% transmittance in the Biolog turbidimeter and with a negative control without bacteria. A solution containing 428 μL H₂O, 120 μL dye D, 1,200 μL 10× seawater medium (160 g NaCl, 32 g NaSO₄, 24 g MgCl₂ · 6H₂O, 4 g KCl, 2 g NH₄Cl, 1.6 g KH₂PO₄, and 1.2 g CaCl₂ · 2H₂O per L), 120 μL 100× NaHCO₃ buffer (1.9 g/100 mL), 120 μL 100× vitamin mix (2 mg biotin [B₇], 20 mg niacin [B₃], 8 mg 4-aminobenzoic acid [B₁₀], and 1 mg thiamine [B₁] per L), and 12 μL 1,000× trace element solution (2.1 g FeSO₄ · 7H₂O, 13 mL 25% HCl, 5.2 g Na₂EDTA, 30 mg H₃BO₃, 100 mg MnCl₂ · 4H₂O, 190 mg CoCl₂ · 6H₂O, 24 mg NiCl₂ · 6H₂O, 2 mg CuCl₂ · 2H₂O, 144 mg ZnSO₄ · 7H₂O, and 36 mg Na₂MoO₄ · 2H₂O per liter) was added to a final volume of 12 mL. Each well of the PM01 plate was inoculated with the cell suspension, and the microplates were incubated at 28°C for 96 h in the OmniLog plate reader. The OmniLog data were processed and analyzed using the R package opm v1.1.0 (70 (link)). Curve parameters were estimated using spline fitting, and the curve maximum was used for plotting the heatmap and comparing the strains.