Omnilog system

Ground poultry meat from above treatment was prepared by addition of sterile phosphate buffered saline (PBS) to make a 10% (v/v) suspension, which was mixed thoroughly by vortex at the maximum speed for 3 min (Fisher Vortex Genie 2, Fisher Scientific, Bohemia, NY, US) at 25 °C. After 10 min on ice, the supernatants were further diluted with PBS to have a final 1% (v/v) concentration [25 (link), 26 (link)] that were then dispensed in 150 μl volumes per well into 96-well EcoPlates™ (Biolog Inc.). The plates with lids were incubated in an OmniLog System (Biolog Inc., Hayward, CA, USA) at 25 °C for 168 h. This typical 96-well microplate consists of 31 unique chemicals and a water control in triplicates. The tetrazolium dye in each well was converted into insoluble violet formazan after bacterial respiration. The concentration of the formazan was proportional to the degree of respiration by the microorganisms in the communities.

The metabolic capability of the two colicin-producing ST10 competitor strains was determined using the BioLog Omnilog system and growth curves as described previously [20 (link)]. Six plates, PM1 to PM4, PM13 and PM14 (http://www.biolog.com/products-static/phenotype_microbial_cells_microPlate_panels_and_media.php) were used. PM1 to PM4 contain carbon, nitrogen, phosphorus and sulphur sources, PM13 and 14 contain antimicrobials and other chemical compounds. The plates were inoculated following the manufacturer’s instructions and grown for 48 h at 37 °C. The experiment was performed using three biological replicates. Data were analyzed using the statistics program R and heat maps were generated taking the area under curve (AUC) value for each well, normalized by the negative control wells [20 (link), 23 (link)].

The metabolic potential of soil communities was determined from community-level physiological profiles (CLPPs) using Biolog EcoPlates (Biolog Inc.; [55 (link)]). For each soil sample, an EcoPlate containing 31 different carbon sources (and one blank with no carbon source) in triplicate was inoculated, and incubated for 196 h at 25 °C in an OmniLog^® system (Biolog Inc.). The rate of carbon source utilization was recorded by the reduction of tetrazolium, a redox color indicator that changes from colorless to purple and is detected at a wavelength of 590 nm. Data were recorded every 15 min of incubation and saved in OmniLog^® units generated by Biolog Data Analysis software (v1.7). Values for each well were calculated by subtracting the blank values from each well of the plate. A single absorbance time point at 50 h was used for the comparisons, as recommended in [84 (link)]. The metabolic potential of the soil microbial communities in each sample, expressed as the average well color development (AWCD), was calculated at the determined time point by dividing the sum of the optical density data by 31 (number of substrates). The total number of wells in a replicate with an absorbance above 25 OmniLog^® units was counted to determine the functional richness of the soil microbial community.

The metabolic potential of soil communities was performed according to the community-level physiological profiles (CLPP) using Biolog EcoPlates^TM (Biolog Inc., Hayward, CA, USA) [40 (link)]. For each soil sample, an EcoPlate containing 31 different carbon sources (and a blank with no C source) in triplicate was inoculated and incubated at 25 °C for 196 h in an OmniLog^® System (Biolog Inc.). The rate of carbon source utilization was indicated by the reduction of tetrazolium, a redox indicator dye, which changes from colorless to purple and is detected at a wavelength of 590 nm. Data were recorded every 15 min of incubation and saved in OmniLog units, generated by the Biolog Data Analysis software (v1.7, Biolog Inc). The values for each well were calculated by subtracting the blank well values from each plate well. Absorbance at a single time point of 70 h was used for comparisons, following the recommendation of Weber et al. [41 (link)]. The metabolic potential of the soil microbial communities of each sample, expressed as average well color development (AWCD), was calculated at the determined time point by dividing the sum of the optical density data by 31 (number of substrates). The total number of wells in a replicate with an absorbance above 25 OmniLog units was counted to define the functional richness of the soil microbial community.

Biolog® GEN III Microbial Identification System (Biolog Inc., Hayward, CA, USA) was used for the biochemical identification of putative N₂-fixers from the isolates of rhizosphere soil and root nodules of test species. The system identifies microorganisms based on their ability to metabolize all major classes of biochemical compounds and determines important physiological properties, such as pH, salt and lactic acid tolerance, reducing power, and chemical sensitivity. Pure bacterial isolates were streaked on biolog universal growth (BUG) agar medium and incubated for 24 hr at 30°C. Subsequently, individual colonies were suspended in IFA inoculating fluid such that the cell density was in the range of 90–98% as per the recommendation of Biolog Inc. A total of 100 μL of the prepared suspension was transferred to each well of a 96-well microplate, which was incubated in an OmniLog incubator at 33°C for 24 h. Colorimetric response was measured using Biolog OmniLog system and compared with the Biolog® database for identity confirmation (GEN III database and characteristics v2.7).

Four autochthonous Lvb. brevis (14.2.10, 30.2.29, 7.8.43, and 3.2.41) and two allochthonous isolates, ATCC14869 (type strain) and ATCC367, were used for phenotype microarray (PM) analysis using the PM01 and PM02 plates of the Omnilog system (Biolog, Hayward, CA) following the manufacturer's instructions. Aliquots of 100 μl of the cell suspensions were added per well and plate. Duplicate PM01 and PM02 plates were inoculated with each isolate. The PM01 and PM02 plates were incubated at 33°C for 48 h in the Omnilog system, as recommended by the manufacturer.
The absorbance as a function of time data from the Omnilog system for each isolate can be found at https://doi.org/10.15482/USDA.ADC/1528683 (Page, 2023 (link)) and was used to calculate the growth rate using a Microsoft Excel spreadsheet and the following equation: = [LOG (End of Log Phase Time)-LOG (Start of Log Phase Time)] × 2.303)/(Absorbance at the End of Log Phase—Absorbance at the Start of Log Phase). The start and end of the log phase time and absorbance were adjusted for each PM plate. Averages and standard deviations were calculated for duplicate plates. Growth rate values for a given substrate that were above the standard deviation of the negative control value were considered utilized.