*3.1. Growth Medium Can Significantly Influence Cell Surface Hydrophobicity and Motility of Bradyrhizobium Strains*

Surface properties of bacterial cells affect interaction with surfaces. We determined surface hydrophobicity of populations cultured in PSY and SESOM using the MATH partitioning assay. Hydrophobicity of the four strains varied, when grown in PSY medium with arabinose. USDA 110 was most hydrophilic while USDA 26 was the most hydrophobic (with USDA126 and USDA3384 displaying intermediate hydrophobicity), indicating strainspecific surface phenotype (Figure 1). When grown in the soil extract medium, SESOM, two of the strains underwent a significant increase in surface hydrophobicity. For USDA 3384, the fraction partitioned to the hydrocarbon phase increased by 700% and for USDA 110 by 300%. For USDA 110, some absorbance values of aqueous phase after partitioning were negative, suggesting minor emulsification due to hydrocarbon droplets into the aqueous phase. However, no significant change was observed for USDA 26 and 126 (Figure 1). These results indicate that surface hydrophobicity of *Bradyrhizobium* has some inherent strainspecific differences, and it is affected by the culture medium, with increased hydrophobicity predicted for cells growing in soil.

**Figure 1.** Surface hydrophobicity of *Bradyrhizobium* strains cultured in either PSY-Arabinose or SESOM to mid exponential phase and partitioned into hexadecane as an apolar solvent. Data were analyzed by Student *t*-test, and *p* values indicate statistical significance (ns not significant, \*\* *p* < 0.01, \*\*\* *p*< 0.001).

To determine whether growth in a soil environment affects motility, strains were inoculated into classical motility low agar plates (0.35%) in either PSY or SESOM. The strains varied in degree of motility in either medium, with USDA 110 and 126 the most motile, USDA 3384 less motile and 26 the least motile (Figure 2). USDA 26, 110 and 3384 showed a significant decrease in motility when grown in SESOM, while 126 remained unchanged (Figure 2). Strains 110 and 3384 showed the largest change. Collectively, USDA 110 and USDA 3384 became more hydrophobic and less motile when cultured in SESOM.

**Figure 2.** Motility radius of *Bradyrhizobium* strains spot-inoculated into either PSY-Arabinose or SESOM solidified with 0.35% agar and incubated for 10 d. Data were analyzed by Student *t*-test, and *p* values indicate statistical significance (ns not significant, \*\* *p* < 0.01, \*\*\*\* *p* < 0.0001).
