Abundance, isolation, and characterization of halotolerant microbes from common oligosaline soils

Abstract

Halotolerant microbes that grow at high NaCl concentrations were studied in common soils with low salinity. The abundance of microbes in common oligosaline (<1.0% salinity) soils was measured by most probable number analyses, where statistically valid replicates of serial dilutions were assessed for growth in liquid Salt Plains medium with 0.1, 10, or 20% (w/v) NaCl. Common Kansas soils were collected from oligosaline agricultural fields (corn, cotton, sorghum, and soybean), pond margin, prairie grassland, and woodland. The abundance of microbes able to grow at 10% NaCl was 0.0025?0.32% of the total counts at 0.1% NaCl. Only marginal growth was observed at 20% NaCl, about 100-fold lower than at 10% NaCl. Bacterial isolates were collected from 20% NaCl enrichments of oligosaline turf soil by repetitive streak-plating. The bacteria were identified by 16S rRNA gene sequencing and characterized. The isolates clustered with low G+C Gram-positive genera (Bacillus, Halobacillus, Staphylococcus, and Virgibacillus). Broad halotolerances (0.1?20% NaCl) were observed for most isolates, but all grew at low salt. Soils were boiled to kill vegetative cells and compared by most probable number analyses with unboiled specimens. The abundance of microbes able to grow at 10% NaCl showed a 3-log decrease with boiling, while microbes able to grow at 0.1% NaCl showed a 5-log decrease in abundance with boiling. It appears that halotolerant microbes in oligosaline soils are cultivable vegetative cells, not perennating structures such as spores. Discrete salt phases are not obvious in oligosaline soils, but could provide a selective advantage to autochthonous halotolerant microbes. Allochthonous microbes associated with salt evaporite particles may be transported by winds, thereby dispersing deliquescent hypersaline microhabitats.