| Summary: | Halotolerant bacteria with biosurfactant (BS) and bioemulsifiers (BE) activity can coexist in Karstic sinkholes with marine influence. Two sinkholes in the Yucatan peninsula were selected to isolate bacteria with BE and BS activity stable in NaCl. The optimal time, the effect of nitrogen and carbon source in the medium, and the conditions (agitation, pH and salinity) for the production of BS and BE compounds in planktonic and sessile (stimulate the formation of biofilms in cell roller) culture were determined. Eighty strains showed the highest emulsification activity (EI<sub>24</sub> ≥ 50%) and drop-collapse ≥ 4 mm. 87% of the strains are moderately halotolerant, and 21% bordered the limit of extreme halotolerance. Twenty-four strains maintained or improved their BS and BE activity under salinity conditions at 5% and 10%, being the most active genera <i>Bacillus</i>, <i>Paenibacillus</i> and <i>Lysinibacillus</i>, identified by sequencing of the 16S rRNA gene. The results show that the nitrogen source positively affects the BS and BE activity, regardless of the type of culture. The sessile culture markedly stimulated BS activity with significant differences. However, we did not find a greater influence on the culture conditions. The results suggest that halotolerant bacteria from sinkholes could be implemented in bioremediation and other biotechnological applications.
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