PHYSICOCHEMICAL CHARACTERIZATION OF BIOSURFACTANT FROM Bacillus

GODOY M; FILIPA M; MASUELLI M; VILLEGAS L B

Modalidad Virtual

Congreso; SAIB-SAMIGE Reunión conjunta; 2021

Surface-active agents or surfactants are amphiphilic molecules that contain hydrophilic and hydrophobic groups in their structure. Because of this nature, surfactants can forming micelles or emulsions. The ability to aggregate and form micelles makes this compound capable of lowering surface tension. Surfactants play an important role in different industries and in bioremediation processes. Current surfactants are chemically synthetized but are toxic and only partially biodegradable. Since science is on the way to shift towards eco-friendly processes and technologies, biosurfactants produced by microorganisms are of great interest. The aim of this work was to describe the physicochemical properties of the surfactant produced by Bacillus atrophaeus. B. atrophaeus was cultivated in Standard Nutrient medium (g/L: NaCl 6; meat peptone 15; yeast extract 3; glucose 1) for 144 h at 30°C and 120 rpm. Samples were taken every 24 h and the cell-free supernatant (CFS) was obtained by centrifugation at 10,000 xg for 10 minutes. Emulsification index with kerosene, drop collapsed and oil spreading assays using corn oil were performed on the CFS to detect the surfactant activity. The highest activity was observed at 72 hours of culture, obtaining an emulsification index of 59%, an oil dispersion area of 14.543 cm2 and a positive result in the collapsed drop assay. Once the preferred cultivation time was found, surfactant precipitation by different methods was attempted but a significant loss in the surfactant activity was observed. Therefore, the CFS was concentrated to 5x at 60°C and several dilutions (2 ml/L to 200 ml/L) were done. Surface tension (ST) using the Du Noüy tensiometer and viscosity using the Ubbelohde viscometer were determined on the different CFS dilutions. Enzymatic and acid digestion assays were performed to partially characterize the obtained biosurfactant using Proteinase K 30 u/mg (50 µl, 60°C, 1 h), Lipolase 100L 700 and 70 u/mg (100 µl, 37°C, 2 h) and concentrated HCl (10 µl, 100°C, 10 minutes). To obtain the critical micellar concentration (CMC) of the surfactant, a ST vs Concentration graph was done, showing a CMC of 14 ml/L(multiplicar por la densidad de la solución y te queda en g/L). To obtain the intrinsic viscosity, a Specific Viscosity/(Concentration) vs (C) graph was done, resulting in 6.6186 g/cm3. Using the Mark-Houwink equation, a molecular weight of 5271.37 g/mol was obtained. The emulsifying activity was lost only after the treatment with HCl, indicating that this biosurfactant is mainly of polysaccharide nature. The surfactant was stable at high temperatures, maintaining emulsifying activity after treatment under autoclave conditions (120°C, 2 atm, 15 min). The results obtained indicate that Bacillus atrophaeus produces a metabolite with interesting characteristics for its application in biotechnological processes. Further studies will focus on testing the ability of this compound to extract heavy metals and the potential use in bioremediation.