Biosurfactants are surface-active compounds mainly produced by bacteria, fungi and yeast. As amphiphilic compounds, they exhibit pronounced surface and emulsifying activities, and comprise a wide range of chemical structures with diverse interfacial properties and physiological functions (Rodrigues and Texeira, 2010). In recent years, there has been an increasing interest in microbial biosurfactants for several reasons: (i) firstly, due its natural origin, biosurfactants are usually reported as relatively nontoxic and as readily biodegradable substances (Silva et al., 2014); (ii) secondly, biosurfactants have unique structures that are just starting to be appreciated for their potential wide range of applications, ranging from biotechnology to environmental clean-up (Soberón-Chavez, 2011), (iii) finally, their stability under extreme conditions of pH, temperature and salinity, allow its applications in several industrial processes. Meanwhile its production in large scale is very limited.
In the first step of this work, we studied the production of surfactin at 37ºC using two different strains of Bacillus subtilis, as well as two different culture media (Vederaman & Ventatesh, 2011, Vaz et al. 2012). All assays were carried out in triplicate, in a batch system, using glucose as substrate. The main goal of this stage was to determine the best experimental conditions, which leads to the highest biosurfactant production. The process of synthesis the surfactin included the following steps: (a) preparation of inoculum; (b) fermentation process; (c) separation of cells from the culture medium by centrifugation; (d) measuring and characterization properties of surfactin recovered. The stocks of microorganisms and the inoculum have been prepared according to the procedure described by Valenzuela-Avila (2017).
The determination of the concentration of surfactin on the culture media allowed us to find the best experimental conditions of the fermentation process to obtain this bioproduct. The biosurfactant recovered at the end of fermentations was used to study its properties, chemical structure and cleaning performance. The interfacial properties of surfactin (surface tension and CMC, among others) were determined as described by Vaz et al. (2012). The chemical of structure of the biosurfactants were determined as indicated by Yang et al. (2015) in the “Centro de Instrumentación Científica (CIC)” of the University of Granada. The surface tension of the fermentation solution (TS), as well as its dilutions 1:10 (TS−1) and 1: 100 (TS−2), has been determined by the method of Wilhelmy's blade on a Kruss-K11® tensiometer. All measurements have been performed at least of three times.
The results of the biosurfactant production tests carried out with the Bacillus subtilis referring to the measurements of pH, surface tension (TS, TS−1 and TS−2), concentration of surfactin in the solution after fermentation (Csurf) are summarized in Table 1. Thus, based on the results, two favorable experimental conditions are detected, which maximize the production of biosurfactants: (i) using strain DSM 3256, after 24 hours of fermentation, (ii) using strain B5, after 48 h of processing.
Table 1 – Surfactin production assays using fermentation
Authors acknowledge the financial support provided by the University of Granada through project PP2017-PIP10. Y. M. acknowledges a scholarship from the Erasmus+ program (European Comission).