## 2 Materials and Methods  
### Materials  
#### 2.1.1 Surfactants and Chemicals Used  
Non-ionic synthetic surfactants, Triton X-100, Tween 80 and Brij 58, were purchased from Sigma-Aldrich, Inc. They were laboratory grade and storable at room temperature. Triton X-100 is characterised by a polyethylene oxide chain as its hydrophilic group and an aromatic hydrocarbon as its hydrophobic group. Tween 80 is a mixture of polyoxyethylene ethers and has emulsifier properties, whereas Brij 58 is polyethylene glycol hexadecyl ether. Biosurfactants were obtained by cultivation of two strains of non-pathogenic sophorolipid-producing yeasts in different conditions. _Yarrowia lipolytica_ was isolated from a crude oil lagoon in the southern Czech Republic by EPS biotechnology s.r.o. (Kunovice, Czech Republic) _Candida bombicola_ CB 2107 was purchased from the AV CR, Czech Academy of Sciences (Prague, Czech Republic). They were cultured on mineral medium, with the addition of different concentrations of polar (glucose) and nonpolar (rapeseed oil) carbon sources and yeast extract as a nitrogen source, to produce 8 different sophorolipid samples (Table 1). To produce the sophorolipids, yeast strains were sub-cultured from agar malt extract to mineral medium (Table 2) along with Difco(tm) Yeast Nitrogen Base (BD Biosciences, Heidelberg, Germany) and 10 g/L of rapeseed oil. The cultures were kept on an orbital shaker (WiseShake SHO-2D, Witeg Labortechnik GmbH, Wertheim, Germany) at 120 rpm (rotation per minute) for 4 days at room temperature. Yeast from these cultures were inoculated again in the mineral medium (\(1.8\times 10^{5}\) cfu/mL for _Candida bombicola_ and \(4.3\times 10^{5}\) cfu/mL for _Yarrowia lipolytica_) and cultured for 4 days at room temperature in a bioreactor (Bioscreen Testing Service, Inc., Torrance, CA, USA) with automatic temperature and pH control. The preculture was incubated for 7 days. Cultivation of the obtained yeast was continued for 168 h in mineral medium containing a non-polar (vegetable oil) and a polar (glucose) carbon source in different ratios (Table 1) at 20 \({}^{\circ}\)C temperature. Shaking was performed on a planar shaker at 120 rpm. The pH was 6, and the oxygen saturation was about 76% throughout the cultivation. The biosurfactants were isolated from the growing media using protocols described before [22; 23; 24]. After dispersion in an ultrasonic water bath for 15 min, the production medium was subjected to triple extraction during each 10 min using ethyl acetate (p.a. grade, Penta s.r.o., Prague, Czech Republic). The default volume ratio of ethyl acetate to aqueous phase was 1:2, but was increased to 3:1 for treatments with poorly separable emulsions. After each extraction step, the organic phase was transferred to a rotary vacuum evaporator flask to separate the crude surfactant from the ethyl acetate phase. Then, the mixture was transferred to a Petri dish and the residual ethyl acetate was left to evaporate at room temperature under the fume hood. Depending on the conditions of pre-treatment of the production medium, the crude surfactant was contaminated with some oil, which was removed by a hexane wash. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**BS 1** & **Producer Yeast** & **Glucose (g/L)** & **Rapeseed Oil (g/L)** & **Place of Growth** & **Yield (g/L) 2** \\ \hline A & _Candida bombicola_ CB 2107 & 1 & 1 & E. Flask.3  & 0  & 0  & 1 & E. Flask & 1.3 \\ C & _Yarrowia lipolytica_ & 10 & 10 & Auto. bio. 4  & ND \\ D & _Yarrowia lipolytica_ & 1 & 1 & E. Flask & 1.2 \\ E & _Yarrowia lipolytica_ & 10  & 10 & E. Flask & 5.2 \\ F & _Yarrowia lipolytica_ & 0  & 10 & E. Flask & 0.1 \\ G & _Yarrowia lipolytica_ & 10  & 10 & Auto. bio. & ND \\ H & _Candida bombicola_ CB 2107 & 10 & 10 & E. Flask & 3.9 \\ \hline \hline \end{tabular}  
* \end{table} 
Table 1: Substrates involved in the production of the sophorolipids. Pure product was formed as deposits on the petridish walls, or in the form of crystals or gel at the bottom of the dish.