Vortex mixer

The chromatographic analysis was performed using an Agilent 1220 Infinity HPLC-UV system (Agilent Technologies, Santa Clara, CA, USA). The system comprised of: (i) the degasser, (ii) the column oven, (iii) the manual injector, and (iv) the UV detector. The OpenLAB software (Agilent Technologies, Santa Clara, CA, USA) and the Method and Run control package were employed to monitor the analysis. The sonication of the samples was performed in a MRC:DC-150-H ultrasonic bath provided by MRC (Essex, UK). A vortex mixer purchased from VELP Scientifica (Usmate Velate, Italy) was used for agitation. Centrifugation was carried out using a 3-16PK centrifuge system supplied by Sigma (Osterode am Harz, Germany). Prior to the injection in the chromatographic system, all samples were filtered through QMax RR 25 mm 0.22 μm PTFE syringe filters purchased from Frisenette ApS (Knebel, Germany).

The extraction method was conducted by adapting the procedures described by De Montijo-Prieto et al. [34 (link)] with slight modifications. For this purpose, previously ground plant material was accurately weighed (0.1 g) and mixed with 5 mL of an 80% methanol/water solution v/v. Extraction was conducted in an ultrasonic bath (Emmi 20 HC, EMAG, Salach, Germany) at 40 °C for 15 min. Every 5 min, the samples were vortexed with a Vortex Mixer (Velp Scientifica, Usmate Velate, Italy) for 15 s. Next, the extracts were centrifuged for 10 min at 9000 rpm. Following this step, the extraction with the solvent was repeated two more times, each time collecting the supernatant. Then, the supernatants were combined and evaporated under reduced pressure.

The chromatographic analysis of the analytes was performed in an Agilent (Santa Clara, CA, USA) 1220 Infinity HPLC-UV, using gradient elution methods. The HPLC system consisted of the following: manual injector, column oven, degasser, and lastly, a UV Detector. In order to monitor the analysis, the Agilent Open Lab software and the package Method and Run Control were used. For data processing, the Data Analysis software package was used to identify and integrate the peaks. A glass vacuum-filtration apparatus, produced by Alltech Associates (Deerfeld, IL, USA), in combination with cellulose nitrate 0.22 μm nylon filters (Whatman Laboratory Division, Maidstone, UK) were utilized for the filtration of the aqueous and organic phase, respectively. QMax RR syringe filters (0.22 μm nylon membrane) were purchased from Frisenette ApS (Knebel, Denmark) and used for filtering the real samples prior to analysis. An ultrasonic bath (MRC: DC-150H) by MRC (Essex, UK) was utilized to remove the template from the MIP as well as for sample preparation. A vortex mixer from VELP Scientifica (Usmate Velate, Italy) was used for the agitation of the samples. A centrifuge system 3–16PK by Sigma (Osterode am Harz, Germany) was operated for centrifugation.

The emulsification activity was carried out using the method used by Uyar and Sağlam [29 (link)].
The emulsifying activity of a biosurfactant is its ability to retain an emulsion of hydrocarbons or oils in water. 5 mL of washed cells and 5 mL of acellular supernatant of each isolate were poured into a test tube containing 5 mL (v/v) of gasoline. The mixture was shaken vigorously for 3 min using a vortex mixer (VELP Scientifica, Italy). The tubes were then incubated at room temperature for 24 h. The height of the emulsion layer and the total height of the mixture were then measured. All the experiments were performed in triplicate, and the emulsification index (E24%) was calculated using the standard formula for E24% (He/Ht) × 100, with He being the emulsion height, Ht being the total height of mixture, and E24% being emulsification percentage after 24 h.

LogD₇.₄ values were determined by slightly modifying a reported procedure [20 (link)]. Namely, an 80 mM solution of each selected EDA analogue in DMSO was diluted in 1-octanol at a final 800 μM concentration. UV–vis spectra were acquired to determine λmax and the necessary dilutions to obtain absorption values for each tested analogue; between 0.5 and 1. 5 mL of each solution were placed in 15 mL conical centrifuge tubes, PBS was added (5 mL, pH 7.4) and the biphasic system was vigorously mixed using a vortex mixer (VELP Scientifica, Usmate Velate, MB, Italy) for 4 min. After centrifugation (2000× g, 30 min), the 1-octanol phase absorption was measured at each compound’ λmax; the procedure was repeated to acquire data in triplicate. The distribution coefficient logD₇.₄ was calculated according to the following equation:
where A_i is the pre-extraction measured absorbance in 1-octanol and A_f is the post-extraction final measured absorbance in 1-octanol.

LogD₇.₄ values were determined by slightly modifying a reported procedure [20 (link)]. Namely, an 80 mM solution of each selected EDA analogue in DMSO was diluted in 1-octanol at a final 800 μM concentration. UV–vis spectra were acquired to determine λmax and the necessary dilutions to obtain absorption values for each tested analogue; between 0.5 and 1. 5 mL of each solution were placed in 15 mL conical centrifuge tubes, PBS was added (5 mL, pH 7.4) and the biphasic system was vigorously mixed using a vortex mixer (VELP Scientifica, Usmate Velate, MB, Italy) for 4 min. After centrifugation (2000× g, 30 min), the 1-octanol phase absorption was measured at each compound’ λmax; the procedure was repeated to acquire data in triplicate. The distribution coefficient logD₇.₄ was calculated according to the following equation:
where A_i is the pre-extraction measured absorbance in 1-octanol and A_f is the post-extraction final measured absorbance in 1-octanol.

The oil-in-water emulsions were prepared by mixing water and oil (diesel, gasoline, sunflower oil, toluene, petroleum ether and hexane) in a volume ratio of 9:1 v/v and rotated by Vortex mixer (VELP Scientifica Srl, Usmate, Italy) at a speed of 3000 rmp for 30 s to obtain a homogeneous milky solution. Note that the prepared oil-in-water emulsions could be stable for at least 60 min under ambient conditions. In order to expand the applications of our membranes in seawater environment, salt was firstly dissolved in DI water (35 g/L) to obtain the saline water. The oil-in-saline water mixtures were then prepared by mixing the saline water and oil (diesel, gasoline, sunflower oil, toluene, petroleum ether and hexane) in a volume ratio of 9:1 v/v with strong shaking.