*2.3. Biodegradation, Colony-Forming Units (CFUs)/mL, pH, and EPS Production* 2.3.1. Biodegradation, Colony-Forming Units (CFUs)/mL, and pH

The biodegrading bacterium (strain RT7) was studied via indirect impedance measurements, performed at 30 ◦C. The aerobic biodegradation was prepared as previously described by Abrusci et al. [35,36]. Minimal growth medium (MGM): g/L: K2HPO4 0.5, KH2PO4 0.04, NaCl 0.1, CaCl2 2H2O 0.002, (NH4)2 SO4 0.2, MgSO4 7H2O 0.02, FeSO4 0.001. Each of the carbon sources was added separately to the medium (MGM): glucose (4 g/L), oleic acid (1 g/L) and surfactants (1 g/L) (polysorbate 80 (Tween 80), and polyethylene glycol (PEG) 200). Polyethylene glycol PEG-200 and oleic acid were used as controls or model compounds in the biodegradation studies. In addition, glucose and Tween 80 were added together (pH adjusted to 7.0).

The bioassay measurements were performed as described by Abrusci et al. [36] in 7 mL bioreactors, introducing 1.5 mL of bacterial suspension. These bioreactors were introduced into disposable cylindrical cells of 20 mL filled with 1.5 mL of 2 g/L KOH aqueous solution, and impedance was measured using four stainless-steel electrodes on a Bac-Trac 4300 apparatus (SY-LAB Geräte GmbH, Neupurkerdorf, Austria). The typical measurement error was 1–2%. The relative change in the KOH solution impedance value was monitored with the device every 20 min and was converted into carbon dioxide concentration by using a calibration curve of variation in impedance against CO2 concentration.

The biodegradation percentage of different carbon sources was computed on the basis of the ratio between the cumulative amount of CO2 actually generated by biodegradation at time t and the theoretical carbon dioxide amount, which assumed that all the carbon in the glucose and polysorbate structures was converted into CO2 (Formula (1)).

$$\% \text{ Biodegradation} = ([\text{CO}\_2] \text{Prod} / [\text{CO}\_2] \text{Theor.}) \times 100\tag{1}$$

To continue with the experiments, the most effective carbon source was chosen. The colony-forming units (CFUs) were evaluated via dilution plating incubated at 30 ◦C for 72 h with a TSA agar medium. A Thermo Orion pH Meter Model 2Star (Thermo Scientific, Asheville, NC, USA) was used to measure the pH values during a 72 h fermentation period.

To evaluate structural changes on Tween 80 after biodegradation, nuclear magnetic resonance (1H-NMR) was recorded in a deuterated chloroform CDCl3 solution on a Varian INOVA-400 instrument (Varian Inc., Palo Alto, CA, USA) at 400 MHz. In the case of the biodegraded products, the compound mixture was filtered to eliminate cells using a centrifuge (0.22 μm Millipore, Merck, Darmstadt, Germany, DEU). The residues were dried and dissolved in CDCl3.

#### 2.3.2. EPS Production and Purification

The strain was inoculated on trypticase soy agar (TSA) medium and incubated for 24 h at 30 ◦C. The strain was later transferred to an MGM medium in 100 mL flasks filled with 20 mL with an initial inoculum of 2.5 × 107 CFU/mL (OD550 nm) measured with a spectrophotometer (Biowave II). The flasks were incubated at 30 ◦C for 24 h at 110 rpm (Orbitek LJEIL model) [27]. Subsequently, 10 mL of the broth was transferred to flasks containing 1000 mL of MGM with glucose–Tween 80. The flasks were incubated at 30 ◦C for 72 h at 110 rpm. The tests were independently repeated three times.

The cultures were centrifuged with a DuPont Sorvall RC-5 centrifuge for 30 min at 4 ◦C at 13.154× *g*. The extracted supernatant was precipitated with three times the volume of ethanol (−80 ◦C). The EPS was then dialysed for 48 h at 4 ◦C with Milli-Q water, and lyophilised with a Flexy-Dry MPTM freeze 150 dryer. Subsequently, the dry weight of the EPS was measured.

The purity of the EPS (10 mL, 10 mg/mL) was evaluated with a DEAE-52 anion exchange column (2.6 × 30 cm), and deionised water was used for elution. For this, the used eluents were different concentrations of NaCl (0.2–1.5 M) at a flow rate of 1 mL/min. The phenol–sulfuric acid method was used to monitor the eluents [37]. The collected fractions were lyophilised, resulting in an EPS that was named EPSRT7.
