2.8.2. Evaluation of Protective EPSRT7 Effect on HeLa Cells from Oxidative Stress

EPSRT7 s ability to protect HeLa cells against oxidative stress was evaluated following the methods described by Huang-Lin et al. [39]. HeLa cells were seeded at a concentration of <sup>5</sup> × <sup>10</sup><sup>4</sup> cell/well and incubated for 24 h. After that, the DMEM solutions were withdrawn and substituted with EPSRT7 diluted in DMEM at different concentrations (25–400 μg/mL). After 1 h, the EPSRT7 solutions were removed, and a new medium containing 2 mM of H2O2 was added and incubated for 1 h more. The MTT method described in Section 2.7.2 was used to determine cell viability. As a positive control, ascorbic acid (20 mg/mL) was used.

HeLa cell viability was calculated with Formula (8):

$$\text{Cell viability [\%]} = (\text{A}\_1/\text{A}\_2) \times 100\tag{8}$$

where A1 refers to cells that were treated with both H2O2 and EPSRT7, and subsequently exposed to the MTT solution; A2 refers to cells that did not receive any treatment and were exposed to the MTT solution.

#### *2.9. Statistical Analysis*

The experiments were conducted three times, and statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS), version 21. The analysis of variance (ANOVA) test was used for statistical comparison, and statistical significance was at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Bacterial Identification, Biodegradation, and EPS Production*

The bacterial strain was isolated as described in Section 2.2, and identified after PCR amplification and sequencing using the 16S rDNA sequence. The 16S rDNA sequences were compared with those in the GenBank database and showed that the isolated strain was *B. amyloliquefaciens* RT7 (accession number, AB300821) with a similarity of 98%.

*Bacillus amyloliquefaciens* RT7 is a Gram-positive, endospore-forming bacterium isolated from the sediments of Rio Tinto (Huelva, Spain), which is one of the most acidic rock drainage fluvial–estuarine systems in the world [48]. This species is ubiquitous and adapts to very different ecological environments. This gives it great versatility in the biodegradation of different compounds, such as waste from the petrochemical industry, where strain *B. amyloliquefaciens* W1 [49] was able to degrade benzene, toluene, ethylbenzene, and xylene (BTEX), also in phenol-contaminated wastewater, which *B. amyloliquefaciens* WJDB-1 was able to biodegrade [50]. In addition, it was successfully used in various technological applications. For example, the *B. amyloliquefaciens* BRRI53 strain could stimulate plant growth [51], and the *B. amyloliquefaciens* BPRGS strain had high flocculant activity [52].

The biodegradation of the different independent carbon sources (glucose, oleic acid, Tween 80, and PEG 200) and the joint biodegradation of glucose–Tween 80 are shown in Figure 1a. The results indicate that the *B. amyloliquefaciens* RT7 strain biodegraded glucose by 27–78%, oleic acid by 27–60%, Tween 80 by 23–58%, PEG-200 by 5–2%, and the combination of glucose–Tween 80 by 46–86% at intervals of 24 and 72 h. The *B. amyloliquefaciens* RT7 strain was the most effective at biodegrading glucose–Tween 80.

The growth of *B. amyloliquefaciens* RT7, pH values, medium biodegradation, and exopolymer production (EPS) at 30 ◦C using the combination of glucose–Tween 80 as a carbon source is shown in Figure 1b. Cell growth peaked (7.53 log CFU/mL) after 30 h. During the process, the medium was not acutely acidified (from pH 7 to pH 6.5).

**Figure 1.** (**a**) Biodegradation study of different carbon sources (glucose, oleic acid, Tween 80, PEG 200 and glucose–Tween 80) from *Bacillus amyloliquefaciens*. (**b**) Optimisation of the production EPS from *B. amyloliquefaciens*. (**c**) Elution curve obtained from the purification of EPSRT7 under MGM with glucose–Tween 80.

To confirm that Tween 80 had undergone structural changes during biodegradation, after the bioassays, the residues were analysed with proton nuclear magnetic resonance (1H-NMR; Figure 2a). The biodegradation of oleic segments decreased the intensity of the peaks of the aliphatic protons (2.5–1.0 ppm), and caused the disappearance of the double-bond signal at 5.35 ppm and the methylene protons next to the oleic ester group at 4.23 ppm. We also found a simplification of the proton signals corresponding to the PEG fragments (HPEG–3.64, 3.77, and 3.99–4.16 ppm), indicating a decrease in the length of the initial PEG fragments after 72 h of the bioassay. This shows that the biodegradation of Tween 80 [53] was effective and likely had a positive effect on the biodegradation of glucose. Similar results were obtained with *Bacillus amyloliquefaciens* [54] when Tween 80 was combined with hydrocarbons, favouring the biodegradation of the latter. This was also confirmed in other species of the *Bacillus* genus, such as *B. subtilis* ZL09-26, where the biodegradation of phenanthrene was more efficient in the presence of Tween 80 [55]. This could have been due to the fact that Tween 80 could influence the permeability of the membrane, improving the expression of the proteins [56] and favouring the more efficient incorporation of organic nutrients such as glucose [57,58].

On the other hand, the biodegradation of this combination (glucose–Tween 80; Figure 1b) with *Bacillus amyloliquefaciens* RT7 resulted in a high production of EPS. The maximal production of the exopolysaccharide, namely, 490 mg/L, occurred at 24 h, during the exponential growth phase. However, this was previously observed as usually taking place at the beginning of the stationary phase [27,39].

The EPS production of the RT7 strain was higher than that for other strains of *B. amyloliquefaciens*. *B. amyloliquefaciens* p16 used a nutrient broth of glucose, peptone, and yeast as an energy source, and only had an EPS production of 223.87 mg/L [59]. The use of Tween 80 by *Bacillus amyloliquefaciens* RT7 as a carbon source not only allowed for greater efficiency in the use of glucose, but also effectively accelerated the synthesis of the exopolysaccharide produced by the RT7 strain. Similar processes were described in the synthesis of natural compounds, such as fengycin, accelerating their production when Tween 80 was used [60]. In addition to this, similar results were found in other genera, such as *Lactobacillus plantarum*, where Tween 80 not only facilitated the entry of nutrients, but also stimulated greater EPS production [61].

The EPS obtained from glucose and Tween 80 as an energy source was purified and showed a single characteristic peak of exopolysaccharides with high purity (Figure 2b). The result of the obtained fraction from the purified exopolymer was named EPSRT7. The estimated molecular weight of EPSRT7 was about 7.0794 × <sup>10</sup><sup>4</sup> Da (Figure 2c), which fell within the typical molecular weight range for heteropolysaccharides (4 × <sup>10</sup><sup>4</sup> and <sup>6</sup> × 106 Da) [62]. EPSRT7 presented a high molecular weight in comparison with that of other EPS produced without the presence of Tween 80, such as strains of *B. amyloliquefaciens* GSBa-1, with an EPS composed of glucose with a molecular weight of 5.4 × <sup>10</sup><sup>4</sup> Da [63], and *B. amyloliquefaciens* 3MS [64], whose EPS comprised glucose, galactose, and glucuronic acid with a molecular mass of 3.76 × <sup>10</sup><sup>4</sup> Da.
