3.2.3. Characterisation of the Thermal Properties of EPSRT7

The thermal stability of EPSs is an important characteristic for their commercial utilisation. The thermal decomposition curve (TGA) of EPSRT7 is shown in Figure 3c. The first step, with initial weight loss of 10.20%, was observed at around 25 and 163 ◦C, mainly due to the moisture loss in EPSRT7. The second step had a weight loss of approximately 55.9% which reached the maximum at around 420 ◦C. The depolymerisation of the polysaccharide and the thermal chemical-bond scission occurred, accompanied by the dehydration of sugar units. Lastly, the EPS presented a gradual weight loss of approximately 12.09%, reaching equilibrium with only 28% of the remaining residue. Similar results associated with thermal decomposition were found in other strains of *B. amyloliquefaciens*. This was the case of the *B. amyloliquefaciens* GSBa-1 strain [63], whose EPS was formed by glucose. In the first step, 7.44% (50 to 160 ◦C) weight loss for water was observed. In the second step, it was 43% (around 400 ◦C) of mass loss. In *B. amyloliquefaciens* BPRGS [52], the first step showed 10% weight loss for water (0 to 180 ◦C), and the second step showed 20.21% weight loss (250 to 550 ◦C). On the other hand, the DSC thermogram of EPSRT7 (Figure 3d) showed two melting peaks at 195.8 and 346.5 ◦C that conferred higher thermostability. The appearance of two melting peaks was directly related to the heterogeneity in the composition of EPSRT7 sugars. This was in contrast with the case of *B. amyloliquefaciens* LPL061 [21]. This strain produced two distinct EPSs, both composed of mannose and glucose (EPS1 and EPS2). The two EPSs had a single melting peak of 224.09 ◦C (EPS1) and 301.09 ◦C (EPS2). This suggests that compositions with more distinct sugars may result in more than one melting points. Therefore, EPSRT7 with various sugars presented an important advantage over polymers with fewer sugars, since its thermostability increased, which is an important factor in various industrial applications [21,76].

#### *3.3. Biotechnological Applications*

#### 3.3.1. Emulsifying Activity

The emulsification behaviour of EPSRT7 was investigated at different concentrations (0.5, 1, and 2 mg/mL), pH levels (7.2, 5.1, 3.1), and at different times (24 h (E24), 48 h (E48), and 168 h (E168)), with natural oils (olive, sunflower, sesame, and coconut) and hydrocarbons (diesel oil, hexane, toluene) (Figures 4 and 5). EPSRT7 was also tested with three commercial emulsifiers (Triton X-100, Tween 20, and SDS).

For natural oils, concentration has a statistically significant effect on emulsifying activity. Concentrations of 0.5 mg/mL resulted in nonsignificant emulsification activity across the board, with the only exception being sesame oil at pH 3.1 ((E24 86.6%), (E48 73.3%)). A concentration of 1 mg/mL generally resulted in emulsifying activity with some exceptions, but the optimal concentration was 2 mg/mL for all oils and pH and time combinations. pH had different effects on emulsifying activity depending on the oil. For olive oil (2 mg/mL), the exopolysaccharide presented significant emulsifying activity at all pH levels and studied times, with pH 3.1 being where the emulsifying activity reached its maximum (E24 (85.7%), E48 (71.4%), E168 (57.1%)). For sunflower oil (2 mg/mL), there was significant emulsifying activity for all pH levels; however, at pH 5.1, emulsification was not maintained at E168 ((E24 (85%), E48 (72%), E168 (Nd)). Sunflower oil had its highest emulsifying activity at pH 3.1 (E24 (87.5%), E48 (86.6%), E168 (62.5%)).

The EPS was initially most effective in emulsifying sesame oil at pH 3.1, although this was not maintained at 168 h (E24 (100%), E48 (82.4%), E168 (46.6%)). However, at pH 7.2, emulsification activity was maintained (E24 (86.6%), E48 (73.3%), E168 (60%)), and it was not successful at a pH 5.1. For coconut oil, emulsifying activity was similar at different pH levels for E24, but for E48, pH 5.1 was most effective, followed by pH 3.1; for E168, only pH 3.1 still presented some emulsification. Similar results were obtained with the EPS of the *B. amyloliquefacens* ZWJ strain [77], where the optimal concentration was 1.5 mg/L for two natural oils (olive oil (96.2%), sunflower oil (76%)). In the case of *B. amyloliquefaciens* LPL061 [21], the emulsifying activity was lower than that presented by EPSRT7. Its EPS was only tested at a concentration of 1 mg/mL. The EPS of this strain showed emulsifying

activity with the natural oils, which did not exceed 66% (olive oil (58.6%), sunflower oil (65.8%), peanut oil (60.3%), rice oil (58.5%)).

**Figure 4.** Emulsifying activity for natural oils with different EPSRT7 concentrations (0.5, 1, 2 mg/mL) and pH levels (7.2, 5.1, 3.1). Emulsion percentages of EPSRT7 with different oils used at 24, 48, and 168 h of study are also shown. \*, statistical differences between oils for each concentration (*p* < 0.05). #, statistical differences between concentrations (*p* < 0.05).

Similarly, for hydrocarbons, a concentration of 2 mg/mL was significantly the most effective for emulsification. Diesel, (pH 7.2 (E24 (57.1%), E48 (53.3%), E168 (53.3%)) and pH 3.1, (E24 (56.6%), E48 (56.6%), E168 (57.1%)), and toluene (pH 3.1 (E24 (53.8%) E48 (50%)) presented the highest emulsification activity, which was maintained across different pH levels. Hexane only presented emulsification activity of over 50 for 2 mg/mL and pH 5.1, measured at E24. There are few studies that found EPSs produced by other strains of *B. amyloliquefaciens* with the ability to emulsify hydrocarbons. The An6 strain of *B. amyloliquefaciens* [78] produced a biosurfactant that had emulsifying activity of 80% with diesel at a pH range of 5.0–9.0, but this biosurfactant was not confirmed as an EPS, as its chemical composition was inconclusive. This ability has previously been found in other genera. *Bacillus subtilis* AF17 [79] produced an EPS that was capable of emulsifying diesel by 17%, hexane by 72%, and toluene by 84% (5 mg/mL). The stabilisation of the EPS emulsions was specific for certain hydrophobic compounds [80,81].

The EPS was compared to commercial emulsifiers (Triton X-100, Tween 20 and SDS) for both natural oils and hydrocarbons, and had similar emulsifying activity to theirs at pH 7.2 (Figure 6). At a concentration of 0.5 mg/mL, both EPSRT7 and commercial emulsifiers were not effective (below 50%) except for Tween 20 for olive oil (E24 87.3%), sunflower oil (E24 50.0%), and coconut oil (E24 55.8%). For the concentration of 1 mg/mL, EPSRT7 presented significantly higher emulsification than that of SDS, except for coconut oil (E24 47.62%) and hexane (E24 52.38%), higher than that of Triton X-100 except for olive oil (E24 86%) and coconut oil (E24 54.5%), and higher than that of Tween 20 except for olive oil (E24 95%), sunflower oil (E24 90%), and coconut oil (E24 60%). For the concentration of 2 mg/mL, EPSRT7 presented significantly higher emulsification than that of SDS except for diesel (E24 59.38%) and hexane (E24 57.89%), higherthan that of Triton X-100 for all natural oils and hydrocarbons, and higher than that of Tween 20 except for olive oil (E24 90%) and coconut oil (E24 66.7%).

**Figure 5.** Emulsifying activity for hydrocarbons with different EPSRT7 concentrations (0.5, 1, 2 mg/mL) and pH levels (7.2, 5.1, 3.1), measured at 24, 48, and 168 h of study. \*, statistical differences between different hydrocarbons for each concentration (*p* < 0.05). #, statistical differences between different concentrations (*p* < 0.05).

**Figure 6.** Comparison of emulsifying activity at different EPSRT7 concentrations (0.5, 1, 2 mg/mL) against commercial emulsifiers (Triton X-100, Tween 20 and SDS) across different natural oils and hydrocarbons. Different letters (a–d) represent the statistical difference between different emulsifiers for each natural oils and hydrocarbons (*p* < 0.05).

The molecular compositions, molecular weight, and functional groups of EPSs have important effects on emulsification. EPSRT7 had good emulsifying activity at high concentrations and the studied pH range, which may be attributed to electrostatic interaction and interactions between hydrophilic groups [82]. A low pH, such as 3.1, has a negative effect on the emulsification activity of commercial polysaccharides such as xanthan and Arabic gum [43]. However, in this case, EPSRT7 emulsifying activity was not affected at pH 3.1. The capability of EPSRT7 to emulsify at different pH levels and thus its ability to bioremediate different environments, and its nontoxicity give it great advantages over other EPSs and commercial emulsifiers.

### 3.3.2. Antioxidant Effect

The EPSRT7 obtained from the biodegradation of the combination of glucose and Tween 80 with *B. amyloquefaciens* RT7 was tested in order to study its potential and benefits, particularly antioxidant and emulsifying activities. To quantify antioxidant activity, the method described in Section 2.6 was used.

DPPH evaluates the radical scavenging activity of nonenzymatic antioxidants. As shown in Figure 7a, the scavenging activity of EPSRT7 on DPPH radicals did not increase in a concentration-dependent manner. EPSRT7 presented its highest scavenging activity of 67% at 7.5 mg/mL, while for the same concentration, the scavenging capability of Vc was 82%. This was close to the one previously presented by the *B. amyloliquefaciens* GSBa-1 strain [63], where the EPS had a DPPH scavenging activity of 76.7% (5 mg/mL/Vc 90%). The potential of EPSRT7 for DPPH inhibition suggests that it had enough proton donors to convert free radicals into stable molecules [83].

The hydroxyl radical is one of the most reactive free radicals in a biological system [84]. This is a type of free radical with the most active chemical properties and that can cause the most harm in comparison to other free radicals, as it damages DNA base sequences [85]. The scavenging activity of the hydroxyl radical is commonly used to evaluate the ability of compounds to scavenge free radicals. Figure 7b shows the results for hydroxyl radical scavenging activity of the EPSRT7. Scavenging activity increased in proportion to the concentration. At a concentration of 5 mg/mL, the scavenging capability of EPSRT7 was 90% and remained constant, whereas the activity of Vc was 98% at the same concentration. This efficacy is similar to that previously obtained by the *B. amyloliquefaciens* GSBa-1 strain: 89.7% (5 mg/mL/Vc 90%) [63]. However, the EPSs obtained from *B. amyloliquefaciens* C-1 [66] were much less efficient than EPSRT7, since they presented hydroxyl radical activity of 60.4% for EPS-1, and less than 10% for EPS-2 at 5 mg/mL (Vc 100%). The high efficiency of EPSRT7 could have been due to the bond dissociation energy of EPSRT7 being relatively weak; therefore, it was easy to provide more electron atoms to bind to the hydroxyl radical [86].

Superoxide radicals can be harmful to cells, and their anions can increment damage to cellular components, as they generate oxidising agents and other free radicals. Figure 7c shows the superoxide anion scavenging activity of EPSRT7. At the concentration of 0.25 mg/mL, the scavenging capability of EPSRT7 was very high, 96.5% (Vc 100%). The superoxidant anion scavenging of EPSRT7 was higher than the previously studied EPS of *B. amyloliquefaciens* GSBa-1 [63] of with 44.8% (5 mg/mL/Vc 99.1%), and both EPS1 and EPS2 produced by the *B. amyloliquefaciens* C1 strain (EPS1: 30.8% (5 mg/mL/Vc 99.1%), EPS2: 8.5% (5 mg/mL/Vc 99.1%)) [66]. The superoxide anion scavenging mechanism was associated with O–H bond dissociation energy [75].

**Figure 7.** Antioxidant tests and toxicity evaluation with different concentrations of EPSRT7. (**a**) DPPH free radical scavenging activity. (**b**) Hydroxyl radical scavenging activity of EPSRT7 (**c**) Superoxide anion scavenging activity. (**d**) Hela cells viability (%) by different EPSRT7 concentrations. (**e**) Hela cells viability (%) against oxidative stress by different H2O2 concentrations. (**f**) Exhibition of EPSRT7 protection on Hela cells viability (%). (\* *p* < 0.05).

3.3.3. Toxicity Evaluation and Antioxidant Ability at the Cellular Level

Figure 7 shows the biocompatibility and antioxidant capacity of EPSRT7 at the cellular level. For all tested polymer concentrations (Figure 7d), HeLa cells did not show statistically significant damage (*p* < 0.05). This implies that EPSRT7 did not cause cytotoxicity in the cell line. Similar results were reported for *B. amyloliquefaciens* amy-1, where cytotoxicity assays revealed that EPSs at 50–800 μg/mL were not toxic to the human enteroendocrine cell line, NCI-H716 cells [87]. On the other hand, other biocompatibility studies for the same species revealed that, for concentrations in the range of 200–800 μg/mL of EPS, HEK293T cells moderately inhibited their growth [88].

Figure 7e shows the cell viability of HeLa cells after being treated to different concentrations of H2O2. Lower cell viability of below 70% was detected due to oxidative stress in the cells resulting from a build-up of reactive oxygen species (ROSs). The antioxidant effect of EPSRT7 on the cell line (Figure 7f) was tested at different concentrations. A statistically significant increase in cell viability was observed in concentrations between 25 and 100 μg/mL. The results demonstrate that EPSRT7 concentrations in the range of 25–100 μg/mL statistically significantly improved cellular viability. These results indicate that EPSRT7 significantly protected HeLa cells from H2O2-induced cytotoxicity. Cells incubated with 25, 50, and 100 μg/mL EPSRT7 grew better (90.2, 89.4, 86.9%, respectively) than cells of the control group did, indicating that EPSRT7 had a growth-promoting effect on normal cells. Similar results were reported with the pretreatment of EPS isolated from *Bacillus amyloliquefaciens* significantly and time-dependently decreasing the ROSs induced by H2O2 for H2O2-treated HepG2 cells [66]. These results suggest that EPSRT7 from *Bacillus amyloliquefaciens* could promote the antioxidant system by stimulating enzymes to have this capacity [89].

#### **4. Conclusions**

*Bacillus amyloliquefaciens* RT7 was isolated from the sediments of Rio Tinto. Biodegradation was effective for the different independent carbon sources (glucose, oleic acid, Tween 80, and PEG 200) and the joint biodegradation of glucose–Tween 80. The latter was the most effective, where high EPS production occurred—490 mg/L at 24 h. Polymer characterisation identified the extracted EPS as a heteropolysaccharide composed of mannose, glucose, galactose, and xylose (molar ratio 1:0.5:0.1:0.1). O–H, C=O, and C–O groups were detected within EPSRT7 using structural analysis. EPSRT7 had an approximate molecular weight of 7.0794 × 104 Da with good thermostability. The EPS also showed emulsifying activity against some natural oils (olive, sunflower, sesame, and coconut) and hydrocarbons (diesel oil, hexane, toluene) when used at a 2 mg/mL concentration and the studied pH range, thus demonstrating its ability to bioremediate different environments. EPSRT7 demonstrated its potential as an antioxidant during in vitro antioxidant assays, as it showed robust radical scavenging activity. It was also nontoxic and showed cellular biocompatibility while providing protection to cells damaged by ROSs. The strong emulsifying activity coupled with its antioxidant effect and lack of cytotoxicity suggest it could have promising applications in bioremediation processes, and offers great advantages over other EPSs and commercial emulsifiers.

**Author Contributions:** E.S.-L.: formal analysis, investigation, writing—original draft; E.H.-L., resources, writing—original draft; R.A.: funding acquisition, project administration, manuscript review; C.A.: conceptualisation, formal analysis, investigation, data curation, funding acquisition, methodology, validation, resources, supervision, writing—original draft, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Spanish Ministry of Science and Innovation for financial support (project PID2019-104812GB-I00) and FUAM, Universidad Autónoma de Madrid, Spain (project no. 820053).

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Data sharing is not available for this article.

**Conflicts of Interest:** The authors declare no conflict of interest.
