**1. Introduction**

The dispersion of large amounts of toxic polluting agents to the environment caused by natural or human activities leads to adverse impacts on population and ecosystem health [1]. Conventional remediation techniques such as the use of surfactants have received great attention when counteracting polluting activities [1].

Surfactants are compounds that reduce surface and interfacial tension at the interfaces of liquids, solids and gases in order to create emulsions with liquids [2]. These compounds are highly used in the industry in order to remediate contaminated sites from environmental pollutants such as hydrocarbons [3]. The most popular surfactants are synthetic chemical surfactants [4], which are generally toxic and lack biodegradability, leading to bioaccumulation [5]. Manufacturing these surfactants and their byproducts can adversely impact the environment. Surfactants can be disposed of in rivers or sewage treatment plants, which results in marine ecosystem pollution [6].

In order to replace these compounds, attention is given to compounds that are kinder to the natural environment, such as bioemulsifiers (BEs), biosurfactants (BSs) and exopolysaccharides (EPSs) [7]. EPSs produced by microorganisms are compounds with significant potential in various commercial applications such as the emulsification of various hydrophobic substrates, food, or the pharmaceutical industry [8,9]. They have significant advantages when it comes to biodegradability and effectiveness [10]. Within this category is a group of highly interesting polymers, such as exopolysaccharides from microorganisms

**Citation:** Sánchez-León, E.; Huang-Lin, E.; Amils, R.; Abrusci, C. Production and Characterisation of an Exopolysaccharide by *Bacillus amyloliquefaciens*: Biotechnological Applications. *Polymers* **2023**, *15*, 1550. https://doi.org/10.3390/ polym15061550

Academic Editor: Shashi Kant Bhatia

Received: 3 March 2023 Revised: 17 March 2023 Accepted: 18 March 2023 Published: 21 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

(microbial exopolysaccharides (EPSs)). Their physicochemical characteristics are especially interesting to researchers, such as their high molecular weight [11], the presence of different groups in their composition, and their thermostability and biocompatibility [12]. Another factor to consider is the environment where contamination can occur; many of these environments are characterised by extreme conditions such as elevated or low temperatures, alkaline or acidic pH, high pressure, or high saline concentrations. The bioremediation of these sites is typically difficult [13].

The importance of looking for microorganisms in extreme environments is due to the immense biotechnological potential of their exopolysaccharides (EPSs) [14], since they would be suitable in extreme environments. One of the most versatile genera is *Bacillus,* which is found in different ecological niches [15] and can propagate under adverse conditions [16], rendering the study of its EPSs very interesting [17]. An example is the case of EPSs produced by *Bacillus vallismortis* WF4 [18] and *Bacillus tequilensis* GM [19], which showed significant emulsifying activity in essential oils. The EPS of *Bacillus megaterium* also showed significant emulsifying activity in hydrocarbons [20]. Emulsifying activity was also found for the EPS produced by *Bacillus amyloliquefaciens* [21].

Some EPSs have wide pharmaceutical application. This is the case of *Bacillus thermoantarcticus* [22] and *Bacillus velezensis* [23], which both presented effective antifungal activity, while the EPSs of *Bacillus subtilis* [24] and *Bacillus aerophilus* [25] demonstrated antioxidant activity.

EPSs produced by different strains of the same species have very diverse pharmaceutical, biotechnological, and industrial applications. For example, different strains of *Bacillus licheniformis* have immunomodulatory [26], antiviral [27], and anticytotoxic activity [28].

Another factor to take into account is the culture medium used to stimulate the production of exopolysaccharides. Among the most used synthetic surfactants is Tween 80, composed of polyoxyethylene glycol sorbitan monooleate. This is an important nonionic surfactant, as it is economical and highly efficient [29]. For medical applications, Tween 80 has been recently included in some vaccines, such as the influenza and AstraZeneca COVID-19 vaccines, or as a food additive, and was widely tolerated [30]. While Tween 80 was degraded by bacteria and stimulated the biodegradation process [29], EPSs arising from degrading Tween 80 have not been reported. The hypothesis of this work is that a potential EPS produced by an extremophilic bacterium biodegrading Tween 80 would result in a polymer that could act in a wide pH range.

This study aims to produce a novel EPSRT7 from the biodegradation of glucose–Tween 80 with the *Bacillus amyloliquefaciens* RT7 strain, and to understand its potential applications. The extremophilic *Bacillus amyloliquefaciens* RT7 strain was isolated from an extreme acidic environment and identified through molecular biology methods. Furthermore, the biodegradation of the strain using different independent carbon sources (glucose, oleic acid, Tween 80, and PGE 200) and the joint biodegradation of glucose–Tween 80 were evaluated with an indirect impedance technique and nuclear magnetic resonance (NMR). EPSRT7 was characterised with different analytical techniques (GC–MS, HPLC/MSMS, ATR–FTIR, TGA and DSC) in order to determine the compositional and structural characteristics, and molecular weight of EPSRT7. Lastly, potential applications of the isolated EPSRT7, such as emulsifying activity against different natural oils (olive, sunflower, sesame, and coconut) and hydrocarbons (diesel oil, hexane, toluene), the stability of the emulsion at different pH levels, times, and concentrations, and emulsion efficiency against different commercial emulsifiers (Triton X-100, Tween 20 and SDS) were compared. In addition, in the in vitro antioxidant assays for different free radicals, we studied the cytotoxicity and antioxidant activity of EPSRT7 at the cellular level.

#### **2. Materials and Methods**
