Based on 16S rRNA phylogenetic studies of Exiguobacterium genus, it has shown that both E. acetylicum and E. profundum display three branch points from the common ancestor with E. aurantiacum. Additionally, E. profundum and E. aurantiacum show privileged homology since they are clustered in a joint node, despite showing higher speciation [3]. At the genome level, the draft sequence of E. aurantiacum, E. profundum [31] and the sequence/assembly of E. acetylicum are available in the databases.
The distinctive feature belonging to this genus is their ability to grow under extreme environmental conditions, including a wide temperature range (-12-55 °C) besides nutrient limitation situations [32, 33]. The robustness and tolerance against harsh conditions showed by Exiguobacterium members turn them in suitable candidates for industrial applications, useful in bioremediation and bioabsorption processes of metals and metalloids [34–37].
Exiguobacterium strains used in this research were previously isolated from different regions in Chile that display a combination of extreme environments such as high salinity, desiccation, high and low temperatures, and volcanic interventions [48]. Most of these factors are responsible for generating oxidative stress to microorganisms, a situation that also occurs often upon bacterial exposure to metal(loid)s. This is corroborated, because in Chile it has been identified and characterized strains of Exiguobacterium obtained from the Salar del Huasco which have arsenic resistance [1, 2].
In this work, resistance for the three elements was higher in the absence of oxygen, a result that was expected because of the absence of ROS, which otherwise would generate oxidative stress [29, 14].
To determine optimal parameters, reduction assays of TeO32-, AuCl4- and Ag+ by crude extracts were carried out at pH values 7.0–9.0, in the presence of NADH or NADPH as the electron donor. Particularly, this study worked in the optimal temperature conditions (37° C), however, it would be interesting to study the minimum and maximum ranges that the system supports, since Exiguobacterium is a polyextremophilic microorganism. Crude extract-mediated reducing activity was higher at pH 9.0 for most tested metal(loid)s, irrespective of the presence of oxygen (Fig. 1). This could reflect the fact that most proteins displaying metal(loid)-reducing activity contain catalytic sites including vicinal cysteine residues that play an important role in the reduction process [38]. Therefore, at basic pH, deprotonation of thiol groups from these cysteines would occur, giving rise to the highly reactive thiolate anion (-S-) [39]. Another possible explanation is that enzymes exhibiting metal(loid)-reducing activity are tolerant to alkali, as has been described for most enzymes of biotechnological interest isolated from Exiguobacterium strains [3]. In turn, E. aurantiacum MF06 crude extracts showed Au(III)-reducing activity at pH 7.0 (Fig. 1B), a situation that may occur because pH can influence metal(loid) speciation. This could result in the formation of complexes and/or deprotonation or protonation of functional groups in amino acids that participate in enzyme-substrate stabilization [40] (Panda and Deepa, 2011). The preference for NADH or NADPH as electron donor could reflect its stabilization at the enzyme´s active site [41].
For Ag+- and TeO32--reducing activities, these were higher under anaerobic conditions, probably due to the limitation of ROS formation in this condition [29, 14]. To date, gold toxicity has not been associated to oxidative stress; the lack of significant differences in AuCl4- reduction by crude extracts from aerobically- or anaerobically grown cells supports this observation.
Assays that were carried out with crude extracts from cells that were previously exposed with sublethal doses of toxicants showed -in general- higher reducing activities than those coming from untreated cells both under aerobic and anaerobic conditions with the exception of Au(III) reduction. Since bacterial Ag and Te resistance is associated to enzymatic reduction [14], the observed results could reflect the expression of genes related to bacterial Ag(I) and/or Te(IV) resistance.
In addition, crude extracts of this genus have been previously used for nanoparticle synthesis. For instance, E. mexicanum extracts were able to synthesize silver nanoparticles of 5–40 nm, a process in which extracellular polymeric substances played a critical role both in silver reduction and nanoparticle stabilization [12]. Because of this, we used Exiguobacterium strains as an ecofriendly way to get NS.
NS synthesis by bacterial crude extracts or purified enzymes has not been widely reported. Indeed, most synthetic procedures are chemical in nature, in which mechanisms of NS formation involve two stages: nucleation and growth, processes that are affected by several factors including thermal energy, metal concentration and reaction rate, among others [42].
AgNS synthesized in aerobic conditions using crude extracts of E. acetylicum MF03 and E. profundum MF08 exhibited larger sizes than their anaerobic counterparts. However, the highest silver-reducing activity was observed precisely in the absence of oxygen. Given that, NS size could be affected by the activity of the enzyme, the following tests considered protein concentration as a critical parameter. Indeed, it was previously observed that during enzymatic synthesis of tellurium-containing NS, particle size was inversely related to enzyme concentration [26]. In addition, NS yield was higher under anaerobic conditions. These results could be explained by a higher metal(loid)-reducing as result of the absence of oxygen that could prevent electron leakage [43].
On the other hand, AuNS generated by E. acetylicum MF03 and E. aurantiacum MF06 did not show significant fluctuations in size or yield both in aerobiosis and anaerobiosis. However, what is relevant about these results is that when working with two different species of Exiguobacterium it is possible to obtain AuNS with different gold content, which from a biotechnological point of view is attractive, for example in the field of medicine.
Finally, aerobically- and anaerobically generated TeNS by E. acetylicum MF03 displayed similar sizes along with elongated morphologies; the exception was anaerobically-synthesized TeNS, which showed dense electron spots. This kind of elongated morphology of tellurium-containing NS was previously reported for Rhodobacter capsulatus [44]. In turn, TeNS generated by E. profundum MF08 (Fig. 5C-D) were much larger in aerobic conditions, which does not correlate with the reducing activity of crude extracts. Similarly, to what was proposed for AgNS, TeNs could be adopting different nucleation/growth processes that could explain this observation [42].
In general, the composition analysis of in vitro synthesized NS included the metal(loid) itself along with other, apparently unrelated elements. These include mainly carbon, oxygen and sulfur, probably indicating the organic origin of NS formation. Consistent with this, previous studies have shown that AuNS can be found in association with the enzyme glutathione reductase [45].
Biological processes for NS synthesis remain a challenge, not solely as a synthetic platform but in green purification techniques for subsequent characterizations. More efforts should be made to expand the characterization techniques applicable to these methods such as XRD, DLS with potential Z, FTIR, among others. When irregular NS with variable size and undefined organic layers are obtained, the results of these analyzes generate errors so they might be not reliable. In our case, XRD analyzes were not possible to perform because the surface of the nanostructures was not clean enough due to the biological processes that were used for the synthesis, this can be seen in the EDS analyzes in which many elements, of organic origin, they are identified, so the background is abundant. However, during TEM observation and navigation, SAED and FFT (Fourier transform) electron diffraction were explored, revealing the polycrystalline character of the samples without identifying preferential growth axes or phenomena of crystallographic and significant interest to report.
All these results allow us to demonstrate the great applicability of the Exiguobacterium genus in processes of resistance, reduction and generation of NS of metals and metalloids, which could be applied to help in developing effective co-cultures to improve the metal(loid) polluted sites like those described in Batool et al. [46]. Moreover, developing new bacterial-assisted techniques for reduced metal(loid) uptake of vegetables in the metal(loid)-contaminated soils [47].