## 4 Discussion  
Spores of the thermophilic _Bacillus horneckiae_ SBP3 possess a high degree of resistance to wet heat and survive better than those of its closest phylogenetic relative _B. horneckiae_ DSM 23495T (BHO) and of the biodosimetry strain and astrobiological model _B. subtilis_ 168 (BSU) [3]. The presence of spore external integuments (exosporium, cortex, coat and inner membrane) can be significant in the resistance to several stresses. The outer layer of _Bacillus_ spores, the exosporium, consisting of glucose, lipids and proteins, constitutes the first barrier between the spore and the environment [36; 37; 38; 39]. However, its presence differs among the different species; for example, the spores of _B. horneckiae_ possess an exosporium, while those of _B. subtilis_ do not (Figure 1), and different factors have been reported to be involved in the spore's resistance to heat [6]. Depending on what can be expected at elevated temperatures in dry conditions, spore inactivation by wet heat treatment has been previously reported not to occur through DNA damage (e.g., depurination) [40], but rather through denaturation processes that target one or more spore proteins [41; 42]. Minor changes in the spore protein structure have been reported before this event, whereas major protein denaturation was detected only after the release of DPA, when the spores were already dead [4; 40; 42]. In this context, our study combined spectroscopy techniques with multivariate analyses to elucidate the biochemical and structural changes in the spectral bands attributed to the principal biomolecules (carbohydrates, lipids and proteins) occurring in spores of three _Bacillus_ strains. FTIR coupled with the wavelength analysis indicated distinctive spore  
Figure 6: PCA plot of untreated and wet-heat-treated (-T) spores from _Bacillus horneckiae_ SBP3 (SBP3), _Bacillus horneckiae_ DSM 23495T (BHO) and _Bacillus subtilis_ 168 (BSU). structures of the two _B. horneckiae_ strains, confirming that this technique can help to identify spores from bacilli that also belong to the same genetic level, as reported previously [43; 44; 45]. The FTIR spectra revealed that the major differences between the untreated spores from _B. horneckiae_ SBP3 and _B. subtilis_ 168 were related to lipids and the secondary structures of proteins (Figure 5). According to [PERSON] et al. [46], the peak shifts present in the amide I region of both SBP3 and BHO spore spectra indicated that the structural proteins were more aggregated and complex than those of BSU. Furthermore, the evident peaks observed at 1040 cm\({}^{-1}\) in the BHO and BSU spectra suggested that their carbohydrates possess similar crystal structures but are different to those of SBP3 spores. To overcome the intrinsic difficulties of the FTIR technique, such as the noise signals of OH stretching, vibrational Raman spectroscopy was used to provide the specific molecular fingerprints (i.e., the number of peaks, shifts and their shape) of spores that survived wet heat treatment, in comparison to untreated spores. As predicted by the FTIR analysis, the PCA plot (Figure 6) presented a clear distinction among the untreated spores, with SBP3 clustered away from BSU, with the maximum distance in the PC1 axis being related to amide I and \(\alpha\)- and \(\beta\)-pleated sheets of secondary proteins, well separated by the PC2 axis (referring to lipids). In the BSU spore spectrum, the peaks observed at 2850 and 2925 cm\({}^{-1}\) (Figure 5), related to the CH2 stretching vibrations in the region referring to lipids, indicated the presence of a longer acyl chain and/or saturated fatty acids [26]. These data suggested that the inner membranes of BSU spores are richer in saturated fatty acids than those of SBP3 and BHO. When compared with the other two strains, the lipidic composition of the SBP3 strain (Table 3) displays the highest content of iso-C15 fatty acids (63%) and the lowest content of anteiso-C15 (nearly 9%) fatty acids.