## 2 Results and Discussion
### VOCs Profile Differentiation in Four Growth Media in the Exponential and Stationary Phase
This has been previously observed in _Trichoderma spp._, where the volatiles profile found by combining three different amino acids in the growth medium was different from the sum of the profiles obtained when each amino acid was supplied individually [42]. The variable importance on projection (VIP) was used to assess the significance of the variables in the differentiation between treatments. Usually, values higher than 1 are considered significant. Twenty-seven VOCs from _M. furfur_ followed this requirement (Figure 5b). Compounds found exclusively in one treatment or medium show the higher VIP values. For example, the hept-3-ene, which was found in the OPE treatment with a value of 1.58, is the compound that better differentiates the four media and the two growth phases, followed by isomer3 of undec-1-ene (VIP = 1.49), a signature compound from _M. furfur_ growing in OPs. Among these 27 compounds, 12 were also signalled as statistically significant using UVA (Table S1 and Figure 2), findings that validate the interpretation of these two types of approximations. Nevertheless, the VIP values from PLS-DA offer more information because they discriminate the experimental treatments by organizing the VOCs in order of importance. On the other hand, this is not possible for the UVA, where each VOC is analyzed separately and not correlated to other compounds of the profile. Nine of the 27 VIP compounds had the higher loadings in the PCA analysis and were also highlighted as relevant in the UVA (carbon dioxide, hexan-1-ol, pentyl acetate, the isomer5 of methyldecane, dimethyl sulphide, undec-5-ene, isomer2 of methylundecane, isomer1 of methyldecane, and 2-methyltetrahydro), whereas the PCA and PLS-DA analysis shared 10 out of these 27 VIP compounds. In addition to the abovementioned nine compounds, 2-methylpropan-1-ol was an important differentiation factor in both analyses. These findings evidence the differences of the multivariate models built under supervised and unsupervised conditions and the limitations of UVA. While the construction of the PCA model does not maximize distances between treatments, the PLS-DA constructs the multivariate components as a function of this response variable (in this case, the medium and phase of growth) [68]. This could explain why the VOCs with the higher VIP (for example, hept-3-ene), found exclusively in OPE, were not recognized as significant in the UVA or the PCA. The ecological meaning of the differentiation produced by the absence of PA and OA on the volatiles profiles of _M. furfur_ need to be further explored. PA is particularly abundant in human sebum, in comparison to other animals [21], so spatial differences in the PA distribution on skin could lead to spatial changes in the VOCs released by _M. furfur_ or other species from human microbiota [10]. Therefore, a homeostasis needs to be established between human skin and microbiota through the production of metabolites. An enzyme of _Malassezia_ that is able to attenuate the formation of biofilms of _Staphylococcus aureus_, an opportunistic bacterium from human skin microbiota [12], is already known; however, volatile metabolites released by these micro-organisms could have similar effects. VOCs functioning as quorum-sensing molecules [36; 44] or antibiotics [58; 69] have been reported in other bacteria and fungi; however, the ecological function of the VOCs released by _M. furfur_ should be studied in the future. These studies show how any alteration in the human skin could be responsible for a disequilibrium in the homeostasis of the total microbiota and may be related to the interconversion of commensal to pathogenic states in _M. furfur_, as well as in other opportunistic species. Figure 5: **(a)** Projection to Latent Structures Discriminant Analysis (PLS-DA) of volatiles profiles of _M. furfur_ growing in eight different experimental treatments. The ellipses in this plot are confidence intervals of 95% using a _t_-distribution. (**b**) Variable importance on projection (VIP) plot showing the most important VOCs from _M.