# FTIR Spectroscopy for Identification and Intra-Species Characterization of _Serpula Iacrymans_
## 4 Discussion
A mycelium can be analysed after liquid culture and harvested by centrifugation, filtration, or freeze-drying, or obtained after growth on the surface of an agar medium or a membrane deposited on an agar medium and harvested from its surface [18; 19; 23; 26; 32]. The advantage of liquid culture is that it prevents the sporulation of many fungi [18; 33]. In the present study, three conditions for obtaining mycelium were compared: growth in broth and harvesting by centrifugation, growth on a microfiltration membrane deposited on the surface of an agar medium, and direct analysis of the mycelium on the membrane or collection of the mycelium from the surface of the membrane by scraping before analysis. The culture time was set at 12 days to obtain the sufficient biomass for slow-growing fungi such as _S. lacrymans_. At longer culture times when there is limited or no growth in the study area, the production of biomolecules tends to decrease in the mycelium leading to an increase in the reproducibility of FTIR analyses [18]. After 2, 3, 4 or 5 days' of growth of moulds on malt extract agar, the highest reproducibility of FTIR spectra was obtained for the longest cultivation time [18]. After the spectral data acquisition from mycelium samples, a pre-processing of the data is necessary before their statistical analysis. In different studies using FTIR spectroscopy for fungal identification, a vector normalisation of the spectral data is performed [19; 20; 22; 26; 34]. Vector normalisation brings the spectra back to the same intensity without altering the spectral profile by performing a second derivative which increases the spectral resolution. In the present study, the results obtained after vector normalisation were compared to the results obtained after min-max normalisation. In the case of min-max normalisation, the baseline of the data was corrected and the data for each spectrum were multiplied so that for each spectrum the minimum was 0 and the maximum was 2. After the normalisation of the spectral data by each of the two methods, the identification of the fungi was carried out using PLS-DA analysis of the spectral data. PLS-DA is a supervised statistical analysis method unlike other statistical methods such as Principal Component Analysis or Hierarchical Ascendant Classification (cluster analysis) [23; 35; 36; 37]. In the case of a supervised statistical analysis method, each spectrum is assigned a class so that quantitative spectral data are supplemented by qualitative data. From the annotated reference IR spectra, the algorithm creates a prediction function of the class to which the analysed spectrum belongs. The results obtained showed that the choice of culture conditions and the type of data normalisation were essential for the identification of _S. lacrymans_ by FTIR spectroscopy. The min-max normalisation was less resolving than the vector normalisation which was widely used for FTIR fungal identification [20; 22; 37]. Regarding the mode of mycelium culture, although liquid culture may be in some cases the simplest to implement [18; 24], it was not relevant for the identification of _S. lacrymans_ by FTIR. Indeed, broth growth was the culture condition that led to the lowest resolution for discriminating different _S. lacrymans_ strains, with even an error at the species level, as a spectrum of the _S. lacrymans_ strain (LRMH-RX-01) was assigned to _R. placenta_ (LRMH-Op-001). In the other mycelium growth conditions tested (on the surface of a microfiltration membrane), only strain affiliation errors within the _S. lacrymans_ species were observed. When the analysis was performed on the IR data obtained after harvesting the mycelium from the membrane, the assignment of IR spectra was consistently correct at the phylum, species and strain level of _S. lacrymans_. This 100% correct assignment percentage must be put into perspective because the number of references we used was small, as the number of strains analysed was limited. [PERSON] et al.