**3.2 Spectral data pre-treatment**

Raman spectra analyzed were collected over dry solid bacterial samples before and after the interaction with different concentrations of metal ions. Therefore, it is highly probable that our measurements include some light scattering effects (background scattering). These effects are in general composed of multiplicative and additive effects (Martens & Naes, 1989).

Spectra collected and analyzed in this section were baseline corrected in order to subtract the fluorescence contribution. To perform this correction, a polynomial function was approximated to the spectrum baseline, and after that, subtracted from the spectrum. Also, the spectra were smoothed using Savitzky-Golay method. Light scattering effects were corrected using the multiplicative scatter correction (MSC) algorithm and then, the spectra were mean centred. Data pre-treatment and multivariate spectra analysis were carried out with Origin version 6.0 from Microcal Company, and The Unscrambler® software version 9.8 from CAMO company.

#### **3.3 Analysis and discussion of PCA results**

PCA was performed on the pre-treated Raman spectra of each bacteria/metal sample in order to correlate metal concentrations with the spectral information.

The criteria used for PCA-scores and loadings interpretation are depicted in the next subsection. Even when the presented data set corresponds to the bacteria/Cd+2 interaction, the same methodology was employed for the analysis of the other bacteria/metal samples.
