**3. PCA of Raman spectra for the determination of heavy metals attached to bacterial surfaces**

Toxic metals are not degradable and tend to accumulate in the exposed organisms causing serious health effects. The use of biological agents to remove or neutralize contaminants (biorremediation) is a very important tool for the removal of such toxics. In particular, the use of inactivated microorganisms as adsorbents (biosorption) has been suggested as an effective and economical way to remove heavy metals from water and food intended to human or animal consumption (Davis et al., 2003; Haltunen et al., 2003, 2007, 2008; Ibrahim et al. 2006; Mehta & Gaur, 2005; Mrvčić et al., 2009; Shut et al., 2011; Volesky & Holan, 1995).

Metal biosorption is usually evaluated by means of analytical methods or atomic absorption spectrometry. These methods allow the quantification of free metal ions in the supernatants of bacterial/metal samples (Ernst et al., 2000; Haltunen et al., 2003, 2007, 2008; Velazquez et al., 2009; Zolotov et al., 1987).

In this sense, a method involving vibrational spectroscopic techniques (*i.e.:* Raman spectroscopy) and multivariate methods (both unsupervised and/or supervised), would represent an advantage over the standard procedures, due to the possibility of quantifying the metal ions directly from the bacterial sample, and at the same time, obtaining structural information (Araujo-Andrade et al., 2004, 2005, 2009; Ferraro et al., 2003; Gerbino et al. 2011; Jimenez Sandoval, 2000).

PCA carried out on the Raman spectra represents the first step in the construction of a calibration model allowing the quantification of metal ions attached to bacterial surfaces. This analysis allows obtaining a correlation between the spectral variations and the property of interest (*i.e.* the metal ion concentration), identifying the optimal spectral region/s for the calibration of quantification models, and also detecting erroneous measurements leading to reduce the predictive ability of the model.

In this section, we present the PCA results obtained from the Raman spectra corresponding to bacterial samples (*Lactobacillus kefir*) before and after the interaction with four heavy metals (Cd2+, Pb2+, Zn2+, Ni2+) in three different concentrations each.

Even when the main objective of this study was to calibrate models for the quantification of metal ions attached to bacterial surfaces using supervised methods (*i.e*.: PLS), the calibration of prediction models goes beyond of the intention of this chapter. For this reason, we focused this section just on the discussion of the PCA results.
