# Adsorption of Heavy Metals from Low Concentration Solutions onto Dried _Chlamydomonas reinhardtii_
## 3 Results  
### Biosorbent Characterization  
Dried _C. reinhardtii_ was fully characterized by different techniques. Upon drying, the surface of the microcalga was characterized by a negative ZP of \(-\)18.8 mV.  
A FTIR (ATR mode) full spectrum of the dried biomass is plotted in Figure 3 and the low frequency region is magnified in Figure S1. Additionally, for clarity, the characteristic bands found by FT-IR analysis and their assignation are summarized in Table S4.  
Analysing the spectrum, different regions can be identified.  
The first is region I (4000-2000 cm\({}^{-1}\)) (Figure 3). In the high frequency region of the spectrum, a broad and complex band at 3400-3200 cm\({}^{-1}\), centred around 3290 cm\({}^{-1}\), is visible due to the overlapping of the -OH and -NH functional groups' stretching modes [35; 36; 42]. The band broadness is typical of the H bonding interaction of the OH groups. The complex bands located around 2955-2855 cm\({}^{-1}\) are assigned to the CH stretching modes [35; 36; 42] while the very small band at 3010 cm\({}^{-1}\) is assigned to the unsaturated =C-H stretching [36].  
The second is region II (2000-1000 cm\({}^{-1}\)) (Figure 3 and Figure S1). In the medium frequency region of the spectrum, the main bands are centred at 1645 cm\({}^{-1}\) and 1539 cm\({}^{-1}\), and they can be assigned to the amide I and amide II bands of the peptide linkage in proteins. The former band is associated mainly with C=O stretching vibration, while the latter band mainly with a combination of N-H in plane bending and CN stretching vibration [35; 42; 51; 52]. The amide III component falls between 1320-1230 cm\({}^{-1}\), thus partially overlapping with other vibrational modes as discussed in the following. A shoulder at 1720 cm\({}^{-1}\) can be related to C=O stretching bonds in carboxylic groups or, more likely, in ester groups in acylglycerols, and thus in the lipid fraction of the biomass [36; 51; 52]. The complex band at 1454 cm\({}^{-1}\) represents the CH\({}_{2}\) and CH\({}_{3}\) bending [35; 52], while the band at 1244 cm\({}^{-1}\) can be assigned to the phosphate P=O group and to the OCO vibrational mode of the ester group [36; 52].  
The third is region III (1000-500 cm\({}^{-1}\)) (Figure 3 and Figure S1). Here, the complex envelope of bands with several maxima at 1030/1000 cm\({}^{-1}\) is due to C-O and C-C stretching modes of the polysaccharide fraction [35; 36]. The bands near 881 cm\({}^{-1}\) refer to out-of-plane OH bending. Thus, the FT-IR spectrum of dried _C. reinhardtii_ matches those found in the literature where the alga cell wall has been described as a \"multi-layered wall\" consisting of hydroxyproline-rich molecules, glycosidically linked to galactose (mainly) and to arabinose [53; 54].  
The morphology of the dried _C. reinhardtii_ was analysed by SEM images to check possible modification of the cell wall upon drying (Figure 4b). In Figure 4a,b the typical spherical structure of CR cells is observed, along with indication of cell disruption, possibly due to the freeze-drying process.  
In the literature it is reported for _Chorella vulgaris_ that signs of pitting and damage on the cell wall, distortion, and collapse are consistent with the drying process [55]. Therefore, similar effects can be inferred regarding pristine C. _reinhardtii_ upon freeze drying, as clearly shown by SEM picture at higher magnification (Figure 4b). To study the chemical  
Figure 3: FTIR in ATR mode spectrum of pristine _C. reinhardtii._  
composition of _C. reinhardtii_, EDX analysis was also performed (Figure S2). The presence of light metals, such as K(I), Ca(II), and Mg(II), and an intense phosphorous (P) peak could be identified. These essential elements are present in all cells and supplied by the growth medium (Table S3) [52].