# Marine Bacteria Associated with Colonization and Alteration of Plastic Polymers
## 3 Results and Discussion
### In Vitro Colonization and Structural Changes of Polymers through FTIR

Table 3: Bacteria growth (in percentages, \(\%\)) over whole and mechanically crushed polymer spheres made of polypropylene (PP) and polystyrene (PS) after one year in culture. spheres compared to those mechanically crushed spheres for _Bacillus cereus_ and _Proteus penneri_ and (ii) these differences were also determined for _P. penneri_ and _Alcaligenes faecalis_ with polystyrene spheres (Table 3). With these results, we infer that specific taxonomic groups show preferences for certain plastic surfaces and that potential metabolic adaptation, such as differential attachment, to plastic may probably occur. Bacteria growth over polymer spheres and EPS production are conducive to polymer alteration, while in turn, alteration could supply nutrient sources for bacteria growth. It is generally accepted that biofilm formation on the polymer surface is a prerequisite for microplastic alteration [42]. In this work, it seems to be confirmed that _M. gitamensis_ both did not produce EPS (Table 2) nor did it grow over plastic spheres (Table 3). Likewise, no changes in FTIR spectra of _M. gitamensis_ are observed as reported below. A definite challenge for this work is to unveil potentially plastic-altering bacteria regardless of environment variables, such as pH and salinity, that can be circumvented as they are under control in in vitro experiments. Bioalteration has been related to changes in FTIR-spectral properties of polymers [15; 16; 17]. Three spectral regions are assumed as fingerprint regions for PP and PS, namely 1100-750 cm\({}^{-1}\), 1600-1000 cm\({}^{-1}\) and 3100-500 cm\({}^{-1}\). Thus spectra of PP and PS spheres with inoculum were superimposed on spectra of polymer without inoculum (control) for analysis. Moreover, as an approach to value changes in spectra, area count reduction was used as a measure of the extent grade of polymer bioalteration and thus we would allow inferring the ability of bacteria to cause the polymer to deteriorate. In this work, polymer bioalteration was characterized for flattening and splitting of peaks and a diminution of vibration bands below 50% (in term of relative area counts) in FTIR spectra through qualitative comparison between spectra of test spheres (with bacteria) and in absence of bacteria (control spheres). Spectra of PP whole spheres inoculated with _Alcaligenes faecalis_ and _B. cereus_ showed peak flattening in a vibration band between 3100 and 2500 cm\({}^{-1}\) (Figure 1 A,B). Flattening in whole PP spectra was associated with a diminution in area counts around 80% and 81% for each bacteria respectively compared to area counts of control. Otherwise, _Alcaligenes faecalis_, _B. cereus_ and _Proteus penneri_ displayed a double peak around 1460 cm\({}^{-1}\) (Figure 1C). Significantly, only _A. faecalis_ was able to alter spectra of mechanically crushed spheres of polypropylene as a diminution in vibration band, in area counts over 75%, was determined between 3100 and 2500 cm\({}^{-1}\) (Figure 2A,B). Peak splitting, around 1460 cm\({}^{-1}\) with three resulting peaks, was also observed (Figure 2C). In all PP spectra, flattening peak consisted of a band located at about 3000 cm\({}^{-1}\) assignable to CH\({}_{2}\) as asymmetric stretching occurs [45]. Furthermore, double and triple peaks at 1000-1600 cm\({}^{-1}\) are indicative of bioalteration of polypropylene as intermediate products, i.e., primary and secondary alcohols and formation of aldehydes and ketones, could have emerged [45]. In particular, a band around 1460 cm\({}^{-1}\) reveals a bending deformation [46]. Despite other bacteria also growing over polymer spheres, both whole and crushed, spectra were similar, with each displaying vibration bands of polypropylene. Concerning polystyrene, although bacteria growth over polymer spheres was also visible and an increment in sphere weight was also determined (Table 3), no changes were observed in FTIR spectra (Figures 3 and 4). Figure 1: FTIR spectra of whole spheres of polypropylene (PP).