# Marine Bacteria Associated with Colonization and Alteration of Plastic Polymers
## 3 Results and Discussion  
### Bacteria Molecular Identification  
The occurrence of microplastics in marine ecosystems is an undisputed fact; however, identification of bacteria associated with these tiny plastic polymers is lacking. It is also worth mentioning that characterization of the bacteria community directly associated with microplastics is not possible as bacteria can be removed during the separation process with a fully saturated salt solution. As a starting point, bacteria sampled and identified in different seasons are assumed as key bacteria species able to alter microplastics. This is because (i) bacteria adhering to marine sand at the drift line above sea water level are prevalently associated with microplastic particles which, in turn, are also deposited the most in the same drift coast line [2, 29, 30]; (ii) bacteria are spread out through covering plastic particles [31]; and (iii) bacteria composition in this drift line can be representative of different plastic surfaces as the bacteria community depends on the kind of polymer and even the sampling period [32]. Different studies have reported that 20% plastic debris in coastal areas is deposited on beaches [33, 34, 35]. Seventeen bacteria pure colonies from marine sand were characterized in culture and identified through the 16S rRNA gene. Four universal primer combinations, used in this work, have been previously validated (Table 1), as they match different positions of the genome, identifying different areas of the 16S rRNA region [36, 37, 38]. Sequences from PCR fragments were assigned to groups based on BLAST similarities. Colony identification was classified with five bacteria genera, _Alcaligenes_, _Proteus_, _Myroides_, _Kcouria_ and _Bacillus_, which contained most species with more than 50% identified clones. Moreover, species from _Kcouria_ genera were doubtfully identified, as the existence of possible chimeras revealed. Other genera were also determined to be less prevalent in the sampling. Bacteria species were molecularly identified as _Myroides gitamensis_, _Proteus pemneri_, _Bacillus_ sp., _Bacillus thuringiensis_, _Bacillus cereus_ and _Alcaligenes faecalis_ in BLAST. The phylogenetic analysis positioned _Myroides gitamensis_ LP5 sequence in a clade within other 16S rRNA Myroides (Figure S1; Table S1). There was a total of 715 positions in the final dataset, of which 516 were conserved, 113 were parsimony-informative and 86 were parsimony-uninformative. The best-fit evolutionary model was \(\mathrm{TPM3E+F+R2}\). This result (Figure S1), with a value of 16S rDNA gene sequence similarity of 78%, demonstrated that _Myroides gitamensis_ could be regarded as representing a species within the genus. The _Proteus pemneri_ dataset was constructed with 15 reference sequences (Table S2). This dataset had a length of 799 characters, of which 738 were conserved, 26 were parsimony-informative and 35 were parsimony-uninformative. The best-fit evolutionary model was \(\mathrm{HKY+F+I}\)[39]. The phylogenetic analysis positioned _P. pemneri LP3_ in a clade within other \(\mathrm{\it P.\ pemneri}\) and had a similarity value of 73% (Figure S2). The tree of _Alcaligenes faecalis_ was constructed with 19 reference sequences (Table S3). This dataset had a length of 873 characters, of which 795 were conserved, 41 were parsimony-informative and 37 were parsimony-uninformative. The best-fit evolutionary model was \(\mathrm{HKY+F+I}\)[39]. _A. faecalis LP6_ was positioned in a clade for the same species with a value of 70% (Figure S3). Significantly, the phylogenetic analysis positioned _Bacillus_ sequences in three clades within _B. thuringiensis (B. thuringiensis LPI; value 76%)_, _B. cereus (B. cereus LP2; value 78%)_ and _Bacillus_ sp. (_Bacillus_ sp. _LP4; value 66%). The dataset, constructed with 15 reference sequences (Table S4), had a length of 968 characters, of which 805 were conserved, 67 were parsimony-informative and 96 were parsimony-uninformative. The best-fit evolutionary model was \(\mathrm{HKY+F}\)[39]. Unlike _B.