Study of actions of Bioplastic Materials on marine microbial communities and effects on marine invertebrate organisms

Bioplastics are nowadays widely considered as an alternative to conventional plastics of petrochemical origin. Considering the high growth rates estimated for their production in the coming years, we want to evaluate whether the environmental problem linked to conventional plastics is not shifting to bioplastics. To do this, we focused on the factors involved in the biodegradation processes of the latter within marine ecosystems, analyzing the two most widespread bio-based and biodegradable biopolymers today: polyhydroxyalkanoates (PHAs) and polylactic acids (PLA). The study of these bioplastics was carried out through the analysis of the bacterial communities constituting a biofilm known as Plastisphere, developed on the surfaces of the biopolymeric materials exposed in marine environment for different times, The research and identification of bacterial degradation products was performed by mass spectrometry analysis (MS). Morover, the possible interactions that the presence of biopolymer micro- and nanoparticles can determine on marine organisms was evaluated on bioindicator model organism Mytilus galloprovincialis by in vitro and in vivo exposure tests. To study the Plastisphere of biopolymers, porous bioplastic scaffolds produced by Thermally Induced Phase reversal process, were exposed in marine environment for 60 and 120 days. The composition of plastispheres was detected by Next Generation Sequencing of V3 and V4 hypervariable region of the 16S rDNA gene. The composition of bacterial communities was related to biodegradable biopolymer and the bacterial diversity was affected by exposition times. In general, the bacterial biomass decreases as time increases and was higher in Poly(DL-lactic Acid)/Poly(L-lactic Acid) (PDLLA/PLLA) scaffold. A high abundance of Proteobacteria was detected in all biopolymers, with Alpha and Gamma Proteobacteria dominant. High abundance of sequences assigned to hydrocarbon-degrading bacteria (i.e. Oleibacter, Labrenzia Alcanivorax) and genera associated with biopolymers was observed (Maricaulis, Oceanicaulis). The genus Vibrio was first detected associated with PLA bioplastic. Tests carried out using minimal mineral media with biopolymer overlays allowed the identification of 5 potentially degrading bacterial strains, capable of growing in the presence of polyhydroxyalkanoate/polyhydroxybutyrate (PHA/PHB) as the sole carbon source, isolated from the plastisphere of scaffolds exposed in a marine habitat. The sequences were assigned to Alcanivorax sp. and Labrenzia sp. genera, suggesting a possible role in degradation of biopolymers. The products of bacterial biodegradation were studied by MS analysis and revealed the presence of products resulting from bacterial degradation of PLA, suggesting an active role of the marine plastisphere in PLA biodegradation. The presence of family and genera know to degrade PLA (belonging to the phyla Actinobacteria, Proteobacteria, Bacteriodetes) detected by sequencing, confirms this hypothesis. The immunobiological investigations, applied here for the first time for the evaluation of the possible effects produced by bioplastics on marine organisms, have highlighted effects determined by exposure to PLA particles. In fact, the haemocytes showed reduced phagocytic activity, compared to an increase in enzymatic activities compared to the control both in the digestive gland sample and in the haemolymph sample. Furthermore, histological analysis of the digestive glands highlighted tissue damage and atrophy with enlargement of the glandular lumens and thinning of the epithelium lining them. The work described here has highlighted actual critical points for the promising environmental compatibility of bioplastics for natural aquatic systems, since they appear to be similar in terms of behavior to plastics of fossil origin and lays the methodological foundations for an in-depth study of the bioplastics problem.