Cross-Feeding Drives Degradation of Phthalate Ester Plasticizers in a Bacterial Consortium

Simone BertoldiSimon KlaesStefanie ClausAlex MarsansHermann J Heipieper^*Christian Eberlein^*

• Helmholtz Centre for Environmental Research, Helmholtz Association of German Research Centres (HZ), Leipzig, Germany

Reports of plastic pollution across diverse ecosystems continue to emphasize the environmental risks associated with the increasing consumption of synthetic polymers. Plastics frequently contain additives such as phthalic acid esters, which are extensively employed as plasticizers to enhance flexibility in plastic materials and as constituents of numerous consumer products. These compounds are not chemically bound to polymers, allowing them to leach into the environment and have been implicated as potential endocrine disruptors in animals. In the present study, the bacterial degradation of selected phthalate esters was examined, with diethyl phthalate (DEP) utilized as a model compound. A bacterial consortium capable of degrading DEP was enriched from a biofilm of a polyurethane tubing. The consortium was capable to mineralize DEP as the sole carbon and energy source at concentrations of up to 4 mM, whereas concentrations above 6 mM inhibited its activity due to DEP toxicity. This degradation was only possible by the whole consortium and not by single isolates. The degradation of DEP as well as the timely occurrence of monoethyl phthalate as degradation intermediate was confirmed by UPLC analysis. Metagenomic sequencing identified the consortium as comprising a Microbacterium sp. strain and two Pseudomonas spp. Metaproteomic analyses of the consortium, performed under varying time points and carbon sources and integrated with complementary growth experiments, facilitated the reconstruction of the degradation pathway and the identification of putative enzymes involved in DEP metabolism. Microbacterium sp. DEP1M initiated the degradation by hydrolysis of DEP into ethanol and monoethyl phthalate, which is then taken up by the cells and further metabolized to ethanol and phthalate. The latter is subsequently oxidized by a dioxygenase and further transformed to the central intermediate 3,4-dihydroxybenzoic acid (protocatechuate). Protocatechuate is then exclusively degraded via the ortho cleavage pathway. Notably, the distribution of enzymatic functions among different community members strongly supports the occurrence of microbial cross-feeding, indicating that DEP mineralization is a cooperative process within the consortium.