Polystyrene (PS), a widely used plastic, contributes significantly to marine plastic pollution. In 2021, approximately 390 million tons of plastic were produced globally, with 5.3% being PS, largely in the form of polystyrene foams (EPS and XPS). The buoyancy and waterproof nature of EPS make it prevalent in aquaculture, leading to substantial environmental release through discarded materials. EPS readily fragments into microplastics (<5 mm), contributing to the growing microplastic problem in oceans. While marine plastic pollution is widespread, its fate and the in situ generation of microplastics are poorly understood. Previous observations of EPS fragments attached to marine invertebrates, including bivalves and crustaceans, suggested a potential role for these organisms in plastic degradation. This study focuses on *Perinereis vancaurica*, a benthic clamworm observed to ingest and fragment EPS, excreting microplastics. Previous research on terrestrial insects, such as the yellow mealworm (*Tenebrio molitor*), has shown that their gut bacteria play a crucial role in PS degradation. However, the role of marine polychaetes in plastic degradation and the contribution of their gut microbiome remain largely unexplored. This study aims to understand the contribution of EPS-eating clamworms to marine microplastic formation and the potential role of their gut microbiome in PS digestion by characterizing the gut microbiome and identifying PS-degrading bacteria.

Several studies have investigated the biodegradation of polystyrene by terrestrial insects and their associated gut microbiota. The yellow mealworm (*Tenebrio molitor*) and its gut bacterium *Exiguobacterium* sp. YT2 have been shown to degrade polystyrene. Similarly, *Acinetobacter* bacteria have been isolated from the gut of *Tribolium castaneum* larvae, also capable of ingesting EPS. *Enterococcus* species have been found in the guts of EPS-feeding larvae of *Galleria mellonella* and *Tenebrio obscurus*, showing in vitro PS degradation capabilities. While these studies provide valuable insights, the role of marine invertebrates in PS degradation and the specific contribution of their gut microbiome remain largely unknown. The few studies exploring similar mechanisms in marine settings highlight the limited understanding of the in situ processes leading to microplastic generation. Studies on the effects of microplastics in marine ecosystems and the bioaccumulation in food chains are also limited. Benthic invertebrates, frequently used as bioindicators of pollution, are crucial components of marine ecosystems and often used as indicators of environmental pollution.

This study involved a multi-faceted approach encompassing field sampling, laboratory analyses, and molecular techniques. Clamworms (*P. vancaurica*) were collected from EPS debris on the east coast of Xiamen Island, China. The clamworms were identified morphologically and through phylogenetic analysis of mitochondrial 16S rRNA and COI genes. The size and morphology of microplastics in the excreted frass (waste) were examined using stereomicroscopy, light microscopy, and scanning electron microscopy (SEM). Gas chromatography-mass spectrometry (GC-MS) was employed to analyze chemical changes in the gut contents and frass. Micro-Fourier transform infrared spectroscopy (µFTIR) was used to assess changes in the chemical functional groups of EPS debris. Gel permeation chromatography (GPC) and proton nuclear magnetic resonance (¹H NMR) were used to determine changes in molecular weight and chemical structure of the frass. 16S rRNA gene sequencing was performed to characterize the bacterial communities in the guts of both EPS-fed and non-EPS-fed clamworms and in the frass. Bacterial isolates from the clamworm gut were cultured, and their ability to degrade PS was assessed using MMC liquid media supplemented with PS films. The degradation capacity of selected isolates was evaluated through weight loss measurements, FTIR, water contact angle (WCA) measurements, and thermogravimetric analysis (TGA). GC-MS analysis was used to identify PS degradation intermediates.

The study found that *P. vancaurica* ingested and fragmented EPS, resulting in the production of microplastics with a mean diameter of 0.6 ± 0.2 mm. SEM analysis revealed surface modifications on the microplastics, suggesting microbial degradation. GC-MS analysis of the clamworm gut contents revealed the presence of long-chain fatty acids, benzene ring-containing substances, and long-chain carboxylic acid esters, indicating metabolic activity. µFTIR analysis showed increased -OH and C=O stretching absorption in EPS fragments from the gut compared to the control, confirming partial biodegradation. GPC analysis showed a decrease in the molecular weight of EPS after passage through the clamworm gut. ¹H NMR analysis confirmed chemical structural changes in the frass. 16S rRNA gene sequencing revealed significant differences in the gut microbiome of EPS-fed and non-EPS-fed clamworms. The EPS-fed group showed a marked increase in *Acinetobacter* abundance, while *Marinobacter* and *Vibrio*, dominant in the non-EPS-fed group, were greatly reduced. Isolation and characterization of gut bacteria identified *Acinetobacter johnsonii*, *Brevibacterium casei*, and *Ruegeria arenilitoris* as effective PS degraders. In vitro experiments with these isolates confirmed PS degradation, showing a slight weight loss (3.3-3.7% in 30 days), changes in chemical functional groups (formation of C=O and C-O groups), increased hydrophilicity (lower WCA), and decreased thermal stability. GC-MS analysis identified various PS monomers, fatty acids, and esters as degradation products, indicating active PS metabolism.

This study provides the first evidence of EPS degradation by a marine benthic polychaete and its associated gut microbiome. The findings highlight the significant role of *P. vancaurica* and its specialized gut microbiota in the fragmentation and partial biodegradation of EPS in marine environments. The dominance of *Acinetobacter* in the gut of EPS-fed clamworms, along with the confirmed PS-degrading ability of *A. johnsonii*, *B. casei*, and *R. arenilitoris*, strongly suggests a causal relationship between EPS ingestion and microbial-mediated degradation. The observed changes in chemical composition, molecular weight, and physical properties of the EPS after passage through the clamworm gut support this conclusion. The results have important implications for understanding the fate of plastic pollutants in marine ecosystems and the potential of marine invertebrates and their gut microbiota in bioremediation strategies. Further research is needed to fully elucidate the specific enzymatic mechanisms involved in PS degradation by these bacteria and the overall contribution of this process to the marine ecosystem.

This study demonstrates that the marine benthic polychaete *P. vancaurica* ingests and fragments EPS, contributing to microplastic production. The clamworm's gut microbiome, notably enriched in *Acinetobacter*, plays a crucial role in this process. Three isolated gut bacteria, *A. johnsonii*, *B. casei*, and *R. arenilitoris*, were confirmed to degrade PS in vitro. The findings highlight the complex interactions between marine invertebrates and their microbiota in the context of plastic pollution. Future research should focus on identifying the specific enzymes responsible for PS degradation and exploring the potential for bioremediation strategies utilizing these bacteria.

The relatively low weight loss (3.3-3.7% in 30 days) observed in the in vitro experiments with the isolated bacteria might underestimate the overall degradation potential in the natural environment. The study focused on a single clamworm species and geographic location; therefore, the generalizability of the findings requires further investigation across different species and locations. The specific environmental factors influencing the in situ degradation rate were not fully explored. Finally, the potential long-term ecological effects of microplastic ingestion and the transfer of microplastics through the food chain need further investigation.