Biodegradation and disintegration of expanded polystyrene by sphaeromatid isopods Sphaeroma via their gut bacteria

1、Introduction：

Plastics, synthetic polymers derived from petroleum, consist of long polymeric chains and are widely used in building insulation, industrial materials, and packaging (Ali et al., 2021; Lv et al., 2024). The global annual plastic production has continually and dramatically increased in the past decades, and consequently over 400 million metric tons after 2020 (Ali et al., 2021). These polymers are classified into six major types, including hydrolyzable plastics with C–O backbone (poly- urethane (PUR), polyethylene terephthalate (PET)), and non- hydrolyzable plastics with C–C backbone (polyvinyl chloride (PVC), polypropylene (PP), high, low, linear-low density polyethylene (HDPE, LDPE, LLDPE) and polystyrene (PS)) (Ali et al., 2021; Lv et al., 2024). Hydrolysable polymers can be hydrolysed via enzymes into monomers due to the presence of ester bonds (Ali et al., 2021; Lv et al., 2024). In

contrast, non-hydrolysable polymers are more resistant to degrade due to their C–C backbone bonds, requiring oxidative cleavage by enzymes with high redox potentials (Ru et al., 2020; Ali et al., 2021; Lv et al., 2024). Among these, expanded polystyrene (EPS) is one of the most frequently used plastic type and a common marine debris item world- wide (Turner, 2020).
Polystyrene (PS) is one of the common aromatic polymers in the environment, consisting of styrene monomer ([–CH– (C6H5) CH2–]n) (Yang et al., 2022). EPS, commonly known as white styrofoam, is lightweight, durable, waterproof, and corrosion-resistant (Jang et al., 2017; Turner, 2020). These properties make it ideal for floating mate- rials (buoys, floats, and pontoons), food containers, construction and packing materials (Jang et al., 2017; Turner, 2020). EPS foam is extensively used as mariculture floating rafts along the west coast of the Taiwan Strait and China's coastline (Jang et al., 2017; Turner, 2020).

However, once released into the environment, plastic debris undergoes fragmentation and aging through physical, chemical, and biotic pro- cesses, such as photo-oxidation (UV radiation), mechanical stress (wave and wind actions), microbial degradation and animal chewing, forming microplastics (MPs, <5 mm) and nanoplastics (NPs, <1 μm) (Turner, 2020; So et al., 2022). It has been assessed that approximately 82–358 trillion MPs litter are floating on the ocean's surface (Kataria et al., 2024). The widespread MPs in the marine environment creates habitats and attachment sites for aquatic biota, and act as vectors by adsorbing pollutants and promoting their bioaccumulation (Kataria et al., 2024). The interactions between organic aggregates, pollutants, microorgan- isms and MPs facilitate microplastic migration through food chains, posing unpredictable biohazards to humans (Amelia et al.,2021; Kataria et al., 2024). Although plastics can persist in the marine environment and resist degradation, bacteria, fungi, algae and mixed microbial cul- tures have been frequently demonstrated plastic-degrading potential (Gao and Sun, 2021; Yang et al., 2022; Zhai et al., 2023; Lv et al., 2024; Xu et al., 2024). Recently, we reported that marine benthic clamworms (Perinereis vancaurica) with specific gut bacteria chew EPS, contribute to PS degradation, but leading to microplastic formation that poses a threat to the health of marine ecosystem (Zhao et al., 2024a, 2024b).
Sphaeroma (Crustacea, Isopod, Sphaeromatidae), wood-boring crus- tacea, named for its ability to curl up into a ball when exposed to external stimuli, is ecologically significant due to its complex digestive habits(Zimmer et al., 2002; Hu et al., 2016; Xu et al., 2021). Sphaeroma is known for its ability to use firm stomatological structures to burrow into numerous substrates for habitat, including intertidal mangrove roots, friable rock, man-made coastal wooden structures and EPS floats in the intertidal zone around the world (Davidson, 2012; Davidson et al., 2014; Yang et al., 2018). Previous studies have shown that the sphaer- omatid isopods are filter-feeders that primarily consume phytoplankton and detritus (Yang et al., 2018). It has been demonstrated that wood- boring isopods achieve lignocellulose degradation due to numerous and diverse carbohydrate-active enzymes (CAZymes) provided by the host and its microbiota(El-Shanshoury et al., 1994; Zimmer et al., 2002). In the marine environment, Sphaeroma frequently perforates the sub- merged surface of the EPS floats used in floating docks and aquaculture facilities, causing economic losses and microplastic pollution (Davidson, 2012). Previous studies have found that marine EPS-borers are suscep- tible to accidental ingestion of microplastic particles, leading to the ubiquitous presence of microfoam in clamworms, isopods and crabs (Zheng et al., 2023). Several studies have revealed the ingestion of EPS by Sphaeroma, however, the capability of depolymerizing/biodegrading PS by Sphaeroma has not been addressed so far.
Given the widespread presence of Sphaeroma and its yet unevaluated power in foam plastic grinding in marine ecosystems, it is critical to understand the mechanism underlying. To this, the behavior of Sphaeroma's EPS ingestion was observed, and plastic fragmentation and biodegradation processes were examined. GPC analysis of EPS debris consumed from wild- and indoor EPS-fed groups of Sphaeroma were performed to determine changes in molecular weight; the oxidative functional groups of EPS particles ingested by Sphaeroma were confirmed by μFTIR. Antibiotic inhibition assays and gut microbiome analysis were conducted to evaluate microbial involvement in PS depolymerization. Further, the key members of gut microbiome were isolated and tested for PS biodegradation and fragmentation potential in vitro. These results will gain insights into the process of marine micro- plastic generation driven by marine animal activity in coastal plastic garbage and discover novel bacterial resources for plastic cycling from the unique niches.

2、Materials and methods：

2.1. Collection of EPS foam and Sphaeroma isopods
EPS floats exhibiting visible surface holes were collected, bagged,

and transported to the laboratory. Then, the blocks were carefully dissected with scissors, and isopods were immediately extracted from the fragmented EPS using forceps (Zhao et al., 2024a, 2024b). We collected only adult Sphaeroma individuals without distinguishing sex or species. Approximately 500 adult isopods were maintained in 20 L transparent plastic boxes containing 10 L seawater at 26 oC. EPS foam was added to serve as both habitat and a potential feeding substrate. The seawater was renewed two days to maintain water quality. The collec- tion site was located on the east coast of Xiamen Island, China (118o 0.10, E, 24o 0.43, N) (Fig. S1). We found high densities of sphaeromatid iso- pods (Sphaeroma terebrans and Sphaeroma retrolaeve) burrowing into the EPS, creating numerous debris-filled burrows that disrupted the struc- tural integrity of the floats (Fig. 1). The isopods were observed under a stereomicroscope (LEICA M80) and identified based on their morpho- logical characteristics. The original EPS around the isopod gnawing holes was also sampled and preserved in the dark as EPS control for subsequent experiments.
2.2. Characterization of physicochemical properties of environmentally derived EPS
To demonstrate the degradation of EPS by Sphaeroma in the wild, 1 g of EPS debris and the EPS control sample were collected in the wild and mixed with 30 % H2O2 solution in a conical flask (24 h, 50 oC) to digest organic matters and then filtered for subsequent analysis of EPS particle size and chemical properties changes (Zhao et al., 2024a, 2024b). EPS debris referred to particles fragmented by Sphaeroma burrowing, while EPS control denoted intact material preserved from gnawing sites. Particle size and morphology of EPS debris were assessed by stereo- microscopy (Leica M80) and measured using ImageJ (Fiji v1.53c). Gel permeation chromatography (GPC) analysis (Agilent 1260, Agilent Technologies Inc., USA) was applied to characterize number average molecular weight (Mn), weight average molecular weight (Mw) and molecular weight distribution (MWD) of the EPS debris (Song et al., 2020). The EPS control and EPS debris samples were dissolved in tetrahydrofuran (THF, purity≥99.8 %, GC) and filtered through 0.20 mm PVDF filters (Woo et al., 2020). The extracted solution was analyzed at a flow rate of 0.7 mL/min at 40 oC (Kim et al., 2020).
The degradation products of EPS released into the gut and fresh EPS debris by the Sphaeroma were detected by gas chromatography–mass spectrometry (GC–MS). Briefly, plastic degradation products in fresh EPS debris were extracted using 5 mL tetrahydrofuran (THF) for 2 h. Plastic degradation products in Sphaeroma tissue fluid containing in- testine were extracted with an equal volume of THF: methanol (2:1) (Lou et al., 2020). Evaporated the solvent using nitrogen gas and redissolved in 1 mL 100 % hexane. Then, 10 μL sample was injected in an GC–MS system (Agilent, QP2010) equipped with a DB–5MS UI (60 m long, 0.25 mm internal diameter, 0.25 μm thickness) chromatographic column. The column temperature was started at 50 oC for 4 min and raised to 270 oC at a rate of 20 oC/min, and finally held for 20 min. The flow rate was set at 1 mL/min with helium as the carrier gas. Potential degradation products were identified by matching them with com- pounds in the NIST14 database (Rong et al., 2024). Thermogravimetric analysis (TGA) (TGA 2, Mettler Toledo, USA) was conducted to assess thermal modifications of EPS after biodegradation by Sphaeroma. EPS debris and EPS control sample in the crucible were heated over a tem- perature range of 30–550 oC (at a rate of 10 oC/min) under a high-purity nitrogen ambience (99.999 %) (at a flow rate of 10 mL/min) (Zhao et al., 2024a, 2024b).
2.3. Chemical changes of environmental EPS particles from the intestine
Isopod individuals in the wild were randomly selected and washed three times with sterile water to remove EPS debris attached to the body surface. To analyze functional group changes in gut-retained EPS par- ticles, ten isopod individuals was digested in 150 mL 30 % H2O2 (24 h,

50 ? C), followed by adding saturated NaCl solutions to float the EPS particles (Revel et al., 2020). The EPS particles were washed with sterile water, vacuum-filtered through 0.8 μm PC filter paper (Merck Millipore Ltd.), dried at room temperature and sealed for further observation. The EPS control performed the same digestion procedure. Particles on the PC filter paper were observed and photographed under a stereomicroscope (Zheng et al., 2023). The EPS particles and the EPS control were picked up to evaluate the modification of the functional groups in the gut using micro-Fourier transform infrared spectroscopy (μFTIR: Nicolet iN10, Thermo Fisher Scientific) following the method described in previous study (Zhao et al., 2024a, 2024b). The absorption peaks in the IR spectra include the methylene (1465 cm_ 1), ester carbonyl bond (1740 cm_ 1), keto carbonyl bond (1715 cm_ 1) and hydroxyl group band (3750 cm_ 1). The carbonyl index (CI) and hydroxyl index (HI) were calculated as a ratio of carbonyl band area and hydroxyl group band area to methylene peak area, respectively, to assess the oxidation of EPS particles in the gut, as reference described (Battulga et al., 2020; Potrykus et al., 2021; Zhao et al., 2024a, 2024b).