Seawater was collected in the Pacific Ocean during the DY79 cruise of COMRA in December 2023, at water depths of 3370 m, located at 19.92°N, 173.33°E ( Supplementary Figure S1 ). Deep-sea seawater was added to MMC medium containing polystyrene microspheres of different particle sizes (60 nm, 600 nm and 1 µm) (Tianjin BaseLine ChromTech Research Centre) and different concentrations (10 mg/L, 50 mg/L and 150 mg/L), and PS plastic pieces (1 × 1 cm) (Sigma−Aldrich, USA), respectively. Subsequently, continuous static incubation was carried out for 50 days at 28 °C. MMC is an artificial seawater medium with PS as the sole carbon and energy source, the formulation of which is described in the Text S1.

The enrichment samples with gradient dilutions of 10^-6, 10^-7, 10^-8 and 10^-9 was spread on marine broth 2216 (BD, DIFCO, USA) agar plates. The plates were incubated in the dark at 28 °C under aerobic conditions for 5 days. All resultant colonies were purified by subcultivation in the same medium. The genomic DNA of the isolated pure culture was extracted as per manufacturer’s instructions using the genome extraction kit (Shanghai SBS Genetech Co., Ltd). Then 16S rRNA gene was amplified by the universal primers: 27 F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492 R (5′-TAC GGC TAC CTT GTT ACG ACT T-3′). The PCR procedure (30 cycles of 45 s at 94 °C, 45 s at 55 °C, and 90 s at 72 °C) was performed using a thermal cycler (Biometra TProfessional Standard gradient 96, Germany) (Liu et al., 2023). Then, the PCR products were sequenced (Shanghai Sangon Biotech Co., Ltd) and the sequence reads were used as queries to search against the EzBioCloud database (http://www.ezbioclound.net). To test the ability of pure strains to utilize PS, cells from 2216 agar plates were collected and washed four times using MMC medium at 3500 rpm for 8 min. Subsequently, a single isolate was cultured in MMC medium with PS and incubated for 30 days at 28 °C, 150 rpm/min. Bacterial growth was observed by light microscope (Nikon 80i, Japan).

Morphological observations were carried out on the bacterial communities exposed to 150 mg/L different particle sizes of microplastics and microplastics released from PS films. Briefly, it was fixed with a 1:2 (v/v) ratio of bacterial culture and 2.5% glutaraldehyde for 2 h. The fixative was removed at 9000 rpm for 10 min using the high-speed freezing centrifuge (Eppendorf Centrifuge 5804R, Germany), and the excess fixative was washed twice with PBS. Subsequently, the cells were dehydrated with ethanol at a gradient of 50%, 60%, 70%, 80% and 90% while they were finally stored in anhydrous ethanol. A little of the liquid was taken and placed on silicon wafer during the experiment and waited for the ethanol to evaporate before spraying platinum. Samples were analysed using SEM (Hitachi SU8600, Japan).

The enriched samples were collected to remove the organic components by digestion. A ratio of 1:10 (v/v) between the sample and 10% KOH was added to 250 mL conical flasks. The reaction was carried out at 60 °C for 24 h until the microorganisms were completely digested. The solution obtained after digestion was density-separated with saturated NaCl and allowed to stand overnight. The supernatant was then filtered through a PC membrane (0.22 µm, 47 mm). Microplastics and nanoplastics were transferred to ultrapure water for following analyses by shaking the PC membrane for 30 s. Morphological changes on the microplastic surface were observed by SEM (Hitachi SU8600, Japan).

The ROS levels in bacterial cells after different treatments were detected using the 2’,7’-dichlorofluorescein diacetate (DCFH-DA) ROS assay kit (UElandy, China). After diluting the bacteria to the same cell concentration (5 × 10⁵/mL), 100 µL bacterial cells were centrifuged and washed three times with 2% NaCl. Then, the bacterial cells were resuspended with 100 µL (10 µmol/L) DCFH-DA and incubated for 30min in the dark at 28 °C. The cells were washed three times with 2% NaCl to remove the excess DCFH-DA. The fluorescence intensity values were measured using a microplate reader (Varioskan LUX, USA) at excitation and emission wavelengths of 504 and 529 nm, respectively. The intracellular ROS levels were expressed as the ratio of fluorescence intensity of the exposed group over the control group (Ning et al., 2022).

The samples enriched for 50 days were filtered through 50 nm PCTE membranes (Whatman, UK) to remove the bacterial community and microplastic particles. DOC was assessed by a total organic carbon (TOC) analyser (Sievers InnovOx, USA) using wet chemical oxidation.

Cells were counted at 40 × light microscope (Nikon 80i, Japan) using a Neubauer-improved haemocytometer ( Supplementary Figure S2 ). The control group (CK) was MMC medium containing sodium acetate (2 g/L) without plastic films/microplastics. The growth rate was calculated as follow.

where C was the cell density of control group and T was the cell density of the treated group.

Samples were collected on 50th day to determine the protein and polysaccharide contents of EPS. The samples pretreatment was as follows: the samples were mixed with phosphate buffered saline (PBS) to make the same cell concentration and subjected to ultrasonic treatment for 3 min using an ultrasonic cleaner (KQ-800DE, China). Finally, the samples were centrifuged with 12000 g (4 °C) for 20 min (Eppendorf Centrifuge 5804R, Germany), and the supernatant was collected for EPS composition analysis. The polysaccharide content of EPS was determined by the phenol-sulphuric acid method and the protein content was determined by BCA protein assay kit (Thermo Fisher Scientific 23225, USA).

Seawater bacterial communities were detected using high-throughput sequencing technology. Bacterial DNA was extracted using the Power Soil DNA Isolation Kit (MoBio, USA) according to the manufacturer’s instructions. The V3 – V4 region of the 16S rRNA genes was amplified using forward primer 338 F (5′-ACTCCTACGGGAGGCAGCAG-3′) and reverse primer 806 R (5′-GGACTACHVGGGTWTCTAAT-3′). DNA sequencing was performed by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Functional and metabolic pathway predictions were analyzed using PICRUSt2, BugBase and FAPROTAX.

In order to analyse the action of the bacterial community on the plastic, the plastic surface was observed using SEM. As shown in Figure 1 , the 60 nm microplastics was broken up, where smaller particles were found on the surface. Compared with the flat and smooth surface of PS microplastics (600 nm and 1 µm) ( Supplementary Figure S3 ), the surface was uneven after 50 days of incubation with the bacterial community. This was consistent that the phenomenon was observed on Rhodococcus ruber C208 degradation of nano-sized PS (Eom et al., 2023). Wrinkles and deep pits were observed on the plastic films surface. The change of microplastic and plastic films surface morphology implied that there are plastic-degrading bacteria in the microbial community. Further to prove this, the samples were diluted and spread on MB agar plates. After several rounds of purification by plate streaking, a total of 16 colonies were isolated. Different colonies were selected for 16S rRNA gene sequencing and uploaded to the EzBioCloud (http://www.ezbiocloud.net) for comparison. The isolated pure strains were cultured in MMC medium with PS as the sole carbon and energy source, and their growth was observed to judge the degradation ability ( Supplementary Figure S4 ). Seven strains were initially found to be able to grow well with plastic ( Table 1 ), where Bacillus cereus and Acinetobacter johnsonii have been reported that they could degrade PS plastics (Yuan et al., 2022; Zhao et al., 2024). Some bacteria were isolated that were unable to degrade plastics, and they may have survived on metabolites of other bacteria. In addition, smaller irregular nanoplastic were found agglomerated together in the medium containing PS films, suggesting that the films could release microplastics and nanoplastics in the medium which could have an effect on the bacterial community.

To determine the interaction between microplastics and microbial communities, the phenomenon was observed by SEM ( Figure 2 ). In these images, PS nanoplastics (60 nm) form aggregates that were tightly clustered around the cells ( Figure 2A ). The agglomeration phenomenon can cause direct physical damage to the cells, such as alteration of the membrane structure or disruption of the cell wall (Zhao et al., 2020; Gong et al., 2023). In contrast, the plastic particles of 600 nm and 1 µm microplastics were relatively spread out without a significant aggregation phenomenon ( Figure 2B, C ). It was also observed that EPS, which consists mainly of protein and polysaccharide, was secreated by microorganisms. EPS plays a key role in the formation of microbial biofilm. Firstly, it helped microorganisms in the environment to be colonized easily, forming a small ecological niche known as the “Plastisphere” (He et al., 2022). Secondly, EPS could protect the microorganisms from toxic external stresses (Gong et al., 2023; Zhao et al., 2023). They could actively adjust their EPS release and composition on response to unfavourable stress conditions when subjected to excessive external stress ( Supplementary Figure S5 ). Large pieces of microplastic were also found in the samples enriched for bacterial communities using plastic films, which had fragmented into nanoplastics and be attached by microorganisms ( Figure 2D ). This phenomenon suggested that microplastics were released by the plastic films into the culture medium, which may be utilized by the bacterial community or affect the growth of the microorganisms.

In order to observe the effects of microplastics and films on the growth of deep-sea bacterial communities, the growth rates of bacterial communities in the enriched samples were examined ( Figure 3A ). In all samples, the growth of the bacterial community was inhibited. The strong size effect resulted in the lowest growth of the bacterial community exposed to 60 nm microplastics, which did not exceed 1%. And there was no significant difference between different concentrations (p > 0.05). Exposed to 600 nm and 1 µm microplastics, the growth rate of the bacterial community increased significantly with increasing microplstic concentration (p< 0.05). This may be due to the presence of DOC in the medium as an additional carbon source for the growth of heterotrophic microbes. The source of DOC in the samples was attributed to two reasons, one is that PS can be degraded by plastic-degrading bacteria of communities to produce water-soluble low molecular weight oligomers. Secondly, plastics could leach DOC in seawater, accompanied by an increase in plastic-derived DOC with the addition of plastic (Romera-Castillo et al., 2018; Chen et al., 2023). Further, plastic biodegradation could lead to aging, which released more DOC than new plastics. For instance, the amount of DOC leached from aging plastics (PE and PP) was up to two orders of magnitude higher than that of virgin plastics (Romera-Castillo et al., 2022). The levels of DOC in all samples were determined during the experiment, as predicted ( Supplementary Figure S6 ). The little growth rate of microorganisms was observed in the medium containing the plastic films, which was considered to be inhibited by nanoplastics released from the films. This was confirmed by SEM of microplastics in the medium within the incubation process ( Figure 2 ).

Oxidative damage caused by ROS generation was usually considered as one of the main toxicity mechanisms of nanoparticles (Ning et al., 2022). In this study, intracellular ROS generation of bacterial communities exposed to microplastics and plastic films was measured at the same cell density. As shown in Figure 3B , ROS level was significantly enhanced in all samples when compared with the control group (p< 0.05). Lower levels of ROS were produced by the bacterial community exposed to 600 nm and 1 µm microplastics for 50 days and there was no significant difference between the different concentrations (p > 0.05). Bacterial community in the medium containing 60 nm microplastic and plastic films produced a higher amount of ROS. Exposure to 60 nm led to more ROS production with increase concentration of nanoplastics (p< 0.05). The possible reason for this was that the external environmental stress generated by the higher concentrations of microplastics resulted in increased level of ROS (Qiang and Cheng, 2021). The ROS was also produced in the medium containing plastic film (B10), which was supposed to be the result of oxidative stress in the microorganisms caused by the small particle size microplastics released from the film. This was consistent with the results of other studies. For example, Escherichia coli and Bacillus sp. produced more ROS exposed to 60 nm for 8-11 h than the larger PS particles (220, 430, 700, 1040, 1700 and 2260 nm) (Kim et al., 2022). The high level of ROS production may result in tolerant microorganisms remaining alive during long-term enrichment of the bacterial community.

The rarefaction curves based on the indices showed smoothness under different conditions, indicating that the sequencing volume was sufficient to cover the microbial community in all samples ( Supplementary Figure S7 ). The specific values are listed in Supplementary Table S1 . The Sobs index was used to indicate community richness. Compared with the control, the addition of PS particles or films resulted in a decrease in the abundance of the biological communities in varying degrees. The lower Sobs values in the 600 nm and 1 µm treatments indicated that larger particle sizes of microplastics had a greater impact on the richness of the microbial communities. The Shannon index was used to indicate the diversity of the biological community. Compared with the control, the diversity of the 60 nm group remained almost unchanged or increased slightly. The diversity of the 600 nm group and 1µm group decreased, implying that the bacterial community was effectively enriched. This further demonstrated that microplastics with larger particle sizes may be better utilized by microorganisms as the sole source of carbon and energy. The growth rate of microorganisms verified this result ( Figure 3A ). It was also worth our attention that the Sobs and Shannon values in the samples enriched by the addition of PS films were in the middle of the values of the 60 nm and 600 nm experimental groups, which may be due to the production of smaller particle size microplastics from PS films during the microbial degradation process. It has been shown that the average particle size of microplastics produced was 614 nm and 496 nm when PS films was biodegraded by Alcanivorax xenomutans and Halomonas titanicae for 30 days, respectively (Lv et al., 2024). PCoA analysis based on OUT levels showed that the community composition of bacteria was significantly separated under the influence of plastic microspheres and films ( Supplementary Figure S8 ).

Microplastics have a selective effect on microbial communities, while microbial community structural characteristics and compositional types play a crucial role in plastics degradation. Overall, Proteobacteria were dominant in all samples. Microbial populations in all samples were relatively similar at the phylum level, but differences in abundance were observed. Compared with the control, the relative abundance of Proteobacteria decreased and the relative abundance of Bacteroidota increased in all samples with plastic as the sole carbon source. The relative abundance of Actinobacteriota (B1, B2, B5, B6, B7, B10) and WPS-2 (B1) increased ( Supplementary Figure S9 ).

As the level of family ( Figure 4 ), Moraxellaceae have the highest relative abundance in the enriched samples, accounting for 48.2%-96.0% of species. It was followed by Alcanivoracaceae (0.1%-21.8%), Rhizobiaceae (0.1%-12.1%). There was a decrease in the abundance of Moraxellaceae and an increase in the abundance of Alcanivoracaceae in the samples exposed to 60 nm and plastic films than that in the samples exposed to 600 nm and 1 µm microplastics. It was evident that the growth of Moraxellaceae would be inhibited due to its relative sensitivity to 60 nm nanoplastics. And the reason for this phenomenon in plastic films is that smaller nanoplastics were released during biodegradation. The increase in the abundance of Alcanivoracaceae may have a certain degree of tolerance to small particle size microplastics. Among the available reports, Alcanivoracaceae occupied considerable abundance in polluted areas containing polycyclic aromatic hydrocarbons (PAHs), oil, etc., which was seen to be adaptive to environmental changes (Chikere et al., 2019; Gosai et al., 2022). In summary, microplastics and nanoplastics may be detrimental to the growth of certain species of microorganisms. Also, microplastics as a carbon source may promote the development of rather complex microbial communities.

To further investigate the functional characteristics of the microplastics or films enriched samples, the KEGG database was used to analyse the metabolic pathways. As shown in Figure 5A , the KEGG level 1 pathways included six pathways. The order of relative abundance was metabolism > genetic information processing > environmental information processing > human diseases > cellular processes > organismal systems. KEGG level 2 analysis revealed a total of 46 secondary functional genes detected ( Figure 5B ). The largest proportions of functional abundance were found with global and overview maps, carbohydrate metabolism, amino acid metabolism, metabolism of cofactors and vitamins, and energy metabolism. Compared with the control, amino acid metabolism and carbohydrate metabolism were slightly decreased by the addition of microplastics and films. This may be caused by two factors, one is that ROS produced by microorganisms are constantly stimulated and scavenged due to the prolonged presence of microplastic, further leading to genotoxicity affecting the metabolic functions of microorganisms (Zhang et al., 2023). The second is that some microorganisms in the community can grow using the products resulting from the plastic biodegradation by other microorganisms as a carbon source (Sun et al., 2022a). In contrast, energy metabolism pathways increased, which was probably due to the fact that microorganisms require more energy to sustain growth in a relatively oligotrophic environment and stress from microplastics (Sun et al., 2022a; Wu et al., 2023). Membrane transport is a protein system that governs unfavorable changes in the bacterial environment and plays an essential part in informing environmental risk assessment (Hamlin et al., 2015). Regarding membrane transport and signal transduction genes, functional abundance was decreased due to the addition of microplastics and films. This result showed that both commercial microplastics and secondary microplastics released from plastic films could impair the ability of biofilms to handle environmental information, inhibiting them from rapidly adapting to environmental changes. Higher cell motility protects against toxic and harmful substances (Gong et al., 2023). The abundance of cell motility decreased when microplastics and films were added, especially exposed to 600 nm and 1 µm microplastics, suggesting that larger particle size microplastics could lead to a decrease in the ability of microorganisms to avoid toxic and harmful substances.

The phenotypic changes of the bacterial community were analysed using BugBase. As shown in Figure 6A , form-biofilm relative abundance phenotypes increased in the enriched samples after microplastic and film exposure, which is consistent with the enrichment of bacterial communities in soil by microplastics (PS, PVC, PE) (Zhu et al., 2022). Exposure to microplastics and plastic films reduced the relative abundance of potentially pathogenic within 50d. This is not consistent with current results that pathogenic bacteria could be enriched by microplastic. The possible reason for this is that pathogenic bacteria can be enriched in the short term and unfavourable under long-term stress, and the results of bacterial communities enriched by PS microplastics in freshwater sediments confirmed our suspicion (He et al., 2024).

FAPROTAX analysis was used to map the 16S rRNA sequencing data to metabolic or other ecologically relevant functions (Sun et al., 2022b). As shown in Figure 6B , it was highly enriched in all samples on chemoheterotrophy, aerobic chemoheterotrophy and aromatic compound degradation. The relative abundance of chemoheterotrophy and aerobic chemoheterotrophy increased suggesting that the bacteria obtained carbon and energy for their growth mainly by oxidizing organic compounds (Zhang et al., 2018; Sun et al., 2022b). PS is a single-use thermoplastic composed of aromatic styrene monomers (Bhanot et al., 2024). The increase on aromatic compound degradation genes indicated that the samples were effectively enriched. Compared with 60 nm and plastic films, 600 nm and 1 µm resulted in higher enrichment of aromatic compound degradation genes. It was believed that larger particles of microplastics would be more susceptible to microbial growth as a carbon and energy source. Notably, the 60 nm microplastics were significantly enriched for plastic degradation gene, although they severely inhibited the growth of microbial communities. This may be due to the fact that most of the plastic-degrading bacteria in the environment can adapt to the toxic chemical microenvironment of microplastics. There is no much enrichment of plastic degradation gene in the medium containing plastic films even though smaller microplastics could be produced. It may be related to its concentration, shape and other factors, which need to be further verified. The functional analysis of the bacterial community in this study was based on 16S rRNA gene, and further metagenomic analyses are required to fully understand the function of the bacterial community in the enriched samples.