# When plastisphere and drilosphere meet: Earthworms facilitate microbiome and nutrient turnover to accelerate biodegradation of agricultural plastic films

Caide Huang, Liuwei Wang, Wei-Min Wu, Yvan Capowiez, Yuhui Qiao, Deyi Hou

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Caide Huang, Liuwei Wang, Wei-Min Wu, Yvan Capowiez, Yuhui Qiao, et al.. When plastisphere and drilosphere meet: Earthworms facilitate microbiome and nutrient turnover to accelerate biodegradation of agricultural plastic films. Environment International, 2025, 196, pp.109309. 10.1016/j.envint.2025.109309. hal-05019812

HAL Id: hal-05019812

https://hal.inrae.fr/hal-05019812v1

Submitted on 3 Apr 2025

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
Full length article

When plastisphere and drilosphere meet: Earthworms facilitate microbiome
and nutrient turnover to accelerate biodegradation of agricultural
plastic films

<figure><img src="image_3.png" /></figure>

Check for
updates

Caide Huang<sup>a</sup>, Liuwei Wang<sup>a,b</sup>, Wei-Min Wu<sup>b</sup>, Yvan Capowiez<sup>c</sup>, Yuhui Qiao<sup>d</sup>, Deyi Hou<sup>a,*</sup> <sup>ID</sup>

ARTICLE INFO

Keywords:

Plastics

Amynthas corticis

Biodegradation

Plastisphere

Dissolved organic matter

A B S T R A C T

Agricultural plastic mulching films have been an environmental concern for decades. The effects of the in-
teractions between the anthropogenic plastisphere and other soil biospheres, particularly that of earthworms, on
the fate of plastics remain poorly understood. Here, we investigated the decomposition of buried nonbiode-
gradable low-density polyethylene (LDPE) versus biodegradable PBTA/PLA copolymers in the presence of
earthworms (Amynthas cortices) in dynamic microcosms. Earthworms significantly enhanced the biodegradation
of plastic films in situ, as confirmed by mass reduction, surface modification, and changes in the molecular
weights of films. Notably, the PBTA/PLA films exhibited a 1.41-fold increase in mass loss and a 5.69% reduction
in the number-average molecular weight when incubated with earthworms. Earthworms influenced the micro-
bial assembly within the plastisphere by increasing both bacterial and fungal biodiversity, as well as their
network complexity. The time-decay patterns in the abundance of keystone degrader taxa, including the genera
Noviherbaspirillum, Rhizobacter, and Mortierella, were mitigated by earthworms over the 60-day period. Addi-
tionally, earthworms preferentially consumed recalcitrant dissolved organic matter in LDPE and PBAT/PLA
plastisphere soils, thereby increasing the bioavailability of components that serve as nutrient supplies for plas-
tisphere microbiomes. Our findings demonstrate that earthworms enhance the decomposition of plastics in soils
via cross-species interplay within the plastisphere and drilosphere, contributing not only to soil conditioning and
biodiversity but also to plastic biodegradation in natural agroecosystems.

1. Introduction

Plastic mulching films are extensively used in agriculture because of
their benefits for soil management and crop yields, sparking a “white
revolution” in agricultural technology worldwide since the 1970 s (FAO,
2021; Zhang et al., 2021). However, overuse and low recycling rates
have turned agricultural soils into major reservoirs of plastic debris,
contributing to pervasive “white pollution” (Hofmann et al., 2023; Liu
et al., 2014). An estimated 12.5 million tons of plastic products are
applied in agriculture annually, including over 4 million tons of plastic

films (Briassoulis, 2023; Hofmann et al., 2023). China accounts for
nearly 60 % of global mulch film consumption, with usage increasing
annually (Khalid et al., 2023; Sun et al., 2020; Xiao et al., 2023). The
accumulation of plastic residues leads to contamination by macro,
micro, and nanoplastics, degrading soil functions by affecting fertility,
microorganisms, and macrofauna (Daghighi et al., 2023; Qian et al.,
2018; Wu et al., 2022). Moreover, plastic debris and chemical additives
have been reported to bioaccumulate and transform through food
chains, posing risks to human health (Li et al., 2020; Qi et al., 2020a; Shi
et al., 2019). To date, plastic mulch film-degrading bacterial cultures

**Abbreviations:** LDPE, Low-density polyethylene; PBAT, Poly(butylene adipate-co-terephthalate); PLA, Polylactic acid; FTIR, Fourier-transform infrared spectroscopy; XPS, X-ray photoelectron spectroscopy; WCA, Water contact angle; AFM, Atomic force microscope; GPC, Gel permeation chromatography; M<sub>n</sub>, Number-average molecular weight; M<sub>w</sub>, Weight-average molecular weight; SEM, Scanning electron microscope; CLSM, Confocal laser scanning microscopy; FT-ICR MS, Fourier-transform ion cyclotron resonance mass spectrometry; DOM, Dissolved organic matter.

<footnote><sup>a</sup> School of Environment, Tsinghua University, Beijing 100084, China</footnote>

<footnote><sup>b</sup> Department of Civil and Environmental Engineering, William & Cloy Codiga Resource Recovery Center, Stanford University, Stanford, CA 94305-4020, United States</footnote>

<footnote><sup>c</sup> INRAE, UMR EMMAH INRAE-Université d'Avignon, Site Agroparc, 84914, Avignon, Cedex 09, France</footnote>

<footnote><sup>d</sup> College of Resources and Environmental Science, China Agricultural University, Beijing 100193, China</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: houdeyi@tsinghua.edu.cn (D. Hou).</footnote>
have been found and isolated, although the degradation rate is slow (Yin et al., 2020). Since the 1990s, biodegradable films, mainly made from poly(butylene adipate-co-terephthalate) (PBAT) and polylactic acid (PLA), have seen a rapid increase in application and are considered promising alternatives to traditional nonbiodegradable or persistent polymers, particularly those made of polyethylene (Albright III and Chai, 2021; Brodhagen et al., 2015; Qi et al., 2020b). Although biodegradable, this plastic can degrade into microplastics and nanoplastics, and incomplete degradation may still pose significant environmental risks (Haider et al., 2019; Mo et al., 2023; Xiong et al., 2024).

Plastic residues provide a durable, carbon-rich habitat for bacterial and fungal colonization, forming a unique ecosystem known as the “plastisphere” (Amaral-Zettler et al., 2020; Li et al., 2021). Originally defined as the microbial community on plastics in aquatic ecosystems, the term has recently been adopted in terrestrial studies and now refers to the entire soil microenvironment immediately influenced by plastics (Rillig et al., 2024; Rüthi et al., 2023). The assembly of microbial communities within the plastisphere niche can dominate the decomposition fates of plastic debris (Miao et al., 2023; Xiang et al., 2022). Previous studies have suggested that the soil plastisphere has greater microbial abundance, greater enzymatic activity and faster nutrient turnover than do bulk soils (Kublik et al., 2022; Xiang et al., 2022; Zhou et al., 2021). Once plastics enter the soil, microorganisms initially adhere to the surface, followed by the selective colonization of functional microbiota that secrete extracellular hydrolases for polymer biodegradation (Lambert and Wagner, 2017; Sander, 2019). Furthermore, some studies have shown that different soil conditions shape distinct plastisphere communities and thus influence the efficiency of plastic biodegradation (Han et al., 2021; Tanunchai et al., 2023). Interactions between the soil plastisphere and surrounding communities inevitably occur, leading to niche overlap or complementarity, which influences plastic fate (Rillig et al., 2015; Wang et al., 2024). Notably, the multiple influences of soil fauna on the functional assembly of the plastisphere remain poorly understood.

Earthworms are soil-dwelling terrestrial invertebrates that belong to the phylum Annelida and are regarded as “soil ecological engineers”. They contribute significantly to the carbon, nitrogen, and mineral cycles in terrestrial environments (Ratsiatosika et al., 2024). Earthworms ingest plastic debris and fragment it into micro- and nanosized particles (Kwak and An, 2021; Meng et al., 2023; Wang et al., 2022). Biofragmentation and biodegradation of compostable polymers, i.e., PLA and PBAT, were confirmed in the presence of earthworm *Lumbricus terrestris* via gel permeation chromatography (GPC) analyses. However, no significant evidence supports LDPE biodegradation by earthworms because of the limitations of the analytical methods used, although the presence of plastic-degrading gut bacteria cannot be ruled out (Carpena-Istan et al., 2024; Huerta Lwanga et al., 2018; Munhoz et al., 2024). Additionally, a frequently overlooked effect is that earthworms, through their bioturbation activities, can modify the immediate environment of buried plastics and facilitate their degradation.

The “drilosphere”, which refers to the soil composition influenced by earthworms, is a key zone for soil microbiota and carbon metabolism (Hoang et al., 2020). It is the portion of soil that has passed through the digestive tract of earthworms or that lines their burrows. As with to the interactions between plants and the soil rhizosphere (Bergmann et al., 2020; Huang et al., 2021), the interactions between the drilosphere and the plastisphere, which are mediated by earthworms could reshape the interspecies collaboration and resource acquisition. The positive effects of earthworms on soil processes and plant growth are well documented, however, their influence on the fate of plastic debris remains unknown. The earthworms *Amynthas corticis* (Kinberg, 1867), which is native to East Asia but widely invasive in various other regions, belong to the family Megascolecidae (Ziemba et al., 2015). They are known as “jumping worms” because of their erratic thrashing behavior when disturbed (Chang et al., 2021). To date, the ability of these jumping earthworms to biodegrade plastics and the relationship between their

drilosphere and the soil plastisphere has not yet been reported.

In this study, we selected jumping earthworms as model species in soil microcosms containing both traditional and biodegradable plastic films, i.e., LDPE versus PBAT/PLA, respectively, to investigate the dynamic interactions within the plastisphere. We hypothesized that earthworms would trigger faster microbial community succession within the plastisphere and enhance the niche advantage of keystone functional microorganisms in plastic decomposition. Plastisphere soil carbon turnover, which is mediated by earthworms, could be an underlying driver of fauna-microbiota collaboration. To verify this, we investigated (1) the dynamic decomposition and biodegradation of plastic films incubated with or without jumping earthworms; (2) biofilm formation, microbial colonization and succession within the plastisphere mediated by earthworms; and (3) alterations in the molecular composition of dissolved organic matter in plastisphere soils and their interplays with the functional microbiota. This study offers novel insights into soil plastisphere research, emphasizing cross-species collaboration dominated by invertebrates in soil ecosystems.

## 2. Materials and methods

### 2.1. Soil, earthworms and plastic films

Soil samples were collected from the top 20 cm layer of a vegetable field in Haidian District, Beijing (40°13′71″ N, 116°18′01″ E) that had never been mulched. The soil samples were air-dried, homogenized, and sieved through a 2 mm mesh for subsequent microcosm incubation. The detailed soil properties are provided in Table S1. Adult earthworms with developed clitellum were sourced from China Agricultural University, Beijing, and identified as the common farmland species *A. corticis* through mitochondrial DNA sequencing (Table S2). *A. corticis* exhibits epi-endogeic behavior, primarily living within the soil, and is capable of ingesting large quantities of both soil and organic matter, as noted in previous studies (Bottinelli et al., 2020; Gong et al., 2023). Earthworms were acclimated to the test soils for two weeks prior to incubation.

LDPE films (> 99 % purity) and Ecovio PBAT/PLA mulching films (85/15, w/w%) were purchased from Yangtai Plastic Products Factory and Jialegmi Technology Co., Ltd. (Zhejiang, China), respectively. Plastic films with a thickness of 0.01 mm were cut into square sheets (20 × 20 mm) and washed three times with 70 % ethanol and Milli-Q ultrapure water. The size of the plastic used in this study was typical of most agricultural residual films and large enough to prevent ingestion by the earthworms (Li et al., 2022; Wang et al., 2022).

### 2.2. Microcosm Experiment

The earthworm-mediated ecosystem microcosm was established in 10 cm³ cubic glass jars. Four paired treatments were designed: LDPE films only (designated LD), LDPE films with earthworms (designated LDE), PBAT/PLA films only (designated PB), and PBAT/PLA films with earthworms (designated PBE) (Fig. 1A). Four replicates were prepared for each treatment, and all the pots were set in duplicate for two sampling periods (Day 30 and Day 60). Each glass chamber was filled with 800 g soil, and four pieces of LDPE or PBAT/PLA plastic film sheets were buried horizontally in the middle layer of the soil column during soil filling (4 cm from the soil surface and from the bottom of the chamber) (Fig. 1A). Four earthworms (1 ± 0.1 g each) were introduced on the soil surface after a 24-h gut depuration in the earthworm coincubation groups. We chose a high but realistic density (400 ind/m²) to ensure that drilosphere bioturbation occurred near the plastic sheets. The soil moisture was initially adjusted to 60 % of its maximum water-holding capacity and soil moisture was maintained by weighing every two weeks. All pots were placed in an artificial climate chamber and incubated in the dark at 25°C for 30 or 60 days.

The incubation soils were carefully removed from each pot on Days 30 or 60. Buried films were collected using sterile forceps, and their
<figure><img src="image_4.png" /><figcaption>(B)</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>(C)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>(D)</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>(E)</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>(F)</figcaption></figure>

<figure><img src="image_9.png" /></figure>

**Fig. 1.** Microcosm experimental design and earthworm contributions to the decomposition of plastic films. (A) Experimental setup for incubation of *A. corticis* earthworms and plastic films, (B) generation of earthworm casts on the surface, (C) visible differences in plastic films from various treatments after soil incubation, (D) bioturbation and vertical migration of mulch films by earthworms were assessed in comparison to their height on Day 0, and (E-F) mass changes and decomposition of LDPE and PBAT films after incubation. Note: LD = LDPE only; LDE = LDPE + worms; PB = PBAT/PLA only; and PBE = PBAT/PLA + worms.
vertical depth was measured and recorded with a caliper. The horizontal movement of the plastic films was not quantified in this study. The soil particles adhering to the plastic films were carefully scraped off, collected, and considered plastisphere soils (Rüthi, 2022; Rüthi et al., 2023). Some of the plastic films were thoroughly cleaned with sterilized phosphate-buffered saline (PBS) to remove residual soil and then prepared for biofilm and microbial tests. The remaining films were ultrasonically washed with ultrapure water and air-dried for mass loss calculations and polymer analysis. The earthworms were collected, washed, and depurated in Petri dishes for 24 h. Biomass loss was determined by calculating the weight difference ((initial weight – final weight)/initial weight). The production of surface casts was also determined to assess earthworm bioturbation (Fig. 1B) (Piron et al., 2017). The cleaned virgin plastic films were used as LD0 and PB0 controls for comparison.

## 2.3. Characterization of plastic decomposition and biodegradation

GPC was used to evaluate the molecular weight changes of the plastic films, as described in Text S1. Stable isotopic analysis (δ¹³C value changes) was used to verify plastic biodegradation, as detailed in Text S2. The degradation of the plastic films was photographed, and surface cracks were observed using scanning electron microscopy (SEM). To examine the mineralization characteristics, Fourier-transform infrared (FTIR) spectroscopy (Nicolet iS50, Thermo, USA) with a wavenumber range of 4000–400 cm⁻¹ and a resolution of 4 cm⁻¹ as well as X-ray photoelectron spectroscopy (XPS) (250Xi, Thermo, USA) with a 0–1200 eV broadband energy range and 90° electron take off angle were used to detect changes in polymer functionality (Wang et al., 2023). The hydrophobicity of the plastic films was measured using a water contact angle (WCA) device (OCA25, DataPhysics, Germany). An atomic force microscope (AFM) (Dimension FastScan, Bruker, USA) was used to determine the surface roughness in peak force tapping mode.

## 2.4. Analysis of biofilm and Microbiome of plastisphere

The plastic films were transferred into sterile centrifuge tubes containing a mixture of fluorophores, including propidium iodide (PI) and SYTO-9 Stain (Thermo, USA), to stain dead cells red and living cells green, respectively. Biofilm observation and image acquisition were performed using confocal laser scanning microscopy (CLSM; IX83, Olympus, Japan). The biofilms on the plastic films were infiltrated and fixed with a 10 % methanol solution, stained with 0.1 % crystal violet, and then decolorized with a 95 % ethanol solution. The absorbance of the decolorized solution was measured at 595 nm to assess the biofilm concentration (Liu et al., 2023). The extracellular protein content was determined using the Coomassie Brilliant Blue method with the eluted cell suspension from the biofilm. Virgin film and Milli-Q water treatments were included as quality controls. Microbial colonization of and attachment to plastic films were visualized using SEM (MERLIN Compact, ZEISS, Germany). Total DNA was extracted from biofilms scraped from the LD and PB plastic films after 30 and 60 days of incubation. The quality and concentration of the DNA were determined using spectrophotometry (NanoDrop, Thermo, USA). PCR amplification of the 16S rRNA and 18S rRNA genes was performed to investigate the bacterial and fungal communities of the plastisphere (Koh et al., 2023; Xiang et al., 2022). The detailed protocols are provided in Text S3.

## 2.5. FT-ICR MS analysis of plastisphere soils

To explore the trade-offs between resource acquisition and community succession of plastisphere microorganisms, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was employed to determine the carbon dynamics of the plastisphere influenced by earthworms (Rummel et al., 2021). DOM from the plastisphere soil (after 60 d of incubation) was extracted using Milli-Q water and then

desalted and concentrated with a solid-phase extraction (SPE) column. Measurements were conducted using a 15.0 T FT-ICR MS (SolariX, Bruker, Germany) instrument equipped with a negative electrospray ionization (ESI) source. The raw spectra were quality controlled and screened to identify seven molecular DOM formulas based on the ratios of oxygen to carbon (O/C) and hydrogen to carbon (H/C) (Zhou et al., 2023). The modified aromaticity index (AI<sub>mod</sub>), double bond equivalents (DBEs), and nominal oxidation state of carbon (NOSC) were calculated to evaluate and compare the characteristics of the DOM formulas. The detailed analytical methods and instrumental parameters are provided in Text S4.

## 2.6. Statistical analysis

Statistical analyses were performed using SPSS 26 (IBM, USA), and data plotting was performed via Prism 10 (GraphPad, USA). The significance of the LD or PB treatments across different time points was tested using a one-way analysis of variance (ANOVA) (*P* ≤ 0.05). Pearson's correlation and linear regression analyses were used to investigate the relationships between plastic biofilms and microorganisms. Microbial co-occurrence and DOM-microbe interaction networks were constructed in R-Studio (v 3.6.0) with the "igraph" and "psych" packages and then refined and visualized using Gephi (v 0.10.1).

# 3. Results and discussion

## 3.1. Time Course of microcosm ecosystem

The results showed that the presence of earthworms significantly affected the vertical transport of the LD and PB films (Fig. 1). After burial in the microcosms, slight changes in surface roughness and color were observed (Fig. 1C). Compared with the LD films, the PB films showed more surface changes, with more pronounced changes observed in the microcosms containing earthworms. Specifically, large cracks formed on the PB films in the PBE group by Day 60 (Fig. 1C). Some film pieces were transported up to 1.50 cm (upward) and -0.80 cm (downward) after a 60-day incubation. As the incubation time increased, the buried plastic films shifted to either the upper or deeper soil layers with the help of earthworms (Fig. 1D). The epi-endogeic ecological category of *A. corticis*, which feeds and casts on the soil surface but lives in the upper layers of the soil, contributes to the plastic film migration (Gong et al., 2023). These findings demonstrate the opportunity for interactions between earthworms and the plastisphere of mulch films.

The average weight of the earthworms decreased over the incubation time due to the lack of an external food supply (Fig. S1). On Day 60, surface cast production increased by 84.9 % and 71.5 % in the LDE and PBE groups, respectively, suggesting active movement and excretion of the earthworm *A. corticis* in the presence of macroplastics (Fig. S2). The mass reduction efficiencies of the LD and PB films were determined on Days 30 and 60. No significant weight changes were observed for the LD or LDE groups, indicating that the LDPE films did not lose measurable mass after 30 or 60 d of incubation regardless of whether the earthworms were present (Fig. 1E). In contrast, the introduction of earthworms significantly enhanced the mass reduction of PB films significantly. The buried films in the PB group experienced a 3.39 % mass loss on Day 30 and 6.21 % mass loss on Day 60, whereas the PBE films experienced a 4.80 % mass loss on Day 30 and 8.77 % mass loss on Day 60, indicating that the activities of the earthworms significantly increased the plastic removal efficiency (*P* ≤ 0.05) (Fig. 1F).

## 3.2. Biodegradation of plastic films Driven by earthworms

Jumping earthworms played key roles in the surface and functional group changes of plastic films (Fig. 2). The SEM images revealed increased roughness and microbial attachments on both LD and PB films. Notably, large cracks were observed on the PB films with the
<figure><img src="image_10.png" /><figcaption>**Fig. 2.** Film surface morphology and biodegradation evaluation of plastic films. (A) SEM images of plastic films after incubation with or without earthworms, red arrows suggest decomposition and cracking of plastic films, whereas green arrows indicate microbial attachment; (B) FTIR spectrum and functional group changes of PB films from different treatments; (C) GPC molecular weight shifts of PB films after incubation with earthworms; (D-E) XPS scanning of PB films; (F) WCA changes in PB films incubated with earthworms; (G-H) AFM roughness characterization of PB films. The detailed changes in the LD plastic films are provided more in the supplementary material. Note: LD = LDPE only; LDE = LDPE + worms; PB = PBAT/PLA only; and PBE = PBAT/PLA + worms. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

introduction of earthworms, which is consistent with the observed mass loss (Fig. 2A). The FTIR spectra displayed characteristic signals at 1092 cm⁻¹ and 1710 cm⁻¹, corresponding to C–O and C=O stretching vibrations, respectively (Fig. 2B; Fig. S3). The oxidation levels of the plastic films increased with prolonged exposure time, particularly in samples incubated with earthworms, which was evidenced by a notable increase in the absorbance intensity of these functional groups. GPC analyses indicated that after 60 d of incubation, the Mₙ values of the LDE60 and PBE60 films decreased by 3.85 % and 5.69 %, respectively, compared with those of the LD60 and PB60 groups, indicating greater plastic biodegradation under the earthworm treatments (Fig. 3C; Fig. S3). An isotopic assay further verified the effects of earthworms on plastic biodegradation by increasing the δ¹³C values, particularly for the

PB films (Fig. S4). The XPS scanning spectra further confirmed the above findings, showing an increase in the relative peak area proportions of C–O for both LD and PB and of C=O for the PB films (Fig. 3D, E; Fig. S5). In summary, the results demonstrated that LD film biodegradation occurred at extremely low rates in the microcosm and was accelerated in the presence of earthworms, although the degradation rate remained slow. The PB film was biodegraded by soil microbes and the degradation rate was significantly increased by the introduction of the earthworms.

Importantly, in this study, commercial mulch films were selected and directly buried in soil to simulate real-world scenarios involving agricultural plastic residues. This approach differs from previous studies that used artificially made films from pure plastic particles created by hand-casting after the raw materials were melted without any exogenous
<figure><img src="image_11.png" /><figcaption>**Fig. 3.** Plastisphere biofilm formation and physiological quantification. (A) Fluorescence microscopy observation of the stained live (green) and dead (red) cells, (B-C) biofilm concentrations of LP and PB films, and (D) extracellular protein contents of the plastisphere biofilm. Each film corresponds to two individual and one merged visual field with a 50 µm scale bar. Note: LD = LDPE only; LDE = LDPE + worms; PB = PBAT/PLA only; and PBE = PBAT/PLA + worms. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

additives. Such studies reported higher degradation and mineralization rates than those observed in our results (Han et al., 2021; Qiang et al., 2021; Wu et al., 2023). Furthermore, unlike studies that focused on the ingestion and gut digestion of earthworms (Meng et al., 2023; Wang et al., 2022), our research used macroplastic-sized pieces that earth-worms cannot ingest directly. In this context, earthworms act as “bio-logical catalysts”, promoting the microbial mineralization of plastic films in soil through their drilosphere activities (including burrowing, casting and mucus deposition) (Hink et al., 2024; Sanchez-Hernandez et al., 2020).

### 3.3. *Dynamic microbial colonization and succession of plastic films*

The CLSM images showed biofilm formation and attachment on the LD and PB films (Fig. 3A). Initially, the biofilms appeared as dots and radial shapes during the early stage (30 d). As the incubation period progressed, the biofilms became more agglomerated and distributed in patches. Additionally, the presence of earthworms enhanced biofilm growth, resulting in more abundant green living extracellular polymeric substances (EPSs). Quantitative analysis indicated that earthworms significantly increased biofilm concentrations by 20.5 % and 26.5 % over the LD60 and PB60 films, respectively ($P \le 0.05$) (Fig. 3B, C). The EPS protein content also increased with incubation time, particularly under coincubation with earthworms. The biofilm protein contents in
the PBE60 and LDE60 groups were 8.94 and 7.11 µg/ml, respectively, which were 1.46 and 1.20 times greater than those in the PB60 and LD60 groups, respectively (Fig. 3D). Moreover, the presence of earthworms resulted in a lower water contact angle and rougher surface (Fig. 2; Table S3) on the plastic films, creating a microhabitat with greater hydrophilicity and better adhesion for plastisphere microorganisms (Wu et al., 2023). These findings provide visual evidence of the positive effects of earthworms on plastic microbial colonization which is essential for the microbial biodegradation of plastics.

Alpha diversity analysis of the sequenced data revealed that the presence of earthworms increased the richness of the plastisphere microorganisms after 60 d of incubation. Both the Shannon and Chao1 indices of bacteria and fungi increased in almost all paired groups when they were coincubated with earthworms except for those in the LDE30 treatments (Fig. 4A-D). The PCoA analysis of beta diversity showed clear spatial separation for bacteria between the control and each type of plastic, suggesting unique microbiota selection under distinct plastisphere habitats (Fig. 4E, F) (Amaral-Zettler et al., 2020). In addition, the microbial abundance of the plastisphere was significantly positively

correlated with the plastic biofilm (Fig. 4G, H) and EPS protein concentrations ($P \le 0.05$) (Fig. S6). The number of unique amplicon sequencing variants (ASVs) was greater for the LDE and PBE films, reaching 7,511 and 962 for bacteria and fungi, respectively, in the PBE60 group (Fig. S7). The heatmap showed significant differences in microbial colonization among the different groups, revealing that *Sphingomonas*, *Rhizobacter*, *Endomyxa* and *Cercozoa*, were the specifically enriched genera (Fig. 4I; Fig. S8). The LDA effect size (LEfSe) analysis supported these results, showing more microbial biomarkers across all groups (Fig. S9).

The interactions among plastisphere microorganisms were influenced and reshaped by the earthworms. The co-occurrence patterns indicated that both the PBE and LDE treatments resulted in more complex networks, with higher densities, average degrees, and clustering coefficients (Table S4; Fig. S10). After coincubation with earthworms for PBE films, the number of nodes and edges greatly increased from 370 to 464 and from 2,800 to 4,674, respectively (Fig. 4J, K). The colonization and succession of plastisphere microbiota are synchronized with the development of surface biofilms, contributing to the mineralization of

<figure><img src="image_12.png" /><figcaption>**Fig. 4.** Biodiversity and structure of the plastisphere microbial community. (A-F) Alpha and beta diversity of the plastisphere bacteria and fungi on the LD and PB films, (G-H) linear correlation between biofilm concentration and microbial abundance, (I) heatmap of the relative bacterial abundance at the genus level, (J-K) co-occurrence network analysis of PB and PBE bacteria at the phylum level. Note: LD = LDPE only; LDE = LDPE + worms; PB = PBAT/PLA only; and PBE = PBAT/PLA + worms.</figcaption></figure>
plastic films (Hong et al., 2024). Core microorganisms were identified through Pearson's correlation between the most abundant bacteria/ fungi and biofilm parameters (Fig. S11). Notably, the bacterial genera *Sphingomonas* and *Noviherbaspirillum*, as well as the fungi genera *Mortierella* and *Sordaria* were significantly correlated. Their roles in polymer degradation were confirmed through a review of related studies (Table S5). For example, the genus *Sphingomonas* was previously isolated from petroleum products and has shown great potential for polyethylene degradation (Padmanabhan et al., 2020).

The abundance changes and time-decay curves of these functional genera are shown in Fig. S12, illustrating the dynamic succession of the core functional microbiota. As observed in previous studies (Woodcock and Sloan, 2017; Xiang et al., 2022), the plastisphere community exhibited a time-specific pattern in which resource competition among microorganisms eliminated some of the initial colonists. Some key genera including *Sphingomonas*, *Noviherbaspirillum*, *Variovoraxc*, and *Ramlibacter*, decreased in relative abundance from Day 30 to Day 60; however, the presence of earthworms delayed this decline or led to a continued increase (Fig. S13). Additionally, the slopes of the time-decay curves for the above keystone taxa were much steeper in the LD and PB groups compared with those in the LDE and PBE groups. These findings reveal that earthworms can influence the microbial assembly processes of the plastisphere and mitigate the time decay of functional microorganisms.

### 3.4. Earthworm-mediated carbon turnover of plastisphere soils

Compared with that in the LD and PB groups, the number of DOM

molecules in the plastisphere soils decreased after 60 d of incubation with earthworms (Fig. 5; Fig. S13). The number of identified formulas declined from 2,718 to 2,473 for the LD plastisphere soils and from 9,395 to 5,317 for the PB treatments. Earthworms tended to consume complex CHONS compounds while increasing the proportion of simpler CHO compounds. According to the van Krevelen diagram, stable compounds such as lignin and tannin were transformed into more easily degradable units such as lipids, proteins, amino sugars, and carbohydrates. For example, the total proportion of lipids and proteins increased from 11.7 % (PB60) to 22.0 % (PBE60) due to earthworm activity (Fig. S14). Earthworms induced the production of 50.8 % "newly produced" (identified only in the earthworm group) DOM molecules relative to PB group, which was 1.44 times greater than the increase in the LD treatment group (Fig. 5A-C). Moreover, in the LD and PB groups, the respective average molecular parameters showed that earthworm introduction decreased the values of AIw (18.2 % and 22.6 %) and DBEw (18.0 % and 21.5 %) but increased the H/Cw ratio (1.50 % and 10.5 %), suggesting that highly aromatic DOM compounds were decomposed by earthworms (Table S6). These results indicated that plant-derived recalcitrant compounds were preferentially consumed by earthworms and converted into molecules with increased microbial availability. The soil aggregates formed through earthworm activity, along with the priming effects due to their gut microbiota, are likely to profoundly influence such carbon turnover.

Limited and slow DOM leaching from the plastic itself results in nutrient limitations for biofilm development and the colonization of functional microorganisms (Qiu et al., 2024; Sheridan et al., 2022). Therefore, the soil surrounding of the plastisphere could serve as a

<figure><img src="image_13.png" /><figcaption>**Fig. 5.** Newly produced, removed, and persistent DOM molecules in plastisphere soils mediated by earthworms. (A-B) Van Krevelen diagrams suggesting the DOM formulas identified by FT-ICR MS, (C) proportions of the three different DOM categories, (D-E) co-occurrence correlation analysis between the DOM formulas and the genera of the top bacterial and fungal taxa based on the DOM and microbial data from Day 60. The connections represent strong (Spearman's R ≥ 0.6) and significant (P-value ≤ 0.05) correlations. Note: LD = LDPE only; LDE = LDPE + worms; PB = PBAT/PLA only; and PBE = PBAT/PLA + worms.</figcaption></figure>
supplementary nutrient source (Rummel et al., 2021; Sun et al., 2022). Through co-occurrence correlation analysis, the interactions between the DOM formulas and the most abundant microbiota were further investigated (Fig. 5D, E). The network generally showed high modularity and denser connections between labile DOM compounds and bacterial/fungal genera. The CHO formulas and protein-like, and carbohydrate-like compounds were positively correlated with relative abundances of the bacteria *Noviherbaspirillum* and the fungus *Farrowia*. Negative correlations were found between core indicator microorganisms and stable DOM, such as between condensed aromatic-like compounds and *Mortierella* as well as between lignin-like compounds and *Bradyrhizobium* (Fig. 5D, E). In other words, the increase in DOM components with increased microbial utilization promoted the succession and sustained abundance of core microbiota (Fig. S14) (Chen et al., 2022; Ling et al., 2022). These findings support our hypothesis that earthworm-mediated DOM turnover in plastisphere soils alleviates carbon limitations on microorganisms, thereby enabling functional taxa to increase their ecological niche advantages. Notably, the positive effects of earthworms on microbial biodiversity and carbon chemodiversity promoted the biodegradation of plastic film residues. The introduction of earthworms can stimulate long-term external nutrient supply to the core microbiota and slow their temporal decay.

### 3.5. Implication and Limitation

Recent peer-reviewed studies, summarized in Table S7, further reinforce the critical role of earthworm species in plastic decomposition and degradation. In conclusion, the dietary ingestion by various earthworm species plays a key role in plastic decomposition and degradation, with most studies emphasizing the processes of plastic fragmentation and aging occurring within earthworm guts and casts. Vermicomposting is another typical research context that focuses primarily on changes in degradable plastics during the earthworm composting process. A common theme across these studies is the attribution of the role of earthworms in plastic degradation to the functions of microorganisms in both the earthworm gut and surrounding soils. In contrast, the current study focuses to the process of earthworm-mediated assembly of indigenous soil microorganisms within the plastisphere, providing a novel perspective on microbial interactions in specific soil microenvironments as opposed to the broader bulk soil context.

This study has several limitations that should be addressed in future research. To date, approximately 7,000 species of terrestrial earthworms have been identified globally. The effectiveness of other major earthworm species on the fate of various plastics, e.g., *Lumbricus terrestris* (Linnaeus, 1758), *Eisenia fetida* (Savigny, 1826), and *Aporrectodea caliginosa* (Savigny, 1826), should also be addressed and characterized in the future. The soil organic matter content in this study was relatively low, representing only one typical agricultural soil condition. Future studies could explore how earthworm activity and plastic degradation dynamics vary across different soil environments. Furthermore, we must acknowledge that the disturbance of residual plastic in the soil caused by earthworms may lead to the fragmentation of larger plastic pieces, thereby posing additional environmental risks. Therefore, the long-term impacts of plastic decomposition and biodegradation induced by earthworms on soil health and crop growth warrant further investigation.

### 4. Conclusion

This study established a microcosm incorporating earthworms and commercial plastic films to explore the potential effects of earthworms on plastic biodegradation. Over a 60-day incubation period, the presence of earthworms significantly enhanced the decomposition of plastic films, particularly the PBAT/PLA polymers. Morphological and chemical alterations were evidenced through plastic mass reduction analyses and advanced techniques such as FTIR, GPC, XPS, AFM, and SEM.

Additionally, the microbial community and soil carbon efficiency within the plastisphere were significantly influenced by the earthworm drilosphere, which accelerated the microbial biodegradation of plastic films. Notably, earthworm activity consumed a substantial proportion of recalcitrant DOM molecules and transformed them into more bioavailable components in plastisphere soils, as revealed by FT-ICR MS analysis. Furthermore, several microbial taxa affected by earthworms, such as *Sphingomonas*, *Rhizobacter*, *Noviherbaspirillum*, and *Mortierella*, were identified as contributors to plastic biodegradation, suggesting promising avenues for future research. These findings provide valuable insights into the fate of plastic mulching films in terrestrial environments and offer new perspectives on nature-based solutions for plastic degradation leveraging soil invertebrates and enhancing microbial activity.

### CRediT authorship contribution statement

**Caide Huang:** Writing – original draft, Methodology, Investigation.
**Liuwei Wang:** Methodology, Investigation. **Wei-Min Wu:** Writing – review & editing, Supervision.
**Yvan Capowiez:** Writing – review & editing, Supervision.
**Yuhui Qiao:** Writing – review & editing, Methodology.
**Deyi Hou:** Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 42225703; No. 42407397) and the China Postdoctoral Science Foundation (No. 2023 M741925).

### Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envint.2025.109309.

### Data availability

Data will be made available on request.

### References

Albright III, V.C., Chai, Y., 2021. Knowledge gaps in polymer biodegradation research. *Environ. Sci. Tech.* 55, 11476–11488.

Amaral-Zettler, L.A., Zettler, E.R., Mincer, T.J., 2020. Ecology of the plastisphere. *Nat. Rev. Microbiol.* 18, 139–151.

Bergmann, J., Weigelt, A., van Der Plas, F., Laughlin, D.C., Kuyper, T.W., Guerrero-Ramirez, N., Valverde-Barrantes, O.J., Bruelheide, H., Freschet, G.T., Iversen, C.M., 2020. The fungal collaboration gradient dominates the root economics space in plants. *Science. Advances* 6, eaba3756.

Bottinelli, N., Hedde, M., Jouquet, P., Capowiez, Y., 2020. An explicit definition of earthworm ecological categories–Marcel Bouché’s triangle revisited. *Geoderma* 372, 114361.

Briassoulis, D., 2023. Agricultural plastics as a potential threat to food security, health, and environment through soil pollution by microplastics: Problem definition. *Sci. Total Environ.* 164533.

Brodhagen, M., Peyron, M., Miles, C., Inglis, D.A., 2015. Biodegradable plastic agricultural mulches and key features of microbial degradation. *Appl. Microbiol. Biotechnol.* 99, 1039–1056.

Carpena-Istan, V., Jurado, M.M., Estrella-Gonzalez, M.J., Salinas, J., Martinez-Gallardo, M.R., Toribio, A.J., Lopez-Gonzalez, J.A., Suarez-Estrella, F., Saez, J.A., Moral, R., 2024. Enhancing earthworm (*Lumbricus terrestris*) tolerance to plastic contamination through gut microbiome fortification with plastic-degrading microorganisms. *J. Hazard. Mater.* 463, 132836.

Chang, C.-H., Bartz, M.L., Brown, G., Callaham, M.A., Cameron, E.K., Dávalos, A., Dobson, A., Görres, J.H., Herrick, B.M., Ikeda, H., 2021. The second wave of earthworm invasions in North America: biology, environmental impacts, management and control of invasive jumping worms. *Biol. Invasions* 23, 3291–3322.
Chen, Y., Sun, K., Sun, H., Yang, Y., Li, Y., Gao, B., Xing, B., 2022. Photodegradation of pyrogenic dissolved organic matter increases bioavailability: Novel insight into bioalteration, microbial community succession, and C and N dynamics. Chem. Geol. 605, 120964.

Daghighi, E., Shah, T., Chia, R.W., Lee, J.-Y., Shang, J., Rodríguez-Seijo, A., 2023. The forgotten impacts of plastic contamination on terrestrial micro- and mesofauna: A call for research. Environ. Res. 231, 116227.

Fao Assessment of Agricultural Plastics and Their Sustainability—A Call for Action. FAO
2021.

Gong, X., Wang, D., Xu, M., Du, Y., Chen, X., Hu, P., Liu, M., 2023. Earthworm ecotype diversity mitigates resource limitations of microbial community in arable soils. Soil Biol. Biochem. 182, 109040.

Haider, T.P., Völker, C., Kramm, J., Landfester, K., Wurm, F.R., 2019. Plastics of the future? The impact of biodegradable polymers on the environment and on society. Angew. Chem. Int. Ed. 58, 50-62.

Han, Y., Teng, Y., Wang, X., Ren, W., Wang, X., Luo, Y., Zhang, H., Christie, P., 2021. Soil Type Driven Change in Microbial Community Affects Poly(butylene adipate-co-terephthalate) Degradation Potential. Environ. Sci. Tech. 55, 4648-4657.

Hink, L., Rohrbach, S., Rehkopf, J., Sehl, E., Agarwal, S., Feldhaar, H., Horn, M.A., 2024. Poly (L-lactide) mineralisation under environmental conditions is enhanced in earthworm guts. Soil Biol. Biochem. 194, 109485.

Hoang, D.T.T., Maranguait, D., Kuzyakov, Y., Razavi, B.S., 2020. Accelerated microbial activity, turnover and efficiency in the drilosphere is depth dependent. Soil Biol. Biochem. 147, 107852.

Hofmann, T., Ghoshal, S., Tufenkji, N., Adamowski, J.F., Bayen, S., Chen, Q., Demokritou, P., Flury, M., Huffer, T., Ivleva, N.P., Ji, R., Leask, R.L., Maric, M., Mitrano, D.M., Sander, M., Pahl, S., Rillig, M.C., Walker, T.R., White, J.C., Wilkinson, K.J., 2023. Plastics can be used more sustainably in agriculture. Commun. Earth Environ. 4, 332.

Hong, J.-K., Lee, T.K., Kim, I., Park, S., 2024. Determinants of microbial colonization on microplastics through wastewater treatment processes: The role of polymer type and sequential treatment. Sci. Total Environ. 914, 170072.

Huang, J., Liu, W., Yang, S., Yang, L., Peng, Z., Deng, M., Xu, S., Zhang, B., Ahirwal, J., Liu, L., 2021. Plant carbon inputs through shoot, root, and mycorrhizal pathways affect soil organic carbon turnover differently. Soil Biol. Biochem. 160, 108322.

Huerta Lwanga, E., Thapa, B., Yang, X., Gertsen, H., Salánki, T., Geissen, V., Garbeva, P., 2018. Decay of low-density polyethylene by bacteria extracted from earthworm's guts: A potential for soil restoration. Sci. Total Environ. 624, 753-757.

Khalid, N., Aqeel, M., Moman, A., Fatima Rizvi, Z., 2023. Impact of plastic mulching as a major source of microplastics in agroecosystems. J. Hazard. Mater. 445, 130455.

Koh, J., Bairoliya, S., Salma, M., Cho, Z.T., Fong, J., Neo, M.L., Cragg, S., Cao, B., 2023. Sediment-driven plastosphere community association on plastic debris in tropical coastal and marine environments. Environ. Int. 179, 108153.

Kublik, S., Gschwendtner, S., Magritsch, T., Radl, V., Rillig, M.C., Schlöter, M., 2022. Microplastics in soil induce a new microbial habitat, with consequences for bulk soil microbiomes. Front. Environ. Sci. 10, 989267.

Kwak, J.I., An, Y.-J., 2021. Microplastic digestion generates fragmented nanoplastics in soils and damages earthworm spermatogenesis and coelomocyte viability. J. Hazard. Mater. 402, 124034.

Lambert, S., Wagner, M., 2017. Environmental performance of bio-based and biodegradable plastics: the road ahead. Chem. Soc. Rev. 46, 6855-6871.

Li, C., Wang, L., Ji, S., Chang, M., Wang, L., Gan, Y., Liu, J., 2021. The ecology of the plastispHERE: microbial composition, function, assembly, and network in the freshwater and seawater ecosystems. Water Res. 202, 117428.

Li, L., Luo, Y., Li, R., Zhou, Q., Peijnenburg, W.J.G.M., Yin, N., Yang, J., Tu, C., Zhang, Y., 2020. Effective uptake of submicrometre plastics by crop plants via a crack-entry mode. Nat. Sustainability 3, 929-937.

Li, S., Ding, F., Flury, M., Wang, Z., Xu, L., Li, S., Jones, D.L., Wang, J., 2022. Macro- and microplastic accumulation in soil after 32 years of plastic film mulching. Environ. Pollut. 300, 118945.

Ling, L., Luo, Y., Jiang, B., Lv, J., Meng, C., Liao, Y., Reid, B.J., Ding, F., Lu, Z., Kuzyakov, Y., Xu, J., 2022. Biochar induces mineralization of soil recalcitrant components by activation of biochar responsive bacteria groups. Soil Biol. Biochem. 172, 108778.

Liu, E., He, W., Yan, C., 2014. 'White revolution' to 'white pollution'—agricultural plastic film mulch in China. Environ. Res. Lett. 9, 091001.

Liu, S., Deng, W.-K., Niu, S.-H., Mo, C.-H., Liao, X.-D., Xing, S.-C., 2023. Doxycycline combined manure microbes to enhances biofilm formation of the soil plastispHERE and increases the surface bio-risk of microplastics vehicle. Chem. Eng. J. 454, 140530.

Meng, K., Lwanga, E.H., van der Zee, M., Munhoz, D.R., Geissen, V., 2023. Fragmentation and depolymerization of microplastics in the earthworm gut: A potential for microplastic bioremediation? J. Hazard. Mater. 447, 130765.

Miao, L., Li, W., Adyel, T.M., Pahl, Y., Deng, Y., Wu, J., Zhou, Y., Yu, Y., Hou, J., 2023. Spatio-temporal succession of microbial communities in plastispHERE and their potentials for plastic degradation in freshwater ecosystems. Water Res. 229, 119406.

Mo, A., Zhang, Y., Gao, W., Jiang, J., He, D., 2023. Environmental fate and impacts of biodegradable plastics in agricultural soil ecosystems. Appl. Soil Ecol. 181, 104667.

Munhoz, D.R., Meng, K., Wang, L., Lwanga, E.H., Geissen, V., Harkes, P., 2024. Exploring the potential of earthworm gut bacteria for plastic degradation. Sci. Total Environ. 927, 172175.

Padmanabhan, L., Varghese, S., Patil, R.K., Rajath, H., Krishnasree, R., Shareef, M.I., 2020. Ecofriendly degradation of polyethylene plastics using oil degrading microbes. Recent Innovations in Chemical Engineering (formerly Recent Patents on Chemical Engineering) 13, 29-40.

Piron, D., Boizard, H., Heddadj, D., Pérès, G., Hallaire, V., Cluzeau, D., 2017. Indicators of earthworm bioturbation to improve visual assessment of soil structure. Soil Tillage Res. 173, 53-63.

Qi, R., Jones, D.L., Li, Z., Liu, Q., Yan, C., 2020a. Behavior of microplastics and plastic film residues in the soil environment: A critical review. Sci. Total Environ. 703, 134722.

Qi, Y., Ossowicki, A., Yang, X., Lwanga, E.H., Dini-Andreote, F., Geissen, V., Garbeva, P., 2020b. Effects of plastic mulch film residues on wheat rhizosphere and soil properties. J. Hazard. Mater. 387, 121711.

Qian, H., Zhang, M., Liu, G., Lu, T., Qu, Q., Du, B., Pan, X., 2018. Effects of soil residual plastic film on soil microbial community structure and fertility. Water Air Soil Pollut. 229, 1-11.

Qiang, L., Cheng, J., Mirzoyan, S., Kerkhof, L.J., Häggblom, M.M., 2021. Characterization of Microplastic-Associated Biofilm Development along a Freshwater-Estuarine Gradient. Environ. Sci. Tech. 55, 16402-16412.

Qiu, X., Ma, S., Pan, J., Cui, Q., Zheng, W., Ding, L., Liang, X., Xu, B., Guo, X., Rillig, M. C., 2024. Microbial metabolism influences microplastic perturbation of dissolved organic matter in agricultural soils. ISME J. 18.

Ratsiatosika, O., Trap, J., Herinasandratva, V., Razafimbelo, T., Bernard, L., Blanchart, E., 2024. Earthworms enhance the performance of organic amendments in improving rice growth and nutrition in poor ferralsols. Soil Biol. Biochem. 195, 109477.

Rillig, M.C., Antonovics, J., Caruso, T., Lehmann, A., Powell, J.R., Veresoglou, S.D., Verbruggen, E., 2015. Interchange of entire communities: microbial community coalescence. Trends Ecol. Evol. 30, 470-476.

Rillig, M.C., Kim, S.W., Zhu, Y.-G., 2024. The soil plastisphere. Nat. Rev. Microbiol. 22, 64-74.

Rummel, C.D., Lechtenfeld, O.J., Kallies, R., Benke, A., Herzsprung, P., Rynek, R., Wagner, S., Potthoff, A., Jahnke, A., Schmitt-Jansen, M., 2021. Conditioning film and early biofilm succession on plastic surfaces. Environ. Sci. Tech. 55, 11006-11018.

J. Rüthi Characterization of the plastisphere microbiome and plastic-degrading microorganisms in cold terrestrial environments 2022 ETH Zurich.

Rüthi, J., Rast, B.M., Qi, W., Perez-Mon, C., Pardi-Comensoli, L., Brunner, I., Frey, B., 2023. The plastisphere microbiome in alpine soils alters the microbial genetic potential for plastic degradation and biogeochemical cycling. J. Hazard. Mater. 441, 129941.

Sanchez-Hernandez, J.C., Capowiez, Y., Ro, K.S., 2020. Potential Use of Earthworms to Enhance Decaying of Biodegradable Plastics. ACS Sustain. Chem. Eng. 8, 4292-4316.

Sander, M., 2019. Biodegradation of Polymeric Mulch Films in Agricultural Soils: Concepts, Knowledge Gaps, and Future Research Directions. Environ. Sci. Tech. 53, 2304-2315.

Sheridan, E.A., Foniville, J.A., Cottingham, S., Zhang, Y., Dittmar, T., Aldridge, D.C., Tanentzap, A.J., 2022. Plastic pollution fosters more microbial growth in lakes than natural organic matter. Nat. Commun. 13, 4175.

Shi, M., Sun, Y., Wang, Z., He, G., Quan, H., He, H., 2019. Plastic film mulching increased the accumulation and human health risks of phthalate esters in wheat grains. Environ. Pollut. 250, 1-7.

Sun, D., Li, H., Wang, E., He, W., Hao, W., Yan, C., Li, Y., Mei, X., Zhang, Y., Sun, Z., 2020. An overview of the use of plastic-film mulching in China to increase crop yield and water-use efficiency. Natl. Sci. Rev. 7, 1523-1526.

Sun, Y., Li, X., Li, X., Wang, J., 2022. Deciphering the Fingerprint of Dissolved Organic Matter in the Soil Amended with Biodegradable and Conventional Microplastics Based on Optical and Molecular Signatures. Environ. Sci. Tech. 56, 15746-15759.

Tanunchai, B., Ji, L., Schröder, O., Gawol, S.J., Geissler, A., Wahdan, S.F.M., Buscot, F., Kalkhof, S., Schulze, E.-D., Noll, M., 2023. Fate of a biodegradable plastic in forest soil: Dominant tree species and forest types drive changes in microbial community assembly, influence the composition of plastispHERE, and affect poly (butylene succinate-co-adipate) degradation. Sci. Total Environ. 873, 162230.

Wang, L., Bank, M.S., Rinklebe, J.R., Hou, D., 2023. Plastic-rock complexes as hotspots for microplastic generation. Environ. Sci. Tech. 57, 7009-7017.

Wang, L., Peng, Y., Xu, Y., Zhang, J., Liu, C., Tang, X., Lu, Y., Sun, H., 2022. Earthworms' Degradable Bioplastic Diet of Polylactic Acid: Easy to Break Down and Slow to Excrete. Environ. Sci. Tech. 56, 5020-5028.

Wang, Y., Jiao, M., Zhao, Z., Wang, Y., Li, T., Wei, Y., Li, R., Yang, F., 2024. Insight into the role of niche concept in deciphering the ecological drivers of MPs-associated bacterial communities in mangrove forest. Water Res. 249, 120995.

Woodcock, S., Sloan, W.T., 2017. Biofilm community succession: a neutral perspective. Microbiology 163, 664-668.

Wu, C., Ma, Y., Wang, D., Shan, Y., Song, X., Hu, H., Ren, X., Ma, X., Cui, J., Ma, Y., 2022. Integrated microbiology and metabolomics analysis reveal plastic mulch film residue affects soil microorganisms and their metabolic functions. J. Hazard. Mater. 423, 127258.

Wu, F., Guo, Z., Cui, K., Dong, D., Yang, X., Li, J., Wu, Z., Li, L., Dai, Y., Pan, T., 2023. Insights into characteristics of white rot fungus during environmental plastics adhesion and degradation mechanism of plastics. J. Hazard. Mater. 448, 130878.

Xiang, Q., Chen, Q.-L., Yang, X.-R., Li, G., Zhu, D., 2022. Soil mesofauna alter the balance between stochastic and deterministic processes in the plastispHERE during microbial succession. Sci. Total Environ. 849, 157820.

Xiao, L., Wei, X., Wang, C., Zhao, R., 2023. Plastic film mulching significantly boosts crop production and water use efficiency but not evapotranspiration in China. Agric Water Manag 275, 108023.

Xiong, L., Li, Z., Shah, F., Wang, P., Yuan, Q., Wu, W., 2024. Biodegradable mulch film enhances the environmental sustainability compared with traditional polyethylene film from multidimensional perspectives. Chem. Eng. J. 492, 152219.
Yin, C.-F., Xu, Y., Zhou, N.-Y., 2020. Biodegradation of polyethylene mulching films by a co-culture of Acinetobacter sp. strain NyZ450 and Bacillus sp. strain NyZ451 isolated from Tenebrio molitor larvae. Int. Biodeter. Biodegr. 155, 105089.

Zhang, Q.-Q., Ma, Z.-R., Cai, Y.-Y., Li, H.-R., Ying, G.-G., 2021. Agricultural Plastic Pollution in China: Generation of Plastic Debris and Emission of Phthalic Acid Esters from Agricultural Films. Environ. Sci. Tech. 55, 12459–12470.

Zhou, J., Gui, H., Banfield, C.C., Wen, Y., Zang, H., Dippold, M.A., Charlton, A., Jones, D. L., 2021. The microplastisphere: biodegradable microplastics addition alters soil microbial community structure and function. Soil Biol. Biochem. 156, 108211.

Zhou, X., Ma, A., Chen, X., Zhang, Q., Guo, X., Zhuang, G., 2023. Climate Warming-Driven Changes in the Molecular Composition of Soil Dissolved Organic Matter Across Depth: A Case Study on the Tibetan Plateau. Environ. Sci. Tech. 57, 16884–16894.

Ziemba, J.L., Cameron, A.C., Peterson, K., Hickerson, C.-A.-M., Anthony, C.D., 2015. Invasive Asian earthworms of the genus Amynthas alter microhabitat use by terrestrial salamanders. Can. J. Zool. 93, 805–811.