Freshwater plastisphere: a review on biodiversity, risks, and biodegradation potential with implications for the aquatic ecosystem health

The plastisphere, a unique microbial biofilm community colonizing plastic debris and microplastics (MPs) in aquatic environments, has attracted increasing attention owing to its ecological and public health implications. This review consolidates current state of knowledge on freshwater plastisphere, focussing on its biodiversity, community assembly, and interactions with environmental factors. Current biomolecular approaches revealed a variety of prokaryotic and eukaryotic taxa associated with plastic surfaces. Despite their ecological importance, the presence of potentially pathogenic bacteria and mobile genetic elements (i.e., antibiotic resistance genes) raises concerns for ecosystem and human health. However, the extent of these risks and their implications remain unclear. Advanced sequencing technologies are promising for elucidating the functions of plastisphere, particularly in plastic biodegradation processes. Overall, this review emphasizes the need for comprehensive studies to understand plastisphere dynamics in freshwater and to support effective management strategies to mitigate the impact of plastic pollution on freshwater resources.

Despite the growing recognition of plastic waste pervasiveness in marine ecosystems and the large body of research focusing on plasticand microplastic-associated biofilms, the freshwater plastisphere remains relatively understudied.Given the critical importance of quality freshwaters to provide essential services to human health and society, this disparity highlights the need for a more comprehensive understanding of plastic pollution in freshwater ecosystems, which can harbor a complex and diverse array of microorganisms differently sensitive to environmental and anthropogenic pollution (Hoellein et al., 2017;Di Pippo et al., 2020, 2022;Eronen-Rasimus et al., 2022;Nguyen et al., 2023).
This study was entailed to synthetically overview the current knowledge of freshwater plastisphere and the main environmental factors potentially influencing its development and microbial community assembly.In particular, we examined the current understanding of how the plastisphere might affect freshwater ecosystems and human health.We also examined its potential for biodegradation and identified critical aspects that require further investigation.
2 Plastisphere biodiversity, taxon composition, and factors affecting microbial community assembly Once dispersed in water, plastics and MPs are rapidly colonized by planktonic microorganisms, which can adhere and grow onto solid surfaces forming complex plastic-associated biofilms whose biodiversity profiles consistently differ from those of the surrounding environments (see Table 1).While there is a shared consensus on the definition of a new plastic-associated micro-ecosystem with distinct microbiota, it is still debated whether the freshwater plastisphere harbors higher or lower biodiversity than planktonic communities and biofilms formed on natural substrates (McCormick et al., 2014(McCormick et al., , 2016;;Hoellein et al., 2017;Arias-Andres et al., 2018;Wu et al., 2019;Wang et al., 2020;Xue et al., 2020;Galafassi et al., 2021;Kelly et al., 2021).
Most of the available information on freshwater plastisphere biodiversity comes from culture-independent methods (Table 1), which allow a comprehensive characterization of plastisphereassociated microbiomes.Among them, the use of high-throughput sequencing methods, both as amplicon sequencing of SSU RNA genes and shotgun metagenomic sequencing, are essential to decipher the taxonomic and functional diversity of samples, thus providing the composition and the metabolic potential of the entire microbial community (Dey et al., 2022;Wani et al., 2023).Recent developments in sequencing techniques have led to sequence very long reads (up to 50 kb) offering multiple cutting-edge options for understanding microbiome structure and functioning (Tedersoo et al., 2021).For example, when applied to amplicon sequencing (e.g., 16S rRNA gene), long-reads can resolve microbial taxonomy at deeper levels rather than short-reads due to the ability to read the entire gene with single nucleotide resolution leading to the identification of sub-species clades or "strains" within the community.Most studies have focused on Bacteria, with very few reports on archaeal and eukaryotic biodiversity (Table 1).
Archaea are likely to represent a minor component of the plasticassociated microbial community (<0.1% of total amplicon sequences) (Mughini-Gras et al., 2021), showing a lower diversity compared to Bacteria.
3 Ecosystem and human health-related issues: occurrence of plastic-associated pathogens and genetic elements of health concern Plastic-associated microbiological elements of health concern are rarely monitored in freshwater ecosystems, despite their fundamental services provided to human health and society (e.g., drinking water supply, agricultural/industrial activities, recreational activities).Plastic debris and associated biofilms have been reported to represent newly available ecological niches that facilitate the accumulation of various harmful microbes.Recent studies have highlighted the presence of potentially pathogenic bacteria in freshwater plastisphere communities, including members of the genera Vibrio, Pseudomonas, Acinetobacter, Arcobacter, Bacillus, Aquabacterium, Mycobacterium, Aeromonas, Tenacibaculum, Escherichia, Klebsiella, and Legionella (Kirstein et al., 2016;McCormick et al., 2016).These bacteria can pose a significant risk to aquatic life and human health by causing infections, skin irritation, and even systemic diseases.In addition to bacterial pathogens, the plastisphere can also harbor eukaryotic microorganisms that can have a potential negative impact.Potentially toxic microalgae and potentially pathogenic fungi (i.e., Chytridiomycota and Cryptomycota species) were found on plastic debris, raising concerns about its role in promoting harmful algal blooms and the spread of water-borne fungal diseases (Barros and Seena, 2021;Di Pippo et al., 2022).More recently, several studies on plastic and MP dispersal in freshwaters showed the co-presence of pathogens and Mobile Genetic Elements (MGEs), including ARGs, thus suggesting a higher probability of antibiotic resistance acquisition mediated by the plastisphere (Junaid et al., 2022; Table 2).Considering the worldwide spread of MPs in the environment, ARGs presence on MPs may exacerbate risk for human to acquire ARGs and-or resistant microorganisms of health concern.Indeed, some studies have revealed that marine microorganisms can uptake MPs from the water environment transferring in the food chain and more recently has been shown that ARGs can transfer through the trophic level into the food chain (Zhu et al., 2019; Figure 1).
ARG enrichment in plastic-associated biofilms is promoted by the proximity and close contact between bacterial cells that facilitate horizontal gene transfer, contributing to long-distance dispersal and long-term persistence of ARGs in the environment (Di Pippo et al., 2022;Du et al., 2022;Rubin and Zucker, 2022;Luo et al., 2023;Chen et al., 2024;Li et al., 2024; Figure 1).Moreover, the presence of plasticadsorbed xenobiotics and metals might enhance ARG occurrence through co-selection processes (Abe et al., 2020;Junaid et al., 2022;Michaelis and Grohmann, 2023).
Unlike in marine environments, studies in freshwater showed limited plastisphere enrichment of ARGs compared to the surrounding waters and natural substrata (Wu et al., 2019;Wang et al., 2020;Xu et al., 2022), with no differences in ARGs and MGEs observed between plastisphere and natural biofilms (Hu et al., 2021).Recent studies on freshwater plastisphere have mainly analyzed the differences in ARG abundance and diversity by comparing (i) surface waters and other natural surfaces (González-Pleiter et al., 2021;Xu et al., 2022;Martínez-Campos et al., 2023), (ii) biodegradable and non-biodegradable plastics (Zhou Q. et al., 2022), (iii) different stages of biofilm development at different contamination levels (Table 2).Although clearly showing the worldwide spread of plastic-associated pathogens and ARGs, the information available on freshwater plastisphere is still limited to properly evaluate human health risks (Manaia, 2017;Zhang et al., 2021).
Notably, the identification of potential pathogens is at the genus level, which does not provide direct evidence of the pathogen's occurrence, infectivity or virulence (Liu et al., 2022).Furthermore, quantitative PCR-based methods are limited to known functional genes and may miss novel or uncharacterized ARGs (Li et al., 2015).Combined omics approaches can provide detailed information on the   et al., 2018).However, due to the large diversity of ARGs and their incomplete coverage by the applied monitoring methods, the plastic-associated ARGs profiles are hardly comparable between different studies.
Recent publications showed how long-reads sequencing can help to overcome these technical limitations (Zhang et al., 2022).Longread sequencing techniques improve the quality and completeness of metagenome-assembled genomes allowing to reduce errors and improving the accuracy of ARG identification and characterization (Table 2).
4 The role of plastisphere in plastic biodegradation processes Once plastic items are transported through the aquatic environment, abiotic factors can cause changes in their mechanical and physico-chemical properties (Luo et al., 2022) and plastisphere microorganisms can modify MP surface properties by degrading additives, secreting MP-modifying/degrading enzymes.The plastisphere-mediated biodegradation of plastic debris and MPs is a complex multifaceted process in which polymers are first bio-fragmented through the secretion of extracellular enzymes.In the subsequent assimilation phase, the small and water-soluble molecules produced during the depolymerization of plastics are transported through the cell membrane.
Once inside the cell, plastic-derived molecules can be used as a carbon source to produce biomass and energy before being mineralized to CO 2 /CH 4 and H 2 O (Tiwari et al., 2020;Yuan et al., 2020;Zeenat et al., 2021;Priya et al., 2022;Zhou Y. et al., 2022;Sun et al., 2023).
Few studies currently available report inconsistent results on the direct involvement of plastisphere in biodegradation processes.The metabolic potential to hydrolyse and use the plastic polymers as carbon sources was not convincingly demonstrated, while plastic materials were mostly used as adhesion surfaces by opportunistic aquatic microbes (Oberbeckmann et al., 2021;Di Pippo et al., 2023).Further investigations are thus needed to provide a deeper understanding of plastisphere role in plastic biodegradation.
Advanced culture-independent approaches based on sequencing technologies are accelerating discoveries in this field.Although still in their infancy, "plastic-omics" (Viljakainen and Hug, 2021) are emerging as important tools for understanding the functional potential of the plastisphere, providing important insights into the identification of potentially degrading bacterial taxa, the factors influencing their enrichment, and the plastic degrading genes/ enzymes, and thus a holistic understanding of the plastic degradation process (Viljakainen and Hug, 2021;Malik et al., 2023).Metatranscriptomics can be a powerful approach to reveal the gene expression profiles and transcriptional activity of microorganisms associated with plastic surfaces, elucidating metabolic pathways and gene regulatory networks involved in plastic biodegradation (Gilbert et al., 2008;Kirstein et al., 2016;Xu et al., 2019;Yang et al., 2019;Lu et al., 2020).Pathways through which plastisphere and plastics may affect ecosystems and human health.Plastics released in freshwater as vectors for both microorganisms and chemicals adsorbed on their surfaces.Once in water, plastics are affected by abiotic factors leading to their fragmentation into smaller particles, that are colonized by the planktonic microbial community (biofouling) with an increase of density of the particles, causing their sedimentation through the water column.The close relationship between the biofilm microorganisms lead to an enhanced horizontal gene transfer (HGT) with the spreading of the ARGs through the plastisphere members.As ubiquitous pollutants, plastic debris travel throughout the environment leading to the spread of antibiotic resistance and adsorbed chemicals into freshwater, finally reaching services provided to human health and society.The persistent plastic debris can also enter the trophic chain.(Images created using BioRender.com.) 10. 3389/fmicb.2024.1395401Frontiers in Microbiology 14 frontiersin.org

Conclusion
This review paper sheds light on the intricate relationship between plastic pollution and microbial communities in freshwater ecosystems, specifically focusing on the freshwater plastisphere.While molecular methods have expanded our understanding of plastisphere biodiversity, fundamental questions regarding the influence of the polymer type and properties and environmental factors on plastisphere structure, biodiversity and on community assembly remain unanswered.The presence of potentially pathogenic microbes and genetic elements of concern within the plastisphere raises important implications for ecosystem and human health.However, the extent of these risks and their impacts are still not fully elucidated, necessitating further research efforts.Advanced sequencing technologies offer promising avenues for uncovering the functional potential of the plastisphere, including its role in plastic biodegradation processes.Overall, the findings underscore the urgent need for comprehensive investigations into freshwater plastisphere dynamics, which are crucial for informing effective management strategies to mitigate the environmental and health impacts of plastic pollution in freshwater ecosystems.

TABLE 1
Main groups of microorganisms found in freshwater plastisphere.

TABLE 2
ARGs and ARBs detected in plastic-associated biofilms in freshwater ecosystems.