Decoding Microbial Plastic Colonisation:
Multi-Omic Insights into the Fast-Evolving Dynamics of Early-Stage Biofilms
Charlotte E. Lee1, Lauren F. Messer1, Ruddy Wattiez2, and *Sabine Matallana-Surget1
1 Division of Biological and Environmental Sciences, Faculty of Natural Sciences, University of Stirling, Stirling, Scotland, FK9 4LA, United Kingdom
2 Laboratory of Proteomics and Microbiology, Research Institute for Biosciences, University of Mons, Place du Parc 20, 7000, Mons, Belgium a
*Corresponding Author: Sabine Matallana-Surget; A/Professor in Environmental Molecular Microbiology, Division of Biological and Environmental Sciences, Faculty of Natural Sciences.
University of Stirling, Stirling, FK9 4LA Phone: 44 (0) 1786 467774 ; Email: sabine.matallanasurget@stir.ac.uk
Abbreviations
AD Alcohol dehydrogenase
Ahp Alkyl hydroperoxide reductase
ALDH Aldehyde dehydrogenase
ArnA Polymyxin resistance protein
ASW+G Artificial seawater + vitamins, trace metals, and glucose
BHB+v Bushnell Haas broth + vitamins, and trace metals
CopA Copper resistance protein A
CSD Cold shock domain-containing protein
D3/D7 Day 3/7
FC Fold change
Fhp Flavohemoprotein
HGT Horizontal gene transfer
Kat Catalase peroxidase
LAD Large adhesive protein
LDPE Low-density polyethylene
MAG Metagenome-assembled genome
Omp Outer membrane protein
PPS Protein precipitation solution
ROS Reactive oxygen species
TelA Toxic anion resistance protein
TerD Tellurium resistance protein
TIISS/TVISS Type- II/VI secretion system
Keywords
Plastisphere; Metaproteome; Marine biofilms; Biofilm formation; Plastic biodegradation
Word count : 6757
Abstract
Marine plastispheres represent dynamic microhabitats where microorganisms colonise plastic debris and interact. Metaproteomics has provided novel insights into the metabolic processes within these communities, however the early metabolic interactions driving the plastisphere formation remain unclear. This study utilised metaproteomic and metagenomic approaches to explore early plastisphere formation on low-density polyethylene (LDPE) over three (D3) and seven (D7) days, focusing on microbial diversity, metabolic activity, and biofilm development. In total, 2,948 proteins were analysed, revealing dominant proteomes from Pseudomonas and Marinomonas, with near-complete metagenome-assembled genomes. Pseudomonas dominated at D3, while at D7, Marinomonas, along with Acinetobacter, Vibrio, and other genera became more prevalent . Pseudomonas andMarinomonas showed high expression of reactive oxygen species (ROS) suppression proteins, associated with oxidative stress regulation, while granule formation, and alternative carbon utilisation enzymes, also indicated nutrient limitations. Interestingly, 13 alkane and other xenobiotic degradation enzymes were expressed by five genera. The expression of toxins, several type VI secretion system (TVISS) proteins, and biofilm formation proteins by Pseudomonas indicated their competitive advantage against other taxa. Upregulated metabolic pathways, including those relating to substrate transport also suggested enhanced nutrient cross-feeding within the biofilm. These insights enhance our understanding of plastisphere ecology and its potential for biotechnological applications.
Significance of the study
Given the increasing plastic pollution in marine environments, understanding early plastisphere assembly is essential from both ecological and biotechnological perspectives. This study advances knowledge by identifying 13 pollutant-degrading enzymes in addition to those found through prior metaproteomic research, shedding light on the bioremediative potential of multi-species biofilms. Their expression by constitutive genera, particularly Marinomonas , also underscores the recognised association of these genera with environmental bioremediation. Additionally, interspecies competition and oxidative stress responses, shaped by resource limitations, were found to govern biofilm dynamics. The selection for generalist species and potential pathogens is concerning due to plastic’s ability to travel through marine ecosystems. However, the cooperative behaviour among plastisphere members supports prior research demonstrating biofilms as resilient microbial solutions. This research not only deepens our understanding of microbial colonisation and interaction but also highlights the utility of metaproteomics in studying complex environmental communities. Insights from this study also contribute to the broader field of plastisphere ecology, offering pathways for future research into managing plastic pollution and developing biotechnological strategies for marine ecosystem resilience.
Introduction
Over 400.3 million metric tonnes of plastic are manufactured, utilised, and discarded annually [1]. Despite diverse waste management strategies implemented by national governments and public initiatives aimed at controlling plastic waste, an estimated 19 to 23 million tonnes of plastic pollution enter the marine environment each year [2, 3]. Marine plastic debris, along with the resulting microplastics, is now anticipated to enter nearly all marine ecosystems, threatening the health of our oceans [4]. Direct studies of marine plastic debris reveal a complex relationship between plastics and the ocean’s most abundant biogeochemical cyclers and producers: microorganisms [5, 6]. As natural biofilm-forming organisms, and secondary surface colonisers, these microorganisms directly interact with marine plastic debris through the formation of the ‘plastisphere’ [7].
Marine plastispheres are complex, multi-species biofilms that consist of both eukaryotic and prokaryotic producers, hydrocarbonoclastic organisms, pathogens, and other biogeochemical cyclers [6-10]. The composition of these communities is influenced by several factors, including the type of plastic, its chemical additives, environmental location, seasonal changes, and the time available for colonisation [6]. Among these factors, the duration of plastisphere development (i.e., the stage at which the plastisphere is collected) appears to exert the most consistent influence across studies. Microbial abundance and diversity are often low in the initial hours following colonisation, peak within 1-2 weeks, and subsequently decline [10-13]. Metaproteomic studies focusing on biofilm formation during these early stages have uncovered both competition and cooperation between species, interactions that are likely to be prevalent within the first days of plastisphere development [14, 15]. This early plastisphere is particularly significant, as it may be the only stage of biofilm development where direct microbe-plastic interactions occur. As biofilms continue to form, new layers often cover older ones, shielding the upper biofilm layers from direct contact with the plastic surface [11].
Prior metaproteomic research on multi-species biofilm formation [14, 15] and established marine plastispheres [8, 16, 17] has offered insights into how microorganisms interact with plastic substrates and with each other. A recent study by our research group highlighted the importance of comparing new versus established plastispheres, demonstrating a significant shift in microbial diversity and composition while maintaining a core assemblage of active Proteobacteria , including Marinomonas , Pseudomonas , andPseudoalteromonas [9]. Additionally, this study highlighted the differential regulation of proteins involved in cellular attachment and energy metabolism during the first 2 weeks of colonisation, offering important context for understanding the evolution of biofilm dynamic. However, the molecular mechanisms driving the functioning of marine plastisphere communities in the earliest stages of development remain poorly characterised at the proteome level.
In the present study, we employ metaproteomics to dissect the molecular basis of plastisphere formation within the first 7 days of colonisation, with a particular focus on interspecies interactions and microbe-plastic dynamics. Our aim was to build on this previous research by providing a deeper understanding of how microorganisms interact with plastic in a marine environment and how these interactions influence community structure and function in the young plastisphere.
As addressed in our prior work [18], metaproteomics poses several challenges, which influenced the experimental design of this study. We collected samples at three (D3) and seven (D7) days post-inoculation to track the development of the nascent plastisphere [11-13]. While limiting the time available for biomass accumulation on the plastic restricts the amount of material for analysis, it is crucial for capturing the earliest stages of plastisphere formation [10]. To increase sample biomass, we enriched the plastisphere communities collected from the environment, prior to plastic inoculation. Low-density polyethylene (LDPE), one of the most widely produced plastics globally (45.7 Mt annually) [1], was selected as the test substrate to ensure relevance to real-world environmental conditions.
  1. Materials and Methods
  2. Recovery of plastisphere stock communities Beached plastics were collected from Oban Bay (56° 24’ 50.4’ ’ N, 5° 28’ 19.2’ ’ W; Scotland) on August 6th, 2022 and washed in artificial seawater [19] to remove loosely attached organisms, prior to preservation at -20℃ until use. To increase plastisphere biomass, transparent plastics were placed into glass containers with 300ml artificial seawater supplemented with vitamins, trace metals, and glucose (ASW+G), and incubated with shaking (65 rpm) at 15℃ for three days, equalling Oban Bay’s highest seawater temperature. Excess ASW+G was carefully removed, and plastics were vortexed in the remaining media and rinsed with additional ASW+G to ensure the biofilm was fully detached. The plastics were then removed and the detached plastisphere cells were pelleted by centrifugation (14℃, 5000 g, 7mins), resuspended in 15ml ASW+G, and preserved with 30% glycerol at -80℃ until further use.
  3. Plastisphere growth
Five pieces of low-density polyethylene (LDPE; 2x10 cm folded in/to 8ths; ET31-FM-000101; Goodfellow, England) per replicate were added to 150ml of Bushnell Haas Broth supplemented with vitamins and trace metals (BHB+v) [20], and inoculated with 50μl of plastisphere stock community. This was then incubated (65 rpm, 15°C) in 250ml glass Erlenmeyer flasks for 3 or 7 days.
All experimental conditions were performed in quadruplicate.