<div>Decoding Microbial Plastic Colonisation:</div><div>Multi-Omic Insights into the Fast-Evolving Dynamics of Early-Stage
Biofilms</div><div>Charlotte E. Lee<sup>1</sup>, Lauren F.
Messer<sup>1</sup>, Ruddy Wattiez<sup>2</sup>, and *Sabine
Matallana-Surget<sup>1</sup></div><div><sup>1</sup> Division of Biological and Environmental Sciences,
Faculty of Natural Sciences, University of Stirling, Stirling, Scotland,
FK9 4LA, United Kingdom</div><div><sup>2</sup> Laboratory of Proteomics and Microbiology, Research
Institute for Biosciences, University of Mons, Place du Parc 20, 7000,
Mons, Belgium <sup>a</sup></div><div>*Corresponding Author: Sabine Matallana-Surget; A/Professor in
Environmental Molecular Microbiology, Division of Biological and
Environmental Sciences, Faculty of Natural Sciences.</div><div>University of Stirling, Stirling, FK9 4LA Phone: 44 (0) 1786 467774 ;
Email: sabine.matallanasurget@stir.ac.uk</div><div>Abbreviations</div><div>AD Alcohol dehydrogenase</div><div>Ahp Alkyl hydroperoxide reductase</div><div>ALDH Aldehyde dehydrogenase</div><div>ArnA Polymyxin resistance protein</div><div>ASW+G Artificial seawater + vitamins, trace metals, and glucose</div><div>BHB+v Bushnell Haas broth + vitamins, and trace metals</div><div>CopA Copper resistance protein A</div><div>CSD Cold shock domain-containing protein</div><div>D3/D7 Day 3/7</div><div>FC Fold change</div><div>Fhp Flavohemoprotein</div><div>HGT Horizontal gene transfer</div><div>Kat Catalase peroxidase</div><div>LAD Large adhesive protein</div><div>LDPE Low-density polyethylene</div><div>MAG Metagenome-assembled genome</div><div>Omp Outer membrane protein</div><div>PPS Protein precipitation solution</div><div>ROS Reactive oxygen species</div><div>TelA Toxic anion resistance protein</div><div>TerD Tellurium resistance protein</div><div>TIISS/TVISS Type- II/VI secretion system</div><div>Keywords</div><div>Plastisphere; Metaproteome; Marine biofilms; Biofilm formation; Plastic
biodegradation</div><div>Word count : 6757</div><div>Abstract</div><div>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 <i>Pseudomonas</i> and <i>Marinomonas,</i> with
near-complete metagenome-assembled genomes. <i>Pseudomonas</i> dominated
at D3, while at D7, <i>Marinomonas,</i> along with <i>Acinetobacter,
Vibrio, and other genera became more prevalent</i> . <i>Pseudomonas</i> and<i>Marinomonas</i> 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 <i>Pseudomonas</i> 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.</div><div>Significance of the study</div><div>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 <i>Marinomonas</i> , 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.</div><div>Introduction</div><div>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].</div><div>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].</div><div>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 <i>Proteobacteria</i> ,
including <i>Marinomonas</i> , <i>Pseudomonas</i> , and<i>Pseudoalteromonas</i> [9]<i>.</i> 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.</div><div>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.</div><div>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.</div><ol><li>Materials and Methods</li><li>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 6<sup>th</sup>, 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.</li><li>Plastisphere growth</li></ol><div>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.</div><div>All experimental conditions were performed in quadruplicate.</div>