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\begin{document}

\title{Decoding Microbial Plastic Colonisation: Multi-Omic Insights into the
Fast-Evolving Dynamics of Early-Stage Biofilms}



\author[1]{Charlotte Lee}%
\author[1]{Lauren Messer}%
\author[2]{Ruddy Wattiez}%
\author[1]{Sabine Matallana-Surget}%
\affil[1]{University of Stirling}%
\affil[2]{University of Mons}%


\vspace{-1em}



  
  \date{March 17, 2025}


\begingroup
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\selectlanguage{english}
\begin{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 and Marinomonas 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.%
\end{abstract}%



\sloppy


Decoding Microbial Plastic Colonisation:

Multi-Omic Insights into the Fast-Evolving Dynamics of Early-Stage
Biofilms

Charlotte E. Lee\textsuperscript{1}, Lauren F.
Messer\textsuperscript{1}, Ruddy Wattiez\textsuperscript{2}, and *Sabine
Matallana-Surget\textsuperscript{1}

\textsuperscript{1} Division of Biological and Environmental Sciences,
Faculty of Natural Sciences, University of Stirling, Stirling, Scotland,
FK9 4LA, United Kingdom

\textsuperscript{2} Laboratory of Proteomics and Microbiology, Research
Institute for Biosciences, University of Mons, Place du Parc 20, 7000,
Mons, Belgium \textsuperscript{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 \emph{Pseudomonas} and \emph{Marinomonas,} with
near-complete metagenome-assembled genomes. \emph{Pseudomonas} dominated
at D3, while at D7, \emph{Marinomonas,} along with \emph{Acinetobacter,
Vibrio, and other genera became more prevalent} . \emph{Pseudomonas}
and\emph{Marinomonas} 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 \emph{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 \emph{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 \emph{Proteobacteria} ,
including \emph{Marinomonas} , \emph{Pseudomonas} ,
and\emph{Pseudoalteromonas} {[}9{]}\emph{.} 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.

\begin{enumerate}
\tightlist
\item
  Materials and Methods
\item
  Recovery of plastisphere stock communities Beached plastics were
  collected from Oban Bay (56\selectlanguage{ngerman}° 24' 50.4'\selectlanguage{english} ' N, 5deg 28' 19.2'\selectlanguage{english} ' W;
  Scotland) on August 6\textsuperscript{th}, 2022 and washed in
  artificial seawater {[}19{]} to remove loosely attached organisms,
  prior to preservation at -20\selectlanguage{english} 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\selectlanguage{english} 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\selectlanguage{english}, 5000 g,
  7mins), resuspended in 15ml ASW+G, and preserved with 30\% glycerol at
  -80\selectlanguage{english} until further use.
\item
  Plastisphere growth
\end{enumerate}

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 50\selectlanguage{greek}μ\selectlanguage{english}l of plastisphere stock
community. This was then incubated (65 rpm, 15\selectlanguage{ngerman}°C) in 250ml glass
Erlenmeyer flasks for 3 or 7 days.

All experimental conditions were performed in quadruplicate.



\textbf{{[}Figure 2{]}}

Key functional categories driving early plastisphere development

The proteins expressed on D3 and D7 were classified into functional
categories, with 'translation, ribosomal structure and biogenesis,'
'energy production and conversion,' and 'amino acid transport and
metabolism' being the most abundant across most genera (Figure 3a).
These categories were then followed by functions associated with `cell
wall/membrane/envelope biogenesis', `cell motility', and biofilm
formation.

Within 'energy production and conversion,' proteins linked to the TCA
cycle were prominent and differentially expressed between time points.
These include succinate dehydrogenase, expressed by \emph{Pseudomonas}
(\textgreater{}D3; FC 2.83), \emph{Marinomonas} (\textgreater{}D7; FC
4.53), \emph{Paracoccus} (\textgreater{}D7; FC
2.72),\emph{Acinetobacter} (\textgreater{}D7; FC 4.27), and
\emph{Vibrio} on both days, as well as by \emph{Rhodobacter} and
\emph{Shewanella} on D7. Additionally, citrate synthase was expressed by
\emph{Pseudomonas} (D3; D7), \emph{Marinomonas} (D7),
\emph{Acinetobacter} (D7), and\emph{Psychromonas} (D7), while malate
dehydrogenase was expressed by\emph{Pseudomonas} , \emph{Marinomonas}
(\textgreater{}D7; FC 3.6),\emph{Paracoccus} (\textgreater{}D7; FC
2.92), and\emph{Pseudoalteromonas} (\textgreater{}D7; FC 4.13) on both
days. Isocitrate dehydrogenase was expressed by \emph{Pseudomonas}
(\textgreater{}D7; FC 3.01 ± 0.79), \emph{Marinomonas}
(\textgreater{}D7; FC 3.21 ±0.52), \emph{Psychromonas}
(\textgreater{}D7; FC 3.85), and\emph{Flavobacterium} (\textgreater{}D7;
FC 4.07) (Supplementary File S3).

Interestingly, proteins associated with 'amino acid transport and
metabolism' were differentially regulated across nine genera (Figure
3a), accounting for 11.5\% (± 1.5) of the total differentially regulated
proteins. These include enzymes involved in glutamate cycling (e.g.
glutamate methylesterase, \textgreater{}D7 \emph{Marinomonas} , FC 3.03;
glutamine synthetase, \textgreater{}D7 \emph{Marinomonas} , FC 2.7;
\textgreater{}D7 \emph{Paracoccus} , FC 2.1), arginine cycling (e.g.
acetylornithine aminotransferase, \textgreater{}D7 \emph{Marinomonas} ,
FC 3.79), and the regulation and transport of branched-chain amino acids
(e.g. acetohydroxy-acid isomeroreductase,
\textgreater{}D7\emph{Flavobacterium} , FC 4.95).

Within the categories of `cell wall/membrane/envelope biogenesis', and
`cell motility',

several membrane proteins facilitating substrate binding and biofilm
formation were expressed. Key adhesive structures include lipoprotein
(expressed by \emph{Pseudomonas} on D3 and \emph{Marinomonas} on D7),
large adhesive protein (LAD; expressed by \emph{Pseudomonas} on D3), and
outer membrane protein A (OmpA; expressed by \emph{Thioclava}
,\emph{Shewanella} , \emph{Pseudomonas} , \emph{Rheinheimera} on D7),
with\emph{Acinetobacter} showing the highest expression on D7 (FC 4.4 ±
0.14; Figure 3). Additionally, Type V secretory adhesin AidA (FC 2.92 ±
0.76) and elements of the Type II secretion system (TIISS) were
expressed by \emph{Pseudomonas} on D3 (Figure 4). Pili were also
expressed by \emph{Pseudomonas} on both D3 and D7, with pili assembly
proteins PilZ (\emph{Pseudomonas} , D3) and FimV (\emph{Marinomonas} ,
D7) similarly expressed. In parallel, the curli assembly/transport
component CsgG was identified in \emph{Marinomonas} on D7 (Figure 4),
indicating potential involvement in biofilm formation. (Figure 4)
Additionally, flagellin, which may facilitate substrate adhesion,
exhibited higher expression on D7 compared to D3. This increase was
particularly noted in\emph{Marinomonas} (FC 4.86 ± 0.79),
\emph{Pseudoalteromonas} (FC 4.5 ± 0.13), \emph{Rheinheimera} (FC 4.02),
and \emph{Pseudomonas} (FC 2.96 ± 0.32). However, certain
\emph{Pseudomonas} species demonstrated elevated flagellin expression on
D3 (FC 3.35 ± 0.31), suggesting variation in flagellar activity between
strains across different time points.

Proteins associated with biofilm arrangement including components of
calcium-gated (EF-hand domain-containing protein, dCache\_2
domain-containing protein, \emph{Pseudomonas} ), and potassium channels
(potassium uptake protein TrkA, \emph{Marinomonas} ) were expressed on
D3 and D7, respectively. One instance of quorum sensing, via the
expression of the autoinducer 2-binding periplasmic protein LuxP, was
found on D7, expressed by an uncharacterised species of \emph{Vibrio} .
Further examples of cell-cell interactions were identified through the
expression of competence protein ComEA (\emph{Pseudomonas} , D3),
involved in horizontal gene transfer (HGT), the OAA-family lectin-sugar
binding domain-containing protein, (\textgreater{}D3 \emph{Pseudomonas}
, FC 4.72), and TIVSS protein (\emph{Marinomonas} , D7). Proteins
involved in purine nucleotide biosynthesis (e.g. bifunctional purine
biosynthesis protein PurH), which are also thought to play a role in
early biofilm formation, were expressed on both days (Figure 3;
Supplementary file S3).

\textbf{{[}Figure 3{]}}

\emph{3.3 Stress-driven resilience and competition in a nascent marine
plastisphere}

Proteins associated with the regulation of oxidative stress, nutrient
limitation, and interspecies competition were abundantly expressed,
reflecting the plastisphere's resilience to environmental stress and the
intense competitive dynamics within these biofilms (Figure 4). Evidence
of nutrient starvation was observed through the expression of cobalamin
biosynthesis protein, CobW (\emph{Marinomonas} ), and stringent
starvation protein (\emph{Marinomonas, Psychromonas} ) on D7.
Additionally, poly(3-hydroxyalkanoate) granule-associated proteins PhaF,
Phal and poly(R)-hydroxyalkanoic acid (Pha) synthase expressed
by\emph{Pseudomonas} on D3 (Figure 4), provided evidence for starvation
responses due to nutrient limitation. Moreover, evidence for alternative
carbon source utilisation was observed, including three enzymes
associated with the alkane degradation pathway. Including,
catalase-peroxidase (Kat) expressed by \emph{Marinomonas}
(\textgreater{}D7; FC 2.96), \emph{Psychromonas} (D7),
and\emph{Pseudomonas} (D3), aldehyde dehydrogenase (ALDH) expressed
by\emph{Pseudomonas} (D3; D7), \emph{Thioclava} (D7), \emph{Rhodobacter}
(D7), and \emph{Marinomonas} (D7), and alcohol dehydrogenase (AD)
expressed by \emph{Marinomonas} (D7; Figure 4). Protein identification
using PlasticDB - a database containing proteins specifically mediating
plastic biodegradation -- identified the polyethylene-degrading enzyme
laccase expressed by \emph{Psychromonas} , on D7 (Figure 2;
Supplementary file S2).

Proteins associated with carbon metabolism, under the functional
classification `carbohydrate transport and metabolism' (Figure 3), were
consistently upregulated by \emph{Pseudomonas} on D3, and
by\emph{Marinomonas} on D7. CreA family proteins and response regulatory
domain-containing proteins were also expressed by \emph{Pseudomonas} on
D3. Tricarboxylic transporters were expressed by \emph{Pseudomonas} on
D3, and by \emph{Pseudomonas, Pseudorhodobacter, Rhodobacter}
and\emph{Marinomonas} on D7 (Supplementary file S3). Interestingly,
enzymes involved in aromatic hydrocarbon biodegradation were
characterised, including dienelactone hydrolase, mostly expressed by
\emph{Marinomonas} on D7 (FC 2.03; Figure 3), and by \emph{Pseudomonas}
on D3. Nine other enzymes associated with this process were discovered
on both days, including 3-oxoadipate-CoA transferase, 3-hydroxyacyl-CoA
dehydrogenase, acetyl-CoA carboxylase, acetyl-CoA acetyltransferase,
acetyl-CoA dehydrogenase, pyruvate aldolase, p-cresol methylhydroxylase
(PCMH)-type protein, thiolase I, and thiopurine s-methyltransferase. Of
these, acetyl-CoA acetyltransferase and acetyl-CoA dehydrogenase were
most expressed on D7 (FC 4.51 and 2.48, respectively). Evidence for
nitrogen cycling was found through the constitutive expression of
nitrate reductase (\emph{Pseudomonas} , D3; \emph{Pseudomonas,
Marinomonas,} D7), nitrous-oxide reductase (\emph{Pseudomonas,} D7), and
nitrogen regulatory protein PII (\emph{Pseudomonas} , D3;
\emph{Pseudomonas, Acinetobacter} , D7) on D3 and D7. Nitrogen and urea
transporters were also expressed by \emph{Pseudomonas, Marinomonas,} and
\emph{Paracoccus} on both days.

In response to intracellular ROS, the plastisphere communities expressed
a variety of stress-response proteins, including alkyl hydroperoxide
reductase (Ahp), catalase (CAT), catalase peroxidase (Kat), glutathione
peroxidase (Gpx), thiol peroxidase (TP), and thioredoxin peroxidase
(TPx) in response to hydrogen peroxide
(H\textsubscript{2}O\textsubscript{2}), flavohemoprotein (Fhp) to
respond to nitric oxide (NO), and superoxide dismutase (SOD) to
neutralise superoxide radicals. The most frequently expressed
stress-response proteins were Ahp (\emph{Pseudomonas} ,
D3;\emph{Pseudomonas} , \emph{Paracoccus} , D7;
\textgreater{}D7\emph{Acinetobacter} , FC 5.83; \textgreater{}D7
\emph{Marinomonas} , FC 4.32), Fhp (\emph{Pseudomonas} , D3;
\emph{Acinetobacter} , D7; \textgreater{}D7 \emph{Marinomonas} , FC
3.5), Kat (\emph{Pseudomonas,} D3; \emph{Psychromonas} , D7;
\textgreater{}D7 \emph{Marinomonas} , FC 2.96), and SOD
(\emph{Paracoccus} , D3; \emph{Acinetobacter} ,\emph{Psychromonas} ,
\emph{Rhodobacter} , D7; \textgreater{}D3\emph{Pseudomonas} , FC 3.92;
\textgreater{}D7 \emph{Marinomonas} , FC 2.74) due to their consistent
expression across multiple genera (Figure 4; Supplementary file S3). In
response to metals and anions,\emph{Marinomonas} (D7), and
\emph{Paracoccus} (D7) expressed copper resistance protein A (CopA), and
toxic anion resistance protein (TelA), respectively, and
\emph{Pseudomonas} (D3) expressed tellurium resistance protein (TerD)
(Figure 4). Osmotic stress proteins OsmC (\emph{Pseudomonas,
Marinomonas,} D7), and glucans biosynthesis protein C
(\emph{Pseudomonas} , D3) were also found. The downstream effects of
stress were apparent in the expression of membrane repair proteins
ATP-dependent zinc metalloprotease (FtsH), \selectlanguage{greek}α-\selectlanguage{english}2 macroglobulin (\selectlanguage{greek}α\selectlanguage{english}2m), and
phage shock protein (Psp). Additionally, transcriptional response
proteins such as HU, RecA, RdgC, cold shock-domain (CSD) proteins, and
DNA repair proteins such as GrpE, Lon protease, and methionine sulfoxide
reductase were also adundant. Further stress mitigation was supported by
thioredoxin/thioredoxin reductase systems, along with a suite of
chaperones and chaperonins (e.g., ClpA, ClpB, DnaJ, DnaK, GroEL, GroES,
HtpG, SurA), and the redox enzyme glucose-6-phosphate dehydrogenase
(Figure 4). Among these, the cold shock-domain protein was the most
abundantly expressed protein, found in the proteomes
of\emph{Pseudomonas} (\textgreater{}D3 FC 3.8), \emph{Marinomonas}
(D7),\emph{Acinetobacter} (D7), \emph{Paracoccus} (D7), and
\emph{Shewanella} (D7). Further protective mechanisms were also
identified, such as dipicolinate synthase expression by
\emph{Pseudomonas} on D3 and capsular biosynthesis protein on D7.
Interestingly, proteins linked to reactive oxygen species (ROS)
generation, including sarcosine oxidase (\emph{Pseudomonas} , D3) and
Na(+)-translocating NADH-quinone reductase (\emph{Marinomonas} ;
\textgreater{} D7 FC 7.01), were also expressed.

Competition for resources was evidenced by the expression of proteins
associated with competitive stress. This included the type VI secretion
system (TVISS) proteins, such as TssM, contractile sheath large subunit,
and secretion proteins Evp, IcmF, and VasK, expressed
by\emph{Pseudomonas} (D3; FC 1.74 \selectlanguage{ngerman}± 0.12), \emph{Pseudoalteromonas} (D7;
FC 3.54), and Acinetobacter (D7) (Figure 4; Supplementary file S3).
Competitive advantage was also indicated by the expression of
hemolysin-related proteins and the actin cross-linking toxin VgrG2
by\emph{Pseudomonas} on D3. In potential response to antibiotic
production, resistance mechanisms were expressed, such as polymyxin
resistance protein ArnA and OprM by \emph{Pseudomonas} on D3, and
\selectlanguage{greek}β-\selectlanguage{english}lactamase by \emph{Pseudomonas} and colicin-I receptor proteins
by\emph{Shewanella} on D7.

Interestingly, the potential pathogens \emph{Pseudomonas}
\emph{syringae} and \emph{Pseudomonas} \emph{aeruginosa} , expressed two
TVISS proteins on D3, while \emph{P.} \emph{syringae} also expressed
tol-pal system proteins and a FeADH domain-containing protein. These
results indicate a complex competition network within the biofilm, where
both resource limitation and interspecies antagonism shape the microbial
community structure and resilience.

\textbf{{[}Figure 4{]}}

Discussion

In this study, we analysed 2,948 non-redundant proteins from the LDPE
plastisphere, providing near-complete proteomes for the two dominant
genera \emph{Pseudomonas} and \emph{Marinomonas} within the first days
of colonisation. This extensive dataset offered valuable insights into
the metabolic activity of these key taxa, as well as the broader
functioning of the young plastisphere community. In particular, we
revealed the intense competition between taxa within the rapidly
developing community and identified the molecular mechanisms
underpinning plastisphere resilience to nutrient limitation and
oxidative stress. Our results also revealed molecular interactions
between the early plastic colonisers and the plastic itself, with
important implications for the modulation of biofilm formation and the
exploration of plastic biodegradation. These results provide new
insights into the dynamics of the nascent plastisphere and their
biotechnological potential.

Dominance of key taxa in the young marine plastisphere

The activity of the young marine plastisphere was dominated by
heterotrophic bacteria, particularly Gammaproteobacteria, consistent
with our previous studies examining plastisphere formation and function
{[}6, 9, 16{]}, and with studies investigating the taxonomic diversity
of the plastisphere {[}7, 28, 29{]}. Herein and in our previous work,
plastisphere samples were collected from the Scottish coast, and
biogeographic location appears to play a significant role in shaping
plastisphere communities, as noted in several studies including our own
{[}6, 9, 16{]}. Community development in the present study mirrored
previous observations demonstrating that plastisphere diversity
increases over time {[}10-13{]}. Indeed, we observed a plastisphere
community whose activity was dominated by \emph{Pseudomonas} after three
days of colonisation, shifting to an active community comprised of
Bacteroidetes after seven days. \emph{Pseudomonas} , the pioneering
genus in this study and one of two high-quality MAGs recovered from the
marine plastisphere, is known for its metabolic versatility and
biotechnological potential. Its species span biogeochemical cyclers,
symbionts, decomposers, denitrifying bacteria, and pathogens {[}30{]}.
This genus is particularly known for its biofilm-forming capabilities,
facilitated by the secretion of specialised membrane proteins and
secretion systems which likely facilitate interactions with plastic
(Section 4.3) {[}31{]}. We have previously observed \emph{Pseudomonas}
within the marine plastisphere, but at \textless{} 25\% of the active
community {[}9, 16{]}, indicating that the functional role
of\emph{Pseudomonas} is most significant within the initial stages of
plastisphere development. As such, the expressed proteome of this
organism provides important insights into microbe-plastic interactions
at the time of most significance, where colonisers specifically
attracted to the plastic surface may utilise it as a substrate {[}32{]}.
Nevertheless, the most abundant and active genera found on
D7,\emph{Marinomonas} , was the second high-quality MAG recovered and is
also a marine-adapted genus typically associated with pollutant
bioremediation {[}33, 34{]}. Its enrichment on the LDPE substrate
suggests that beached plastic provided an ideal niche for colonisation,
but likely as a secondary coloniser. Consistent with this, in our
previous work, \emph{Marinomonas} was also the dominant and active taxa
following 1-2 weeks LDPE colonisation, but it was less important within
the mature marine plastisphere. Additionally, further bioremediative
genera such as \emph{Thioclava} {[}28{]} were active on D7 (relative
abundance 1.5\%), alongside the presence of potential pathogens \emph{P.
aeruginosa,} and \emph{P. syringae} which represented 4.6\% and 17.5\%
of all annotated species, equalling 285 non-redundant proteins {[}35,
36{]}. Indeed, \emph{P. syringae} was the most annotated species in the
D7 plastisphere, indicating a concerning enrichment of potential
pathogens within the portion of the plastisphere (19\%) that could be
annotated to this level. \textbf{The observed} diversity within this
early-stage plastisphere highlights the biofilm as a dynamic
environment, initially dominated by one or two taxa, likely shaped by
significant competitive interactions.

Biofilm formation

Biofilm formation is a key survival strategy for microbial proliferation
in challenging, nutrient-poor marine environments {[}37, 38{]}. Several
proteins involved in surface adhesion were found to be repetitively
identified across different taxa known for their propensity to form
biofilms. In this way, biofilm formation might have been promoted
in\emph{Pseudomonas} , \emph{Marinomonas} , \emph{Acinetobacter} ,
and\emph{Vibrio} through the expression of secretion systems I, II, and
IV, as well as flagellin, adhesin, curli, Omps, lipoprotein, and LAD
{[}39-41{]}. These surface adhesion mechanisms were predominantly
expressed by \emph{Pseudomonas} , potentially aiding their dominance and
rapid colonisation of plastic at D3. The succession of\emph{Marinomonas}
after the initial colonisers further supports their biofilm-forming
ability, which is well-documented in marine environments {[}34{]}. The
detection of quorum sensing molecules expressed by\emph{Vibrio} on D7
could indicate that this genus was involved in processes potentially
related to surface adhesion, such as the regulation of membrane sorting
proteins, which are often associated with coordinated biofilm
development {[}42{]}.

Biofilm formation may have also been supported by the upregulation of
purine biosynthesis, the TCA cycle, arginine, and glutamate cycling
proteins, which are crucial for energy and resource allocation within
the biofilm community {[}37, 38, 43{]}. Moreover, our previous findings
highlighted the critical role of glutamine in biofilm proliferation
{[}16{]}. Its regulation was found to be closely linked with ammonium
availability and oxidative stress within low-nutrient environments,
with\emph{Marinomonas} showing distinct glutamine metabolism within the
plastisphere {[}9{]}. This suggested a differential nutrient strategy
between biofilm-associated and planktonic communities, further
supporting the notion of a resilient and metabolically adaptive
plastisphere biofilm {[}16{]}. The observed increase in microbial
diversity over time implies that the resources produced by pioneering
species (e.g., carbon, amino acids) may have facilitated the growth of
subsequent colonisers, creating a more complex and resilient biofilm
{[}15, 44{]}.

Importantly, plastisphere biofilms offer distinct advantages to
microorganisms in these environments. For instance, the expression of
TIVSS and a competence protein suggests the potential for HGT within the
plastisphere {[}39{]}, allowing microbes to share adaptive traits.
Moreover, the expression of calcium-gated channels (e.g., EF-hand and
dCache\_2 domain-containing proteins) and potassium uptake proteins
(e.g., TrkA) points to a potential cooperative exchange of nutrients
between neighbouring cells, further enhancing the biofilm's capacity to
thrive under nutrient-limited conditions {[}45{]}. Moreover, nitrogen
sufficiency is suggested by the expression of nitrogen cycling proteins
like nitrate reductase, nitrogen regulatory protein PII, and glutamine.
Carbohydrates and amino acids were metabolised and transported within
the community, indicating possible resource sharing through symbiosis or
cross-feeding, contributing to the overall resilience and stability of
the biofilm {[}15, 44{]}.

However, it is important to note that biofilm communities are not only
cooperative but also competitive, as microorganisms compete for
dominance and resources within these complex ecosystems.

Resilience and competition

Proteins associated with the regulation of metabolic stressors were
among the most abundant expressed non-housekeeping proteins found in our
datasets. Enzymes responsible for mitigating oxidative stress were
frequently expressed by multiple genera (e.g., \emph{Pseudomonas}
,\emph{Marinomonas} , \emph{Acinetobacter} , \emph{Paracoccus}
,\emph{Psychromonas} , and \emph{Rhodobacter} ), with nearly the entire
process of reactive ROS suppression (antioxidant production - DNA
regulation - DNA repair) being characterised {[}46{]}. Oxidative stress
in multi-species biofilms may be triggered by spatial constraints,
forced oxygen gradients, competition, nutrient limitation, and the
accumulation of abiotic stressors such as metals (e.g., CopA, TelA,
TerD) {[}6, 47{]}. The plastic itself could also contribute to oxidative
stress through the accumulation of ROS under UV-light exposure {[}48,
49{]}. The multifunctionality of proteins identified here, such as the
frequently expressed CSD protein (\emph{Pseudomonas} ,\emph{Marinomonas}
, \emph{Acinetobacter} , \emph{Paracoccus} ,\emph{Shewanella} ),
suggests that they may have been induced by a combination of these
stressors. Indeed, in addition to their role in cold adaptation, cold
shock proteins are crucial for maintaining protein stability and proper
folding under stress conditions, extending their protective function
against oxidative and metabolic stress {[}50{]}.

Granule storage, competition, and the expression of
nutrient-limitation-related proteins highlight nutrient stress during
both sampling days. Interestingly, granule formation is often triggered
by the scarcity of other essential nutrients outside carbon sources
{[}51{]}. Additionally, we found evidence for the utilisation of diverse
potential carbon sources through the expression of enzymes associated
with alkene and consequentially polyethylene degradation (AD, ALDH, Kat,
laccase), also as noted by Delacuvellerie et al.
(2022).\emph{Pseudomonas} enriched at D3 are also known to be associated
with the biodegradation of polyethylene {[}52-54{]}. Enzymes related to
aromatic hydrocarbon metabolism, such as dienelactone hydrolase {[}55,
56{]}, were also detected, though no definitive evidence of plastic or
aromatic hydrocarbon biodegradation was found. The synthesis of granules
and the presence of carbohydrate metabolism proteins suggest an
available carbon source, though potentially through cross-feeding.

Nutrient limitation is known to cause dysregulation of core metabolic
processes, leading to the accumulation of ROS, as well as an increased
competition for resources {[}57, 58{]}. In support of this, the
expression of hemolysin and VgrG2 toxins by \emph{Pseudomonas} may
suggest competitive interactions within the plastisphere biofilm. The
production of these toxins, alongside other virulence factors, could
potentially induce membrane damage in neighbouring microorganisms
through the action of the TVISS {[}59, 60{]}. Interestingly, we
identified a near-complete annotation of the TVISS machinery (i.e. TVISS
proteins IcmF, VasK, VipB, membrane subunit TssM, contractile sheath
small subunit, and large subunit, Hcp1 family TVISS effector, EvpB
family TVISS protein) expressed by active members of the marine
plastisphere {[}8, 16, 17{]}. These findings suggest that interspecies
competition within the biofilm could be a significant factor in shaping
plastisphere community structure and dynamics.

Using the TVISS as an indication of virulence
{[}61{]},\emph{Pseudomonas} were most competitive on D3 within a
predominantly single genera biofilm. Previously, \emph{Pseudomonas} ,
and\emph{Pseudoalteromonas} have also been found to outcompete other
microbial species through the secretion of toxins {[}62-64{]}, perhaps
actively antagonising other members of the biofilm. The expression of
proteins used to convey antibiotic resistance (i.e. b-lactamase, efflux
RND transporters, capsule protein) {[}39, 64, 65{]} by\emph{Pseudomonas}
and \emph{Marinomonas} on D7, highlight the intense competition within
the diversified plastisphere community. These mechanisms are important
for maintaining cell integrity in a stressful environment, but their
expression puts great demand on the cell's intracellular resources
{[}39, 57, 61, 62{]}, including those which may be used in key metabolic
processes. The deregulation of these processes can further precipitate
oxidative stress -- similar to nutrient deprivation {[}57, 58{]} -- and
limit the growth of impacted bacteria {[}46{]}. Interspecies competition
may have therefore contributed to this plastisphere's function and
composition, confirming the observations made by Herschend \emph{et al.}
(2017) and Guillonneau\emph{et al.} (2018) in model biofilm communities
{[}14, 15{]}.

Metabolic versatility for plastic degradation

The proteins expressed here which relate to the utilisation of
alternative carbon sources are of interest within the context of plastic
biodegradation. As stated in \emph{Section 4.3} , we do not find this to
be conclusive evidence of pollutant biodegradation within these
plastispheres. However, it is interesting to note that the two enzymes
expressed here which align with Delacuvellerie's (2022) depiction of the
alkene degradation pathway (AD, ALDH) are expected to be involved in the
initial depolymerisation process of polyethylene degradation by Tao
(2023) {[}8, 66{]}. Catalase peroxidase, also found here, but not in the
alkane degradation pathway, is expected to play a role in the first
steps of polyethylene biodegradation. Our recent paper on the late-stage
plastisphere also revealed the expression of acyl-CoA dehydrogenase
{[}16{]}, another element of the alkene degradation pathway, which was
also expressed in Delacuvellerie's (2022) study alongside ferredoxin and
ferredoxin reductase {[}8{]}. In combination, five enzymes associated
with the alkene degradation pathway have now been annotated from within
the marine plastisphere, plus an additional enzyme which may directly
depolymerise polyethylene.

In alignment with our previous investigation, 10 proteins involved in
fatty acid beta-oxidation (i.e. acetyl-CoA carboxylase,
acetyltransferase, and dehydrogenase) were found in these plastispheres
{[}16{]}. This process, like the alkene degradation pathway, has been
linked to both plastic biodegradation and the biodegradation of other
xenobiotic hydrocarbons due to the need to dismantle their similarly
structured hydrocarbon base {[}67{]}. Metabolites from xenobiotic
degradation are also likely funnelled directly into the fatty acid
beta-oxidation pathway once initially depolymerised {[}66{]}.
Dienelactone hydrolase is not linked to this pathway but is associated
with the final stages of aromatic hydrocarbon (chlorophenol) degradation
{[}55, 56{]}. The discovery of such enzymes is interesting because no
such xenobiotics were present in these samples, and the plastics used
were purportedly free of additives. Another trend of note is the
upregulation of these proteins by \emph{Marinomonas} . Even if these
organisms did not actively biodegrade aromatic hydrocarbons in these
samples, this may reveal a little of their adaptability to other
xenobiotics such as crude oil in the marine environment {[}33{]}.

The combined expression of enzymes which may be used by bacteria to
biodegrade plastics, and other noted xenobiotics here may not indicate
that plastic biodegradation is occurring in this scenario. However, it
may suggest that organisms found here and in other marine plastispheres
have the capacity to perform this biodegradation. Interestingly, the
incomplete annotation of these processes can rarely be attributed to a
singular genus. Instead, several genera express these enzymes in tandem.
This may have significant implications for the field of plastic
bioremediation, encouraging the exploration of complex microbial
communities rather than relying on single species for more effective
plastic degradation.

Conclusion

To date, the molecular mechanisms underpinning the initial days of
microbial colonisation of marine plastic debris have remained
uncharacterised. To resolve the interactions between the earliest
colonisers and plastic surfaces, we utilised multi-omics to investigate
the activity of the marine plastisphere community grown on LDPE for 3
and 7 days. Our findings reveal a complex relationship between
pioneering and secondary colonisers, shaped by stressors such as
reactive oxygen species (ROS) and nutrient limitation, while also
highlighting the interplay of cooperation and competition among
microorganisms. Consistent with previous research, the majority of these
nascent plastisphere microorganisms were primarily composed of
heterotrophic bacteria, indicative of marine plastisphere communities
found in colder environments. The near-complete assembly of
metagenome-assembled genomes (MAGs) for \emph{Pseudomonas}
and\emph{Marinomonas} significantly enhanced our ability to characterise
the associated proteomes and elucidate their precise functions within
the dynamic young plastisphere. In this study, \emph{Pseudomonas,} the
pioneering genera, was likely aided through its known ability to form
biofilms, while the genera detected in the later plastisphere
diversified over time, potentially fuelled by metabolites produced
within these biofilms. Proteins related to the biodegradation of plastic
and other xenobiotics were expressed by \emph{Pseudomonas}
,\emph{Marinomonas} , and three additional genera, highlighting the
potential for these microorganisms to contribute to plastic
biodegradation processes. Further studies exploring the functioning of
the nascent plastisphere are required, to determine their role in those
processes. Overall, this study significantly enhances our understanding
of the early formation of the plastisphere and its interactions with
plastic, providing valuable insights essential for addressing the
growing environmental challenge of plastic pollution.

Associated data

The mass spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium
(https://proteomecentral.proteomexchange.org/) via the MassIVE partner
repository {[}68, 69{]} with the dataset identifier PXD056358 (for
reviewers only: ftp://MSV000095986@massive.ucsd.edu).

Acknowledgements

The authors would like to thank Dr. Johannes Werner for the updated
version of mPies.

Funding

This research was funded by the joint~UKRI~Natural Environment Research
Council (NERC)~and the~National Research Foundation Singapore (NRF),
project, ``Sources impacts and solutions to plastics in South-East Asia
coastal environments''. L.F.M and S.M.-S were supported by
the~UKRI~NERC/NRF~project (NERC~Award No.~NE/V009621/1,~NRF~Award
No.~NRF-SEAP-2020-0001). The Bioprofiling platform used
for~proteomic~analysis was supported by the European Regional
Development Fund and the~Walloon Region, Belgium.

CL is the recipient of a studentship funded by the NERC Scottish
Universities Partnership for Environmental Research (SUPER) Doctoral
Training Partnership (DTP) (Grant reference number NE/S007342/1).
Additionally, CL received a travel mobility fund through the MASTS-SFC
Saltire Emerging Researcher Scheme (MASTS-SERS) to support her travel
expenses to Belgium for her research visit at UMons.

\emph{The authors declare no financial or intellectual conflicts of
interest regarding the publication of this paper.}

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\textbf{Figure legends}

\textbf{Figure 1} . Three- and seven-days growth of a marine microbial
community on LDPE, and analysis of biofilm structure and functioning.
ASW+G, artificial seawater supplemented with vitamins, trace metals, and
glucose; BHB+v, Bushnell Haas Broth supplemented with vitamins and trace
metals.

\textbf{Figure 2.} Relative abundance of microbial species found within
the metaproteome at each time point. Calculated using the number of
proteins associated with each genus relative to all the proteins found
in that condition. Values used for doughnut plot shown in table below,
as well as corresponding metagenomic data.

\textbf{Figure 3.} Total differentially regulated proteins
(FC\textgreater{}1.5, P-adj. \textless{}0.05) according to function and
taxonomic classification. Positive fold change (purple) in a indicates
greater abundance of proteins on D7 (purple), and negative fold change
(orange) indicates greater expression on D3.

\textbf{Figure 4.} Graphic representation of biologically relevant
processes found within this study's most abundant bacterial genera from
relative quantification and qualitative analysis of the metaproteomic
dataset. Purple = more expressed on day 7, orange = more expressed on
day 3, black = qualitative results, * = qualitative results with 1
entry, grey = not found, but can be assumed. Colour code indicates all
basal processes active within the genus at that time (\textgreater{}1
protein each). All basal processes active in the largest, annotated
microorganisms. Acronyms: \textbf{Figure elements} : H2O2, hydrogen
peroxide; HGT, horizontal gene transfer; NO, nitric oxide; O2,
superoxide; PE, polyethylene; PHA, polyhydroxyalkanoate granule; Xn,
xenobiotic. \textbf{Stress} (osmotic, oxidative, heat/cold, interspecies
interaction, xenobiotic): Ahp, alkyl hydroperoxide reductase; ArnA,
polymyxin resistance protein; \selectlanguage{greek}α\selectlanguage{english}2M, alpha-2-macroglobulin; \selectlanguage{greek}β-\selectlanguage{english}L,
\selectlanguage{greek}β-\selectlanguage{english}lacatamse; Cap, capsular biosynthesis protein; CAT, catalase; CopA,
copper resistance protein A; CSD, cold shock domain containing protein;
Fhp, flavohemoprotein; FtsH, ATP-dependent zinc metalloprotease; G6PD,
glucose-6-phosphate dehydrogenase; Gpx, glutathione peroxidase; GrpE,
DnaK nucleotide exchange factor; GSH, glutathione; HU, transcriptional
regulator HU; Kat, catalase peroxidase (also PE degradation) ; LonP, lon
protease; MsrP, methionine sulfoxide reductase; OP, 5-oxoprolinase;
OprM, outer membrane efflux system protein; Osm, osmotically inducible
protein; Psp, phage shock protein; RdgC, recombination-associated
protein; RecA, SOS response protein ; SOD, superoxide dismutase; OpuAC,
substrate binding domain of ABC-type glycine betaine transport system;
TerD, tellurium resistance protein; TP, thiol peroxidase; TPx,
thioredoxin peroxidase; Trx, thioredoxin; TrxR, thioredoxin reductase;
Chaperones- ClpA, ClpB, DnaJ, DnaK, HtpG, SurA; Chaperonins- GroEL,
GroES. \textbf{Virulence and biofilm formation} : 1B, hemolysin
co-regulated protein; BepC, outer membrane efflux protein; Ccda, control
of cell death protein; ComEA, competence protein; EL-Tu, elongation
factor Tu; hcp, hemolysin-coregulated protein; Hly, hemolysin
activation/ secretion protein; Omp, outer membrane protein; RND, efflux
resistance-nodulation-division (RND) transporter; SecYEG, translocon;
TISS, type-I secretion system; TIISS, type-II secretion system; TIVSS,
type-IV secretion system; TVISS, type-VI secretion system; VgrG2b, actin
cross-linking spike protein; Translocons- IcmF, Tld/PmbA, Vask,
VipB.\textbf{Starvation} : CobW, cobalamin biosynthesis protein; PhaF,
polyhydroxyalkanoate (Pha) granule-associated protein F; Phal, Pha
granule-associated protein L; PHAs, PHA synthase; SSP, stringent
starvation protein. \textbf{Adhesion} : AidA, adhesin; CGC,
calcium-gated channel; CsgG, curli assembly protein G; LAD, large
adhesive protein; LP, lipoprotein; OAA, OAA-family lectin sugar binding
domain containing protein; TrkA, Potassium uptake protein.
\textbf{Aromatic hydrocarbon degradation} : 3-OCoAT, 3-oxoadipate-CoA
transferase; 3-HA-CoAd, 3-hydroxyacyl-CoA dehydrogenase; ACC, acetyl-CoA
carboxylase; A-CoAAT, acetyl-CoA acetyltransferase; A-CoAD, acetyl-CoA
dehydrogenase; CMBL, dienelactone hydrolase; PA, pyruvate adolase;
PCMH-p, p-crestol methylhydroxylase (PCMH) type protein; TI, thiolase I;
TMPT, thiopurine s-methyltransferase. \textbf{PE degradation} : AD,
alcohol dehydrogenase; ALDH, aldehyde dehydrogenase.

\textbf{Supplementary files}

\textbf{Supplementary File\_S1} Overview of the total metaproteome
identified at D3 and D7 showing the taxonomic annotation (LCA score
\textgreater{} 80\%) obtained from mPies using our Combined-Metagenome
Database.

\textbf{Supplementary File\_S2} Protein identification obtained from the
publicly available databases for antimicrobial resistance (CARD),
plastic metabolism (PlasticsDB), and virulence factor (VFDBs).

\textbf{Supplementary File\_S3} Fold-change analysis summary using
Skyline when comparing protein abundance ratio at D7 and D3.

\textbf{Supplementary File\_S4} Peptide Summary Exports generated by
Protein Pilot, including peptide sequences, and associated metadata for
each condition and replicate.

\textbf{Supplementary File\_S5} Protein Summary Exports generated by
Protein Pilot, and associated metadata for each condition and replicate.

\textbf{Hosted file}

\verb`Figure.1.pptx`  available at \url{https://authorea.com/users/840919/articles/1231089-decoding-microbial-plastic-colonisation-multi-omic-insights-into-the-fast-evolving-dynamics-of-early-stage-biofilms}

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\verb`Figure.2.pptx`  available at \url{https://authorea.com/users/840919/articles/1231089-decoding-microbial-plastic-colonisation-multi-omic-insights-into-the-fast-evolving-dynamics-of-early-stage-biofilms}

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\verb`Figure.4.pptx`  available at \url{https://authorea.com/users/840919/articles/1231089-decoding-microbial-plastic-colonisation-multi-omic-insights-into-the-fast-evolving-dynamics-of-early-stage-biofilms}

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