# The biofilm matrix: multitasking in a shared space

Hans-Curt Flemming, Eric van Hullebusch, Thomas Neu, Per Nielsen, Thomas Seviour, Paul Stoodley, Jost Wingender, Stefan Wuertz

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## To cite this version:

Hans-Curt Flemming, Eric van Hullebusch, Thomas Neu, Per Nielsen, Thomas Seviour, et al. The biofilm matrix: multitasking in a shared space. Nature Reviews Microbiology, 2023, 21, pp.70-86. 10.1038/s41579-022-00791-0. hal-03903210

HAL Id: hal-03903210

https://cnrs.hal.science/hal-03903210v1

Submitted on 3 Oct 2025

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# The biofilm matrix: multitasking in a shared space

Hans-Curt Flemming¹✉, Eric D. van Hullebusch², Thomas R. Neu³, Per H. Nielsen⁴, Thomas Seviour⁵, Paul Stoodley⁶,⁷, Jost Wingender⁸ and Stefan Wuertz⁹¹

**Abstract** | The biofilm matrix can be considered to be a shared space for the encased microbial cells, comprising a wide variety of extracellular polymeric substances (EPS), such as polysaccharides, proteins, amyloids, lipids and extracellular DNA (eDNA), as well as membrane vesicles and humic-like microbially derived refractory substances. EPS are dynamic in space and time and their components interact in complex ways, fulfilling various functions: to stabilize the matrix, acquire nutrients, retain and protect eDNA or exoenzymes, or offer sorption sites for ions and hydrophobic substances. The retention of exoenzymes effectively renders the biofilm matrix an external digestion system influencing the global turnover of biopolymers, considering the ubiquitous relevance of biofilms. Physico-chemical and biological interactions and environmental conditions enable biofilm systems to morph into films, microcolonies and macrocolonies, films, ridges, ripples, columns, pellicles, bubbles, mushrooms and suspended aggregates — in response to the very diverse conditions confronting a particular biofilm community. Assembly and dynamics of the matrix are mostly coordinated by secondary messengers, signalling molecules or small RNAs, in both medically relevant and environmental biofilms. Fully deciphering how bacteria provide structure to the matrix, and thus facilitate and benefit from extracellular reactions, remains the challenge for future biofilm research.

Biofilms are ecosystems in which microbial cells are embedded in a matrix of extracellular polymeric substances (EPS). This matrix represents a structured and shared space, which is generated and controlled by the organisms within the biofilm. The composition, properties and dynamics of the matrix influence the biofilm mode of life. EPS is the standard term in biofilm research¹, but ‘extracellular matrix’ (ECM) is sometimes used as an alternative. However, the latter term includes not only microbial but also eukaryotic matrices², which causes confusion when discussing biofilms in tissue culture or clinical specimens³. Most recently, the term ‘matrixome’ has been suggested, defining the entire inventory of currently known bio-molecules, and their molecular, structural and functional diversity⁴. In this Review we use the term ‘EPS matrix’ for the EPS produced by the microorganisms in a biofilm.

This matrix surrounds and immobilizes the biofilm cells. It comprises water-soluble polysaccharides, proteins, extracellular DNA (eDNA) and water-insoluble compounds, such as cellulose⁵, amyloids⁶ and non-amyloid proteinaceous fibres⁷ as well as lipids. Humic-like microbially derived refractory substances have also been reported as matrix components⁸.

The matrix can contain multifunctional membrane vesicles that are shed by the biofilm cells⁹,¹⁰. It can sequester material from external environments, thus acquiring components such as dissolved nutrients, mostly plant-derived humic substances, transparent exopolymer particles¹¹, colloids, biotic and abiotic particles¹² from environmental, technical and clinical settings, or host-derived eDNA¹³.

The EPS matrix provides the infrastructure for an extremely flexible, versatile and adaptable form of multicellular microbial life and demonstrates emergent properties that are not shared by single planktonic or sessile cells. As early as 1987, Costerton et al. likened biofilms to multicellular tissues¹⁴. Later, biofilms were classified as collective biological systems, such as forests, bee hives or coral reefs¹², and likened to extended organisms¹⁵. Biofilms display a high level of diversity¹⁶ and a clear division of labour, as exemplified in *Bacillus subtilis* biofilms¹⁷. The situation is highly complex: organisms within a biofilm produce diverse EPS components at various points in time during their growth, which can be influenced by other microbial species present in the biofilm. This evolution of the matrix takes place in response to the amount and nature of nutrients available and the environmental conditions they encounter,

**Extracellular polymeric substances**

(EPS). Microbial biopolymers such as polysaccharides, proteins, extracellular DNA (eDNA) and others, forming the biofilm matrix.

**Extracellular matrix**

The non-cellular component present within all tissues and organs, sometimes also used as an alternative to the term extracellular polymeric substances (EPS); frequently used in the medical context.

<footnote>✉e-mail: hc.flemming@uni-due.de
https://doi.org/10.1038/s41579-022-00791-0</footnote>
**Amyloids**

Aggregates of proteins
in fibrillar morphology.
Pathogenic amyloids form
by misfolding of previously
normal structures. In biofilms,
amyloids fulfil many functions,
including, for example, matrix
stabilization, nutrient storage,
desiccation resistance and
others.

Humic-like microbially
derived refractory
substances

Remains of bacterial cells that
are not readily degraded after
cell death. As high molecular
weight compounds they remain
present within microbial
communities, contributing to
the polymeric matrix.

Humic substances

The organic components of
humus. Humic substances are
hetero-polycondensates based
on a motif of aromatic nuclei
with phenolic and carboxylic
substituents linked together.
They can form aggregates,
provide cation complexation
sites and regulate the
bioavailability of metal ions.

Transparent exopolymer
particles

Extracellular acidic polysaccha-
rides produced by phytoplank-
ton and bacteria in saltwater,
freshwater and wastewater;
they are extremely abundant
and play a significant role in
biogeochemical cycling of
carbon and other elements
in water.

Collective biological systems

Systems such as forests,
beeches, coral reefs or kelp
fields that show emerging
properties which exceed those
of the sum of the single
individuals. Also known as
'extended organisms'.

such as hydrodynamic shear stress, pressure, salt con-
tent, temperature, light regime and grazers. In fact, the
matrix exists in a continuum of physical states ranging
from dissolved to dense gels¹⁸, which sometimes con-
tain biogeominerals, either precipitated in or sequestered
by the matrix, such as manganese oxides or calcium
carbonate¹⁹.

The EPS matrix is heterogeneous in space and time,
even in single-species biofilms grown under controlled
conditions in the laboratory, frequently studied in the
medical context. Much less is known about its hetero-
geneity in microbial communities in natural environ-
ments, man-made structures and certain medical and
oral habitats. Millions of bacterial species coexist in
those biofilms, most of which are uncultured and unde-
scribed, all producing numerous EPS components. As
an example, several million types of exopolysaccha-
rides can be expected to exist²⁰. The physico-chemical
characterization of various individual EPS components
in mixed-species biofilms has made great progress.
Nevertheless, it is still challenging to identify which
member in a given biofilm community produces cer-
tain components and for what function and when. In
addition, current methods of analysis have revealed
snapshots of the matrix only, not enabling the follow
up of temporal dynamics and interactions of differ-
ent community members and their individual matrix
compounds. New methods such as the combination
of catalysed reporter deposition-fluorescence in situ
hybridization with fluorescent lectin-binding analysis
begin to enable the identification of glycoconjugates and
their 3D localization in relation to the microorganisms
that produce them²¹. More recently, stable isotope trac-
ing combined with 2D correlation spectroscopy linked
carbon and nitrogen turnover to distinct matrix polysac-
charides and matrix proteins²². Furthermore, optotrac-
ing, a method that is currently under development, was
used for selective fluorescence-based monitoring of
the location and the dynamics of curli and cellulose in
*Salmonella enterica* biofilms²³. However, what triggers
or inhibits the production of individual EPS molecules,
to what extent and how they can be engineered remains
largely unexplored.

It is thus not surprising that EPS have been termed
'the dark matter of biofilms', representing an intractable
component<fnref n="8" />, and consequently an 'identity crisis' was

declared<sup>24</sup>. Very important and underappreciated are the
interactions among EPS molecules and the cells which
they embed.

In this Review, we revisit the enormously growing
interest and progress in analysing and understanding
the matrix of microbial biofilms as compared with our
understanding more than a decade ago<sup>25</sup>. We focus on
the diversity of the EPS components and novel aspects
of the mechanisms and consequences of their func-
tional interactions. These are central to understanding
the resilience and the evolutionary success of microbial
biofilms in both environmental and medical settings.

Furthermore, we acknowledge that the contribution
of eukaryotic microorganisms to EPS composition,
function and dynamics in biofilms can be substantial.
Microalgae produce large amounts of matrix com-
pounds in aquatic environments<sup>26</sup>. In addition, fungi are
known to produce EPS compounds in medically impor-
tant biofilms (for example, *Candida* spp.<sup>27</sup>), but are also
considered relevant in environmental biofilms<sup>28</sup>. Beyond
the scope of this Review are the manifold ecological,
technical and medical aspects of the biofilm matrix,
which will also not be addressed.

The matrix

Much information on chemical interrogation of
components in the matrix depends on the isolation
method used; however, there is no universal tech-
nique. Carbohydrates, proteins and nucleic acids are
consistently found with various methods when dif-
ferent physical and chemical isolation techniques are
compared (Supplementary Table 1). Surprisingly, these
methods have not changed throughout the past decade,
with the exception of ionic liquids for EPS extraction
(Supplementary Box 1). By contrast, the various meth-
ods to analyse the matrix components chemically and
microscopically have greatly expanded over recent years
(Supplementary Tables 2 and 3).

The components of the biofilm matrix determine the
architecture, function, mechanical stability and dynam-
ics of the EPS matrix in various ways. This is the result
of different and dynamic interactions of a multitude of
macromolecules produced by cells as the biofilm devel-
ops. Using confocal laser scanning microscopy, diffu-
sion coefficients of molecules ranging from 10,000 to
240,000 Da were determined and found to be linearly
proportional to the cube roots of the molecular weights
and markedly lower than diffusion coefficients in bulk
water, regardless of biofilm thickness²⁹. Interestingly,
pores in the matrix can be generated by cells that actively
move through the matrix ('swimmers'). These pores
remain open for a sufficiently long time to facilitate the
diffusion of macromolecules throughout an established
biofilm³⁰,³¹.

Many EPS components can now be imaged non-
destructively using specific fluorescent dyes and
probes. For example, the location and distribution of
lectin-specific glycoconjugate linkages as well as polysac-
charides having a β-(1 → 3) or β-(1 → 4) linkage such as
cellulose could be analysed in a river biofilm. By means
of such combinations, the complex, coral reef-like struc-
ture of environmental biofilms can be visualized (FIG. 1).

Author addresses

<footnote n="1">¹Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Singapore.</footnote>

<footnote n="2">²Université Paris Cité, Institut de Physique du Globe de Paris, CNRS, Paris, France.</footnote>

<footnote n="3">³Department of River Ecology, Helmholtz Centre for Environmental Research — UFZ, Magdeburg, Germany.</footnote>

<footnote n="4">⁴Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark.</footnote>

<footnote n="5">⁵Aarhus University Centre for Water Technology, Department of Biological and Chemical Engineering, Aarhus University, Aarhus, Denmark.</footnote>

<footnote n="6">⁶Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA.</footnote>

<footnote n="7">⁷Department of Orthopaedics, The Ohio State University, Columbus, OH, USA.</footnote>

<footnote n="8">⁸University of Duisburg-Essen, Biofilm Centre, Department of Aquatic Microbiology, Essen, Germany.</footnote>
<figure><img src="image_4.png" /><figcaption>Fig. 1 | **The matrix of microbial biofilms.** Confocal laser scanning microscopy micrograph of an epilithic river biofilm. **a** | Multichannel image data set (five channels) visualized as 3D volume projection. This example demonstrates the complexity and heterogeneity of an environmental biofilm landscape showing only two of the many potential matrix components. **b–e** | Individual channels and the biochemical nature of the signal (on the right): volume of bacterial cell distribution (panel **b**); autofluorescence of chlorophyll **a** indicating algae (panel **c**); massive volume of lectin-specific glycoconjugates containing fucose (panel **d**); volume of β-(1 → 3) and β(1 → 4) polysaccharides such as cellulose (panel **e**). Please note that the combined image in panel **a** diminishes parts of the individual signals due to their 3D location and the dense biofilm structure. Colour allocation: white, mineral reflection; green, SybrGreen nucleic acid stain; blue/purple, autofluorescence of phototrophic microorganisms; red, AAL-lectin stained glycoconjugates; yellow, CalcofluorWhite-stained polysaccharides. Grid size: 50 µm. Images courtesy of U. Risse-Buhl (sample), U. Kuhlicke (confocal laser scanning microscopy) and T. R. Neu (visualization), all from Helmholtz Centre for Environmental Research — UFZ.</figcaption></figure>

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b

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c

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d

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e

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**Polysaccharides as structural matrix elements**

For most of the history of biofilm research, polysaccharides were thought to be the major and most important matrix component, originally likened to the glycocalyx of eukaryotes³². Purification, separation, quantification and identification of polysaccharide constituents are not trivial tasks. Polysaccharides are highly diverse, not only in their monosaccharide constituents but also in glycosidic linkages, branching and substitution with non-carbohydrate residues and charges (for example, acetate, pyruvate, succinate and phosphate, among others), resulting in a nearly unlimited range of structures²⁰,³³,³⁴. Different microbial species produce different polysaccharides, and even single strains produce multiple polysaccharides at different stages of biofilm development and depending on changing environmental conditions. For example, *Pseudomonas aeruginosa* produces at least three polysaccharides — Psl, Pel and alginate. Polysaccharides have distinct functions in adhesion and microcolony formation, in the development of the structure and architecture of monospecies biofilms as well as in defined mixed-species biofilms. Thereby, polysaccharide composition substantially contributes to the emergent properties of such communities as, for example, collective protection from environmental stresses such as desiccation, immune effectors or predators³⁵,³⁶. The serine protease inhibitor *ecotin* binds to Psl and, thus, protects *P. aeruginosa* from neutrophil elastase-mediated killing³⁷.

Post-excretion modifications of matrix polysaccharides are well known, including changes in length as well as addition or removal of substituents. Pel and poly-N-acetylglucosamine are partially N-acetylated, generating a heterogeneous backbone that, depending on the organism, can influence synthesis, translocation and adhesiveness³⁸. In *Rhizobium leguminosum*, a glycanase determines the length of the acidic polysaccharide and strongly influences the assembly and stability of the biofilm matrix. The surface-associated cellulase CelC2 has a key role in cellulose biosynthesis
Psl

An extracellular polysaccharide of *Pseudomonas aeruginosa* and an important structural and functional feature of *P. aeruginosa* biofilms. Psl is rich in galactose and mannose.

Pel

An extracellular polysaccharide of *Pseudomonas aeruginosa* and an important structural and functional feature of *P. aeruginosa* biofilms. The structure of Pel is not fully characterized but it is a cationic polysaccharide, differing from Psl and alginate.

Ecotin

A protease inhibitor, formed in the periplasmic space of Gram-negative bacteria, inhibiting neutrophil elastase.

Anammox

(Ammonium oxidation).
The reaction of nitrite and
ammonium ions leading
directly to dinitrogen and
water.

by modulating the length of the cellulose fibrils that
mediate firm adhesion to root tips³⁹.

Research on the EPS matrix of aerobic granular sludge
revealed the role of α-1,4-glucans and proteins as key
polymers for granule cohesion, bridged by divalent cati-
ons as the major aggregative mechanism⁴⁰. Recently, hya-
luronic acid and sulfated glycosaminoglycan-like (GAG)
polymers were found in aerobic granular sludge⁴¹. They
seem to be widespread, contributing to the stability of
biofilm systems, although it remains unclear whether
they are formed within the granules or are imported
from wastewater. Glycosylation of matrix proteins is
now recognized as a way to modify functionality. An
example is sialic acid, which has been found as an EPS
component of seawater-adapted aerobic granular sludge,
but also in pathogenic bacteria. Sialic acid seems to be
involved in the protection of sugar residues of glycopro-
teins against glycosidases⁴². Glycoproteins are suggested
to be strongly involved in the structure of anaerobic
ammonium-oxidizing (anammox) granules, having a sim-
ilar role to glycans in the extracellular matrix of eukary-
otic organisms, for example in vertebrates, in which they
are an important constituent of the connective tissue⁴¹,⁴³.

Cellulose — an insoluble matrix polysaccharide

Water-insoluble polysaccharides, in particular cellulose,
have been identified as fundamental matrix components,
influencing the architecture and morphology of microbial
biofilms. Although bacterial cellulose was first described
by Brown as long ago as 1886 (REF.⁴⁴), it has only recen-
tly been acknowledged to be of geochemical, agricultural,
biotechnological and medical importance, providing
both structure and protection to microbial biofilms³⁵,⁴⁵.
The ubiquity and abundance of cellulose in a wide range
of microbial species led to the suggestion that cellulose is
one of the most prevalent exopolysaccharides present in
the biofilm matrix⁵. Cellulose consists of polysaccharide
chains of glucose units with (1 → 4) β-glycosidic bonds.
The backbone of the β-glycosidic bonds imparts high
crystallinity and considerable rigidity on the cellulose
molecule, provided by the hydrogen-bonding network
present among hydroxyl groups of glucosyl residues⁴⁴.
These hold the chains firmly together and result in fibrils
with a mechanical stress resistance comparable with
steel⁴⁶. Cellulose has a central role in cellular aggregation
of *Escherichia coli*, *Salmonella typhimurium*, *Pseudomonas
spp.*, *Vibrio cholerae*, *B. subtilis* and others in liquid
cultures⁵. In *Aliivibrio fischeri* biofilms, cellulose affects
both the morphology and the elasticity of the matrix⁴⁷.

Cellulose can be ‘decorated’ by phosphoethanolamine (pEtN) substituents after synthesis (pEtN cellulose)⁴⁸. pEtN cellulose can form nanocomposites with amyloid fibres in the EPS matrix of biofilms, resulting in tissue-like cohesion and elasticity. When, macrocolonies give way under mechanical stress, due to the pEtN nanocomposites, they fold into macroscopic morphological patterns of agar-grown colonies termed ‘wrinkled’, ‘rugose’ or ‘rdar’ (for red, dry and rough)⁵.

Proteins

Enzymes. One of the most obvious protein functions
in the matrix is the enzymatic catalysis of biochemical

reactions. Their global relevance cannot be overest-
mated: the primary function of extracellular enzymes
is the degradation of macromolecules to low molecular
weight products small enough to be transported into
the cells as carbon and energy sources for microbial
metabolism. Comparing activity in microbial aggre-
gates versus that in the bulk water, specific activities were
usually higher in attached bacteria⁴⁹. Comprehensive
overviews of matrix enzymes and their functions are
available⁵⁰,⁵¹. As the enzymes can be retained, for exam-
ple, by polysaccharides⁵² or humic substances⁵³, they are
kept in close proximity to the biofilm cells, resulting in
an external digestion system¹².

The role of extracellular enzymes is not restricted
to polymer degradation; they are important for
the modulation of the matrix, particularly through
polysaccharide-degrading hydrolases, esterases, pro-
teases and lyases, which enables continuous restruc-
turing (BOX 1). Thus, enzymes modulate structural
dynamics of the matrix by causing localized modifi-
cations with concomitant changes in the chemical and
physical properties such as gelation behaviour, ion bind-
ing and mechanical stability²⁵. Moreover, extracellular
enzymes can function as virulence factors; an example
is the strong interference of biofilm-associated proteases
with wound healing after infection⁵⁴, although host pro-
teases are also involved. Enzymes are also crucial for the
detachment and dispersal of biofilms⁵⁵,⁵⁶.

In natural ecosystems, the turnover of biopolymers,
which is performed by extracellular enzymes, represents
the rate-limiting step in the mineralization and cycling
of particulate and dissolved organic matter. The major-
ity of microorganisms on Earth live in biofilms, driving
all biogeochemical cycles⁵⁷. Their extracellular enzymes
transform enormous quantities of dissolved polymers
globally and render particulate substrates bioavail-
able, thus these enzymes represent the bottleneck of the
planetary self-purification processes⁵⁷⁻⁵⁹.

**_The ubiquitous functional amyloids._** Amyloids are highly ordered water-insoluble proteins composed of a vast variety of amino acids but all have in common a strongly conserved quaternary structure of cross-β-strands. Their ordered structure results in oligomers which adopt a β-sheet structure that is non-covalently bound by hydrogen bonds⁶⁰. They can be stained by dyes such as Congo red or Thioflavin T⁶⁰. The term amyloid entails a biophysical, and not a biological, description. The morphology and biophysical characteristics of all amyloid fibrils are remarkably similar, although the primary structure of amyloid proteins may differ strongly⁶¹. Strikingly, amyloids are self-assembling in a nucleation-dependent process from their monomeric constituents given the right environmental conditions⁶². In contrast to amyloids as misfolded plasma proteins associated with brain diseases in humans, amyloids in biofilms fulfil manifold biological functions. Amyloids show a structural, biochemical and functional versatility that microorganisms exploit in different contexts and for different purposes⁶³.

Amyloids represent an important and frequent struc-
tural component of biofilm matrices⁵ that help main-
tain homeostasis¹⁵. Amyloids can stabilize the biofilm
Box 1 | Matrix dispersion and degradation: leaving the house and eating it

The biofilm matrix is a dynamic space with continuous production and degradation of all extracellular polymeric substances (EPS) components, which turns them into a nutrient source. The many structural components are produced, transformed and degraded by various extracellular matrix housekeeping enzymes, either produced by the producer itself or by other biofilm microorganisms<sup>183</sup>; ultimately, they can function as ample carbon sources, as reported in activated sludge<sup>184</sup>, soil<sup>185</sup> or the deep sea<sup>186</sup>.

The degradation of matrix components is of particular importance for the dispersion of bacteria from the biofilm and of practical relevance for anti-biofouling measures. Many enzymes have been tested for single-species and mixed-population biofilm removal. The mechanisms are diverse and affected by numerous factors, both intrinsic and extrinsic to the biofilm<sup>55,56</sup>. In general, the dispersion coincides with the expression of genes encoding matrix-degrading enzymes, primarily for the degradation of self-produced adhesins, DNA and polysaccharides<sup>55</sup>. Several recent examples are described, including galactosidase from *Mycobacterium tuberculosis*<sup>187</sup> and glycosidase from *Desulfovibrio vulgaris*<sup>188</sup>. Endonuclease is expressed to degrade the DNA present in the matrix and glycoside hydrolases for degradation of the polysaccharides Psl and PeI<sup>189</sup>. The surface-associated protein CdrA is matrix-stabilizing, and when released from cells it impedes the dispersion<sup>55</sup>. *Staphylococcus aureus* produces an arsenal of exoenzymes that have multiple purposes such as facilitating dispersal, maintaining or changing matrix or biofilm properties and degrading its own or other EPS components as nutrients<sup>139</sup>.

Phages are also involved in biofilm dispersal. EPS-degrading enzymes can be encoded in bacteriophages, and they are believed to facilitate access for phages to cells within the biofilms<sup>190</sup>. These include depolymerases degrading sialic acid, levan, xylan, dextran, rhamnogalacturonan, poly-γ-glutamate, hyaluronate, alginate and triacylglycerol, and can, besides various biotechnological applications, also be used for biofilm disruption<sup>190</sup>.

Our knowledge about the degradation of polysaccharides in environmental biofilms is limited as most bacteria in these communities are uncharacterized and the structure of the polysaccharides is extremely variable<sup>34,191</sup>. Enzymes that break down glycans are called CAZymes (carbohydrate-active enzymes). The high specificity of many of these enzymes can provide information about the structure of the degraded glycan<sup>191</sup>. Bacteria belonging to the phylum Bacteroidetes use hundreds of enzyme combinations to degrade glycans<sup>191</sup>. They encode glycan-degrading systems termed polysaccharide utilization loci (PULs), and it seems that each PUL can break down specific glycan structures<sup>191</sup>. Bacteroidetes have surface-linked carbohydrate-binding proteins, sugar transporters and a sugar sensing apparatus detecting early degradation products<sup>192</sup>. Members of the Bacteroidetes (for example, the families Cytophagaceae and Saprospiraceae) are abundant in environmental biofilms (for example, in soil and wastewater systems) and have an important role in the degradation of exopolysaccharides<sup>193</sup>.

Cellulose is also degraded by numerous bacteria and, besides the Bacteroides, well-known bacteria within a biofilm such as *Bacillus*, *Acinetobacter* and *Pseudomonas* spp. can do so, but the mechanism is best described in relation to degradation of plant material (cellulose<sup>194</sup>, lignocellulose<sup>195</sup>).

Extracellular proteins are generally easily degraded by a few conserved families of proteases that are excreted by various bacterial species, and they have been successfully applied to eradicate specific biofilms (for example, *Bacillus pumilus* biofilms<sup>196</sup>). Most bacteria excrete multiple proteases. For example, *Pseudomonas aeruginosa* secretes several proteases that target both biofilm and host proteins<sup>197</sup>. Bacterial functional amyloids are characterized by their extreme robustness, also to enzyme attack, and no specialized amyloid-degrading microbial enzymes are described today. Microbial keratinases are known to also degrade β-amyloids related to human disease<sup>198</sup>.

Extracellular DNA (eDNA) can be degraded by a range of extracellular nucleases, for example, promoting the degradation of the biofilm matrix of several pathogens such as *P. aeruginosa* and *Vibrio cholerae*, and leading to dispersal of the biofilm<sup>199</sup>. In marine sediments, some bacterial species seem to be specialized in degrading eDNA, some producing up to five different nucleases<sup>200</sup>.

functional amyloids in *P. aeruginosa*<sup>67</sup>, the extracellular protein TasA<sup>68</sup>, biofilm-associated proteins (baps)<sup>69</sup> and phenol-soluble modulins (PSMs)<sup>70</sup> have all been comprehensively described. Some methanogens even use amyloids as a sheath attributing cell stability and stiffness<sup>71</sup>.

**Hydrophobic regions in a hydrophilic matrix.** Although highly hydrated, the matrix can still contain hydrophobic regions. Hydrophobic matrix proteins (hydrophobins) have been studied mostly in *B. subtilis*. The biofilm surface layer protein A (BslA) secreted by *B. subtilis* can form a hydrophobic film that coats the biofilm surface, thus rendering it water-repellent<sup>72,73</sup>. This molecule undergoes an environmentally responsive conformational change upon partitioning from an aqueous to a hydrophobic environment, for example, when encountering a hydrophobic surface or the air-water interface, and has been termed a 'hydrophobic raincoat'<sup>73,74</sup>. Once at an air-liquid interface, BslA self-assembles into an ordered rectangular 2D paracrystalline lattice that forms a macroscopically visible elastic protein layer<sup>75</sup>. Three categories of BslA and its variants based on their wrinkle relaxation behaviour were proposed<sup>72</sup>: surfactant-like; transiently wrinkling biofilm formers; and rigid biofilm formers. It was suggested that the hydrophobic surface of a biofilm helps prevent erosion of cells and, at the same time, helps retain humidity in the underlying matrix, and thereby protects biofilm cells from water stress<sup>76</sup>.

Although the EPS matrix is generally considered to be a hydrophilic environment, activated sludge flocs from wastewater treatment plants can accumulate hydrophobic pollutants<sup>77</sup>. It was shown that they were associated with the matrix and not the cells, clearly indicating the existence of hydrophobic domains within the EPS matrix<sup>25,78</sup>. Proteins can interact with amyloids, resulting in hydrophobic pockets<sup>79</sup>. BslA can self-assemble into an elastic hydrophobic film, coating the interface of *B. subtilis* biofilms<sup>75</sup>. In addition to proteins such as Bap1 and BslA being hydrophobic<sup>72,73,76</sup>, single-stranded eDNA was also shown to increase the hydrophobicity of the EPS matrix<sup>80</sup>. The remains of lipid-containing compounds such as bacterial cell envelopes may also contribute to the hydrophobic domains within the EPS matrix<sup>81</sup>, together with rhamnolipids<sup>82</sup>. This could explain the finding of hydrophobic domains in *Shewanella oneidensis* as measured with amphiphilic Q-dots in combination with confocal laser scanning microscopy<sup>83</sup>. Nevertheless, a deeper mechanistic understanding of hydrophobic domains in the biofilm matrix is still lacking.

**The manifold functions of eDNA**

eDNA has been identified as an important feature of the biofilm matrix, representing much more than merely an inert structural element<sup>84</sup>. It is one of the most frequently found matrix polymers<sup>80</sup>. Different mechanisms are involved in eDNA release, such as autolysis and active secretion as well as release via membrane vesicles. The origin of eDNA seems to be genomic DNA<sup>80</sup> and mostly lysis-related<sup>85</sup>. However, eDNA is not always structurally or compositionally identical to genomic DNA<sup>86</sup>. In several bacteria, there is a relationship between eDNA

Rhamnolipids

Amphiphilic glycolipids consisting of a monosaccharide or disaccharide connected by a glycosidic bond to a fatty acid; they act in various roles in the EPS matrix.

matrix or be involved in cell division, nutrient storage, surface-tension modulation, cell cycle regulation, cell-cell communication, adhesion, flocculation, desiccation resistance, cytotoxicity, virulence<sup>64</sup> and evasion of the host immune system<sup>65</sup>. Furthermore, they contribute to resistance against mechanical, thermal and chemical stressors of biofilms<sup>5</sup>. Amyloids such as curli in *E. coli*<sup>5,66</sup>,
Necromass

The mass of dead biological
material, including
microorganisms.

release and bacterial competence to take up DNA from
the environment⁸⁵.

The biological functions of eDNA include roles in
adhesion and primary stages of biofilm development, as
a scaffold component, in gene transfer and DNA damage
repair, and as a source of organic carbon, nitrogen and
phosphate⁸⁷. The analysis of G-quadruplex eDNA struc-
tures using ionic liquid extraction (Supplementary Box 1)
revealed viscoelastic networks in *P. aeruginosa* that con-
tribute to matrix stability⁸⁸. eDNA might also provide a
genetic pool for bacteria that can take up genetic mate-
rial and recombine it with homologous DNA fragments
by natural transformation⁸⁹,⁹⁰.

Interactions between DNA and other biomole-
cules on the cell surface and in the EPS matrix seem
to be mediated by unspecific electrostatic interactions,
aided by divalent cations⁸⁰. eDNA was found to bind
to proteins of the DNABII family in single-species
and mixed-species biofilms. These proteins stabilize
the eDNA scaffold and are localized at the vertices
of the eDNA lattice structure and have a high affinity
for branched DNA structures which include Holliday
junctions⁹⁰. Holliday junctions are single-stranded
crossover intermediates of homologous recombination.
Mature biofilms are resistant against DNAses, which has
been suggested to be due to the formation of left-handed
Z-form eDNA; this molecule seems to have an important
structural role in biofilms by interacting with DNABII
proteins⁹¹.

Furthermore, some DNA-amyloid complexes mod-
ulate the mechanical resistance of *Bacillus licheniformis*
biofilms⁹², and eDNA provides a shielding function
against charged antibiotics and cationic antimicrobial
peptides through electrostatic binding to the molecules⁸⁴.
It further contributes to antibiotic tolerance by directly
inactivating cationic antibiotics⁹³. In *Clostridioides diffi-
cile*, eDNA is essential for biofilm formation and struc-
tural integrity⁹⁴. *Staphylococcus aureus* biofilms have
been reported to be stabilized by positively charged
cytoplasmic proteins that are released into the extra-
cellular environment, where they interact electrostati-
cally with the negatively charged cell wall and eDNA⁹⁵.
eDNA-binding lipoproteins contribute to *S. aureus* bio-
film formation by linking bacterial cells through cross
links with eDNA. With the biofilm maturing, eDNA
enters a nuclease-recalcitrant state, probably to further
protect the resident bacteria in the biofilm⁹⁶. In addi-
tion, extraction of matrix components with ionic liquids
(Supplementary Table 1) revealed substantial amounts
of external RNA in the matrix of *P. aeruginosa*⁸⁸.
Interestingly, *S. aureus* biofilms could sequester external
RNA from Brain Heart Infusion medium and use it as
contribution to the structural integrity of the matrix⁹⁷.

Membrane vesicles

Membrane vesicles are produced by all domains of
life. They consist of lipid bilayers that form spheres
20–500 nm in diameter and are released by bacteria,
archaea and eukaryotic membrane vesicles⁹⁸. Bacteria
produce physiologically heterogeneous types of mem-
brane vesicles⁹⁹, and the same species can release dif-
ferent types of membrane vesicles¹⁰⁰. Outer membrane

vesicles (OMVs) derived from the outer membrane of
Gram-negative bacteria have been found to be ubiq-
uitous in the matrices of bacterial biofilms, including
laboratory biofilms (for example, *P. aeruginosa* bio-
films) as well as biofilms in natural and technical aque-
ous environments¹⁰¹. OMVs can be considered to be
'nano-parcels' that travel across the matrix, containing
cytoplasmic material¹⁰² such as nucleic acids, toxins,
lipoproteins and enzymes¹⁰³, effector proteins, DNA¹⁰⁴,
antibiotics¹⁰, both hydrophilic¹⁰⁵ and hydrophobic¹⁰⁶ sig-
nalling molecules, lipids, adhesins, virulence modulating
factors¹⁰⁷ or ATP¹⁰⁸. OMVs of Gram-negative bacteria
are involved in bacterial survival under stress and regu-
late the interactions among members of Gram-negative
bacterial communities¹⁰⁷. OMVs have been described as
instrumental for stepwise cellulose hydrolysis¹⁰⁹. In an
oxacillin-sensitive methicillin-resistant *S. aureus* strain,
antibiotic stress (exposure to ceftazidime) induced
biofilm formation; the produced membrane vesicles
functioned as structural components and the increase
in hydrophobicity promoted intercellular junctions
within the biofilms¹¹⁰. Release of OMVs from cells
enhances biofilm formation of *Aeromonas* strains¹¹¹.
Gram-positive bacteria can also shed membrane
vesicles¹⁰³. Phage-encoded endolysin generated holes in
the cell walls of *B. subtilis* through which cytoplasmic
membrane material protruded into the extracellular
space, released as membrane vesicles¹¹², which induced
OMV formation in neighbouring Gram-negative
bacteria.

Humic substances and recalcitrant compounds

Humic substances are known as the components
of soil organic matter that have non-specifically
auto-assembled in supramolecular associations and are
composed of a vast variety of macromolecules¹¹³. It is
debatable whether humic substances should be included
in the definition of EPS and whether they can be mod-
ified within the matrix. In this Review, we consider
humic substances as common constituents present in
the matrix under environmental conditions when bio-
films come into contact with exogenous compounds
(such as water and soil). Several sources for humic sub-
stances must be considered: the microbial carbon pump
in the ocean¹¹⁴ and in soil¹¹⁵; dissolved organic matter,
particulate organic matter¹¹⁴ and soil organic matter¹¹⁵;
and humic-like microbially derived refractory com-
pounds and necromass¹¹⁶. The findings of these studies
suggest that the microorganisms are also responsible
for the production of long-term stable constituents and
particulates, all of which will contribute to recalcitrant
compounds and microbially derived humic substances
present in the biofilm matrix.

How and why matrix components interact

Structure, functions, mechanical stability and dynamics
of the matrix are based on various interactions among a
multitude of macromolecules and metabolites produced
by the cells during their life cycle in the biofilm (FIG. 2).
The matrix is not simply a passive scaffold and a protec-
tive space but can actively and reactively influence the
cells in the biofilm, and thus the dynamics of biofilm
<figure><img src="image_13.png" /><figcaption>b Transport, protection</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>c Matrix cross-linking</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>d Protection of ecotin
from protease</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>e Retention and protection
of exoenzymes (extracellular
digestion system)</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>f Stabilization of eDNA lattice</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>g Enhanced electron
transfer with eDNA</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>h Matrix stabilization
with eDNA and cations</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>i Matrix stabilization and
protection of eDNA</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>j Anchoring matrix to
biofilm cells</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>k Promoting cell aggregation</figcaption></figure>

<figure><img src="image_23.png" /></figure>

Fig. 2 | Interactions of extracellular polymeric substance components and their functions. **a** | Polysaccharides interact with each other, which increases matrix stability. **b** | Outer membrane vesicles (OMVs) transport matrix components, such as extracellular DNA (eDNA), enzymes, signalling molecules or ATP. **c** | Polysaccharide chains (PS1 and PS2) are cross-linked by interactions with proteins such as the microbial surface-associated protein CdrA. **d** | Interaction between the protease inhibitor ecotin and matrix exopolysaccharides (Pel) protects against host proteases. **e** | Polysaccharides aid in the retention of exoenzymes, which renders the

biofilm matrix an external digestion system. **f** | eDNA binds to proteins of the DNABII family, which stabilizes the eDNA lattice. **g** | Phenazine enhances electron transfer along the eDNA molecule. **h** | eDNA forms matrix-stabilizing complexes with cations. **i** | Interactions between amyloids and polysaccharides protect eDNA and stabilize the matrix. **j** | eDNA and lipoprotein (which is anchored in the membrane) connect the matrix to microbial cells within the biofilm. **k** | The interactions between eDNA and the exopolysaccharide PIA can promote cell aggregation. See Supplementary Table 4. AHL, N-acyl-L-homoserine lactone.

development (Supplementary Table 4). Functional inter-
actions turn the matrix into a feedback system with the
biofilm¹¹⁷, regulating proliferation, physiological charac-
teristics, intra-biofilm migration and differentiation of
the resident cells. Those functional interactions generate
many of the emergent properties of biofilms.

The matrix represents a confined space in which the
inherently high concentration of EPS molecules strongly
promotes various physico-chemical interactions, and
can be likened to molecular crowding inside microbial
cells¹¹⁸. These interactions result in the viscoelastic, but
mechanically stable and highly adaptable, structure that
develops during biofilm formation. Not all interactions
are cohesive; steric repulsion, another aspect of macro-
molecular crowding¹⁹, prevents the collapse of the
matrix, together with electrostatic repulsion of substit-
uents of the same charge, and the dilution by the high
water content.

A comprehensive view of the temporal and spa-
tial dynamics of biofilms is difficult to obtain. One
of the few successful examples is the development of
*V. cholerae* biofilms. Multiresolution imaging revealed
the complementary architectural role of four essential
matrix components: the protein RbmA mediates cell–
cell adhesion; the protein Bap1 provides adhesion of
developing biofilms to surfaces; and, together, *Vibrio*
polysaccharide (VPS), RbmC and Bap1 form dynamic,
flexible and structured envelopes that integrate biofilm
cell clusters¹²⁰,¹²¹.

The interactions of EPS components fulfil many
crucial functions. An example is the retention and
stabilization of degradative enzymes (for example,
lipase) by bacterial polysaccharides (for example, alg-
inate), preventing their loss into the environment⁵².
eDNA can interact with polysaccharides¹²²,¹²³ and pro-
teins, modulating and strengthening the EPS matrix⁹⁵.
In *P. aeruginosa* biofilms, the cross-linking of the exopolysaccharide Psl¹²⁴ with eDNA provides protection against enzymatic attack from DNase¹³. Of particular interest is the function of the exopolysaccharide Psl as an intercellular signalling molecule that actively stimulates cellular cyclic-di-guanosine-5'-monophosphate (c-di-GMP) production, thus amplifying the matrix biosynthesis in a positive feedback circuit¹²⁵. EPS molecules can retain iron ions, which promote biofilm formation by downregulating the synthesis of rhamnolipids, which in turn increases Psl production in *P. aeruginosa*¹²⁶. In *P. aeruginosa*, amyloids in the matrix can temporarily store and accumulate the signalling molecules, including N-acyl-L-homoserine lactones (AHLs) and *Pseudomonas* quinolone signal (PQS)¹²⁷. In mucoid *P. aeruginosa*, overproduction of the exopolysaccharide alginate interferes with the response to PQS, but not to AHLs¹¹⁷. In addition to its mechanical role, eDNA has been shown to facilitate the production of amyloid fibres in the EPS matrix of *S. aureus* biofilms by promoting the polymerization of PSMs⁷⁹. To add further complexity, the same study found that amyloid fibres induced autolysis and the release of eDNA, which can be interpreted as a positive feedback loop of EPS production⁷⁹. These examples indicate that EPS components can modulate cell-cell communication, and a new function was ascribed to EPS: the ability to function as signalling molecules that the cells can receive and respond to¹¹⁷,¹²⁵. OMVs¹²⁸ and proteins⁹⁰ have been shown to bind to eDNA to increase cellular aggregation and biofilm structural stability, and protect eDNA from degradation¹²⁹. In *P. aeruginosa*, phenazine as well as pyocyanine¹³⁰ interact with eDNA, which results in enhanced electron transfer among biofilm components, better eDNA binding to cell surfaces, increased biofilm viscosity¹³⁰ and enhanced electron transfer among biofilm components¹³¹. Furthermore, Mg²⁺ ions sequestered by eDNA trigger the expression of resistance genes in *P. aeruginosa*⁸⁰.

Thus, the matrix can be considered to be a continuously interactive, self-regulating, shared space — way beyond just a passive structure and protective shell for biofilm cells. There is increasing evidence that EPS components can be versatile in their multiple and sometimes redundant biological functions based on interactions with diverse ions, signalling molecules and biomers in response to the prevailing conditions of biofilm composition and development.

## Regulation of EPS matrix synthesis

EPS matrix formation, development and maintenance are highly regulated by multiple, species-specific systems through both genetic control at the cellular level and environmental factors. Most of the information has been gained from medically relevant single-species biofilms in laboratory settings, already demonstrating a wide diversity of strategies; multiple-species environmental communities will be even more complex. Certainly, there is no single regulatory mechanism for all EPS components in all biofilms at all development stages and in all environmental situations, but there is a huge variety of regulation pathways and networks with which different microorganisms are genetically equipped and in which

they can respond to environmental factors. However, some common principles can be identified (FIG. 3).

Different species use distinct regulatory pathways, and extrapolations of the insights gained from single-strain studies to mixed-species biofilms is questionable. Consequently, it is not yet feasible to manage biofilms in technical systems with the aim to overproduce or underproduce EPS, with the possible exception of biogranulation¹³². Abiotic factors such as nutrient levels, temperature, osmolarity or shear forces will also influence regulation. Furthermore, biofilms are rarely homogeneous and, instead, are characterized by heterogeneous cellular physiology and architecture, because of gradients of nutrients and terminal electron acceptors, such as oxygen, that are further accentuated during biofilm growth. A consequence is the heterogeneous production of matrix components. In general, a fully developed biofilm is not only the result of a specific genetic programme of developmental differentiation but also of growth and transport limitation¹³³, all of which lead to a spatially differentiated multicellular environment.

The control of matrix components by intracellular and extracellular signals is achieved transcriptionally and post-transcriptionally, as well as by environmental factors¹³⁴. Commonly, the control pathway starts with sensors, followed by signal transmitters, and ends with effectors which, in turn, execute production or

## Regulation of biofilm matrix components

<figure><img src="image_24.png" /></figure>

Fig. 3 | **Regulation of matrix synthesis.** Major factors that influence the formation and release of matrix compounds such as exopolysaccharides, proteins, extracellular DNA (eDNA) and membrane vesicles. At the genetic level, assembly is mostly coordinated by secondary messengers such as cyclic-di-guanosine-5'-monophosphate (c-di-GMP), signalling molecules such as quorum sensing molecules, or small RNAs, depending on the species involved. In addition, several environmental factors also control biofilm formation, such as oxygen, temperature, nutrients, osmolarity, redox potential, cell density, phages, antimicrobials, toxic compounds and mechanical stress. The mechanisms control matrix synthesis in both medically relevant and environmental biofilms.

**Phenazine**

The chemical description of the class of dibenzo annulated pyrazine; it embraces pyocyanine as a subclass in which one of the nitrogen atoms is substituted with a methyl group.
modulation of factors involved in biofilm formation, maintenance, and dispersion and detachment¹³⁵. In general, quorum sensing signalling molecules called autoinducers, the intracellular second messenger c-di-GMP and small RNAs are considered to be the main bacterial regulators as transmitters in the signal transduction pathway.

In *P. aeruginosa*, biofilm formation is regulated by three known systems¹³⁵. First, quorum sensing molecules and quinolone systems sense cell density and activate LasI and RhII, and thus their effectors LasR and RhIR. This results in the synthesis of rhamnolipids and the secretion of eDNA. Second, c-di-GMP is sensed by receptors such as WspA. The signal is transmitted by diguanylate cyclases and phophodiesterases to effectors and repressors such as Alg44, which initiates the synthesis of adhesins and exopolysaccharides and inhibits motility. Last, small non-coding RNA molecules are involved in a regulatory pathway that involves sensor kinases such as GacS and its cognate response regulator GacA, which activate the transcription of the small RNAs *rsmY* and *rsmZ*. In turn, *rsmY* and *rsmZ* reduce the activity of the effector protein RsmA, which results in the inhibition of Psl exopolysaccharide synthesis and enhanced motility¹³⁵,¹³⁶. In *P. aeruginosa*, LasB could promote biofilm formation partly through rhamnolipid-mediated regulation¹²⁶. The functional diversity of polysaccharides at different time points is controlled by complex regulatory mechanisms to ensure that the correct polysaccharide is produced at the right time and location within the biofilm³⁶.

An additional signalling function was discovered for the matrix exopolysaccharide Psl¹²⁶. It stimulates the production of c-di-GMP, and the increased intracellular c-di-GMP concentrations then lead to the increased production of Psl. These self-regulation mechanisms are based on a positive feedback regulatory loop linking production of the exopolysaccharides to their concentration in the biofilm. The results indicate the potential of matrix exopolysaccharides to act as short-range signalling molecules and function as a common good in the biofilm community¹²⁵,¹²⁶.

The formation of *V. cholerae* biofilms and the production of EPS in *V. cholerae* are well understood¹²⁰,¹²¹,¹³⁷. The production of VPS, which is the main constituent of the matrix, is mediated by proteins that are encoded by 12 genes, including the enzymes involved in producing the nucleotide sugar precursors, glycosyltransferases, polymerization and export proteins, acetyltransferases and phosphotyrosine protein phosphatase. The matrix proteins RbmA, RbmC and Bap1 are produced and secreted at various times during biofilm formation. A type II secretion system, composed of multiple export proteins, is responsible for the secretion of RbmA, RbmC and Bap1. In aquatic environments, salinity and osmolarity fluctuations as well as phosphate limitation can affect VPS gene expression and biofilm formation. As a key signalling molecule, c-di-GMP controls the motility and biofilm formation of *V. cholerae*.

Elegant work has shown in *E. coli* biofilms that a matrix of proteins, amyloid fibres, exopolysaccharides such as pEtN cellulose and eDNA form a robust network with pronounced stress resistance and

spatial, physiological differentiation in the supracellular architecture⁵,¹⁵. Gradients of nutrients, oxygen, waste products and signalling molecules determine whether the cells grow and proliferate or enter a stationary phase according to microscale spatial conditions. The heterogeneity of the matrix influences the architecture, physical properties and morphology of the macrocolonies. The extracellular matrix components, pEtN cellulose and amyloid curli fibres are major targets of the underlying control network. c-di-GMP specifically controls pEtN cellulose production, which enables the cells to alter the ratio of pEtN cellulose to amyloid curli fibres and, thus, to adjust the mechanical properties of the matrix in different zones of the biofilms⁵,¹⁵.

In *S. aureus*, biofilm development and virulence are regulated through numerous two-component systems. Genes in the accessory gene regulator (agr) system¹³⁸, which regulates the production of proteases and PSMs involved in biofilm assembly and disassembly, are best described. Interestingly, the expression of secreted virulence factors is inversely correlated to biofilm formation. In *S. aureus*, initially eDNA is the main EPS component, which is released through autolysis. Cells can adhere to eDNA through DNA-binding lipoproteins, providing stability⁹⁵. In this early stage, bacterial cells can be released from the biofilm by the production of nucleases (a stage that has been termed the 'exodus' stage)¹³⁸. In the early stages of development, PSMs reduce biofilm formation through their surfactant effect. PSMs can also have a direct positive role in EPS production through their polymerization into amyloid protein fibrils; in this later developmental stage the EPS matrix is increasingly proteinaceous¹³⁹. Active release of cells from the biofilm at this stage is achieved through the production of proteases. Complex structural and regulatory interactions among EPS components in *S. aureus* are likely. The presence of eDNA has been shown to promote the formation of amyloid fibres by phenol-soluble peptides⁷⁹. To add further complexity, the same study found that amyloid fibres induced autolysis and increased the release of eDNA, possibly indicating a positive feedback loop. The regulation of multiple-species biofilms and how biofilm organisms influence each other spatio-temporally remain open questions in biofilm research.

## Biofilm architecture

The architecture of microbial biofilms is determined by various factors. The major drivers are the hydrodynamics, substratum, temperature, nutrient limitation and ratios of available carbon, nitrogen and phosphorus, as well as trophic grazing and susceptibility to bacteriophages. The observer's perception of a given biofilm is influenced by the microscopic technique applied. In fact, the real architecture is only revealed by visualizing the fully hydrated biofilm. For this purpose, laser scanning microscopy (confocal/one-photon and multiphoton) evolved as a key analytical technique¹⁴⁰. More recently, optical coherence tomography and selective plane illumination microscopy have also been embraced¹⁴¹,¹⁴². Since the first substantial report¹⁴³, numerous publications of biofilm micrographs show the architecture of laboratory-grown single-species and multiple-species
<table><caption>Table 1 | Approaches and techniques established and needed to investigate the biofilm matrix</caption><thead><tr><th>Status</th><th>Approaches and techniques</th><th>Information provided</th></tr></thead><tbody><tr><td rowspan="5">Established</td><td>Traditional extraction and analysis</td><td>Biochemical composition of overall matrix</td></tr><tr><td>Proteomics</td><td>Identification of matrix proteins</td></tr><tr><td>Confocal microscopy</td><td>3D multichannel imaging of hydrated biofilm architecture</td></tr><tr><td>Optical coherence tomography</td><td>3D large-area single-channel reflection imaging</td></tr><tr><td>Modelling</td><td>Simulation of biofilm — development and dispersion, cellular interactions, matrix production</td></tr><tr><td rowspan="5">Needed</td><td>New extraction and analysis</td><td>Identification of new polysaccharide types</td></tr><tr><td>Glycomics</td><td>Large-scale glycan structure—function relationship</td></tr><tr><td>D-SPIM, mesolens</td><td>3D multichannel large-area imaging of biofilm architecture</td></tr><tr><td>Image scanning microscopy</td><td>Similar to confocal microscopy but at double resolution</td></tr><tr><td>Nanoscopy</td><td>Local high-resolution imaging, cell–matrix association, matrix–matrix interactions</td></tr><tr><td></td><td>Metabolomics (metabolic labelling)</td><td>Biosynthesis and dynamics of matrix constituents</td></tr></tbody></table>

D-SPIM, dual-view selective plane illumination microscopy.

### Glycomics

The study of all glycan structures in biology and a subset of glycobiology. Glycomics focuses on the identification of structure and function of the total collection of glycans (the glycome) produced by biological systems under specified conditions of time, space and environment.

### Mesolens

A novel microscope objective lens that combines a high numerical aperture with a large field of view of up to 6 mm combined with high spatial resolution.

### Nanoscopy

A term describing light microscopy techniques at a resolution across the diffraction limit of light. The techniques include localization or blink microscopy, stimulated emission depletion microscopy and, more recently, MinFlux. By exploiting switchable fluorochromes, achieving a resolution of 20–10 nm down to a few nanometres becomes possible.

biofilms, as well as biofilms found in the natural and built environment. Taken together, they demonstrate how variable and diverse biofilms are, but delineating their features microscopically depends on the visualization technique applied, the magnification and how the undis- turbed biofilm is prepared and mounted. Although the illustrations of biofilms derived from such images show a clearly defined biofilm border, in reality the biofilm edge represents a gradient in EPS concentration. Biofilms are accordingly mostly ragged at their periphery. In some cases, exposed microbes at the biofilm–water interface do not even seem to show a matrix around them.

Examples of environmental biofilms provide some insight into the architecture of the matrix microenvi- ronment. In most biofilm systems, water and EPS con- stituents are not homogeneously distributed within the matrix as there are pores and channels as well as clus- ters of matrix polymers. Three zones of EPS typically form: cell-associated, intercellular and an outer layer of EPS. This arrangement provides a scaffold that can seg- regate extracellular activities at the microscale as well as differentially binding contaminants<sup>144,145</sup>. Thus, the biofilm matrix is highly hydrated and displays certain spatial features, especially in thick gel-like biofilms. For example, methanol-fed biofilms developed in rotating annular reactors grew up to 4 mm thickness due to extensive production of a nearly transparent matrix<sup>146</sup>. The matrix examined with a range of probes showed globular features with bacteria both inside and outside the globules. In a different habitat, matrix-dominated biofilm structures found in semi-artificial caves show highly structured matrix features not seen before<sup>147</sup>.

Imaging of cells and EPS by means of confocal laser scanning microscopy in combination with fluorescent lectin-binding analysis<sup>148</sup> revealed extended capsular features and globular structures often linked to globu- lar strands with voids, but also multilayered spherical globules with higher levels of organization. The globu- lar matrix structures, depending on their biochemical composition, may influence the flux of low and high molecular weight compounds in and out of the biofilm.

Molecular techniques such as genomics, transcrip- tomics and proteomics focused, in part, on the extra- cellular space. So far, several omics techniques applied to microorganisms in the biofilm and biofilm matrices have revealed numerous proteinaceous constituents with new and unknown functions<sup>149</sup>. Only glycomics is missing to complete the omics approach. In fact, the information content of glycans is several orders of mag- nitude larger at a log scale if compared with genome, transcriptome and proteome data<sup>33</sup>. Nevertheless, inte- gral analysis of the structure and function of glycans is necessary to understand the different glycoconjugate constituents of the biofilm matrix. For this purpose, a range of approaches such as functional genetics, glycan microarrays, structure characterization, biochemical analysis of interactions and bioinformatics is required<sup>150</sup>. In contrast to many eukaryotic studies<sup>151</sup>, all of these tools have not yet been used in biofilm matrix research, very likely due to the complexity and missing expertise in glycomics and glycobiology.

In addition to the glycomic approaches mentioned, imaging the glycome represents another tool. Traditional methods applied fluorescently labelled antibodies (pure bacterial cultures) or lectins (environmental commu- nities) in combination with confocal laser scanning microscopy. Furthermore, metabolic labelling with fluorescent chemical reporters enables visualization of glycoconjugate dynamics in living cells<sup>152</sup>. This tech- nique, already applied in the field of microbiology, has demonstrated its potential<sup>153,154</sup> although it has not yet been implemented in biofilm matrix research. In terms of new technical developments in matrix visualization, image scanning microscopy with double resolution rel- ative to confocal microscopy may supplant traditional laser microscopy techniques<sup>155</sup>. Light sheet microscopy in a special configuration (dual-view selective plane illumination microscopy (D-SPIM); TABLE 1) may ena- ble 3D multichannel imaging of large biofilm areas, including the biofilm matrix landscape<sup>156</sup>. Similarly, mesolens microscopy will provide insights into both large areas as well as fine structures in biofilms<sup>157</sup>. Furthermore, laser-based ‘nanoscopy’ techniques applied to fully hydrated biofilms have great potential to pro- vide information on the dynamics and spatial inter- actions between EPS components and microbial cells. This approach has been applied to *V. cholerae* biofilms and showed the interaction of VPS with matrix proteins at high resolution<sup>120</sup>. Thus, nanoscopy techniques offer large potential in imaging different matrix constituents and interactions, and their application, even if currently hampered by the limited availability and access to the relevant instrumentation, can facilitate new insights into matrix architecture.
Mechanical properties of the EPS matrix

Viscoelasticity is a corollary of the self-assembly of matrix components¹⁵⁸,¹⁵⁹. Biofilm mechanics is an important field providing clues about how biofilms respond and adapt to physical forces. This is highly relevant for attached biofilms exposed to shear stress and free-floating aggregates prone to collisions. Adaptability in mechanical response is of particular importance for the survival in environments where physical forces can fluctuate greatly¹⁶⁰.

Mechanically, biofilms are generally considered to be highly hydrated hydrogels¹⁶¹. Their macroscopic rheological properties are determined at the molecular level, for example by electrostatic interactions and entanglement between the EPS polymers and charged molecules or ions. More recently, complex eDNA network structures, including those mediated by extracellular nucleotide-binding proteins⁸⁸,¹⁶², have been found to also have a mechanical function. Although the microbial cells produce the matrix polymers, in conceptual models explaining mechanical properties they are generally not considered to be influencing the bulk mechanical properties due to their relatively low volume fraction in the biofilm, which for many biofilms is estimated to be around 1% (see, for example, REF. ¹⁶³). Particle fillers are used in engineered materials to increase the stiffness of elastomeric polymers, and the required volume fraction is generally above 5%¹⁶⁴, which is above the volume

fraction of cells in biofilms. Astonishingly, however, no studies have been published so far about the actual contribution of the cells to matrix stability, and surprises appear to be possible.

A key feature of the reported mechanical properties of biofilms is their viscoelastic nature, even though the degree of elasticity (how much the biofilm stretches and springs back) and viscosity (how much the biofilm flows) under mechanical stress vary over many orders of magnitude, even for single-species biofilms¹⁶⁵. There are two main reasons that explain such variability. The first, and biologically most interesting, is that biofilms respond to different nutrient and hydrodynamic conditions structurally and mechanically over very short time spans (milliseconds)¹⁶⁶, or adapt gradually (days to weeks)¹⁶⁷. In principle, the cohesion of the matrix is based on four kinds of non-covalent bonds (hydrogen bonds, ionic bonds, electrostatic interactions and hydrophobic interactions) as well as on entanglement of polymers²⁵ (FIG. 4a). Each of these polymers has many potential binding partners, for example OH, acetyl, amino, pyruvate, sulfate or phosphate groups³⁴, that can engage in these interactions, adding mechanical stability to the EPS matrix. The rheological behaviour of biofilms can range from resembling elastic solids¹⁶⁸ to low viscosity liquids¹⁶⁹, providing an enormous range of survival functionality. It makes sense in terms of adaptive responses that covalent bonds among the EPS

<figure><img src="image_25.png" /><figcaption>Fig. 4 | Mechanical properties of the biofilm matrix. **a** Five non-covalent forces keep matrix polymers together: hydrogen bonds; ionic interactions; electrostatic interactions; hydrophobic interactions; and entanglement. The matrix polymers have substituents capable of all four interactions, and the length is suited for entanglement. A linear polysaccharide is shown, but the matrix components can also include branched polymers with various substituents³⁴ as well as proteins and nucleic acids. The 'hydrophobic moiety' is provided by hydrophobic substituents or hydrophobic parts of partner molecules. **b** Biofilm behaviour under shear stress, which might be imposed by a moving overlying fluid. In the absence of stress, the biofilm structure, biofilm-associated cells and extracellular polymeric substances (EPS) are undisturbed. At low shear stress, the biofilm can immediately</figcaption></figure>

stretch out and spring back when the stress is removed, which is mediated by intermolecular forces among matrix components. The biofilm behaves similar to a viscoelastic solid. As shear increases, the biofilm flows more as bonds begin to break and polymers move past each other, and when the shear stress is removed the remaining bonds slowly pull the biofilm back but it never regains its original form; patches of the biofilm can be torn off. The biofilm behaves similar to a highly viscous liquid. After exceeding a critical shear force, many intermolecular forces are broken, the polymers are gliding past each other and the biofilm flows similar to a low-viscosity liquid, sometimes in ripples. When the shear stress is removed, the biofilm does not regain any of its form but new intermolecular bonds form providing stability to the new form.
components have not been observed as such bonds would make the matrix more rigid, less dynamic and less capable to rapidly respond to changing physiological and environmental conditions. FIGURE 4b schematically depicts the behaviour of the matrix under stress, beginning with the viscoelastic state and the rapid transition from elastic solids to highly viscous fluids depending on the magnitude of applied mechanical stresses<sup>25,170</sup>. These interactions occur among polysaccharides, proteins, eDNA and all other biopolymers in the matrix and can be controlled by the water content through regulation of polymer density<sup>171</sup>. Multivalent cations added to the surrounding liquid or even cationic polymers produced by the bacteria themselves can cause cross-linking of negatively charged polymers, such as alginate and eDNA, within the biofilm, causing biofilms to become more solid-like<sup>172</sup>. However, it is not clear whether the degree of such ionic cross links is controlled by the bacteria in the biofilm or whether this is incidentally due to the chemistry of the local environment.

The second reason for variability is that biofilms are nonlinear materials<sup>173</sup>, and the measured mechanical properties are highly dependent on the test methods<sup>174</sup>. At first glance, testing seems to be a simple process of applying a mechanical force and measuring a physical change in length, shape or volume, or vice versa. However, how sample preparation is done and whether the testing is done under shear, compression or tension, as well as the rates, duration and magnitudes of the applied perturbations and the scale of the testing, will influence the outcome.

The testing method should relate to the forces the biofilm may experience in its natural environment. The mechanical properties of biofilms can be considered multi-scalar, ranging from single-cell adhesive interactions to the bulk properties of whole biofilms treated as a continuum material<sup>175</sup>. Even if the bacteria in the biofilm are killed, the material properties of the matrix may nevertheless remain unchanged<sup>176</sup>. The matrix can be shared with other bacteria which did not contribute to its synthesis and assembly. However, in *B. subtilis* it was shown that the matrix remained partially controlled by the producer subpopulation, which can ‘stick together’ even under shear stress<sup>67</sup>. Higher levels of structural order emerge as whole biofilms form bridging networks<sup>177</sup>, which will also influence the mechanical properties of the overall structure. There is a link between mechanical properties and antibiotic susceptibility<sup>178</sup>. It is not surprising that these correlate as the EPS-cell-water relationships that determine the mechanical properties will also be important for determining the rates of transport of antimicrobial agents and nutrients (and therefore cellular metabolic activity) through the biofilm. Thus, although the biofilm mechanics, due to promotion of persistence of the cells, can be considered a virulence and environmental survival factor in its own right<sup>158,160</sup>, it belongs to a subset of factors governed by the combined physics and chemistry of the EPS. In addition, it was found that the contributions of exopolysaccharides to viscoelasticity of *P. aeruginosa* biofilms were different between biofilms grown in vitro and in vivo, which seems to arise from the incorporation of host material,

most notably collagen, into the matrix of the in vivo grown biofilm<sup>179</sup>.

## Conclusions and outlook

In the past decade, great progress has been made in understanding the pivotal role of the EPS matrix for the biofilm mode of life both in the environment and in medicine. This is reflected in a broader definition of biofilms which embraces the widely diverse phenomena of microbial aggregates, all sharing common features and the special environment provided by the EPS matrix<sup>180</sup>. The complexity of the matrix has been demonstrated; it is not just a blob of slime made of polysaccharides but provides a dynamic, functional and protective shared space for biofilm cells. The synthesis, export and assembly of matrix components is determined by multiple interconnected systems, and bacterial species may differ in terms of matrix components synthesized and regulatory pathways.

Much progress has been made in the analysis of the EPS components and the fine structure of the matrix, and a shift of focus has unfolded the paramount role of proteins. Extracellular enzymes in the matrix are the tools with which biopolymer transformations are accomplished, and amyloids are ever present as structural, protective and functional EPS constituents. eDNA has also been recognized as having multiple and pivotal functions in the matrix. Finally, as the largest constituent of the matrix, water has found appropriate attention<sup>181</sup> (Supplementary Boxes 2 and 3). Of particular interest are the functional interactions of EPS molecules, which extend beyond influencing the mechanical stability of the matrix. Surprisingly, the influence of the cells on the mechanical properties of biofilms seems still unresearched.

Of course, there are technical, environmental and medical aspects to be addressed, such as, for example, the role of EPS in the tolerance of biofilm organisms to antimicrobial agents. Among the less well studied but deserving aspects ranks the effect of EPS on the stability and movement of sediments in rivers and estuaries and their ecological function as a ‘microbial skin’ of streams and rivers<sup>59</sup>. The EPS matrix functions as a sink and source for sorbed organic and inorganic contaminants, which can interfere with the agricultural use of sewage sludge. EPS are involved in the resistance to erosion and humidity balance of soils, and thus have a direct impact on agriculture. In water and wastewater treatment, EPS enable the retention of biofilms in biofilters, or act as the ‘glue’ that keeps activated and granular sludge together, which enables the separation of biomass from water. In medicine, EPS are involved in tolerance against antimicrobials (BOX 2). Furthermore, biotechnological aspects of EPS deserve attention. For example, their adhesion properties and mechanical stability have to be overcome in cleaning and other antifouling strategies. Finally, they are also involved in microbially influenced corrosion.

What further remains open is how to control EPS production and composition, which would be very attractive in terms of environmental engineering — for example, for carbon sequestration or bioremediation
One of the defining traits of bacterial biofilms is the high degree of tolerance to antibiotics and other antimicrobial agents, regardless of the bacterial species or class of antimicrobials<sup>201</sup>. Usually, this tolerance is lost upon dispersal of the biofilm cells and is different from resistance, which is genetically encoded<sup>202</sup>. The matrix contributes to antimicrobial tolerance in two distinct ways, both of which provide multiple protection mechanisms for the bacteria by concentration gradients of antimicrobials due to interaction with matrix components, and/or by adaptive responses of the cells within the biofilm.

The matrix contributes to tolerance by the establishment of diffusion-limited gradients, which are the result of chemical and/or enzymatic reactions of the matrix with antimicrobials, an effect termed ‘diffusion-reaction inhibition’<sup>203</sup>. Generally, the matrix is negatively charged due to the presence of molecules such as anionic polysaccharides, teichoic acids, acidic proteins and extracellular DNA (eDNA) which can bind, for example, cationic antibiotics such as vancomycin<sup>204</sup> and tobramycin<sup>205</sup>. In addition, the Pel polysaccharide was shown to protect *Pseudomonas aeruginosa* biofilms from aminoglycoside antibiotics by facilitating aggregation by cell-to-cell interactions<sup>206</sup>. Matrix components can also bind host defence molecules such as antibodies, functioning as a sink before they reach the cells. This was exemplified by poly N-acetyl glucosamine binding IgG in a *Staphylococcus epidermidis* biofilm<sup>207</sup> and eDNA binding human β-defensin 3, which is a cationic antimicrobial host defence protein<sup>208</sup>. Oxidizing disinfectants are first consumed by matrix components, which then act as ‘sacrificial material’ before they can target the microbial cells, with the underlying chemical reaction having an important role. For example, bacterial alginate reacts with chlorine and substantially reduces its concentration. This effect is due to the acetyl substituents of alginate, whereas non-acetylated alginate did not neutralize the biocide<sup>209</sup>. The protective effect of the matrix can also be mediated by enzymatic reactions; for example, catalase was shown to neutralize hydrogen peroxide in biofilms<sup>210</sup>.

The extracellular polymeric substances (EPS) matrix may also function as a physical barrier for particles, not just slowing down but preventing penetration. Diffusion studies and particle tracking have shown that channels within the biofilm matrix are usually around 100–200 nm, but sometimes even in the range of 100 µm wide<sup>144,146,211</sup>. As both channels and charge are important to consider when designing delivery agents such as nanoparticle carriers<sup>211</sup>, these factors should be taken into consideration with respect to managing biofilm penetration.

Antibiotics at subinhibitory concentrations have been shown to induce matrix synthesis<sup>217</sup> and biofilm formation in several species<sup>213</sup>. Oxygen limitation in biofilms containing aerobic or facultative aerobic species has been well characterized through microelectrode studies<sup>214</sup>; oxygen is sparingly soluble in aqueous solution and consumed by respiration faster than it can diffuse into the biofilm. Under anaerobic conditions, tolerance towards antibiotics is increased<sup>215</sup>. Acidogenic bacteria can cause the pH to drop within the biofilm<sup>218</sup> and low pH has been associated with the more antibiotic-tolerant small colony variant formation of *Staphylococcus aureus* in biofilms<sup>217</sup>. The cells within biofilms can substantially slow down their growth under nutrient limitation, preventing interference of antibiotics with metabolism<sup>218</sup>. Upon slow growth conditions, for example, due to nutrient limitation, the cells can enter states of dormancy<sup>219</sup> and become *persisters*<sup>220</sup>, or develop the newly identified antibiotic-tolerant, poetically termed, ‘phoenix phenotypes’<sup>221</sup>.

Transfer of resistance genes from eDNA or other cells is facilitated by high cell density in biofilms; therefore, biofilms have been termed ‘hotspots’ of horizontal gene transfer<sup>59,222</sup>. A further protective mechanism that the matrix provides is through providing a physical shelter from disruption, whether by mechanical forces exerted, for example, during attempted engulfment by phagozytes<sup>220</sup> or by an applied fluid shear<sup>160</sup>.

and in technical engineered systems such as wastewater treatment or fuel cell development. As an example, increasing the EPS concentration in soils could improve humidity retention. Conversely, minimizing EPS production, or decreasing their adhesion strength or increasing their permeability and cleanability, could constitute an antifouling strategy in membrane processes.

We must bear in mind that most chemical and functional information about EPS has been generalized from a relatively small number of experimental laboratory models, often microorganisms of clinical relevance, and these bacteria are uncommon in biofilms found in nature or the built environment, such as wastewater treatment plants<sup>25</sup>. We need to isolate more relevant and ecosystem-specific bacteria to study their EPS components in the laboratory, particularly using representatives from poorly studied phyla. The use of next-generation DNA sequencing combined with other omics tools seems to be a promising way to identify signature EPS components in many environmental biofilms and may help understand their regulation. To this end, in situ approaches will be important. Chemical imaging becomes an increasingly helpful tool

to analyse complex microbiota and connect this with EPS chemistry<sup>25</sup>. New opportunities will arise when glycomics is further applied as the complexity of polysaccharides can be studied and better understood. High-resolution imaging will reveal the fine structure and progress in metabolomics will enable the detailed study of matrix biosynthesis and dynamics.

Not surprisingly, the deeper we peer into the structure, function and regulation of components in the EPS matrix, the more its complexity is revealed. An exciting new aspect is that the maintenance of biofilms is discernible as an active process; preliminary research on *P. aeruginosa* indicates that the maintenance of EPS matrix regulation is mainly performed by transcriptional regulation and secondary messenger signalling<sup>182</sup>. Furthermore, it is still unclear whether the functionality of respective EPS components is similar across different biofilm systems. After all, the matrix evolved over billions of years, enhancing the odds of survival for sessile cells living at interfaces, often under extreme environmental conditions.

Published online 20 September 2022

**Persisters**

A subpopulation of transiently antibiotic-tolerant bacterial cells that are often slow growing or growth arrested, and are able to resume growth after a lethal stress.

**Phoenix phenotypes**

Phoenix colonies that grow out of the zone of clearance of antibiotic-loaded beads from lawn biofilms while there are still very high concentrations of antibiotic present, suggesting an antibiotic-resistant phenotype.
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**Acknowledgements**

The authors are grateful to I. C. H. Tan and C. Mayer for help with designing the drafts for Figs. 2 and 5a, respectively.

**Author contributions**

H.-C.F., T.R.N., P.H.N., T.S. and P.S. researched data for the article. H.-C.F., E.D.v.H., T.R.N., P.H.N., T.S., P.S., J.W. and S.W. contributed substantially to discussion of the content. H.-C.F., T.R.N., P.H.N. and P.S. wrote the article. H.-C.F., T.R.N., J.W. and P.S. reviewed and/or edited the manuscript before submission. H.-C.F. assembled the team and coordinated the writing process.

**Competing interests**

The authors declare no competing interests.

**Peer review information**

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