Chapter 1

Viewing Biofilms within the Larger Context of Bacterial

Aggregations

Olena V. Moshynets and Andrew J. Spiers

Additional information is available at the end of the chapter

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Abstract

The ‘Microbial Cities’ vision of bacterial biofilms has dominated our understanding of
the development and functioning of bacterial aggregations for the past 20 years, during
which active sludge, clumps, colonies, flocs, mats, pellicles, rafts, slimes, zooglea, etc.
have been largely forgotten or ignored. Although the medically inspired developmen‐
tal model of human pathogen biofilms has merits including providing a rationale for
the development of anti-biofilm therapeutics, it fails to provide links to other types of
bacterial aggregation that are commonly found in a wide range of natural and man-
made environments. Possibly as a result, applied and environmental microbiologists
tend to avoid the term ‘biofilm’ and use others such as ‘microbial mats’ instead. Here
we challenge the simplistic planktonic (independent and free-swimming bacteria)-
biofilm (sessile and co-operative bacteria) dichotomy, and consider biofilms within the
larger context of bacterial aggregations. By placing biofilms into context, which we see
as a continuum of aggregations or communities with varying abiotic and biotic
properties, fundamental physical, biological, and evolutionary ecological processes that
effect community development and function can no longer be considered unique to
biofilms, but may also be important in other aggregations that develop over time and
change in nature depending on prevailing conditions. By doing this, we will be better
able to distinguish those processes which govern bacterial colonisation and ecological
success in a wider sense from those that are unique to particular environments and
specialised strategies.

Keywords: Bacterial aggregations, Biofilms, Colonies, Communities, Planktonic and

sessile bacteria

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4 Microbial Biofilms - Importance and Applications

1. Introduction

Modern biofilm research often acknowledges the seminal reviews of Costerton et al. [1,2] in
which a conceptual model of biofilm development and structure was first presented. In this
model, biofilm development is described as a series of linked events, from the attachment of
free-swimming planktonic bacteria to a submerged solid surface, the growth of microcolonies
in simple conical structures, and subsequent maturation as larger mushroom-shaped struc‐
tures which have been envisioned as ‘Microbial Cities’ (this appellation may derive from reviews
entitled ‘City of Microbes’ and ‘Microbial Metropolis’ [3,4], but we are unsure). Equally
important to this description was the dichotomous differentiation between independent free-
swimming planktonic bacteria with the co-operative and co-ordinated communities of sessile
bacteria forming biofilms, and the somewhat teleological suggestion that surface-attached
communities allowed growth in harsh conditions which planktonic bacteria could not sur‐
vive [2]. This view of complex bacterial behaviour and growth strategies was in contrast with
the apparently contemporary idea that bacteria were unsophisticated organisms [5].

Since the publication of the Costerton et al. reviews, our understanding of biofilms has
developed through the study of model bacteria as well as of natural communities forming
multispecies biofilms (we direct the reader to the reviews cited in the following sections as a
means of accessing recent biofilm research and current understanding). Model human
pathogens forming biofilms important for virulence include Escherichia coli [6], Pseudomonas
aeruginosa [7], Salmonella enterica [8], Staphylococcus aureus [9], Vibrio cholera [10], etc., though
the archetype is probably P. aeruginosa, an opportunistic pathogen of the human respiratory
tract and a key factor in cystic fibrosis patient mortality [11]. As a result of an understanding
of pathogen biofilm formation, critical points in the developmental processes are now being
scrutinised as possible targets for anti-biofilm therapeutics [7]. Biofilms are also recognised as
having importance in a range of other natural and man-made environments, impacting on
crop productivity, food technology, metal corrosion, veterinary medicine, etc. [12–15], and
microbial mats, a term seemingly preferred by applied and environmental microbiologists, are
found on rock surfaces, in caves, wetlands, sediments, salt marshes, lakes and seas, thermal
springs, hypersaline ponds and lagoons, methane and petroleum seeps, oil wells, etc. [16–21].
Comparisons between pathogenic and environmental biofilm-forming bacteria highlighting
commonalities suggest that biofilm developmental pathways or responses may not be unique
to species or particular environments.

Investigations of biofilm-forming bacteria have revealed key sensory-regulatory pathways,

including intercellular communication and intracellular regulation, required to control biofilm
development by altering motility and attachment behaviour, physiology and metabolism, the
production of extracellular polymeric substances (EPS) forming the matrix of biofilms, and
dispersants required to release bacteria from mature structures [22–27]. The use of a variety of
different experimental systems, including in vitro flow cells, microtitre plates, static micro‐
cosms, etc., as well as animal models [28–31], has also identified the impact of abiotic factors
such as liquid flow and mass transport; O2 and nutrient diffusion; surface physical-chemistry
and topology; and biotic interactions between bacteria, surfaces, and matrix components; and
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so on, on biofilm formation and structure [23–26,32]. In addition, research focussed more on
medical and environmental microbiology, rather than on biofilm formation per se [33–37], as
well as on evolutionary ecology and social microbiology [11,38–41], are increasingly providing
explanations for the role or function of biofilms in different environments and evidence of their
ecological success.

Despite the obvious diversity in biofilm research as evidenced by publications in journals

covering a wide range of disciplines including microbiology, microbial biotechnology,
environmental science, and medical microbiology, our general understanding of biofilms is
nonetheless dominated by a few human pathogens and the submerged solid-surface interface
biofilms they produce in flow cells or microtitre plates [28,29,31] (here we refer to these as
liquid-solid surface (L-S) interface biofilms to differentiate them from other types of biofilms
or bacterial aggregations). Of these, the medically inspired developmental model of P.
aeruginosa, often chosen to represent the ‘Microbial Cities’ vision, is perhaps the most persua‐
sive, with bacteria growing in these structures almost exclusively compared to free-swimming
planktonic bacteria. We are growing concerned that this vision is beginning to dominate
biofilm research in a negative manner.

2. A continuum of aggregations

In this opinion piece, we challenge the simplistic planktonic-(sessile) biofilm dichotomy and
advocate the inclusion of biofilms within the larger context of bacterial aggregations. We
believe that by recognising biofilms within a continuum of aggregations or communities with
varying properties, it will enable a more extensive investigation of bacterial colonisation, and
in particular, allow us to distinguish those processes governing general colonisation and
ecological success from those unique to particular environments and specialised strategies.

Costerton et al. [2] defined biofilms as ‘matrix-enclosed bacterial populations adherent to each
other and/or to surfaces or interfaces ... (and) includes microbial aggregates and floccules and
also adherent populations within the pore spaces of porous media’. Although this definition
is broad (i.e. sensu lato), there is a presumption by current researchers that biofilms are those
structures formed on submerged solid surfaces (i.e. at the L-S interface) and that other
structures associated with surfaces or interfaces are somehow different or inconsequential. We
would suggest that L-S interface biofilms as observed in flow-cells and microtitre plates are a
means to investigate biofilm formation independently of natural environments or context, as
it is difficult or impossible to extrapolate from these simple in vitro systems to the more
complex natural environments from which the bacteria of interest were first isolated [29–31].
We note that in some later reviews, the description of biofilms is extended with more examples.
However, this has also lead to a more relaxed (sensu amplo) definition in which ‘biofilm’ is
frequently used as a synonym of ‘aggregation’, even though the former is often defined by the
latter (e.g. [42]). As a matter of etymology, ‘aggregation’ which originates in late Middle
English (1150–1500 AD) should take precedence over ‘biofilm’ whose usage largely stems from
the 1990s.
6 Microbial Biofilms - Importance and Applications

As an example of how the biofilm definition has been extended following the definition of
Costerton et al. [1,2], we consider the inclusion of air-liquid (A-L) interface biofilms and agar
plate-grown colonies as suggested by Branda et al. [42].

3. Biofilms at the air-liquid interface

Material, including bacteria, accumulates at the air-liquid interface of sea or fresh water to
form surface films often subject to highly variable conditions [19]. A-L interface biofilms also
form on the surface of static liquids in experimental microcosms, and are sometimes referred
to as pellicles or floating biofilms. These are produced by a wide range of bacteria, including
Bacillus subtilis [43], Gluconacetobacter xylinus (formerly known as Acetobacter xylinum and since
reclassified as Komagataeibacter xylinus) [44], as well as numerous enteric bacteria and pseu‐
domonads [44–46].
Although A-L interface biofilms may look superficially similar, the diversity of bacteria which
form them would suggest that they vary in structure and other characteristics as well. We have
been investigating this by comparing biofilms produced by environmental Pseudomonas spp.
isolates, using relatively large static microcosms in 30-ml glass universal vials containing liquid
growth medium [44,47,48]. These allow us to undertake combined biofilm assays which
determine growth, attachment to the vial walls, and biofilm strength [49–51]. Using this
approach, we have been able to quantitatively differentiate biofilms produced at the meniscus
and A-L interface [50,52]. These include biofilms limited to the meniscus region, attached
biofilms which extend across the A-L interface, and unattached ‘floating’ biofilms (as well as
‘invisible’ attached biofilms too thin or transparent to see by eye [52]). It is possible that the
floaters and attached biofilms represent substantially different colonisation strategies, with the
former recruiting planktonic cells directly from the liquid column to the A-L interface and
growing from multiple loci, and the latter developing from sessile cells attaching in the
meniscus region and subsequently growing out across the A-L interface [52].
Although floating biofilms have been reported in which buoyancy is the result of trapped
CO2 released by respiration (e.g. G. xylinus [44]), the two different A-L interface biofilms
produced by our model environmental pseudomonad, P. fluorescens SBW25, known as the
viscous mass and Wrinkly Spreader biofilms, are not buoyant per se and readily sink when
disturbed [49,53,54]. It is likely that they are maintained at the A-L interface by hydrophobic
cell surfaces, matrix components, and surfactant which pierce or weaken the A-L interface
[53,54]. Interestingly, we have recently found that for the Wrinkly Spreader, a class of adaptive
mutants of the wild-type strain which evolves in static microcosms, drip-fed glass bead
columns, and soil [48,51,55], attached A-L interface biofilm growth can be seamlessly linked
to swarming motility and colony growth using ‘transitional microcosms’ in which a layer of
agar is set along the side of the vial, providing both liquid and dry agar surfaces for colonisation
(C. Immoor, O. Moshynets , A. Spiers, Unpublished Observations).
In these transitional microcosms, we wonder whether there is more than just planktonic
bacteria growing in the liquid column and a single, distinct structure colonising both the liquid
and agar surfaces in these simple environments, as suggested by the simplistic planktonic-
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(sessile) biofilm dichotomy. Instead, we think it is likely that there are different types of
aggregation including L-S interface biofilms attached to the vial walls at the meniscus, attached
biofilms extending out across the liquid surface, confluent colonies growing on the agar
surface, and microcolonies developing in the swarm-front as bacteria move up the agar surface
and away from the liquid media. We are interested in identifying which abiotic and biotic
factors drive growth at different points across the transitional zone, and understanding how
these might alter behaviour and gene expression patterns to produce the structures we can
observe within a few hours and over several days.

4. Colonies are not biofilms

Whilst we approve of the inclusion of A-L interface biofilms in the sensu amplo Costerton et al.
[1,2] definition, we resist the suggestion that colonies grown on agar plates should be included
too, despite the fact that investigations of colony morphology have been presented as biofilm
research (e.g. [43,56–61] etc.). In contrast, we have investigated the colony morphologies of
Wrinkly Spreader mini-Tn mutants on agar plates as a means to identify the genes required
for biofilm formation; importantly, we also tested the mutants in static microcosms to deter‐
mine the impact on biofilm formation to confirm the identity of these genes as important in
biofilm formation [48].

Our objection to the inclusion of colonies as biofilms is based on a consideration of the O2 and
nutrient gradients established in these aggregations, as well as liquid flow (Figure 1). Rather
than argue that these are insignificant differences, we would suggest that colonies, A-L and L-
S interface biofilms would be better presented within a larger continuum of aggregations in
which O2 and nutrient gradients (and other chemicals including communication signals and
waste, etc.), and liquid flow can be used to differentiate between types of aggregation. In this
way, it now becomes reasonable to ask whether the parallel O2 and nutrient gradients observed

Figure 1. Bacterial aggregations include biofilms and colonies with significant similarities and interesting differen‐
ces. Liquid-solid surface (L-S) interface biofilms (middle) are subject to physical stress and establish various chemical
gradients. Similarly, air-liquid interface (A-L) biofilms (left) or colonies (right) also experience physical stress and es‐
tablish gradients. In each type of aggregation, a layer of cells is attached to the solid surface (Zone 1) with distal re‐
gions are held in place by cell and matrix component interactions (Zone 2). In A-L interface biofilms, cells and matrix
components may also break through the interface (Zone 3). However, nutrients are supplied by capillary flow (mass
transport) from beneath colonies, whereas in A-L and L-S interface biofilms, they are transported or diffuse from the
surrounding liquid. (A, air; L, liquid; PS, permeable or porous solid; S, solid.)
8 Microbial Biofilms - Importance and Applications

in L-S interface biofilms present a substantially different set of conditions for bacteria than the
opposing gradients found in colonies. Similarly, it would be interesting to compare liquid flow
within biofilms subject to external liquid currents, with the evaporation- and capillary-driven
liquid flow within colonies subject to different drying regimes.
Notably, colonies show different growth patterns, have highly structured morphologies, are
sometimes surrounded by EPS, and may show co-operative behaviour, so it is not unreason‐
able to consider that they are responding to abiotic and biotic conditions as do biofilms [6,42,
43]. In soil, water availability, often described by the matrix potential, is a significant factor
restricting bacterial motility, the formation of aggregations, and colonisation through the pore
network [62–64]. In such systems it is highly likely that contiguous bacterial populations
colonise non-permeable solid surfaces covered or linked by thin films of water with slimes or
swarms; permeable solids through which water is available with microcolonies and colonies;
and partially and fully saturated pores with A-L and L-S interface biofilms, slimes, and
planktonic bacteria.

5. Aggregations respond to different conditions

We would argue that bacteria colonising a range of environments should develop into different
aggregations in response to local conditions and opportunities. These aggregations might
appear to be superficially similar (e.g. A-L and L-S interface biofilms) or substantially different
(cf. a colony), depending on bacterial responses and colonisation strategies aimed at maxi‐
mising ecological success, as well as on our ability to recognise which abiotic and biotic factors
have the greatest impact on the developing population.
Our observations of linked Wrinkly Spreader A-L interface biofilms and colonies in the
transitional microcosms might suggest that these are very similar aggregations used to colonise
two interfaces (the liquid and agar surfaces) which do not pose significantly different chal‐
lenges to bacterial growth. However, competitive fitness assays in each environment suggests
that Wrinkly Spreaders achieve substantially different levels of ecological success in colonising
liquid and agar surfaces: they have a fitness advantage in static microcosms but are at a
disadvantage in colonies compared to wild-type P. fluorescens SBW25 [65–68]. These fitness
differences suggest that environmental conditions probably change across the transitional
zone, with the initial Wrinkly Spreader population responding to these changes to colonise
the A-L interface and agar surfaces in different manners.
We speculate that the fitness advantage of Wrinkly Spreaders is determined by the subtle
trade-off in energy expenditure needed to produce A-L interface biofilms in static microcosms
and the increased access to O2 biofilm formation allows. The growth of P. fluorescens SBW25 is
limited by O2 and it drives the evolution of the Wrinkly Spreaders [69]. Relative small numbers
of wild-type colonists rapidly generate an O2 gradient through respiration, converting the
homogeneous liquid column into a shallow upper zone having normal levels of O2 and a
deeper lower zone with rapidly diminishing O2 levels (these colonists are in effect environ‐
mental engineers) [69]. As this population rapidly expands, random mutation results in
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Wrinkly Spreader genotypes which are recruited to the A-L interface through the expression
of attachment factor and cellulose which provides the main matrix component for the biofilm
[49,53,68]. Those bacteria localised to the A-L interface have access to higher levels of O2,
compared with those lower down, and consequently grow faster [69]. Higher levels of O2 might
also induce a SOS response via reactive oxygen species (ROS) leading to the expression of an
error-prone DNA polymerase, as in the case of P. aeruginosa PA01 [70], increasing mutation
rates and the appearance of Wrinkly Spreader mutants.

As a result of access to higher levels of O2, Wrinkly Spreaders have a fitness advantage over
non-biofilm-forming competitors [69]. We speculate that in colonies where Wrinkly Spreaders
still express attachment factor and cellulose, these components have no essential function and
therefore pose a fitness cost to the growing population, explaining why the Wrinkly Spreaders
are poorly adapted to growing on agar surfaces. Interestingly, improved O2 access has also
been suggested as an explanation for the wrinkled colonies produced by P. aeruginosa PA14.
However, it appears that a more complex level of redox control regulating the use of different
electron donors, including O2 diffusing into the colony from above and redox-active phena‐
zines produced by bacteria located at the base of the colony, may govern metabolism in P.
aeruginosa PA14 and other bacteria producing wrinkled colonies including B. subtilis [57,71,72].

More generally, O2 gradients determine the distribution of aerobic and anaerobic bacteria in
a wide range of environments, including water columns, sediments and soils, where it effects
growth, gene expression patterns and metabolism, and along with other competing electron
acceptors, help define aerobic, micro-aerobic (transitional) and anaerobic niches [73–76]. It is
therefore not surprising if O2 gradients also played a central role in the development and
function of a wider range of bacterial aggregations, and not just in biofilms and colonies.

6. Other terminology

As a slight digression, we present a non-exhaustive selection of vernacular and scientific

terminology used to describe bacterial aggregations which includes active sludge, biofilms,
clumps, colonies, flocs, mats, pellicles, rafts, slimes, zooglea, etc. (Table 1). We would expect
that a more extensive review of the early microbiology literature, including French, German,
and Russian publications, and of current microbiology, microbial biotechnology, environ‐
mental science, and medical microbiology publications, would result in more terms being
identified. Although the first observations of biofilms (dental plaque) were made by van
Leeuwenhoek (1683–1708) [34], microbial mats appeared much earlier, and are identified
today as the fossilised remains of 3.5-billion-year-old stromatolites [77]. Arguably the first
observation by a microbiologist was made by Pasteur (1864) [34], and by the end of the
nineteenth-century, environmental microbiologists were investigating them as well (we list
several early observations following Pasteur in Table 2). For example,Winogradsky (1895) [78]
described jelly-like masses of bacteria as ‘zooglea’, whilst Egunov (1895) [79]) and Sorokina
(1938) [80] more obviously referred to biofilms in the current sense, using terms that translate
into bacterial ‘plate’ or ‘plane’, and ‘film’, respectively.
10 Microbial Biofilms - Importance and Applications

Abscesses, inclusion A mass of bacteria growing within another structure, including sediments, soils, plant and
bodies and metastasis animal tissues.
Active fluids Formed by motile bacteria moving together at high density in a liquid.
Active sludge A complex mixture or community of bacteria and other microorganisms produced and used in
wastewater treatment.
Aggregates, blobs, A mass of bacteria (and other microorganisms) having some sort of physical cohesion; these
clumps, colloids, may have developed by growth, or they may be the result of physical mixing or disturbance.
lumps and masses
Bacterial plates A layer of bacteria formed in a water column at a certain depth depending on oxygen levels.
Biofilms A mass of bacteria enclosed in a protective matrix of EPS and associated with a surface or
interface; most often used to refer to liquid-solid surface (L-S) interface biofilms such as those
developing in flow-cells and in microtitre plates.
Biolaminites, Living and fossilised microbial mats which may trap and bind sediments and/or cause mineral
microbialites, precipitation, sometimes intercalated by sediment laminae; sometimes referred to as microbial-
stratifera and induced sedimentary (MIS) structures.
stromatolites
Clusters A zone of physiologically synchronised bacteria within a lager aggregation, or a small mass of
bacteria such as a colony or floc.
Collapsed cakes Formed by the collapse of clumps or flocs on membranes during filtration.
Colonies, macro- and A mass of bacteria having some sort of physical cohesion and having developed by growth on
micro-colonies a solid dry surface; micro-colonies are those associated with the biofilm development process
on submerged solid surfaces, or small aggregations of bacteria not noticed as colonies unless
observed with magnification on leaf surfaces, detritus, or agar plates.
Communities and A complex mixture of multiple bacterial species (or genotypes) and possibly other
consortia microorganisms in which biotic interactions define structure and function.
Crusts, dust Microbial communities developing on the surface of soils and dessert sand; also the dried
particulates and remnants of colonies etc.
aerosols
Deposits and Mass or body of bacteria that accumulate on dry or submerged surfaces due to wind or water
sediments movement.
Desert varnish A dark stain or coating covering rock surfaces and colonised by bacteria.
Filamentous Long strands of material stretching out from the main mass of a biofilm subject to liquid flow.
structures and
streamers
Films, layers, planes, A thin layer or volume containing bacteria which may or may not be physically connected to
plates, volumes and one another, solid surfaces, or other interfaces, and occurring in liquids, porous or permeable
zones solids.
Flocs and snow Masses of bacteria formed by growth, self-association, hydrophobic interactions, or by
attachment to suspended inert particles; flocs in sea water are referred to as snow.
Floaters Biofilms at the air-liquid interface having no appreciable attachment to a solid surface; these
may be localised at the liquid surface by buoyancy, penetration of the interface, or by
hydrophobic surfaces.
Foams Air-water emulsions containing high concentrations of bacteria and compounds such as
polymers and surfactants, which may help stabilise the structure.
Granules A mass of bacteria growing on small solid particles.
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Jellies and A slimy mass of bacteria encased in a gel-like material, often found floating in water or found
zooglea (or zoogloea) on plant stems or leaf litter.
Laminae Layers of microbial mat generations, or microbial mats overgrowing sediments.
Mats Microbial communities, often with clear layering or stratification, found in streams, lake or sea
beds; these may also contain, algae and plants, and trap small particles including sand and
stones.
Meniscus growth A mass of bacteria adhered to a solid surface in the meniscus region of static liquids (the air-
liquid-solid surface (A-L-S) interface).
Microcenosis, Microbial communities formed in a particular niche or site.
microbial cenosis
Micro-zones and Microbial communities growing in thin layers structurally segregated with different
pelogens characteristics or activities in sediments or silt.
Pellicles A thin film or gel-like coating surrounding of individual bacteria, as well as air-liquid (A-L)
interface biofilms.
Phlegm balls Flocs found in underground streams.
Plaque Dental biofilms formed largely by anaerobic bacteria on the surfaces and cavities in teeth.
Rafts Flat sections on the edges of colonies, often associated with twitching motility, or flat pieces of
un-attached biofilm found at the air-liquid interface.
Remains and A mass of bacteria and cellular debris found at a site at which they probably did not develop
remnants or a portion of a larger mass which has been removed by physical disturbance, predation or
decay.
Sediments Bacteria from liquids no longer in suspension, or microbial communities developing in
sediments or silt.
Slimes, Viscous liquids or regions of a larger volume of liquid containing high densities of bacteria and
glycocalyx and EPS.
viscous liquids
Snottites and Pendulous or dripping masses of bacteria developing on cave walls or at the bottom of
snoticles stalactites, especially limestone cave speleothems.
Swarms Bacteria showing a particular form of surface-associated motility, moving in high densities
across moist or wet surfaces.

This is a non-exhaustive list where terms, meanings and usage varies between contexts, and grouped terms may not be
synonymous.

Table 1. Vernacular and scientific terminology used to describe bacterial aggregations

1864 Pasteur describes slimy material called Mother of Vinegar [34].

1887 Winogradsky observed bacterial growth in ring-like structures in a liquid microcosm containing H2S and
covered with a glass slide to restrict O2 diffusion [86].
1893 Beijerink observed bacteria growing in zones of enriched water microcosms, the positions of which could be
altered depending on O2 and H2 levels. Described ‘Bakterienniveau’ or ‘niveau’ as an aggregation formed by
motile bacteria [87].
1895 Winogradsky observes bacterial zoogleas on potato slices [78].
12 Microbial Biofilms - Importance and Applications

1895 Egunov observed bacterial plates forming in microcosms containing Black Sea sediments, the position of which
depended on anaerobic conditions, O2 and H2S levels. Some of these bacteria were motile, and Egunov asked
what forces drove them to form a stationary aggregation [79,88].
1900 Egunov describes bacterial attachment during plate formation, and his ‘bioanisotropy’ concept for
environments (continuous matter exchange between an organism and its surroundings) [89].
1914 Isachenko describes ‘pink water’ caused by the aggregation of purple bacteria in sea water as well as bacteria
forming cloud-like structures in liquid microcosms [36].
1933 Henrici observed that bacteria mostly grow on submerged surfaces, not in free flowing water [34].
1935 Zobell describes marine bacteria attachment to surfaces [34].
1938 Sorokina describes a bacterial film forming on a submerged slime surface in a liquid microcosm [80].

Table 2. Early observations of bacterial aggregations.

More recently, pendulous and dripping snottites have been described on cave walls and
stalactites [81] and collapsed cakes are a problem in filtration [82]. More interesting, perhaps,
are the reports that bacterial remains have been misidentified as dinosaur soft tissues [83] and
desert varnishes are being used to train sensors for future planetary explorations [84].
Regrettably, we also note that a chance to create a more evocative science fiction term for
biofilms on the International Space Station was missed [85].

We argue that such an extensive collection of terms used to describe bacterial aggregations
should not be considered a plethora, but rather an indication that the diversity of aggregations
we are aware of may reflect the multitude of ways bacteria to respond to differing ecological
opportunities. However, we do not suggest that each of the terms are unique, as quite evidently
different types of aggregations may be associated closely or more distantly from one another,
depending on which abiotic or biotic factors are considered.

7. Key features linking aggregations

Bacteria interact with abiotic and biotic factors by responding to physical and chemical cues
according to behavioural and adaptive strategies under constant selection to maximise fitness.
When these cues and strategies are considered in the context of a larger continuum of aggre‐
gations, it is possible to identify key features that link aggregations within a larger continuum
(Table 3). Here we briefly indicate how physical interactions, diffusion radii, and increasing
genetic diversity might be used to compare different types of aggregations.

Chemical gradients Controlling the behaviour of individuals and groups, defining zones of optimal and restricted
growth, providing information about local conditions; of nutrients, O2 and other electron
acceptors and metabolites, chemosensory, regulatory and communication compounds, and
waste.
Competition Between genotypes or lineages for resources, drives adaptation, allows investment in public
goods but also results in cheaters.
Complexity Of abiotic and biotic interactions, of genotypes, metabolism, structures, etc.
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Communication, co- Linking individuals into a group through the exchange of communication signals and/or
operation and co- response to the same environmental signals, resulting in similar behaviours or activities, and
ordination the production of common goods such as EPS and other secreted products.
Developmental Guiding the behaviour individuals and groups through a series of defined stages and resulting
pathway in a specific type of aggregate; responding to abiotic and biotic factors including
communication signals.
Environmental Abiotic chemical and physical factors, biotic factors; niche, opportunities, substrates, resources,
conditions and stress, variation and instability; local, large-scale and irreversible changes, depletion,
modification contamination.
Gene expression Controlling behaviour, communication, metabolism, the production of compounds required
for the formation of aggregations, etc.
Genotypes and Aggregations may arise from a single individual or founder population of the same genotype,
diversity but diversity will develop over time with radiation and immigration; single or multi-species
aggregations; cheaters, invaders and persisters.
Liquid flow Around and within aggregations, effecting the development of the physical structure,
deformation and breakage, boundary layers, mass transport and diffusion of molecules, as
well as the movement of bacteria.
Mobility Of individual bacteria, small groups and larger aggregations, over small and large-scale
distances, across interfaces, surfaces and volumes, and through environments.
Physical interactions With interfaces, surfaces, bacteria, EPS, etc., in terms of strength, elasticity or resilience,
distances, duration and reversibility.
Resilience To chemical and physical stress, external competition, predation, and in terms of physical
structure.
Sensory zones The ability of individuals to detect the presence of others using altered chemical gradients,
metabolites and communication signals (diffusion radii), and the distance separating
individuals or groups.
Stratification From homogeneous mixtures to clustered or layered differences resulting from age,
metabolism, diversity and function.
Structure and Aggregations having properties similar to Newtonian liquids, visco-elastic gels and solids.
rheology  
Succession The diversity and function of aggregations will develop over time and with changing
environmental conditions.
Time The time-scale for the development, presence or persistence of aggregations may vary
considerably, effecting population size, radiation, succession, aggregation structure and
stratification, and environmental impact.

Table 3. Features linking bacterial aggregations within a larger continuum.

Physical interactions involving bacterial surface coatings, appendages and matrix, solid
surfaces and interfaces within A-L and L-S interface biofilms are known to be complex (Figure
2), but there is no reason to believe that all types of interactions are unique to these particular
aggregations. We therefore propose that one feature of the continuum of aggregations could
be expressed by a scale going from no interactions with solid surfaces or other interfaces, as
in the case of a population of planktonic bacteria, through short-term, weak, or distant physical
contacts with surfaces, interfaces, or material, to the complexity of interactions seen in A-L and
L-S interface biofilms, and presumably in other aggregations such as flocs, granules, and snow.
14 Microbial Biofilms - Importance and Applications

Figure 2. Bacterial aggregations are constructed by numerous and varied physical interactions. Bacterial cells within
aggregations will interact with one another through close, intermediate, and long-range interactions involving surface
coatings and extended appendages (e.g. flagella), matrix components, solid surfaces and interfaces (e.g. the air-liquid
interface). (Key: A, air; L, liquid; S, solid; Matrix components or fibres are artistically depicted by dashed lines and
bacteria as bacilli with a single flagella.)

Interactions with surfaces and interfaces also clearly limit the ability of an aggregation to
develop, so the ability to expand across surfaces or to penetrate volumes, along with altered
mass transport and diffusion characteristics, also present other scales with which to compare
aggregations (Figure 3). In particular, diffusion radii will determine the ability of individual
bacteria to detect the proximity of others and to respond competitively or with co-operation.
Clearly, low-density planktonic and surface-attached bacteria may be beyond detection
distances, but as bacterial numbers increase, their individual and collective impacts on local
environmental conditions will lead to a situation where they are now within the same micro-
environment, and similar conditions may result in coordinated changes in gene expression

Figure 3. Bacterial aggregations develop at interfaces, within liquids, porous solids, and visco-elastic materials. Col‐
onising bacteria may develop into communities restricted primarily by diffusion or liquid flow (mass transport) of
chemicals and the ability to expand across interfaces or into spaces (radial arrows). Shown here (left to right) are colo‐
nies growing on and into a solid, biofilms growing on a solid submerged surface, flocs developing from a suspended
particle, floaters forming at the air-liquid interface, and meniscus growth occurring at the air-liquid-solid surface inter‐
face. (Key : A, air; IPS, impermeable solid; L, liquid; PS, permeable or porous solid; S, solid; arrows indicate expansion
radii and the opposite direction indicates diffusion gradients that limit growth.)
 Viewing Biofilms within the Larger Context of Bacterial Aggregations 15
http://dx.doi.org/10.5772/62912

patterns, behaviour, and metabolism. We propose that separation distance, scaled in terms of
chemical gradients, will also be a useful means of comparing and differentiating bacterial
aggregations.

Aggregations developing over significant periods of time will also gain diversity by radiation
and immigration, leading to multispecies communities subject to ecological succession, driven
by internal competitive and co-operative interactions and changing environmental conditions
(Figure 4). We propose that both diversity in terms of genotype or species composition and
community function could also be used to consider bacterial aggregations, and this perspective
is complementary to understanding the physical interactions and the molecular biology
underlying the development of these structures.

Figure 4. Bacterial aggregations gain diversity over time. An aggregation developing from a single genotype will gain
diversity through radiation (mutation), immigration, and succession. Stochastic events and changing selective pres‐
sures will result in different genotypes within the community; some genotypes may become extinct and diversity may
fall. (Key: Time progresses from left to right; colonising genotypes are shown as circles; mutation events as vertical
lines; successful genotypes are indicated by arrows and an extinction event by a truncated line.)

By considering the ability of individual bacteria to respond to their local environments via
different growth and colonisation strategies, the impact of abiotic conditions on individuals
and the structures they create, and the longer-term development of the community, it is
possible to speculate how altered environmental conditions and circumstance can lead to the
cycling between different types of aggregation (Figure 5). The ability of bacteria to move
between different types of aggregation with changing conditions or to exploit new opportu‐
nities will clearly differentiate those able to colonise a wide range of environments with those
adapted to very specific niches.

Figure 5. Bacterial aggregations change nature with altering environmental conditions. Although aggregations may
develop and persist in one site, conditions may change resulting in an aggregation with a substantially different na‐
ture. The archetypal L-S interface biofilm developmental process is shown on the left. Biofilms may dry out to form
slimes and colonies, and further drying might result in dust particulates which may rehydrate to form aggregations.
(Key: A, air; L, liquid; S, solid; bacteria are artistically depicted as bacilli with a single flagella.)
16 Microbial Biofilms - Importance and Applications

8. Conclusion

In this opinion piece, we advocate the inclusion of biofilms within the larger context of a
continuum of bacterial aggregations. We do this because we are growing concerned that the
‘Microbial Cities’ vision originating from the seminal reviews by Costerton et al. [1,2] is
beginning to dominate biofilm research, and that such a narrow view limits our ability to better
understand bacterial colonisation of a variety of different environments. In this continuum,
we consider A-L and L-S interface biofilms to be biofilms, but argue that other aggregations
such as colonies are significantly different and should not be referred to using this particular
term. It is also possible that applied and environmental microbiologists prefer to refer to
microbial mats rather than to biofilms, because the latter is too closely associated with
experimental L-S interface biofilms produced in flow-cells or microtitre plates, and too far
removed from the aggregations found in natural and other man-made environments.

We believe that the advantages of taking a wide view will allow us to distinguish those
processes governing general colonisation through the formation of aggregations and ecolog‐
ical success, from those unique to particular environments and specialised strategies (as an
apologia, we remind readers of the sensu lato definition of biofilms, secundum Costerton et
al., which was inclusive of a number of different aggregations). By suggesting that biofilms
are better considered as one of a variety of different aggregations, the simplistic planktonic-
(sessile) biofilm dichotomy is also challenged, and perhaps the best reference or comparator
for different aggregations will not always be logarithmic phase planktonic bacteria.

Author details

Olena V. Moshynets1 and Andrew J. Spiers2*

*Address all correspondence to: [email protected]

1 Cell Regulatory Mechanisms Department, Institute of Molecular Biology and Genetics of the
National Academy of Sciences of Ukraine, Kiev, Ukraine

2 School of Science, Engineering and Technology, Abertay University, Dundee, United

Kingdom

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