**1. Introduction**

Our research interests have focussed on air-liquid (A-L) interface biofilmformation by the model pseudomonad *Pseudomonas fluorescens* SBW25 and the adaptive Wrinkly Spreader in experimental microcosms (see our recent reviews [1, 2]), and we have recently begun to extend our investigations into biofilm-formation by communities dominated by similar biofilm-competent pseudomonads. Our research has also developed from a molecular biology perspective [1] towards a more evolutionary and ecological understanding [2] of why biofilms are such a successful colonisation strategy used by bacteria in a wide variety of environments.

In contrast to our changing perspectives, we realise that although biofilm research is interdisciplinary, it appears dominated by molecular biologists working with medically relevant model species with a focus on a mechanistic understanding of biofilm-formation which has remained unchanged from that of the early biofilm pioneers [3, 4]. However, contemporary biofilm research includes a wide range of other disciplines, including evolutionary ecology which provides a framework for understanding how the cooperation needed between bacterial cells to produce biofilms is established and maintained, how bacteria diversify and adapt within these structures, and how biofilm communities respond to changing environmental conditions.

We note that although biofilm reviews addressing evolutionary ecology are published regularly, evolutionary ecology content is negatively correlated with molecular biology and medical content in those reviews with a wider focus.1 This should be of concern, as any mechanistic understanding of biofilms lacking an evolutionary ecological element will not be able to evaluate the importance of these structures nor make long-term predictions about persistence or function in a wide range of medical, biotechnological and industrial contexts. Furthermore, these negative correlations suggest that the medical molecular microbiology community is ignoring or is unaware of the contributions evolutionary ecology could make towards understanding and mitigating the impact of biofilm-associated disease.

## **2. Importance of an eco-evolutionary perspective in biofilm research**

Evolutionary ecology seeks to understand how ecological interactions can affect selection and adaptation and the consequences of evolutionary change [5–7]. These interactions occur within and between populations, as well as with the environment, and ecological processes involving these interactions explain community dynamics and succession. In contrast, evolutionary processes are usually considered as driving lineages through time, and when subject to selection can result in adaptive changes and ultimately speciation (we use the term lineage here to include mutations, alleles and genotypes, individuals and mutants, and species, all of which can be followed through time and across generations to investigate ecological interactions or evolution). However, ecological and evolutionary changes are directly linked and can occur on the same time-scale [8, 9]. Such eco-evolutionary dynamics are especially important in bacterial populations and communities, where growth rates and numbers are high and selective pressures can be extreme, leading to the rapid fixation of adaptive mutations and striking changes in phenotype or community function.

Evolution research should not therefore be limited to examining fossils or contemporary ecosystems but can be undertaken over relatively short time-scales

**329**

**Figure 1.**

*Extending an Eco-Evolutionary Understanding of Biofilm-Formation at the Air-Liquid Interface…*

Two significant eco-evolutionary processes are particularly relevant to biofilm research. The first of these are ecological interactions which help assemble, stabilise or change community structure [21–23] (community change is often referred to as succession). The main two-way interactions between members of a community are mutualism, commensalism, competition and predation. Cooperation, one example of mutualism where both partners benefit, is usually considered an intraspecific or within-lineage interaction, though it can also occur between closely related lineages or lineages with very similar phenotypes as in the case of community biofilms. External forcing such as physical disturbance can alter ecological interactions (**Figure 1a**) and the impact of this can be measured in terms of system stability and productivity, and possibly even by a change in function. Evolutionary processes, including selection, speciation, drift and dispersal also effect community composi-

The second significant eco-evolutionary process relevant to biofilm research is adaptive radiation [5], the evolution of diversity through random mutation and selection (**Figure 1b**), which in the context of bacteria, can happen very rapidly within a few generations. Developing populations accumulate mutations or diversify, and those mutants with a fitness advantage over their competitors can be considered successful or adaptive. Although evolution is normally thought of as the slow accumulation of mutations with small additive effects on fitness, bacterial microcosms are usually dominated by the first adaptive lineage to appear or by adaptive lineages which appear early on in the process of diversification [14]. Adaptive lineages often make use of new ecological opportunities with key innovations that allow them to interact with the environment in a fundamentally different way [5, 25, 26]. Ecological interactions also occur between lineages and will result in the fixation or loss of particular mutations. These interactions clearly link community change and adaptive radiation, as they help determine the importance of novel ability, such as biofilm-formation, brought in by immigration or key innovation resulting from mutation. In terms of the cooperation required for

*Eco-evolutionary processes involve ecological interactions and adaptive radiation. Basic ecological interactions determine community dynamics which can change over time, for example, by the immigration (dashed line) of a new member with a novel ability (arrows and bars linking nodes represent positive and negative interactions between community members, respectively) (a). Adaptive mutations occurring in diversifying populations established by a common ancestor can lead to new lineages with key innovations which then compete with other* 

*lineages (vertical lines represent mutations giving rise to new lineages) (b).*

in experimental evolution studies using microbial populations and microcosms [10–20]. In particular, the ease with which bacterial populations can be cultured, short generation times and large population sizes which allow mutations to accumulate (diversification) and be identified, the ability to freeze isolates indefinitely, and undertake genetic analyses, make bacteria an ideal model to explore aspects of

*DOI: http://dx.doi.org/10.5772/intechopen.90955*

evolutionary ecology.

tion and diversity [21, 23, 24].

<sup>1</sup> We have assessed changing interests in biofilm research by undertaking a simple content analysis of open access reviews published between 2000 and 2004 and 2014–2019 listed by Google Scholar and PubMed on 10 October 2019 (*n* = 40), scoring each for medical (M), molecular biology (MB), and evolutionary or ecological (EE) content. No significant differences were seen in each content type between dates (Wilcoxon, *P* < 0.05) or between contents for each date (Kruskal-Wallis, *P* < 0.05). In early publications we found a significant correlation between M & MB (Spearman ρ = −0.83, *P* < 0.0001), but not between M & EE (*P* = 0.12) or MB & EE (*P* = 0.96). In recent publications there were significant correlations between M & EE (ρ = −0.74, *P* = 0.0002) and MB & EE (ρ = −0.43, *P* = 0.06), but not between M & MB (*P* = 0.49).

### *Extending an Eco-Evolutionary Understanding of Biofilm-Formation at the Air-Liquid Interface… DOI: http://dx.doi.org/10.5772/intechopen.90955*

in experimental evolution studies using microbial populations and microcosms [10–20]. In particular, the ease with which bacterial populations can be cultured, short generation times and large population sizes which allow mutations to accumulate (diversification) and be identified, the ability to freeze isolates indefinitely, and undertake genetic analyses, make bacteria an ideal model to explore aspects of evolutionary ecology.

Two significant eco-evolutionary processes are particularly relevant to biofilm research. The first of these are ecological interactions which help assemble, stabilise or change community structure [21–23] (community change is often referred to as succession). The main two-way interactions between members of a community are mutualism, commensalism, competition and predation. Cooperation, one example of mutualism where both partners benefit, is usually considered an intraspecific or within-lineage interaction, though it can also occur between closely related lineages or lineages with very similar phenotypes as in the case of community biofilms. External forcing such as physical disturbance can alter ecological interactions (**Figure 1a**) and the impact of this can be measured in terms of system stability and productivity, and possibly even by a change in function. Evolutionary processes, including selection, speciation, drift and dispersal also effect community composition and diversity [21, 23, 24].

The second significant eco-evolutionary process relevant to biofilm research is adaptive radiation [5], the evolution of diversity through random mutation and selection (**Figure 1b**), which in the context of bacteria, can happen very rapidly within a few generations. Developing populations accumulate mutations or diversify, and those mutants with a fitness advantage over their competitors can be considered successful or adaptive. Although evolution is normally thought of as the slow accumulation of mutations with small additive effects on fitness, bacterial microcosms are usually dominated by the first adaptive lineage to appear or by adaptive lineages which appear early on in the process of diversification [14].

Adaptive lineages often make use of new ecological opportunities with key innovations that allow them to interact with the environment in a fundamentally different way [5, 25, 26]. Ecological interactions also occur between lineages and will result in the fixation or loss of particular mutations. These interactions clearly link community change and adaptive radiation, as they help determine the importance of novel ability, such as biofilm-formation, brought in by immigration or key innovation resulting from mutation. In terms of the cooperation required for

### **Figure 1.**

*Bacterial Biofilms*

conditions.

has also developed from a molecular biology perspective [1] towards a more

colonisation strategy used by bacteria in a wide variety of environments.

evolutionary and ecological understanding [2] of why biofilms are such a successful

In contrast to our changing perspectives, we realise that although biofilm research is interdisciplinary, it appears dominated by molecular biologists working with medically relevant model species with a focus on a mechanistic understanding of biofilm-formation which has remained unchanged from that of the early biofilm pioneers [3, 4]. However, contemporary biofilm research includes a wide range of other disciplines, including evolutionary ecology which provides a framework for understanding how the cooperation needed between bacterial cells to produce biofilms is established and maintained, how bacteria diversify and adapt within these structures, and how biofilm communities respond to changing environmental

We note that although biofilm reviews addressing evolutionary ecology are published regularly, evolutionary ecology content is negatively correlated with molecular biology and medical content in those reviews with a wider focus.1

should be of concern, as any mechanistic understanding of biofilms lacking an evolutionary ecological element will not be able to evaluate the importance of these structures nor make long-term predictions about persistence or function in a wide range of medical, biotechnological and industrial contexts. Furthermore, these negative correlations suggest that the medical molecular microbiology community is ignoring or is unaware of the contributions evolutionary ecology could make towards understanding and mitigating the impact of biofilm-associated disease.

**2. Importance of an eco-evolutionary perspective in biofilm research**

<sup>1</sup> We have assessed changing interests in biofilm research by undertaking a simple content analysis of open access reviews published between 2000 and 2004 and 2014–2019 listed by Google Scholar and PubMed on 10 October 2019 (*n* = 40), scoring each for medical (M), molecular biology (MB), and evolutionary or ecological (EE) content. No significant differences were seen in each content type between dates (Wilcoxon, *P* < 0.05) or between contents for each date (Kruskal-Wallis, *P* < 0.05). In early publications we found a significant correlation between M & MB (Spearman ρ = −0.83, *P* < 0.0001), but not between M & EE (*P* = 0.12) or MB & EE (*P* = 0.96). In recent publications there were significant correlations between M & EE (ρ = −0.74, *P* = 0.0002) and MB & EE (ρ = −0.43, *P* = 0.06), but not between

Evolutionary ecology seeks to understand how ecological interactions can affect selection and adaptation and the consequences of evolutionary change [5–7]. These interactions occur within and between populations, as well as with the environment, and ecological processes involving these interactions explain community dynamics and succession. In contrast, evolutionary processes are usually considered as driving lineages through time, and when subject to selection can result in adaptive changes and ultimately speciation (we use the term lineage here to include mutations, alleles and genotypes, individuals and mutants, and species, all of which can be followed through time and across generations to investigate ecological interactions or evolution). However, ecological and evolutionary changes are directly linked and can occur on the same time-scale [8, 9]. Such eco-evolutionary dynamics are especially important in bacterial populations and communities, where growth rates and numbers are high and selective pressures can be extreme, leading to the rapid fixation of adaptive mutations and striking changes in phenotype or community function. Evolution research should not therefore be limited to examining fossils or contemporary ecosystems but can be undertaken over relatively short time-scales

This

**328**

M & MB (*P* = 0.49).

*Eco-evolutionary processes involve ecological interactions and adaptive radiation. Basic ecological interactions determine community dynamics which can change over time, for example, by the immigration (dashed line) of a new member with a novel ability (arrows and bars linking nodes represent positive and negative interactions between community members, respectively) (a). Adaptive mutations occurring in diversifying populations established by a common ancestor can lead to new lineages with key innovations which then compete with other lineages (vertical lines represent mutations giving rise to new lineages) (b).*

biofilm-formation, kin selection ensures that cost of construction, often considered in terms of public goods or common pool resources such as the extracellular polymeric substances (EPS) which provide the main structural element of biofilms, is spread across all members who then all share in the benefits [15, 27].

Cooperation is further stabilized in biofilms by spatial separation of producers and cheaters who do not contribute to the cost of construction and a reduction of distance over which the benefits of cooperation act [19]. It is important to note that where external forcing or selection occurs, or where there is an ecological opportunity, community structures will change and lineages continue to adapt, until the theoretical end-point of evolution in a community known as an evolutionary stable community is achieved [28].

### **3. The SBW25 model system**

*Pseudomonas fluorescens* SBW25 was originally isolated from the sugar beet (*Beta vulgaris* subsp. *vulgaris*) phyllosphere and has been used in experimental evolution studies where the appearance of mutant lineages with altered colony morphologies (these are sometimes referred to as morphotypes or morphs) in diversifying populations have allowed the dynamics of diversification and the fitness of adaptive mutations to be readily investigated [1, 2, 13, 14, 19, 29]. In this system, competitive trade-offs between lineages result in negative frequency-dependent selection and indicate that the major driver of adaptive radiation is competition for limited resources [29].

Fitness, a measure of an individual's reproductive success, is determined at the population level in such microcosms. For bacteria, fitness is readily assessed by comparing the maximum growth rate (*v*Max) of one population with that of a second, reference population. Simple growth rate comparisons are typically used to infer the success of mutations for which enzymatic or regulatory changes are being investigated, but a more meaningful ecological comparison can be made by growing the two populations together, allowing them to interact with one another and to compete for limiting resources. Competitive fitness [14, 30, 31]<sup>2</sup> can be readily determined using co-cultures if the two populations produce different colony morphologies allowing viable number counts to be made on agar plates, or if they can be labelled using fluorescent markers, to allow more rapid enumeration with automated cell counters.

The evolutionary consequences of ecological processes are readily studied using microcosms. They provide defined environments for bacterial growth, and because they are reproducible, treatments can be replicated, experiments are repeatable, and selective pressures can be changed by altering resources or inocula. Nonetheless, the use of microcosms in evolution studies faces some criticisms, including the fact that they are unnatural and very simple environments, and that these studies are essentially contrived [15]. However, although populations may be founded in these synthetic environments, evolutionary and ecological dynamics are interpreted in terms of recent evolutionary history which may span 10–60,000

**331**

**Figure 2.**

*for more biofilm images). Photographs: A. Spiers.*

*Extending an Eco-Evolutionary Understanding of Biofilm-Formation at the Air-Liquid Interface…*

generations in 1 day–25 years (for example, in our system which is described in the following sections, and Lenski's long-term evolution experiment (LTEE) [32]) during which the populations adapt to these environments. Microcosms are not used to replicate the complexity of natural environments but are rather models in which key factors involved in the process of adaptive radiation can be tested [15]. These studies are of course contrived, in the sense that they are designed and in some cases the outcomes are inevitable, but the value of such an approach is that they can be initiated at any point along the evolutionary process and are not limited by the initial diversity or time (for example, the fitness of an adaptive lineage or mutant compared to the ancestral strain can be immediately explored by using genetic manipulation to produce the mutation without having to wait until it appears

In liquid cultures, wild-type SBW25 populations diversify as random mutations occur, dividing the initially homogeneous or isogenic population into a number of related but diversified lineages. One re-occurring lineage frequently found in static microcosms was the Wrinkly Spreader (WS) mutant class, named after the wrinkled and flat colonies produced on agar plates which are readily distinguished from the smooth and rounded colonies produced by wild-type SBW25 (**Figure 2a**) [29] (quantitative aspects of the WS phenotype are referred to as wrinkleality [1, 35]). WS mutants are further distinguished by an altered niche preference in static microcosms, where they form a robust and well-attached physically cohesiveclass biofilm [36] at the air-liquid (A-L) interface, rather than growing throughout the liquid column like wild-type SBW25 (**Figures 2b** and **3b**) [29] (A-L interface

Wrinkly Spreaders are considered to be adaptive (evolved) lineages because they have a competitive fitness advantage over their ancestor, wild-type SBW25, which does not normally form biofilms in static microcosms [29, 38]. However, in shaking microcosms WS mutants are disadvantaged because they cannot form biofilms [38] and on agar plates the WS phenotype is genetically unstable [39]. Biofilm-formation by Wrinkly Spreaders and SBW25 [40] is neither unusual nor peculiar, as many other soil, plant and water-associated pseudomonads form A-L interface biofilms in

*Ancestral SBW25 and adaptive Wrinkly Spreaders. Wild-type SBW25 and Wrinkly Spreader colonies are readily identified on agar plates (a). In static microcosms (b), wild-type SBW25 grows throughout the liquid column (left microcosm) and the Wrinkly Spreader forms a robust biofilm at the A-L interface (right microcosm). These microcosms are 28–30 ml glass vials containing 6 ml growth medium; they are incubated with shaking which provides a homogeneous and unstructured environment with good aeration, or statically which leads to a heterogeneous and structured environment dominated by an O2 gradient [29, 33]. When tipped out, the WS biofilm retains shape (c) demonstrating just how robust these structures are (see Figure 4*

*DOI: http://dx.doi.org/10.5772/intechopen.90955*

biofilms are sometimes referred to as a pellicle [37]).

static microcosms under the same conditions [36].

naturally) [15].

<sup>2</sup> The competitive fitness (*W*) of one population (A) compared to a reference population (B) is determined as the ratio of Malthusian parameters (*m*A/*m*B) where *m* = *ln* [final numbers/initial numbers] for each population over the period of the assay [30] (*m* is scaled here for generation time using *ln* as a correcting factor [31]). When *W*A,B is greater than one, A has a competitive advantage over B (and B is at a disadvantage), when *W*A,B is equal to one, the two populations are neutral, and when *W*A,B is less than one, A is at a disadvantage (and B has a competitive advantage). As *W* might be dependent on the initial ratios of the two competing populations, it can show a frequency-dependent response. The selection coefficient (*s*) is also often used as a measure of survival and success (*s* = 1 – *W*).

### *Extending an Eco-Evolutionary Understanding of Biofilm-Formation at the Air-Liquid Interface… DOI: http://dx.doi.org/10.5772/intechopen.90955*

generations in 1 day–25 years (for example, in our system which is described in the following sections, and Lenski's long-term evolution experiment (LTEE) [32]) during which the populations adapt to these environments. Microcosms are not used to replicate the complexity of natural environments but are rather models in which key factors involved in the process of adaptive radiation can be tested [15]. These studies are of course contrived, in the sense that they are designed and in some cases the outcomes are inevitable, but the value of such an approach is that they can be initiated at any point along the evolutionary process and are not limited by the initial diversity or time (for example, the fitness of an adaptive lineage or mutant compared to the ancestral strain can be immediately explored by using genetic manipulation to produce the mutation without having to wait until it appears naturally) [15].

In liquid cultures, wild-type SBW25 populations diversify as random mutations occur, dividing the initially homogeneous or isogenic population into a number of related but diversified lineages. One re-occurring lineage frequently found in static microcosms was the Wrinkly Spreader (WS) mutant class, named after the wrinkled and flat colonies produced on agar plates which are readily distinguished from the smooth and rounded colonies produced by wild-type SBW25 (**Figure 2a**) [29] (quantitative aspects of the WS phenotype are referred to as wrinkleality [1, 35]). WS mutants are further distinguished by an altered niche preference in static microcosms, where they form a robust and well-attached physically cohesiveclass biofilm [36] at the air-liquid (A-L) interface, rather than growing throughout the liquid column like wild-type SBW25 (**Figures 2b** and **3b**) [29] (A-L interface biofilms are sometimes referred to as a pellicle [37]).

Wrinkly Spreaders are considered to be adaptive (evolved) lineages because they have a competitive fitness advantage over their ancestor, wild-type SBW25, which does not normally form biofilms in static microcosms [29, 38]. However, in shaking microcosms WS mutants are disadvantaged because they cannot form biofilms [38] and on agar plates the WS phenotype is genetically unstable [39]. Biofilm-formation by Wrinkly Spreaders and SBW25 [40] is neither unusual nor peculiar, as many other soil, plant and water-associated pseudomonads form A-L interface biofilms in static microcosms under the same conditions [36].

### **Figure 2.**

*Bacterial Biofilms*

community is achieved [28].

automated cell counters.

**3. The SBW25 model system**

biofilm-formation, kin selection ensures that cost of construction, often considered in terms of public goods or common pool resources such as the extracellular polymeric substances (EPS) which provide the main structural element of biofilms, is

Cooperation is further stabilized in biofilms by spatial separation of producers and cheaters who do not contribute to the cost of construction and a reduction of distance over which the benefits of cooperation act [19]. It is important to note that where external forcing or selection occurs, or where there is an ecological opportunity, community structures will change and lineages continue to adapt, until the theoretical end-point of evolution in a community known as an evolutionary stable

*Pseudomonas fluorescens* SBW25 was originally isolated from the sugar beet (*Beta vulgaris* subsp. *vulgaris*) phyllosphere and has been used in experimental evolution studies where the appearance of mutant lineages with altered colony morphologies (these are sometimes referred to as morphotypes or morphs) in diversifying populations have allowed the dynamics of diversification and the fitness of adaptive mutations to be readily investigated [1, 2, 13, 14, 19, 29]. In this system, competitive trade-offs between lineages result in negative frequency-dependent selection and indicate that the major

Fitness, a measure of an individual's reproductive success, is determined at the population level in such microcosms. For bacteria, fitness is readily assessed by comparing the maximum growth rate (*v*Max) of one population with that of a second, reference population. Simple growth rate comparisons are typically used to infer the success of mutations for which enzymatic or regulatory changes are being investigated, but a more meaningful ecological comparison can be made by growing the two populations together, allowing them to interact with one another and

determined using co-cultures if the two populations produce different colony morphologies allowing viable number counts to be made on agar plates, or if they can be labelled using fluorescent markers, to allow more rapid enumeration with

The evolutionary consequences of ecological processes are readily studied using microcosms. They provide defined environments for bacterial growth, and because they are reproducible, treatments can be replicated, experiments are repeatable, and selective pressures can be changed by altering resources or inocula. Nonetheless, the use of microcosms in evolution studies faces some criticisms, including the fact that they are unnatural and very simple environments, and that these studies are essentially contrived [15]. However, although populations may be founded in these synthetic environments, evolutionary and ecological dynamics are interpreted in terms of recent evolutionary history which may span 10–60,000

<sup>2</sup> The competitive fitness (*W*) of one population (A) compared to a reference population (B) is determined as the ratio of Malthusian parameters (*m*A/*m*B) where *m* = *ln* [final numbers/initial numbers] for each population over the period of the assay [30] (*m* is scaled here for generation time using *ln* as a correcting factor [31]). When *W*A,B is greater than one, A has a competitive advantage over B (and B is at a disadvantage), when *W*A,B is equal to one, the two populations are neutral, and when *W*A,B is less than one, A is at a disadvantage (and B has a competitive advantage). As *W* might be dependent on the initial ratios of the two competing populations, it can show a frequency-dependent response. The selection

coefficient (*s*) is also often used as a measure of survival and success (*s* = 1 – *W*).

can be readily

spread across all members who then all share in the benefits [15, 27].

driver of adaptive radiation is competition for limited resources [29].

to compete for limiting resources. Competitive fitness [14, 30, 31]<sup>2</sup>

**330**

*Ancestral SBW25 and adaptive Wrinkly Spreaders. Wild-type SBW25 and Wrinkly Spreader colonies are readily identified on agar plates (a). In static microcosms (b), wild-type SBW25 grows throughout the liquid column (left microcosm) and the Wrinkly Spreader forms a robust biofilm at the A-L interface (right microcosm). These microcosms are 28–30 ml glass vials containing 6 ml growth medium; they are incubated with shaking which provides a homogeneous and unstructured environment with good aeration, or statically which leads to a heterogeneous and structured environment dominated by an O2 gradient [29, 33]. When tipped out, the WS biofilm retains shape (c) demonstrating just how robust these structures are (see Figure 4 for more biofilm images). Photographs: A. Spiers.*

### **Figure 3.**

*The success of the Wrinkly Spreader in static microcosms can be understood from an evolutionary ecological perspective. The ecosystem engineering of the initial wild-type SBW25 colonists produces an O2 gradient (dotted curve) which creates an O2-rich upper zone (the Goldilocks zone) and a lower depleted zone (a). Wildtype SBW25 and Wrinkly Spreaders show different niche preferences with the WS colonising the top of the Goldilocks zone at the A-L interface (b). The WS biofilm-forming strategy is a more efficient use of resources than constant aerotaxis (swimming) to counter Brownian motion, microcurrents and vibrations which would move cells away from the optimal growth zone (c) (cell tracks indicate (i) aerotaxis towards the goldilocks zone and (ii) displacement from this region; WS biofilms (iii) are formed at the A-L interface).*

The distinctive WS colony morphology allowed an investigation of the genes required for biofilm-formation, as mini-transposon mutants of the archetypal WS with wild-type-like colony morphologies were also defective in biofilmformation [38]. This approach identified the cellulose biosynthesis (*wss*) operon required for the production of partially acetylated cellulose which was the primary biofilm matrix or EPS [38, 41]. However, the WS colony morphology and biofilm also involves an additional EPS, poly-β-1,6-N-acetyl-D-glucosamine (PNAG), as well as lipopolysaccharide (LPS), and interactions between cellulose, PNAG, LPS, and cells are required to maintain biofilm strength and integrity [42, 43]. Mini-transposon analysis also identified a chemotaxis-like (*wsp*) operon with a diguanylate cyclase (DGC) response regulator [38, 44–46]. Subsequent sequence analysis of this operon from the archetypal WS determined the presence of a single nucleotide mutation changing one amino acid residue in the methylesterase subunit [45] which acts as a negative regulatory component of the system. This results in the over-activation of the DCG, leading to increased c-*di*-GMP levels and the activation of the cellulose synthase complex. Mutations in other Wsp subunits, regulators and DGCs activated the WS phenotype in a series of independently isolated mutants [35, 43, 45, 47–49].

This understanding of the underlying molecular biology of the WS phenotype allowed a mechanistic link to be made between adaptive mutation and fitness [45] and demonstrated how easily perturbations c-*di*-GMP homeostasis could result in a key innovation through the activation of a system normally repressed in wildtype SBW25 [1, 2]. The relative ease of recovering WS lineages from diversifying populations of wild-type SBW25, demonstrating a change in niche preference and determining the competitive fitness advantage compared to the ancestral strain, also makes the SBW25 system a model for demonstrating evolution in laboratory classes [50, 51].

The microcosm system has therefore since been used to examine how wild-type colonists modify their environment [33], cells access the A-L interface [52], different environmental conditions drive WS evolution, phenotype and fitness [35, 53], and whether quorum regulation might be involved in biofilm-formation [54]. In the following subsections, we describe how the ecosystem engineering of the colonists provides the ecological opportunity and creates the niche for adaptive WS lineages and explain why biofilm-formation is the better strategy for colonizing this new niche.

**333**

*Extending an Eco-Evolutionary Understanding of Biofilm-Formation at the Air-Liquid Interface…*

Sterile static microcosms have a uniform O2 distribution throughout the liquid column. However, after inoculation the metabolic activity of wild-type SBW25 cells rapidly produces a steep O2 gradient, with less than 0.1% normal levels of dissolved O2 below 1 mm after 5 h [33]. The ecosystem engineering by these early colonists is driven by O2 uptake levels which exceed the O2 flux from the air above into the liquid column, and as a result the initially spatially homogeneous and unstructured environment is divided into an upper high-O2 zone and a lower O2-depleted zone (**Figure 3a**). The transition between the two zones is arbitrary but reflects a significant change in growth by wild-type SBW25. Further growth makes the O2 gradient even more extreme, with less than 1% O2 found below the top 200 μm layer of the

This depletion of O2 is an example on a bacterial scale of the social dilemma known as the tragedy of the commons. In this, O2 is a shared and limiting resource known as the commons, and if used selfishly and without restraint by members of the community it will be depleted and eventually destroyed [55]. Despite the growing difference between high and low-O2 zones, wild-type SBW25 cells remain distributed throughout the liquid column though there is an appreciable accumulation of cells at the top [52]. Growth rates will be higher in this region which we

down, as growth is limited by O2 availability rather than by nutrient levels in this

The ecosystem engineering of the initial colonists is also an example of niche creation (niche construction or biogenic habitat formation) [19, 56], as the high-O2 zone now represents an ecological opportunity [5, 25, 26] for any adaptive lineage capable of colonizing this region more successfully than the initial colonists. Adaptive radiation and niche creation are inter-linked [5, 19, 25, 26, 57], and in this system the high-O2 zone is colonized primarily by the Wrinkly Spreaders by biofilm-formation at the A-L interface (**Figure 3b** and **c**). Single-cell confocal Raman spectroscopy has demonstrated that WS cells recovered from within the biofilm have the same spectral profile as those grown under high-O2 conditions, while cells recovered from the liquid column below the biofilm are more similar to those grown under low-O2 conditions [58]. WS cells under high-O2 conditions also grow faster than those under low O2-conditions [33]. However, although WS cells do not grow faster than wild-type SBW25 cells under high O2-conditions [33], their rapid domination of the A-L interface and subsequent population growth displaces the wild-type colonists from this region in a process known as niche exclusion. WS growth at the A-L interface further reduces O2 flux into the lower parts of the liquid column in a density dependent manner, effectively limiting the growth of any non-biofilm-forming competitor and WS biofilms have more impact on niche divergence as populations

As the WS biofilm population increases, the division between the high and low-O2 zones also moves up into the biofilm [33], allowing further niche differentiation within the biofilm structure itself. Substantial fitness variation has been observed

<sup>3</sup> 'Goldilocks and the Three Bears', written by Robert Southey, is a tale about a girl called Goldilocks who enters the home of a family of bears while they are away. She tests their chairs, beds and breakfast porridge, always choosing the one most favourable for her, before eventually being chased away when the bears return. The 'Goldilocks zone' is also used to refer to the habitable zone around a star where the temperature is just right for liquid water to exist on an orbiting planet. Here we use the term, stricto

sensu, to mean the A-L interface plus the high-O2 zone immediately below it.

of optimal growth [2, 53], rather than lower

**4. Ecosystem engineering, ecological opportunity and niche creation**

*DOI: http://dx.doi.org/10.5772/intechopen.90955*

liquid column after 5 days [33].

have described as the Goldilocks zone3

lacking WS produce shallower O2 gradients [59].

microcosm system [33, 53].

*Extending an Eco-Evolutionary Understanding of Biofilm-Formation at the Air-Liquid Interface… DOI: http://dx.doi.org/10.5772/intechopen.90955*
