**6. Biofilms are not equivalent structures or of equal value**

Although biofilm-formation has been extensively investigated for a wide range of model bacteria, SBW25 is the only strain for which multiple A-L interface biofilms with qualitatively different phenotypes have been reported. Wild-type SBW25 produces a cellulose-based but fragile and poorly attached 'viscous mass' (VM) [36] biofilm when induced by exogenous Fe [40], and a genetically modified strain over-expressing the *wss* operon produces a similarly fragile biofilm [38]. In addition to the Wrinkly Spreaders, Fuzzy Spreaders have also been recovered from diversifying populations of SBW25 in static microcosms [29]. Though these were initially thought to be adaptive mutants which grew at the bottom of static microcosms and were adapted to anoxic conditions, they have subsequently been shown to produce fragile and short-lived A-L interface biofilms in which cells aggregate because of altered LPS expression [67]. A range of other biofilm-forming mutants have also evolved from genetically manipulated strains of SBW25, including the CBFS and PWS mutants which utilise PNAG as the primary biofilm matrix [43, 68].

WS and WS-like phenotypes are often caused by loss-of-function mutations affecting negative regulators, less frequently by promoter activation or gene-fusion mutations, and finally by rare mutations resulting in intragenic gain-of-function [47]. In general, these biofilm-forming lineages have a fitness advantage compared to non-biofilm-forming competitors [38, 43, 47, 49]. However, possible negative pleiotropy and epistasis effects [11, 14] might contribute to a lower-than-expected fitness advantage in some cases, and the accumulation of additional mutations not associated with the WS phenotype may also have a negative effect on fitness in a process known as Muller's ratchet [14].

In order to better understand the links between WS mutation, phenotype, and fitness, it has been necessary to develop quantitative assays to describe WS biofilms and an experimental approach to test the effect of physical disturbance on biofilmformation and fitness. Variations in WS phenotype or wrinkleality [1, 35], including microcosm growth, biofilm strength and attachment levels, can be determined using a combined biofilm assay [69] that can quantitatively differentiate WS isolates recovered from different environments, whilst careful use of orbital shakers can provide intermediate levels of disturbance between static and shaking conditions.

Using this approach, we can differentiate CBFS, VM and WS biofilms on the basis of competitive fitness compared to a non-biofilm-forming strain. Under static conditions CBFS fitness is greater that either VM or WS biofilms, suggesting that the CBFS biofilm is the most cost-effective solution to colonising the A-L interface.

**337**

**Figure 6.**

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

However, as the level of physical disturbance increases, CBFS and VM biofilms fail and sink before the more resilient WS biofilms. As a result, their fitness decreases before WS fitness (**Figure 6**). At maximum levels of disturbance where no biofilm can form, VM and WS fitness is lower than CBFS fitness. This suggests that the VM and WS phenotypes which continue to produce cellulose but cannot form biofilms are more costly than the CBFS phenotype which does not utilise this particular EPS. We noted that in these microcosms CBFS aggregates accumulated on the vial walls above the liquid line. Stranded cells may have better access to O2 than those remaining in the liquid column and this may further increase competitive fitness. We are also able to differentiate CBFS, VM and WS biofilms on the basis of structure and rheology, which, when combined with our fitness analyses, suggests that the CBFS biofilm is the most cost-effective structure allowing the colonisation of the A-L interface. It falls between the more costly and over-engineered WS and barely adequate VM biofilms and provides a greater fitness advantage because the levels of physical disturbance static microcosms are subject to will neither increase, which might favour the WS biofilm, nor fall, which may favour the VM biofilm.5

*The adaptive advantage of biofilms is dependent on levels of physical disturbance. Competitive fitness assays were used to assess the adaptive advantage of CBFS, VM and WS biofilms compared to a non-biofilm-forming competitor across a range of levels of physical disturbance from static to shaken conditions. Means are shown with standard errors. Dotted lines suggest trends and differences between means were investigated by Tukey-Kramer HSD; means sharing the same letters are not significantly different (α = 0.05). Data and analyses will* 

**7. Community biofilm-formation in static microcosms**

occupation of the Goldilocks zone in our microcosms.

As the evolutionary dynamics of diversifying SBW25 populations and the fitness advantages of biofilm-formation in static microcosms are increasingly well understood, we have begun to consider the drivers of biofilm-formation in community-based multi-species biofilms [70–72]. Communities artificially established in microcosms from mixed inocula are particularly interesting as strong

<sup>5</sup> This 'neither too much nor too little' evaluation suits Red Queens who choose to compete for the

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

*be reported in full elsewhere (A. Koza and A. Spiers).*

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

### **Figure 6.**

*Bacterial Biofilms*

A-L interface, than constant aerotaxis.

process known as Muller's ratchet [14].

microcosms in which we had added low concentrations of agar or polyethylene glycol to increase viscosity, as diffusion is inversely dependent on liquid viscosity [52]. Both wild-type and WS cell localization improved with increasing viscosity and, furthermore, WS competitive fitness was found to decrease with increasing viscosity (**Figure 5**) [52]. This indicates that WS biofilm-formation is a better strategy allowing the colonization of the high-O2 zone and more specifically, of the

We argue that the need to remain in place at the top of the liquid column efficiently in order to make use of greater O2 availability is the fundamental explanation for the success of A-L interface biofilm-formation in static microcosms by motile aerobic such as the pseudomonads [36] where growth is limited by O2-availability rather than by nutrients [53]. The success is determined by a cost-benefit trade-off, in which resource costs required for biofilm-formation by the community or con-

Although biofilm-formation has been extensively investigated for a wide range of model bacteria, SBW25 is the only strain for which multiple A-L interface biofilms with qualitatively different phenotypes have been reported. Wild-type SBW25 produces a cellulose-based but fragile and poorly attached 'viscous mass' (VM) [36] biofilm when induced by exogenous Fe [40], and a genetically modified strain over-expressing the *wss* operon produces a similarly fragile biofilm [38]. In addition to the Wrinkly Spreaders, Fuzzy Spreaders have also been recovered from diversifying populations of SBW25 in static microcosms [29]. Though these were initially thought to be adaptive mutants which grew at the bottom of static microcosms and were adapted to anoxic conditions, they have subsequently been shown to produce fragile and short-lived A-L interface biofilms in which cells aggregate because of altered LPS expression [67]. A range of other biofilm-forming mutants have also evolved from genetically manipulated strains of SBW25, including the CBFS and

stant aerotaxis by individual cells are balanced against population gains.

PWS mutants which utilise PNAG as the primary biofilm matrix [43, 68].

WS and WS-like phenotypes are often caused by loss-of-function mutations affecting negative regulators, less frequently by promoter activation or gene-fusion mutations, and finally by rare mutations resulting in intragenic gain-of-function [47]. In general, these biofilm-forming lineages have a fitness advantage compared to non-biofilm-forming competitors [38, 43, 47, 49]. However, possible negative pleiotropy and epistasis effects [11, 14] might contribute to a lower-than-expected fitness advantage in some cases, and the accumulation of additional mutations not associated with the WS phenotype may also have a negative effect on fitness in a

In order to better understand the links between WS mutation, phenotype, and fitness, it has been necessary to develop quantitative assays to describe WS biofilms and an experimental approach to test the effect of physical disturbance on biofilmformation and fitness. Variations in WS phenotype or wrinkleality [1, 35], including microcosm growth, biofilm strength and attachment levels, can be determined using a combined biofilm assay [69] that can quantitatively differentiate WS isolates recovered from different environments, whilst careful use of orbital shakers can provide intermediate levels of disturbance between static and shaking conditions. Using this approach, we can differentiate CBFS, VM and WS biofilms on the basis of competitive fitness compared to a non-biofilm-forming strain. Under static conditions CBFS fitness is greater that either VM or WS biofilms, suggesting that the CBFS biofilm is the most cost-effective solution to colonising the A-L interface.

**6. Biofilms are not equivalent structures or of equal value**

**336**

*The adaptive advantage of biofilms is dependent on levels of physical disturbance. Competitive fitness assays were used to assess the adaptive advantage of CBFS, VM and WS biofilms compared to a non-biofilm-forming competitor across a range of levels of physical disturbance from static to shaken conditions. Means are shown with standard errors. Dotted lines suggest trends and differences between means were investigated by Tukey-Kramer HSD; means sharing the same letters are not significantly different (α = 0.05). Data and analyses will be reported in full elsewhere (A. Koza and A. Spiers).*

However, as the level of physical disturbance increases, CBFS and VM biofilms fail and sink before the more resilient WS biofilms. As a result, their fitness decreases before WS fitness (**Figure 6**). At maximum levels of disturbance where no biofilm can form, VM and WS fitness is lower than CBFS fitness. This suggests that the VM and WS phenotypes which continue to produce cellulose but cannot form biofilms are more costly than the CBFS phenotype which does not utilise this particular EPS. We noted that in these microcosms CBFS aggregates accumulated on the vial walls above the liquid line. Stranded cells may have better access to O2 than those remaining in the liquid column and this may further increase competitive fitness.

We are also able to differentiate CBFS, VM and WS biofilms on the basis of structure and rheology, which, when combined with our fitness analyses, suggests that the CBFS biofilm is the most cost-effective structure allowing the colonisation of the A-L interface. It falls between the more costly and over-engineered WS and barely adequate VM biofilms and provides a greater fitness advantage because the levels of physical disturbance static microcosms are subject to will neither increase, which might favour the WS biofilm, nor fall, which may favour the VM biofilm.5

### **7. Community biofilm-formation in static microcosms**

As the evolutionary dynamics of diversifying SBW25 populations and the fitness advantages of biofilm-formation in static microcosms are increasingly well understood, we have begun to consider the drivers of biofilm-formation in community-based multi-species biofilms [70–72]. Communities artificially established in microcosms from mixed inocula are particularly interesting as strong

<sup>5</sup> This 'neither too much nor too little' evaluation suits Red Queens who choose to compete for the occupation of the Goldilocks zone in our microcosms.

selection would be expected to play a role in community assembly with a rapid loss of redundant members who do not contribute to the new system. There are simple organising principles in microbial communities especially where competitive interactions are dominant [73, 74]. However, with the exception of WS-like biofilms initiated through mutation, cell-to-cell communication is thought to co-ordinate biofilm-formation and ensure that all members contribute to the cost of production without cheating [15, 27]. As a result, biofilm-formation is seen largely as a cooperative undertaking by closely related lineages, yet this appears to conflict with the view that competitive interactions generally dominate microbial communities.

In order to investigate the relative importance of cooperation and competition in community biofilms, we have developed a model system using soil-wash inocula which include biofilm-competent pseudomonads [36] and our static microcosms in which O2 is the growth-limiting factor. This typically resulted in very fragile and poorly attached VM-like biofilms within 2–3 days with substantial growth also occurring throughout the liquid column. Preliminary trails suggested that growth levels were sensitive to different media and aeration conditions, and treatment with antibiotics, copper and perchlorate had differential effects on growth, biofilm strength and attachment levels, demonstrating that different selective pressures could alter community productivity and biofilm-formation.

We then undertook a serial transfer experiment selecting for biofilm-formation by transferring biofilm samples using a wire-loop across a series of 10 microcosms for a total of 60 days of incubation. Under such selection, we expected to see replicate communities dominated by robust WS-like biofilms and a decrease in strain diversity as non-biofilm-formers and uncompetitive strains were lost. We also expect to see a significant reduction in the number of bacteria growing below the biofilm in the liquid column, as competition for access to O2 should drive ecological change and result in more 'effective' biofilm-formation.

However, replicate communities continued to produce weak biofilms despite their physically cohesive appearance [36], suggesting that the selective pressure for biofilm-formation was not particularly strong. Nonetheless, a significant loss of diversity was observed, and an analysis of random isolates suggested that the proportion of biofilm-formers increased, and a phenotypic shift occurred between the initial and final selected communities (**Figure 7**), confirming that these communities were subject to selective pressure. Although we expected to see the selected communities dominated by one or a few 'super' biofilm-formers, they appeared to be dominated by a mix of lineages with very similar phenotypes. This is perhaps not surprising, as our preparation of the soil-wash inocula would have selected for fast-growing aerobic and biofilm-competent bacteria such as *Pseudomonas* spp. from the original soil community (environmental filtering within the soil would also have selected for related lineages and lineages with similar phenotypes). Such mixes may be stable, as the coexistence of related lineages and the coexistence of unrelated lineages with similar phenotypes, is possible because they may not exhibit significant levels of negative interactions and might even facilitate one another [75].

We also found significant levels of growth in the liquid column below the biofilms, suggesting that lineages were colonising the A-L interface and low-O2 region from the biofilm transfer samples and that migration was occurring between these two zones. It is possible that biofilm-competent lineages might avoid competition at the A-L interface by choosing a less competitive niche lower down the liquid column in a biochemical trade-off [76] in which lower growth rates resulting from O2-limitation are balanced by the cost of biofilm-formation which would have been required at the A-L interface.

**339**

**8. Conclusions**

**Figure 7.**

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

Although this research is still on-going and will be published in full elsewhere, our current focus is to better understand the levels of competition occurring within the community biofilm and the role of the low-O2 region in maintaining diversity in selected communities. A future goal is to investigate the dynamics of diversification of wild-type SBW25 in these communities in order to see how competition within

*Serial transfer of biofilm samples results in changes in biofilm characteristics of individual community members. Isolates were sampled from the initial (light grey circles) and final communities (dark grey circles) after serial transfer of biofilm material by wire loop across 10 microcosms over 60 days. Principal component analysis (PCA) of isolate biofilm characteristics, including total microcosm growth, biofilm strength and attachment levels, shows a phenotypic shift occurring between initial and final communities. Data and analyses* 

Biofilm research is interdisciplinary but is increasingly fragmented and polarised, with interest still dominated by molecular biologists working with medically relevant model species and a mechanistic focus on biofilm-formation. This perspective limits our understanding of more complex community-based biofilms, as ecological interactions and evolutionary processes play important roles in the development and success of these structures, with immigration and adaptive radiation introducing novel abilities or key innovations which may have a significant impact on community function. Biofilm research is now at the stage where an ecoevolutionary perspective should be included to produce a more comprehensive and holistic understanding of biofilms in a wide range of contexts, from model systems

the community biofilm effects WS evolution and fitness.

*will be reported in full elsewhere (R. Jerdan and A. Spiers).*

to biofilm-associated disease, biotechnology and industry.

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

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

#### **Figure 7.**

*Bacterial Biofilms*

selection would be expected to play a role in community assembly with a rapid loss of redundant members who do not contribute to the new system. There are simple organising principles in microbial communities especially where competitive interactions are dominant [73, 74]. However, with the exception of WS-like biofilms initiated through mutation, cell-to-cell communication is thought to co-ordinate biofilm-formation and ensure that all members contribute to the cost of production without cheating [15, 27]. As a result, biofilm-formation is seen largely as a cooperative undertaking by closely related lineages, yet this appears to conflict with the view that competitive interactions generally dominate microbial communities. In order to investigate the relative importance of cooperation and competition in community biofilms, we have developed a model system using soil-wash inocula which include biofilm-competent pseudomonads [36] and our static microcosms in which O2 is the growth-limiting factor. This typically resulted in very fragile and poorly attached VM-like biofilms within 2–3 days with substantial growth also occurring throughout the liquid column. Preliminary trails suggested that growth levels were sensitive to different media and aeration conditions, and treatment with antibiotics, copper and perchlorate had differential effects on growth, biofilm strength and attachment levels, demonstrating that different selective pressures

We then undertook a serial transfer experiment selecting for biofilm-formation by transferring biofilm samples using a wire-loop across a series of 10 microcosms for a total of 60 days of incubation. Under such selection, we expected to see replicate communities dominated by robust WS-like biofilms and a decrease in strain diversity as non-biofilm-formers and uncompetitive strains were lost. We also expect to see a significant reduction in the number of bacteria growing below the biofilm in the liquid column, as competition for access to O2 should drive ecological

However, replicate communities continued to produce weak biofilms despite their physically cohesive appearance [36], suggesting that the selective pressure for biofilm-formation was not particularly strong. Nonetheless, a significant loss of diversity was observed, and an analysis of random isolates suggested that the proportion of biofilm-formers increased, and a phenotypic shift occurred between the initial and final selected communities (**Figure 7**), confirming that these communities were subject to selective pressure. Although we expected to see the selected communities dominated by one or a few 'super' biofilm-formers, they appeared to be dominated by a mix of lineages with very similar phenotypes. This is perhaps not surprising, as our preparation of the soil-wash inocula would have selected for fast-growing aerobic and biofilm-competent bacteria such as *Pseudomonas* spp. from the original soil community (environmental filtering within the soil would also have selected for related lineages and lineages with similar phenotypes). Such mixes may be stable, as the coexistence of related lineages and the coexistence of unrelated lineages with similar phenotypes, is possible because they may not exhibit significant levels of negative interactions

We also found significant levels of growth in the liquid column below the biofilms, suggesting that lineages were colonising the A-L interface and low-O2 region from the biofilm transfer samples and that migration was occurring between these two zones. It is possible that biofilm-competent lineages might avoid competition at the A-L interface by choosing a less competitive niche lower down the liquid column in a biochemical trade-off [76] in which lower growth rates resulting from O2-limitation are balanced by the cost of biofilm-formation which would have been

could alter community productivity and biofilm-formation.

change and result in more 'effective' biofilm-formation.

and might even facilitate one another [75].

required at the A-L interface.

**338**

*Serial transfer of biofilm samples results in changes in biofilm characteristics of individual community members. Isolates were sampled from the initial (light grey circles) and final communities (dark grey circles) after serial transfer of biofilm material by wire loop across 10 microcosms over 60 days. Principal component analysis (PCA) of isolate biofilm characteristics, including total microcosm growth, biofilm strength and attachment levels, shows a phenotypic shift occurring between initial and final communities. Data and analyses will be reported in full elsewhere (R. Jerdan and A. Spiers).*

Although this research is still on-going and will be published in full elsewhere, our current focus is to better understand the levels of competition occurring within the community biofilm and the role of the low-O2 region in maintaining diversity in selected communities. A future goal is to investigate the dynamics of diversification of wild-type SBW25 in these communities in order to see how competition within the community biofilm effects WS evolution and fitness.

### **8. Conclusions**

Biofilm research is interdisciplinary but is increasingly fragmented and polarised, with interest still dominated by molecular biologists working with medically relevant model species and a mechanistic focus on biofilm-formation. This perspective limits our understanding of more complex community-based biofilms, as ecological interactions and evolutionary processes play important roles in the development and success of these structures, with immigration and adaptive radiation introducing novel abilities or key innovations which may have a significant impact on community function. Biofilm research is now at the stage where an ecoevolutionary perspective should be included to produce a more comprehensive and holistic understanding of biofilms in a wide range of contexts, from model systems to biofilm-associated disease, biotechnology and industry.
