**3. Archetypal 'flow-cell' biofilms**

2 Cellulose – Medical, Pharmaceutical and Electronic Applications

plant-pathogenic environmental pseudomonads [15]. However, the ecological role of

*Rhizobium* 

*Escherichia Salmonella* 

*Rhodospirillales Acetobacteraceae\* Gluconacetobacter* Rotting fallen

*Pseudomonadales Pseudomonadaceae Pseudomonas* Water, soil and

intestine commensals

fruits

Plant pathogens Plant symbionts

human pathogens

plant-associated, including plant, fungal and animal

pathogens

}Mammalian } intestinal } commensals and pathogens

cellulose and the fitness advantage it confers to these bacteria is poorly understood.

*α-Proteobacter Rhizobiales Rhizobiaceae Agrobacterium* 

*γ-Proteobacter Enterobacteriales Enterobacteriaceae† Enterobacter* 

**Table 1.** Cellulose-expressing bacterial genera

**2. Bacterial assemblages and biofilms** 

raised in this review.

Class Order Family Genus Key habitat *Clostridia Clostridales Clostridiaceae Sarcina* Mammalian

*β-Proteobacter Burkholderiales Alcaligenaceae Alcaligenese* Opportunistic

Adapted from [2-3]. **\*,** Also known as the acetic acid bacteria; **†,** Referred to here as the enteric bacteria.

Here we provide a review of our work focussing on biofilm-formation and cellulose expression by SBW25 and other environmental pseudomonads. We do not provide an extensive list of primary literature or current reviews, but hope that the citations we have made will allow others to access the growing wealth of publications relevant to the subjects

The formation of biofilms by bacteria is a key strategy in the colonisation of many environments, though biofilms are only one of a range of bacterial assemblages involved in this process. Bacterial assemblages range from isolated surface-attached bacteria, monolayers of associated bacteria forming micro-colonies, larger and more complex structures including differentiated biofilms, as well as poorly-attached or free-floating flocs and slime. At times the differences between assemblage types may be minor and will depend on local environmental conditions. These differences are frequently ignored by many who prefer the simple dichotomy of individual, free-swimming planktonic bacteria verses the structurally complex and genetically-determined biofilms. Here we use the term 'biofilm' to include Biofilm research has largely focussed on submerged, solid-liquid (S-L) interface biofilms to provide archetypal models of biofilm structure, function and allow genetic investigation (e.g. *Pseudomonas aeruginosa* PA01 flow-cell biofilms). In these, a surface-attached exopolysaccharide (EPS) polymer matrix-based structure develops away from the solid surface, into the flow of a nutrient and O2-rich growth medium, and where fluid flow and mass transfer affects biofilm development, structure and rheology (for reviews, see [19, 28-29]).

Biofilm formation begins when planktonic bacterial cells initiate attachment to a solid surface. Attached bacteria start to move across the surface, grow and form micro-colonies, which then develop slowly into the mature biofilm structure in which bacterial cells are embedded in an exopolysaccharide polymer matrix. When conditions become unfavourable within the biofilm, single bacteria or large lumps of biofilm material detach

and move away to colonise new surfaces in more favourable environments (reviewed in [22]). Biofilms of mixed bacterial communities and of individual species that develop on solid surfaces exposed to a continuous flow of nutrients form a thick layer generally described as consisting of differentiated mushroom and pillar-like structures separated by water-filled spaces.

Cellulose Expression in *Pseudomonas fluorescens* SBW25 and Other Environmental Pseudomonads 5

depending on the growth medium [40]. In contrast, the *B. xylinum* isolate, which would probably now be recognised as a *Gluconacetobacter* spp. produced a 'vinegar plant' described as a jelly-like transparent mass at the bottom of the liquid, but under favourable conditions it could also produce a robust gelatinous A-L interface biofilm up to 25 mm

Vinegar plants are generally a consortia of acetic acid bacteria and yeasts which produce a zoogleal mat or mixed-species biofilm, and were traditionally used to produce vinegar from beer, cider or wine. Acetic fermentation is initiated by a starter culture known as the 'mother' and obtained from a previous vinegar in a process known as back-slopping [41]. A similar starter often referred to as a 'tea fungus' is used today to produce Kombucha, a carbonated cider-like drink from a sugary solution containing black tea (see the description given in [42]). Acetic acid bacteria, including *Gluconacetobacter* spp., can be isolated from these and similar consortia where they are responsible for the cellulose matrix-based biofilm (see an early review of the acetic acid bacteria by [43]). These artificially-maintained *Gluconacetobacter* spp. are probably better adapted to growth in static liquid conditions than environmental isolates recovered from rotting fallen fruit [44] and under the right conditions, some can produce a gelatinous 'plug' up to 20 mm deep in 10-12 days [45]. In these, cellulose expression and probably growth, is restricted to a thin 50-100 μm deep zone at the top, where it is limited by O2 diffusing from above and nutrients diffusing through the mature biofilm from below [45]. The growing biofilm is maintained in position by the accumulation of small CO2 bubbles and by pressing against the walls of the container as it

We expect that smaller-scale A-L interface biofilms might also occur in a wide range of natural environments, such as the partially-saturated fluid-filled pore networks of soils, in temporary puddles collecting on plants and other surfaces after rainfall, water-logged leaf tissues, or in small protected bodies of water such as ponds where the surface is not disturbed by wind or currents. In these environments, biofilm development would be restricted by a combination of nutrient availability, O2 diffusion, physical disturbance, as

A-L interface biofilms are readily produced in experimental static liquid-media microcosms [5, 11, 15], and an example of the *P. fluorescens* SBW25 Wrinkly Spreader A-L interface biofilm is shown in Figure 1. In a survey of environmental pseudomonads using nutrientrich liquid King's B microcosms, we categorised A-L interface biofilms on the basis of phenotype and physical robustness into the physically cohesive (PC), floccular mass (FM), waxy aggregate (WA) and viscous mass (VM)-class biofilms [15, 46]. The characteristics of these biofilm-types are summarised in Table 2 (see also Figure 2). A-L interface biofilm formation appears to be an evolutionary deep-rooted ability amongst bacteria, presumably with significant ecological advantages. In experimental microcosms, increases in competitive fitness of biofilm-formers have been observed compared to non-biofilm– forming strains, whilst the cost to being a biofilm-forming mutant in an environment not

well as microbial competition and predation by protists and nematodes.

suited to these structures is also measurable [5, 47-49].

thick [1].

develops.

A defining feature of many biofilms is the exopolysaccharide polymer 'slime' that encapsulate the bacteria and provide the main structural component or matrix of the biofilm [20, 22, 24-25]. Although generally assumed to be primarily composed of polysaccharides, e.g. alginate, PEL (a glucose-rich polymer) and PSL (a repeating pentasaccharide containing d-mannose, d-glucose and l-rhamnose) produced by *P. aeruginosa* PA01, PIA (a 28 kDa soluble linear (1-6)-N-acetylglucosamine) and related PNAG polymer produced by *Staphylococcus aureus* MN8m and *S. epidermidis* 13-1, and PIA-like polymers produced by *Escherichia coli* K-12 MG1655, biofilm matrices can also contain proteins and nucleic acids having significant structural roles (reviewed in [30]). Exopolysaccharides are typically viewed as a shared resource that provides a benefit to the biofilm community by maintaining structure, facilitating signalling, and protecting residents from predation, competition, and environmental stress [20, 22, 32-35].

A second characteristic common to many S-L interface biofilms has been the involvement of quorum sensing in micro-colony development, exopolysaccharide expression, and dispersal. For example, the quorum signalling molecule, acyl-homoserine lactone (AHL), functions as a signal for the development of *P. aeruginosa* PA01 and *Pseudomonas fluorescens* B52 biofilms [36-37]. However, mathematical models based on O2 and nutrient transport (diffusion) limitation result in similar biofilm architecture (reviewed in [38]), suggesting biofilm development is equally sensitive to environmental conditions as it may be to geneticallydetermined regulation. Although quorum sensing is important in the development of some biofilms, the bacterial community will exploit all available mechanisms to adapt to local environmental conditions. In order to further understand the development and role of biofilms, the local environment should be considered in terms of ecological landscape theory in which the spatial configuration of the biofilm biomass is shaped by multiple physical and biological factors [39]. It is therefore likely that biofilm formation is the net result of many independent interactions, rather than the result of a unique pathway initiating attachment and terminating with dispersal of mature biofilm communities.
