Ian W. Sutherland

Biofilm exopolysaccharides: a strong and sticky framework

Biofilms probably comprise the normal environment for most microbial cells in many natural and artificial habitats, and as such are complex associations of cells, extracellular products and detritus either trapped within the biofilm or released from cells which have lysed as the biofilm ages (Christensen, 1989). The main ‘cement ’ for all these cells and products is the mixture of polysaccharides secreted by the cells established within the biofilm. Probably the nearest analogy is processed food, in which a mixture of macromolecules of all types interact in variousways to form a recognizable structure. Within such a structure, cells, water, ions and soluble low-and high-molecular-mass products are trapped. In many biofilms, as in food, the hydrated polysaccharides may be in a semi-solid state. The major component in the biofilm matrix is water – up to 97% (Zhang et al., 1998), and the characteristics of the solvent are determined by the solutes dissolved in it. The exact structure of any biofilm is probably a unique feature of the environment in which it develops. As pointed out by Stoodley et al. (1999a), nutritional and physical conditions greatly affect the nature of laboratory biofilms and this is equally true for other types. Wimpenny & Colasanti (1997) have also suggested that biofilm structure is largely determined by the concentration of substrate. They further postulated that such differences also validate at least three conceptual models of biofilms – heterogeneous mosaics, structures penetrated by water channels, and dense confluent biofilms.

The biofilm matrix – an immobilized but dynamic microbial environment

The biofilm matrix is a dynamic environment in which the component microbial cells appear to reach homeostasis and are optimally organized to make use of all available nutrients. The major matrix components are microbial cells, polysaccharides and water, together with excreted cellular products. The matrix therefore shows great microheterogeneity, within which numerous microenvironments can exist. Although exopolysaccharides provide the matrix framework, a wide range of enzyme activities can be found within the biofilm, some of which will greatly affect structural integrity and stability.

Novel and established applications of microbial polysaccharides.

Microbial exopolysaccharides such as xanthan and dextran have been commercial products for many years; the search for new gelling agents has yielded gellan. Exopolysaccharides have many other novel properties to offer, and the discovery of immune modulation and tumouristasis by beta-D-glucans, and the use of bacterial cellulose in audio membranes and of hyaluronic acid in cosmetics provide some novel applications. Semisynthetic polymers and polysaccharides as sources of oligosaccharides and as enzyme substrates in the determination of enzyme specificity should further increase the interest in these macromolecules.

Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase

Biofilm bacteria Enterobacter agglomerans 53b and Serratia marcescens Serr were isolated from a food processing factory. A bacteriophage (SF153b), which could infect and lyse strain 53b, was isolated from sewage. This has been shown to possess a polysaccharide depolymerase enzyme specific for the exopolysaccharide (EPS) of strain 53b. Using batch culture and chemostat-linked Modified Robbins Device systems it was observed that SF153b could degrade the EPS of a mono-species biofilm (strain 53b) and infect the cells. The disruption of the biofilm by phage was a combination of EPS degradation by the depolymerase and infection and subsequent cell lysis by the phage. Strain Serr biofilms were not susceptible to the phage and the biofilm EPS was not degraded by the phage glycanase, with the result that the biofilm was unaffected by the addition of SF153b phage. Scanning electron microscopy confirmed that specific phage could extensively degrade susceptible biofilms and continue to infect biofilm bacteria whilst EPS degradation was occurring.

Microbial polysaccharides from Gram-negative bacteria

The structures of many microbial polysaccharides from a wide variety of bacterial species and from some algae, fungi and yeasts have been determined. A number of bacterial polysaccharides have either been adopted as commercial products or have the potential for commercialisation. The biosynthetic mechanisms for production of some of these polymers have also been elucidated and the effects of genetic manipulation have been studied. There is now also a much greater knowledge of structure/function relationships in microbial polysaccharides. While almost all this non-structural data has accumulated from the study of Gram negative species, this background information provides a sound basis from which polysaccharides from Gram positive bacteria can now be developed.

The Role of Exopolysaccharides in Adhesion of Freshwater Bacteria

SUMMARY: Cultures of two strains of freshwater bacterial isolates adhered readily to inert glass surfaces exposed in the growth medium. The process of microbial film formation could be followed by a new staining technique based on congo red, a dye specific for carbohydrate material. In conjunction with a chemical assay for total carbohydrate, the association of extracellular polysaccharides with attached cells was demonstrated. Under optimal growth conditions, the involvement of exopolysaccharide in the adhesion process appeared to follow the initial attachment of bacterial cells, leading to the formation of microcolonies enmeshed in polysaccharides. A non-polysaccharide-producing mutant attached to glass slides in numbers similar to the wild-type bacteria, but did not form microcolonies. Growth conditions such as glucose or Ca2+ limitation which affected polysaccharide synthesis in the wild-type prevented microcolony formation, but not cell attachment. It is proposed that exopolysaccharide production is involved in the development of the surface films, but possibly not in the initial adhesion-process. In those strains which do produce polysaccharide, the cells which attach develop into microcolonies.

The interaction of phage and biofilms.

Biofilms present complex assemblies of micro-organisms attached to surfaces. they are dynamic structures in which various metabolic activities and interactions between the component cells occur. When phage come in contact with biofilms, further interactions occur dependent on the susceptibility of the biofilm bacteria to phage and to the availability of receptor sites. If the phage also possess polysaccharide-degrading enzymes, or if considerable cell lysis is effected by the phage, the integrity of the biofilm may rapidly be destroyed. Alternatively, coexistence between phage and host bacteria within the biofilm may develop. Although phage have been proposed as a means of destroying or controlling biofilms, the technology for this has not yet been successfully developed.

Biosynthesis of Microbial Exopolysaccharides

Publisher Summary This chapter surveys current knowledge of the production and synthesis of microbial exopolysaccharides. Although the emphasis is on bacterial polymers, relevant information is included on similar products from yeasts, fungi, and other eukaryotes. The chapter covers specific aspects such as capsule synthesis, regulation of biosynthesis, and industrial applications of the products. The chapter explores that numerous microorganisms produce exopolysaccharides—that is, polysaccharides found outside the cell wall, either attached to it in the form of capsules or secreted into the extracellular environment in the form of slime. Such polymers vary considerably in their chemical structures. The three groups of exopolysaccharides, which have been most studied are those produced by Enterobacter aerogenes, Escherichia coli and Streptococcus (Dipfococcus) pneumoniae. The chapter considers that exopolysaccharides belongs to five distinct groups: (1) dextrans and related polysaccharides, (2) group of polymers, (3) number of microorganisms produce extracellular homopolysaccharides from any of several carbon substrates, (4) heteropolysaccharides, and (5) bacterial alginate.

Polysaccharases for microbial exopolysaccharides

Microbial exopolysaccharides (EPS) are the substrates for a wide range of enzymes most of which are highly specific. The enzymes are either endoglycanases or polysaccharide lyases and their specificity is determined by carbohydrate structure with uronic acids often playing a major role. The presence of various acyl substituents frequently has little effect on the action of many of the polysaccharases but markedly inhibits some of the polysaccharide lyases including alginate and gellan lyases. The commonest sources of such enzymes can be either microorganisms or bacteriophages. These specific polysaccharide-degrading enzymes can yield oligosaccharide fragments, which are amenable to NMR and other analytical techniques. They have thus proved to be extremely useful in providing information about microbial polysaccharide structures and were routinely used in many such studies. Complex systems containing various mixtures of enzymes may also be effective in the absence of single enzymes but may be difficult to obtain with reproducible activities. Such preparations may also cause extensive degradation of the polysaccharide structure and thus prove less useful in providing information. Commercially available enzyme preparations have seldom proved capable of degrading microbial heteropolysaccharides, although some are active against bacterial alginates and homopolysaccharides including bacterial cellulose and curdlan.

Comparison of the acid-base behaviour and metal adsorption characteristics of a gram-negative bacterium with other strains

Thermodynamic parameters for proton and metal adsorption onto a gram-negative bacterium from the genus Enterobacteriaceae have been determined and compared with parameters for other strains of bacteria. Potentiometric titrations were used to determine the different types of sites present on bacterial cell walls. Stability constants for adsorption of Pb, Cu and Zn to specific sites were determined from batch adsorption experiments at varying pH with constant metal concentration. Titrations revealed 3 distinct acidic surface sites on the bacterial surface, with pK values of 4.3±0.2, 6.9±0.5 and 8.9±0.5, corresponding to carboxyl, phosphate and hydroxyl/amine groups, with surface densities of 5.0±0.7×10−4, 2.2±0.6×10−4 and 5.5±2.2×10−4 mol/g of dry bacteria. Only carboxyl and phosphate sites are involved in metal uptake, yielding the following intrinsic stability constants: Log Kcarboxyl: Zn=3.3±0.1, Pb=3.9±0.8, and Cu=4.4±0.2, Log Kphosphoryl: Zn=5.1±0.1 and Pb=5.0±0.9. The deprotonation constants are similar to those of other strains of bacteria, while site densities are also within an order of magnitude of other strains. The similarities in surface chemistry and metal stability constants suggest that bacteria may be represented by a simple generic thermodynamic model for the purposes of modelling metal transport in natural environments. Comparison with oxide-coated sand shows that bacteria can attenuate some metals to much lower pH values.