International Biodeterioration & Biodegradation (1994) 203-221 Copyright © 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0964-8305/94/$7.00 ELSEVIER 0964-8305(94)00017-8 B i o f i l m s in B i o d e t e r i o r a t i o n - - a Review L. H. G. M o r t o n & S. B. S u r m a n Department of Applied Biology, University of Central Lancashire, Preston, Lancashire, PR1 2HE, UK ABSTRACT This review defines the various types of biodeterioration processes and discusses the role that microbial films play in the biodeterioration of a number of materials of economic importance. A review of the way in which biofilms may form and attach to surfaces is presented and the occurrence and nature of biofilms is considered. Included in this review is an account of biodeterioration problems asso- ciated with water distribution systems, biocorrosion, plastics, hydrocarbons, paints and coatings and buildings and monuments. The micro-organisms involved include bacteria,fungi and algae, whiehform members of the biofilm communities responsiblefor the biodeterioration problems described. INTRODUCTION Biodeterioration m a y be defined as the 'Study of the deterioration of materials o f economic importance by micro-organisms' (Huek, 1965). Biodeterioration is due to any undesirable change in the properties of a material caused by the vital activities of organisms. It can be described as the net loss in value o f a product o f natural or manufactured origin; examples of biodeterioration can be found in both domestic and industrial situations. The processes involved in biodeterioration have been classified as follows: (1) Mechanical processes, where the material is damaged as a direct result of the activity of an organism, such as its movement or 203 204 L. H. G. Morton, S. B. Surman growth. An example of this form of biodeterioration is the damage caused to cabling as a result of insect or rodent attack. (2) Chemical assimilatory biodeterioration, perhaps the most c o m m o n form of biodeterioration. It occurs when a material is degraded for its nutritive value. The breakdown of cellulosic materials by cellu- lolytic micro-organisms, is an example of this type of biodeteriora- tion. (3) Chemical dissimilatory biodeterioration, which occurs when meta- bolic products damage a material by causing corrosion, pigmenta- tion, or by the release of toxic metabolites into a substance. The poisoning of grain by mycotoxins is an example of this process. (4) Soiling/biofouling, the form of biodeterioration which occurs when the mere presence of an organism or its excrement renders the product unacceptable. The biofouling of ships' hulls, the formation of slime in fuel lines and corrosion within water pipelines are examples of this form of biodeterioration. Microbial biodeterioration may be defined as the deterioration of materials by micro-organisms. Microbial hydrolytic enzymes often play an important role in these decay processes. BIOFILMS The term 'biofilm' in the context of biodeterioration gives rise to a number of fundamental questions: (i) What are biofilms? (ii) How are they formed? (iii) Where are they found? (iv) What is their role in biodeterioration? W h a t is a biofilm? Microbial biofilms are extremely complex microbial ecosystems that are difficult to study by conventional microbiological techniques. They may consist of complex consortia of bacteria, algae, and grazing protozoa which may display morphological features not usually associated with the organisms when grown in pure culture. It is therefore acknowledged to be very difficult to produce a biofilm which is truly representative of that found in a particular environment. The diversity of the biofilm microflora and microfauna and their interspatial relationships are extremely difficult to reproduce. Biofilms in biodeterioration 205 Biofilm formation Hamilton and Characklis (1989) described the phases of biofilm develop- ment as follows: (l) the transport of organic molecules and cells to the surface, (2) the adsorption of organic molecules to give a 'conditioned' surface, (3) the adsorption of cells to the conditioned surface, (4) the growth of adsorbed cells with associated synthesis of expoly- meric substances (EPS). Colonisation is one of the first steps leading to the subsequent forma- tion of a biofilm on a material, resulting at best in a reduction of its performance and, at worst, in its destruction. Colonisation is the process by which micro-organisms adhere to surfaces. They do so by means of extracellular polysaccharide substances, EPS, secreted by the cells. This EPS is also called the glycocalyx. In recent years it has become apparent that in the natural and industrial environments, bacterial adhesion is mediated by the glycocalyx. This is a hydrated polyanionic polysaccharide matrix produced by polymerases affixed to the lipopolysaccharide component of the cell wall (Costerton et al., 1981, 1985). In aqueous environments bacteria with the ability to generate glycocalyces abound. Several factors contribute to the preferential selection of such micro- organisms in these environments: • Organic and inorganic nutrients are concentrated at the solid-liquid interface; organisms able to secure themselves in this niche are clearly at an advantage (Costerton et al., 1981). • The glycocalyx acts as an ionic exchange matrix, trapping nutrients that are then transported into the cell by highly efficient permeases (Costerton & Geesey, 1979). • The glycocalyx conserves and concentrates the digestive enzymes released by the bacteria, thus increasing the metabolic efficiency of the cells (Costerton et al., 1978). • The glycocalyx constitutes a physical barrier that affords partial protection from antibacterial agents. (Costerton et al., 1981). Where are biofilms found? Surface associated microbial activity and colonisation, or biofilm forma- tion is a p h e n o m e n o n that occurs in both natural and man-made envir- onments, even in nutrient limited conditions. Biofilms may exist as beneficial epilithic communities in rivers and streams. They are also found 206 L. H. G. Morton, S. B. Surman in waste water treatment plants on trickling filter beds and in the alimen- tary canal of mammals (Costerton et al., 1986; Bryers & Characklis, 1982). Biofilm are truly ubiquitous. What is their role in biodeterioration? In many industries the formation of biofilms within pipework, cooling systems, heat exchangers and filters can cause problems. The resulting losses of efficiency due to increased frictional resistance in pipes (McCoy et al., 1981) or decrease in heat exchange capabilities (Trulear & Char- acklis, 1982; Shariff & Hassa, 1985) can result in decreased production rates and increased costs. Biofilms, however, are not confined to solid/liquid interfaces, they can also be found at solid/air interfaces. Airborne pathogens and deteriogens have been shown to be important factors in the biodeterioration of surface coatings. Microorganisms such as algae and fungi (rather than bacteria) often play the major role (Lloyd, 1987). Biofilms found at liquid/liquid interfaces are implicated in hydrocarbon degradation, which includes fuel oils and industrial coolants. Relationships exist between humans and biofilm micro-organisms; for example, in the gut and in the female urethra; these relationships are often symbiotic (Geesey et al., 1992). However, biofilms can also be detrimental to humans, for example, when occurring as dental plaque on teeth and dentures causing human caries and gum disease (Keevil et al., 1987). Biofilms also occur on medical prostheses, including pacemakers, replacement joints and indwelling catheters where colonisation causes chronic infections in the surrounding tissue. Often these tissues tend to be resistant to broad spectrum antimicrobial drugs and this can lead to septicaemia. In many cases the removal of the infected prostheses is required to prevent recurrent life-threatening infections. Biofilm forma- tion has also been implicated in gallstone formation and in the plugging of biliary stents used in the treatment of biliary cancer (Sung et al., 1992). Water distribution systems The occurrence of biofilms within domestic and industrial water distribu- tion systems is well documented (Costerton et al., 1987; Le Chevallier et al., 1988; Colbourne et al., 1988). Sloughing and erosion of the biofilm surface results in an increase in planktonic micro-organisms which may include potential human pathogens e.g. Legionella pneumophila (Alary & Joly, 1991; van der Wende, 1988; Rowbotham, 1980; Keevil et al., 1989), Cryptosporidia and Gardia spp. (Reasoner, 1988). Biofilms in biodeterioration 207 Legionella pneurnophila, the main aetiological agent responsible for Legionnaires' disease, is a micro-organism which is widespread within the environment. It is an opportunistic human pathogen found in high numbers in both natural and man-made aquatic environments (Grimes, 1991). Sources of infection have been found to include hot water systems, especially in large institutions such as hospitals (Hsu et al., 1984; Stout et al., 1992; Bezanson et al., 1992), cooling towers and evaporative condensors (Tobin et al., 1981; Breiman, 1993; Bentham, 1993), fountains, machine cutting coolant, misting devices, spa baths (Anon., 1991) and nebulisers (Agrawal et al., 1991). Factors which predispose artificial man- made environments to infection with L. pneumophila include: the temperature of the system, stagnation which often occurs in the dead ends of the distribution system pipework and in storage tanks (Verissimo et al., 1990), and the presence of certain nutritional sources. These may include the material of the system itself, scale, sediments and non-legionellaceae micro-organisms (Anand et al., 1984; Barbaree et al., 1986; Vickers et al., 1987; Anon., 1991; Nahapetian et al., 1991; Lfick et al., 1991; Stout et al., 1985, 1992; Rogers et al., 1993; Breiman et aL, 1993). The presence of other micro-organisms in the system is important. Legionella pneumophila appears to be capable of thriving with many different micro-organisms. The association of Legionella pneumophila with different species isolated from aquatic sources is well documented. These include protozoa, cyano- bacteria, algae and other bacteria (Rowbotham, 1980; Tison et al., 1980; Tesh & Miller, 1981; Fliermans et al., 1981; Bohach & Snyder, 1983; Wadowsky & Yee, 1983, 1985; Grimes, 1991; Pope et al., 1982; Hume & Hann, 1984). Biofilms in man-made aquatic environments therefore provide ecological niches ideally suited to Legionella pneumophila survival and growth. Other problems associated with biofilms within water distribution systems include deterioration of water quality (Le Chevallier & McFeters, 1985) and corrosion of distribution system pipework (Lee et al., 1980; Walker et al., 1991). The fact that L. pneumophila can derive nutrients from sources other than the surface supporting its growth explains why L. pneumophila is frequently isolated from water distribution systems (Dennis, 1993). The use of polymeric materials to replace traditional plumbing materials has been on the increase for many years. Organic compounds which leach from the surfaces of these components provide a nutrient source for the micro-organisms which colonise them (Ashworth & Colbourne, 1987a, b). Biofilm formation on such surfaces may lead to a rapid decrease in water quality leading to a failure to meet the required standards (Anon., 1982, 1983). L. pneumophila has been found to colonise rubber sealing washers and gaskets within hot water systems (Dennis, 208 L. H. G. Morton, S. B. Surman 1993). Rogers et al. (1990) compared a range of different materials used in plumbing systems for their ability to support the growth of micro-organ- isms, copper was the most resistant to biofilm formation whilst ethylene- propylene and latex were the most susceptible. Vess et al. (1993) showed that the colonisation of PVC pipes by micro-organisms present in the water can lead to an increased resistance to antimicrobial agents in these micro-organisms. Biocorrosion The mechanisms by which biofilms contribute to corrosion are influenced by the availability of oxygen in the environment. Under aerobic conditions, localised biofilm deposits can cause the formation of anodic and cathodic areas on the surface of a metal. These areas become a series of differential chemical cells, each inducing the transfer of electrons with loss of cations (Videla, 1990). Under aerobic conditions the utilisation of the hydrocarbons of diesel fuel and aviation kerosene by Hormoconis resinae, results in the production of organic acids which are corrosive to metals (Hendey, 1964; Parberry, 1968; Hedrick, 1970). Sulphur oxidising bacteria produce sulphuric acid in quantities sufficient to bring about the corrosion of metals and concrete (Engvall, 1986). Bacteria and fungi may accelerate corrosion indirectly by utilising corrosion inhibitors (Prince & Morton, 1988). Under anaerobic conditions sulphate reducing bacteria are the major cause of corrosion in low oxygen or oxygen-free environments. Gaylarde (1989), lists the various mechanisms by which sulphate reducing bacteria induce metal disolution. They include: (1) cathodic depolarisation brought about by the bacterial enzyme hydrogenase, (2) the production of corrosive iron sulphides, through the reaction of ferrous metals with the hydrogen sulphide released during bacterial metabolism, (3) sulphide-induced stress corrosion cracking, (4) hydrogen-induced cracking or blistering, (5) oxidation of biogenic hydrogen sulphide to corrosive elemental sulphur; this can occur when oxygen is available in the environ- ment. The cell walls of Gram-positive bacteria, the outer membranes of Gram-negative bacteria and their capsules all contain polysaccharides in various amounts (Gaylarde & Beech, 1989). Polysaccharides in the form of lipopolysaccharides (LPS) form side chains, which project from Gram- Biofilms in biodeterioration 209 negative bacterial cell surfaces, and form the interface between other adjacent cells and/or the surrounding environment (Peterson & Quie, 1981). LPS may selectively bind extracellular cations. This chelating property has been implicated in the biocorrosion of metals (Beech & Gaylarde, 1991). The initial conditioning of the surface of a metal by inorganic and organic molecules is followed by the adhesion of bacterial cells (Videla, 1990). Extracellular polysaccharide substance (EPS), which is secreted from some bacterial cells, may enhance the adhesion of these cells and of those in the immediate vicinity leading to the formation of a biofilm (Beech & Gaylarde, 1991). EPS has also been implicated in chela- tion of metal ions (Lieve et al., 1968; Ferris et al., 1987). Attachment of bacteria such as the sulphate reducer Desulfovibrio vulgaris and the marine bacterium Vibrio alginolyticus to a metal surface has been shown to result in rapid metal corrosion (Gaylarde & Johnson, 1980; Gaylarde & Videla, 1987). Hamilton (1985) describes the corrosion of steel drilling platform legs mediated by sulphate-reducing biofilm micro-organisms releasing hydrogen sulphide, which attacked the metal. Keevil et al. (1989), in a survey of a hot water system supplied with soft water, found microbial biofilm activity associated with copper corrosion. Both aerobic bacteria and anaerobic sulphate-reducing bacteria (SRB) were implicated, causing pitting and subsequent perforation of copper pipes. Plastics Plastics possess a broad range of chemical and physical properties often tailored to meet the particular requirements of industry. Specifically they have been formulated for increased durability and to resist weathering and therefore to resist microbial biodeterioration. They are both cheap and relatively easy to produce and consequently they have replaced many traditional materials such as wood, metal and rubber. Materials which are commonly considered as plastics include natural and synthetic rubbers, regenerated and modified celluloses, regenerated proteins, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyamides, polyesters and polyurethanes. Plastics are of great economic importance, with varied uses such as packaging material and in the manufacture of furniture, adhesives and inks. The transportation and construction industries are now using increasing amounts of plastics. There is a wide range of poly- mer formulations available with differing mechanical and physical prop- erties. As far as commercial plastics are concerned, it is the plasticisers and fillers used in the formulations which render them susceptible to attack. The susceptibility of some plastics is shown in Table 1. This attack usually manifests itself in the form of a surface biofilm which causes little adverse 210 L. H. G. Morton, S. B. Surman TABLE 1 The Susceptibilityof Some Plastics and Rubber to Microbial Attack Materials A t tacked by Natural rubber Bacteria and fungi Synthetic rubbers, e.g. Actinomycetes butadiene polyisoprene butyl rubber Regenerated and modified celluloses Bacteria, fungi and actinomycetes Bulk polymers (commercialplastics), plasticisers and Bacteria and fungi fillers in: polyethylene polyvinylchloride Polyamides Non-biodegradable Polyesters -- alkyd resins only Fungi and bacteria Polyurethankes -- polyester polyurethanes only Fungi and bacteria effect to the physical or chemical integrity of the material. In some cases, however, severe cracking can occur, in e.g. in PVC. Polyester polyurethanes are particularly susceptible to microbial attack. These are used in many industrial and commercial applications, for their strength and their resistance to oxidation, oil and ozone. Their resistance to impact at decreased temperatures is also a useful property (Ossefort & Testroet, 1966). However, there is conclusive evidence that polyester polyurethanes are readily biodegraded by micro-organisms under certain conditions (Ossefort & Testroet, 1966; Darby & Kaplan, 1968; Rei, 1978; Inoue, 1982; Seal & Pathirana, 1982; Pathirana & Seal, 1983, 1984a, b; Wales & Sagar, 1985, 1987; Kay et al., 1993). The term biodegradation rather than biodeterioration is used here because the material is broken down to an environmentally acceptable state (Anon., 1983). The degra- dation processes are thought to be due to hydrolysis cleaving the main chain ester group and fungal breakdown of the polymer. Kay et al. (1991) demonstrated the role of bacterial biofilms in the degradation of poly- urethane foams. They measured changes in the physical integrity of the material and observed the breakdown of the foam using light microscopy. Interestingly, the breakdown of the polyurethane was considerably enhanced by the addition of supplementary nutrients. It has been sugges- ted (Tokiwa & Suzuki, 1974, 1977; Tokiwa et al., 1976; Fields & Rodri- guez, 1975; Cook et al., 1981; Pathirana & Seal, 1983, 1984b, 1985a, b; Wales & Sagar, 1985, 1987; Cameron et al., 1986, 1987; Cameron & Costas, 1987) that esterase enzyme action is responsible for the microbial degradation of alkyd resins and polyester polyurethanes. Degradation of Biofilms in biodeterioration 211 those plasticisers derived from long chain fatty acids has also been shown to be due to esterolytic action (Berk et al., 1957; Klausmeier, 1966; Williams et al., 1969; Williams & Dale, 1983). The polyether poly- urethanes have been shown to be very resistant to attack (Darby & Kaplan, 1968; Dixit et al., 1971; Pathirana & Seal, 1985a). Hydrocarbons Microbial degradation of hydrocarbons occurs in formulations where the hydrocarbon component is intended to come into contact with water, as is the case in 'soluble' metal-working fluid emulsions; it also occurs in fuels and lubricants where water gains access into the fuel or lubricating system, allowing microbial activity to flourish (Smith, 1990). In such systems there are effectively four components: water, oil, air and the hydrocarbon- degrading micro-organisms. Growth in the presence of a water-insoluble substrate presents problems to such organisms. It is thought that there are two general types of interaction, pseudosolubilisation which involves the uptake of fine droplets of oil (less than 1 #m in diameter) from the bulk phase and that which occurs when there is direct contact between cells and large oil droplets (Erikson & Nakahara, 1975). Oil-in-water emulsions may be formed by hydrocarbonoclastic micro-organisms growing on hydrocarbons (Erikson & Nakahara, 1975; Zajic & Panchal, 1976; Margaritis et al., 1979; Cooper & Zajic, 1980). Emulsions are formed either by direct surface action of the micro-organisms or by the produc- tion of extracellular bioemulsifiers; both actions have the effect of break- ing down the hydrocarbon to small droplets. This increased mixing of the oil and water phases increases the accessibility of the hydrocarbon to these microorganisms. Bioemulsifier-producing micro-organisms will often be found forming a dense film at the oil-water interface, utilising the emul- sified hydrocarbon. Prince and Morton (1989) have suggested that in a two-phase system, a film of micro-organisms developing at the interface should be regarded as a true biofilm. Biodeterioration of bioemulsifiers and associated coupling agents caused by microbial contamination in oil emulsions often results in degradation of the emulsion associated with an increase in the size of the oil droplets and ultimately the separation of the two phases (Hill et al., 1976; Hill, 1977). A worrying factor concerning the bacterial contamination of soluble metal-working fluids is the ability of pathogens to flourish in such systems (Rossmoore, 1981). The organism most often implicated in the biodeterioration of hydrocarbon fuels is Hormoconis resinae (Lindau) de Vries, syn. Cladosporium resinae (Lindau) de Vreis, the imperfect form of Amorphotheca resinae, Parberry. The biodeterioration of fuel oils in turbine engines, including marine engines 212 L. H. G. Morton, S. B. Surman and also jet fuels has been ascribed to H. resinae (Parberry, 1971; Park, 1975; Houghton & Gage, 1979; Smith & Crook, 1980; May & Niehof, 1981). Water which remains in the small crevices and seams in fuel tanks provides an ideal environment for H. resinae to colonis e, spreading over the oil-water interface, providing ideal conditions for further water catchment (Elphick & Hunter, 1968). The removal of H. resinae from the tanks is not an easy task, the mycelia being firmly attached to the surfaces. Fuel oils may be subject to biodeterioration by H. resinae (Cofone et al., 1973; Teh & Lee, 1974). This ability to metabolise such a range of hydro- carbons (Cooney & Proby, 1971; Walker & Cooney, 1973), can result in the development of thick mycelial mats formed by the growth of H. resi- nae at the oil-water interface. The acid produced may lead to an increase in the rate of corrosion of the fuel tanks due to the decrease in pH. However, because of the rapid rate at which the fuel is used and the large volumes that the tanks hold, little discernible deterioration of the fuel itself is observed (Hill, 1978). The main problem resulting from this contamination is the blockage of fuel lines and filters and the shorting of the capacitive probes of fuel gauges (Williams & Lugg, 1980). More recently it has been observed that the synthetic metal working fluids may become contaminated by fungi. Fungal biofilms resulting in free floating biomass often cause real problems in industry today. Prince and Morton (1988) have shown that certain susceptible components of a formulation are readily attacked by fungi including pathogenic forms. Biofilms on the surfaces of buildings and monuments Two main groups of microorganisms, algae and fungi are known to colo- nise the external surfaces of buildings and monuments giving the surface a dirty, neglected and unsightly appearance (Perrichet, 1987). In addition, they are considered to be the forerunners of lichens, mosses and higher plants capable of extensive corrosive activity. In terrestrial environments, epiphytic algal growths will occur on surfaces where conditions of damp- ness, warmth and light are conducive for their growth (Hueck-van der Plas, 1968; Whitely, 1973; Richardson, 1973; Springle, 1979; Morton, 1979). The chelation of cations by microbially produced organic acids is believed to play an important role in the deterioration of stone building materials (Wainwright et al., 1993). Whilst algal growths are not believed to play a significant role in the assimilative biodeterioration of the formulation components of external coatings, they may have profound effects on the soundness of a building. The majority of algal genera involved belong to the Chlorophyceae and Cyanophyceae with species of Pleuro¢occus, Stichococcus, Trentepohlia, Oscillatoria and S c y t o n e m a Biofilms in biodeterioration 213 being particularly implicated (Gillatt & Tracey, 1987). Apart from the obvious undesirable effects on the appearance of the surface, major problems may occur due to algal water retention during winter months. Repeated freezing and thawing of the hydrated algae contribute further to the deterioration of a surface and this may be a contributory factor to causing damp in such a building. Biofilms on painted surfaces and surface coating On internal coatings, however, fungi have been recognised as being the main deteriorgens. The genera most often incriminated include Aureobasidium, Cladosporium, Aspergillus and Penicillium (Gillatt & Tracey, 1987). The initial disfigurement of interior decorative surfaces by these organisms may have far-reaching consequences, because as they are able to metabolise some of the components of the interior coating formu- lations they may expose underlying structural materials, such as timber and metal, leaving these open to attack by a range of micro-organisms (Morton, 1987), these may include pathogenic, or more correctly aller- genic fungi. The health-related aspects of such fungal colonisation in damp, ill-ventilated dwellings is becoming an increasing cause for concern. Oligotrophic fungi may also grow on painted surfaces obtaining nutrients not from the surface itself but from airborne dust and organic substances, this occurs particularly in areas of high humidity. CONCLUSION Biofilms form when adherent micro-organisms colonise surfaces. Bacterial adhesion is by means of a glycocalyx, a hydrated polyanionic poly- saccharide matrix, which can act as an ionic exchange matrix. 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