International Biodeterioration & Biodegradation 53 (2004) 177 183

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Corrosion of technical materials in the presence of biolmscurrent understanding and state-of-the art methods of study
Iwona B. Beech
School of Pharmacy and Biomedical Sciences,University of Portsmouth, St. Michaels Building White Swan Road, Portsmouth P01 2DT, UK

Abstract It is documented that biolms are capable of in uencing electrochemical processes at the metal surface, often leading to deterioration of metals referred to as biocorrosion or microbiologically in uenced corrosion. Biolms typically consist of microbial cells and their metabolic products including extracellular polymers, and inorganic precipitates. Interaction of biolms and exopolymers with metal ions has long been proposed as one of the mechanisms of metal biodeterioration. This paper presents an overview of the application of modern microscopy and surface analysis techniques in studying the involvement of biolms and extracellular polymeric material in the biocorrosion process of metals and their alloys. ? 2003 Elsevier Ltd. All rights reserved.

1. Biolms and biodeterioration The term biolm refers to the development of microbial communities on submerged surfaces in aqueous environments (Characklis and Marshall, 1990). The growth of biolm is considered to be a result of complex processes involving transport of organic and inorganic molecules and microbial cells to the surface, adsorption of molecules to the surface (formation of the conditioning layer) and initial attachment of microbial cells followed by their irreversible adhesion facilitated by production of extracellular polymeric substances (EPS), often referred to as the glycocalyx or slime (Costerton et al., 1978). The biofouling and biodeterioration of man-made materials, including metal and their alloys, due to the biolm formation has great environmental and economical implications. Many industrial sectors such as gas, oil, nuclear power, shipping, aircraft, chemical and civil engineering su er potential pollution problems, health and safety hazards and substantial nancial losses as a result of biolm development (Hamilton, 1985; Flemming, 1996). Several models are proposed to explain possible mechanisms by which biolms can in uence deterioration of metals (Borenstein, 1994). It has been documented that degradation of metal surfaces, also termed biocorrosion or microbially in uenced corrosion (MIC), occurs when the contact

between microbial cells, or products of their metabolism such as EPS, and the surface is established (Gaylarde and Johnston, 1982; Gaylarde and Videla, 1987). 2. Interaction of bacterial exopolymers with metals It is unequivocally accepted that microbial exopolymers, including lipopolysaccharides (LPS), play an important role in the process of cell attachment to metal surfaces (Beech and Gaylarde, 1989; Flemming et al., 1998). The EPS consist of lipids, polysaccharides, proteins and nucleic acids (Wingender et al., 1999). The content of these macromolecules in EPS varies, depending on bacterial species and growth conditions (Sutherland, 1985; Beech et al., 1991a; Beech et al., 1999a). Although in planktonic bacteria, EPS are often present as a capsule, exopolymers do not have to be associated with cells but can also be released into the bulk phase of surrounding liquid. The free EPS material can compete with bacterial cells for binding sites at the metal surface and thus needs to be considered when investigating biocorrosion (Paradies et al., 1992). One of the important properties of EPS is their ability to complex with metal ions (Geesey and Mittelman, 1985). Most of the available information on the EPS-mediated mechanism of metal accumulation has been generated in waste treatment research. Many purpose-built systems for metal removal from wastewaters use immobilized living

Tel.: +44-1705-842147; fax: +44-1705-842147. E-mail address: iwona.beech@port.ac.uk (I.B. Beech).

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microorganisms. In some of these systems, bacterial cells colonize support materials such as glass, stainless steel wire, wood shavings, reticulated foams and polyvinyl chloride to form biolms. Such biolms have been shown to be very e cient in immobilizing metal ion species (Macaskie and Dean, 1987). Although the role of exopolymers present within these biolms in the removal of metal ion species has not been considered, it is likely that EPS could contribute to this process. As already stated, one of the functions of EPS is to enhance the ability of cells to adhere to surfaces (Fletcher and Floodgate, 1973; Marshall, 1992). Nutrients, which are scarce in aquatic environments, are concentrated on surfaces (Characklis and Marshall, 1990). The colonization of the surface would therefore prove to be highly advantageous for cells capable of attaching to it. Studies on EPS production by Pseudomonas atlantica showed the expression of EPS to be variable and controlled by genome rearrangement (Bartlett et al., 1988). The authors speculated that recombinal switches to generate diversity in a population exist, with the result that a small fraction of the population could take advantage of favourable circumstances. The presence of a nutrient rich surface could provide that trigger. Thus, cells would regulate the secretion of EPS in response to the presence of the surface (Beech et al., 1991b). Evidence has been presented to support this theory by the revelation that expression of certain genes can be modied following association of bacteria with a surface (Davies et al., 1994). Examination of protein proles in EPS of marine sulphate-reducing bacteria (SRB) isolates, revealed the appearance of new bands in exopolymers harvested from cultures grown in the presence of mild steel surfaces, thus further conrming the e ect of surface on cell metabolism (Zinkevich et al., 1996). It can be proposed that interaction between bacterial surface molecules such as LPS and/or EPS and metal ions released at the metal/liquid interface can be regulated by the abundance of metal ion species (Gaylarde and Beech, 1988). All bacteria require metal ions for their growth. The availability and the type of ions (type of metal species and their reactivity determined by the metal oxidation state) are likely to have an e ect on the colonization of a metal surface (Beech et al., 1993). The chemistry of the surface could affect bacterial population positively or adversely, in the rst case by facilitating the attachment of cells to the surface via binding of metal ions by EPS and/or LPS (Beech, 1990 and references therein), and in the second case by preventing surface colonization due to the toxic e ect of some of the metal ion species, e.g. Cr (Percival et al., 1997). The former phenomenon would favour attachment and development of a biolm, often leading to severe fouling and deterioration of metal. The latter case would result in the partial inhibition of bacterial adhesion and decrease in fouling, consequently limiting the degree of material damage. The interactions between bacterial EPS and metal ions are documented (Ford et al., 1988). Many exopolymers of aquatic microorganisms act as polyanions under natural

conditions, carrying a charge that promotes ionic and electrostatic binding of counterions, including metals. Several classes of polymeric molecules participate in EPS/metal interactions by formation of salt bridges with carboxyl groups on acidic polymers, i.e. polysaccharides containing uronic acids, and by forming weak electrostatic bonds with hydroxyl groups on polymers containing neutral carbohydrates. A large number of metals have been reported to cross-link polysaccharides (Geesey and Jang, 1989). In acid mine drainage waters heterotrophic bacteria, often referred to as acid streamers, produce copious amounts of slime consisting of a brillar polymer network. This extracellular material has been reported to be a mixture of neutral and acidic polysaccharides and RNA (Johnson and Kelso, 1981). Further studies by Johnson et al. (1993) showed that the acid streamers participated in oxido-reduction of iron, and that iron was bound within the exopolymer matrix of the streamers. It can be speculated that carboxylic groups originating from the carbohydrate component of the EPS and phosphate groups liberated during nucleic acid degradation could play a part in the binding of metals, e.g. iron, increasing the overall metal binding capacity of EPS. The latter property of bacterial EPS contributes to the formation of metal concentration cells and promotes galvanic coupling, thus in uencing the electrochemical behavior of metal (Ford et al., 1987; White et al., 1985). Indeed, evidence exists that exopolymers exhibit great selectivity in complexing metal ions, and a direct involvement of EPS in the biodeterioration of metal surfaces such as stainless steel, copper and carbon steel has been shown (Angell et al., 1995; Thies et al., 1995; Beech et al., 1996). There is, however, a controversy regarding the type of EPS fraction playing a key role in interactions with metals. Some reports indicate that proteins are involved (Angell and Chamberlain, 1991), while others emphasize the role of the polysaccharide component of EPS in metal/bacteria interaction (Geesey and Jang, 1990). Despite all the research e ort focusing on the involvement of exopolymers in metal deterioration, very little (if any) information is available on the synthesis and chemical structure of bacterial EPS within biolms formed on metal surfaces and the types of EPS macromolecule that are involved in interacting with metal ions at the surface. E orts are therefore made to apply surface analysis techniques to gain a new insight into these phenomena (Gubner and Beech, 2000; Beech et al., 2000a). 3. The use of microscopy and surface science to study biolm/metal interactions The forms of deterioration which can be stimulated by the interaction of biolms with metals are numerous and a variety of techniques are employed to link biodeterioration to microbial activities at surfaces (Beech, 1999; Beech and Coutinho, 2003). Chemical spectroscopy at surfaces o ers qualitative and semi-quantitative information on the nature

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of the deterioration products that have accumulated on a metal surface. Spatially resolved surface chemistry obtained with spectroscopic instrumentation should then be related to spatially resolved microbiology at the same location, a procedure that requires image registration and alignment. The latter has only recently been applied to processes involving interaction of biolms with metals (Geesey et al., 1996; Pendyala et al., 1996; Suci et al., 1997; Beech et al., 1999b). 3.1. Microscopy The contributions of biolms to metal deterioration is usually qualitatively assessed using scanning electron microscopy (SEM), environmental scanning electron microscopy (ESEM) and atomic force microscopy (AFM). Re ected di erential interference contrast microscopy (DIC) can also be employed where the metal surface provides su cient re ective properties to resolve attached microorganisms (Surman et al., 1996). Recently, confocal scanning laser microscopy (CSLM) has also been used to provide three-dimensional information on the biolm/metal surface interaction (Walker et al., 1998). Microscopy provides information about the morphology of microbial cells and colonies, the distribution of microbial colonies on the surface, presence of EPS and the nature of corrosion products (crystalline or amorphous). It also reveals the type of attack by revealing changes in metal microstructure after removal of the biolm (Little et al., 1991; Wagner and Ray, 1994; Beech et al., 1996 and references therein). Recent review on the use of microscopy techniques for the study of biolms is provided by Beech et al. (2000b, 2002). 3.2. Surface analysis Surface chemical analysis allows determination of the chemical composition of corrosion products and microbiological deposits, and thus the opportunity to gain insight into the electrochemical reactions involved in the biolm-in uenced metal deterioration process. X-ray di raction (XRD) and energy dispersive X-ray analysis (EDS or EDX) have been widely used to obtain elemental information on corrosion products on metal surfaces (Marquis, 1989). XRD has limited application to biocorrosion, as it does not possess the high spatial resolution to detect localized attack. EDX assesses elemental abundance at high spatial resolution over a sampling depth of approximately 1 m. Depending on the thickness of the corrosion deposit, EDX may provide elemental information on not only corrosion products but also the underlying bulk material. Particle-induced X-ray emission spectroscopy (PIXE) is a form of nuclear microprobe (NM). In the NM a focused beam of MeV light ions (most commonly protons) is scanned across the sample surface, and the strength of the

relevant analytical signal is measured at each position in the scanned area to generate an image of the sample. PIXE analysis measures X-rays produced from the sample. Unlike EDX, PIXE uses MeV protons rather than keV electrons, thus generating less secondary radiation. This makes PIXE a more sensitive technique than EDX for imaging trace element distribution. Applications of PIXE in biology are numerous, ranging from studies of calcium and barium uptake in green algae, or investigation of elemental variation and metal accumulation in a germinating fungus, to elemental analysis of diseased human tissue (Breese et al., 1992.). Because of its resolving power and sensitivity, this technique is currently being applied to the study of metal distribution within the biolms developed by marine Pseudomonas sp. on stainless steel surfaces (Beech, unpublished). Auger electron spectroscopy (AES) has been used to investigate biocorrosion of condenser tubes (Chen et al., 1988). The technique gives elemental abundance only in the top few nanometers of the surface at very high spatial resolution. This allows mapping of corrosion products across a metal surface that has experienced localized attack. Surface contamination by organics and other material can interfere with detection of the underlying corrosion products when using such a surface-sensitive technique. Mossbauer spectroscopy can be applied to detect iron-containing compounds. It has been employed to detect green rust 2 among corrosion products of steel exposed to marine sediments containing SRB (Olowe et al., 1991, 1992). Since samples for XRD, EDS, AES and XPS analysis must be dehydrated as they are analysed in vacuo, some distortion and reorientation of surface-associated material can be anticipated. Care must also be taken to avoid changes in the oxidation state of some corrosion products during dehydration. Attenuated total re ectance Fourier transform infrared spectroscopy (ATR/FT-IR) has been applied to quantify the rate of dissolution of a thin metal lm deposited on an internal re ectance element exposed to either owing or stagnant aqueous media containing microorganisms and products of their metabolism. The method is based on the observation that water absorbance in the infrared region increases as the thickness of the thin lm decreases due to metal deterioration. Changes in the thickness of the metal lm corresponding to a few atomic layers can be detected and the measurements are obtained non-destructively in real time. This approach has been used to demonstrate the participation of a microbial biolm in the localized attack on copper lm (Bremer and Geesey, 1991); it revealed correlation between the onset of corrosion and the synthesis of EPS during biolm formation on the lm. ATR/FT-IR has also been employed to demonstrate that the exopolysaccharide produced by the marine bacterium Pseudoalteromonas (Pseudomonas) atlantica has an in uence on the corrosion of copper (Jolley et al., 1989). Most of the investigations of metal binding by either microbial biomass or EPS material employ techniques of

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analytical chemistry such as atomic absorption spectrometry, polarography and inductively coupled plasma spectrometry (ICP). Such methods often require a substantial quantity of sample (mg) in order to detect parts-per-billion (ppb) levels of metals, thus demanding large-scale purication of samples, which is often expensive and not always feasible. In contrast, modern techniques of surface science such as time-of- ight secondary ionization mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS) are well suited to study interaction of metals with biological material using a small (ng) sample size (Muddlman et al., 1994; Baty et al., 1996; Beech et al., 1998a). 3.2.1. X-ray photoelectron spectroscopy XPS or electron spectroscopy for chemical analysis (ESCA) provides direct chemical characterization of the samples surface layer (25 nm depth). XPS involves exposure of the sample to an X-ray beam in order to emit electrons. The kinetic energy of the emitted core electrons (photoelectrons) is analysed, and their binding energy, EB , determined according to the photoelectric e ect EB =h Ee , where h is the energy of the incident radiation (X-rays) and Ee is the measured kinetic energy of the photoelectron. The spectrum is obtained as a plot of the number of electrons per energy interval vs. their binding energy. The changes in the core level EB , or chemical shifts, are due to alterations in the electron density of the valence shell. A more highly oxidized species gives rise to a higher EB , while a more reduced species shows the opposite e ect. Each peak of the recorded spectrum is characteristic of a given energy level of an element which is in uenced by the chemical state of the element, mainly the electron density resulting from chemical bonds made with neighbouring atoms. Most elements have major photoelectron peaks below 1100 eV, and so a scan range from 1100 0 eV binding energy is usually su cient to identify all detectable elements (a survey scan). For the purpose of chemical state identication, for quantitative analysis of minor components and for peak deconvolution, detailed scans are obtained (high-resolution scans or multiplex scans). Quantitative data is obtained from peak heights or peak areas, and identication of chemical states is made from exact measurements of peak positions and separations, as well as from certain spectral features such as the shape of the peaks. XPS has been applied to investigations of microorganisms, proteins and microbial adhesins on non-metallic surfaces (Rouxhet et al., 1994 and references therein; Sosa et al., 1994). However, the technique has limited ability to provide detailed molecular information, particularly when atoms are in a wide variety of chemical states, as is the case for macromolecules such as carbohydrates and proteins. XPS is often employed to study bonding in metal compounds (Carver et al., 1972 and references therein); however, its application in the eld of biodeterioration

of metals has, so far, been limited to studies involving biocorrosion of copper (Geesey et al., 2000) and stainless steel (Pendyala et al., 1996; Beech et al., 1998b, 1999a). The technique has been employed to study the effect of bacterial biolms and exopolymers on the composition of passive layers formed on AISI 304 and 316 stainless steel. The thickness of the passive layers, the distribution of the alloying elements within the layers, and the level of layer hydration, changed signicantly when the biolm was present, compromising corrosion resistance of steel. 3.2.2. Time-of- ight secondary ionization mass spectrometry TOF-SIMS is a high vacuum (HV) technique employing a pulsed ion beam of, e.g., Cs or Ga (primary ions) to sputter the outermost surface of the sample (approximately 1 nm depth). The majority of the sputtered material is in the form of neutral atoms and molecules, but a certain percentage of this material is emitted as either positively or negatively charged ions. These secondary ions generated by a primary ion pulse on the target surface are electrostatically collected, accelerated into a ight tube, and their mass determined by monitoring the time at which the ions reach the mass detector. The exact masses are known with such accuracy that particles with the same nominal mass can be distinguished from one another. Mass resolution of 0.000X (four decimal points) atomic mass units (amu) and a mass range from 0 to 10 000 amu can be obtained. The data is displayed in a secondary ion signal intensity vs. amu format. The spectra produce unique patterns that vary according to the structure of the material. Although SIMS o ers greater chemical selectivity and surface sensitivity than XPS and has been widely used to study organic compounds (Siegbahn et al., 1967; Hercules, 1993), it has only recently been considered in applications such as characterization of microbial cells and bioadhesives. Techniques of XPS and TOF-SIMS helped to evaluate binding of Fe ions originating from mild steel by exopolymers recovered from cultures of two marine SRB species (Ind1 and Al1 strains) of the genus Desulfovibrio. Previous work demonstrated that under identical growth conditions the two SRB strains, although having similar growth rates and sulphate-reduction rates, di ered in their aggressiveness towards mild steel (Videla et al., 1996). Biolms formed by strain Ind1 caused greater deterioration of steel than those of strain Al1 (Cheung, 1995). One of the marked di erences between the Ind1 and Al1 was the yield and composition of their EPS (Zinkevich et al., 1996). Furthermore, the exopolymer of Ind1 was corrosive towards mild steel, causing increased Fe dissolution, which resulted in extensive pitting of steel (Beech et al., 1998c). The application of TOF-SIMS and XPS helped to reveal the di erences in Fe binding by the EPS produced by both strains, demonstrating that of the two, Ind1 EPS had much higher a nity for Fe uptake. In

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addition, XPS spectra strongly indicated that in each type of EPS, Fe was present in a di erent chemical environment within the organic matrix. This implied that di erent types of macromolecule were participating in the Fe binding process. Furthermore, iron released from the surface of steel as Fe2+ was associated with EPS as Fe3+ ion (Beech et al., 1999c). 4. Summary Microbial biolms form on all metal surfaces exposed to environmental conditions conducive for the formation of biolms. The attack on metal, often in a form of localized damage, occurs as a result of the activity of physiologically diverse microbial species within such biolms. These include the consumption of oxygen, production of acids, sulphides and enzymes that promote the establishment of localized chemical gradients at the metal surface. The presence of such chemical gradients facilitates the development of electrochemical cells, which in uence anodic and/or cathodic reactions, leading to the loss of metal from the discrete locations on the surface. Information obtained by techniques suited to provide both topological and chemical information at high level of resolution such as advanced microscopy combined with surface spectroscopy, should certainly aid in better understanding of the elusive surface biochemical reactions, which give rise to the electrochemistry driving the corrosion process. It is undeniable that the knowledge of fundamental mechanisms that govern interaction of biolms with metals in marine, fresh water and terrestrial environments is essential with respect to the avoidance of environmental problems caused by biofouling and biocorrosion. References
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