BIOFILM

A biofilm is an aggregate of microorganisms in which cells are stuck to each other and/or to a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm EPS, which is also refered to as "slime," is a polymeric jumble of DNA, proteins and polysaccharides. Biofilms may form on living or non-living surfaces, and represent a prevalent mode of microbial life in natural, industrial and hospital settings . The cells of a microorganism growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which by contrast, are single-cells that may float or swim in a liquid medium. Microbes form a biofilm in response to many factors, which may include cellular recognition of specific or non-specific attachment sites on a surface, nutritional cues, or in some cases, by exposure of planktonic cells to sub-inhibitory concentrations of antibiotics. When a cell switches to the biofilm mode of growth, it undergoes a phenotypic shift in behavior in which large suites of genes are differentially regulated

Formation

Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. These first colonists adhere to the surface initially through weak, reversible van der Waals forces. If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures such as pili.

The first colonists facilitate the arrival of other cells by providing more diverse adhesion sites and beginning to build the matrix that holds the biofilm together. Some species are not able to attach to a surface on their own but are often able to anchor themselves to the matrix or directly to earlier colonists. It is during this colonization that the cells are able to communicate via quorum sensing using such products as acetyl homoserine lactone (AHL). Bacteria have fascinating and diverse social lives. They display coordinated group behaviors regulated by quorum-sensing systems that detect the density of other bacteria around them. A key example of such group behavior is biofilm formation, in which communities of cells attach to a surface and envelope themselves in secreted polymers. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment. The final stage of biofilm formation is known as development, and is the stage in which the biofilm is established and may only change in shape and size. This development of biofilm allows for the cells to become more antibiotic resistant.

Development

There are five stages of biofilm development (see illustration at right).

1. initial attachment2. irreversible attachment

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3. maturation I4. maturation II5. dispersion

Five stages of biofilm development. Each stage of development in the diagram is paired with a photomicrograph of a developing P. aeruginosa biofilm. All photomicrographs are shown to same scale.

Quorum Sensing

Cell-to-cell signaling, known as quorum sensing, has been shown to play a role in biofilm formation. Modulating quorum sensing processes—for example, by enzymatic degradation of the signaling molecules—will prevent biofilm formation or possibly weaken established biofilms. Bacterial gene expression in some bacterial species may be regulated by quorum sensing, a cell density-dependent signaling system mediated by chemical autoinducer molecules produced by bacteria. The autoinducer molecules bind to the appropriate transcription regulator(s) when the bacterial population reaches the quorum level (that is, the signal concentration reaches a threshold concentration sufficient to facilitate binding to the receptor). Binding of the autoinducers is followed by activation or repression of target genes. Thus, quorum sensing allows bacteria to display a unified response that benefits the population. Bacterial quorum sensing systems enhance access to nutrients and more favorable environmental niches, and they enhance action against competing bacteria and environmental stresses. Examples of cellular processes modulated by quorum sensing are symbiosis, transfer of conjugative plasmids, sporulation, antimicrobial peptide synthesis, regulation of virulence, and biofilm formation.

Though planktonic cells secrete chemical signals (HSLs, for homoserine lactones), the low concentration of signal molecules does not change genetic expression. Biofilm cells are held together in dense populations, so the secreted HSLs attain higher concentrations. HSL molecules then re-cross the cell membranes and trigger changes in genetic activity.

Courtesy, MSU-CBE.

There are several different quorum sensing autoinducer systems in bacteria. For example, in Gram-negative bacteria, the quorum sensing system is dependent on homologues of the Vibrio fischeri LuxI-LuxR regulatory proteins (Miller and Bassler

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2001). Synthesized by the LuxI-like proteins, the autoinducer compounds are acylated homoserine lactones (AHLs), which are also known as autoinducer 1. Gram-positive bacteria also regulate a number of cellular processes through quorum sensing. However, the autoinducer compounds in Gram-positive bacteria are secreted after translation as modified peptides. Similar to AHLs, the concentration of peptides secreted to the external environment increases with the increase in bacterial populations.

A large number of bacteria have a common quorum sensing system mediated by autoinducer 2 (AI-2), which is found in both Gram-negative and Gram-positive bacteria.

Role of Quorum Sensing in Biofilm Formation

There is evidence that in some bacteria, biofilm formation is a carefully orchestrated process controlled by quorum sensing. While most research supports the role of quorum sensing in biofilm formation and in the resulting characteristics of the biofilm community, few studies indicate that quorum sensing does not affect the formation of biofilms.

Genetic Transfer within Biofilms

The frequency of gene transfer in planktonic cells is probably lower than that seen in cells found within biofilms. Interfering with Quorum Sensing

Countermeasures to cell-to-cell signaling have been explored in an attempt to reduce the ability of cells to form biofilms, attenuate virulence, and modulate other processes influenced by quorum sensing. Inhibition of quorum sensing can be accomplished in several ways, which include (1) enzymatic degradation of the signal molecule, (2) blocking signal generation, and (3) blocking signal reception.

Biofilm dispersal

Dispersal of cells from the biofilm colony is an essential stage of the biofilm lifecycle. Dispersal enables biofilms to spread and colonize new surfaces. Enzymes that degrade the biofilm extracellular matrix, such as dispersin B and deoxyribonuclease, may play a role in biofilm dispersal. Biofilm matrix degrading enzymes may be useful as anti-biofilm agents. Recent evidence has shown that a fatty acid messenger, cis-2-decenoic acid, is capable of inducing dispersion and inhibiting growth of biofilm colonies. Secreted by Pseudomonas aeruginosa, this compound induces dispersion in several species of bacteria and the yeast Candida albicans.

Biofilm development factorsSurface materialSurface material has little or no effect on biofilm development. Microbes will adhere to stainless steel or plastics

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with nearly equal enthusiasm.Surface areaSurface area is one primary factor influencing biofilm development. Plumbing systems, unlike most natural environments (lakes and rivers), offer a tremendous amount of surface area. RO membranes, DI resins, storage tanks, cartridge filters, and piping systems all provide surfaces suitable for bacterial attachment and growth.SmoothnessAlthough smoother surfaces delay the initial buildup of attached bacteria, smoothness does not significantlyaffect the total amount of biofilm on a surface after several days.Flow velocityHigh flow will not prevent bacteria attachment nor completely remove existing biofilm, but it will limit biofilm thickness. Regardless of the water velocity, it flows slowest in the zone adjacent to pipe surfaces. Even when water flow in the center of the pipe is turbulent, the flow velocity falls to zero at the pipe wall. The distance out from the pipe wall in which the flow rate is not turbulent is called the laminar sublayer. This distance can be considered equal to the maximum biofilm thickness.Limited nutrientsLike other living creatures, bacteria require certain nutrients for growth and reproduction. Limiting these nutrients will limit bacteria growth, but even minute amounts of organic matter will support many bacteria. Theoretically, just 1 ppb of organic matter in water is enough to produce 9,500 bacteria/ml! Current technology cannot reduce nutrient levels completely, so total control of bacteria is not achievable by simply controlling nutrients. Similarly, very small quantities of oxygen will adequately support luxurious bacterial growth. Although it won’t eliminate bacteria, nutrient-poor reverse osmosis water will support less biofilm than regular tap water supplies.

Properties-key characteristics of biofilms

1. Biofilms are complex, dynamic community structures

2. Genetic expression is different in biofilm bacteria when compared toplanktonic bacteria

3. Biofilm cells can coordinate behavior via intercellular "communication" using biochemical signaling molecules

4. Biofilms make bacteria less susceptible to antimicrobial agents

Biofilms are usually found on solid substrates submerged in or exposed to some aqueous solution, although they can form as floating mats on liquid surfaces and also on the surface of leaves, particularly in high humidity climates. Given sufficient resources for growth, a biofilm will quickly grow to be macroscopic. Biofilms can

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contain many different types of microorganism, e.g. bacteria, archaea, protozoa, fungi and algae; each group performing specialized metabolic functions. However, some organisms will form monospecies films under certain conditions.

Researchers from the Helmholtz Center for Infection Research have investigated the strategies used by biofilms. They discovered that biofilm bacteria apply chemical weapons in order to defend themselves against disinfectants and antibiotics, phagocytes and our immune system.

The lead researcher, Dr. Carsten Matz, began a serious investigation in order to find why phagocytes cannot annihilate the biofilm bacteria. He analyzed the marine bacteria, which defend themselves against the amoebae, the behavior of which copies the behavior of phagocytes. The amoebae behave in the sea just like the immune cells in human body: they search for and feed on the bacteria. When bacteria are alone and separated in the water, they become an easy catch for the attackers. However, when they attach to a surface and join other bacteria, the amoebae cannot assault them.

The researcher stated that biofilms may be seen as a source of new bioactive agents. When bacteria are organized in biofilms, they produce effective substances which individual bacteria are unable to produce alone.

Extracellular matrix

The biofilm is held together and protected by a matrix of excreted polymeric compounds called EPS. EPS is an abbreviation for either extracellular polymeric substance or exopolysaccharide. This matrix protects the cells within it and facilitates communication among them through biochemical signals. Some biofilms have been found to contain water channels that help distribute nutrients and signalling molecules. This matrix is strong enough that under certain conditions, biofilms can become fossilized.

Bacteria living in a biofilm usually have significantly different properties from free-floating bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. In some cases antibiotic resistance can be increased 1000 fold.

The concept that biofilms are more resistant to antimicrobials is not completely accurate. For instance the biofilm form of Pseudomonas aeruginosa has no greater resistance to antimicrobials, when compared to stationary phase planktonic cells. Although, when the biofilm is compared to logarithmic phase planktonic cells, the biofilm does have greater resistance to antimicrobials. This resistance to antibiotics in both stationary phase cells and biofilms may be due to the presence of persister cells.

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Examples

Biofilm in Yellowstone National Park. Longest raised mat area is about half a meter long.

Biofilms are ubiquitous. Nearly every species of microorganism, not only bacteria and archaea, have mechanisms by which they can adhere to surfaces and to each other.

Biofilms can be found on rocks and pebbles at the bottom of most streams or rivers and often form on the surface of stagnant pools of water. In fact, biofilms are important components of food chains in rivers and streams and are grazed by the aquatic invertebrates upon which many fish feed.

Biofilms grow in hot, acidic pools in Yellowstone National Park (USA) and on glaciers in Antarctica.

Biofilms can grow in showers very easily since they provide a moist and warm environment for the biofilm to thrive.

Biofilms can develop on the interiors of pipes leading to clogging and corrosion, especially in engineered systems. Biofilms on floors and counters can make sanitation difficult in food preparation areas. Biofilms in cooling water systems are known to reduce heat transfer. Biofilms in marine Systems, such as pipelines of the offshore oil and gas industry, can lead to substantial corrosion problems. Corrosion is mainly due to abiotic factors, however, at least 20% is caused by microorganisms (i.e., microbially influenced corrosion) that are attached to the metal subsurface.

Bacterial adhesion to boat hulls serves as the foundation for biofouling of seagoing vessels. Once a film of bacteria forms, it is easier for other marine organisms such as barnacles to attach. Such fouling can inhibit vessel speed by up to 20%, making voyages longer and requiring additional fuel. Time in dry dock for refitting and repainting reduces the productivity of shipping assets, and the useful life of ships is also reduced due to corrosion and mechanical removal (scraping) of marine organisms from ships’ hulls.

Biofilms can also be harnessed for constructive purposes. For example, many sewage treatment plants include a treatment stage in which waste water passes over biofilms grown on filters, which extract and digest organic compounds. In such biofilms, bacteria are mainly responsible for removal of organic matter (BOD); whilst protozoa and rotifers are mainly responsible for removal of suspended solids (SS), including pathogens and other microorganisms. Slow sand filters rely on biofilm development in the same way to filter surface water from lake, spring or river sources for drinking purposes. What we regard as clean water is a waste material to these

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microcellular organisms since they are unable to extract any further nutrition from the purified water.

Biofilms can help eliminate petroleum oil from contaminated oceans or marine systems. The oil is eliminated by the hydrocarbon-degrading activities of microbial communities, in particular by a remarkable recently discovered group of specialists, the so-called hydrocarbonoclastic bacteria (HCB).

Biofilms are also present on the teeth of most animals as dental plaque, where they may become responsible for tooth decay and gum disease.

Biofilms are found on the surface of and inside plants. They can both contribute to crop disease or, as in the case of nitrogen fixing Rhizobium on roots, exist symbiotically with the plant. Examples of crop diseases related to biofilms include Citrus Canker, Pierce's Disease of grapes, and Bacterial Spot of plants such as peppers and tomatoes.

Biofilms and infectious diseases

Biofilms have been found to be involved in a wide variety of microbial infections in the body, by one estimate 80% of all infections. Infectious processes in which biofilms have been implicated include common problems such as urinary tract infections, catheter infections, middle-ear infections, formation of dental plaque, gingivitis, coating contact lenses, and less common but more lethal processes such as endocarditis, infections in cystic fibrosis, and infections of permanent indwelling devices such as joint prostheses and heart valves.. More recently it has been noted that bacterial biofilms may impair cutaneous wound healing and reduce topical antibacterial efficiancy in healing or treating infected skin wounds

It has recently been shown that biofilms are present on the removed tissue of 80% of patients undergoing surgery for chronic sinusitis. The patients with biofilms were shown to have been denuded of cilia and goblet cells, unlike the controls without biofilms who had normal cilia and goblet cell morphology. Biofilms were also found on samples from two of 10 healthy controls mentioned. The species of bacteria from interoperative cultures did not correspond to the bacteria species in the biofilm on the respective patient's tissue. In other words, the cultures were negative though the bacteria were present.

Biofilms can also be formed on the inert surfaces of implanted devices such as catheters, prosthetic cardiac valves and intrauterine devices.

New staining techniques are being developed to differentiate bacterial cells growing in living animals, e.g. from tissues with allergy-inflammations .

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Pseudomonas aeruginosa biofilms

The achievements of medical care in industrialised societies are markedly impaired due to chronic opportunistic infections that have become increasingly apparent in immunocompromised patients and the aging population. Chronic infections remain a major challenge for the medical profession and are of great economic relevance because traditional antibiotic therapy is usually not sufficient to eradicate these infections. One major reason for persistence seems to be the capability of the bacteria to grow within biofilms that protects them from adverse environmental factors. Pseudomonas aeruginosa is not only an important opportunistic pathogen and causative agent of emerging nosocomial infections but can also be considered a model organism for the study of diverse bacterial mechanisms that contribute to bacterial persistence. In this context the elucidation of the molecular mechanisms responsible for the switch from planctonic growth to a biofilm phenotype and the role of inter-bacterial communication in persistent disease should provide new insights in P. aeruginosa pathogenicity, contribute to a better clinical management of chronically infected patients and should lead to the identification of new drug targets for the development of alternative anti-infective treatment strategies.

Dental plaque

Dental plaque is the material that adheres to the teeth and consists of bacterial cells (mainly Streptococcus mutans and Streptococcus sanguis), salivary polymers and bacterial extracellular products. Plaque is a biofilm on the surfaces of the teeth. This accumulation of microorganisms subject the teeth and gingival tissues to high concentrations of bacterial metabolites which results in dental disease.

Legionellosis

Legionella bacteria are known to grow under certain conditions in biofilms, in which they are protected against disinfectants. Workers in cooling towers, persons working in air conditioned rooms and people taking a shower are exposed to Legionella by inhalation when the systems are not well designed, constructed, or maintained.

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BiofoulingBiofouling or biological fouling is the undesirable accumulation of microorganisms, plants, algae, and/or animals on wetted structures.ImpactBiofouling is especially economically significant on ships' hulls where high levels of fouling can reduce the performance of the vessel and increase its fuel requirements. Biofouling is also found in almost all circumstances where water based liquids are in contact with other materials. Industrially important examples include membrane systems, such as membrane bioreactors and reverse osmosis spiral wound membranes cooling water cycles of large industrial equipments and power stations. Biofouling can also

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occur in oil pipelines carrying oils with entrained water especially those carrying used oils, cutting oils, soluble oil or hydraulic oils.Anti-foulingAnti-fouling is the process of removing the accumulation, or preventing its accumulation. In industrial processes, bio-dispersants can be used to control biofouling. In less controlled environments, anti-fouling coatings which contain biocides or non-toxic coatings which prevent organisms from attaching can be used. [BiocidesBiocides are chemical substances that can deter or kill the microorganisms responsible for biofouling. Biocides are incorporated into an anti-fouling surface coating, typically physical adsorption or through chemical modification of the surface. Biofouling occurs on surfaces after formation of a biofilm. The biofilm creates a surface onto which successively larger microorganisms can attach. In marine environments this usually concludes with barnacle attachment. The biocides often target the microorganisms which create the initial biofilm, typically bacteria. Once dead, they are unable to spread and can detach. Other biocides are toxic to larger organisms in biofouling, such as the fungi and algae. The most commonly used biocide, and anti-fouling agent, is the tributyltin moiety (TBT). It is toxic to both microorganisms and larger aquatic organisms. It is estimated that TBT derived anti-fouling coatings cover 70% of the world's vessels. The prevalence of TBT and other tin based anti-fouling coatings on marine vessels is a current environmental problem. TBT has been shown to cause harm to many marine organisms, specifically oysters and mollusks. Extremely low concentrations of tributyltin moiety (TBT) causes defective shell growth in the oyster Crassostrea gigas (at a concentration of 20 ng/l) and development of male characteristics in female genitalia in the dog whelk Nucella lapillus (where gonocharacteristic change is initiated at 1 ng/l)The international maritime community has recognized this problem and there is planned phase out of tin based coatings, including a ban on newly built vessels. This phase out of toxic biocides in marine coatings is a severe problem for the shipping industry; it presents a major challenge for the producers of coatings to develop alternative technologies. Safer methods of biofouling control are actively researched.Copper compounds have successfully been used in paints and continue to be used as metal sheeting (for example Muntz metal which was specifically made for this purpose), though there is still debate as to the safety of copper.Non-toxic Coatings

Non-toxic anti-fouling coatings prevent any attachment of microorganisms thus negating the use of biocides. Further, these coatings are usually based on polymers and researchers are able to design self-healing coatings.There are two classes of non-toxic anti-fouling coatings. The most common class relies on low friction and low surface energies. This results in hydrophobic surfaces. These coatings create a smooth surface which can prevent attachment of larger microorganisms. For example, Fluoropolymers and silicone coatings are commonly used. These coatings are ecologically inert but have problems with mechanical strength and long term stability. Specifically, after days biofilms (slime) can coat the surfaces which buries the chemical activity and allows microorganisms to attach.The second class of non-toxic anti-fouling coatings are hydrophilic coatings. They rely on high amounts of hydration in order to increase the energetic penalty of removing water for proteins and microorganisms to attach. The most common example of these coatings are based on highly hydrated zwitterions, such as

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glycine betaine and sulfobetaine. These coatings are also low friction but are superior to hydrophobic surfaces because they prevent bacteria attachment, preventing biofilm formation. These coatings are not yet commercially available and are being designed as part of a larger effort by the Office of Naval Research to develop environmentally safe biomimetic ship coatingsTypesBiofouling is divided into microfouling — biofilm formation and bacterial adhesion — and macrofouling — attachment of larger organisms, of which the main culprits are barnacles, mussels, polychaete worms, bryozoans, and seaweed. Together, these organisms form a fouling community.Individually small, accumulated biofoulers can form enormous masses that severely diminish ships' maneuverability and carrying capacity. Fouling causes huge material and economic costs in maintenance of mariculture, shipping industries, naval vessels, and seawater pipelines. Governments and industry spend more than US$ 5.7 billion annually to prevent and control marine biofouling.

MICROBIOLOGICALLY INFLUENCED CORROSION (MIC)

Bio-corrosion is one of the direct consequences of microbial film formation on the surface of water distribution pipes. It is one of the major contributors to water quality and environmental contamination. Bio-corrosion causes severe economic losses in water distribution systems. Corrosion of iron and steel pipes can occur as a result of variety of chemical reactions that establish an electrochemical gradient, leading to loss of metal from the pipe due to electrolysis. The physical presence of microbial cells on a metal surface, as well as their metabolic activities, can cause Microbiologically Influenced Corrosion (MIC) or bio-corrosion, The forms of corrosion caused by bacteria are not unique. Bio-corrosion results in pitting, crevice corrosion, selective de-alloying, stress corrosion cracking, and under-deposit corrosion. Biofilms provide the localized environmental conditions (e.g. decreased pH; differential oxygen cells) for initiating or propagating corrosion activities. The metabolic capabilities of microorganisms are being harnessed to improve the recovery of metals and petroleum from the environment. Sulphur- oxidising thiobacilli are commercially employed in bioleaching operations for the recovery of copper and uranium. Microorganisms play both beneficial and detrimental roles in the mining and mineral processing of metals.

Classification of Microorganisms in Corrosion

Microorganisms can be categorized according to oxygen tolerance:

Strict (or obligate) anaerobes, which will not function in the presence of oxygen.

Aerobes, which require oxygen in their metabolism.

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Facultative anaerobes, which can function either in the absence or presence of oxygen.

Microaerophiles, which use oxygen but prefer low levels.

Another way of classifying organisms is according to their metabolism:

o The compounds or nutrients from which they obtain their carbon for growth and reproduction.

o The chemistry by which they obtain energy or perform respiration.

o The elements they accumulate as a result of these processes.

A third way of classifying bacteria is by shape. These shapes are predictable when organisms are grown under well defined laboratory conditions. In natural environments, however, shape is often determined by growth conditions rather than pedigree.

Examples of shapes are:

"Vibrio," for comma shaped cells. "Bacillus," for rod shaped cells.

"Coccus," for round cells.

"Myces," for fungi like cells.

CAUSES OF CORROSION Corrosion is caused by anyone or more of the following mechanisms. Cathodic depolarization, whereby the cathodic rate limiting step is accelerated by micro-biological action.Formation of occluded surface cells, whereby microorganisms form "patchy" surface colonies. Sticky polymers attract and aggregate biological and non-biological species to produce crevices and concentration cells, the basis for accelerated attack.Fixing of anodic reaction sites, whereby microbiological surface colonies lead to the formation of corrosion pits, driven by microbial activity and associated with the location of these colonies.Underdeposit acid attack, whereby corrosive attack is accelerated by acidic final products of the MIC "community metabolism", principally short-chain fatty acids.Oxygen Influencing Corrosion Non-uniform (patchy) colonies of biofilm result in the formation of differential aeration cells where areas under respiring colonies are depleted of oxygen relative to surrounding non-colonised areas. Having different oxygen concentrations at two locations on a metal causes a difference in electrical potential and consequently corrosion currents. Under aerobic conditions, the areas under the respiring colonies become anodic and the surrounding areas become cathodic. Oxygen depletion at the surface of stainless steel can destroy

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the protective passive film. Since stainless steels rely on a stable oxide film to provide corrosion resistance, corrosion occurs when the oxide film is damaged or oxygen is kept from the metal surface by microorganisms in a biofilm. MIC-associated bacteria are grouped on the basis of their mode of attack on ferrous and non-ferrous metals. The most common MIC groups include sulphate-reducing, iron-oxidising, acid-producing, sulphur- oxidising and nitrate-reducing bacteria. Acid production, hydrogen sulphide generation, tubercle formation and the subsequent development of differential aeration cells can lead to deterioration and failure of mild steel, copper, stainless steel, and other ferrous and non-ferrous metals used as construction materials. Oxygen depletion at the surface also provides a condition for anaerobic organisms like sulphate-reducing bacteria (SRB) to grow. This group of bacteria is one of the most frequent causes for bio-corrosion. The metabolic activities of anaerobic sulphate-reducing bacteria result in the formation of iron hydroxides which are corrosion products. Sulphur bacteria obtain energy by reducing or oxidising inorganic sulphur compounds that are present in feed waters. The bacteria most often associated with MIC in water systems belong to the anaerobic sulphate- reducing (SRB) group, which includes Desulfovibrio desulphuricans.Direct attack of ferrous and non-ferrous metals by their hydrogen sulphide metabolic by-product is a significant problem in many industries. Reduction of sulphate to H2S (addition of electrons) results in cathodic depolarisation. Sulphate reducing bacteria accelerate the electrolytic corrosion process by promoting depolarisation of the anodic (+) and cathodic (-) surface during the anaerobic corrosive reaction. H2S reacts with ferrous ion to convert it to ferrous sulphide - effect of this reaction is anodic depolarisation. Additionally, a very active hydrogenase associated with Desulfovibrio species removes the protective layer of hydrogen that surrounds submerged iron pipes, exposing the underlying iron to corrosive attack. Aerobic bacteria near the outer surface of the biofilm consume oxygen and create a suitable habitat for the sulphate-reducing bacteria at the metal surface. Sulphur oxidising bacteria, such as Thiobacillus species, are aerobic microorganisms that can produce sulphuric acid. This group of organisms often lives in close association with SRB. SRBs can grow in water trapped in stagnant areas, such as dead legs of piping. Symptoms of SRB-influenced corrosion are hydrogen sulphide (rotten egg) odour, blackening of waters, and black deposits. The black deposit is primarily iron sulphide.

Nitrate Reducing Bacteria Nitrate reducing bacteria (NRB) can utilise nitrogen containing organic compounds in feed waters, producing significant quantities of ammonia. In addition to odour problems, ammonia production is associated with stress corrosion cracking of copper alloys. Nitrite based corrosion inhibitors may be a source of nitrogen for this group of MIC bacteria. Acid-producing Bacteria

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Bacteria can produce aggressive metabolites such as organic or inorganic acids. For example, Thiobacillus thiooxidans produces sulphuric acid and Clostridium aceticum produces acetic acid. Acids produced by bacteria accelerate corrosion by dissolving oxides (the passive film) from the metal surface and accelerating the cathodic reaction rate. Hydrogen-producing Bacteria Many microorganisms produce hydrogen gas as a product of carbohydrate fermentation. Hydrogen gas can diffuse into metals and cause hydrogen embrittlement.Iron Bacteria Iron-oxidising bacteria obtain energy through oxidation of reduced ferrous species to the ferric state. Iron oxidation by bacterial species in this group usually results in the formation of ferric hydroxide, Fe(OH)3' which is precipitated in their slime. Iron-oxidising bacteria, such as Gallionella, Sphaerotilus, Leptothrix, and Crenothrix are aerobic and filamentous bacteria which oxidise iron from a soluble ferrous (Fe2+) form to an insoluble ferric (Fe3+) form. The dissolved ferrous iron could be from either the incoming water supply or the metal surface. The ferric iron these bacteria produce can attract chloride ions and produce ferric chloride deposits which can attack austenitic stainless steel. For iron bacteria on austenitic stainless steel, the deposits are typically brown or red-brown mounds. Anaerobic Microbial Corrosion This type of corrosion of cast iron causes graphitisation, a process in which a pipe loses much of its iron thereby becoming soft and brittle. Steel and aluminium pipes are also subjected to anaerobic corrosion. Anaerobic microbial corrosion of steel results in localised pitting which sometimes causes perforation of the pipe. Pitting Corrosion Pitting corrosion is a localised form of corrosion; the bulk of the surface remains unattacked. Pitting is often found in situations where resistance against general corrosion is conferred by passive surface films. Localised pitting attack is found where these passive films have broken down. Pitting attack induced by microbial activity, such as sulphate reducing bacteria (SRB) also deserves special mention. Within the pits, an extremely corrosive micro-environment tends to be established, which may bear little resemblance to the bulk corrosive environment. For example, in the pitting of stainless steels in chloride-containing water, a micro-environment essentially representing hydrochloric acid may be established within the pits. The pH within the pits tends to be lowered significantly, together with an increase in chloride ion concentration, as a result of the electrochemical pitting mechanism reactions in such systems. The detection and meaningful monitoring of pitting corrosion usually represents a major challenge. Pitting failures can occur unexpectedly, and with minimal overall metal loss. Furthermore, the pits may be hidden cruder surface deposits, and/ or corrosion products. Monitoring pitting corrosion can be further complicated by a distinction between the initiation and propagation phases of pitting processes. The highly sensitive electrochemical noise technique may provide early warming of imminent damage by characteristic signals in the pit initiation phrase.

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Pipe failures resulting from microbiologically influenced corrosion (NAIC) have been widely recognized in petrochemical, gas and nuclear power industries, but only recently has this phenomenon been associated with failures in fire protection systems (FPS). MIC results in mechanical blockages of piping and sprinkler heads, as well as through-wall penetration of ferrous and non-ferrous metals. FPS are designed for the life of the structures in which they reside; however, reports of new systems developing NAIC- associated through-wall leaks within months of installation are becoming more prevalent. Pitting corrosion occurring under deposits in FPS can be initiated or propagated by these microbial activities. Through-wall penetration of can-bon steel and copper has been reported within months after a new pipeline has been brought into service. This extensive tuberculation can cause occlusion of pipelines, sometimes completely blocking flow in six-inch diameter pipelines. These problems become more critical as pipe diameter decreases posing a potential threat to proper sprinkler head mechanical functioning.

In addition, FPS make-up waters are typically stagnant, soft (relatively' low in hardness), acidic and devoid of antimicrobial agents such as the sodium hypochlorite that is used for microbial control in potable waters. These characteristics predispose FPS to biological fouling and MIC. Regulatory requirements that dictate periodic testing can also contribute to development of MIC in FPS when make-up waters are replaced with oxygenated and nutrient-rich waters. MIC-associated microorganisms can use these nutrients as growth sources, leading to fouling of affected systems. The most serious consequence of MIC in FPS is mechanical blockage of piping and sprinkler heads. MIC-associated organisms can attach to the metallic surfaces of FPS, forming corrosion deposits that are termed tubercles Tubercles can completely occlude pipes, and more significantly, these deposits can break off and block sprinkler head flow channels. Localised pitting-type attack can also occur underneath tubercles, resulting in through-wall penetration. The resulting acid production, hydrogen sulphide generation and development of differential aeration cells can lead to the loss of essential metallic properties of mild steel, copper, stainless steel and other ferrous and non-ferrous metals. Protection from Corrosion Pipes can be protected from corrosion by following the procedures given

By increasing the pH to 9.5 pipelines can be protected against the action of sulphate reducing microbes.

Buried pipes can be coated to prevent contact between metal surface, water and soil microbes.

Electric currents can be applied to the pipe to preclude corrosion processes. Various bacterial inhibitors can be employed to control microbial corrosion.

For example, alkyl substituted amine and quaternary ammonium compounds are toxic to microbes. Various bacterial inhibitors can be employed to control microbial corrosion. For instance, alkyl substituted amine and quaternary ammonium compounds are toxic to many bacteria like Desulfovibrio sp.a bacterium of major importance in the corrosion process.

MICROBIALLY INDUCED CONCRETE CORROSION

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Microbially Induced Concrete Corrosion is an important biological or chemical phenomenon that is having extreme effects on the infrastructure of our cities. We are conducting research that is designed to provide more insight into the biochemical and chemical reactions occurring, the microbial ecology of concrete corrosion as well as to allow us to develop process based models of concrete corrosion and develop control mechanisms to prevent or con~~l concrete corrosion. It is found that aerobic heterotrophs and neutrophilic and acidophilic sulphur oxidizers are the dominant microbes. There are ~o SRB, anaerobic heterotrophs, nitrate reducing bacteria, and ammonia oxidising bacteria present in-some of the samples. The corrosion of concrete pipes is a consequence of a cyclic process caused by microbial sulphur metabolism. Two types of sulphur metabolizers are involved in the cycle of sulphur in the environment. One is an anaerobic process in which H2S is produced by anaerobic bacteria; the other is an aerobic process in which the H2S is oxidized to elemental sulphur (S) or sulphuric acid (H2S04). This cyclic process exists as a natural method for the cycling of sulphur compounds in the environment and may also exist in sewage collection systems. During the transport of raw sewage from the top of the sewage collection system to the treatment plants, the organism~ in the sewage start to degrade the abundant organic compounds present in the raw sewage. This .often results in a depletion of 02 from the sewage. This results in the creation of anaerobic or anoxic conditions which allow the growth of sulphate reducing bacteria (SRB) which grow only in the absence of oxygen and obtain energy by utilizing small organic compounds or H2 as energy sources and transferring the electrons produced to sulphate, thus reducing it to sulphide. The sulphide produced eventually partitions into HS- and H2S. The H2S is a gas and evolves into the headspace of the sewer pipes, reaching the crown of the pipe. The crown of the pipe is exposed to an aerobic environment which supports the growth of sulphur oxidising bacteria. The sulphur oxidising bacteria grow on and within the concrete of the pipe, oxidising the H2S present and producing H2S04. The sulphuric acid dissolves the CaOH and CaC03 in the cement binder, thus causing corrosion of the concrete pipes. There have been only a few species of thiobacilli (the largest genera of organisms that oxidise H2S to H2SO4 ) described by researchers. These are T.novellus, T. neopolitanus, T. intermedius, and T. thiooxidans. The first four organisms are important for establishing the conditions necessary for corrosion to occur, while the acid loving T. thiooxidans grows in conditions of very low pH and produces H2S04 in copious amounts, thus lowering the pH even more.

Many industries are affected by MIC:

Chemical processing industries: stainless steel tanks, pipelines and flanged joints, particularly in welded areas after hydrotesting with natural river or well waters.

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Nuclear power generation: carbon and stainless steel piping and tanks; copper-nickel, stainless, brass and aluminum bronze cooling water pipes and tubes, especially during construction, hydrotest, and outage periods.

Onshore and offshore oil and gas industries: mothballed and waterflood systems; oil and gas handling systems, particularly in those environments soured by sulfate reducing bacteria (SRB)-produced sulfides

Underground pipeline industry: water-saturated clay-type soils of near-neutral pH with decaying organic matter and a source of SRB.

Water treatment industry: heat exchangers and piping

Sewage handling and treatment industry: concrete and reinforced concrete structures

Highway maintenance industry: culvert piping

Aviation industry: aluminum integral wing tanks and fuel storage tanks

Metal working industry: increased wear from breakdown of machining oils and emulsions

Marine and shipping industry: accelerated damage to ships and barges

Methods of Detection of MIC Populations:

1. Direct Inspection

Direct inspection is best suited to enumeration of planktonic organisms suspended in relatively clean water. In liquid suspensions, cell densities greater than 107 cells cm-3 cause the sample to appear turbid. Quantitative enumerations using a phase contrast microscopy can be done quickly using a counting chamber which holds a known volume of fluid in a thin layer.

2. Growth Assays

The most common way to assess microbial populations in industrial samples is through growth tests using commercially available growth media for groups of organisms most commonly associated with industrial problems. These are packaged in a convenient form suitable for use in the field. Serial dilutions of suspended samples are grown on solid agar or liquid media.

3. Activity Assays

Whole Cell

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Approaches based on the conversion of a radioisotopically labelled substrate can be used to assess the potential activity of microbial populations in field samples.

4. Enzyme Based Assays

An increasingly popular approach is the use of commercial kits to assay the presence of enzymes associated with microorganisms suspected to cause problems.

5. Metabolites

An overall assessment of microbial activity can be obtained by measuring the amount of adenosine triphosphate (ATP) in field samples.

6. Cell Components

Biomass can be generally quantified by assays for protein, lipopolysaccharide or other common cell constituents but the information gained is of limited value.

7. Fatty Acid Profiles

Analyzing fatty acid methyl esters derived from cellular lipids can fingerprint organisms rapidly. Provided pertinent profiles are known, organisms in industrial and environmental samples can be identified with confidence.

8. Nucleic Acid Based Methods

In principle, probes could be developed to detect all possible sulfate-reducers but application of such a battery of probes becomes daunting where large numbers of field samples are to be analyzed.

Prevention Microbiologically influenced corrosion, or microbial corrosion or biological corrosion can be prevented through a number of methods:

Regular mechanical cleaning if possible Chemical treatment with biocides to control the population of bacteria Complete drainage and dry-storage

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