CHEMOLITHOTROPHIC BACTERIA viewSeveral groups of soil and aquatic bacteria are able to use inorganic compounds or ions (ammonium, nitrite, sulphide, thiosulphate, sulphite, and ferrous

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CHEMOLITHOTROPHIC BACTERIA

Several groups of soil and aquatic bacteria are able to use inorganic compounds or ions (ammonium, nitrite, sulphide, thiosulphate, sulphite, and ferrous' iron) as well as elemental sulphur, hydrogen, or carbon monoxide as electron or hydrogen donors, gaining energy and reducing equivalents for synthetic processes by their oxidation. Energy is usually obtained by respiration with oxygen as the final electron acceptor. Only a few 'specialists' in this group can grow with nitrate, nitrite, or nitrous oxide as hydrogen acceptors, i.e., anaerobic respiration. This mode of life with inorganic hydrogen donors is called chemolithotrophy. Most of the bacteria belonging to this metabolic type grow with carbon dioxide as their sole, or major, source of carbon for cell synthesis. They are therefore also autotrophs (chemolithoautotrophs). All the chemolithotrophic bacteria so far examined assimilate their carbon from co2by fixation via the ribulose-bisphosphate cycle; the mechanism of this CO2fixation will therefore be discussed later in this chapter. In this respect some chemolithotrophic bacteria are obligate; others have the alternative of growing as chemo-organoheterotrophs, i.e., they are facultatively chemolithotrophic. Many ofthe chemolithoautotrophs are highly specialised and occupy a decided monopoly position. The oxidation of ammonium, nitrite, and inorganic sulphur compounds in nature is due in the first place to the activities of the nitrifying and sulphur-oxidising bacteria.

AMMONIUM AND NITRITE OXIDATION: NITRIFICATION

In the course of aerobic or anaerobic degradation of nitrogen-containing substances, the nitrogen is liberated in the form of ammonium.

The conversion of' ammonium to nitrite, i.e., nitrification, is carried out in soil by nitrifying bacteria. There is no known bacterium that converts ammonium directly to nitrate;two kinds of bacteria. are always involved jn this oxidation: the ammomum oxidisers form mtrite and the nitrite oxidisers produce nitrate. The best-known species are Nritrosomonas europaea and Nitrobacter winogradskyi. According to more' recent investigations, however,

Nitrosolobus species, rather· than Nitrosomonas, are the amammonium-oxidising organisms in agricultural soils.

Ammonium-oxidising (nitroso-) NH4

++ 1½O2 →N02-+ 2H++ H20

Nitrosomonas europaea Nitrosococcus oceanus Nitrosospira briensis Nitrosolobus multiformis

Nitrite-oxidising (nitro-) N02

-+½O2→NO3-

Nitrobacter winogradskyi Nitrobacter agilis Nitrospira gracilis Nitrococcus mobilis

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Reaction pathway in the oxidation of ammonium:The following inter- mediate steps are most probably involved in the oxidation of ammonium:

NH3→ NH20H→ (NOH)→ N02→N03- The first step is endergonic and is catalysed by an ammonium mono- oxygenase; the oxygen atom ofNH20H comes from molecular oxygen. The second step is catalysed by a hydroxylamine oxidoreductase. In the oxidation of nitrite the electrons are transferred to a cytochrome a1 . Only the oxidation steps from hydroxylamine to nitrite and from nitrite to nitrate yield utilizable energy.

The role of nitrification in soils. The ammonium ions liberated by the mineralization of nitrogen-containing substances are rapidly oxidized in well-aerated soils. The conversion of a cation to an anion effects an acidification of the soil and hence leads to an increased solubility of minerals (potassium, calcium, magnesium, and phosphates). The nitrifying microflora was therefore regarded' as a significant factor in soil fertility. This view has undergone changes, however. Ammonium ions are much more tenaciously retained by soils

than nitrate, especially by adsorption to clay minerals and more or less tight binding to, and in, humus components. Nitrate, on the other hand, is easily leached out. Thus, there have been attempts recently to suppress nitrification in areas of agricultural use, and to search for agents that specifically inhibit the growth of nitrifying bacteria. Such substances, it is thought, could serve as nitrogen-conserving agents in agricultural practice. On the other hand, growth and metabolism of the autotrophic nitrifying bacteria have a narrow pH optimum between 7 and 8, The pH range of the complete nitrification of ammonium to nitrate is therefore quite narrow,since free ammonia (in alkaline soils) and nitrous acid (in the acid range) are toxic for Nitrobacter. The concentrations of free NH, and free HN02are known to be pH dependent. The nitrifying bacteria are also indirectly involved in the destruction of limestone and cement (e.g., in motor ways, buildings) because they oxidise any ammonium derived from the atmosphere or animal excreta to nitric acid. Reverse electron transport and cell yields. The oxidation of ammonium, nitrite, sulphur compounds, or iron by autotrophic bacteria is energetically very unfavourable. Their substrates have a very positive redox potential. The normal potential E' 0 for NH4 + /NH20H is + 899m V; for N03-/N02-,+420mV; for N02-/NH20H,+66mV; for Fe3+/ Fe2+,+770mV; in comparison to -320mV for NAO+/NAOH2. Therefore, the oxidation of these substrates cannot be directly coupled to the reduction of NAD+.But reduced NAD+is essential for the reduction of CO2in the ribulose-bisphosphate cycle. There is evidence that the electrons from the oxidation of inorganic substrates enter the respiratory chain at the level of cytochrome c or a; the energy yield is correspondingly small because only one phosphorylation step (of the respiratory chain) can be utilised. Moreover, a part of this energy is then used to drive electrons entering at the cytochrome level in the reverse direction of the respiratory chain to the level of the pyridine nucleotides which are thus reduced. This reverse electron transport is an essential mechanism for these bacteria to gain reducing equivalents that are required for synthetic processes.The very small amounts of energy that can be obtained by the oxidation of the inorganic substances mentioned are in agreement with the very low cell yields. The synthesis of one gram of cellular dry weight requires the consumption of far greater amounts of substrates than is known for other organisms. Several of these bacteria

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also carry out uncoupled oxidation reactions, i.e., they oxidise substrates without simultaneous synthesis of cell material ('metabolic idling'). It is therefore not surprising that nitrification reactions in soil and water are carried out by relatively small populations of bacteria.

Oxidation of reduced sulphur compounds The

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ability to obtain energy by oxidation of reduced sulphur compounds is the property of a group of gram-negative bacteria with polar flagella; these bacteria constitute the genus. Thiobacillus.Quite recently, a bipolarly flagellated spirillum (Thiomicrospirai and a non-motile thermophilic bacterium (Sulfolobus) with this capacity were discovered. Most of the thiobacilli can oxidise several sulphur compounds and form sulphate as the end-product.

Sulphur-oxidising bacteria

Species pH of growth Electron donor

Thiobacillus thiooxidans 2-5 S2-, S203-2 ,S

Thiobacillus ferrooxidans 2-6 Fe2+, S203-2, S

Thiobacillus thioparus 6—8 CNS-, S203-2 .S

Thiobacillus denitrificans 6--8 CNS-,S203-2 ,S

Thiobacillus intermedius 2-6 S203-2 , S, glutamate

Thiobacillus novellus 6—8 S203-2 ,S, glutamate

Thiomicrospira pelophila 6—8 S2 -, S203-2 ,S

Sulfolobus acidocaldarius 2-3 S, glutamate, peptone

S2-+2 O2 → SO42-

S + H20 + 1½O2 → SO42-+ 2H+

S2O32- + H20 + 2 O2 → SO4

2-+ 2H+

OXIDATION OF IN ORGANIC COMPOUNDS,BIOFILM AND MICROBIAL INFLUENCED CORROSION 5

Most of the thiobacilli (T. thiodxidans, T. thioparus, T. denit;ificans) are obligate chemolithoautotrophs and depend on CO2fixation. Others can also grow with organic compounds as energy and carbon sources (T. novellus, T. intermedius).T. thiooxidans produces large amounts of sulphuric acid and is specifically adapted to growth at low pH; it can tolerate I N sulphuric acid. This acidification can be exploited in a number of ways. Addition of elementary sulphur can be used for de-alkalisation of chalky soils; the sulphuric acid produced by thiobacilli converts calcium carbonate to the more soluble calcium sulphate, which is readily leached out.In a similar way, the acidophobic organism that causes potato scab can be combatted. Whereas the above-named thiobacilli live aerobically, T. denitrificans can utilise nitrate as hydrogen acceptor and thus carry out anaerobic respiration. This organism denitrifies nitrate but cannot carry out assimilatory reduction to ammonium; hence it is dependent on ammonium salts as nitrogen source.Sulfolobus acidocaldarius and Caldariella acidophila are occupants of extreme ecosystems. They inhabit acid hot springs where mainly volcanic hydrogen sulphide is oxidised. S. acidocaldarius is a thermophilic, facultatively chemolithotrophic bacterium that oxidises' elemental sulphur to sulphuric acid and grows optimally at pH 2-3 and' temperatures of 70--75 'C, though it can tolerate even 90 'c.

Reaction pathways in the oxidation of sulphur compounds. It has been difficult to establish the steps in the reaction pathway of sulphur oxidation because hydrogen sulphide, as well as sulphur in aqueous solutions, is oxidised non-biologically, albeit slowly. The scheme shown in Figincludes the most important enzyme-catalysed reactions. The yellow sulphur (flowers of sulphur) exists as an eight- membered ring .(S8) which is moderately soluble in water (0.176 mg sulphur / L).It is assumed that the electrons obtained in the oxidation of sulphite to sulphate enter the respiratory chain at the level of cytochrome c. At least some thiobacilli (T. thioparus, T. denitrificans) can utilise the 'energy available from the oxidation of sulphite to sulphate by a substrate-level phosphorylation.

ADP Sulfurylase (2) APS + P ADP + SO4

2-

adenylate kinase(3) 2ADP ATP + AMP


Hydrogen Oxidation

Many organisms are capable of using hydrogen (H2) as a source of energy. While several mechanisms of anaerobic hydrogen oxidation have been mentioned previously (e.g. sulfate reducing- and acetogenic bacteria) hydrogen can also be used as an energy source aerobically. In these organisms hydrogen is oxidized by a membrane-bound hydrogenase causing proton pumping via electron transfer to various quinones and cytochromes. In many organisms, a second cytoplasmic hydrogenase is used to generate reducing power in the form of NADH, which is subsequently used to fix carbon dioxide via the Calvin cycle. Hydrogen oxidizing organisms, such as Cupriavidus necator (formerly Ralstonia eutropha), often inhabit oxic-anoxic interfaces in nature to take advantage of the hydrogen produced by anaerobic fermentative organisms while still maintaining a supply of oxygen.

Hydrogen Bacteria

Gain energy by oxidizing hydrogen gas: H2 + NAD+ -------(hydrogenase enzyme)------> NADH + H+

alternative: electrons can be donated directly to ETS chain, bypassing NAD Note: only need one special enzyme to carry this step out: hydrogenase. Many different genera of bacteria include members that can induce hydrogenase. When hydrogen

disappears, back to heterotrophic life. Hydrogen bacteria are usually facultative chemolithotrophs.

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Ferrous iron (Fe2+) Oxidation

Ferrous iron is a soluble form of iron that is stable at extremely low pHs or under anaerobic conditions. Under aerobic, moderate pH conditions ferrous iron is oxidized spontaneously to the ferric (Fe3+) form and is hydrolyzed abiotically to insoluble ferric hydroxide (Fe(OH)3). There exists, therefore, three distinct types of ferrous iron-oxidizing microbes. The first are acidophiles, such as the bacteria Acidithiobacillus ferooxidans and Leptospirrillum ferrooxidans, as well as the archaeon Ferroplasma. These microbes oxidize iron in environments that have a very low pH and are important in acid mine drainage. The second type of microbes oxidize ferrous iron at neutral pH along oxic-anoxic interfaces. Both these bacteria, such as Gallionella ferruginea and Sphaerotilus natans, and the acidophilic iron oxidizing-bacteria are aerobes. The third type of iron-oxidizing microbes are anaerobic photosynthetic bacteria such as Chlorobium, which use ferrous iron to produce NADH for autotrophic carbon dioxide fixation. Biochemically, aerobic iron reduction is a very energetically poor process which therefore requires large amounts of iron to be oxidized by the enzyme rusticyanin to facilitate the formation of proton motive force. Like during sulfur oxidation reverse electron flow must be used to form the NADH used for carbon dioxide fixation via the Calvin cycle.


Iron Bacteria

Curious discovery: Ferrobacillus ferrooxidans. Carries out oxidation of iron: Fe++ (ferrous) > Fe+++ (ferric) + e-

Originally thought bacteria get energy from oxidation, make ATP. But redox potential of Fe oxidation is + 0.78 v., and redox potential for oxygen is + 0.86 v., so delta Eo' for aerobic respiration is only -0.08 v., calculated delta Go' is much less than the 7.3 kcal/mole needed to make ATP.

It only grows in very acidic habitats, pH less than 3. Found with Thiobacillus thiooxidans, bacterium that produces sulfuric acid. Ferrobacillus lives off the pH gradient created by acidic pH. This maintains very high proton gradient. As H+ flows in, ATP gets made. But need to get rid of H+ inside, keep internal pH at 7. Use Fe++ as electron donor to oxygen, combine with H+ to form water, get rid of outside cell. Iron functions as electron supplier to get rid of protons. Cells process an enormous amount of iron for very small yields of energy. Fe+++ reacts with OH- ions to form insoluble precipitate, Fe(OH)3, reddish yellow color.

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BIOFILM

A Biofilm is a complex aggregation of microorganisms growing on a solid substrate. Biofilms are characterized by structural heterogeneity, genetic diversity, complex community interactions, and an extra cellular matrix of polymeric substances.

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 molecules 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. Only some species are able to attach to a surface on their own. Others are often able to anchor themselves to the matrix or directly to earlier colonists. Once colonization has begun, the biofilm grows through a combination of cell division and recruitment. 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.

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 or the cells to become more antibiotic resistant.

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 foodchains 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.

In industrial environments, biofilms can develop on the interiors of pipes, which can lead to clogging and corrosion. Biofilms on floors and counters can make sanitation difficult in food preparation areas. Biofilms in cooling water systems are known to reduce heat transfer and harbour Legionella bacteria.

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.

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 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.

Biofilms consist of many species of bacteria and archaea living within a matrix of excreted polymeric compounds. This matrix protects the cells within it and facilitates communication among them through chemical and physical signals. Some biofilms have been found to contain water channels that help distribute nutrients and signalling molecules. This matrix is strong enough that in some cases, biofilms can become fossilized.

Bacteria living in a biofilm can have significantly different properties from free-floating bacteria, 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 extra cellular matrix and the outer layer of cells protect the interior of the community.

Biofilms are common in nature, as bacteria commonly have mechanisms by which they can adhere to surfaces and to each other. Dental plaque is a biofilm. In industrial environments, biofilms can develop on the interiors of pipes and lead to clogs and corrosion. In medicine, biofilms spreading along implanted tubes or wires can lead to pernicious infections in patients. Biofilms on floors and counters can make sanitation difficult in food preparation areas.

A biofilm can be formed by a single bacterial species, but more often biofilms consist of many species of bacteria, as well as fungi, algae, protozoa, debris and corrosion products.

Microbial infections can form on biomaterials that are totally inside the human body or partially exposed to the outside. Escherichia coli, staphylococci, and Pseudomonas species are among the most common invading bacteria.

Formation

5 stages of biofilm development. Stage 1, initial attachment; stage 2, irreversible attachment; stage 3, maturation I; stage 4, maturation II; stage 5, dispersion. 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.

Significance of Biofilms:


The negative effects of biofilms includes such problems as the infection of an artificial heart. This colonization may present the need for additional operations, amputation, or it may even lead to death.

Individual (planktonic) bacterial cells have the ability to adhere to surfaces. Other planktonic bacteria can then attach to the adhered bacteria. This process of continued adhesion eventually leads to multilayers of bacteria on the surface. A large amount of extracellular polymeric substances (EPS) accompany the bacterial cells, creating a matrix throughout the biofilm.

Microbial biofilms on surfaces cost the nation billions of dollars yearly in equipment damage, product contamination, energy losses and medical infections.

Conventional methods of killing bacteria (such as antibiotics, and disinfection) are often ineffective with biofilm bacteria. The huge doses of antimicrobials required to rid systems of biofilm bacteria are environmentally undesirable (and perhaps not allowed by environmental regulations) and medically impractical (since what it would take to kill the biofilm bacteria would also kill the patient!). So new strategies based on a better understanding of how bacteria attach, grow and detach are urgently needed by many industries.

Conversely, microbial processes at surfaces also offer opportunities for positive industrial and environmental effects, such as bioremediating hazardous waste sites, biofiltering industrial water, and forming biobarriers to protect soil and groundwater from contamination.

Biofilm - Biofilm formation, or more specifically microbial film formation, is caused by adhesion of bacteria to surfaces. The film consists of an accumulation of cells, extracellular products and inorganic and organic debris.Within a water distribution system, the biofilm acts as an inoculum for the rest of the piping system.Another important consequence of biofilm is that it provides a protective environment for many microorganisms, including pathogens, from disinfection and cleaning techniques.

Gallionella, an iron-oxidising bacterium, and Pedomicrobium manganicum, a bacterium that is important in oxidising and depositing manganese are the important bacteria in the water distribution biofilms.The consequences of iron and manganese oxides deposition are turbercle formation, resulting in restricted water flow, biocorrosion and decreased water quality as the deposits slough off to form dirty water. Sulfate - and nitrate - reducing bacteria, nitrite oxidisers and numerous unidentified heterotrophs are also found.

Pigmented bacteria also appear to be an important microbial community in the biofilm of water distribution systems.The major genus of pigmented bacteria is Flavobacterium.Other genera include those with pigmented species such as Serratia, Corynebacterium, Microbacterium and Chromobacterium.Biofilm formation also present serious problems of hygiene, odor and taste in distribution systems. Waterborne pathogenic and opportunistically pathogenic bacteria include species of Pseudomonas, Campylobacter, Clostridium, Flavobacterium and Legionella.

Of these Legionella pneumophila is the ideal example of a biofilm pathogen.Free living pathogenic amoebae are also important, of which Acanthamoeba causes chronic eye infection and Naegleria causes meningo encephalitis.


BIOFOULING

Biofouling or biological fouling is the undesirable accumulation of microorganisms, plants, algae, and/or animals on wetted structures.

Impact

Biofouling 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 occur in oil pipelines carrying oils with entrained water especially those carrying used oils, cutting oils, soluble oil or hydraulic oils.

Anti-fouling

Anti-fouling is the process of removing the accumulation, or preventing its accumulation. In industrialprocesses, 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.

[Biocides

Biocides 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 oysterCrassostrea gigas (at a concentration of 20 ng/l) and development of male characteristics in female genitalia in the dog whelkNucella 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 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.

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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. 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 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 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.

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 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 areinvolved 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 inthe 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 energyby utilizing small organic compounds or H2as 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 ofthe pipe, oxidising the H2S present and producing H2S04. The sulphuric acid dissolves the CaOH and CaC03in the cement binder, thus causing corrosionof 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 lovingT. thiooxidans growsin 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.

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

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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CHEMOLITHOTROPHIC BACTERIA viewSeveral groups of soil and aquatic bacteria are able to use inorganic compounds or ions (ammonium, nitrite, sulphide, thiosulphate, sulphite, and ferrous