BACTERIAL EXOPOLYSACCHARIDES AND THEIR ROLE IN

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STUDY ON

BACTERIAL EXOPOLYSACCHARIDES AND THEIR ROLE IN

ADHESION AND CORROSION

Ph. b. Thesis By

FRA bbRY b'SOUZA

/1 7 ²⁰⁰⁴

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57 D` so t STUDY ON BACTERIAL EXOPOLYSACCHARIDES

AND THEIR ROLE IN

ADHESION AND CORROSION

THESIS SUBMITTED TO GOA UNIVERSITY FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

-01\7E4- ktx MICROBIOLOGY

UNDER THE GUIDANCE

S.

OF

DR. N. B. BHOSLE SCIENTIST

MARINE CORROSION AND MATERIAL RESEARCH DIVISION NATIONAL INSTITUTE OF OCEANOGRAPHY

DONA PAULA - 403 004, GOA, INDIA

JUNE 2004 BY

FRADDRY D'SOUZA

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CERTIFICATE

This is to certify that the thesis entitled "Study on bacterial exopolysaccharides and their role in adhesion and corrosion" submitted by Mr. Fraddry D'Souza for the award of the degree of Doctor of Philosophy in Microbiology is based oh his original studies carried out by him under my supervision. The thesis or any part thereof has not been previously submitted for any other degree or diploma in any university or institution.

Place: Dona Paula Dr. N.B. Bhosle

Date : J u^,

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STATEMENT

I hereby declare that:

a) this thesis has been composed by me, b) the work is my own,

c) the work has not been submitted for any other degree or professional qualification.

(Fraddry D'Souza)

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.ACK,WOWL ^ED GE W ENTS

I am deeply indebted to my guide Dr. N. B. Bhosle, Scientist, National Institute of Oceanography, Goa, whose help, stimulating suggestions and encouragement helped me in all the time of research work and writing of this thesis. I also thank him for his enthusiasm and integral view on research and his mission for providing 'high quality work' has made a deep impression on me I owe him lots of gratitude for having me shown this way of research.

I thank Dr. S. R. Shetye, Director, National Institute of Oceanography, Goa, for extending me research facilities. So also I gratefully acknowledge Dr. E. Desa, Ex- Director, NIO, for his permission and encourage me to go ahead with my thesis.

I express my sincere thanks to Dr. A. C. Anil, Scientist, and Dr. S.S. Sawant, Scientist, for extending their help and timely support whenever I required I also gratefully

acknowledge Mr. A. P. Selvam for his invaluable assistance during the whole work of this thesis. A special thanks goes to Ms. Anita Garg, whom I have know for more than six years now and who showed to be a kind, mostly helpful and trustful friend I also express

my sincere thanks to Mr. K. Venkat, Dr. C. Venugopal, Mr. Shyam Naik, Mr. N.S.

Prabhu and Mr. P.R. Kurle for their ever-helping attitude.

I also acknowledge the help given by Dr. S.G Dalal, Scientist, in statistics and Dr. C. G.

Naik, Scientist, for analyzing the IR and NMR samples.

I am thankful to the staff of Drawing section, Library, SEM Lab, Workshop specially Mr.

V. N. Chondankar and Electroplating Section, Mr. B. Madasami.

I would also like to thank the members of my PhD committee who monitored my work and took effort in reading and providing me with valuable comments: Prof (Retd) S.

Mavinkurve, Dr. I. Furtado and Dr Sarita Nazereth, all from Department of

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Micorbiology, Goa University, I thank you all. I also sincerely thank Dr S. Bhosle and Dr. S. Garg, Department of Micorbiology, Goa University for their timely help.

My colleagues of the department all gave me the feeling of being at home at work and their help during various stages of my thesis: Dr. P. V. Bhaskar, Evonne Cardozo, Rakhee Khandeparker, Asha Griyani, Dr. Dattesh Desia, Y. Vishwakiran, Dr. N. L.

Thakur, Dr. Jagadish Patil, Dr. Lidita Khandeparker, Dr. Smita Mitbavkar, Loreta Fernandes, Shripad Kunkolienkar and Michelle Velho.

My other colleagues in the department Sangeeta Jadav, Ranjita Harji, Priya D'Coast, Leena Prabhudesai, Preeti Revankar, Ramila Furtado, Shamina D'Silva, Sahana Hegde,

Chelan Gaonkar, Anand Jain, Vishwas Khodse, many thanks for being your colleague

I had the pleasure to supervise and work with several students who did their dissertation work in our project: Sana Amir, Lalan Borkar, Xavier Bocquillion and Nicolas Defayolle, I thank you all for the help.

I also sincerely appreciate the help and support rendered by my friends from the institute, Sweta Harihar, Dr. Manguesh Gauns, Dr. Damodar Shenoy, JayS4nkar De and Prasant Pathange.

I thank CSIR for awarding me the Senior Research Fellowship and providing me partial financial support in this thesis work

I also thank Department of Ocean Development, Govt. of India for awarding me the Junior Research Fellowship.

Last but not the least, I am very grateful for the endless love and support of my parents and brothers.

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DEDICATED TO MY

PARENTS'

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Chapter 1 General Introduction

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Chapter 1 I) Biofilm

Most of the bacteria in the environment live in micro-ecosystems filled with hundreds of different microorganisms. In aquatic environment most of the microorganisms are not free floating but are attached to surfaces as complex communities called biofilm (Mayer-Riel, 1994; Brunke & Gonser, 1997; Decho, 2000; Beech et al, 2002; Bressel et al, 2003; Chang et al, 2003).

Biofilm is formed when bacteria adhere to surfaces in aqueous environments and begin to excrete a slimy, glue-like substance that can anchor them to all kinds of surfaces such as metals, plastics, soil particles, medical implants and tissue. A biofilm can be formed by a single bacterial species, but more often biofilm consist of many species of bacteria, as well as fungi, algae, protozoa, debris and corrosion products. Essentially, biofilm may form on any surface exposed to environment containing bacteria and some amount of water.

Biofilm Formation

How are biofilms formed? The adhesion of primary population of microorganism to marine surfaces has been studied extensively (Marshall, 1978; Corpe, 1977;

Rice et al, 2000). Generally, it is believed that clean surfaces placed in the aquatic environments sorbs bacteria within hours, while development of community to a critical population size to yield growth may take days or weeks.

Formation of biofilm appears to involve the following five stages (Cooksey &

Characklis, 1983; Mitchell & Kirchman, 1984; Maki et al, 1990).

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i) Initial conditioning stage

ii) Chemicals attraction of motile bacteria iii) Reversible adsorption

iv) Irreversible stage

v) A final stage of development of micro-flora

i) Initial conditioning stage

Solid surfaces placed in seawater are immediately coated with a monolayer of polymeric material. This process results in the formation of a conditioning film or molecular film (Baier, 1980; Fletcher & Marshall, 1982; Basis et al, 1999; Taylor et al, 1997). A conditioning film is primarily composed of glycoproteins (Baier, 1973), humic material (Loeb & Neihof, 1975) and/or unspecified macromolecules (Zaidi et al, 1984). Recent studies based on modern surface analytical tools such as the time-of-fight-secondary ion mass spectrophotometry (ToF-SIMS) and Fourier Transform infrared reflection absorption spectroscopy (FT-IRRAS) suggested that proteins are the first to conditioned the surfaces followed by carbohydrates (Pradier et al, 2000; Compere et al, 2001; Poleunis et al, 2002, 2003). The deposition of polymeric material affect surface tension, surface charges and the wettability of a solid surface thereby influencing the settlement of primary bacterial film (Fletcher & Marshall, 1982; Loeb, 1985).

ii) Chemical attraction of motile bacteria

Bacteria get attracted to surfaces for a number of reasons. One may be gravity because of which the organism may just settle out and end up resting on a surface. Bacteria with a negative charge on their outer envelope may be

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attracted to the positively charged inorganic surfaces. However, motile bacteria have chemoreceptors, which allow them to move either toward or away from minute concentration (10-6 molar) of attractants or repellants (Berg and Brown, 1972; Adler, 1975). Many surfaces attract and concentrate nutrients, which may help chemotactic bacteria to form biofilm.

iii) Reversible adsorption or adhesion

During initial stages of biofilm formation, bacteria are weakly held close to a surface by physical attractive forces such as van-der-Waals forces, hydrogen bonding and electrostatic interactions (Dempsey, 1981; Kelley, 1981; Meyer-

Reil, 1994). During reversible adhesion bacteria exhibit Brownian motion. As the bacteria are held only weakly to the surface and can be removed easily, it is called as reversible adhesion.

iv) Irreversible adhesion

The irreversible sorption is a firmer adherence and is primarily due to exopolymeric substances (EPS) and/or other cellular component like flagella, fimbrie and pilli (Weiss, 1973; Rosenberg et al, 1982; Marshall, 1985). With the help of EPS or cell appendages bacteria attach very strongly to the surface.

Such attached bacteria are difficult to remove so such adhesion is called as irreversible attachments. Microbial attachment (adhesion) is discussed in more details later in this chapter.

v) Secondary microbial populations

The production of slimy layer by irreversible attached bacteria enables them to

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trap other microorganisms and debris. The rate of secondary microbial population development depends on the nutrient status of the aquatic environment. It may take a week or so to form biofilm in open ocean but only days in coastal waters.

Microbial Adhesion

ZoBell, (1943) was the first to study microbial adhesion. He suggested that adhesion is a biphasic phenomenon, with initial reversible adhesion followed by irreversible binding of microorganisms to surfaces. Microbial adhesions are of two type i.e. specific adhesion and non-specific adhesion

Non-specific Adhesion

Non-specific microbial adhesion involves macromolecules on the surface of microorganisms. These macromolecules may interact with either the surface of the substratum or the macromolecules present on that surface. Ionic, dipolar,

hydrogen bonding and hydrophobic interactions may be involved. The number of interactions and strength of the interaction may vary from organisms to organisms or within the same organism to different surfaces. Such microbial interactions with surfaces are not specific and hence non-specific adhesion.

Specific Adhesion

The requirement for specific adhesion is the presence of some form of stereochemical components, which brings more than one pair (normally several) of neighboring interacting group on microorganism and on the substratum into contact. For example, sugar residual-protein interactions on two complementary

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polymers, where a lock-and-key mechanism is involved. The presence of adhesin molecules on the surface of the cell and the receptor on the substratum can follow specific adhesion. In certain special cases it may be possible to have a receptor-like molecule on both cell and substratum surfaces and free adhesin- like molecules that bind the two together.

Mechanism of Bacteria Adhesion

Two types of models have been suggested to explain the adhesion of microorganism to surfaces. These are electrokinetic and thermodynamics models, both have some success in describing bacterial adhesion to surface.

Electrokinetic's model

The electrokinetic model is primarily concerned with electrostatic forces occurring between the bacteria and the surfaces. Such interactions are described in DLVO theory (Derjaguin & Landau, 1941; Verwey & Overbeek,

1948).

The DLVO theory in its simplest form looks at the two main forces acting on charged colloidal particles in a solution. These two forces are

1) electrostatic repulsion

2) Van der Waals attractive forces.

Later, microbiologists suggested that the DLVO theory might well be a useful model to explain bacterial adhesion to surfaces. Bacteria have a net negative charge at physiological pH values. Similarly, most surfaces are also negatively charged. This will result in electrostatic repulsion due to an overlap between the electrical double layers associated with the charged groups on the surfaces.

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The only significant attractive force known to be present is Van der Waals force that is due to an interaction between oscillating dipoles on the surface molecules. Van der Waals attractive force is a very powerful force but it only operates over a small distances (5-10nm). However, it is less than the repulsive force. Nevertheless, if the cells can get close enough to the surfaces, Van der Waals force is very strong to hold it very tightly.

Bacteria do not have enough kinetic energy to overcome the repulsive force.

However, DLVO theory suggests that as the radius of the particle decreases, the repulsive energy barrier decreases. Thus, when cells are able to reduce their effective radii as in the production of filopodia (e.g. Mammalian cells) or fimbriae and exoploymers (bacteria), they may overcome the repulsive forces.

Further, EPS produced by the bacteria act as a cementing material by forming bridges across the repulsion zone thereby helping the cells to develop contact with the surfaces.

Thermodynamic models

Although numerous studies have shown that ionic interactions can affect bacterial adhesion, there are also studies suggesting that the attachment is not related to charge differences (Abbott et al, 1980). This is because the DLVO theory does not consider involvement of short-range interactions such as hydrogen bonding, dipole and hydrophobic interactions. Such interactions are considered in the thermodynamic theory, which evaluate bacterial adhesion in terms of free energy changes. Theoretically, if the total free energy of a substratum containing a cell and adjacent substratum is reduced by contact, then the adhesion of the cells to the substratum takes place (Characklis &

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Cooksey, 1983). Absolom et al, (1984) reported a close relationship between adhesion and the surface free energy of the solid substratum. Similarly, the surface tension of the substratum is related to adhesion (Fletcher, 1983). It is much more difficult to evaluate surface free energy of bacteria as they do not constitute flat surfaces or deformable liquids. However, it is much simpler to measure free energy or hydrophobicity using contact angle on lawn of cells (Absolom et al, 1984). The measurement of hydrophobicity showed good relationship with the adhesion properties of the cells.

Succession in Biofilm.

The succession of biofilm formation on solid surfaces submerged in seawater seems to follow a course that can be divided into roughly three phases.

a) Primary periphytes

It includes the primary attachment of the common chemorganotrophic bacteria, which readily use organic nutrients adsorbed to the solid substratum.

Pseudomonas species were recognized as the principle periphytic bacteria that attach initially. Other bacteria such as Flavobacterium and Achromobacter species were also commonly encountered as primary colonizers (Corpe 1973;

Fletcher & Marshall, 1982; Sonak & Bhosle, 1995). The extent of their growth seems to be related to the nutrient concentration and probably more specifically to the amount of nutrient adsorbed to the solid substratum.

b) Secondary periphytes:

The initial attachment of primary periphytes are followed by attachment and

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growth of somewhat more nutritionally specialized, stalked and/or filamentous forms, which become the predominant periphytic flora. Bacteria, which attach secondarily and become the predominant flora, include Caulobacter, Hyphomicrobium and Saprospira (Marshall et al, 1971; Corpe 1973; Fletcher &

Marshall, 1982). It seems likely that the primary colonizers may provide specialised nutrients for secondary colonizers as secretions, by cell leakage or upon cell lysis at death.

c) Microalgae and Protozoa:

Generally, once the bacterial films have been established, diatoms, other microalgae, fungi and sessile protozoa appear in large numbers (Shanker &

Mohan, 2001). The appearance of protozoa may be quite dependent on the bacterial film to supply the amount and kind of nutrients, which favor their growth. The degradation of organic materials by enzymes from the periphytes would be expected to make the surfaces a rich source of nutrients and hence sites of intense biological activity.

Factors Affecting Biofilm Formation

Many factors may influence biofilm formation. Some of these are described below.

1) Dispersed microbial cell and nutrient concentrations in the bulk liquid. In a eutrophic environment (high cell numbers and nutrient concentrations), biofilm formation rates are high (Fletcher, 1977; Brayers & Characklis, 1981; Trulear &

Chrarcklis, 1982).

2) Temperature: Biofilm formation rate increases with temperature up to some

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critical temperature dictated by the microbial species present and above this critical temperature biofilm formation rate decreases (Characklis, 1980;

Stathopoulos, 1981).

3) Fluid shear stress at the liquid-solid interface: Fluid shear stress increases detachment rate of the biofilm and, therefore decrease biofilm formation.

However, if liquid phase mass transfer is controlling biofilm nutrient removal or transport of cells to the surface, shear stress will increase the rate of biofilm formation (Bolt & Pinheiro, 1977; Brayers & Characklis, 1981; Trulear &

Characklis, 1982).

4) Characteristics of substratum surface: Characteristics of the substratum surface are important in the adhesion process. Wettability, or critical surface tension, is the property used most frequently to describe surface characteristics in microbial attachment studies (Loeb & Neihof, 1975; Fletcher & Loeb, 1979).

In seawater, cell attachment increases with increasing critical surface tension (glass, copper, polyethylene, teflon). Both macro- and microroughness of the substratum influence biofilm rates. Roughness influence rate of transport to the substratum of cells and nutrients. Roughness partially shield cells at the surface from fluid shear stress and increase the effective area for adsorption.

5) Suspended or particulate material in the water also can influence accumulation rate (Bhosle et al, 1990). Light, small particles in a low velocity flow adsorb easily to the sticky biofilm surface, increasing the accumulated deposit mass. Heavier, large particles in a highly turbulent flow can scour the surface, thus reducing the rate of accumulation.

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Effects of Biofilm

Biofilms can be beneficial or deleterious, depending on species composition and where they are found. Some of the advantages and disadvantages of biofilm are discussed below.

Advantages of Biofilm

Some of the examples where biofilm can be beneficial are presented below:

1) The bacteria within the biofilms can break down contaminants in soil and water. Efforts are on to optimize the potential of biofilm for bioremediation of soils and water contaminated with toxic substances (Zhang et al, 1995;

Guieysse & Mattiasson, 1999; Haddox & Cutright, 2003).

2) When oil is being extracted from the ground there is often the danger of an oil spill near the bottom of the well. These types of spills are very difficult to contain and remove. The possibility of the spill being transported, due to ground water flow, into a river or stream creates a problem. One method to alleviate this problem is the use of biofilms as 'bio-barriers'. That is, if a wall of biofilm can be put in place downstream to the ground water flow, the contamination can be contained. Moreover, if the biofilm consists of bacteria that can convert the contaminant into a less toxic product, the spill can be removed as well.

Disadvantages of Biofilm

Once bacteria begin to colonize surfaces and produce biofilms, numerous problems begin to arise, these include loss in heat transfer efficiency, fouling, corrosion, and scale. When the biofilm activity is involved in economic losses it is termed as microfouling or microbial fouling.

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Heat transfer efficiency

Microbial biofilms in industrial systems reduce heat transfer efficiency. For example, the output of the power station heat exchangers is affected by biofilms that form on the surface in contact with river, estuarine or seawater used for cooling (bott, 1992; Nair & Venugopalan, 1996). Transfer of heat is due to presence of biofilm that affects heat exchange between the liquid phase and the cooling surface (Lappin-Scott & Costerton, 1989).

Fouling

When biofilms develop in low flow areas, such as cooling tower; they may initially go unnoticed, since they will not interfere with flow or evaporative efficiency. After time, the biofilm becomes more complex, often with filamentous development. The matrix developed will accumulate debris that may impede or completely block flow. Algal biofilms may foul cooling tower distribution decks, tower fill, and basins. When excessive algal biofilms develop, portions may break loose and transport to other parts of the system, causing blockage as well as providing nutrient for accelerated bacterial and fungal growth. Fouling on the ship hulls increase the frictional drag thereby reducing the speed and decreasing fuel efficiency. Biofilms can also impair the working of filters, ion exchange and underwater sound equipments.

Scale

Biofilms often lead to the formation of mineral scales, which are calcium and magnesium oxides or hydroxide on the surface. Calcium ions are fixed into the biofilm by carboxylate functional groups of the polysaccharides. A typical

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example of biofilm-induced mineral deposit is the calcium phosphate scale on the teeth. When biofilms grow on tooth surfaces, they are referred to as plaques. Similarly, on heat exchangers may form mineral scale and reduce the heat transfer efficiency.

Role of microorganisms in corrosion

The growth of bacteria and formation of biofilms on surface may influence corrosion. Microbiological corrosion or microbially induced corrosion is defined as the corrosion that is influenced by the growth of microorganisms, either directly or indirectly. Microbial corrosion is discussed in more details later in this chapter.

Biofilm Control

Various methods have been suggested for controlling biofilm. There is no single, elegant solution to the overall problem of fouling. The choice of preventive methods depends on the site and/or the structure to be protected. Some of the methods used to control biofilm are briefly discussed below.

1) Physical method

Physical methods of combating fouling includes manual cleaning, hydraulic pressure, compressed air, water jets, sand blasting and high velocity ice particles (Partridge, 1981; Bain, 1981; Fishcher et al, 1984)

2) Electrolytic method

In this method electric current is used for cleaning the fouled structure. One of

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such methods includes placement of metal bar (as anode) close to the structure (as cathode) in an electric circuit. When the current is supplied, a large amount of hydrogen is liberated from the surface under the corrosion product, breaking up rust and removing it from the surface. Thus, the fouling organisms along with the corrosion products are removed (Anon, 1977). In another method, the fouling is prevented by generating insitu electrolytic chlorine.

3) Ultrasonic and radiation method

Ultrasonic and UV radiation methods have also been used for controlling fouling (Aras, 1980). However, UV radiations interfere with the cellular components, causing the rupture and/or death of the cells.

4) Biological method

The biological method to prevent fouling includes use of marine organisms, which feed on the sedentary organisms and algae onto the surface to be cleaned (Wahl, 1989). Some oil companies have attempted biological control of fouling growth but attained very limited success. This type of antifouling method

has advantages over others, as it is environmentally safe.

5) Chemical method

Chemical methods appear to be more effective in controlling biofilm settlement, and therefore they are used extensively. These methods include application of biocides and biodispersant. Application of biocides can be achieved in two ways. It can be added either directly in the environment (injectable biocides) or can be applied in the form of paint onto the surface to be protected from fouling.

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Hypochlorous acid is most widely used biocide in the running water pipes. Other biocides include aeroline, soluble copper salts, bromine, iodine, bromine chloride, ozone, carbondioxide and various trialkyl organotin compounds (Fisher et al, 1981; Fishcher et al, 1984). Trialkyl organotin compounds are widely used in antifouling paints. However, it is evident that some of these biocides such as tributyltin (TBT) are highly toxic to many non-target organisms (Beaumont &

Newman, 1986; Wester et al, 1990). For this reason, international Maritime Organization (IMO) has imposed restrictions on the use of these compounds in most of the advanced countries. A complete ban on the use of organometallic compound, especially TBT is imposed in Japan, while in Britain, its use on small boats is prohibited since 1988 (Clare, 1995).

Biofilm control programs can be made more effective through the utilization of a biopenetrant and/or dispersant products. Products that penetrate and loosen the biopolymer matrix will not only help to slough the biofilm but will also expose the microorganisms to the effects of the biocide. These products are especially effective when dealing with systems that have a high total organic carbon loading and a tendency to foul. These products are typically fed in slug additions prior to micro-biocide feed. Low-level continuous feed may not be as effective, since it often takes a certain threshold amount to produce the desired effect.

Recent developments in biodispersant technology are making this approach more effective and popular than ever before (Cloete et al, 1998).

Enzyme technologies that will break down the extracellular polysaccharides and destroy bacterial attachment structures (fimbriae) are currently being developsd and patented (Johansen et al, 1997). These technologies, although expensive, may provide effective biofilm control where micro-biocide use is environmentally

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restricted.

II) Microbial Exopolymers

Microorganisms that are growing within the biofilm are often encased in a matrix of extracellular polymeric substances (EPS). The production of EPS generally occurs in both prokaryotic (bacteria, archaea) and in eukaryotic (algae, fungi) microorganisms. The EPS may be defined as the extracellular organic polymers (biopolymers) that are frequently responsible for binding cells and other particulate materials together and to the substratum in biofilm system (Characklis & Wilderer, 1989). In earlier research the term EPS has been frequently used as abbreviation for "exopolysaccharides" or "extracellular polysaccharides" as it was assumed that polysaccharide is the most abundant components (Costerton et al, 1981). However, recent studies indicates that protein and nucleic acids (Platt et al, 1985; Frolund et al, 1996; Nielsen et al, 1997; Dignac et al, 1998) as well as (phospho) lipids (Neu 1996; Takeda et al, 1998; Sand & Gehrke, 1999) may have accounted for a significant proportion of the EPS or even the predominate component of the EPS. In view of this, in recent years the term "extracellular polymeric substances" (EPS) is used to represent different classes of organic macromolecules such as polysaccharide, proteins, nucleic acids, (phospho) lipids and other polymeric substances that are secreted extracellularly by biofilm organisms.

The EPS are classified into two major types based on their general physical structures i.e. capsular and slime exopolymers. The capsular exopolymers are tightly bound to the cells forming an envelope around the cell, whereas slime exopolymers are loosely bound to the cells and often form a film around the

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cells and diffuse into immediate environment (Decho, 1990, 1994). The quality and the chemical composition of these exopolymers are influenced by growth phase and nutrient status of surface associated bacteria (Decho, 1990).

Composition and Structure of EPS

EPS are organic macromolecules that are formed by polymerization of similar or identical building blocks, which may be arranged as repeating units within the polymer molecules. The major organic fractions of the EPS are carbohydrates, proteins and humic substances (Morgan et al, 1991; Nielsen & Jahn, 1999;).

Proteins can be glycosylated with oligosaccharides to form glycoproteins (Horan

& Eccles, 1986; Morgan et al, 1991) or can be substituted with fatty acids to form lipoproteins. Significant amount of DNA and RNA are also found (Nielsen &

Jahn, 1999). In addition EPS may contain nonpolymeric substituents of low molecular weight compounds, which greatly alter their structure and physicochemical properties. Thus, extracellular polysaccharides often carry organic substituents such as acetyl, succinyl, or pyruvyl groups or inorganic substituents such as sulphate.

Since polysaccharides are identified as major or common constituents of bacterial EPS, the term "glycocalyx" is often used for description of polysaccharide-containing structures of bacterial origin (Costerton et al, 1981, 1992). The term glycocalyx encompasses different EPS containing structures such as capsules, sheaths, and slimes. Capsules are normally firmly associated with the cell surface as discrete structure with distinct outlines. Capsular polymers are attached to the cell surface by non-covalent interactions, but may also be covalently bound to phospholipid or lipid-A molecules at the surface

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(Roberts, 1996). Filamentous bacteria such as certain cyanobacteria or bacteria of the genera Leptothrix and Sphaerotilus (Takeda et al 1998) possess sheaths as linear EPS containing structures surrounding chains of cells. EPS may only be loosely attached to the cell surface as peripheral capsules and can be shed into the surrounding environment as less organized slime.

Most of the microbial exopolysaccharides are highly soluble in water or dilute salt solution. On the other hand, some exopolysaccharides are virtually

insoluble in water or form very rigid gels, which are commonly found in biofilms.

Structurally, these polysaccharides usually contain a backbone in which there is a predominance of either 1,3 or 1,4 linkages in either the a - or more commonly the [I - configuration. The actual composition of microbial polysaccharides shows an almost infinite range of possibilities. Some are simple homopolysaccharides containing a single moiety of monosaccharide and containing single linkage type. For example, the 1,3 linked glucans mutan from Streptococcus mutans and curdlan from Agrobacterium radiobacter and other species, or the amino sugar – containing 1,6 - -glucan from S. epidermidis (Mack et al, 1996).

CurdIan

[ –(1--43) -8-D-Glcp —(1-43) -13-D-Glcp –(1-] - -4

Mutan

--> [ -3-a-D-Glcp –(1--43) -a-D-Glcp —(1-43) -a-D-GIcp –(1-] - -->

More complex polymers i.e. heteropolysaccharides may be composed of three 18

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to five different monosaccharides and repeat units of hexa-, hepta-, or octasacchrides. The complexity is increased further by the presence of various acyl groups such as acetyl, succinyl, or pyruvyl groups. An example of complex heteropolysaccharide structure can be seen in colanic acid. This exoploysaccharide is secreted by many strains of Escherichia coli and also other enteric species such as Enterobacter cloacae. The repeat unit of this polymer is a hexasaccharide composed of four sugars in which both acetyl and pyruvate groups are present. An example of simpler heteropolysaccharide is acidic polysaccharide alginate secreted by gram-negative bacterium Pseudomonas aeruginosa (Doggett, 1969). This extracellular substance is composed of 13 ^-1,4 —linked D — mannuronic acid and L-guluronic acid.

Colanic acid

—> ( —(1--3) -13-D-Fucp —(1—>4) -a-D-Fucp —(1-] - -->

'I\

(3^-D^-Ga1p=pyr

Alginate

[ ^-4^-I3^-D^-ManUoA P^-D^-ManUoA^—(1^-44)^-a^-D^-G1cU^—(1^-] ^-

Synthesis and Secretion of EPS

By definition, EPS are located at or outside the cell surface independent of their origin. The extracellular localization and their composition may be the results of different processes: active secretion, shedding of cell surface material, lysis,

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and adsorption from the environment. Thus, the sites of synthesis, release, and ultimate localization of EPS components are not necessary identical.

The most common mechanism of polysaccharide biosynthesis is intracellular, only the neutral polysaccharide dextran or levan are known to be synthesized extracellular (Sutherland, 1990). Intracellular biosynthesis starts from activated monosaccharides (nucleoside diphosphate sugar or less often nucleoside monophosphate sugars). In this form the sugars are sequentially transferred to a lipid acceptor (polyisoprenyl phosphate) on which the assembly of sugars residues to complete repeating units and their polymerization occurs. Non- carbohydrates substituents may be added to the oligosachaarides repeating units attached to the lipid carrier. For example, biosynthesis of xanthan by bacterium Xanthomonas campestris occurs by stepwise assembly of repeating pentasaccharides units and their subsequent polymerization to yield the complete polysaccharide. The non-carbohydrate substituents acetyl and pyruvate residues are attached to the lipid-linked pentasacharide level from acetyl-CoA and phophoenolpyruvate as donors respectively (Sutherland, 1990;

Becker et al, 1998). In some cases the polymerization of polysaccharide chain may be modified in the periplasm or after secretion in the extracellular environment. For example, in mucoid strains of P. Aeruginosa, alginate is 0- acetylated at the C2- and/or C3- on its mannuronate residue in the periplasmic space; extracellular epimerization of polymannuronate to the final alginate by an extracellular epimerase has been observed in A. vinelandii (Rehm & Valla, 1997; Gacesa, 1998; Davies, 1999).

EPS may be actively secreted by living cells. Various discrete export machineries involving the translocation of EPS across bacterial membranes to

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the cell surface or into the surrounding medium has been described for bacterial proteins (Binet et al, 1997; Hueck, 1998; Filloux et al, 1998) and polysaccharides (Sutherland, 1990; Leigh & Coplin, 1992; Roberts, 1995, 1996;

Rehm & Valle, 1997; Becker et al, 1998; Jonas & Farah, 1998). One of the mechanisms that commonly occur in gram-negative bacteria is the spontaneous liberation of integral cellular components such as lipopolysaccharides (LPS) from the outer membrane, which occurs through formation of outer membrane derived vesicles (Cadieux et al, 1983; Beveridge et al, 1997; Li et al 1998).

Another mechanism of release of extracellular polymers is through the pore proteins in the outer membrane. For example, P. aeruginosa produces a porin- like outer membrane protein, AIgE, which is involved in the secretion of alginate to the cell surface (Grabert et al, 1990; Rehm et al, 1994). Death and lysis of cells also contribute to the release of cellular high molecular weight compounds onto the medium and entrapment within the biofilm matrix and may thus become part of EPS.

Functions of EPS

The production of extracellular polymeric substances (EPS) by microorganisms involves a significant investment of carbon and energy. This expenditure of energy (in some cases more than 70 % -see Harder & Dijkhuizen, 1983) is likely to hold benefits to the producers of EPS, as well as those organisms associated with them. The importance of EPS has long been recognized and a variety of functions have been attributed to EPS. Some of the functional roles of EPS are elucidated below.

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Role of EPS in cellular associations

Microorganisms exist in the environment as free floating cells or attached to surfaces. The relative importance of each state differs from one environment to another. Microorganisms produce EPS during both suspended and sessile (attached) growth. Evans et al, (1994) found little difference between EPS production by suspended cells and attached cells when growth rates were high.

However, the attached cells produced significantly more EPS than their suspended counterparts during slow growth rates. Coaggregation and consortial activities are required for many microbial processes, which are not possible with single species population (Geesey & Costerton, 1986). A typical example is the utilization of recalcitrant molecules as an energy source, which are resistant to degradation by single species. EPS play an important role in these interactions by facilitating communication between cells through participating in cell-cell recognition, by serving as an adhesin, and the establishment of favorable micro- environment (Swift et al, 1996; Decho, 1999).

Role of EPS in biofilm formation

EPS are considered as important mediators in the adhesion of bacteria and other microorganisms to surfaces (Sutherland, 1984; Marshall, 1985; Stotsky, 1985; Quintero & Weiner, 1995; Becker, 1996). The involvement of capsular EPS during the initial stages of attachment has been suggested by several researchers (Geesey et al, 1977; Morris & McBride, 1984; Marshall et al, 1989;

Cowan et al, 1991). Furthermore, McEldowney & Fletcher (1988) indicated that initially attachment may occur without the requirement for the production of new adhesives when attractive interaction takes place between polymers already

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present on the cells and the attachment surfaces. In addition to initial attachment of cells during biofilm formation, the EPS is also involved in maintaining the structural integrity of biofilm (Costerton et al, 1981; Marshall, 1984; Vandevivere & Kirchman, 1993), and therefore the overall stability of biofilm community (Uhlinger & White, 1983).

Genetic transfer

In aquatic environment DNA form complexes with clay minerals, quartz, feldspar, humic substances, minerals and suspended particles. It appears that this adsorption of extracellular DNA is for protection against DNAases.

Considering the importance of EPS in the attachment of cells to surfaces and the fact that the attached cells produce more EPS then suspended cells (Vandevivere & Kirchman, 1993), it could not be ruled out that EPS also played role, at least indirectly in exchange of the genetic material between cells.

Horizontal gene transfer may be facilitated by several mechanisms such as conjugation, transduction and natural transformation because of the close contact of aggregated cells in the biofilm. A few studies have demonstrated gene exchange in pure culture biofilms (Angles et al, 1993; Lisle & Rose, 1995).

For instance, Lorenz et al, (1988) demonstrated that in sand-filled flow through column that competent Bacillus subtilis cells were able to take up extracellular chromosomal DNA and found out that transformation efficiency is 50 times higher in solid/liquid interface than the liquid interface.

Role of EPS in nutrition

Microorganisms often live under oligotrophic condition in natural environments

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and thus they needed to evolve adaptive strategies to live under such condition.

The production of EPS may be one of the strategies that ensure microorganism to survive under nutrients limited condition. Little work has been done to determine the extent to which nutrients in bacterial EPS can be utilized, either by the same organism, and by cross feeding between species. Patel and Gerson (1974) demonstrated the capacity of Rhizobium strain to re-utilzed its own EPS. Dudman (1977) suggested that the majority of EPS-producing organisms are unable to utilize their own EPS as carbon sources. The presence of EPS-degrading enzymes in consumer animals suggests that the EPS utilization may occur. Despite the earlier conclusion that EPS do not serve as reserve sources of carbon and energy, there appears at least two way where EPS may function as food reservoirs: 1) production of EPS as nutrients reserve and 2) accumulation of nutrients by EPS.

EPS and micro-environment

Biofilms can be viewed as ecosystem consisting of a micro-environment in which the fluctuation of the physical and chemical changes are less severe than that in the external environments. Further, such micro-environment regulates the exchange of molecules within their environment. This regulation of molecules flow is likely due to the physical properties of EPS matrix.

EPS as a physical barriers to solute translocation

Single cell, either attached or in suspension is in close contact with environment and hence transport of nutrients to the cells is direct. However, due to the presence of cell-EPS matrix in the biofilm, diffusion becomes a rate-limiting

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step. Diffusion through the cell-EPS matrix of biofilm occurs generally at a slower rate than through the water and has been termed hindered diffusion (Bohrer et al, 1984; Korber et al, 1995). The resistance of biofilms to antibiotics and biocides is of a significant importance to industry and medicine. Hindered diffusion and the inactivation of antimicrobial agents are two mechanisms, which are possible ways in which EPS are involved in increased resistance to toxic chemicals (Nichols, 1989; Samrakandi et al, 1997).

Protection against predation/digestion

The physical and chemical properties of the EPS influence the tropical interaction between the microbial cells and the consumer animals (Decho &

Lopez, 1993). For example, the predation of the ciliate is selective and based on the surface chemistry of the EPS of the prey (Snyder, 1990). Decho and Lopez (1993) indicated that capsular polymer is significantly less digestible to consumers then the slime polymers, providing evidence that the capsules protect the cells against digestion.

Applications of Exopolysaccharides

The bacterial EPS has many and varied applications in agriculture, oil and chemical industries, in food processing and medical. EPS are used in medicine for tumor control, eye and joint surgery, anticoagulants heparin analogues and as wound dressing (bacterial cellulose). In food industries it is used as emulsion stabilizers, foam stabilization (beer, fire-fighting fluids), gelling agents and food coatings. Other important applications includes a hydrating agents in cosmetics and pharmaceuticals, oil recovery and oil drillings 'muds' in oil industries and

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suspending agents in paper coating, agrochemical pesticides and sprays.

Further new products and expansion of the market for microbial EPS depends on its novel application or unique biological properties. For example, the bacterial species such as Lactobacillus sp., is already accepted bacterium in food industries, and now the research is switched to its EPS for use in food industry.

Ill) Microbial Corrosion

The close association between the microorganism and the metal surface in aquatic environments results in corrosion or, in some cases, passivation and protection of metal. The main cause of corrosion of metals is their spontaneous tendency to return to their thermodynamic stable state. Corrosion can be defined as the destruction or deterioration of a metal or an alloy because of its chemical or electrochemical reaction with its environment resulting in formation of thermodynamically stable compound. Corrosion is one of the two forms of metal deterioration, the other being the mechanical loss of the metal by erosion, abrasion or wear.

The corrosion of metals can be divided in two types, dry corrosion and wet corrosion. In the former, corrosion occurs at a gas/metal interface and water plays little role in the reaction. In wet corrosion, the interface is metal/solution. In both cases, the basic reaction is electrochemical oxidation and involves the removal of one or more electrons from the metal resulting the formation of positive ions. The metal zone at which the metal get oxidized is called anode, thus anode is the area at which metal is lost. The electrons given up by the

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metal flow to the cathode to be consumed in a reduction reaction. Thus any corrosion reaction is always accompanied by a flow of electricity (electrons) from one metallic area (anode) to another area (cathode) through a medium. It is possible to have on same metal, many cathodic and anodic zones because of heterogeneity of the structure of metal.

Bacterial corrosion is one of the wet type corrosion. In microbial corrosion it must be emphasized that corrosion by bacteria does not involve any new type of corrosion process. Microbiological corrosion is also electrochemical corrosion where in some manner the presence of the microorganisms is having some influence in the acceleration or inhibition of the corrosion processes. The deterioration of metals by corrosion process occurs directly or indirectly, as a result of metabolic activity of microorganisms. Alternatively, microbial corrosion may be due to production of corrosive metabolites to render the environment corrosive or to precipitate directly the metal into the solution. The presence of microbial growths or deposits on a metal surface encourage the formation of different aeration cells with anodic and cathodic sites as a result of uneven distribution of biofilm or deposits on the metal surface (King & Miller, 1971; Luty, 1980; Deshmukh et al, 1992; Lee et al, 1993; Walker et al, 1998).

Bacterial corrosion is electrochemical in nature just like other corrosion reaction, excepts for the fact that one never knows the exact chemicals or mechanism involved in the corrosion process. For examples, corrosion may be enhanced biologically, through direct enzymatic action of bacteria, or abiotically, as a results of reaction with metabolic byproducts or changes in local conditions (e.g.

pH) by the bacterial activity. Based on the metabolism of the organisms the mechanisms of bacterial corrosion are classified as follows

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i. Absorption of nutrients (oxygen) by microorganisms.

ii. Liberation of corrosive metabolites or end products of fermentative growth.

iii. Production of sulphuric acid.

iv. Interference in the cathodic process under oxygen-free conditions by obligate anaerobes.

Although this above classification is useful, the mechanisms of microbial corrosion are discussed in terms of either aerobic or anaerobic environmental condition.

Mechanisms of Microbial Corrosion

When a metal is immersed in natural waters, damp soil or other aqueous environments, bacteria and other microorganisms get attached to the surface.

The irreversible adhesion with subsequent multiplication and EPS production leads to formation of the biofilm. The bacteria growing within the biofilm create electrochemical condition at the metal interface that are vastly different from those of the ambient environment. For example, the uptake of oxygen by actively growing microbial colony in the biofilm results in the depletion of oxygen concentration. The poorly aerated surface thus becomes the anode of the cells and the better aerated regions provide the cathodic zone. Thus the mechanism of corrosion is simply the formation of a differential aeration cell.

The anodic reaction, the first reaction that occurs in the dissolution of metal is M —> Mn+ + ne-

For example in case of iron (Fe) Fe --> Fe2+ + 2e-

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These electrons are consumed at nearby cathodic site by the balancing reaction. There are several cathodic reactions, which are possible according to the environmental condition.

Under aerobic condition, the reduction of oxygen to hydroxyl ions occurs, this increases the pH on the surface of the iron, hence hydroxyl and Fe' form an oxide of Fe(OH)2, which is commonly know as rust.

2H20 + 02 + 4e 40H"

Under anaerobic condition or absence of oxygen, the usual cathodic reaction is the reduction of hydrogen

2W + 2e H2

Another mechanism of anaerobic corrosion is the utilization of cathodic hydrogen for the dissimilatory reduction of sulphate by sulphate reducing bacteria (Hassan et al, 1990; McKenzie & Hamilton, 1992; Little et al, 1993;

Videla, 2000). The reaction occurs are as follows.

4 Fe 4 Fe2+ + 8e- anodic reaction 8 H 2O 8 W + 8 OH" dissociation of water 8 W + 8e 8 H cathodic reaction

SO4 ` + 8 H + 4H20 cathodic depolarization by bacteria Fe" + FeS corrosion product

3 Fe" +

6or-r

3 Fe(OH)2 corrosion product

4 Fe + SO4`- + 4 H2O 3 Fe(OH)2 + FeS + 20H" ----overall reaction

Another important corrosion is due to acidic environments, for example an environment enriched by Thiobacillus sp. the microorganism produce highly

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corrosive sulphuric acid in presence of oxygen and water. The chemical reactions are

2 H2S + 2 02 ---> H2S203 + H2O

5 Na2S2O3 + 4 02 + H2O --> 5 Na2S2O3 + H2SO4 + 4 S 4S+602+4H20-->4H2SO4

The sulphuric acid thus produce is responsible for metallic corrosion.

Monitoring and Prevention of Corrosion

In a field of corrosion measurement, control and prevention covers a very broad spectrum of technical activities. With the area of corrosion control and prevention there are technical options such as cathodic and anodic protection, material selection, chemical dosing and the application of internal and external coatings. Corrosion measurement employs a variety of techniques to determine how corrosive the environment is and at what rate metal loss is being experienced. Corrosion monitoring is the practice of measuring the corrosivity of process stream conditions by the use of "probes" that are immersed into the process stream. Corrosion monitoring "probes" can be mechanical, electrical or electrochemical devices. A large number of corrosion monitoring techniques exist. The following are the most common techniques, which are used in industrial application.

i) Weight loss method ii) Electrical resistance (ER) iii) Potentiodynamic monitoring iv) AC impedance

v) Galvanic monitoring

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vi) Biological monitoring vii) Sand/Erosion

viii) Hydrogen penetration monitoring

Corrosion measurement is the quantitative method by which the effectiveness of corrosion control and prevention techniques can be evaluated and this provides the feedback to optimize the corrosion control and prevention. The methods or combination of methods used to deal with corrosion problems are largely dependents on economic consideration, as well as, the practical situation where these methods are used. There are several methods for protection against aqueous corrosion, which are derived from electrochemical principles. Others perform the obvious task of separating the metal from the environment. The success of the latter depends upon the chemical resistance of the protective layer and its chemical properties.

The methods are

i) Use of metals of high purity and special alloy conditions ii) Modification of the corrosive environment

iii) Applications of inhibitors iv) Cathodic protection v) Anodic protection

vi) Use of protective coating like anticorrosive paints, metallic coating, chemical coating etc.

Thus there are numerous ways to protect metals from the corrosion, however the effect of these methods in preventing corrosion last for only short time.

Moreover, some of these control methods do not give proper results, which depend on the compositions of the metal, whereas others are too costly.

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Recently, few studies have dealt with exploiting the use of bacteria in controlling the corrosion. A new approach of corrosion prevention based on regenerative biofilms appears to be a useful tool in controlling the corrosion. The corrosion control using bacterial biofilm emerges as new approach to control MIC.

Aim and Scope of the Present Research

Numerous studies have been carried out on biofilm development during the last few years. However, most of these studies have dealt with microscopic biological observations or the analysis of routine biomass parameters such as dry weight, POC, PON, ATP and protein. Although useful, these studies provide little information on the dynamics of organic matter of biofilms at molecular level.

As compared to this, a few studies have used molecular tools to assess the development of biofilm on metal surfaces immersed in natural environment.

Since such studies provides a better picture of biofilm development, one of the aims of this study was to employ a number of biomarkers such as monosaccharides, amino acids, fatty acids and hydrocarbons to assess the development of biofilm on stainless steel, a commonly used engineering material for marine application.

Generally, bacteria do not attach to surfaces directly. Most often they produce exopolymeric substances so as to remain attached to surfaces. Although a number of functions have been ascribed to EPS, little is know about their production, isolation and characterization. So the other aspect of the biofilm research was to isolate and characterize the exopolysaccharides produced by marine fouling bacteria. This information was then used to assess the role of EPS in microbial adhesion. Further, a few laboratory studies based on a single

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organism grown in nutrient rich media have suggested that EPS production is induced in attached bacteria. However, in natural environment such high nutrients and abundance of a single species is seldomly observed. This question was addressed by analyzing exopolysaccharides of the natural biofilm developed on stainless steel panels.

Biofilm bacteria and their EPS may play an important role in either accelerating or inhibiting the corrosion of steel in marine waters. Corrosion inhibiting biofilms or their metabolic products offers a solution to control corrosion of metal.

Therefore, another aspect of the research presented here was to isolate the corrosion inhibiting bacteria and their metabolite. The research presented in the thesis provides some basic aspects of microbial fouling and corrosion. It is believed that such studies will provide a solid foundation to understand the biofilm formation. This information may be further used to develop suitable methods to control biofouling and corrosion of economically important metals and alloys.

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