REVIEW OF LITERATURE

CHAPTER 3

REVIEW OF LITERATURE

2.1 Culturable Aerobic Heterotrophic Bacteria

Microorganisms present in coastal waters carry out many ecologically

important functions. The primary role of the heterotrophic bacteria is to serve as

carbon source to a variety of grazers and to decompose and mineralize

dissolved and particulate organic matter. They play an important role in the

utilization of organic carbon in the sea (Porneroy, 1974; Azam et ai., 1983;

Ducklow et a/., 1993). Changes in microbial activities caused by changes in

environmental conditions may therefore, cause significant impact on coastal

marine ecosystems.

2.2 Effect of temperature

Temperature is one of the most important factors which is known to

influence the growth and metabolism of aquatic organisms. Kinne (1 970)

reported that an increase in temperature of sea water resulted in an increased

rate of metabolism of the organisms and a reduction of its dissolved oxygen

content.

Information on the impact of thermal effect on marine and estuarine

organisms in temperate waters abounds literature (Videau et a!,, 1979; En'ckson

and Hawkins, 1980; Margreay et a/., 1981; Hall et at., 1982.; Sellner and Kachur,

1987; Langford, 1990; Karas, 1992; Mallin et al., 1994). Like wise, there are

extensive studies describing the distribution of bacterial densities depending on

changes in water temperature, salinity, abundance of nutrients, and physico-

chemical parameters (Jorgensen and Des Marais, 1988; Henneke and d e

Lange, 1990; Fong ef a/., 1993).

Choi et al., (2002) investigated the effects of thermal effluents on HNF

near Hadong Power Station, Korea. Additionally they have carried out

manipulation experiments at relatively high temperature (40°C). Increased

temperature had somewhat inhibiting effects on bacterial abundance and

bacterial population. After 1 h of exposure at 4U°C, bacterial abundance was

reported to decrease substantially (21 % reduction) compared to the control and

was still lower after 2 h of incubation. Further, they were of the view that thermal

effluents inhibited bacterial production and HNF grazing rates and abundance.

The increase in bacterial production, HNF abundance and HNF grazing rates

with distance from the power station were reported to be more pronounced in

summer than winter.

On the other hand Jiao et a/., (2005) while studying the dynamics of

autotrophic picoplankton and heterotrophic bacteria in the East China Sea,

reported that no correlations existed between temperature and heterotrophic

bacteria either in the winter or in the summer.

2.3 Nitrate reducing bacteria

Nitrogen is a major element of many biological metabolites and structural

molecules (amino acids, proteins, nucleic acids). In the marine environment, the

availability of nitrogen often is believed to be key in limiting growth and

productivity (Ryther and Dunstan, 1 971 ). Both f resh water and marine studies

have shown that bacteria may rely on both N H ~ + and NO< for growth and

biomass synthesis and overall they may be significant consumers of inorganic

nitrogen. Mean consumption values of 30% and 40% have been reported for

N H ~ and NO; respectively (Kirchrnan, 2000). Bacterial NOY assimilation is not

a pathway currently considered in pelagic carbon and nitrogen cycle models

(Fasham et al., 1990; Bissett et a/., 1999; Haupt ef al., 1999). Nitrogen can be

lost from ecosystems through the activities of microorganisms that use oxidized

forms of nitrogen as electron acceptors. Paerl and Zehr (2000) reported

bacterial respiration that results in the release of nitrous oxide and N2 gas and

nitrogen fixation to balance this loss.

Denitrifiers utilize nitrate and nitrite as respiratory substrates they can

respire using these oxides of nitrogen as electron acceptors in place of oxygen,

in the process converting, nitrate and nitrite to nitric oxide and nitrous oxide, and

finally to dinitrogen gas (Tiedje, 1988). Although denitrification involves several

semi independent steps that need not function together all the time, denitrifiers

begin the sequence with nitrate and produce varying amounts of the other

products depending on the environmental conditions (Ferguson, 1994).

Ogilvie et a/., (1 997) have studied NRB from estuarine sediments and

reported that the patterns of seasonal dominance within the NRB community

were due to the differential effects of temperature on the affinities for NO3 of the

summer and winter NRB populations.

2.3.1 Molecular identification of nitrate reducing bacteria

Of late molecular approaches have been successfully used to detect and

characterize bacteria and the genes that are important in several aspects of the

nitrogen cycle, including nitrification, dentrification, and nitrogen fixation (Scala

and Kerkhof, 1988; Zehr and McReynolds, 1989; Kirshtein ef al., 1991 ; Voytek

and Ward, 1995; Voytek et a]., 1997; Zehr and Paerl, 1997; Zehr and Hiorns

1998; Scala and Kerkhof 1999; Voytek et al., 1999; Zani et a!., 2000). Allen et

al., (2001) have employed a PCR approach to construct a database of nasA

genes to detect the genetic potential for heterotrophic bacterial nitrate utilization

in marine environments. A nested PCR primed by four degenerate

oligonucleotides was used (Gregory ef al., 2000) to amplify fragments of the

narG gene from Pseudomonas sp.

The narGHJl genes encoding the membrane-bound nitrate reductase

had been described from the Gram negative bacterium E. coli (Blasco et a/.,

1990) and the Gram positive bacterium B. subfilis (Hoffman et al., 1995). The

genes narZYMV encoding an isoenzyme of the E. coli nitrate reductase was

described by Blasco et a/., (1990).

nap provides the biochemical apparatus allowing nitrate respiration to

proceed in the presence of oxygen. In other cases nap is known to catalyse the

first step of an anaerobic denitrification pathway (Berks eta/., 1995; Bedzyk and

Ye, 1999). Flanagan (1999) used PCR based protocol for the detection of the

napA gene encoding the catalytic subunit of nap.

2.4 16s rDNA sequencing

The 16s rDNA gene encodes an RNA molecule that gives structure to

the small subunit of the eubacterial ribosome. The sequence of this gene

serves as a popular model for studies of bacterial evolution and taxonomy. The

DNA sequences of thel6S rDNA gene of many clinical and environmental

isolates are available over the internet through the National Center for

Biotechnology Information and the Ribosomal Database Project. These sites

also provide search algorithms to compare new sequences with their database

(Olsen, 1986).

2.4.1 Phylogenetic relationship

One major issue that haunts microbiologists is how to tackle the

identification of microorganisms. According to Hillis and Dixon (1 991 ) molecular

identification techniques involving the sequencing of the prokaryotic 16s rRNA

gene are useful in establishing coherent phylogenetic relationships at the

species level or higher. However Normand et at., (1996) were of the view that it

often lacks accuracy in discriminating strains within the same species and

sometimes even different species within the same genus (Martinez et a/., 2001).

2.5 Occurrence of P. ruthenica, Staphylococcus sp. and Bacillus sp.

Palaniappan and Krishnamurthy (1 985) reported that Bacillus sp. was

dominant in both water and sediment samples of the Bay of Bengal and Arabian

sea. Prabu et a/., (1991) reported that 7% of the total bacterial counts from Bay

of Bengal were constituted by Bacillus sp. Cavallo et al., (1999) have invariably

encountered Bacillus sp. in t h e Mar Piccolo of Taranto (lonian sea, Italy).

However, Staphylococcus aureus and Pseudomonas aeruginosa were reported

to be occasionally found in water and sediment during the sampling period from

October 1996 to September 1997.

Pujalte et a/., (1999) in their studies on the seasonal fluctuations of the

main heterotrophic bacterial groups in Mediterranean sea water samples

observed Pseudoalteromonas sp . to be predominantly present at higher

temperature regimes.

2.6 Heat shock proteins

HSPs play a key role in cellular metabolism under all growth conditions,

assisting the folding, assembly and translocation of cellular proteins (Craig et

a/., 1993; Georgopoulos and Welch, 7 993; Hartl et al., A 994).

Ellis (1996) defined molecular chaperones as 'proteins that assist the

correct non-covalent assembly of other protein-containing structures in v i m but

are not permanent components of these structures when they are performing

their normal biological functions. However, Hendrick and Hartl (1 993)

alternatively defined HSP as a molecular chaperone that binds to and stabilizes

an otherwise unstable conformer of another protein, and by controlled binding

and release of the substrate protein facilitates its correct fate in vivo, be it

folding, oligomeric assembly, transport to another sub cellular compartment, or

controlled switching between activelinactive conformations. The precise nature

of peptide binding and the factors involved appeared to be chaperone

dependent, whereas ATP binding is important for the release of HSP9O

peptides (Grenert et a/., 1999). Pockley (2001) had reasons to believe that not

all stress proteins function as molecular chaperones and all those that fulfilled

an essential intracellular role and in the extracellular compartment, have t h e

capacity to mediate the induction of peptide-specific immunity.

Besides heat shock, other physiological stimuli also lead to the synthesis

of heat shock proteins. HSP synthesis in Listeria mesenteroides was found to

be stimulated in response to ethanol treatment. Ethanol induced HSPs in

diverse organisms as E. coii, yeast and mammalian cells (Travers and Mace,

1982; Lindquist, 1986). Production of heat shock proteins GroEL (65 kDa) and

DnaK (75 kDa) by Enterococcus faecium at temperature above 52°C was

identified by western blotting analysis (Laskowska et al., 2003). Cold shock

(exposure to 10°C) also was found to induce GroEL and DnaK homologues of

L. mesenteroides. Laskowska et a/., (2003) have reported the induction of DnaK

and DnaJ, CipB and IPB AIB HSPs in E. coli cells by trimethoprim.

Holmquist et al., (1993) reported induction of DnaK and GroEL

homologous proteins by heat shock and long term starvation in Vibrio

vulnificus, Vibrio sp. strain14 and Vibrio sp. DWI. In each Vibrio strain, 60 kDa

protein was reported to react with antibodies against E. coli - GroEL and two

proteins DnaK (69 kDa) and Sis 1 (60-62 kDa) reacted with antibodies against

E. COIL

2.7 Exopolysaccharides

Exopolysaccharides can be classified into two main groups viz., capsular

and slime polysaccharides, based on their general physical structures. Capsular

exopolymers are tightly bound to the cells forming an envelope, whereas slime

exopolymers are loosely bound to the cells and often diffuse into the immediate

environment. Capsular, complex extracellular polymorphic substances have

multiple industrial applications. For example Xanthomonas campestris,

produces xanthan gum which finds application in a number of different food and

industrial applications. Similarly marine biopolymers represent a vast resource

of untapped potential, for only a proportion of marine prokaryotes have been

cultivated (Sandford, 1 984).

EPS can function variously in surface adhesions, in protection from predation

and desiccation, in ionic interactions as enzyme stabilizers or concentrators as

storage or nutrient reserves and as dispersants to release bacteria from nutrient

spent surfaces (Dudrnan, 1977, Weiner et a/., 1995). Frolund et a/., (1995)

showed that EPS are composed of a variety of organic substances:

carbohydrates (Cescutti et al., 1999) and proteins (Dignac et a/., 1998) as major

constituents and humic substances (Frolund et al., 1995), uronic acids and

nucleic acids (Nielson et al., 1996) in minor quantities. Moreillo et al., (2003)

reported that EPS produced by Geobacillus sp. contains hexose: mannose:

Glucose and Galactose in the relative ratio of 0.5:0.0:0.3. EPS are usually

composed of high molecular weight capsular polysaccharides and have many

important functions in nature, including protecting parasitic organisms from

phagocytosis. Their major function in biofilm composition and physiology has

recently gained much attention (Poetra, 1996).

2.8 Properties of EPS

Rosenberg and co-workers (1983) have worked with several EPS with a

number of interesting properties. One group of EPS, isolated from bacteria that

colonize fish, was shown to have drag-reduction properties probably

responsible for enhanced fish mobility. Another type of EPS are ernulsan

(Zuckerberg et a/., 1979) produced most notably by Acinefobacter, believed to

adsorb oil-degrading bacteria from 'spent' hydrocarbon droplets (Rosenberg et

al., 1983). Applications for both of these polymers by industries including

petroleum and shipping are shown in Table I.

Table I. Biopolymers from marine bacteria with current or potential

commercial application*.

Polymer - - - - - - - -

Potential application(s)

Pigments

Polyesters

Complex polysaccharides Adhesions Underwater surface

and related extracellular coatings, bioadhesives

Polymeric substances Drag reducers Oil cleaning and viscosity

(EPS; normally capsular) Emulsan reduction.

Surfactant Dispersing agent,

grinding aid

Alg inate Food, textile, etc.

Metal-binding EPS

Melanins Biotechnology, reporter

gene, cosmetics, dyes,

colorings, sun screens

Poly 3-hydroxy Biodegradable plastics

(e.g., polyhydroxy

alkanates butyrate)

*Adopted from Weiner (1 997)

2.9 Mechanism of adhesion

It is generally accepted that one of the mechanisms by which

microorganisms adhere irreversibly to surfaces or substrata for survival and

growth is production of EPS (Sutherland, 1983; Read and Costerton, 1987; Abu

et a/., 1991). Polysaccharides may serve to anchor the producing bacteria to

substrata by participation of their polyhydroxy groups.

Two theoretical models have been proposed based on the colloid and

surface chemistry to evaluate and understand the interactions between the

surfaces. I. The DLVO (Derjaguin- Landau and Vewey - Overbeek) theory,

which takes into account the attractive Van der Walls interactions and repulsive

electrostatic interactions. 2. Thermodynamic models, in which the adhesive

interaction is treated as an equilibrium process and is described in terms of the

surface free energies of the bacterium substratum, and separating liquid.

2.10 Effect of carbon sources on EPS production

Linton (1990) reported that the amount of EPS production by organisms

was influenced by the carbon sources. Rodrigues and Bhosle (1991) have

reported that among various carbon sources such as glucose, fructose.

rnannitol and glycerol employed in the medium, glucose supported maximum

EPS production. However, Moriello et a/., (2003) reported that sucrose was the

best carbon source compared to glucose, fructose, galactose, trehalose,

mannose and cellobiose for maximal EPS production by Geobacillus sp.

Bacillus thermantarcticus was strongly induced for the production of EPS when

mannose was used as sole carbon source (Nicolaus et al., 2004).

2.1 1 EPS composition

Fletcher and Floodgate (1973) proposed the involvement of two

polysaccharides in t h e adhesive process of a marine pseudomonad. The first or

primary polysaccharide was believed to be produced in trace amounts and was

involved in t h e initial stages of adhesion, whilst the second or secondary

polysaccharide provided a firm attachment to the surface.

Sutherland (1 985) and Whitfield (1 988) reported that chemically,

bacterial EPS are highly heterogeneous polymers containing a number of

distinct monosaccharide and non-carbohydrate constituents, many of which are

strain specific and vary in size from l o 3 to 10'. Depending upon the

components of the repeat units, polysaccharides are usually negatively

charged, sometimes neutral or rarely positively charged (Tait et al., 1986) which

in turn can affect their capacity to interact with other polymers and cations

(Sutherland, 1990).

2.12 EPS purification

In recent years there has been a growing interest in the isolation and

identification of new marine polysaccharides that might have wider applications

in many industrial sectors. Many workers (Boyle and Reade, 1983; Read and

Costerton, 1987; Rodrigues and Bhosle, 1991 ; Bejar et a/., 1996) have shown

that monomeric sugar constituents present in EPS, bonding properties, group

distribution etc., greatly influence the physical and chemical properties of the

EPS.

In all the methods described by Abu et a/., (1991 ), Vincent et a/., (1994)

and Moriello et a/., (2003) the bacterial cells were pelleted by centrifugation and

the EPS in the supernatant was precipitated by addition of 95% ethanol. For

purification and characterization the EPS was subjected to gel filtration

chromatography and GC analysis.

2.13 Viscometric analysis of the EPS

Ha et al., (1991) reported that the apparent viscosity of the EPS from

Butyrivibrio fibrisolvens strains decreased by only 50 to 60%, when the shear

rate was increased from 20 to 1000 s"'.

Vincent et al., (1 994) noted that the EPS produced by Alteromonas strain

HYD 1545 was a polyelectrolyte and the viscosity of its solutions depended on

the ionic strength. The decrease in viscosity observed with increasing NaCl

concentrations and the effect of ca2' in decreasing the viscosity at low ca2'

concentrations were thus claimed to support a model in which the

polysaccharide carries anionic groups.

Bejar et a/., (I 996) reported that a moderately hemophilic eubacterium

Vibrio eurihalina produced EPS that showed pseudoplastic non-newtonian

behaviour quite resistant to high ionic strength and temperature. Rheological

behaviour of the EPS was reported to depend on sugar residues, linkage

pattern and occurrence of intra and intermolecular interactions. They also

reported that viscosity and pseudo plasticity of the polymer solutions were

influenced by the nutritional conditions in which the microorganism was grown.

Vicente-Garcia et a/. , (2004) reported that the cyano bacterium Phormidium 94a

synthesized EPS that showed Newtonian behavior at lower EPS concentrations

and pseudoplastic nature at higher concentrations.

2.14 Effect of ionic concentration on the viscosity of EPS

The EPS produced by HYDI-1545 strains showed stabilization and

increase in viscosity with increase in calcium chloride concentrations d u e to

inter molecular bonds or the polymer conformational change. Decrease in

viscosity due to addition of sodium chloride to EPS solution was reported by

van den Berg et a/., (1 995) for the EPS produced by Lactobacillus sake 0-1.

Calvo et a/., (1998) have studied the effects of monovalent and divalent

cations on the rheological behaviour of Halomonas eurihalina EPS and showed

the highest viscosity value. A loss of sulfate content seemed to correlate with

the increase in viscosity. Addition of hydrophobic substrates to culture media

were reported to lead to changes in chemical composition and emulsifying

activity of the EPS.

2.1 5 Structural studies

Compositional and structural studies of pure EPS produced by different

microorganisms can shed more light on the mechanism involved in the

adhesion process. FTl R spectra of EPS produced by Alteromonas macleodii

subsp. fiiensis exhibited a broad 0-H stretching variation (Raguenes et a/.,

1996). Based on FTlR spectral analysis Abu eta/., ('l991) reported that the EPS

produced by S. colwelliana contained polyhydroxy compounds. Ha et a/. , (1 991 )

reported that FTlR spectrum of the EPS produced by Butyrivibrio fibrisolvens

strains (F2d, CF3, CF3a, CFS, and H l Ob) exhibited a broad 0-H stretching, a

minor C-H stretching and several C-0 stretching and COOH structure. The

FTl R spectrum of the EPS produced by Pseudomonas alcaligenes showed

strong peaks for 0-H stretching, C=O stretching and C-0 stretching, indicating

t h e presence of these functional groups in the EPS. Vincente- Garcia et a/.,

(2004) have reported that the IR spectrum of the EPS produced by Phormidium

9423 exhibited OH stretching and CH stretching frequency and carboxylated

group. Similar observation was also reported by Nichols et al., (2004) in an

Antarctic marine bacterium Pseudoalteromonas sp.

Abu et a/., (1991) reported that EPS produced by S. co/welliana

predominantly consisted of glucose. EPS produced by V. fischeri was shown

(Rodrigues and Bhosle, 1991) to consist of glucose, galactose, mannose,

arabinose and rhamnose in t h e ratio of 3.4:3.7:1.5:0.6:0.7. Bejar ef al., (1 996)

noted that the relative sugar composition of the EPS produced by H. eurihalina

was greatly influenced by the composition of the growth medium. The EPS

contained rhamnose and glucose, mannose in a lower proportion and an

unknown additional compound. Wai et a/., (1998) analysed the EPS produced

by V. cholerae and reported it to be composed of N-acetyl D glucosarnine, D-

mannose, 6-deoxy D-galactose and D-galactose in the molar ratio of

7.4:10.2:2.4:3.0. EPS produced by A. infernus was found to be composed of

galactose, glucose, rhamnose, mannose, glucoseUA galactoseUA, lactose

glucoseUA in the ratio of I 5.2: 16.3:0.2:0.0: 10.9:6.3:0.0 (Raguenes et a/. ,

2003). Nichols et a/., (2004) have reported that EPS produced by Geobacillus

sp. isolate CAM 2005 and isolate CAM 036 composed of arabinose, ribose,

rhamnose, fructose, galactose A, mannose, galactose, glucose, galactoseNAc

in the molar ratio of 1.0:0.3:1 . I :0.4:6.4:0.3:13.3:12.8:0.1 and 1.0:0.0:0.0:7.2:

5.8:0.4:6.1:2.6 respectively.

2.16 Biofilms

Biofilms can be regarded as microbial consortia in which different

microbial strains and species efficiently cooperate in order to protect

themselves against environmental stresses, and to facilitate efficient nutrient

uptake and utilization. Most often, biofilms are unwanted and related to diverse

problems as microbially induced corrosion of oil-rigs and food, drinking water

contamination, (Notermans e f a/., 1991 and Flint et a/., 1997) dental caries and

periodontal diseases (Marsh and Martin, 1992) and a variety of biomaterial

centered infections in man, pipelines and reduced heat transfer efficiency,

(Turetgen, 2004). Biofilms most often help in nutrient cycling of carbon and

minerals such as N, P and sulphur ions. In microorganisms capable of nitrogen

fixation, biofilms protect the nitrogenase enzyme from inactivation by

atomospheric oxygen (Chan et a/., 1997; Bazylinski et al., 2000).

Bacterial colonization is known to occur on both living and non living

surfaces like contact lenses, ship hulls, dairy and petroleum pipelines, rocks in

streams and all varieties of biomedical implants and transcutaneous devices

(Costerton et a/., 1987; Carpentier and Cerf, 1993; Elder et a/., 1995; Costerton

eta/ . , 1995; 1999). In many circumstances, the surface might also be a nutrient

source, such as cellulose in the paper industry.

2.17 Mechanism of biofilm formation

Gottenbos et al., (2000) have described various steps involved in the

formation of biofilm. The mechanisms involved are: 1. When organic matter is

present, a conditioning film of adsorbed components is formed on the surface

prior to the arrival of the first organisms. 2. Microorganisms are transported to

the surface through diffusion, convection, sedimentation or active movement. 3.

Initial microbial adhesion occurs. 4. Attachment of adhering microorganisms is

strengthened through exopolymer production and unfolding of cell surface

structures. 5. Surface growth of attached microorganism and continued

secretion of exopolymers. 6. Localized detachment of biofilm organisms caused

by occasionally high fluid shear or other detachment forces operative. Three

basic ingredients of biofilm reported are microbes, EPS and surface. If one of

these components is removed from the mix, a biofilm does not develop.

By using phase-contrast microscopy to analyze a collection of mutants

that are defective for surface attachment, flagella and type-lV pili were shown to

play important roles in the early events in biofilm development by P. aeruginosa.

These observations are consistent with previous studies that strongly hinted at

the roles of these cell surface structures in the adhesion of bacteria to a wide

variety of surfaces (De Wager et a/., 1987; Doig et a/., 1988; Ramphal et a/.,

1991 ; Simpson et a/., 1992; Prince, 1992; Grant et a/., 1993; Fletcher, 1996;

Smyth eta/., 1996). In addition to flagella and pili, Makin and Beveridge, (1996)

have reported that changes in lipopolysaccarides result in altered attachment

behavior. In particular, it was shown that in P. aeruginosa strains that make two

types of lipopolysaccarides (A and B band), loss of B-band LPS by mutation

reduced attachment to hydrophilic surfaces and increased attachment to

hydrophobic surfaces.

Watnick et a/., (1999) reported that V. cholerae require flagella-mediated

motility, the mannose-sensitive type-lV pilus encoded by the mshA locus, and

synthesis of the major EPS to form a wild-type biofilm. Further, they have

shown that flagella and type-IV pili were not absolutely required for initial

attachment to the surface, but they greatly facilitated this process in

V. cholerae.

Recent studies by Cramton (1999) and colleagues have shown that

S. aureus, like S. epidermidis, has the ica locus, which encodes the functions

required for intracellular adhesion. These data suggest that the early stages in

biofilm formation may be similar between these two organisms.

Enterococci are important pathogens in device-related infections and like

other Gram-positive organisms, the formation of enterococcal biofilms on

medical implants is an increasing clinical issue (Barie et a/., 1990; Stickler et a/.,

1993; Venditti et a/., 1993). As for other organisms, growth in a biofilm confers

Enterococcus sp. with an increased resistance to antibiotics (Foley and Gilbert,

1997).

2.18 Surface conditioning

Primary contact generally occurs between an organism and a

conditioned surface and the hydrophobicity of the latter can vary greatly

depending on the molecules in the conditioning film. Wang et a/., (1993)

demonstrated that primary adhesion of S. epidermidis to polyethylene disks was

enhanced in the presence of surface-activated platelets and reduced by

adsorbed plasma proteins relative to uncoated polyethylene. Using

polymethylmethacrylate as a substratum, Herrmann et a/., (1988) showed that

the adhesion of coagulase-negative Staphylococci was enhanced when the

surface was coated with various plasma proteins, including fibronectin.

A net repulsion between two surfaces can be overcome by specific

molecular interactions mediated by adhesins located on structures extending

from the cell surface, such as pili (An et al., 2000; Boland et a/., 2000). The

longevity of primary adhesion was reported to depend on the sum total of all

these variables, but surface chemistry pushed the equilibrium in favor of

adhesion by predicting that organic substances in solution did concentrate near

a surface and that microorganisms tended to congregate in nutrient-rich

environments.

2.19 Biofilm estimation

Biofilm formation may be determined in several different ways but most

frequently it was done by the tube test (Christensen eta/., 1982; 1985) in which

the bacterial film lining a culture tube is stained with a cationic dye and visually

scaled, or by the microtiter plate test (Christensen et a/., 1985) in which the

optical density of the stained bacterial film is determined spectrophotometrically.

Stepanovic ef a/., (2000) have quantified the biofilm formation of

Staphylococcus sp. using tube test and microtiter plate test wells. The growth of

31 coded Listeria monocytogenes strains were measured by their growth in

PVC microtiter plate wells and biofilm formation was indirectly assessed by

staining with 1% crystal violet and measuring crystal violet absorbance, using

destaining solution. Since cellular growth rates and final cell densities did not

correlate with biofilm formation, they have indicated that differences in biofilm

formation under the same environmental conditions were not due to growth rate

differences (Djordjevic etal., 2002). Vasudevan ef al., (2003) have reported that

the individual strains of S. aureus varied in their biofilm forming ability on the

microtiter plate wells. Narisawa et a/., (2005) estimated biofilm formation by the

cell number of E. coli in microtiter plate wells.

2.20 Structure of biofilms

With the advent of inexpensive high-speed computers and increasingly

sophisticated software, great strides have been made leading to transformation

of traditional microscopic observation into the discipline of analytical imaging. Of

the many recent digital imaging devices now available, CLSM. (Shotton, 1989)

has proven particularly well suited for the study of microbial biofilms. CLSM

allows the nondestructive, in situ analysis of living, fully hydrated biofilms

without the need for harsh chemical fixation or embedding techniques. This

feature, coupled with the ability to digitally extract optical thin sections from

specific biofilm locations, free from out-of-focus optical interference, permits the

direct examination of complex xy and xz spatial and chemical relationships that

exist between bacteria, their extracellular products, and their environment

(Wilson, 1990).

Of late, CLSM is increasingly used to provide detailed information on cell

morphology, cellular metabolism, cell phylogeny, microenvironment chemistry,

as well as the physical architecture and chemistry of the biofilm matrix and

associated polymers. Earlier studies using both light and electron microscopy

had suggested that microbial biofilms were heterogeneous in structure (Bakke

and Olson, 1986; Costerton ef a/., 1995). The episcopic nature of CLSM also

enables the examination of biofilms cultivated on non transparent surfaces

(Lawrence et a/., 1991 ; Caldwell et a/., 1992).

2.20.1 Biofilm architecture

The biofilm "architecture" (Lawrence et a/., 1991), is often species-

specific for pure-culture biofilms, as well as substrate-specific for certain

microbial consortia (Wolfaardt et at., 1994). Notably, heterogeneous biofilm

architecture does not appear to be the sole function, or the result, of mixtures of

different organisms with variable growth habits inhabiting a similar niche. Pure-

culture biofllms also accommodate many of the heterogeneous features found

in mixed-culture biofilms, suggesting a fundamental relationship between biofilm

structure and in situ function (Venugopalan eta/. , 2005). Many workers (Korber

et a/., 1993; 1993; 1994; 1994; Lawrence and Korber, 1994) have claimed that

basic functional requisites underlie biofilm structure, and that structural diversity

actually reflects the adaptation of unicellular organisms to a diverse range of

physical, chemical, and communal circumstances on surfaces.

2.20.2 Biofilm thickness and Coverage analysis

Lawrence et a/., (1991) in their elegant studies have shown that

variability in the distribution of biomass of 24 h P. fluorescens, P. aeruginosa,

and V. parahaemolyticus biofilms was governed by the tendency for biofilm

bacteria to form aggregates at different horizontal and vertical sites and the

area of highest cell density varied among different species. P. aeruginosa

biofilm was characterized by a dense cell mass located near the biofilm base,

whereas V. parahaemolyticus biofilm exhibited an inverted pyramid structure,

with the most biofilm biomass located nearest the biofilm-liquid interface.

Significantly, studies of this nature (Lawrence et a/., 1991; Korber ef a/., 1992;

Korber ef a/., 1994) have repeatedly confirmed that living biofilms were highly

hydrated, with 50-90°h of the total area at each sectioning depth consisting

either of polymer and / or void space.

Sectional analyses of each biofilm layer ( I-pm thickness) carried out by

Takenaka ef a/., (2001) demonstrated the three dimensional development of

biofilms and revealed that the biofilms were 9 pm in height after 12 h. The

biofilm thickness of Citrobacter sp. on glass slides showed 10.5 pm after 72 h,

45.3 pm after 96 h and 121.8 pm after 120 h measured by CLSM (Allan ef a/.,

2002).

2.21 Biofilm formation on metallic and non metallic surfaces

Sonak and Bhosle (1 995) reported the growth of biofilm formation on four

surfaces. The number of Vibrio cells attached to aluminum panels was

reportedly high, probably because of the roughness of the aluminum panels. It

was their observation that rough surfaces being more conducive always

supported better bacterial attachment than smooth surfaces. Bott (1993)

reported that electro polished stainless steel and fluorinated enzyme propylene

and glass tubes were less amenable to biofilm growth than untreated stainless

steel 31 6.

The biofilm formation of L. monocytogenes on both hydrophilic (stainless

steel) and hydrophobic (Polytetrafluroethylene, PTEF) surfaces at different

temperature and growth phases was analyzed (Chavant et a/., 2002) and the

biofilm growth was reported to be faster on the hydrophilic surfaces compared

to hydrophobic surfaces and were negatively correlated with growth

temperature.

Martino et a/., (2003) reported that the Klebsiella pneumoniae (F. 3051)

reference strain expressing only type I fimbriae adhered slightly to glass and

polystyrene. K. pneumoniae IA 565 and CF3 3097 reference strains producing

type 1 and type 3 fimbriae showed efficient adherence to both glass and

polypropylene and biofilm formation on polystyrene. A correlation was observed

between adherence properties to glass or polypropylene and biofilm

development on polystyrene indicating that bacterial adherence to non

biological surfaces constituted the first step in K. pneumoniae biofilm

development.

2.22 Chlorine

Chlorine is generally used as a powerful popular disinfectant and is

easily available for control of bacterial growth in drinking water and cooling

water systems. Sodium hypochlorite is the oldest and most widely used of the

chlorine compounds employed in chemical disinfection. It has been shown

experimentally that the bactericidal action of chlorine releasing agents results

from an oxidative interaction with the sulfydryl groups on certain enzymes in the

cell membrane or protoplasts.

Chlorine is known to exert disruptive effects on subcellular processes

(Haas and Gould, 1979) including the following (i) in vitro formation of

chlorinated derivatives of purine and pyrimidine nucleotide bases (Dennis et a/.,

1979). (ii) oxidative decarboxylation of amino acids (Pereira et a/., 1973) and

other naturally occurring carboxylic acids (Larson and Rockwell., 1979). (iii)

lnhibition of enzymes in void in intermediatory metabolism (Camper and

Macfeters, 1979). (iv) lnhibition of protein biosynthesis (Benarde et al., 1967) (v)

Introduction of single and double stranded lesions in the bacterial chromosome

(Shih and Lederberg, 1976). (vi) Production of bacterial mutations. (vii)

lnhibition of membrane mediated active transport processes and respiration

activity (Camper and Mcfeters, 1979) and (viii) Uncoupling of oxidative

phosphorylation accompanied by leakage of macromolecules from the cell

(Wenkobachar et a/., 1975; 1977).

2.22.1 influence o f added chlorine on detachment of P. ruthenica cells

Pitts et a/., (2003) developed a quantitative spectrometric method to

measure the removal and killing efficacy of antibiofilm agents using the

respiratory indicator 5-cyano-2, 3-ditolyl tetrazolium chloride. The percentage

reductions at 100 and 1000 mg/l concentrations were significantly greater than

at the 1 mg/l concentration for both Pseudomonas and Staphylococcus biofilm.

2.22.2 Biofilm detachment and viable counts

Both for P. aeruginosa and S. epidermidis the analysis of variance

reportedly showed that the mean log reductions at the 100 and I000 mg/l were

statistically significantly greater than at the 1 mgll concentration. Gilbert ef a/.,

(2001) used two cationic biocides phenoxy ethanol and cetrimide for removal of

biofilm grown in microtiter plate wells by end turbidity endpoint and viable count

method. Pitts et a/., (2003) reported that sodium hypochlorite was used for the

removal of biofilm attached to the microtiter plate by viable count method. The

conventional log reduction response based on viable cell counts provided

essentially the same trends as the percentage reduction in respiratory dye 5-

cyano-2-3 ditolyl tetrazolium chloride absorbance.

2.23 Observations of biofilm detachment by CLSM

Takenaka et a/., (2001) reported that when 12 h old biofilms were

exposed to ciprofloxacin at minimum bactericidal concentration of 6.26 pglml for

90 min, all the organisms were killed, but some organisms in 24 h old biofilms

with thicker and denser structure were still alive after exposure for 120 min.

Lindsay ef a/., (2002) reported the differential efficacy of a chlorine

dioxide against single and binary biofilms of a dairy associated Bacillus cereus

and P. fluorescens. In their work the survival of P. fluorescens M2 cells after

exposure to chlorine dioxide was observed to be enhanced by the presence of

B. cereus in binary biofilms. However, 5. cereus apparently showed increased

susceptibility to chlorine dioxide in the presence of P. fluorescens.

According to Ren et a/., (2002) furanone was found to inhibit both the

growth of B. substilis and its swarming motility in a concentration dependent

way. In addition, as shown by CLSM, furanone was reported to inhibit the

biofilm formation of B. substilis. At 40 pg ml-', furanone decreased the biofilm

thickness by 25%, decreased the number of water channels, and reduced the

percentage of live cells by 63%.

Lomander et a/., (2004) examined the effectiveness of chlorine as a

killing and or biofilm removal agent on stainless steel surfaces of three different

morphologies. They have found that overall the growth trends of the biofilm on a

polished stainless steel surface followed those on a scribed surface.

2.24 Planktonic biofilrn inhibition

Cochran et al., (2000) reported that biofilm of P. aeruginosa attached to

alginate gel beads in sparse, thin biofilm exhibited reduced susceptibility to

monochloramine and hydrogen peroxide compared with same plantkonic cells

of the microorganisms. Disinfection rate coefficient for planktonic bacteria

averaged 0.551 mg I-' ml-' for monochloramine. The corresponding values for

24 h old biofilms reportedly were 0.291 mg/l and 9.2 X l o 5 mg-' mi-' for

monochloramine and hydrogen peroxide respectively. They have reported no

correlation between biofilm susceptibility and biofilm initial area cell density.

While studying the efficacy of alkaline hypochlorite and chlorosulfamates

on two species of biofilms of P. aeruginosa and K. pneumoniae, Stewart eta/. ,

(2001) observed superior penetration by chlorosulfamate due to reduced

reaction with constituents of the biofilm. Poor biofilm killing despite

measurement of effective typical penetration of the antimicrobial agent into the

biofilm, they claimed, demonstrated that bacteria in the biofilm were protected

by some mechanism other than simple physical shielding by the biofilm matrix.

Turetgen (2004) compared the efficacy of free residual chlorine and

monochloramine against biofilms in model and full scale cooling towers and

reported that monochloramine was significantly more effective than free chlorine

against cooling water biofilm. Scher ef a/., (2005) investigated the role of a

biofilm at the inter surface between air and pellicle in the susceptibility of

S. enferica serovar typhimurium to stress conditions. They reported that pellicle

cells were significantly more resistant to chlorination, wherein surrounding

matrix conferred protection against the reactive sodium hypochlorite.

2.25 Biofilm detachment by chemical biocide

Ortho-phthalaldehyde (OPA) is a new type of disinfectant that is claimed

to have a highly potent bactericidal and sporicidal activity (McDonnel and

Russel, 1999; Cabrera-Martinez ef a/, 2002). Simes ef a/., (2003) used OPA to

control biofilms formed by P. fluorescens on stainless steel surfaces. The higher

concentrations of OPA and longer exposure were needed to inactivate

P. fluorescens than planktonic populations, thus denoting that sessile bacteria

have a reduced susceptibility to OPA.

2.26 Biofilm detachment by laser treatment

The impact of laser assessment in a biofilm forming bacterium

Pseudoalteromonas carrageenovora was studied by Nandakumar et a/., (2003)

using flow cytometric system and the results revealed that in a flowing system,

low power pulsed larger irradiations could reduce the bacterial attachment

eventhough it did not cause significant mortality.

Bott (2000) demonstrated that control of biofilm formation and the

removal of established biofilms on the inside of tubes could be achieved by

ultrasound technology.

2.27 Effect of copper complexes on biofilm formation

Copper and copper alloys have long been successfully used in marine

applications because of good mechanical strength and workability, corrosion

resistance, good thermal conductivity and resistance to macro fouling.

Application includes distribution and piping systems, heat exchangers and

intake screens (Moreton and Glover, 1980; Blunn, 1987; Kirk, 1987; Little et a/.,

1996).

Teitzel and Parsek, (2003) reported the effect of heavy metals copper,

lead and zinc on biofilm and planktoinc P. aeruginosa. It was reported that

biofilms were anywhere from 2 to 600 times more resistant to heavy metal

stress than free swimming cells. When planktonic cells at different stages of

growth were examined it was found that logarithmically growing cells were more

resistant to heavy metals than stationary phase cells. However, biofilm were

observed to be more resistant to heavy metals than either stationary phase or

logarithmically growing planktonic cells. The exterior of the biofilm was

preferentially killed after exposure to elevated concentrations of copper and the

majority of the swimming cells were near the substratum. A potential

explanation offered by Teitzel and Parsek. (2003) for this phenomenon was that

the extracellular polymeric substances that encase a biofilm may be responsible

for protecting cells from heavy metal stress by binding the heavy metals and

retarding their diffusion within the biofilm.

Barranguet eta/. , (2000) while studying effects of heavy metal on aquatic

biofilms, showed that Cu affects the physiology of phototrophic organisms.

Massieux et a/., (2004) investigated the effects of copper on the structure and

physiology of fresh water biofilm microbial communities. For this purpose,

biofilms that were grown during 4 weeks in a shallow, slightly polluted ditch

were exposed to the copper concentrations. Denaturing gradient gel

electrophoresis revealed changes in the bacterial community in all aquaria. The

extent of change was related to the concentration of copper applied, indicating

that copper directly or indirectly caused the effects. However, they were of the

opinion that copper did affect the physiology of the phototrophic community, as

evidenced by the fact that a decrease in photosynthetic activity was detected in

the treatment with the highest copper concentration.