Biofouling

ISSN: 0892-7014 (Print) 1029-2454 (Online) Journal homepage: http://www.tandfonline.com/loi/gbif20

Influence of Surface Characteristics and

Microstructure on Adhesion of Bacterial Cells onto
a Type 304 Stainless Steel

R P George , P Muraleedharan , K R Sreekumari & H S Khatak

To cite this article: R P George , P Muraleedharan , K R Sreekumari & H S Khatak (2003)

Influence of Surface Characteristics and Microstructure on Adhesion of Bacterial Cells onto a Type
304 Stainless Steel, Biofouling, 19:1, 1-8, DOI: 10.1080/08927010290031017

To link to this article: http://dx.doi.org/10.1080/08927010290031017

Published online: 09 Sep 2010.

Submit your article to this journal

Article views: 139

View related articles

Citing articles: 21 View citing articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=gbif20

Download by: [Charles Darwin University] Date: 08 July 2017, At: 01:19
Biofouling, 2003 Vol 19 (1), pp 1–8
Influence of Surface Characteristics and Microstructure on
Adhesion of Bacterial Cells onto a Type 304 Stainless Steel
R P GEORGEa,*, P MURALEEDHARANa, K R SREEKUMARIb and H S KHATAKa
a
Corrosion Science and Technology Division, Materials Characterization Group, Indira Gandhi Centre for Atomic Research, Kalpakkam 603 102, India;
b
Joining and Welding Research Institute, Osaka University, Japan 567-0047
(Received 24 June 2001; in final form 8 March 2002)
A study was carried out to understand the influence of the
surface characteristics/microstructure of a type 304
stainless steel on bacterial adhesion by exposing sol-
ution-annealed, sensitized and air-oxidized stainless steel
specimens in a culture of Pseudomonas sp. in dilute
nutrient broth. Epifluorescence microscopy of the exposed
surfaces revealed that the pattern of adhesion as well as
number density of bacterial cells was different depending
on the metallurgical condition of the substratum. Among
the specimens with different microstructures, the sensi-
tized specimens had the highest bacterial density,
followed by the solution annealed and the oxidized
specimens. The same trend was shown by the total viable
counts on the various surfaces, estimated by a plate count
technique. The study assumes significance in the context
of the widely reported observation of preferential attack of
the welded region during microbiologically influenced
corrosion of fabricated components.
Keywords: microbial biofilm; bacterial adhesion; 304 stainless steel;
microstructure; sensitization; oxidation
INTRODUCTION
Austenitic stainless steels (SS) are prone to micro-
biologically influenced corrosion (MIC) when in
contact with natural waters. This is especially true in
cooling water systems that provide ideal conditions
such as highly oxygenated water, good exposure to
sunlight and air, temperatures in the range 27 –608C,
and a pH between 6 and 9 for the growth of micro-
organisms (Dexter, 1992; Borenstein, 1994). The MIC
is caused by the presence and activities of micro-
organisms on the metal surface. The most important
feature resulting from microbial colonization of a
*Corresponding author; fax: þ91-4114-80301, 80060, 80081; e-mail: rani@igcar.ernet.in
ISSN 0892-7014 print/ISSN 1029-2454 online q 2003 Taylor & Francis Ltd
DOI: 10.1080/08927010290031017
metal surface is the formation of biofilm (Fleming &
Geesey 1991; Rao et al., 1997). Such films and
associated bacteria form complex biological systems
that can cause several chemical changes at the
metal/biofilm interface (Costerton et al., 1987;
George et al., 2000). The biofilm substantially alters
the chemistry at the metal surface, often lowering the
pH and reducing oxygen tension. Therefore, bulk
solution chemistry provides little information con-
cerning chemical events at the interface between the
solution and the metal. In other words, microorga-
nisms can ‘engineer’ a thermodynamic state con-
ducive for corrosion at the metal/biofilm interface
(Walsh et al., 1994). In addition, they can alter the
kinetics of corrosion reactions at the surface of the
material. Microbes localize and accelerate corrosion
in several ways, e.g. they form concentration cells
and differential aeration cells, they reduce polari-
zation and directly oxidize/reduce metallic atoms/
ions, they produce corrosive metabolic by-products
and promote the passive co-accumulation of anions
that disrupt passivating films. MIC is manifested in
many forms of localized corrosion, which include
pitting, crevice corrosion, under deposit corrosion
and stress corrosion cracking (Little et al., 1999).
Corrosion is influenced by the microstructure,
therefore it is expected that microstructure also plays
a role in MIC. This is evident from the analyses of
many failures due to pitting corrosion in power
plants, chemical processing plants and pulp and
paper mills, which indicate that the welded regions
of SS components are preferentially attacked by
microbes (Syrette & Colt, 1983). Pits in such cases
often exhibit a special morphology having very small
2 R P GEORGE et al.
entrance and exit penetrations with very large
subsurface cavities. Pits may be associated with the
attack of weld material, generally near the fusion line
or in the heat-affected zone. Sensitization has been
recognized as a factor related to the extent of pitting
in the heat-affected zone (Ibars et al., 1992). Two-
phase weld metal appears to be the most susceptible,
although the relative susceptibilities of the two
phases, austenite and delta ferrite are not clearly
definable. As shown in the investigations referred to
above, in some situations the austenite is preferen-
tially attacked. In other situations, the delta ferrite is
removed and the austenite is not attacked. Of the
seven cases showing pitting in the weld metal
studied by Borenstein (1988), two demonstrated
preferential attacks in the austenite, whereas five
others demonstrated preferential attack in the ferrite.
The behaviour of weld metal also has been discussed
by other authors (Soracco et al., 1988; Licina, 1989).
The question often asked is ‘why do welds
undergo preferential attack during MIC?’ It is well
known that metal surfaces are extremely hetero-
geneous even in wrought conditions. Regional
differences (at all scales, from sub-micron to
macroscopic) are so well defined that local anodes
and cathodes form and corrosion progresses even in
apparently monolithic structures. Welding alters the
material characteristics. Topological, chemical and
microstructural changes caused by welding may
increase the corrosion susceptibility and introduce
the potential for galvanic corrosion or other electro-
chemical events at the welded region. The altered
surface condition seems to influence, at least in the
initial stages, preferential attack of the weld during
MIC. The results of electrochemical tests carried out
on welds of different superficial finishes (Buchanan
et al., 1990) using different bacterial concentrations
have shown that the ‘as-welded’ state is most
sensitive to attack.
Attempts have been made to correlate residual
stress, cold work and sensitization with MIC
susceptibility. Stein (1991) pointed out that sensiti-
zation of type 304 and 316 SSs does not affect their
susceptibility to MIC. However, type 304 SS is
susceptible to MIC when it is cold worked. It has also
been noted that, when high annealing temperatures
are used to eliminate or reduce deformation effects,
this improves the resistance of austenitic stainless
steels to MIC. However, tests which were carried out
in the laboratory (Mele et al., 1991; Videla et al., 1991)
showed that there is a clear relationship between the
sensitization state of a type 304 SS and the pitting
potential measured in a biogenic sulphide-contain-
ing electrolyte (SRB metabolites). However, all these
studies have considered the problem from the
corrosion angle only.
It is worthwhile to consider whether the micro-
structural and microchemical heterogeneity present
TABLE I Chemical composition of type 304 SS
Element C Si Mn Cr Ni Mo Fe
Wt% 0.07 0.48 1.55 18.5 9.3 – Bal*
*Bal ¼ balance
in a weld facilitates preferential attachment and
proliferation of microorganisms on the metal surface.
Since adhesion of microbial cells is the first step in
MIC, it is very important to examine whether initial
attachment is influenced by any of the metallurgical
features of the metal substratum as well as
subsequent growth and proliferation. Several
workers have reported the importance of motility
in bacterial transport to the surface (Piette & Idziak,
1991; Mueller et al., 1992; Camper et al., 1993). Once
bacteria reach the surface, subsequent attachment
can be influenced by surface topography and surface
chemistry. The goal of this investigation was to
understand how bacterial adhesion on stainless steel
surfaces was affected by metallurgical changes such
as sensitization and oxide formation introduced
during welding.
MATERIALS AND METHODS
Specimen Preparation
The chemical composition of the AISI type 304 SS
used in the study is given Table I. Coupons ð30 £
15 £ 1:6 mmÞ were used in the following conditions:
i) solution-annealed, 11008C/30 min, water quench;
ii) sensitized, 6258C/24 h, air cool; iii) oxidized,
9008C/5 min, air cool. The solution-annealed and
sensitized specimens were ground to a diamond
finish after heat treatment while the oxidation was
carried out on specimens that were polished to a
diamond finish. The coupons were cleaned using a
detergent solution, degreased with ethanol, and air-
dried.
Evaluation of Degree of Sensitization
The degree of sensitization in the specimens heat
treated at 6258C for 24 h was evaluated using the
electrochemical potentiokinetic reactivation (EPR)
technique (Cihal, 1980). The sample was anodically
polarised in 0.5 M H2SO4 and 0.01 M NH4SCN
solution from the open circuit potential (OCP) to
þ 200 mV, SCE at a scan rate of 6 V h21 and after
holding at the passivation potential for 2 min, the
scan was reversed back to OCP. The presence of a
current peak in the reverse scan indicated that the
material was sensitized and the ratio of the peak
current density in the reverse scan to that in the
forward scan gave a measure of the degree of
 ADHESION OF BACTERIA TO SS 304
TABLE II Results of morphological and biochemical tests
Tests Response
Gram staining Gram-negative small rods
Motility þ
Oxidase þ Short term and long term exposure studies
Catalase þ
Growth at 458C þ For short term studies three coupons each of
Nitrate reduction þ (forming NO2/NH3)
Citrate utilization þ solution-annealed, sensitized and oxidized stainless
Growth on cetrimide agar þ steel were exposed in the ‘phosphate buffer’
Indole production þ maintained at 32 ^ 28C and were withdrawn after
sensitization. An EPR test was also conducted on
oxidized specimens.
Inoculum Preparation and Exposure of Specimens
The strain of bacterium (Pseudomonas sp.) used in the
study was isolated from a natural biofilm formed on
Type 304 SS coupons exposed to fresh water in an
open reservoir. The biofilm on the SS coupon was
dispersed into sterile water using a water bath
sonicator as described by Mittleman and Geesey
(1987). The length of sonication for optimum re-
covery of cells was found to be 5 min. Pseudomonas sp.
was isolated from the biofilm suspension using
Pseudomonas isolation agar. The colony morphology
on common nutrient medium was smooth, circular
and transparent. The blue green colouration of the
colonies was attributed to the production of
pyoverdin and pyocyanin pigments (Palleroni,
1984). Identification was carried out by morphologi-
cal and physiological characteristics and by bio-
chemical tests (Table II) (Krug & Hold, 1984). All the
morphological and biochemical characteristics clo-
sely matched those of Pseudomonas aeruginosa.
However, this species was referred to as Pseudomonas
sp. in this paper, as positive identification was not
done.
A dilute nutrient culture for exposure studies was
prepared by inoculating 0.1% nutrient broth with
0.1 ml of 24 h culture of Pseudomonas sp. in 100%
nutrient broth. This culture in dilute nutrient
medium was recultured and used for long term
exposure and henceforth it will be referred to as
‘nutrient culture’. A dilute nutrient medium was
used for the study in order to avoid pelagic growth of
bacteria and to favor biofilm formation (White et al.,
1996).
For short term exposure studies, a suspension of
bacterial cells in phosphate buffer (0.0425 g KH2PO4,
0.19 g MgCl2 l21) was used. One hundred and fifty
ml of 24 h Pseudomonas sp. culture (100% nutrient
broth) with a bacterial density of 5:2 £ 107 cfu ml21
were cooled to 48C and centrifuged at 12,000 rpm
ðg value , 15; 000Þ for 30 min to pellet the cells. The
pellet was washed twice with buffer and finally
resuspended in 150 ml of buffer. The density of cells
3
was 5 £ 107 cfu ml21 ; confirming retention of viabi-
lity. This medium will be referred to henceforth as
‘phosphate buffer’.
1 h and 6 h. For long term studies 4 coupons each of
solution-annealed, sensitized and oxidized stainless
steel were exposed in the ‘nutrient culture’ main-
tained at 32 ^ 28C to study attachment, subsequent
growth and biofilm formation of Pseudomonas sp. on
these surfaces. These coupons were retrieved for
analysis at various intervals ranging from 24 h to
168 h.
Post Exposure Evaluation
Bacterial density
The bacterial density on the coupons after various
durations of exposure was estimated using a plate
count technique. Three coupons of each metallurgi-
cal condition (triplicate experiments) were used for
total viable count (TVC) estimation (APHA, 1989).
The coupons were removed from the medium and
gently washed to remove loose cells and the bacterial
cells on the coupons were dispersed into 15 ml sterile
phosphate buffer by ultrasonication for 5 min. Serial
dilutions of the bacterial cell suspension were
prepared and 0.1 ml of each dilution was plated
onto nutrient agar. The plates were incubated for
24 –48 h and the number of colonies counted. Mean
TVC values were calculated for each coupon.
One coupon of each metallurgical condition
exposed in nutrient culture was used for direct
microscopic observation. The coupons were gently
washed with sterile water and air-dried in a sterile
chamber and a few drops of acridine orange (0.1%
solution in distilled water) were placed on the
coupon. After 2 min, the excess stain was drained off
and the coupons were washed in sterile water, dried
and observed under a Nikon Eclipse E600 epifluor-
escence microscope (excitation filter BP 490; barrier
filter O 515). Cells of Pseudomonas sp. (and other
microbes such as fungi, diatoms) fluoresced green/
orange and were easily differentiated from inorganic
particles and detritus. Acridine orange (AO) is a
metachromatic dye which differentially stains
double stranded (ds) and single stranded (ss) nucleic
acids. When AO intercalates into dsDNA it emits
green fluorescence upon excitation at 480– 490 nm
and when AO complexes with single stranded
nucleic acid i.e. RNA (Mah & O’Toole, 2001)
orange-red fluorescence is obtained. Cells on the
4 R P GEORGE et al.
FIGURE 1 EPR curves showing the degree of sensitization.
coupons exposed to nutrient culture were not
counted since the density was high. However, direct
counts were carried out on coupons exposed for
short periods in phosphate buffer (ASTM, 1985). Ten
different fields were selected randomly and oran-
ge-red fluorescing cells counted. Results are
expressed as number of cells cm22. Statistical
analysis of the data was carried out using MYSTAT
Software. Three replicates were analyzed for each
experimental condition. A Tukey-Kramer Multiple
Comparison test was performed to assess signifi-
cance between bacterial counts on different surfaces.
Qualitative assay of siderophores by chrome
azurol S (CAS) agar
A chrome azurol S agar assay was carried out to
detect production of siderophores by Pseudomonas sp.
used in this study. To prepare 1 l of agar, 60.5 mg
chrome azurol S dye were dissolved in 50 ml of water
and mixed with 10 ml of iron (III) solution (1 mM
FeCl3·6H2O, 10 mN HCl). This solution was added to
cetrimide (HDTMA) agar solution under continuous
stirring. The pH was maintained at 6.8. The resulting
agar medium was autoclaved and CAS agar plates
prepared. The Pseudomonas sp. was streaked on the
agar surface and incubated for 24 h at 32 ^ 28C: The
Fe (III) – chrome azurol S complex tints the agar a rich
blue color. The siderophores produced by iron-
starved microorganisms displace the chrome azurol
S dye causing a change of colour to orange.
TABLE III Density of Pseudomonas sp. on 304 SS with different microstructure/surface characteristics in long term exposure studies
Sensitized
Time of exposure TVC ^ SD
24 h 4.5 £ 104 ^ 4 £ 103
72 h 8.4 £ 104 ^ 8 £ 103
168 h 2.2 £ 105 ^ 1 £ 104
TVC ¼ total viable counts of bacteria; SD ¼ standard deviation
FIGURE 2 Adhesion of Pseudomonas sp. on 304 SS with different
microstructure and surface characteristics in short term exposure
studies (DAOC counts). Bars ¼ SD of the mean.
RESULTS
Evaluation of Degree of Sensitization
Figure 1 shows the activation and reactivation curves
of sensitized and oxidized specimens in the EPR test.
The reactivation peak is only present in the case of
the sensitized specimen. The absence of any
reactivation peak for the oxidized specimen indicates
that oxidizing heat treatment did not result in any
sensitization of the material.
Short Term Exposure Studies
Direct acridine orange counts (DAOC) on solution-
annealed, sensitized and oxidized 304 SS specimens
after short term exposure showed maximum
adhesion on the sensitized specimens, followed by
solution-annealed and oxidized specimens (Figure 2).
Tukey-Kramer Multiple comparison tests showed
that variation in DAOC counts on the different
surfaces were statistically significant. The P value for
sensitized vs solution-annealed specimens was
, 0.01, for sensitized vs oxidized specimens , 0.001
and for solution-annealed vs oxidized speci-
mens , 0.05.
Solution-annealed Oxidized
TVC ^ SD TVC ^ SD
1.5 £ 104 ^ 2.0 £ 103 4.6 £ 103 ^ 5 £ 102
2.0 £ 104 ^ 2.5 £ 103 8.4 £ 103 ^ 1 £ 102
6.0 £ 104 ^ 3.0 £ 103 1.1 £ 104 ^ 1.6 £ 103
 ADHESION OF BACTERIA TO SS 304
FIGURE 3 The plot of log10 (TVC) on various metallurgical
surfaces. Bars ¼ SD of the mean.
Long Term Exposure Studies
Table III and Figure 3 present the results of long term
exposure studies using solution-annealed, sensitized
and oxidized 304 SS specimens. In all cases,
maximum adhesion was found on the sensitized
specimens. Observation by epifluorescence and
scanning electron microscope (not shown) showed
a higher density of cell aggregation and more biofilm
coverage on sensitized specimens (Figure 4). Again,
the P values were , 0.05, showing that the measured
variations in bacterial density on the different
surfaces were statistically significant.
CAS Agar Assay
CAS agar plates streaked with Pseudomonas sp.
showed orange color along the streaked lines and
around isolated colonies (Figure 5) indicating side-
rophore production by the species used in the
present study.
DISCUSSION
Although microbial colonization of metal surfaces is
considered to be a crucial step in initiating MIC, the
5
factors determining cell adhesion are still obscure.
The existing literature is confusing and contradictory
with respect to the mechanism of bacterial adhesion
to hard surfaces. It is reported that surface
characteristics of the materials such as wettability
and charge, in tandem with the physico-chemical
factors of the bacterial cell surface affect cell
adhesion to surfaces (Boulange-Petermann et al.,
1997, Busscher et al., 1990). However, Sjollema and
Busscher (1990) suggested that theories based on
attractive and repulsive electrostatic forces are not
sufficient to explain deposition phenomena, because
negatively charged cells are found deposited on
negatively charged surfaces. It is also well known
that bacteria change their surface composition,
including charge, in response to the environment
(Cowan et al., 1992). The thermodynamic theory of
adhesion based on surface free energy of the cell and
substratum may also not explain the mechanism, as
secretion of adhesives can negate this theory
(Busscher et al., 1989). It is also reported that the
cell surface hydrophobicity of an organism varies in
different environments (Van der Mei et al., 1992).
Thus, the changeable nature of the charge and
hydrophobicity of bacterial surfaces indicate that
cells cannot be strictly considered as colloids.
However, many workers (Mueller et al., 1992;
Camper et al., 1993) agree that transport of cells to the
surface of a material dominates the physico-chemical
effects in predicting cell attachment in flowing
systems. Before bacteria can interact with the surface,
they must be transported there. Piette and Idziak
(1991) have shown that flagellated cells of Pseudo-
monas fluorescens attach in greater numbers than
deflagellated cells. A motile, Gram-negative, rod-
shaped Pseudomonas sp. was used in this study and
hence the factors contributing to transport are
constant under all experimental conditions. The
present study concentrates on factors that contribute
to subsequent attachment and growth, specifically
surface topography and surface chemistry. It has
been recognized in the literature that, in general,
electrochemically active surfaces favour bacterial
adhesion, probably because of the favourable
topography produced by the presence of corrosion
products on the surface.
The aim of this study was to provide direct
evidence for the influence of metallurgical charac-
teristics such as sensitization and oxidation on
microbial adhesion. The bacterial density and the
degree of aggregation and biofilm coverage were
highest in the case of sensitized coupons followed by
solution-annealed and oxidized coupons. Epifluo-
rescence micrographs showed most of the cells
attached to sensitized and solution-annealed SS
specimens which fluoresced orange-red compared to
the majority of cells that fluoresced green on the
oxidized surfaces. Mah and O’Toole (2001) reported
6 R P GEORGE et al.
FIGURE 4 Epifluorescence photographs showing adhesion,
growth and biofilm formation of Pseudomonas sp. after exposure
for 7 d to 304 SS specimens with (a) sensitized, (b) solution-
annealed, and (c) oxidized surfaces. The aggregations of cells in (b)
are embedded in an extensive matrix of EPS.
FIGURE 5 Chrome azurol S agar plates streaked with
Pseudomonas sp. showing orange halos around colonies
indicating siderophore production by this species.
that AO bound to nucleic acids forms the RNA-AO
complex fluorescing orange-red, while the DNA-AO
complex fluoresces green. Thus, it appears that
bacterial cells attached to sensitized and solution-
annealed SS surfaces have a high RNA content
compared to those cells attached to the oxidized
surface of the same material. This
observation suggests that the cells on the sensitized
SS were more metabolically active than those on the
solution-annealed SS and the oxidized SS.
A difference in the colonization pattern was also
apparent with sensitized and solution-annealed
coupons which showed bacterial cells in a biofilm
embedded in extraceullar polymeric substances
(EPS) whilst oxidized coupons showed scattered
attached cells with little evidence of EPS and
establishment of a biofilm (Figure 4).
The differences in the colonization behaviour of
the SS surfaces under different metallurgical con-
ditions suggest that attachment and proliferation of
bacterial cells is influenced by the microstructure/
chemistry of the surface. Since most bacterial cells
were observed on the sensitized surface, it is possible
that the susceptibility of this surface to intergranular
corrosion, and thereby release of iron, favoured
bacterial attachment. It is well known that release of
iron ions from sensitized surfaces will be at a
maximum, as the chromium-depleted grain boun-
dary zones have no protective Cr-rich oxide film.
Release of iron from the oxidized surfaces is
expected to be the lowest.
Iron plays a fundamental role in microbial growth
and metabolism (Carroll, 1989). Barnes et al. (1999)
studied the effect of milk proteins on adhesion of
bacteria to SS surfaces and reported that individual
milk proteins on SS surfaces reduce adhesion of
Pseudomonas sp. and an increase in the iron signal
from the surface increased bacterial attachment.
Neilands (1995) in his mini-review on siderophores
clearly explains the requirement of iron by bacteria.
 ADHESION OF BACTERIA TO SS 304
Microorganims growing under aerobic conditions
need free ferric ion for a variety of functions
including reduction of oxygen for synthesis of ATP,
reduction of ribotide precursors of DNA, for
formation of heme and for other essential purposes.
A level of at least 1 mM iron is needed for optimum
growth. However, the maximum amount of uncom-
plexed ferric ion in solution at biological pH is
probably not greater than 10218 M. The aerobic
atmosphere of the planet has caused the surface iron
to become converted to oxyhydroxide polymers of
very sparing solubility. These environmental restric-
tions and biological imperatives have required
microorganism to form specific molecules known
as siderophores (Kilz et al., 1999) that can compete
effectively with hydroxyl ion for the iron in the ferric
state, a nutrient which is abundant but essentially
unavailable. This special characteristic of producing
siderophores is used as a tool for identifying
(siderotyping) Pseudomonas aeruginosa. Therefore,
detection of siderophores in a culture of Pseudomonas
sp., was strong evidence for the iron requirement of
these bacteria.
The present study has also shown that irrespective
of the culture medium, i.e. nutrient culture or
phosphate buffer, adhesion of this Pseudomonas sp.
was greater on sensitized 304 SS surfaces and this is
attributed to the increased release of iron ions from
the sensitized surfaces. The present results are
significant in the context of preferential corrosion of
weldments during MIC of service components.
Microstructural and surface topographical changes
that take place during welding make the weld more
anodic, causing preferential corrosion even in the
absence of microorganisms. The increased abiotic
corrosion of the weld and the resultant corrosion
products attract microbial settlement, which in turn
accelerates corrosion in the region. Since sensitization
is one of the metallurgical changes that can take place
during the welding of SS, it can facilitate colonization
of microorganisms on weldments, thus favoring
corrosion of the weld. This study has demonstrated
for the first time that there is an increased attachment
of bacteria on sensitized surfaces compared to
solution-annealed and oxidized surfaces.
Acknowledgements
The authors acknowledge Dr V S Raghunathan,
Associate Director, Materials Characterization
Group, for useful suggestions and fruitful discus-
sions. They sincerely acknowledge Dr Baldev Raj,
Director, Materials, Chemistry and Reprocessing
Group, for his keen interest in the study and constant
encouragement.
7
References
APHA (1989) Standard methods for the examination of water and
waste water, 14th Edition. APHA, USA
ASTM (1985) Enumeration of aquatic bacteria by epifluores-
cence microscopy counting procedure. Water 11.02: 632 –635,
American Society for Testing and Materials, Philadelphia
Barnes L -M, Lo M F, Adams M R, Chamberlain A H L (1999)
Effects of milk proteins on adhesion of bacteria to stainless
steel surfaces. Appl Environ Microbiol 65: 4543–4548
Borenstein S W (1988) Microbiologically influenced corrosion
failures of austentic stainless steel welds. Mater Perf 27: 62 –66
Borenstein S W (1994) Microbiologically Influenced Corrosion Hand-
book. Woodhead Publishing Limited, Cambridge, England
Boulange-Petermann L, Rault J, Bellon-Fontaine M N (1997)
Adhesion of Streptococcus thermophilus to stainless steel with
different surface topography and roughness. Biofouling 11:
201 –216
Buchanan R A, Zhang X, Li P, Stansbury E E, Dowling N J E, Hall T,
Lindberg A (1990) Effects of surface condition on suscepti-
bility to microbially influenced corrosion: stainless steel
weldments and carbon steel. In: Dowling N J, Mittleman M W,
Danko J C (eds) Microbially Influenced Corrosion
and Biodeterioration. University of Tennessee, Knoxville,
pp 3/99–3/102
Busscher H J, Bellon-Fontaine M N, Mozes N, Van der Mei H C,
Sjollema J, Cerf O, Rouxhet P G (1990) Depositon of
Leuconostoc mesenteroides and Streptococcus thermophilus to
solid substrata in a parallel plate flow cell. Biofouling 2: 55 –63
Busscher H J, Weercamp A H, Van der Mei H C, Van Steenberghe
D, Quirynen M, Pratt I H, Marecchal M, Rouxhet P G (1989)
Physico-chemical properties of oral streptococcal cell surfaces
and their relation to solid substrata in vitro and in vivo. Colloids
Surf 42: 345– 353
Camper A K, Hayes J T, Sturman P J, Jones W L, Cunningham A B
(1993) Effects of motility and absorption rate coefficient on
transport of bacteria through saturated porous media. Appl
Environ Microbiol 59: 3455–3462
Cihal V (1980) A potentiokinetic reactivation method for
predicting the ICC and IGSCC sensitivity of stainless steels
and alloys. Corrosion Sci 20: 737–744
Carroll L (1989) Metal toxicity. In: Hughes M N, Poole R K (eds)
Metals and Microorganisms. Chapman and Hall, pp 252–302
Costerton J W, Geesey G G, Jones P A (1987) Bacterial biofilms in
relation to internal corrosion monitoring and biocides.
Corrosion/87, Paper No 57, NACE, Houston, TX
Cowan M M, Van der Mei H C, Rouxhet P G, Busscher H J (1992)
Physico-chemical and structural properties of the surface of
Peptostreptococcus micros and Streptococcus mitis as compared
to those of mutans streptococci, Streptococcus sanguis and
Streptococcus salivarius. J Gen Microbiol 138: 2707– 2714
Dexter S C (1992) Localized biological corrosion. In: Corrosion,
ASM Hand Book 13. ASM International, USA, pp 114 –122
Fleming H C, Geesey G G (1991) Biofouling and Biocorrosion in
Industrial Water Systems. Springer-Verlag, New York, 162 pp
George R P, Muraleedharan P, Parvathavarthini N, Khatak H S,
Rao T S (2000) Microbiologically influenced corrosion of
AISI Type 304 SS under fresh water biofilms. Werkstoffe Korr
51: 1–6
Ibars J R, Moreno D A, Ranninger (1992) MIC of stainless steels: a
technical review on the influence of microstructure. Int
Biodeterior Biodegrad 29: 343– 355
Kilz S, Lenz Ch, Fuchs R, Budzikiewiz H (1999) A fast screening
method for the identification of siderophores from fluorescent
Pseudomonas sp. by liquid chromatography/electrospray
mass spectrometry. J Mass Spectr 34: 281–290
Krug N R, Hold J G (eds) (1984) Bergey’s Manual of Systematic
Bacteriology, Volume 1. Williams and Wilkins, Baltimore
Licina G J (1989) An overview of microbiologically influenced
corrosion in nuclear power plant systems. Mater Perf 28: 55–60
Little B, Ray R, Wagner P, Jones-Meehan J, Lee C, Mansfeld F (1999)
Spatial relationships between marine bacteria and localized
corrosion on polymer coated steel. Biofouling 13: 301 –321
Mah T C, O’Toole G.A (2001) Mechanisms of biofilm resistance to
antimicrobial agents. Trends Microbiol 9: 34–39
8 R P GEORGE et al.
Mele M F L, de Moreno D A, Ibars J R, Videla H A (1991) Effect of
inorganic and biogenic sulphide on localized corrosion of heat
treated type 304 stainless steel. Corrosion 47: 24–30
Mittleman M W, Geesey G G (1987) Sample collection and pro-
cessing. In: Biological Fouling of Industrial Water Systems: a
Problem Solving Approach. Water Micro Associates, USA,
pp 269–288
Mueller R F, Charackilis W G, Jones W L, Sears J T (1992)
Characterization of initial events in bacterial surface
colonization by two Pseudomonas species using image
analysis. Biotechnol Bioeng 39: 1161–1170
Neilands J B (1995) Siderophores: structure and function of
microbial iron transport compounds. J Biol Chem 270:
26723–26726
Palleroni N J (1984) Gram-negative aerobic rods and cocci. In:
Bergey’s Manual of Systematic Bacteriology, Volume 1.
Williams and Wilkins, Baltimore, pp 140 –219
Piette J P G, Idziak E S (1991) Role of flagella in adhesion of
Pseudomonas aeruginosa to tendon slices. Appl Environ Microbiol
57: 1631–1639
Rao T S, George R P, Venugopalan V P, Nair K V K (1997) Biofilm
formation in a fresh water impoundment under experimental
‘photic’ and ‘aphotic’ conditions. Biofouling 11: 265 –282
Sjollema J, Busscher H J (1990) Deposition of polystyrene particles
in a parallel plate flow cell. 1. The influence of collector
surface properties on the experimental deposition rate.
Colloids Surf 47: 323–336
Soracco R J, Pope D H, Eggers J M, Effinger T N (1988)
Microbiologically influenced corrosion investigations in
electric power generating stations. Corrosion/88, NACE,
Houston, TX, Paper 83
Stein A A (1991) Metallurgical factors affecting the resistance of
300 series stainless steels to microbiologically influenced
corrosion. Corrosion/91, NACE, Houston, TX, Paper 107
Syrette B C, Colt R L (1983) Causes and prevention of power plant
condenser tube failures. Mater Perf 22: 44–49
Van der Mei H C, Cowan M M, Genet M J, Rouxhet P G, Busscher
H J (1992) Structural and physicochemical surface properties
of Serratia marcesens strains. Can J Microbiol 38: 1033–1041
Videla H A, Mele M F L, de Moreno D A, Ibars J R, Ranninger C
(1991) Influence of microstructure on the corrosion behaviour
of different stainless steels. Corrosion/91, NACE, Houston,
TX, Paper 104
Walsh D, Willis E, Diepen T V, Sanders J (1994) The effect of
microstructure on Microbial interaction with metals–accent
welding. Corrosion/94, NACE, Houston, TX, Paper 612
White D C, Arrage A A, Nivens D E, Palmer R J, Rice J F, Sayler G S
(1996) Biofilm ecology: on-line methods bring new insights
into MIC and microbial biofouling. Biofouling 10: 3 –16