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EPS COMPOSITION
COMPOSITION::
PROTEINS
(glycoproteins)
POLYSACCHARIDES
(neutral carbohydrates, amino-sugars, uronic acids)
NUCLEIC ACIDS
(DNA, RNA)
LIPIDS
TYPE OF EPS
CAPSULAR
PLANKTONIC / FREE
BIOFILM
EPS
BIOFILM MATRIX
Ind
Ind EPS
EPS
BIOFILM
BIOFILM RESISTANCE
RESISTANCE TO
TO ANTIMICROBIAL
ANTIMICROBIAL AGENTS
AGENTS
PROPOSED MECHANISMS
OF BIOCORROSION
EXTRACELLULAR POLYMERIC
SUBSTANCES ?
PROPOSED MECHANISM
OF EPS-INFLUENCED CORROSION
METAL BINDING
ENZYMATIC ACTIVITIES
EPS
Bacterial Cell Wall
Bacterial cell
= metal ions
= metal ion/EPS complex
RESEARCH FOCUS:
Marine biocorrosion of Cu-Ni alloys
in coolers
1m
1 m
Bacterial cells
1 m
Cu
Ni
Ca
Cu
PIXE mapping
of bacterial cells
Scan area
2000 x 2000 micron
EPS
Cell Wall
= MeHPO4
= Phosphatase
RESEARCH FOCUS:
EPS of sulphate-reducing bacteria (SRB)
and their importance in cell/surface
interactions
Why SRB ?
Anaerobic colonisers of metallic and non-metallic surfaces
in marine and fresh water environments
- contribute to fouling, as well as corrosion problems and
health and safety hazards due to H2S production.
Present in human intestine and in colon:
- thought to be involved in ulceritive colitis, Crohn disease
and, possibly, in colon cancer.
D. indonesensis
Isolated from a sever marine corrosion failure
Slower coloniser than Ala
Able to induce severe corrosion under laboratory conditions
Aggressive strain
Attachment of D. alaskensis and D. indonensiensis mixed population to the AISI 316 stainless
steel surface after 4 h (a) and 12 h (b) of exposure.
DNA-DAPI-staining shows in blue all attached cells.
D. indonensiensis cells (arrows) are identified by double staining in green (FITC) and blue
(DAPI). Superposed images of FITC and DAPI (insets) show that D. alaskensis (green) colonises
surface more rapidly than D. indonesiensis.
Bar =1
m.
INVESTIGATION OF Fe BINDING
BY EXOPOLYMERS OF
D. indonesiensis and D.alaskensis
Ind 1
Al 1
INVESTIGATION OF Fe BINDING
BY EXOPOLYMERS OF
D. indonensiensis and D. alskensis
XPS
ToF-SIMS
Sample
Ind1
EPS-C
Al1
C 1s
N 1s
O 1s
P 2p
Fe 2p
61.85
14.30
21.96
0.36
1.53
0.68
0.15
0.23
0.04
0.09
62.13
14.59
22.14
0.68
0.46
0.67
0.16
0.28
0.56
0.04
Quantification
Quantification of
of Fe
Fe in
in crude
crude (EPS-C)
(EPS-C) exopolymer
exopolymer samples
samples
from
from Ind1
Ind1 and
and Al1
Al1 SRB
SRB cultures
cultures grown
grown
with
with mild
mild steel
steel coupons
coupons based
based on
on the
the analysis
analysis
of
of high
high resolution
resolution XPS
XPS spectra.
spectra.
(b)
(a)
O2
O2
Oxygenated biofilm
Oxygenated biofilm
1/2O2+2H++2e- => H2O
EPS
Fe3+
e-
O2
Fe3+(EPS)
Fe2+
e-
e
Cathode
CP
Cathode
Fe2+(EPS) Fe3+(EPS)
Cathode
e-
Anode
Ferrous metal
Ferrous metal
CP
Anode
INVESTIGATION OF Fe BINDING
BY EXOPOLYMERS OF
D. indonesiensis and Desulfovibrio alaskensis
ToF-SIMS
XPS
Sample
Level of Fe
type
15
30
45
60
90
120
Buffer
57
212
419
564
1049
5137
10
10
(ppm SD)
In the 10 l
CCF
2876
3640
4774
6575
7632
12281
Droplet
sample
16
12
22
50
49
Theoretical
Buffer
0.16
0.29
0.38
0.38
0.48
1.75
0.02
0.01
0.00
0.00
0.00
0.00
rate of
Corrosion
CCF
4.85
3.07
2.68
2.77
2.15
2.59
mm y-1 SD)
sample
0.03
0.00
0.00
0.00
0.01
0.01
Sample
Ind1
EPS-C
Al1
C 1s
N 1s
O 1s
P 2p
Fe 2p
61.85
14.30
21.96
0.36
1.53
0.68
0.15
0.23
0.04
0.09
62.13
14.59
22.14
0.68
0.46
0.67
0.16
0.28
0.56
0.04
Planktonic
Biofilm matrix
Cells cm -2 / 107
40.00
35.00
30.00
25.00
20.00
Water
Capsular
EPS 0.1
mg/ml
Capsular
EPS 0.5
mg/ml
Planktonic
EPS 0.1
mg/ml
Attachment Studies
No difference in attachment was observed
between untreated 304 and 316 coupons.
Each type of steel exhibited differences in
bacterial attachment when conditioned with
different types of EPS.
Each type of steel exhibited differences in
bacterial attachment when conditioned with
different concentrations of EPS.
Attachment Studies
96% of attached cells were metabolically active.
The type and concentration of EPS had a significant
influence on numbers of attached cells.
Following the EPS treatment the grade of stainless
steel had a significant influence of numbers of cells
attached.
Interaction between the EPS and steel grade
influenced cell attachment.
Attachment Studies
The data indicate that the chemistry of the
substratum influences the interaction
between the type of cell and the conditioning
layer
Colorimetric Assays
Neutral Carbohydrates:
Biofilm EPS: 11.5 %
Planktonic EPS: 13.3%
Capsular EPS: 13.3%
Protein Content:
Biofilm EPS: 9.6%
Planktonic EPS: 9.3%
Capsular EPS: 14.1%
Allose
Mannose
Glucose
Galactose
Glucoseamine
Mannoseamine
Galactoseamine
Galacturonic acid
yes
yes
yes
yes
yes
yes
yes
yes
Planktonic EPS
yes
yes
yes
yes
Capsular EPS
yes
yes
yes
yes
yes
yes
yes
yes
SDS-Page Gel
Each type of EPS has own
characteristic protein profile with
fewer distinct bands at 100, 60 and
35 kDA for the planktonic EPS
X-ray Photoelectron
Spectroscopy
Carbon
The peak shape
for the C1s
peak is
different for
each type of
EPS.
Company Name
575.20 min
EPSBF4H2.SPE
16000
C-C,H
14000
C-O,N
12000
O-C=O
c/s
10000
8000
6000
4000
2000
292
290
288
286
Binding Energy (eV)
284
282
The peak
shape for the
C1s peak is
composed by
different
components
AFM images in water of Ala EPS native and Ala EPS/Ca on mica
Interaction
Interaction between
between hydrophilic
hydrophilic tip
tip and
and
Cytochrome
Cytochrome CC
and
and native
native Al-EPS
Al-EPS
File: ccinwatrk01fv.000, Curve: 242, slope/av. slope= 1.0263, Area above= 2.0253, Area below: 11.6365
0.5
X0(Approach)= 127.8398
L= -0.14093, F= -0.12568
X0(Retraction)= 41.6222
L= 17.6975, F= -0.18398
Slope(Retraction)= -0.010136
L= 33.3937, F= -0.086477
X intercept(Retraction)= 41.8569
L= 49.6318, F= -0.22483
0.4
0.3
FORCE (nN)
0.2
0.1
-0.1
-0.2
-0.3
50
100
150
PIEZO DISPLACEMENT (nm)
200
250
300
C
omparison between AFM force curves
Comparison
for a model polysaccharide and EPS
Interaction
ydrophilic tip
Interaction between
between hhydrophilic
tip and
polysaccharide
extran
polysaccharide ddextran
Interaction
ydrophilic tip
Interaction between
between hhydrophilic
tip and native
Ala
Ala EPS
EPS
0.8
X0(Approach)= 96.4668
L= 83.7689, F= -0.010819
X0(Retraction)= 66.321
L= 92.879, F= -0.015333
Slope(Retraction)= -0.019668
L= 130.4863, F= -0.0043808
X intercept(Retraction)= 48.5137
L= 138.1734, F= -0.44691
0.6
L= 148.5159, F= -0.40795
L= 181.6848, F= -0.066153
FORCE (nN)
0.4
0.2
-0.2
-0.4
-0.6
-0.8
50
100
150
PIEZO DISPLACEMENT (nm)
200
250
300
Representative
Representative data
data for
for interactions
interactions of
of
hydrophilic
hydrophilic surface
surface (i.e.
(i.e. tip)
tip) with native
Ala
Ala EPS
EPS
Representative
Representative data
data for
for interactions
interactions of
of
hydrophilic
hydrophilic surface
surface (i.e.
(i.e. AFM
AFM tip)
tip) with
2+
Ala
Ala EPS
EPS enriched
enriched with
with Ca
Ca2+
Results
EPS of Desulfovibrio alaskensis comprise macromolecules that
bind to hydrophilic surfaces, as well as macromolecules that
adhere preferentially to hydrophobic surfaces.
Using AFM in force volume mode, the presence of
polysaccharides and proteins in the EPS material can be
demonstrated.
The ddata
ata reveals that the D. alaskensis EPS is more likely to
adhere to a hydrophobic surface than to a hydrophilic surface
surface,,
as more events, more stretching in the form of multiple events,
longer polymer extension and higher work of adhesion have
been observed using the hydrophobic tip.
LSI
ESI
Abundance
300
200
142
100
0
500
1000
m/z
1500
Acumulation of radionucleides
Radionucleides accumulated in biofilms on steel :
60Co, 65Zn
2500
60 Co
1000
3000
500
141Ce
144Ce
80
das
106
das
142
das
170
das
254
das
365
das
Das de inmersin
57Co
58Co
60Co
59Fe
51Cr
Titanium
Titanio
600
500
400
300
200
100
0
26 das
2500
134Cs
2000
Bq/cm2
Bq/cm2
1500
51
das
Bq/cm2
y 54Mn
Titanium
UNS S44200
Acero inoxidable
UNS
S44200
2000
y 54Mn
1500
1000
500
137Cs
60 Co
59Fe
59 Fe
95 Nb
62 das
90 das
174 das
Das de inmersin
285 das
54Mn
65Zn
0
0
100
200
Das de inmersin
300
400
FUTURE PERSPECTIVES
for the study of biofilm / materials interactions