El papel de los exopolmeros celulares en

los procesos de formacin, persistencia

y control de las biopelculas

EXTRACELLULAR POLYMERIC SUBSTANCES

EPS
(SLIME !!!)

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

AFM image in air of a 7 day old, wet biofilm of Desulfovibrio

indonesiensis (Ind) on the surface of AISI 316 stainless
steel
Ind
Ind cells
cells

Ind
Ind EPS
EPS

Massive biofouling in a paper mill

Hole in paper due to EPS (slime)

BIOFILM
BIOFILM RESISTANCE
RESISTANCE TO
TO ANTIMICROBIAL
ANTIMICROBIAL AGENTS
AGENTS

SURFACE SCIENCE TECHNIQUES

(imaging and chemical analysis)
SEM / ESEM
EDX
XRD
XPS / Auger
ToF-SIMS and biochem MS
ATR - FTIR
AFM

IRON SULPHIDES AND CORROSION

PROPOSED MECHANISMS
OF BIOCORROSION

EXTRACELLULAR POLYMERIC
SUBSTANCES ?

PROPOSED MECHANISM
OF EPS-INFLUENCED CORROSION

METAL BINDING
ENZYMATIC ACTIVITIES

Metal ion binding by bacterial EPS

EPS
Bacterial Cell Wall
Bacterial cell

= metal ions
= metal ion/EPS complex

= metal ion binding sites

within EPS

RESEARCH FOCUS:
Marine biocorrosion of Cu-Ni alloys
in coolers

1m

1 m

Bacterial cells

1 m

Cu
Ni

ESEM image of bacterial cells grown

in the presence of CU

Ca

Cu

PIXE mapping
of bacterial cells

Scan area
2000 x 2000 micron

Metal Binding by EPS

Organic Phosphate HPO4-2 + Me2+  MeHPO4 

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.

DIFFERENT GENERA AND SPECIES

OF SRB VARY IN THEIR
AGGRESSIVNESS TOWARDS
METALLIC SUBSTARTUM
SPECIES SPECIFICITY?

Why Ala and Indo?

D. alaskensis
Isolated from a soured oil reservoir in Alaska
Fast anaerobic coloniser of abiotic and biotic surfaces
Non-corrosive behaviour in laboratory tests (protective?)
Non-aggressive strain
(based on testing biofilm and EPS efect on steel surfaces and in human
epithelial tissue culture studies)

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.

Light micrograph of the surface of mild steel coupon exposed to

(A) sterile buffer for 90 min,
(B) EPS sample in buffer for 60 min,
(C) 2% nitric acid in ethanol for 15 min
(D) EPS sample in buffer for 45 min, revealing pit formation
( black arrows).

Mild Steel Corrosion by Exopolymer

of D. indonesiensis

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

Relative element concentration (%) SD

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.

 EPS FROM DIFFERENT STRAINS

OF SRB VARY IN THEIR ABILITY
TO BIND Fe
DEPENDING ON THE STRAIN OF SRB
DIFFERENT MACROMOLECULES WITHIN
EPS MATRIX MEDIATE Fe-BINDING

(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

Light micrograph of the surface of a mild steel coupon

exposed to
(A) sterile buffer for 90 min,
(B) EPS sample in buffer for 60 min,
(C) 2% nitric acid in ethanol for 15 min
(D) EPS sample in buffer for 45 min, revealing pit formation
(black arrows).

MILD STEEL CORROSION by EPS

of D. indonesiensis

Sample

Level of Fe

Time of exposure [minutes]

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

S.D. - standard deviation

The total concentration of iron detected in buffer and

In Ind EPS sample (CCF) and calculated theoretical values
of corrosion rates.

Sample

Relative element concentration (%) SD

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 of Fe in crude (EPS-C) exopolymer

samples from Ind1 and Al1 SRB cultures grown
with mild steel coupons based on the analysis
of high resolution XPS spectra.

EPS FROM DIFFERENT STRAINS

OF SRB VARY IN THEIR ABILITY
TO BIND Fe

The effect of EPS on attachment of Pseudomonas

NCIMB 2021 to AISI 304 and 316 stainless steel
Capsular

Planktonic

Biofilm matrix

The effect of exopolymers on Pseudomonas NCIMB

2021 attachment to AISI 316 stainless steel
45.00

316 DAPI stained

316 CTC stained

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

Planktonic Biofilm EPS Biofilm EPS

0.1 mg/ml
0.5 mg/ml
EPS 0.5
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

What are the differences in the

chemistry of the different types of
EPS?

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%

Gas Chromatography with Mass Detector

Biofilm EPS

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

Small-spot X-ray Photoelectron

Spectroscopy
(PHI Model 5600ci)
at Montana State University

Carbon
The peak shape
for the C1s
peak is
different for
each type of
EPS.

XPS peak deconvolution

EPSBF4H2.SPE: conditoned for 4 h, 45 degree
98 Feb 17 Al mono 300.0 W 0.0 45.0 23.501.5872e+004
eV
max
C1s/Full/1 (Shft)

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

Atomic Force Microscopy

Atomic Force Spectroscopy

AFM study of adhesive properties

of Desulfovibrio alaskensis EPS
(NASA,
SA)
(NASA, ONR
ONR sponsored
sponsored research,
research, carried
carried out
out at
at MSU
MSU in
in Bozeman,
Bozeman, UUSA)

Tutorial: Principle of force curve measurement

(a) approach, (b) contact with surface and binding, (c) pull
out, and (d) pull further until unbinding is achieved. An
example force curve (left). It took ~100 pN force to break the
molecular binding.

AFM images in water of Ala EPS native and Ala EPS/Ca on mica

Comparison between AFM force curves

for a model protein and native Ala EPS
Interaction
Interaction between
between hydrophilic
hydrophilic tip
tip

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

File: dex2%inseawaterk03fv.002, Curve: 64, X 0(Approach.): 96.4668, X0(Retraction)= 66.321

1

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 data for hydrophilic surface

(i.e. as received silicon nitade tip) /Ala EPS
ionteraction

Representative data for hydrophobic surface

(i.e. functionalized AFM tip) / Ala EPS
interaction

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.

Negatively charged ion fragments of Ala EPS

revealing pattern characteristic to inorganic salts
400

LSI
ESI

Abundance

300

200

142

100

0
500

1000

m/z

1500

Acumulation of radionucleides
Radionucleides accumulated in biofilms on steel :

60Co, 65Zn

Radionucleides accumulated in biofilms on titanium:

60Co, 59Fe, 95Nb, 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

Take Home Message

Biofilms can cause corrosion
Biofilms are not just our enemies
they can also be our friends

FUTURE PERSPECTIVES
for the study of biofilm / materials interactions

Spatial and temporal Correlation between

microbial processes and interfacial chemistry
-omics
Mass Spectrometry Analysis and Imaging