effect of mixed species biofilm on corrosion of cast iron

• By • Farhad Batmanghelich

• Youngwoo Seo

• Department of Chemical and Environmental Engineering

• University of Toledo

September 2015

Effect of Mixed Denitrifying and Sulfate Reducing Bacterial

Biofilms on Corrosion Behavior of Cast Iron

Outline

2

Introduction and Theoretical background

Scope of Study Materials and

Methods Results and Discussions

Conclusions

Outline

3




Conclusions

Corrosion

4

Corrosion is the deterioration of a material due to the reactions with its surrounding environment

5

Corrosion Economic Impact

Utilities

47.9 billion $

Production and

manufacturing

17.6 billion $

Government

20.1 billion $

Infrastructure

22.6 billion $

Transportation

29.7 billion $

b 43

Why knowing about corrosion mechanisms is

important?

6

Utilities

47.9 billion $

Production and

manufacturing

17.6 billion $

Government

20.1 billion $

Infrastructure

22.6 billion $

Transportation

29.7 billion $

b 43

Corrosion mitigation strategies:

1- Mechanism identification

2- Occlude those mechanisms

3- Monitor

Corrosion in Water Systems

7

Decrease in water capacity………(Energy Loss)

Holes and leakages………………(Structural Integrity)

Drinking water quality…………...(Health Problems)

Red water………………………...(Aesthetic problems)

Odor……………………………...(Aesthetic Problem)

The Cycle of Corrosion

8

Ore

Thermodynamically

stable state Energy

Reduction

Metals &

Alloys Products

Corrosion

Electrochemistry

Corrosion is an interfacial electrochemical process

Electrochemical rxns are redox rxns in which:

Electron transfer over extremely large distances

In a redox rxn oxidation state of the reactants change

9

Interface (Double

layer)

Bulk

Surf

ace

Electrochemistry

10

Full reaction Half reactions

(electrode reactions)

Electrochemistry

11

Anodic half

reaction

Cathodic half

reaction

Equilibrium Thermodynamics (Nernst equation)

Non-equilibtium Kinetics (Butler-Volmer; Conttrell, etc.)

The rate of an electrochemical reaction is expressed

in terms of electric current.

Electrochemical Techniques

12

Basis of all electrochemical techniques:

A deliberate perturbation in one of the parameters of the system and

monitoring the response of the system.

(a) Potential Step : { chronoamperometry, chronopotentiometry, chronocoulometry, etc.}

(b) Potential sweep : { linear scan voltammetry, cyclic voltametry, etc.}

Potentiodynamic polarization

Electrochemical Impedance Spectroscopy

(EIS)

Electrochemical Impedance Spectroscopy (EIS)

13

Nyquist Bode

Electrochemical Impedance Spectroscopy

(EIS)

14

The sine AC energy can be either stored or dissipated

at the interface by different layers.

The interface of a metal and its surroundings can

be modeled based on physical geometry and properties

of the surface.

The model can then be fitted to the impedance data

to acquire physical values such as interfacial resistance

and capacitances.

Biocorrosion (the role of biofilm)

Any material or phenomena that

changes the interface of a metal and

its environment is able to affect

electrochemical reactions, thence

alters corrosion behavior.

15

Biofilms form on metal surfaces

Biofilms change corrosion behavior

Biofilms can either

exacerbate or alleviate

corrosion hazard.

In studying corrosion;

effect of bulk

metabolites and biofilms

should be considered

separately.

Sulfate Reducing Bacteria (SRB)

SRB species such as Desulfovibrio Vulgaris reduce

SO42-

to S2- and eventually produce H2S.

H2S is corrosive….

SRB are anaerobes.

16

Venzlaff, corr.sci, 66, 2013

Pseudomonas aeruginosa

Pseudomonas aeruginosa (PAO1) is an opportunistic pathogen with a high level of

adaptability to various environments.

It is a facultative bacteria and can survive in both aerobic and anaerobic environments

Under anaerobic conditions it can respire through denitrification

Denitrification:

Reduction of nitrate (NO3-) to nitrite (NO2

-), ammonia and N2.

17

PAO1 wild type

Mixed Species Corrosion

Bacteria usually live in consortia.

Denitrifying and Sulfate Reducing Bacteria (SRB) are abundant in aqueous environments.

Under corrosion tubercles there is no oxygen, therefore PAO1 and SRB can exist together

in that area.

Mixed species consortia can exacerbate or alleviate corrosion.

18

19




Conclusions

Scope of Study

Main objective of this study is to evaluate effect of mixed bacterial biofilms and cultures

on corrosion behavior of cast iron.

(i) What impact mixed model bacteria solutions containing corresponding metabolites have

on corrosion of cast iron?

(ii) How biofilm composition and morphology contributes to corrosion of cast iron in the

absence of bacterial metabolites?

20

Outline

21




Conclusions

Materials and Methods, Bacteria Culture

Preparation

D. vulgaris and P.aeruginosa were

grown anaerobically in an anaerobic

chamber. 95% N2- 5% H2

22

Materials and Methods, Bacteria Culture

Preparation

D. vulgaris and P.aeruginosa were grown

anaerobically in an anaerobic chamber. 95%

N2- 5% H2

All the seed preparations were made in a sterile

clean hood under UV light with ethanol

sterilization. Coupons were also sterilized by

70% ethanol and subsequent UV irradiation.

23

Coupon Preparation

24

PAO1 and SRB seeds

SRB produces Hydrogen Sulfide (H2S). Iron reacts with H2S to

form black FeS precipitates. A procedure for checking SRB

growth is to add iron salts to grown SRB cultures to see if black

precipitates form.

25

PAO1 SRB

Anaerobic Hungate

tubes

Overview of experiments

26

In-situ electrochemical

experiments

• EIS and potentiosynamic polarization experiments on coupons that have been immersed for 1, 3 and 7 days in bacterial solutions of PAO1, SRB and mixed species.

• Corrosion weight loss : 1, 3 and 7 days immersion bacteria cultures; ASTM D-2688.

Ex-situ electrochemical

experiments

• EIS and potentiodynamic polarization tests in 3.5% NaCl solution (pH = 4), on coupons that have been immersed in bacterial solutions of PAO1, SRB and mixed for 1, 3 and 7 days.

• Corrosion weight loss : 1, 3 and 7 days immersion in bacteria + immersion in 3.5% NaCl solution (pH = 4) for 4 days; ASTM D-2688.

Biofilm quantification

and observation

• EPS extraction [Cationic Exchange Resin (CER) method] and quantification [phenol sulfuric acid (PSA) for polysaccharides; Coomassie blue method for proteins].

• Fluorescence microscopy

• Atomic Force Microscopy (AFM)

Corrosion Intrumentation (ex-situ)

27

For in-situ electrochemical experiments: A multiportTM

Corrosion Cell kit (Gamry) connected to a Reference 600

potentiostat (Gamry) was used.

For ex-situ electrochemical experiments: A cell was

designed to maintain anaerobiosis during the course of ex-

situ eperiments.

Corrosion Intrumentation (in-situ)

28

For in-situ electrochemical experiments: A multiportTM

Corrosion Cell kit (Gamry) connected to a Reference 600

potentiostat (Gamry) was used.

For ex-situ electrochemical experiments: A cell was

designed to maintain anaerobiosis during the course of ex-

situ eperiments.

Outline

29




Conclusions

Results and Discussion

30

In-situ electrochemical

experiments

• EIS and potentiosynamic polarization experiments on coupons that have been immersed for 1, 3 and 7 days in bacterial solutions of PAO1, SRB and mixed species.

• Corrosion weight loss : 1, 3 and 7 days immersion bacteria cultures; ASTM D-2688.

Ex-situ electrochemical

experiments

• EIS and potentiodynamic polarization tests in 3.5% NaCl solution (pH = 4), on coupons that have been immersed in bacterial solutions of PAO1, SRB and mixed for 1, 3 and 7 days.

• Corrosion weight loss : 1, 3 and 7 days immersion in bacteria + immersion in 3.5% NaCl solution (pH = 4) for 4 days; ASTM D-2688.

Biofilm quantification

and observation

• EPS extraction [Cationic Exchange Resin (CER) method] and quantification [phenol sulfuric acid (PSA) for polysaccharides; Coomassie blue method for proteins].

• Fluorescence microscopy

• Atomic Force Microscopy (AFM)

EIS in-situ (day 1)

31

Nyquist

Bode

EIS in-situ (day 3)

32

Bode

Nyquist

EIS in-situ (day 7)

33

Nyquist Bode

EIS modeling for in-situ

34

RS(QP(RP(QCTRCT))) model:

RS = solution resistance

QCT = charge transfer CPE

RCT = charge transfer resistance

QP = biofilm pores CPE

RP = biofilm pore resistance

CPE = constant phase element

RS (ohm) CPEP n (0<n<1) RP CPECT NCT

0<n<1

RCT

PAO1 day 1 9.94 6.999×10-4 0.8926 8.92 6.716×10-4 0.8797 18020

PAO1 day 3 10.18 6.432×10-4 0.9173 10.21 6.934×10-4 0.9143 31560

PAO1 day 7 10.44 1.833×10-4 0.9532 12.48 6.967×10-4 0.8078 80560

SRB day 1 5.49 4.345×10-4 0.9878 4.64 8.125×10-4 0.8189 6952

SRB day 3 6.84 1.236×10-4 0.9938 3.24 3.048×10-4 0.9972 6592

SRB day 7 6.11 1.551×10-4 0.8597 1.03 9.493×10-5 0.8696 3603

Mixed day 1 8.38 9.061×10-4 0.8362 5.24 3.899×10-4 0.8018 12130

Mixed day 3 9.24 1.585×10-4 0.8231 8.15 4.334×10-4 0.8213 17680

Mixed day 7 8.73 4.933×10-4 0.8723 6.92 4.231×10-4 0.8419 15020

Interfacial Resistances (RCT and RP) : in-situ

35

RP RCT

Potentiodynamic polarization : in-situ

36

Ecorr (mV) Icorr (µA/cm2) βa βc

PAO1 -812 0.118 43.2 × 10-3 82 × 10-4

SRB -894 0.761 68.4 × 10-3 46.1 × 10-3

Mixed -845 0.424 73 × 10-4 52 × 10-4

The reduction in RCT and Icorr for mixed bacteria

cultures compared to SRB can be attributed to

(i) Electron transfer competitions between NO3- & SO4

2- in the presence of PAO1

(ii) Inhibitory effects of denitrification pathway products (ex. Nitrite) for SRB

EIS ex-situ (day 1)

37

Nyquist Bode

EIS ex-situ (day 3)

38

Bode Nyquist

EIS ex-situ day (7)

39

Nyquist Bode

EIS ex-situ modeling

40

RS(QP(RP(QCTRCT))) model:




QP = biofilm pores CPE

RP = biofilm pore resistance


RS CPEP n 0<n<1 RP CPECT nCT 0<n<1 RCT

PAO1 day1 1.524 1.79×10-7 0.9436 1.582 9×10-3 0.9328 60.63

PAO1 day 3 1.237 2.18×10-7 0.9081 1.327 1.4×10-2 0.907 88.13

PAO1 day 7 1.069 9×10-7 0.9274 1.365 3.46×10-2 0.9992 88.94

SRB day 1 0.8252 9.7×10-8 0.8694 42.56 8.4×10-4 0.9931 234.6

SRB day 3 0.8502 1.1×10-7 0.8607 18.2 1.3×10-2 0.8571 197.2

SRB day 7 0.9869 8.7×10-8 0.8932 63.1 9.3×10-3 0.8777 247.1

Mixed day 1 0.7114 9.7×10-8 0.8432 4.45 5.4×10-3 0.87 207.4

Mixed day 3 2.746 1.65×10-7 0.9438 5.261 7.2×10-3 0.9962 65.23

Mixed day 7 0.9632 7.8×10-8 0.931 7.611 5.6×10-3 0.917 152.8

Interfacial Resistances (RCT and RP) : ex-situ

41

RP RCT

Potentiodynamic polarization : ex-situ

42

Ecorr (mV) Icorr (µA/cm2) βa βc

PAO1 -814 9.43 87.6×10-3 33.35×10-2

SRB -730 3.77 48.4×10-3 57.5×10-3

Mixed -773 7.82 53.6×10-3 73.8×10-3

EIS ex-situ sterile

43

EIS ex-situ sterile modeling

44

RS(QCTRCT) model:





Days RS CPE n (0<n<1) RCT

Day 1 0.86 2.94×10-3 0.9629 278.5

Day 3 0.89 1.62×10-3 0.9267 193.6

Day 7 0.81 3.78×10-3 0.9639 174

SRB biofilm protects the surface since RCT,SRB>RCT,sterile

PAO1 biofilm cannot protect the surface R

Fluorescence microscopy and EPS

quantification 45

0

10

20

30

40

50

60

70

80

90

SRB Mixed PAO1

% Area Polysaccharide

Protein

Total coverage

SRB

PAO1

Mixed

Fluorescence microscopy and EPS

quantification, cont’d

46

Cultures

Polysaccharide

(µg / cm2 coupon)

Protein

(µg / cm2 coupon)

PAO1 23.4 ± 2.8 7.6 ± 1.3

SRB 59.2 ± 2.9 86.8 ± 18.1

Mixed 47.7 ± 7.7 76.22 ± 34.3

Protein and polysaccharide content as well as

Surface coverage is higher for SRB compared to

mixed species and PAO1 biofilms respectively

At high iron concentrations on the surface of

coupons PAO1 attachment to iron surface decreases

resulting in patchy and scanty biofilms

0

10

20

30

40

50

60

70

80

90

SRB Mixed PAO1

% Area Polysaccharide

Protein

Total coverage

Atomic Force Microscopy (AFM)

AFM was used to:

(i) Compare the influence of bacterial metabolites in PAO1 and SRB solutions on

corrosion behavior of cast iron (in-situ)

(ii) Study effect of biofilms on corrosion behavior of cast iron in a standard chloride

solution (3.5% NaCl solution, pH = 4, ex-situ).

7 days of biofilm development followed by biofilm removal by Clark’s solution.

47

Atomic Force Microscopy (AFM) :

metabolites effect, in-situ

48

PAO1;

Roughness

SRB;

Roughness

Pits

Ra = 14.94 nm

Rq = 23.45 nm

Ra = 38.36 nm

Rq = 66.24 nm

Atomic Force Microscopy (AFM) :biofilm

effect, ex-situ

49

PAO1;

Roughness

SRB;

Roughness Ra = 664.9 nm

Rq = 845.4 nm

Ra = 110.5 nm

Rq = 146.3 nm

Atomic Force Microscopy (AFM)

High surface roughness and distorted surface morphology after PAO1 biofilm removal in

coupons that have been immersed for further 4 days in 3.5% NaCl solution compared to

SRB biofilms. This reveals weaker ability of PAO1 biofilms compared to SRB in

protecting the surface.

Pits and higher roughness for coupons that have been immersed in SRB solutions for 7

days without immersion in NaCl. This reveals detrimental effect of SRB metabolites (such

as H2S) in its bacterial solutions compared to PAO1.

50

Weight loss

Weight loss was used to grasp an idea about the actual extent of material loss due to

corrosion. ASTM D-2688.

It was conducted to study effect of bacterial metabolites in solution and biofilms alone.

7 days of biofilm development on coupons followed by biofilm removal:

(a) without immersion in 3.5% NaCl solution Influence of bacterial metabolites.

(b) immersion in 3.5% NaCl solution for 4 days Effect of biofilm.

51

Weight loss

52

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Day 1 Day 3 Day 7

Weight loss

mg/cm2

SRB

PAO1

Mixed

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Day 1 Day 3 Day 7

Weight loss

(mg/cm2)

SRB

PAO1

Mixed

Bacterial metabolites effect (in-situ) Biofilm effect (ex-situ)

Outline

53




Conclusions

Conclusions

Mixing denitrifying PAO1 with SRB lead to a decrease in the extent of corrosion of cast

iron (higher electrical resistances, less weight loss) compared to SRB monocultures which

can be attributed to reduced SRB activity in the presence of PAO1.

Although SRB bacterial solution exerted higher corrosion on coupons, SRB biofilms

protect the surface because of their high surface coverage and weight in the surface of cast

iron.

Surface roughness and pitting is higher in SRB solutions mostly because of H2S, however

SRB biofilms could protect the surface against attack of an aggressive electrolyte.

54

Acknowledgements

I appreciate:

Dr Youngwoo Seo (My Adviser)

This research was conducted under the support of NSF award # 1236433.

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