Biofilm formation by sulfate reducing bacteria and biocorrosion of metals in seawater

BIOFILM FORMATION BY SULFATE REDUCING BACTERIA
AND BIOCORROSION OF METALS IN SEAWATER

VU PHUONG THANH
(B.ENG., HANOI UNIVERSITY OF TECHNOLOGY)

A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF ENGINEERING

DEPARTMENT OF CHEMICAL
AND BIOMOLECULAR ENGINEERING

NATIONAL UNIVERSITY OF SINGAPORE
2009


Acknowledgements

ACKNOWLEDGEMENTS

My deep and sincere gratitude goes to my supervisor Professor Ting Yen Peng
for allowing me to join his team, for his expertise, kindness, and for his patience.
Without his guidance and support during two years of the master course, I might not
finish my research work. I would like to thank Dr. Serena Teo for providing facilities
for my isolation of new sulfate reducing bacterium from local marine and Professor
Hong Liang for allowing me to use the equipment in his lab.
I would like to express my recognition and appreciation to our lab officers Ms.
Sylvia Wan and Ms. Jamie Sew for their support and assistance in my experiments. I
would like to acknowledge the advice and guidance of Ms. Samantha Fam, Dr. Yuan
Ze Liang, Mr. Chia Phai Ann and Dr. Rajarathnam Dharmarajan on the operation of
AFM, XPS, SEM and HPLC equipment. I also thank Mr. Ng Kim Poi for supplying

and fabricating metal coupons for the corrosion study.
I have furthermore to thank my lab members for their friendship and
encouragement. My special thank goes to my friend, Ms. Pham Van Anh, for her
understanding and helpfulness during my time in Singapore.
I would like to thank my family members, especially my husband, Nguyen Ngoc
Chien, for supporting and encouraging me to pursue this degree.
This work received the assistance of Tropical Marine Science Institute
(Singapore) National University of Singapore and Data Storage Institute (A-Star).

i


Table of Contents

TABLE OF CONTENTS
ACKNOWLEDGEMENTS .......................................................................................... i
TABLE OF CONTENTS ............................................................................................. ii
SUMMARY ................................................................................................................... v
LIST OF TABLES ...................................................................................................... vii
LIST OF FIGURES ...................................................................................................viii
NOMENCLATURE..................................................................................................... xi
CHAPTER I: INTRODUCTION ................................................................................ 1
1.1 Importance of microbiologically influenced corrosion........................................ 3
1.2 Microorganism in biocorrosion ............................................................................. 5
1.3 Objective and Scope of the project........................................................................ 6
CHAPTER II: LITERATURE REVIEW .................................................................. 8
2.1 Fundamental aspects of corrosion......................................................................... 8
2.2 Biofilm formation and proposed mechanisms of MIC ...................................... 10
2.2.1 Biofilm formation process ................................................................................... 10
2.2.2 Biocorrosion mechanisms.................................................................................... 12

2.3 Sulfate reducing bacteria and its influence on the corrosion of metals ........... 15
2.4 Titanium and titanium corrosion ........................................................................ 18
2.5 Continuous systems in biocorrosion study ......................................................... 20
CHAPTER III: MATERIALS AND METHODS.................................................... 23
3.1 MIC study of titanium in continuous and semi-continuous system ................. 23
3.1.1 Metal coupons preparation................................................................................... 23
3.1.2 Bacteria ................................................................................................................ 24
3.1.3 Bacterial culture for MIC studies......................................................................... 25
3.1.3 SEM sample preparation and operation............................................................... 26
3.1.4 AFM sample preparation and operation .............................................................. 27

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Table of Contents
3.1.5 EIS sample preparation and operation ................................................................. 27
3.1.6 XPS sample preparation and operation................................................................ 28
3.1.7 ICP-MS sample preparation and operation.......................................................... 29
3.2 SRB Isolation and Characterization ................................................................... 30
3.2.1 Isolation................................................................................................................ 30
3.2.2 Morphological characterization ........................................................................... 31
3.2.3 Physiological characterization ............................................................................. 32
3.2.4 HPLC sample preparation and operation............................................................. 33
3.2.5 Phylogenetic classification................................................................................... 34
3.2.6 MIC study of the new isolate ............................................................................... 34
CHAPTER IV: RESULTS AND DISCUSSION...................................................... 36
4.1 Biocorrosion behavior of titanium grade 5 in semi continuous system ........... 36
4.1.1 Experiment conditions ......................................................................................... 36
4.1.2 Biofilm formation and detachment ...................................................................... 39
4.1.3 Pit observation and measurement ........................................................................ 43

4.1.4 Electrochemical study of corrosion behavior ...................................................... 45
4.1.4.1 Modeling of control coupons ...............................................................46
4.1.4.2 Modeling of coupons exposed to Desulfovibrio desulfuricans and
Desulfovibrio singaporenus in semi-continuous cultures................................49
4.1.5 ICP analysis for confirmation of bacterial effect on metal dissolution ............... 53
4.1.6 Verification of titanium biocorrosion mechanism using X-ray photoelectron
spectroscopy.................................................................................................................. 57
4.1.7 Summary .............................................................................................................. 59
4.2 Biocorrosion behavior of titanium grade 5 in continuous system .................... 60
4.2.1 Experiment conditions ......................................................................................... 61
4.2.2 Biofilm formation and detachment ...................................................................... 62
4.2.3 Pit observation and measurement ........................................................................ 64
4.2.4 EIS data for corrosion behavior and evidence of pitting corrosion ..................... 67
4.2.4.1 Modeling of control coupons ...............................................................67
4.2.4.2 Modeling of coupons exposed to Desulfovibrio desulfuricans
continuous culture............................................................................................69

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Table of Contents
4.2.5 ICP analysis ......................................................................................................... 73
4.2.6 Summary .............................................................................................................. 75
4.3 Isolation and characterization of new SRB from local marine......................... 76
4.3.1 Bacterial cell morphology.................................................................................... 76
4.3.2 Physiological properties....................................................................................... 77
4.3.3 The growth of TA1L in lactate and sulfate containing medium.......................... 80
4.3.4 Phylogenetic properties........................................................................................ 82
4.3.5 MIC study of stainless steel 316, mild steel, aluminum alloy 6064 and titanium
grade 5........................................................................................................................... 86

4.3.6 Summary .............................................................................................................. 89
CHAPTER V: CONCLUSION AND RECOMMENDATION .............................. 91
5.1 Conclusion ............................................................................................................. 91
5.2 Recommendation................................................................................................... 92
REFERENCES............................................................................................................ 94

iv


Summary

SUMMARY

The project focuses on titanium and its corrosion behaviors under the influence
of sulfate reducing bacteria in seawater. The major focus of the project is a study on
the corrosion of titanium in semi-continuous and continuous systems. The isolation of
a local marine SRB is also reported.
The corrosion behavior of titanium grade 5 in seawater was investigated under
the influence of two axenic sulfate reducing bacterial cultures of Desulfovibrio
singaporenus (EF178280) and Desulfovibrio desulfuricans (ATCC 27774) in semicontinuous conditions, using electrochemical impedance spectroscopy technique.
Observations using scanning electron microscope (SEM) showed that the bacteria
quickly colonized on titanium surface and biofilm formation occurred within the first
week of exposure. Biofilm detachment was also observed during the period. Pitting
corrosion was noted after 3 months, and surface pits were revealed using atomic force
microscopy. Analysis using inductively coupled plasma mass spectroscopy (ICP-MS)
showed higher concentration of titanium in the bacterial culture medium for both D.
singaporenus and D. desulfuricans cultures than in the control without the bacteria.
All the data suggested that titanium dissolution occurred in seawater, with the bacteria
playing a role in its corrosion.
A continuous system based on a reactor provided by Centre of Disease Control

(CDC reactor) was established using the sulfate reducing bacterium D. desulfuricans.
Bacterial colonization and biofilm formation rapidly occurred as in the semicontinuous system. The biofilm formed in the continuous system was more compact
and less patchy, and covered the metal surface with fewer voids. AFM data showed
different section profile of pits on titanium surface after 3 months and 4 months in the

v


Summary
continuous culture. On the surface of the control coupon, some holes were also found
but it is unlikely that they were corrosion pits because of their small number and
shallow depth. EIS was successfully used in modeling the behavior of titaniumbacterium-medium interaction. DC polarization data showed some evidence of
biocorrosion. ICP data confirmed the influence of the bacterium in titanium corrosion
as bacterial culture consistently showed higher metal concentration than the control in
the aqueous system.
A novel sulfate reducing bacterium, named TA1L, was isolated from a biofilm
on titanium substratum in the local marine environment in Singapore. The cell was
Gram negative, rod curve shaped, motile and non-spore forming. The bacterium is
halophilic and is capable of using many common electron acceptors as sulfate
reducing bacteria. The bacterium also uses sulphite, thiosulphate as electron
acceptors. Optimal condition for cell growth was 35oC, pH 7.5 and a salinity of 2.5%.
Desulfoviridin test as well as partial 16S-rRNA encoding gene sequence analysis
showed that the bacterium is a member of Desulfovibrio genus. Phylogenetic tree
indicated that the new isolate is a close relation to Desulfovibrio bizertensis and
Desulfovibrio singaporenus. Microbiologically influenced corrosion studies suggested
the role of TA1L in initiating and promoting the corrosion of SS316, mild steel,
aluminum and titanium grade 5 in seawater.

vi

List of tables

LIST OF TABLES

Table 3.1: Modified Barr’s Medium for D. desulfuricans ........................................... 24
Table 3.2: Postgate medium B for D. singaporenus .................................................... 24
Table 3.3: Artificial seawater ....................................................................................... 25
Table 4.1.1 Value of model parameters obtained by fitting the two models [Rs(QbRb)]
and [Rs(Qb[Rb(QpRp)])] to EIS data for the control coupons in semi-continuous system.
....................................................................................................................................... 48
Table 4.1.2 Value of fitted parameters for EIS data of D. desulfuricans coupons in
semi-continuous system ................................................................................................ 51
Table 4.1.3 Value of fitted parameters for EIS data of D. singaporenus coupons in
semi-continuous system ................................................................................................ 51
Table 4.1.4 Titanium concentration (mg/L) in bacterial cultures and controls immersed
with 3-month old and 4-month old coupons in semi-continuous conditions. (n denotes
the number of samples).................................................................................................. 54
Table 4.2.1 Value of fitting parameters obtained by fitting model [Rs(QbRb)] and
model [Rs(Qb[Rb(QpRp)])] to EIS data of control coupons in continuous system. ....... 68
Table 4.2.2 Value of fitted parameters obtained by fitting the two models
[Rs(Qb[Rb(QpRp)(QfRf)])] and [Rs(Qb[Rb(QfRf)])] to EIS data of control coupons in
semi-continuous system. ............................................................................................... 71
Table 4.2.3 Titanium concentration of bacterial cultures and controls immersed with
3-month old and 4-month old coupons in continuous conditions................................. 74
Table 4.3.1 Morphology and physiological properties of SRB isolated from titanium
sample. .......................................................................................................................... 79
Table 4.3.2 Comparison of cell morphology and physiological properties of TA1L and
its two close relatives Desulfovibrio bizertensi and Desulfovibrio singaporenus ........ 85

vii


List of figures

LIST OF FIGURES

Figure 1.1 Corrosion cell (Properties of Internal Pipe Corrosion)................................. 9
Figure 2.1 The biofilm life cycle (Biofilm hypertextbook). ........................................ 12
Figure 3.1 CDC reactor system set up (picture from manufacturer) ........................... 26
Figure 4.1.1 Batch growth of ( ) D. singaporenus and ( ) D. desulfuricans in ASW
at 25oC under anaerobic condition................................................................................ 38
Figure 4.1.2 pH value and cell concentration of ( ) D. singaporenus and ( ) D.
desulfuricans cultures during two 6-day replenishment periods (6th day was chosen as
replenishment point). .................................................................................................... 39
Figure 4.1.3 D. desulfuricans biofilm at week 10 (a) before and (b) after
replenishment................................................................................................................ 39
Figure 4.1.4 AFM images of (a) D. desulfuricans and (b) D. singaporenus cells and
their EPS on titanium surface ....................................................................................... 40
Figure 4.1.5 (1) D. desulfuricans and (2) D. singaporenus biofilm formed on titanium
surface after (a) 3 days, (b) 7 days and (c) 14 days ...................................................... 41
Fig. 4.1.6 Metal surfaces with (a1) D. desulfuricans biofilm at week 5; (a2) D.
desulfuricans biofilm detachment at week 6; (b1) D. singaporenus biofilm at week 6
and D. singaporenus biofilm detachment at week 7..................................................... 42
Figure 4.1.7 Section profile of pits on (1) D. desulfuricans and (2) D. singaporenus (a)
3-month and (b) 4-month samples by AFM. ................................................................ 44
Figure 4.1.8 Electrical circuits for model (a) [Rs(QbRb)] and model (b)
[Rs(Qb[Rb(QpRp)])] for semi-continuous control samples. ........................................... 47
Figure 4.1.9 (a) Nyquist, (b) Bode modulus and (c) Bode phase plots for control
coupons and their fitted data with two models [Rs(Qb[Rb(QpRp)])] and [Rs(QbRb)] .... 48

Figure 4.1.10 Electrical circuits for (a) model [Rs(Qb[Rb(QpRp)(QfRf)])] and (b) model
[Rs(Qb[Rb(QfRf)])] for semi-continuous D. desulfuricans samples. ............................. 49
Figure 4.1.11 (1) Nyquist and (2) Bode phase plots for (a) D. desulfuricans and (b) D.
singaporenus coupons and their fitted data with two models [Rs(Qb[Rb(QfRf)])] and
[Rs(Qb[Rb(QpRp)(QfRf)])].............................................................................................. 50
Figure 4.1.12 Cyclic polarization curves of titanium coupons exposed to semicontinuous culture (a) D. desulfuricans, (b) D. singaporenus and (c) seawater after 14
weeks............................................................................................................................. 53
Figure 4.1.13 Comparison of average value of total titanium concentrations in D.
desulfuricans and D. singaporenus 3-month samples and controls.............................. 55
viii


List of figures
Figure 4.1.14 Comparison of average value of total titanium concentrations in D.
desulfuricans and D. singaporenus 4-month samples and controls.............................. 55
Figure 4.1.15 Average values of titanium concentration in 3-month and 4-month
samples.......................................................................................................................... 57
Figure 4.1.16 XPS spectra of titanium and sulfur on the Ti coupon exposed to D.
singaporenus culture after removal of biofilm. ............................................................ 58
Figure 4.2.1 Desulfovibrio desulfuricans growth curve in artificial seawater at 25oC,
pH 7, and under ambient conditions ............................................................................. 62
Figure 4.2.2 AFM images of Desulfovibrio desulfuricans colonization and (a)
exopolymer substances after 1 week and (b) biofilm after 4 weeks ............................. 62
Figure 4.2.3 SEM images of biofilm on titanium surface under continuous condition
after (a) 3 days, (b) 1 week, (c) 2 weeks and (d) 10 weeks. ......................................... 63
Figure 4.2.4 Section profile of pits on (a) 3 month and (b) 4 month continuous
coupons ......................................................................................................................... 66
Figure 4.2.5 AFM image of a shallow hole on control coupon after 4 months in
continuous system......................................................................................................... 66
Figure 4.2.6 (a) Nyquist, (b) Bode modulus and (c) Bode phase plots for control

coupons and their fitted data with model [Rs(QbRb)] and model [Rs(Qb[Rb(QpRp)])] .. 68
Figure 4.2.7 (a) Nyquist, (b) Bode modulus and (c) Bode phase plots for coupons
exposed to D. desulfuricans continuous culture and their fitted data with model
Rs(Qb[Rb(QfRf)])] and model [Rs(Qb[Rb(QpRp)(QfRf)])] .............................................. 71
Figure 4.2.8 Cyclic polarization curves of titanium coupons exposed to continuous (a)
D. desulfuricans culture and (b) control after 14 weeks............................................... 72
Figure 4.2.9 Comparison of titanium concentration in 3rd month and 4th month
controls and samples exposed to continuous bacterial culture ..................................... 74
Figure 4.3.1 FESEM images of TA1L cells and biofilm on SS316 coupon after 2
weeks of exposure to seawater...................................................................................... 77
Figure 4.3.2 Growth curve of TA1L in term of ( ) logarithm of cell concentration.
( ) Lactate consumption, ( ) acetate production and (x) pyruvate production during the
cell growth. ................................................................................................................... 81
Figure 4.3.3 Partial 16S-rRNA encoding gene of TA1L ............................................. 83
Figure 4.3.4 Phylogenetic tree based on 16S-rRNA gene of new isolate TA1L and
other bacteria from Desulfovibrio genus. The scale bar represents 7 nucleotide
substitutions per 100 nucleotides. ................................................................................. 84
Figure 4.3.5 Bacterial colonization on (a) SS316, (b) mild steel, (c) aluminum and (d)
titanium after 5 days...................................................................................................... 86

ix


List of figures
Figure 4.3.6 Potentiodynamic scanning curves of (—) control and (—) D.
desulfuricans sample of (a) SS316 coupon after 7 days, (b) mild steel coupon after 5
days, (c) Al coupon after 1 day, (d) Cu coupons after 4 weeks and Ti coupon after 3
months........................................................................................................................... 88
Figure 4.3.7 SEM images of pits found on (a) aluminum surface after 5 days, (b)
stainless steel after 7 weeks and (c) mild steel after 1 week......................................... 89

x


Nomenclature

NOMENCLATURE
AFM

Atomic force microscopy

ASW

Artificial seawater

CBR

Centre of Disease Control Biofilm Reactor

CDC

Centre of Disease Control

CPE

Constant phase element

Epit

Breakdown potential (V)

Epp

Passivation potential (V)

Ecorr

Corrosion potential (V)

EASW

Enriched artificial seawater

EIS

Electrochemical impedance spectroscopy

EPS

Extracellular polymeric substance

HPLC

High performance liquid chromatography

ICP

Inductively coupled plasma

ICP-MS

Inductively coupled plasma-Mass spectrometry

MIC

Microbiologically influenced corrosion

MPN

Most probable number method

ppm

Part per million

OCP

Open circuit potential (V)

Qb

Capacitance of the compact oxide barrier (µF)

Qedl

Capacitance of the electrical double layer (µF)

Qp

Capacitance of the porous oxide film (µF)

Rb

Resistance of the compact oxide barrier (k )

Rct

Charge transfer resistance (k )

xi


Nomenclature
Rp

Resistance of the porous oxide film (k )

Rs

Solution resistance ( )

n

Constant phase angle of CPE (rad)

SD

Standard deviation

SEM

Scanning electron microscope

SRB

Sulfate-reducing bacteria

XPS

X-ray photoelectron spectroscopy

ZCPE

Impedance of the constant phase elements ( )

icorr

Corrosion current density (A)

µmax

Specific growth rate (h-1)

Yo

Constant of CPE (µ

-1 n

s)

xii


Introduction

CHAPTER I: INTRODUCTION

The term microbiologically influenced corrosion (MIC) is usually understood as
the deterioration of metal which is promoted and enhanced by the effect of
microorganisms. Microorganisms in general, and bacteria in particular, are the main
factor in MIC, apart from other requirements such as energy source, carbon source,
electron donor, electron acceptor and water (Javaherdashti, 2008). However
microorganism not only cause the corrosion of metals but may indeed inhibit this
phenomena (Jayaraman et al., 1997; Zuo, 2007), and the term “biofouling” is more
generally used. When a metal is exposed to water, the water-borne microorganisms
colonize on its surface and form biofilm through a series of steps. Problems which
occur when the biofilm builds up are termed biofouling. Microbial fouling not only
causes MIC but may also lead to other severe consequences such as product
contamination, reduction in a transfer or mechanical blockages.
Microorganisms are ubiquitous; as long as the environment contains water,
bacteria can form biofilm and biofouling occurs. Metal corrosion is one of the most
important aspects of biofouling. Although metal corrosion can occur without
microorganism, microbial activities accelerate the reactions rate (Horn et al., 2002).
Metabolic products such as organic acids, sulfide, protein and polysaccharides affect
the kinetics of cathodic and/or anodic reactions and also modify the properties of the
metal surface as well as passive layers which are formed on the metal surface (Beech
et al. 2004a).
Microbiologically influenced corrosion (MIC) or biocorrosion has long been
recognized and is a topic of interest due to the complexities resulting from the

involvement of living organisms (Coetser et al. 2005). From the pioneer work of

1


Introduction
Wolzogen Kuhr and Van de Flugt in 1934 (Von Wolzogen Kuhr et al. 1934) until the
1960s, only few studies were published. The first stage in the development of our
understanding of MIC is the discovery and connection between the presence of
bacteria and the corrosion phenomena. During the 1960s and early 1970s, publications
were focused on explaining the role of sulfate reducing bacteria (SRB) and the use of
some electrochemical techniques for MIC investigation. During this period, the major
factors of biocorrosion, bacterial colonization and biofilm settlement, were not widely
investigated (Videla, 1991a).
The 1980s is considered the booming stage in MIC studies with efforts to
establish a mechanism for the corrosion process. Videla and his colleagues (1991b)
considered this stage a progress of understanding MIC. Various hypotheses were
proposed to interpret the hidden mechanisms of MIC. Understanding of biocorrosion at
that point of time created an industrial awareness of this phenomenon. From the
1990’s, many modern applications have been introduced in the detailed investigation
of MIC; these include microscopy techniques and electrochemical techniques (Beech,
2004b). Modern microscopy techniques such as atomic force microscopy (AFM),
scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM)
reveal the metal surface covered with biofilm, microorganism products such as
extracellular polymer substances (EPSs) and corrosion products. Energy Dispersive Xray Analysis (EDX), X-ray photoelectron spectroscopy (XPS), X-ray diffraction
(XRD) and other methods were used in the analysis of corrosion products and the
establishment of corrosion mechanisms. Quantitative assessments of MIC including
electrochemical techniques have improved, and numerous new approaches such as
Electrochemical Impedance Spectroscopy (EIS) and Electrochemical Noise (ECN)
have been widely utilized. These electrochemical techniques, in addition to traditional

2


Introduction
weight loss measurement, are effective methods for calculating corrosion rates. Other
techniques such as Attenuated Total Reflectance/Fourier Transform Infrared
Spectroscopy (ATR/FTIR), Inductively Coupled Plasma spectrometry (ICP) and TimeOf-Flight Secondary Ionization Mass Spectrometry (TOF-SIMS) are also applied in
this field. Over the years, research has intensified with detailed examination with
regard to corrosion materials, environments as well as the microorganisms involved.
1.1 Importance of microbiologically influenced corrosion
MIC occurs in all aqueous environments such as fresh water, sea water, and
humid soil and in industrial systems. Therefore, its sphere of influence is very
extensive, from bridges, buildings and infrastructures on the ground to pipelines and
rails underground, from marine ships, harbors and offshore systems to airplanes and
aerospace devices. Biocorrosion as well as other types of corrosion attack are
responsible for a large number of economic losses. The following cases are some of
the negative impact of MIC.
MIC reportedly contributes 10- 20% of the total corrosion damage of all metals
and building materials out of 4% GNP loss in developed countries (Geesey et al.,
2000). In England, it has been estimated that biocorrosion contributes up to 10% of
corrosion cases (De Romero et al., 2000). Annually, the direct cost of MIC is estimated
at 30-50 billion US dollar. In USA alone, industries spend about 1.2 billion US dollar
annually on biocides to mitigate MIC (Flemming, 1996).
In the oil and gas industry, biocorrosion accounts for 15-30% of the corrosion
cases. In USA alone, overall loss to oil and gas industry due to MIC has been reported
to be more than $100 million per annum (Maxwell et al., 2004). Underground
corrosion of steel gas or water pipes which cost 0.5 to 2 billion US dollars per year

3

Introduction
(Greathouse et al., 1954) is the best known economic disaster engendered by sulfate
reducing bacteria (SRB), a group of bacteria involved in MIC.
Microorganisms have been held to be the major or minor cause of accidents
such as oil spill, building and bridge collapses, etc. MIC-inducing microorganisms are
also involved in another significant issue: they cause severe contaminations to
industrial products, and their metabolic products excreted poison the reservoirs. The
black color of the Black Sea is believed to be the result of the activities of SRB. SRB
have also been reported to be responsible for many other environmental problems such
as massive fish kills, death of sewer workers by H2S generation, and the formation of
poisonous fogs, etc. (Singleton, 1993).
In recent years, the use of metal components for human implants has been
increasing. In-vivo biocorrosion of these implants by human parasitic and other
microorganisms and by body fluids are issues of concern. Singh and Dahotre (2007)
reported that corrosion of metallic implants due to the body environment and bacteria
cause many problems: it may release undesirable metal ions and/or corrosion products
which are non-biocompatible to human body; it may shorten the life of implant device
which leads to another costly surgery, and even shorten the patient’s life.
Singapore is the third biggest oil refinery centre in the world. The quantity of
metals used in infrastructure, including offshore pipelines, and vessels is extremely
high. These metal-based systems are highly susceptible to marine biocorrosion.
Understanding MIC helps us to protect our systems by applying protection methods
such as coating and alloying the metals, and the use of biocides and inhibitors. This
approach will significantly reduce the cost of damage to both the material and product.
At the same time, the structure integrity of the infrastructure will not be compromised.

4

Introduction
Titanium is well known to be a highly resistant material. The term titanium
biocorrosion is relatively new as titanium has been shown to be not susceptible to MIC
under natural conditions. However, the corrosion of titanium in human fluids as well
as in industrial environment has been reported (Yen et al., 1998, Hsu et al., 2004, Rao
et al., 2005). Over the years, titanium has been increasingly used. As such, the role of
bacteria in the corrosion of titanium over the long term should be investigated.
1.2 Microorganism in biocorrosion
Many bacteria have been shown to be associated with the process of
biocorrosion since 1960s. According to Beech (2003), sulfate reducing bacteria (SRB),
iron/manganese oxidizing bacteria, iron reducing bacteria, sulfur oxidizing bacteria
and bacteria which produce organic acids and slime are the dominant types of
microorganism involved in MIC in terrestrial and aquatic habitats. These microbes are
found in natural biofilm as they all contribute to the complex consortia of biofilm
(Beech et al., 2004a).
Among others, SRB is not only the first group of interest in biocorrosion study
(Videla et al., 1991b) but has also been recognized as the most significant contributors
to MIC (Coetser et al., 2005). In the early stage of MIC study, SRB were thought to be
the only bacteria causing biocorrosion (Horn et al., 2002). Overtime, however, it is
recognized that this group is facilitated in natural biofilm owing to the activities of
aerobic bacteria which form reducing conditions as well as nutrient source for SRB.
Currently, SRB continues to be the most published group of bacteria in the field of
MIC. SRB has been widely investigated in the corrosion of iron-based alloys,
aluminum-based alloys, copper-based alloys, etc.
The study of bacteria in their natural environment helps us to understand the
natural consortia and provides us effective approaches to exploit their advantages as
5

Introduction
well as minimize their disadvantages in engineered systems. In MIC studies, SRB as
well as other bacteria involved are usually isolated from field sites and construction
materials. The isolation and characterization of new bacteria are necessary for the
investigation of underlying corrosion mechanisms as there is no universal mechanism
that fully explains the phenomenon. In addition, study on the activities of bacteria in
their axenic cultures and in mixed cultures is important in understanding the role of an
individual bacterium as well as synergistic effects of different bacteria.
Many SRB have been isolated from field sites such as Desulfovibrio halophilus
sp. nov. from Solar Lake in Sinai (Caumette et al., 1991); Desulfovibrio vietnamensis
from oil storage tank in Vietnam (Dang et al., 1996); Desulfotomaculum sp. from oilwater in Turkey (Cetin et al., 2007); Desulfovibrio marinus sp. nov. from marine
sediments in Tunisia; etc. In our lab, a new SRB named Desulfovibrio singaporenus
was isolated from corroded stainless steel 316 surface in natural seawater. The
bacterium has been shown to cause the corrosion of SS316 in the marine environment
(Sheng et al., 2007a). Its role in other metals including titanium should be investigated.
1.3 Objective and Scope of the project
The project focuses on titanium and its corrosion behavior under the influence of
SRB. The SRB biofilm formation on its surface in different conditions is also an aspect
of this work. Based on the above objectives, the focus of this work is as follows:
(i). Study on the influence of two SRB D. desulfuricans (ATCC 27774) and D.
singaporenus on the corrosion of titanium grade 5. The formation of biofilm on metal
surface in artificial seawater (ASW) is observed. The morphology of the corrosion and
type of attack are revealed. The presence of metal ions and precipitate in the culture media
is used to confirm the dissolution of metal under the impact of the bacteria. The nature of
corrosion products such as elemental composition and oxidation states is examined.
6


Introduction
(ii). Study on influence of Desulfovibrio desulfuricans (ATCC 27774) on the

corrosion of titanium in a flow-through system. Under more realistic conditions (i.e.
continuous flow and ambient conditions), a CDC reactor system is operated, and the
corrosion behavior of titanium is investigated. Biofilm formation and detachment are
observed. The morphology of microbial cell, their distribution on the surface and the
presence of EPS are also described. Evidence of corrosion pits, morphology and type
of corrosion attack are investigated.
Due to the diversity of SRB in different water sources and different locations, it
is necessary to isolate and investigate the effect of local bacteria on the corrosion of
some commonly used metals. Understanding of local bacteria MIC would help to
establish more effective metal protection strategy resulting in lower cost of
replacement. Hence, another objective of this study is to investigate the biocorrosion of
some metals under the influence of a local marine SRB which is isolated from a
titanium substratum. The following works are conducted for that purpose.
(iii). Isolation of new SRB from natural seawater in St. John’s Island. The SRB
is then characterized and classified to confirm its SRB origin. The morphology,
physiology and phylogenesis of the cell are investigated. The influence of the new
bacterium on the corrosion of different metals is examined in batch cultures, using
seawater as the culture medium.
This study would contribute to the understanding of titanium biocorrosion which
has not been widely investigated. The use of CDC reactor for corrosion study over a
long time period would provide more realistic conditions compared to other studies in
batch and semi-continuous conditions. The study would also provide an insight into
MIC by a SRB isolated locally in laboratory condition.

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CHAPTER II: LITERATURE REVIEW

2.1 Fundamental aspects of corrosion
Corrosion is a natural phenomenon. According to ISO 8044 standard, corrosion is
defined as a “physicochemical interaction between a metal and its environment which
results in changes in the properties of the metal and which may often lead to impairment
of the function of the metal, the environment, or the technical system of which these form
a part” (Stearns, 2004). In nature, corrosion is the result of a series of redox reactions
between a metal-based material and its surrounding medium which bring about the
deterioration of the metal or its properties. The reduction of an electron acceptor (often
but not always oxygen) located adjacent to the metal surface, in tandem with the oxidation
of the metal is the ultimate cause of the metal dissolution.
There are many underlying agents that increase the likelihood of corrosion in an
aqueous system, such as the dissimilarity of metals or alloys, soil variances, naturally
corrosive soils, atmospheric corrosion, environmental contamination and microbial
corrosion (Stearns, 2004). However, in order for corrosion to happen, the following
components must be present (Barton et al., 2007):
+ an anode site,
+ a cathode site,
+ an electrolyte,
+ a cathodic reactant, and
+ an anodic reactant.
Corrosion reactions are electrochemical in nature, and take place when there is
contact between an oxidizing agent and a reducing agent. The overall corrosion reaction
comprises two half reactions as follows:
(i) oxidation of metal or anodic reaction:

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Meo å Men+ + ne(ii) reduction of chemical species in contact with the metal or cathodic reaction, for
example, the reduction of oxygen:
½ O2 + H2O +2e- å 2OHThe overall reaction is:
2Meo + ½ nO2 + nH2O å 2Men+ + 2nOHThe anodic and cathodic reactions can occur separately at distant locations on the
metal surface. When anodic sites are sparse and irregularly distributed on the surface, the
attack is localized corrosion in the form of pitting and/or crevice corrosion. On the other
hand, when the two half-reactions are close to each other, they form a corrosion cell
(Figure 1.1) and may cause evenly distributed corrosion if the corrosion cells are dense
(Beech et al., 2007).

Figure 1.1 Corrosion cell (Properties of Internal Pipe Corrosion)

If a thermodynamic driving force exists and promotes one or both of the halfreactions, corrosion will take place (Mattsson, 1989). The corrosion rate (or rate of metal
dissolution) is directly proportional the anodic reaction rate or the magnitude of anodic
current. However, the anodic current and the cathodic current must be equal due to the
tendency to obtain electrical neutrality. Hence, the corrosion rate depends on the cathodic

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reaction rate or on the surrounding environment. A reductive environment is usually
aggressive to the metals.
When metals and alloys are exposed to an oxygen-containing environment, or when
a passivation process is applied to protect them, a passive film forms on the surface of these
materials. The passive layers are usually in the form of metal oxide and consist of not only
the major metal but also the alloying metals. For some metals and alloys such as stainless
steels and titanium, the passive layers are strong and highly resistant to corrosion. The
corrosion of these materials only occurs when this protective layer is damaged by an
external factor such as a mechanic force or aggressive chemicals (Beech et al., 2007).

An important concept in corrosion study is “polarization”. When a metal is exposed
to an aqueous environment, there is an equilibrium between the oxidation of metal atoms
to metal ions and the reduction of metal ions to metal atoms:
Men+ + ne-

Me

At the equilibrating state, no electrode reactions occur and the electrode potential is
called equilibrium potential. If a current is applied to change the electrode potential and
equilibrium no longer exist, we call the process polarization. Polarization curve is a graph
of electrode potential when current is altered. Polarization is composed of anodic
polarization and cathodic polarization. Understanding the process and its components
helps us to comprehend kinetics of the corrosion, calculate the corrosion rate as well as
determine a proper strategy for metal protection.
2.2 Biofilm formation and proposed mechanisms of MIC
2.2.1 Biofilm formation process
Although MIC has been studied for many years and many mechanisms have been
proposed, none of them fully explain the phenomenon of biocorrosion. Nonetheless, it is

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generally accepted that the key to surface alterations and enhancement or inhibition of
corrosion is biofilm formation (Videla, 1996). The presence of biofilm on a metal surface
often establishes new electrochemical reaction pathways or promotes reactions which are
not normally favored in the absence of microorganisms. This may result in the increase of
corrosion rates and undesired effects on the performance of metal (Lewandowski et al.,
1997).
Biofilm formation on metal surface is an accumulation process that starts

immediately after metal immersion in an aqueous environment. This process consists of
different stages (Figure 2.1). In the first stage, the deposition of inorganic and organic
high molecular mass compounds from the aqueous environment alters the electrostatic
charges and wetability of the substratum, thus facilitating the colonization of bacteria. In
the second stage, microbial growth and extracellular polymeric substance (EPS)
production results in biofilm formation. The presence of EPS in aqueous media makes
them slimy. Biofilm are thought to contain up to 95% water and this is attributed to the
presence of EPS (Guiamet et at, 1999). EPS is believed to protect the biofilm from the
attack of external factors such as shear stress, and also improve the nutrient absorbability
of biofilm. Subsequently, the biofilm thickening via transport processes and chemical
reactions produce the third step of biofilm formation. Biofilm may be thin (10-12µm) or
as thick as 100µm which prevents the diffusion of ions between metal surface and bulk
fluid (Guiamet et at, 1999). The forth stage, detachment, is the least understood process of
biofilm accumulation. Detachment refers to the release of a patch of cells and their
associated EPS from the biofilm to the bulk fluid. Hunt et. al. (2004) listed some of the
factors that have been suggested to be important in biofilm detachment as matrixdegrading enzymes, microbially generated gas bubbles, nutrient levels and microbial
growth status, availability of multivalent cross-linking cations, fluid shear stress, contact

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attrition, quorum-sensing signals, and the activation of a lytic bacteriophage. Among
others, accumulation of a metabolic product and the depletion of a metabolic substrate are
the most potential factors (Stoodley et al., 2001; Kaplan et al., 2003; Mai-Prochnow et al.,
2004; Parsek et al., 2004). Detachment provides inoculum for further biofilm
accumulation and this phenomenon has been hypothesized to be a factor in the promotion
of MIC (Stewart, 1993). The development and detachment of biofilm are influenced by
many factors such as nutrient sources, flow conditions, surface roughness, temperature
and pH, etc. (Coetser et al., 2005).

Figure 2.1 The biofilm life cycle (Biofilm hypertextbook).
1: Individual cells populate the surface. 2: EPS is produced and biofilm is formed. 3 and 4: biofilm thickens
via transport processes and chemical reactions. 5: Biofilm detachment and inoculation of new generation of
biofilm.

2.2.2 Biocorrosion mechanisms
Videla and his co-workers stated that any biological effect that either facilitates or
impedes one of the anodic or cathodic reactions or permanently separates anodic and
cathodic sites will increase corrosion (Videla et al., 1991b). Based on this principle, many
possible mechanisms of MIC have been proposed.
The first mechanism, proposed by Wolzgen Kuhr and Van der Vlugt (1934), was
cathodic depolarization. It was suggested that the dissociation of hydrogen from the
cathodic site is the rate-limiting step in the corrosion process, and the consumption of
hydrogen by SRB depolarizes the cathode and thus accelerates the corrosion.

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