ii

EXTERNAL CORROSION GROWTH FOR BURIED STEEL PIPELINE IN

ENVIRONMENT CONTAINING SULFATE REDUCING BACTERIA

ARMAN ABDULLAH

A thesis submitted in fulfilment of the

requirements for the award of the degree of
Doctor of Philosophy (Civil Engineering)

Faculty of Civil Engineering

Universiti Teknologi Malaysia

NOVEMBER 2016
 iii

Every challenging work needs self efforts as well as guidance of elders especially
those who very close to our heart.

My humble effort I dedicate to my sweet and loving,

Father, Mother, My Wife and Ainul

Whose affection, love, encouragement and pray of day and night make me able to get
such success and honour

Along with hard working and respected

Lecturers
 iv

ACKNOWLEDGEMENT

Allhamdulillah, all praises to Allah S.W.T. for the strengths and His love and
blessing in completing this thesis. I am deeply grateful to my supervisor, Professor
Dr. Nordin Yahaya and co-supervisor Associate Professor Dr. Norhazilan Md. Noor,
for their constant encouragement, immense enthusiasm for this work, guidance and
for being there when it mattered most. Without their supervision and constant help
this thesis would not have been possible.

My sincere thanks to all my friends; Lim Kar Sing, Siti Rabeah, Akima Abu
Bakar, Mardhiah Ismail, and other RESA members such as Mimi, Fariza and others
for their kindness, help and support in completing this research. My
acknowledgement also goes to all lecturers and technician of FKA‟s, FKM‟s, and
FS‟s for the help.

Last but not least, my deepest appreciation to my beloved wife Maria Alfah,
my daughter Ainul Mardhiah, my mother, brothers, and sisters for all the sacrifices
that you have made on my behalf. Your prayer for me that made me sustained this
far. Thank you.
 v

ABSTRACT

Microbiological-induced corrosion (MIC) by the presence of sulfate reducing

bacteria (SRB) is a serious threat to the long-term integrity of steel pipelines.
Previous studies on the influence of SRB towards corrosion progress in Malaysia
were focused more on marine environment. Considering that Malaysia has
underground gas pipelines of about 2,500 km in length, serious efforts are needed to
conduct detail investigations on the underground SRB threat. The aim of this
research is to conduct an in-depth investigation on the performance of a local
underground SRB strain from Sungai Ular area and benchmark this against
ATCC7757TM strain in terms of bacterial growth and its influence on metal loss
using derived empirical models. Two empirical models associated with MIC were
derived using multilinear regression (MLR) and response surface method (RSM).
The models acquired important corrosion data from field work and laboratory
examination. A total of 48 steel coupons were buried at Sungai Ular site and these
were retrieved every three months for a total of 18-month period. Metal loss and
selected soil parameters were recorded throughout the research duration. Results
have confirmed that the site was highly corrosive by referring to the established
corrosion index. Analysis of the site data using Principal Component Analysis
(PCA) and MLR model with high correlation, R2 of 0.86 showed that the SRB-
related parameters of pH, organic content, and sulfide have significant influences
towards the corrosion rate. The field work results were later confirmed by laboratory
experiments using an isolated local SRB strain from Sungai Ular based on one-
factor-at-a-time-approach (OFAT) and RSM model whereby the pH was found to be
the most dominant parameter that influenced bacterial growth and high corrosion
rate. The DNA test of Sungai Ular SRB strain was found identical to the genus of
ATCC7757TM. A comparative study has identified the Sungai Ular SRB strain as
more aggressive due to the highest corrosion rate recorded at 6.2 mm/year, 11.4%
higher than ATCC7757TM strain. The findings have successfully identified the SRB-
related parameters as the main contributor to the high corrosivity of Sungai Ular site
using a comprehensive field work and laboratory experiment exercises. These can be
highly beneficial guidelines to identify the main parameters that governed the
mechanism of underground corrosion. The derived empirical models can be utilised
by pipeline operators to design a better pipeline maintenance and structure integrity
assessment against MIC threat.
 vi

ABSTRAK

Pengaratan disebabkan oleh mikroorganisma pengaruh karat (MIC) dengan

kehadiran bakteria-penurun-sulfat (SRB) adalah satu ancaman yang serius kepada
integriti talian paip keluli dalam jangka panjang. Kajian terdahulu terhadap SRB di
Malaysia banyak ditumpukan kepada persekitaran marin. Memandangkan Malaysia
mempunyai kira-kira 2,500 km talian paip gas bawah tanah, usaha serius perlu
dilakukan sebagai pemangkin kepada kajian menyeluruh terhadap ancaman SRB
bawah tanah. Tujuan kajian ini adalah untuk menjalankan kajian terperinci terhadap
prestasi SRB bawah tanah baka tempatan bagi kawasan Sungai Ular dan
perbandingan terhadap baka ATCC7757TM, berkaitan pertumbuhan bakteria dan
pengaruhnya ke atas kehilangan logam menggunakan model empirikal. Dua model
empirikal berkait dengan MIC telah diperolehi dengan menggunakan kaedah Regresi
Linear Berbilang (MLR) dan Kaedah Tindakbalas Permukaan (RSM). Model-model
ini menggunakan data-data pengaratan dari kerja lapangan dan makmal. Sebanyak
48 keping kupon keluli telah ditanam di lapangan Sungai Ular dan kupon-kupon
diambil semula setiap tiga bulan sepanjang 18 bulan. Kadar kehilangan logam dan
parameter tanah terpilih telah direkodkan sepanjang tempoh penyelidikan.
Keputusan kajian telah mengesahkan bahawa lokasi kajian adalah sangat mengakis
berdasarkan kepada perbandingan dengan indeks hakisan. Kolerasi R2 yang tinggi
(0.86) dari analisis lapangan kajian menggunakan Prinsip Analisis Komponen (PCA)
dan model MLR menunjukkan parameter yang berkaitan SRB, iaitu pH, kandungan
organik dan sulfida, mempunyai pengaruh penting terhadap kadar kakisan. Hasil
kajian lapangan kemudiannya disahkan oleh kajian makmal berdasarkan kaedah
satu-faktor-pada-satu-masa (OFAT) dan model RSM menggunakan SRB baka
tempatan dari Sungai Ular yang mana, didapati pH merupakan parameter yang
paling utama yang mempengaruhi pertumbuhan bakteria dengan kadar karatan yang
tinggi. Ujian DNA juga mendapati baka SRB dari Sungai Ular didapati dari keluarga
yang sama dengan baka ATCC7757TM. Satu kajian perbandingan mendapati baka
SRB Sungai Ular adalah lebih agresif dengan kadar kakisan yang tertinggi dicatatkan
sebanyak 6.2 mm/tahun, 11.4 % lebih tinggi berbanding baka ATCC7757TM. Hasil
kajian ini juga telah berjaya mengenal pasti parameter-parameter berkait dengan
SRB yang menjadi penyumbang utama terhadap kadar menghakis yang tinggi di
lapangan. Model empirikal yang diperolehi boleh digunakan oleh pengendali talian
paip untuk merekabentuk kaedah penyelenggaraan dan penilaian integriti talian paip
yang lebih baik terhadap ancaman MIC.
 vii

TABLE OF CONTENTS

CHAPTER TITLE PAGE

DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES xiv
LIST OF FIGURES xviii
LIST OF ABBREVIATIONS xxiii
LIST OF SYMBOLS xxv
LIST OF APPENDIX xxvii

1 INTRODUCTION 1
1.1 Introduction 1
1.2 Problem Background 2
1.3 Research Problem 3
1.4 Aims and Objectives of the Research 4
1.5 Research Scopes 5
1.6 Research Importance 5
1.7 Brief Methodology of Study 6
1.8 Structure of the Thesis 7
 viii

2 LITERATURE REVIEW 12
2.1 Introduction 10
2.2 Gas Transmission Pipelines in Malaysia 10
2.3 Corrosion 12
2.4 Background of MIC 12
2.5 Characters of MIC 13
2.6 Mechanism of MIC 15
2.6.1 Creating Differential Concentration Cells 15
2.6.2 Generating Corrosive Substance 16
2.6.3 Alteration of Anion Ratio 18
2.6.4 Reducing the Effectiveness of Corrosion
Inhibitor 18
2.7 Background and Characters of SRB 19
2.7.1 Mechanisms of SRB 21
2.7.2 Corrosion by Hydrogen Sulfide (H2S) 21
2.7.3 Corrosion by Cathodic Depolarisation 21
2.7.4 Alternative Corrosion Mechanism 22
2.8 Bio-corrosion on Steel Pipelines 22
2.8.1 Bio-corrosion Issues in Malaysia 23
2.9 Corrosion Models 25
2.9.1 Mechanistic Model 25
2.9.2 Semi-Empirical Model 26
2.9.3 Empirical Model 27
2.9.4 Corrosion Prediction in Soil 27
2.9.4.1 Soil Corrosion Assessment 28
2.9.5 MIC Corrosion Model 33
2.10 Microbial Growth Requirements 34
2.10.1 Presence of Water 35
2.10.2 Nutritional Requirements 35
2.10.3 Oxygen Level and Redox Potential 36
2.10.4 Temperature 36
2.10.5 pH 37
2.10.6 Salinity 37
2.10.7 Ferrous Ion Concentration (Fe 2+) 38
 ix

2.10.8 Effect of Sulfate Concentration (SO 42-) 38

2.11 Operating Condition Factors Affecting MIC 39
2.11.1 Pressure 39
2.11.2 Impact of Flow Rate 39
2.11.3 Impact of Debris on the Bottom of the
Pipeline 40
2.11.4 Impact of Pigging Frequency 40
2.11.5 Impact of Biocide Treatment 40
2.12 Experimental Design, Mathematical Model
and Statistical Analysis 41
2.12.1 The Concept of Factorial Experiments 41
2.12.2 Main and Interactive Effects on Variables 42
2.12.3 Linear Model for Factorial Experiment
Results 43
3
2.12.4 Full Factorial Experiments 2 for
Composite Design 45
2.12.5 Quadratic Response Surface and
Calculation for Variable Phase
Experiments 46
2.12.6 Verification Model using Orthogonal
Factor 48
2.13 Concluding Remarks 52

3 RESEARCH METHODOLOGY 53
3.1 Introduction 53
3.2 Coupon Preparation 55
3.3 Chemical Composition and Morphology of
API-5LX-70 57
3.4 Weighing and Labelling Process 58
3.5 Methodology for Site Study 58
3.5.1 Site Selection 58
3.5.2 Research Area Selection 59
3.5.3 Installation of Coupons 60
3.5.4 Coupon Retrieval, On-Site Measurement
 x

and Soil Sampling 60

3.5.5 Coupon Cleaning After Retrieval 62
3.5.6 Calculation of Corrosion Rate According
to ASTM 63
3.6 SRB Bacteria 63
3.6.1 Site SRB Bacteria 64
3.7 Molecular Characterisation of SRB 65
3.7.1 DNA Extraction 65
3.7.2 PCR Amplification of 16S rRNA Fragment 65
3.7.3 Agarose Gel Electrophoresis 66
3.7.4 rDNA Fragments Sequencing and Analysis 66
3.8 Media Preparation 67
3.8.1 ATCC 1249 Medium 67
3.8.2 Postgate‟s C Medium and Postgate‟s B
Medium 68
3.9 Corrosion Testing Using Electrochemical Methods 69
3.9.1 Open-circuit Potential Measurement (OCP) 70
3.9.2 Tafel Slope Analysis and Corrosion Rate
Experiment 71
3.10 Analytical Methods 73
3.10.1 SRB Enumeration 73
3.10.2 Sulfate (SO42-) Ions Concentration 73
3.10.3 FESEM Observation 73
3.10.4 Ferrous (Fe2+) Ions Concentration 74
3.10.5 pH Value 74
3.11 Weight Loss Techniques 74
3.11.1 Weight Loss in Anaerobic Vials 75
3.12 Response Surface Method (RSM) 76
3.12.1 Selecting Factors 77
3.12.2 SRB Bacteria Sample 77
3.12.3 Incubation Period 78
3.12.4 Incubation Temperature 79
3.12.5 Test Matrix for Other Parameters 80
3.12.6 SRB Corrosion Model using RSM 81
 xi

3.13 Concluding Remarks 82

4 DATA ANALYSIS AND DEVELOPMENT OF 83

SITE CORROSION MODEL
4.1 Introduction 83
4.2 Site Selection Description 83
4.3 Preliminary Data Analysis 85
4.4 Experimental Results 86
4.5 Data Screening 90
4.5.1 Normality Test 90
4.5.2 Mann – Whitney U Test 91
4.5.3 Outliers Detection 92
4.6 Corrosion Growth Pattern 98
4.7 Linear Correlation Analysis (LCA) 100
4.8 Power Law Equation 107
4.9 Principal Component Analysis (PCA) 107
4.10 Multiple Linear Regressions (MLR) 111
4.11 Development of On-Site Corrosion Model 118
4.12 Conclusion 120

5 ANALYSIS OF CORROSION PARAMETERS 122

5.1 Introduction 122
5.2 Chemical and Microstructure Analysis of Carbon
Steel Coupons 122
5.3 SRB Bacteria 123
5.3.1 SRB Isolation 124
5.4 Characterization of SRB from Sungai Ular 125
5.4.1 DNA Amplification and Sequencing 125
5.4.2 16s rDNA Sequence Analysis 127
5.5 Bacteria Growth Curve 129
5.6 Open Circuit Potential 130
5.7 Corrosion by Isolated SRB 132
5.8 Microscopic Observation 134
 xii

5.9 Screening the Optimum Parameter Range

Using One-Factor-at-a-Time (OFAT) Method 140
5.9.1 Effect of Incubation Temperature on
Corrosion Rate in Bio-corrosion 141
5.9.2 Effect of Medium pH on Corrosion
Rate in Bio-corrosion 143
5.9.3 Effect of SO42- Concentration on
Corrosion Rate in Bio-corrosion 146
5.9.4 Effect of Fe2+ Concentration on
Corrosion Rate in Bio-corrosion 148
5.10 Conclusion 150

6 RESPONSE SURFACE METHOD ANALYSIS 151

6.1 Introduction 151
6.2 Response Surface Methodology for Sungai Ular
Isolated SRB 152
6.3 Response Surface Plots for Sungai Ular
Isolated SRB 157
6.4 Response Surface Methodology for ATCC7757TM
SRB 161
TM
6.5 Response Surface Plots for ATCC7757 SRB 166
6.6 Conclusion 170

7 DISCUSSION 171
7.1 Introduction 171
7.2 Field Work Approach 171
7.2.1 Soil Parameter Data 172
7.2.2 Corrosion Growth Pattern 175
7.2.2.1 Parameter Classification Using PCA 176
7.2.2.2 Multi Linear Regression Model 176
7.3 Laboratory Works 177
7.3.1 Bacteria Identification 178
7.3.2 Influence of Stipulated Parameters from
OFAT Analysis 178
 xiii

7.3.3 Influence of Stipulated Parameters from

RSM Model 179
7.4 Relationship between Field Work, OFAT and RSM
Analysis 179
7.5 Comparison between ATCC7757 TM and Sungai
Ular SRB Strain 180

8 CONCLUSION 182
8.1 Introduction 182
8.2 Conclusion 182
8.3 Research Contribution 184
8.4 The Recommendations for Future Work 184

REFERENCES 186
Appendices A - B
 xiv

LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Mechanisms of microbiologically-influenced corrosion 13

2.2 Most common micro-organisms implicated in MIC 14

2.3 30
Table of AWWA soil test evaluation
2.4 Soil corrosivity classification index by Nazim (2015) 31
2.5 Comparison between AWWA index and Nizam Soil
classification index 32
2.6 Overview of factors for various influencing parameters
34
in MIC
3.1 3 sizes of coupons used in this study 55

3.2 Site locations and their corresponding description 59

3.3 Composition of ATCC 1249TM‟s medium 67

Composition of modified Postgate‟s C medium (Cortas

3.4
et al. 2012) 69

Composition of modified Postgate‟s B medium

3.5
(Tanner, 1989). 69

Summary of SRB species and used medium in SRB

3.6 research 78

Summary of incubation period by other researchers in

3.7
bio-corrosion research 79

Summary of incubation temperature by other

3.8
researchers in bio-corrosion research 80

3.9 Test matrix of OFAT study for SRB 81

Full factorial experimental design matrix with four

3.10
variables (coded and natural) 82
 xv

4.1 Selected site location information (Site C) 84

Results of laboratory analysis of soil for Sungai Ular

4.2
site 86

Results of soil analysis for Sungai Ular site after data

4.3 87
treatment

Sungai Ular soil corrosiveness based on AWWA and

4.4
Nazim (2015) at 3 months of exposure. 88

Metal loss (ML) and corrosion rate (CR) data at 0.5 m

4.5 89
depth

Metal loss (ML) and corrosion rate (CR) data at 1.0 m

4.6 90
depth

4.7 Normality test results 91

Hypothesis testing for metal loss data at depths 0.5 m

4.8 92
and 1.0 m

Metal loss (ML) and corrosion rate (CR) data at 0.5m

4.9 92
depth

Metal loss (ML) and corrosion rate (CR) data at 1.0m

4.10 92
depth

Outliers calculation using Dixon‟s test, Grubbs‟ test

4.11 94
and box plot

Metal loss (ML) and soil parameters (outliers‟ values

4.12 95
are bold)

Metal loss (avg) after removal of outliers at 0.5m

4.13 96
depth

Metal loss (med) after removal of outliers at 0.5m

4.14 96
depth

Metal loss (max) after removal of outliers at 0.5 m

4.15 97
depth

Metal loss (avg) after removal of outliers at 1.0m

4.16 97
depth

Metal loss (med) after removal of outliers at 1.0m

4.17 97
depth

Metal loss (max) after removal of outliers at 1.0m

4.18 97
depth
 xvi

Metal loss (ML) and corrosion rate (CR) for after

4.19
removal of outliers for both depth (0.5m and 1.0 m) 98

Spearman‟s rho correlation of metal loss (med) with

4.20 soil parameters 101

Pearson‟s correlation of metal loss (med) with soil

4.21
parameters 102

Spearman‟s rho correlation of metal loss (avg) with

4.22
soil parameters 103

Pearson‟s correlation of metal loss (avg) with soil

4.23
parameters 104

Spearman‟s rho correlation of metal loss (max) with

4.24
soil parameters 105

Pearson‟s correlation of metal loss (max) with soil

4.25
parameters 106

k and n of power law model according to average,

4.26
median and maximum data 111

4.27 Possible combination of k and n constant 113

Multiple regression analysis for constant k and n

4.28
(average metal loss data) 114

Multiple regression analysis for constant k and n

4.29
(median metal loss data) 115

Multiple regression analysis for constant k and n

4.30
(maximum metal loss data) 116

Summary of R2 from the proposed model for average

4.31
metal loss data 117

Summary of R2 from the proposed model for median

4.32
metal loss data 117

Summary of R2 from the proposed model for

4.33
maximum metal loss data 118

Predicted metal loss data from the proposed model

4.34
compared with experimental result 119

Chemical components of carbon steel coupon for API

5.1
5L X-70 using GDS technique 123
 xvii

Comparison of corrosion rate for ATCC 7757TM and

5.2 isolated SRB (Sungai Ular) inoculated at 37°C for 30
days 132

5.3 Corrosion parameters obtained from Tafel plots for

ATCC7757TM and Sungai Ular strains for 7 and 21 133
days of exposure

5.4 CR for OFAT study for Sungai Ular SRB incubated at 141
different temperature for 30 days by (Ismail, 2015)

Summary of Tafel plot of OFAT for different

5.5 incubation temperatures for 14 days 142

CR of OFAT study for Sungai Ular SRB incubated at

5.6 different pH for 30 days by (Ismail, 2015) 144

Summary of the Tafel plot of OFAT for different pH

5.7 incubated at 37°C for 14 days 145

Summary of the Tafel plot of OFAT at different initial

5.8 sulfate concentrations incubated at 37°C for 14 days 147

Summary of the Tafel plot of OFAT at different initial

5.9
Fe2+ concentrations incubated at 37°C for 14 days 149

Experimental range and coded level of independent

6.1
variables 152

Experimental designs, experimental results and

6.2 predicted responses using RSM for Sungai Ular
isolated SRB 154

ANOVA summary of RSM for Sungai Ular SRB

6.3
inoculated at selected parameters 155

Experimental designs, experimental results and

6.4
predicted responses using RSM 163

6.5 ANOVA summary of RSM for ATCC7757TM SRB

164
inoculated at selected parameters

7.1 Assessment of soil corrosiveness using AWWA and

173
Nazim (2015) corrosivity index
 xviii

LIST OF FIGURES

FIGURE NO. TITLE PAGE

1.1 Flow chart of the research structure 7

2.1 Peninsular Gas Utilisation network (Hamid, 2008) 11

2.2 Two morphologies of SRB. Arrow indicates a) vibro,

and b) spiral 20

2.3 Corrosion growth pattern in a function of time

(Velazquez et al. 2009) 28

3.1 Flow chart of methodology 54

3.2 Carbon steel pipe cut to small size before being sent to
UTM for subsequent cutting process 55

3.3 Cutting machine used to remove heat-affected zone

(HAZ) 56

3.4 Schematic diagram of carbon steel coupon mounted in

polyester resin for electrochemical experiment 56

3.5 Carbon steel coupons used in anaerobic vials (10 mm

× 20mm × 7mm) 57

3.6 The Wenner four-electrode method in soil resistivity

measurement 62

3.7 Anaerobic workstation used for isolation and

manipulates bacteria under anaerobic condition 65

3.8 Transferring the medium to anaerobic vials in the

laminar flow 68

3.9 OCP test conducted in an electrochemical cell

inoculated with SRB 70

3.10 Autolab PGSTAT 30 with GPES system software 71

3.11 The differences between inoculated and control vials 76

 xix

4.1 Carbon steel coupon retrieved from Sungai Ular site 85

4.2 Summary of box plot for metal loss data 93

4.3 Plot of average metal loss against exposure period 99

4.4 Plot of median metal loss against exposure period 99

4.5 Plot of maximum metal loss against exposure period 100

4.6 Extracted component of soil variables for metal loss

(med data) 108

4.7 Extracted component of soil variables for time factor

(med data) 109

4.8 Extracted component of soil variables for metal loss

(avg data) 109

4.9 Extracted Component of soil variables for time factor

(avg data) 110

4.10 Extracted component of soil variables for metal loss

(max data) 110

4.11 Extracted component of soil variables for time factor

(max data) 111

4.12 Experimental metal (Actual) loss vs. predicted metal

loss 120

5.1 Microstructure of carbon steel coupon under

microscope 123

5.2 Sani SRB detection kit 124

5.3 Colony of Sungai Ular SRB grown on agar medium

after 7 days of incubation at 37°C anaerobically 125

5.4 Agarose gel electrophoresis of purified plasmid DNA

for SRB SgU 1 (line 1), SgU 2 (line 2) and SgU3 (line
3) 126

5.5 Agarose gel electrophoresis of purified PCR product

16S rDNA for SRB SgU 1 (line 1), SgU 2 (line 2) and
SgU 3 (line 3) 126

5.6 Partial 16SrDNA sequence of Sungai Ular 1 strain

using BLASTN analysis 127

5.7 Phylogenetic dendrogram based on 16SrDNA gene

sequences indicating the position of Sungai Ular A
 xx

strain among closely related members 128

5.8 Growth curve of SRB bacteria incubated at 37°C for

60 days in anaerobic vials 130

5.9 Plots of open circuit potential for carbon steel with

Sungai Ular and ATCC 7757TM 131

5.10 Tafel plot from Autolab software for ATCC 7757TM

and Sungai Ular SRB for 7 and 21 days of incubation
period 133

5.11 FESEM images of the biofilms and SRB incubated at

37°C for 30 days for isolated Sungai Ular SRB 134

5.12 FESEM images of the biofilms incubated at 37°C for

30 days for isolated Sungai Ular SRB 135

5.13 FESEM images of the biofilms incubated at 37°C for

30 days for ATCC7757TM SRB 136

5.14 FESEM images of the biofilms incubated at 37°C for

30 days for ATCC7757TM SRB 137

5.15 SEM micrographs of the morphology of the corrosion

for a) Sungai Ular strain and b) ATCC 7757TM strain 138

5.16 SEM images for (a) control coupon and (b) coupon
with Sungai Ular SRB after removal of biofilms for 20
days of inoculation at 37°C 138

5.17 SEM images of biofilms inoculated at 37°C observed

after 30 days 138

5.18 EDS analysis of the corrosion products on the coupon

surface after exposure to SRB for 30 days 139

5.19 EDS analysis of biofilms for ATCC7757TM and

Isolated Sungai Ular SRB bacteria inoculated at 37°C
for 30 days 140

5.20 Corrosion rate (mm/yr) of the carbon steel coupons

(X-70) exposed to SRB bacteria at 5°C, 20°C, 30°C,
40°C, 50°C and 60°C 141

5.21 Tafel plot of OFAT for different incubation

temperature for 14 days 142

5.22 Anaerobic vials for experiments conducted at different

pH 143
 xxi

5.23 OFAT for pH value incubated at 37°C for ATCC

7757TM and Sungai Ular SRB 144

5.24 Tafel plot of OFAT for different pH values incubated

at 37°C for 14 days 145

5.25 OFAT for SO42- concentration incubated at 37°C for

30 days 146

5.26 Tafel plot of OFAT at different initial sulfate

concentrations incubated at 37°C for 14 days 147

5.27 OFAT for Fe2+ concentration incubated at 37°C for 30

days 148

5.28 Tafel plot of OFAT at different initial Fe2+

concentrations incubated at 37°C for 14 days 149

6.1 Images of the weight loss experiment conducted in

anaerobic vials at different study parameters.
153

6.2 Parity plot for of Sungai Ular isolated SRB inoculated

with selected parameters 156

6.3 Pareto chart for CR of Sungai Ular isolated SRB

inoculated in selected parameters 156

6.4 Response surface plot as a function of pH and

temperature for corrosion rate of Sungai Ular isolated
SRB 157

6.5 Response surface plot as a function of pH and SO4-2

concentration for corrosion rate of Sungai Ular isolated
SRB 158

6.6 Response surface plot as a function of pH and initial

Fe2+ concentration for corrosion rate of Sungai Ular
isolated SRB 159

6.7 Response surface plot as a function of temperature and

SO4-2 concentration for corrosion rate of Sungai Ular
isolated SRB 159

6.8 Response surface plot as a function of temperature and

initial Fe2+ concentration for corrosion rate of Sungai
Ular isolated SRB 160

6.9 Response surface plot as a function of SO4-2

concentration and initial Fe2+ concentration for
corrosion rate of Sungai Ular isolated SRB 161
 xxii

6.10 Parity plot for of ATCC7757TM SRB inoculated in

selected Parameters 165

6.11 Pareto chart for CR of ATCC7757TM SRB inoculated

in selected parameters 165

6.12 Response surface plot as a function of pH and

temperature for corrosion rate of ATCC7757TM SRB 166

6.13 Response surface plot as a function of pH and SO4-2

concentration for corrosion rate of ATCC7757TM SRB 167

6.14 Response surface plot as a function of pH and initial

Fe2+ concentration for corrosion rate of ATCC7757TM
SRB 168

6.15 Response surface plot as a function of temperature and

SO4-2 concentration for corrosion rate of ATCC7757TM
SRB 168

6.16 Response surface plot as a function of temperature and

initial Fe2+ concentration for corrosion rate of
ATCC7757TM SRB 169

6.17 Response surface plot as a function of SO4-2

concentration and initial Fe2+ concentration for
corrosion rate of ATCC7757TM SRB 170
 xxiii

LIST OF ABBREVIATIONS

AFAT - All Factors at a Time

ANOVA - Analysis of Variance

API X-70 - Grades of Steel Pipelines

API - American Petroleum Institute

ASTM - American Society of Testing of Materials

ATCC - American Type Culture Collection

AUEB - Alberta Energy and Utilities Board
AWWA - American Water Works Association
BDO - Baram Delta Operation
BLAST - Basic Local Alignment Search Tool
CCD - Central Composite Design
CDT - Cathodic Depolarisation Theory
CP - Cathodic Protection
CPD - Critical Point Drying
CR - Corrosion Rate
DNA - Deoxyribonucleic Acid
EDS - Energy Dispersive Spectroscopy
EDTA - Ethylenediaminetetraacetic
EIS - Electrochemical Impendence Spectroscopy
EPS - Extracellular Polymeric Substances
ESEM - Environmental Scanning Electron Microscopy
FESEM - Field Emission Scanning Electron Microscopy
GDS - Glow Discharge Spectrometry
 xxiv

GPES - General Purpose Electrochemical System

HAZ - Heat Affected Zone
IRB - Iron Reducing Bacteria
LPR - Linear polarization resistance

MC - Moisture Content

MIC - Microbiologically Influenced Corrosion

ML - Metal loss

NACE - National Agency of Corrosion Engineer

OC - Organic Content

OCP - Open Circuit Potential

OFAT - One Factor at a Time

PCA - Principle Component Analysis

PCR - Polymerase Chain Reaction

PGU - Peninsular Gas Utilisation

RNA - Ribonucleic Acid
RSM - Response Surface Method
SCE - Saturated Calomel Electrode

SEM - Scanning electron microscope

SKO - Sarawak Operation

SRB - Sulfate–Reducing Bacteria
USA - United State of America
UTM - Univerisiti Teknologi Malaysia
XRD - X-ray Diffraction
 xxv

LIST OF SYMBOLS

% - percentage
µA.cm-2 - micro ampere per centimeter square
µm - micrometer
0
C - Degree Celsius

A - Area of sample in cm2

Al - Aluminum

CFU/mL - colony forming unit per mililiter

Cl- - Content of chloride ion

cm2 - Square centimeter

Cr - Chromium

Cu - Copper

D - Metal density in g /cm3

Ecorr - Corrosion Potential

Eh - Redox potential

Fe2+ - Ferrous iron

g - gram
g/cm3 - gram per cubic centimeter

g/L - gram per liter

H2O - Water

Icorr - Corrosion Current

k - Constant regression parameters

Kg - Kilogram
 xxvi

mg - milligram
mg.cm-2 - milligram per centimeter square
mg/L - milligram per liter
mL - milliliter
mm/y - Millimeter per year

Mn - Manganese

mVSEC - millivolts of Saturated Calomel Electrode

Ni - Nickel

O2 - Oxygen

P - Phosphorus

Pb - Lead

ppm - part per million

S2- - Sulfide
Si-C - Silica - Carbide

SO42- - sulfate
T - Temperature

t - Time

W - Weight loss

X70 - Grade of Steel Pipelines

Μm - Micrometer
 xxvii

LIST OF APPENDICES

APPENDIX TITLE PAGE

A List of data for site Sungai Ular 198

B Tafel Plot for OFAT Study data 203

 1

CHAPTER 1

INTRODUCTION

1.1 Introduction

Corrosion is a major threat to industries, causing gradual degradation and

continual damage to metallic facilities and civil infrastructures. Based on a study
conducted by the National Association of Corrosion Engineers (NACE) in 2002, the
industrial operating loss due to corrosion in the United States had increased to
US$137.9 billion in 2001, out of which 34.7% were attributed to utility service
providers, including gas, water, electricity and telecommunications (Arteaga et al.,
2008). Therefore, corrosion preventive measures are urgently needed to curb
industrial monetary losses from snowballing.

Corrosion is a natural phenomenon of metal, a normal course of an

electrochemical reaction between the metal and its surrounding substances.
Corrosion occurs in the presence of electrolytes, which allow the formation of ions
and the release of electrons at an anode, where they subsequently lead to oxidation
and deterioration of metal. Simultaneously, the cathodic surface, or electrode, will
recognize the electrons generated at the anode with an equivalent rate of reaction.
These continuous reactions will then result in corrosion and decay, causing strength
reduction in the material serving as the anode. The effect of corrosion in this instance
is recognized in several forms, including general corrosion, galvanic, crevice, pitting,
inter-granular, erosion and stress corrosion cracking.
 2

1.2 Problem Background

The corrosion rate can further accelerate if accompanied by the presence of

micro-organisms, appearing when bio-film forms on the surface of metal. This type
of corrosion, caused directly or indirectly by activities of micro-organisms, is known
as microbiologically-influenced corrosion (MIC), or bio-corrosion. MIC is the
catalyst which causes metal to corrode uncontrollably. MIC is a unique culprit
capable of adapting to various types of materials, such as metal, mineral, organic
material and plastic. The issue of MIC is widely discussed and researched in material
science, chemistry, microbiology and engineering study disciplines. In cases of
underground pipelines, for example, sulfate-reducing bacteria (SRB) have always
been identified as a micro-organism associated to the MIC.

MIC can produce unexpected problems, such as premature failures and costly
repairs. The by-products, resulting from metabolic activities of micro-organisms in
bio-films on metal surfaces, promote several forms of corrosion, including pitting,
crevice corrosion and under-deposit corrosion (Pots, 2002). Also known as microbial
corrosion, MIC is actually not a new type of corrosion, but one that is normally
triggered by macro- and micro-organisms rich in nutrients and other elements found
in soil or water. In turn, this type of micro-organisms prefers to attach themselves
and live on the surface of metal structures. Their biological activities can alter the
local environment chemistry, causing corrosion to the metals. In the oil and gas
(O&G) industry, MIC attacks on pipelines have been identified as a significant cause
to increasing operating cost. Consequently, O&G pipeline owners have initiated a
life-span integrity programme for such structures.

Corrosion in the pipeline has been recognized as a significant problem in the

O&G industry. The estimated annual cost of corrosion has increased from US$840
million in 1996 (Buck et al., 1996) to US$13.4 billion in 2001 (Koch et al., 2001).
Various research efforts have been initiated to reduce such cost by utilizing new and
more effective materials, corrosion prevention methods and corrosion management
strategies. Corrosion modelling, for example, is one of the methods in corrosion
 3

management strategies. This predictive corrosion model can be used in reducing the
cost of corrosion by optimizing maintenance cost and planning.

The O&G industry is a major economic contributor in Malaysia. As an O&G-

producing country, there are more than 2,500km of pipelines laid underground for
transporting natural gas to the designated areas (Petronas Gas Bhd, 2011). For safety,
these transporting pipelines are normally buried underground, and therefore, are
exposed to various types of soil. One of the types of corrosive problems faced by
these underground pipelines is MIC, due to the presence of SRB (Ozman, 2012).
Some researchers (Budiea, 2015, Othman, 2015, Sahrani et al., 2008) highlighted
that MIC attacking underground structure and pipeline is a serious threat to the
industries.

1.3 Research Problem

Although a high number of studies on MIC have been carried out locally
(Fatah et al., 2013, Bakar et al., 2013, Sahrani et al., 2008, Allison et al., 2008), yet
majority of these works revolve around the effect of SRB in a marine environment.
In addition, those studies were focusing on finding the influence of SRB in terms of
growth and corrosive effects, but paid less attention to modelling the MIC
mechanism based on specific local bacteria strain in soil. As a result, researches
incorporating microbial corrosion subject to specific local bacterial strain for
underground environment are very limited in Malaysia.

Researchers frequently used commercially available strains of SRB such as

ATCC7757TM in their studies especially for laboratory experiments due to their
availability and simplicity (Ismail, 2015). Previous researches have the tendency to
use commercially available pure-SRB strains as opposed to isolated SRB from sites.
However, there is no solid evidence proving that the commercially available SRB
can closely simulate the performance of SRB strain isolated from local site. In fact,
future research regarding modelling and sensitivity analysis can be misled by the
 4

difference of performance between a commercially available and isolated SRB

strains, unless the performance can be proven comparable. Therefore, undertaking a
strong empirical evident is crucial to reaffirm this statement. On that note, a
comparative study on MIC rate and performance between commercially available
and isolated SRB strains; based on one-factor-at-a-time (OFAT) and Response
Surface Method (RSM) toward selected parameters; is a necessity towards more
reliable information in regards to MIC mechanism.

1.4 Aims and Objectives of the Research

The aim of this research is to investigate the performance of local

underground SRB strain in terms of bacterial growth and influence on metal loss as
experienced by buried gas pipeline in peninsular Malaysia by developing empirical
models of metal loss. The investigation was based on information acquired from
field work (long term corrosion) and laboratory works utilizing One-factor-at-a-time
(OFAT), all-factor-at-a-time (AFAT) and response surface method (RSM)
approaches. The local underground SRB strain is coupled with commercially
available SRB strain of ATCC7757TM due to its matched DNA for comparative
study purposes. The following objectives have been identified as the steps to
undertake towards achieving the research aim:

a) To determine the level of corrosiveness of Sungai Ular site and the influence
of MIC-related parameters on long term metal loss of steel coupon buried
underground.

b) To identify the optimum growth of SRB strains and its governance over
MIC progress subject to stipulated environmental parameters of pH,
temperature, ferum and sulfate content using open circuit potential (OCP),
Tafel slope and weight-loss method.

c) To determine the correlation between corrosion rate and environmental

 5

parameters using empirical models based on Multi-Linear Regression

analysis (MLR) and Response Surface Method (RSM).

1.5 Research Scopes

Corrosion rate measurement was based on physical metal loss to represent

underground corrosion process based on in situ experiment at a selected local site
with suspected SRB activities and laboratory experiment. Metal loss data was
collected by installing the API 5 L X-70 carbon steel coupons underground for a
long term exposure up to 18 months and laboratory experiments. Samples of SRB
were isolated from the collected soil sample and cultured in the laboratory for DNA
identification. A comparative statistical analysis was conducted between locally-
isolated SRB versus commercially available SRB from ATCC7757TM bacteria. The
study focuses on the selected SRB-related parameters which are pH value,
temperature, sulfate (SO42-) concentration and initial iron (Fe2+) concentration to
develop the empirical models to identify the performance of the isolated SRB and
ATTCC7757TM specifically for underground environment.

1.6 Research Importance

MIC topic has been broadly investigated in order to understand and uncover
suitable preventive measures in arresting microbiologically-influenced corrosion
while maintaining reliability of steel structures. Several MIC models have been
developed worldwide to assist prediction of the degradation timeline of pipeline
structures caused by micro-organisms attack.

However, such study for a Malaysian soil environment is scarcely available

due to limited efforts carried out locally. If the pure-commercially available strains
of SRB could not represent local SRB, then the established MIC models need to be
improvised, taking into account the performance of local SRB. On this note, the
 6

outcome of this research will provide profound understanding of underground MIC

mechanism due to local strain SRB and illustrate the difference, in terms of
performance between pure-commercially available and local SRB strains against the
corrosion growth projection.

With a reliable MIC model, pipeline operators will be able to determine the
appropriate corrosion mitigation techniques, inspection and maintenance program,
such as coating, cathodic protection, inhibitors, the frequency of pipe cleaning
(pigging) and materials selection, design improvements or pipeline relocation
planning. Through this research, the model for microbiologically-influenced
corrosion can be implemented based on tested parameters. This model can
subsequently be used to obtain the corrosion growth rate and to some extent, to
predict the remaining lifespan of corroding internal pipelines.

1.7 Brief Methodology of Study

This research was divided into two parts which are field works and
laboratory experiments. Both parts are required to produce metal loss data for
empirical model development. Field work involved installation of steel coupon
underground at local site for 18-month period and the coupons were periodically
retrieved every 3 months. Empirical model of metal loss is developed using Principle
Component Analysis (PCA) and Multi-linear regression technique (MLR). SRB
sample was isolated from the soil sample of the local site and later cultured for DNA
identification. The laboratory experiment involved response surface methodology
(RSM) method to produce another empirical model for local strain SRB. The results
were benchmarked against the ATCC 7757TM strain from American Type Culture
Collection for comparison purposes. Figure 1.1 illustrates the connection of the
methodology with the stated objectives.
 7

Research Aim
Field Work
Investigate the performance of local
 Site Selection underground SRB strain as experienced
 Buried coupons by buried gas pipeline in peninsular
Malaysia
 Retrieved every 3 Months
 Soil analysis

Objective 1
Lab Work To determine the level of corrosiveness of
 Weight Loss Method Sungai Ular site and the influence of MIC-
related parameters on long term metal loss of
 Tafel Plot steel coupon buried underground
 Open Circuit Potential (OCP)
 One Factor at a Time (OFAT) Objective 2
 Respond Surface Method (RSM) To identify the optimum growth of SRB
strains and its governance over MIC progress
subject to stipulated environmental
Analysis Method
parameters of pH, temperature, ferum and
 Liner correlation analysis (LCA) sulfate content using open circuit potential
(OCP), Tafel slope and weight-loss method.
 Principal Component Analysis
(PCA)
Objective 3
 Multiple Liner Regration (MLR)
To determine the correlation between
 Respon Surface Methodology corrosion rate and environmental parameters
(RSM) using empirical models based on Multi-
Linear Regression analysis (MLR) and
 SEM Response Surface Method (RSM).
 EDX

Conclusion
The performance of local
underground SRB benchmarked
against ATCC7757TM

Figure 1.1: Flow chart of the research structure

 8

1.8 Structure of the Thesis

This thesis is divided into eight chapters. The organization of the thesis
structure is as follows:

i) Chapter One provided the introduction of the study by introducing the

research background, research problem, aim of this research, objectives,
scopes and the importance of this research. A brief methodology of this
research was also outlined.

ii) Chapter Two contained the literature review related to the SRB topic. It
covered the issues of corrosion faced by O&G industries worldwide. It
began with an overview of buried gas transmission pipelines in Malaysia,
followed by general corrosion, MIC and its mechanism. One of the
microbiological causal for corrosion, SRB and their influenced parameters
were also discussed in this chapter. The chapter concluded with some
explanation on the currently available corrosion models and statistical
analysis methods used to derive the corrosion models.

iii) Chapter Three illustrated the research plan and methodology of the
experiments in this thesis. The proposed research methodology consists of
field work and laboratory experiment to model the MIC mechanism using
metal loss data. Field work experiment involves installation of API 5 L X-70
steel coupon underground at the selected site. The AFAT technique including
Multi-linear regression was utilised to model the metal loss due to
underground exposure. Meanwhile, the laboratory experiment utilised OFAT
technique and RSM to produce another empirical model of metal loss due to
SRB.

iv) Chapter Four presented the analysis of the result from field work. In this
chapter, justification on the selection of the site was discussed and the
analysis of weight loss data for the buried carbon steel coupons and site
corrosion parameters was presented. It was then followed by data screening
 9

processes including normality and hypothesis testing and outliers detection.

Linear correlation analysis (LCA), principal component analysis (PCA) and
multiple linear regressions (MLR) were then carried out to define the
relationship among the examined factors. Model optimization was conducted
to obtain the best prediction result with the most accurate values.

v) Chapter Five consists of the analysis of the experimental result from the
laboratory experiment. The chapter delivered the analysis of laboratory
work, identification of the bacteria, the bacteria growth curve profile, OFAT
study, SEM images of bio-films and corrosion, Tafel slop testing and
corrosion analysis.

vi) In Chapter Six, the outcome from the laboratory work was presented. This
chapter focused on the RSM model from the experimental results for isolated
SRB and ATCC7757TM bacteria. In this chapter, the RSM model for
ATCC7757TM and isolated SRB were proposed.

vii) Chapter Seven discussed in detail the main results from the field work and
laboratory experiments as well as highlighting the importance of the
proposed models. This chapter discussed the relationship between the
parameters and corrosion rate. It also illustrated how the proposed empirical
models based on MLR and RSM techniques can be utilized and comparative
studies were also made with other researchers.

viii) A summarized conclusion, discussions and the contributions of the research

was presented in chapter eight. It also highlighted a number of
recommendations for future studies related to this research.
 10

CHAPTER 2

LITERATURE REVIEW

2.1 Introduction

This chapter reviews relevant literatures related to the Malaysian gas

pipeline, types of corrosion, MIC and its mechanisms, sulfate- reducing bacteria
(SRB) and case studies using corrosion modelling. The bacteria growth requirements
for the bacteria and the fundamental of response surface analysis are also discussed
in this chapter.

2.2 Gas Transmission Pipelines in Malaysia

Pipelines are one of the most inexpensive ways to transport fluids, especially
in the O&G industry. Pipeline can be defined as a line of connected pipes used to
transport liquid or gas over long distance. In 2014, there were 3.5 million kilometers
of pipelines installed in 120 countries all over the world, with 65% of them being in
the United States, followed by Russia (8%) and Canada (3%). Collectively, slightly
more than 75% of all global pipelines were located in these three countries (The
World Factbook, 2013). Typically, O&G pipelines are made of steel or plastic tubes,
and usually buried underground.

According to Hamid (2008), Malaysia has 75 trillion cubic feet of natural gas
reserves as of January 2007. In Peninsular Malaysia, the natural gas reserves are
 11

mainly for domestic consumption, and not so much for export. Crude gas from
offshore Terengganu is piped to the gas processing plant (GPP) in Kerteh via the
Peninsular Gas Utilisation (PGU) system, which has the capacity to transport over
200 million standard cubic feet of natural gas per day, before it is sold to consumers.
Beginning in 1984, Petronas Gas Bhd was given the task to initiate and maintain the
PGU system. The infrastructure project to facilitate effective natural gas
transmission took nearly 17 years of construction and was finally completed in 2001
(Petronas Gas Bhd, 2011). Figure 2.1 shows the Peninsular Gas Utilisation (PGU)
network of gas transmission and lateral pipelines in Peninsular Malaysia, which
spans over 2,500km.

Figure 2.1: Peninsular Gas Utilisation network (Hamid, 2008)

 12

2.3 Corrosion

Deterioration of materials due to their reaction to the environment is

commonly accepted as the definition of corrosion. Scientifically, corrosion is a
chemical interaction (an electrochemical nature) between a metal and its
environment, resulting in changes in the properties of the metal and often leads to
functional impairment of the metal (Revie and Uhlig, 2008).

Corrosion has also been referred as an electrochemical process involving

three main components; anode, cathode and electrolyte. The metal surface that
corrodes is called the anode, while the cathode is the area of the metal surface that
does not dissolve. An oxidation reaction is defined as the process when metal loses
electrons, whereas reduction reaction occurs when metal gains electrons. The
material corrosion impact can be divided into three categories: energy and material
waste, economic loss and environmental and health impacts.

2.4 Background of MIC

Bio-corrosion has an elaborate history, in terms of electrochemical processes.

Bio-corrosion was first reported in 1891 by Gaines in his publication on the subject
of corrosion of a lead-covered cable. He attributed the corrosion to the metabolites of
bacterial activity. By 1910, Gaines had defined the problem clearer by producing
evidence indicating that iron and sulfur bacteria were partly responsible for corrosion
of buried ferrous metals (Gaines, 1910).

In 1934, Von Wolzogen Kuhr and van der Vlught suggested the removal of
atomic hydrogen from steel surface as a result of SRB activity under anaerobic
conditions, providing significant evidence on the anaerobic corrosion. They named
the finding as “cathodic depolarization theory (CDT)”, also known as the classical
theory (Kuhr et al., 1934).
 13

In 1940, Starkey and Wight indicated that oxidation-reduction (redox)

potential was the most reliable indicator for bio-corrosion (Javaherdashti, 2008).
Thirty years later in the 70s, Videla initiated a research on bio-corrosion devoted to
either objecting or validating corrosion by SRB, as formulated by CDT (Videla,
2007). In the same decade, King and Miller minimized the role of SRB corrosion by
focusing the emphasis on the corrosion product (iron sulfide) instead (King et al.,
1971).

Notwithstanding the above studies, it was subsequently discovered that MIC

would cause more serious problems as it widely occurred in sub-surface and
underground materials, such as steel and brass pipe (Patenaude, 2003 and Alanis,
1985), power plant cooling systems (Bibb, 1986) and oil production and offshore
systems (Bethencourt et al., 2006).

2.5 Characters of MIC

The National Agency of Corrosion Engineer (NACE) used the term MIC or
microbial corrosion or bio-corrosion, interchangeably, to address the corrosion
caused by the contribution of microbial activity, directly or indirectly. Table 2.1
highlighted four possible mechanisms in microbiologically-influenced corrosion.

Table 2.1: Mechanisms of microbiologically-influenced corrosion

No. Mechanisms
1. Creating differential concentration cells
2. Generating corrosive substance
3. Creating alteration of anodic/cathodic reactions
4. Reducing the effectiveness of corrosion inhibitor

Selected numbers of bacteria are repeatedly reported as causing corrosion in

the environment. Table 2.2 shows some of the most common bacteria related to
MIC.
 14

Table 2.2: Most common micro-organisms implicated in MIC

pH Oxygen
Genus/Species Metals Affected Mechanism
Range Requirement
Use hydrogen in
Iron, steel, reducing SO42- to
Desulfovibrio also
stainless steels, S2- and H2S;
known as 4–8 Anaerobic
aluminium, zinc, promote the
D.desulfuricans
copper alloys formation of
sulfide films.
Iron ,steel and Reduce SO42- to
Desulfotomaculum 6–8 Anaerobic
stainless steels S2- and H2S.
Reduce SO42- to
Desulfomonas - Anaerobic Iron and steel
S2- and H2S.
Oxidizes sulfur
and sulfides to
Iron, steel,
Acidithiobacillus form H2SO4;
0.5 – 8 Aerobic copper alloys,
thiooxidans damages
and concrete
protective
coatings.
Oxidizes ferrous
Iron, steel, and (Fe2+) to ferric
Gallionella 1–7 Aerobic
stainless steels (Fe3+) ions and
Mg2+ to Mg3+.
Iron and carbon
Siderocapsa - Low Oxygen Oxidizes iron.
steel

MIC is not a new types of corrosion processes. Most MIC is localized

corrosion from pitting, crevice corrosion, under-deposit corrosion and de-alloying, in
addition to enhancing galvanic and erosion corrosion. There are many factors and
contributing organisms for MIC that can vary between metals and alloys, and
operating conditions, for the same materials.
 15

The main character of MIC is the formation of bio-film on the metal surface.
The morphology of the bio-film has a significant influence on the corrosion effect.
Bio-film does not only entrap deleterious metabolites secreted by bacteria, but also
creates gradients of pH, dissolves oxygen and chloride. Over time, this condition can
influence the immediate surroundings of the metal surface, leading to localized
corrosion of the metal.

2.6 Mechanisms of MIC

There are a few numbers of MIC mechanisms proposed to explain their roles
in causing corrosion. These proposed mechanisms occur simultaneously or
consecutively depending on their environment, metal composition and micro-
organisms involved, either in aerobic or anaerobic condition (Tiller, 1986).

2.6.1 Creating Differential Concentration Cells

One of the well-known literary on corrosion mechanisms is the existence of

differential concentration in cells. This mechanism occurs when a metal is exposed
to two or more different concentrations of solution in the same electrolyte. France
(1972) classified these mechanisms into three categories, namely oxygen
concentration cells, metal ion concentration cells and active-passive cells.

An oxygen concentration cell occurs when different oxygen concentration at

two or more points exists in one system. The metal area with a low oxygen
concentration will be the anodic side, whereas the higher oxygen concentration is at
the cathodic. In the presence of micro-organism, this mechanism can easily exist
due to the formation of bio-films. Non-uniform bio-films formation will encourage
the existence of differential concentration cells. The bio-films will create layers to
prevent oxygen diffusion to the metal. These non-uniform bio-film layers will
generate coated and uncoated areas that are distributed over the surface of the metal.
 16

Micro-organisms can also create aerobic respiration that might be able to contribute
to the formation of different oxygen concentration. Aerobic respiration creates the
anode side of the metal under the colonies whereas the cathode side is created at the
areas outside the respiratory colonies with higher oxygen concentration. This
mechanism in differential oxygen concentration cells has been highlighted by many
researchers, including Herro et al., (1991), Ray et al., (2009), Tiller et al., (1982) and
Rao et al., (2000).

The bio-films initiated by the micro-organism are also known as extracellular

polymeric substances (EPS). EPS consists of different chemical properties and
functions. The difference in chemical properties can contribute to generating
differing affinities with the contact metal. The chemical properties underneath the
EPS with low and high affinities of ion-metal will act as cathode and anode,
respectively. This MIC mechanism was highlighted by Dong et al., (2011) in
understanding the effect of isolated EPS on the corrosion of low-alloy steel material.

Corrosion can also be accelerated by the formation of active-passive layer

cells. This mechanism occurs when the passive layer consisting oxide film is broken
and the active layer underneath is exposed to the corrosive environment.
Electrochemical reaction occurs when a passive layer acts as at the cathode side
while the active layer underneath act as an anode. The micro-organism can also
break the passive layer by producing a chelating agent that can promote iron oxide
dissolution. Some micro-organisms are also able to create dissimilar metal-reducing
bacteria that can directly degrade passive films. Videla et al., (2008) in their study
proved this mechanism by using iron-reducing bacteria in dissolving insoluble ferric
oxide.

2.6.2 Generating Corrosive Substance

The most popular classical theory on MIC mechanism was introduced by

Von Wolzogen Kuhr and Van Der Vlugt in 1934 (Kuhr and Vlught, 1934). They
proposed that some species of the bacteria were able to produce corrosive substance.
 17

In their study of anaerobic bacteria, SRB could be used as terminal electron

acceptors. Corrosive reactions occurred by removing hydrogen from the cathode,
stimulating a cathodic reaction. As a result, the corrosive substance of hydrogen
sulfide was produced. This mechanism works according to the following reactions:

Anodic reaction 4Fe  4Fe2+ +8e-

Water dissociation 8H2O  8H+ +8OH-
Cathodic reaction 8H+ + 8e- 8H
H2S + e- HS- + ½ H2
Bacterial consumption SO42- + 8H  S2- +4H2O
Corrosion product Fe2+ + S2-FeS
Corrosion product 3Fe2+ + 6OH- 3Fe(OH)2
Overall reaction 4Fe + SO42- + 4H2O  3Fe(OH)2 + FeS +2OH- (Eq 2.1)

Hydrogen sulfide can also be a corrosive agent as it can react with metal to
form metal sulfide and atomic hydrogen, which force more iron to dissolve at the
anode. Iron sulfide produced at the surface of the metal also creates new active-
passive electrochemical cells that can accelerate the corrosive process. In this cell,
iron sulfides act as the cathode while exposed metal as the anode (Lewandowski and
Beyenal, 2009).

Sulfur-oxidizing bacteria and SRB are two examples of micro-organisms that

are capable of producing corrosive substance or agent. Fungal species, such as
Fusarium sp.,Penicillium sp. and Hormoconis sp. were proven by Little et al., (2001)
capable of producing corrosive organic acids, such as formic and acetic acids. Sand
(1997) also proved that some micro-organisms are capable of metabolizing organic
solvents, such as ethanol, propanol, butanol and acetone. These organic substances
are capable of dissolving metal.
 18

2.6.3 Alteration of Anion Ratio

Several studies have shown that the anion ratio of sulfate plays an important
role in influencing the rate of corrosion. Wu and Wu (1995) reported that sulfate
anion has a significant effect on the corrosion of aluminum-alloy metal in sodium
chloride solution. In their study, the corrosion rate was reduced due to the
competition between sulfate anion and chloride. Little and Lee (2007) highlighted
the important behavioral role of anion in the electrolyte. The ratio of aggressive to
inhibitive anions is the key parameter in predicting corrosion aggressiveness.

2.6.4 Reducing the Effectiveness of Corrosion Inhibitor

Corrosion is a natural process that is almost impossible to prevent, but can

still be controlled. A corrosion inhibitor is one of the chemical methods usually used
to control the corrosive process and a chemical substance capable of raising
resistance to corrosion. This inhibitor can exist naturally in the system or can be
added purposely. There are many mechanisms of corrosion inhibitor to slow down
the corrosive process, namely reducing the rate of anodic oxidation, decreasing the
ionic conductivity of the solution, pacifying of the metal surface and neutralization
of moisture (Davis, 2000). However, the presence of micro-organisms with their bio-
film layer can reduce the effectiveness of corrosion inhibitor. The bio-film layer may
prevent the penetration of corrosion inhibitor to react with metal surface. In certain
instance, some species of micro-organisms even use corrosion inhibitor as their
nutrients. For example, sodium nitrite is being used as a corrosion inhibitor in the
cooling water system, but some nitrifying bacteria are able to metabolize sodium
nitrite to sodium nitrate (Lundgrend and Krikszens, 1959).
 19

2.7 Background and Characters of SRB

SRB are among the micro-organisms most frequently associated with MIC of
iron, copper and ferrous alloys. It is reported to be the most troublesome groups of
micro-organisms in MIC in O&G and shipping industries (Cord-Ruwish et al., 1987,
Tardy-Jacquenod et al., 1996, and Fonseca et al., 1998). SRB derive their energy
from organic nutrients and they are anaerobic bacteria. In other words, they do not
require oxygen for growth and activity. As an alternative, SRB uses sulfate to
produce sulfide (Javaherdashti, 1999). However, SRB can survive for a long period
of time in the presence of oxygen (Iverson, 1987) and some of SRB genera can
tolerate oxygen (Abdollahi and Wimpenny 1990, Hardy et al., 1981).

SRB are defined as obligate anaerobes, which obtain energy for growth from
the reduction of organic substances using sulfate as the external electron acceptor,
reducing sulfate to sulfide (Postgate, 1984). Corrosion related to SRB is primarily
recognized as a localized attack, normally in the form of pitting corrosion. SRB can
grow in conditions within a pH range of 5.0 to 10.0 and temperatures from 5°C to
50°C (Javaherdashi, 1999).

Most genera of SRB are gram negative. However, some species are also
reported to be gram positive. The term gram negative/positive is indicative of the
thickness of a special protective layer of peptidoglycan, which helps the microbes to
survive in dry environments (Hamilton and Lee, 1995; Odom and Singleton, 1992).

Generally, SRB is distinguished by their ability to respond in sulfate

reduction, using sulfate as the terminal electron acceptor, reducing it to sulfide
(Iverson 1987; Mudryk, et al., 2000). The function of dissimilatory sulfate reduction
is performed by different species of SRB. Stackebrandt et al., (1995) had classified
these organisms into several sub-groups as follows:
 20

Subgroup 1: Spore forming SRB. Example: Desulfotomaculum

Subgroup 2: Non-spore forming SRB: Perform incomplete electron donor oxidation

to acetate. Examples: Desulfobulbus, Desulfomicrobium, Desulfivibrio and
Thermodesulfobactterium.

Subgroup 3: Non-spore forming SRB: Perform complete electron donor oxidation

to CO2. Examples: Desulfobacter, Desulfobacterium, Desulfonema and
Desulfosarcina.

Furthermore, each genus may contain different species of bacteria. For

example, the genus Desulfobulbus of Subgroup 2 includes Desulfobulbus elongates
and Desulfobulbus propionicus. Each different species of bacteria may have its own
characteristic shape, size, capability of utilizing different substrates and other
optimum performance parameters like temperature, pressure and pH. However, all
SRB are grouped under their common function of reducing sulfate. Figure 2.2 (a)
and (b) show two different morphologies of SRB (Javaherdashti, 2008).

Figure 2.2: Two morphologies of SRB. Arrow indicates a) vibro and b) spiral
 21

2.7.1 Mechanisms of SRB

The presence of MIC in corroding metal does not introduce a new type of
corrosion, but it may influence the occurrence and rate of all currently known types
of corrosion. SRB are always associated at the sites where anaerobic corrosion of
iron occurs (Hamilton and Lee, 1995). The corrosiveness of these organisms is partly
due to their metabolic product hydrogen sulfide H2S (Widdel 1992 and Lee at al.,
1995) and partly due to cathodic depolarization (Kuhr and Vlught, 1934). SRB-type
corrosion can be divided into four general categories, namely production of organic
and inorganic acids as metabolic by-products; production of sulfide under anaerobic
conditions; introduction of new redox reactions; and production of oxygen or
chemical concentration cells.

2.7.2 Corrosion by Hydrogen Sulfide (H2S)

It has been demonstrated that the rate of chemical corrosion is proportional to

the concentration of H2S added (Videla, 2000). H2S accelerates iron corrosion by
acting as a source of bound protons (Eqn. 1) and by precipitation of Fe2+ as FeS
(Eqn. 2) (Lee et al., 1995):

Fe + H2S  FeS + H2 (Eq 2.2)

Fe2+ H2S  FeS + 2H+ (Eq 2.3)

The formed H2 may be further utilised by SRB or by other H2-scavenging micro-

organisms.

2.7.3 Corrosion by Cathodic Depolarisation

The most frequently discussed mechanism of corrosion influenced by SRB is

depolarization via oxidation of cathodic hydrogen as formulated in the cathodic
 22

depolarization theory (Kuhr and Vlught, 1934). The mechanisms of metal corrosion
in the presence of SRB are complex. For example, in an anaerobic environment,
SRB uses sulfate as the electron acceptor and reduces it to sulfide, and hence,
described as cathodic depolarization. Based on this theory, SRB consumes the
atomic hydrogen accumulated at cathode by a hydrogenous enzyme.

2.7.4 Alternative Corrosion Mechanism

Booth et al. (1968) studied cathodic depolarization of steel with

Desulfovibrio desulfuricans growing on fumarate as an electron acceptor and in the
presence or absence of chemically prepared FeS. The result from the study revealed
that the corrosion rate was significantly lower without added FeS when compared
with the one added, thereby providing evidence for the significance of FeS in
cathodic depolarization. These mechanisms are also agreed upon by other
researchers that solid FeS formed on metal surface becomes the cathodic site
whereas hydrogen evolution from electrons and protons occurs more easily than on
metal (King and Miller, 1971; Widdel, 1992).

2.8 Bio-corrosion on Steel Pipelines

Iron is the most widely used metal in the world due to economic reason and
available technology. According to the reports by the International Iron and Steel
Institute (IISI), the total steel production and consumption in 2001 was 845 million
tons. According to the statistics by the US Office of Pipeline Safety, internal
corrosion caused approximately 15% of O&G transmission pipeline incidents in the
country between 1994 and 2000. According to a Canadian report by the Alberta
Energy and Utilities Board (AUEB), the total number of failures in the province due
to internal corrosion in both gathering and transmitting pipelines (gas and liquid) for
the past 20 years was more than 5,000 cases (Sooknah et al., 2008).
 23

The most well-known accident involving MIC was the accident of

transmission pipelines in Carlsbad, New Mexico in 2003, which resulted in 12
casualties (Sooknah et al., 2008) On March 2, 2006, internal corrosion at a pipeline
owned by BP Exploration in Alaska caused 267,000 gallons of crude oil to spill into
Prudhoe Bay (Jacobson, 2007). Environmentalists described the spill as the largest
ever in Alaska‟s North Slope region. Another case of MIC attack was in Lost Hills
Oilfield in California, operated by Chevron USA. The new oil and water pipeline
system experienced pinhole leaks in various segments just 18 months after starting
up. The result from the internal inspection of the leaking pipeline showed severe
microbial attack had occurred (Agostini and Yong, 1996). Similar incident took
place at the water injection system, operated by Petrolite Corporation. The corrosion
rate recorded exceeded 100mpy (millimeter per year) at several sections (Strickland
et al., 1996).

Another important case involving MIC is the Brazilian offshore water

injection system. The site was studied by installing online monitoring device
BioprobeTM, in real conditions. The result of the study showed the degree of
corrosiveness was not caused by the seawater alone. SEM observations also revealed
the SRB were involved in the process (Videla et al., 1992).

2.8.1 Bio-corrosion Issues in Malaysia

As one of the progressive oil producing countries, pipeline failure due to

MIC threat has become one of the major integrity issues in Malaysia. One of the
issues that have drawn great attention among researchers is MIC threat towards
offshore transmission line in Miri, Sarawak. Allison et al. (2008) has studied the
relationship between level of microbial activity and MIC severity. The Baram Delta
Operation (BDO) has also reported that majority of the crude oil pipelines operations
contain more than 40% seawater and different levels of microbial activities. The
findings suggested that a comprehensive management program to control MIC threat
on the pipeline at Baram Delta should be developed. Recently, there are numbers of
publication on SRB and MIC in various areas in Malaysia (Sahrani, 2008; Kakooei
 24

et al., 2012; Bakar et al., 2013; Fatah et al. ,2013). Sahrani (2008) investigated
marine SRB strain isolated from deep subsurface water in Pasir Gudang. In 2011, the
general manager of Petronas Carigali Sarawak Operation (SKO) presented an
integrated approach to manage the integrity of pipelines operated by them (Amin,
2011). Internal corrosion due to MIC leads to a very high corrosion rate and
structural failure. In order to identify and manage MIC risk, SKO has implemented
several structured approaches including SKO Wide Risk Assessment, inspection and
condition assessment, Integrity FFS Assessment and development of appropriate
mitigation actions. However, these approaches are only practical for marine
structures. Corrosion of carbon steel with the presence of MIC poses a significant
problem to the O&G industry. Although high-cost corrosion-resistant alloys (CRAs)
have been developed to address the problem, carbon steel is still regarded as the
most cost-effective material.

Further study on the behaviour of microbial corrosion with the presence of SRB
strain was carried out by Bakar et al., (2013). Yet, the study only incorporated single
pure strain and the corrosion behaviour was based on fixed environmental parameters.
Later, Fatah et al., (2013) investigated the effects of SRB metabolic species on carbon
steel by applying abiotic chemistry approach. The study aimed to find the reliable
prediction of corrosion growth pattern by SRB on the carbon steel structure by
considering the statistical approach. However, this study focused on marine environment
and commercially available SRB strain, namely Desulfovibrio thermophiles.

Latest study on the MIC for buried pipeline incorporating SRB species produced
by Ismail et al., (2015). In this study, only statistical analysis on metal loss data due to
SRB was presented without any effort of modelling the corrosion process. Despite the
reliable numbers of research on MIC for marine structure in Malaysia, there were little
efforts for the underground areas. Research on external corrosion underground mostly is
focussing on the material for coating and corrosion characterisation on the carbon steel.
This is quite a concern because Malaysia has thousands of kilometres of buried pipelines
transporting crude oil and natural gas which may exposed to MIC environment.
Therefore, a study on external corrosion due to MIC for buried pipeline is necessary to
be conducted.
 25

2.9 Corrosion Models

There are many corrosion models available in the O&G industry. Corrosion
models can be classified under three categories; mechanistic, semi-empirical and
empirical. These models are classified according to the significance of the model
compared to the theory.

Mechanistic models have a strong theoretical background. According to

Nesic, (2007), when calibrated on a reliable experimental database, this model can
generally produce accurate and physically realistic results. Semi-empirical models
are developed for practical purposes and partially based on theory. Therefore, some
of the semi-empirical models are close to theory, while others may just be best-fit
parameters. Empirical models have little theoretical background and almost all of the
constants have less physical mean and just-fit parameters to the available
experimental data. Even though the empirical models have less theoretical
prediction, the result is still reliable. The empirical model is still the most simplified
model and easy to understand.

2.9.1 Mechanistic Model

Corrosion is a complex phenomenon in which several simultaneous and

interacting processes occur, such as electrochemical reactions, chemical reactions
and transport processes. A mechanistic model should incorporate all of these aspects
to get a good model that can fit into all processes.

De Waard and Milliams (1975) corrosion model is one of the most well-
known models in the O&G industry. Based on their experimental data from
electrochemical studies, they presented a correlation between the corrosion rate and
temperature and carbon dioxide (CO2) partial pressure.

2320
Log (Vcor )  7.96   5.55103 T  0.67 log( pCO2 ) (Eq. 2.4)
T  273
 26

Where Vcor = corrosion rate in mm/year,

T = temperature in °C, and
Pco2 = partial pressure of CO2 in bar (105 Pa).

The model has since been revised based on the experimental results from
Dugstad et al. (1994) and the following expression was obtained:

1710
Log(Vcor )  5.8   0.67 log( pCO2 ) (Eq. 2.5)
273 T

This equation is normally referred to as de Waard-Milliams equation. This

model has been used extensively and the result is reasonably satisfactory. However,
it still has serious limitations. The model assumes all hydrogen ions in the solution
come from the dissociation of carbonic acid and the value of pH is dependent only
on CO2 partial pressure. In a real environment, this phenomenon is not true as
corrosion product and ferrous ions could also change the pH value of the solution.
Consequently, the original de Waard-Milliams model was modified by taking into
consideration various correction factors, such as velocity, pH, pressure of the system
and some other parameters. Corrosion rate is then calculated by multiplying the
original value with a series of corrosion factors. This modification is typically a
semi-empirical method.

2.9.2 Semi-Empirical Model

The original de Waard and Milliams model have since been modified on
several occasions, such as by de Waard, et al.,(1991) and de Waard and Lotz (1993).
The modified model introduces some correction factors to consider the effects of
various parameters. Corrosion rate is calculated by multiplying the “base value” with
a series of correction factors, such as protective films, system pressure, flow
velocity, oil wetting, water sweep-out and others.
 27

Dugstad et al., (1994) also came out with a semi-empirical model in studies
of CO2 corrosion. According to the authors, the corrosion rate was assumed to be a
function of CO2 partial pressure, pH, temperature, shear stress, steel chromium
content and film properties. The model provide a useful tool in estimating CO2
corrosion rates under different service conditions. However, the model by Dungstad
et al., requires knowledge of shear stress, which makes this model difficult to be
used in practice.

2.9.3 Empirical Model

A few purely empirical CO2 corrosion models exist in open literature. Adam
et al., (1991) proposed a multi-variable regression model for CO2 corrosion.
However, this model is questionable, especially when interpolation and extrapolation
are involved. Even though purely empirical models essentially disregard the
corrosion mechanism, all its reaction and processes, a simple model could still give a
satisfactory result.

2.9.4 Corrosion Prediction in Soil

There are widely covered literatures reporting on the evaluation of corrosion

rate growth for metals in soil. Valor et al., (2007) suggested a model for the
commencement and evolution of corrosion based on Stochastic modelling of pitting
corrosion. The model was expected to be able to mimic pitting corrosion of different
materials in diverse corrosive soils. A mathematical model used to estimate the
localized corrosion damage spread for structures buried in soil have been developed
by Alamilla et al. (2009) using environmental parameters. Caleyo et al., (2009) have
presented a new Markov chain model for pitting corrosion which was developed and
endorsed using both synthetic and experimental pitting corrosion data for
underground pipelines. The process of developing a predictive model for corrosion
in buried O&G pipelines has been provided by Velazquez et al., (2009). The model
established a physically sound correlation between the pitting model parameters and
 28

the soil and pipe variables. The model considers the significant chemical and
physical properties of soil using a power law formula of maximum pit depth
proposed as the following:

P = Ktn (Eq 2.6)

Where, P is dependent variables (metal weight loss/pit depth) to be predicted

by independent t (time exposure). The constant K is the regression coefficient
whereas n is the power coefficient. Power law pattern consists of two types, concave
and convex. Concave pattern occurs when n value is less than 1.0 while convex
when n exceeds 1.0. The concave and convex curves specify the rate of corrosion
slowdown or acceleration as the time proceeds as shown in Figure 2.3.

Figure 2.3: Corrosion growth pattern in a function of time (Velazquez et al., 2009)

2.9.4.1 Soil Corrosion Assessment

Soil is a material composed of broken rock particles that have been altered by
chemical and mechanical processes, including weathering and erosion processes. It
is also described as a natural body comprised of three mediums consisting of solid,
liquid, and gas, all of which can serve as electrolytes which occur on the land surface
and occupy space. It is a medium that consists of lots of elements that can act as
 29

catalysts for structural damage, especially corrosion problems. Just like any
environment, soils can be very corrosive. From the corrosion point of view, soils are
very complicated systems (Rodhe and Harrera, 1988). In corrosion engineering, this
becomes an issue because a wide variety of structures that people and the industry
rely on are buried under the ground, such as vessels, storage tanks and pipelines.
Corrosion in soil mostly occurs due to the influence of the soil material and its
characteristics on the buried metal. Soil structure and conditions will not only
threaten the cathodic protection of pipeline systems, but it also may contribute to the
creation of a corrosive environment. In this case, soils act as an electrolyte solution
which reacts with buried metal performing the corrosion process.

It is recognized that there are four general types of corrosion in soil. These
are corrosion in disturbed soil, corrosion in undisturbed soil, bacterial corrosion, and
corrosion by stray currents (Escalante, 1989). Corrosion of metals in soil can vary
from relatively rapid material loss to negligible effects. It is very important to
understand the corrosivity of particular soil as it is a multi-scale process and it can
minimize such soil corrosion of buried material structures. Muhlbauer (2004) states
that underground corrosion is further complicated by the fact that corrosion activity
is normally deduced only from indirect evidence, but the fact is direct observation
are a rather limited option. The response of carbon steel to soil corrosion depends
primarily on the nature of the soil and certain other environmental factors, such as
the availability of moisture and oxygen.

Until now, several of indexing types were designed to determine the soil
corrosivity phase. Previous developments of corrosion index assessments are
produced by the American Water Work Association (AWWA). AWWA has
developed a specific standard that is used to evaluate and score the soil corrosivity
cumulative rate of cast-iron pipes for underground water pipelines. There are five
soil parameters that have been assigned with specific points: soil resistivity, pH,
sulfide content, redox potential, and moisture content as shown in Table 2.3. The soil
corrosivity condition was determined by summing all points for each parameter. The
soil is categorized as corrosive when the cumulative total score is above 10 marks
 30

(>10), while it is categorized as non-corrosive when the cumulative total marks are
lower than 10 marks (<10).

Table 2.3: Table of AWWA soil test evaluation (Bonds et al., 2005)

Soil Characteristics Points

Resistivity (Ω.cm)
< 700 10
700 – 1000 8
1000 – 1200 5
1200 – 1500 2
1500 – 2000 1
> 2000 0
pH
0–2 5
2–4 3
4 – 6.5 0
6.5 – 7.5 0
7.5 – 8.5 0
> 8.5 3
Redox Potential
> + 100mV 0
+ 50mV to + 100mV 3.5
0 mV to + 50mV 4
Negative 5
Sulfide
Positive 3.5
Trace 2
Negative 0
Moisture
Poor drainage, continuously wet
Fair drainage, generally moist 2
Good drainage, generally dry 1
0

The soil corrosivity index was developed by Nazim (2015) to evaluate the
corrosiveness for pipeline buried underground. There are six quantitative and three
qualitative soil parameters used to calculate the soil corrosiveness using the Soil
Corrosivity Index Equation Model developed in the research. The corrosivity
conditions were classified into four conditions consisting of “very corrosive” with a
total mark of 0 – 25%, “corrosive” for 26 – 50%, “mildly corrosive” for 51 – 75%,
and “not corrosive” for 76 – 100%. 0% indicates the worst corrosive condition,
while 100% represents the most non-corrosive condition. The quantitative
 31

parameters involve are soil resistivity, moisture content, pH, chloride content, sulfate
content and sulfide content while soil type, water access and disturbance factor are
the qualitative parameters. The details of the soil corrosivity classification and their
scored are shown in Table 2.4.

Table 2.4: Soil corrosivity classification index by Nazim (2015)

PARAMETER VALUE MARKS

>10,000 10
5,000 ≤ Ω.cm≤ 10,000 7
Soil Resistivity (Res)
3,000 ≤ Ω.cm < 5,000 4
(Ω.cm)
1,000 ≤ Ω.cm < 3,000 2
Ω.cm < 1,000 0
0 – 10 % 10
Moisture Content (Mc)
11 – 25 % 7
(%)
26 – 40 % 4
41 – 60 % 0
0 – 2.9 0
3 – 4.9 3
pH (pH) 5 – 6.9 6
7 – 8 (neutral) 10
>8 7
100 < Cl < 500 10
Chloride Content (Cl) 500 ≤ Cl < 1500 6
(ppm) 1,500 ≤ Cl ≤ 5,000 4
Cl > 5,000 0
0 ≤ SO4 < 0.25 10
2.5 ≤ SO4 < 5 6
Sulfate Content (SO4)
5.0 ≤ SO4 < 7 5
(ppm)
7 ≤ SO4 < 10 2
SO4 ≥10 0
0 – SO3 – 500 10
Sulfide (SO3) 500 ≤ SO3 < 1,500 6
(ppm) 1,500 ≤ SO3 ≤ 10,000 5
SO3 >10, 000 0
Clay soil (high moisture content) 1
Silt soil (high moisture content) 2
Soil Type Loamy and lateritic soil (high moisture) 6
Sandy soil (good drainage) 8
Gravelly soil (excellent drainage) 10
No existing river 10
Water Access Existing fresh water river 6
Existing swampy mangrove 2
Undisturbed 10
Hilly and non-developed 7
Disturbance Factor
Developed 4
Hilly and developed 2
 32

Table 2.5: Comparison between AWWA index and Nizam Soil classification index

Parameter AWWA Soil Remarks

Index Classification
Index by Nazim
Soil √ √ Both soil corrosivity indexes considered the soil
Resistivity
resistivity value in their indexes. AWWA index give
a highest point for the soil resistivity factor. This
show the soil resistivity is the most significant
parameter in determination of the soil corrosivity.
For example, if the resistivity value is less than 700
(Ω.cm) the point obtained is 10. The total score 10
and above is considered as corrosive in AWWA
index. Meanwhile in Nizam classification index the
weightage for soil resistivity is same with other soil
parameters.
pH √ √ Both soil corrosivity indexes give high point for
acidic condition and less/no point at neutral pH
conditions.
Moisture √ √ Both soil corrosivity indexes give score for the
Content
moisture content. In AWWA index the point given
qualitatively based on the drainage and moisture
conditions. While in the Nizam classification index,
the moisture content is quantitatively measured in
percentage (%).
Sulfide √ √ Sulfide concentration also been considered in the
Content
both soil corrosivity indexes. The sulfide content not
quantitatively measured in the AWWA index. The
sulfide content only classified as positive, trace and
negative. While in the Nizam classification index
the sulfide content is measured in part per million
(ppm). High score was given for the sulfide
concentration more than 10,000 ppm.
Redox √ N/A Redox potential is only been considered in the
Potential
AWWA index. Soil condition with negative redox
potential value will btained high score and no score
given if the redox potential value is more than 100
mV.
 33

Table 2.5 shows the comparison between AWWA index and Nizam
classification index. Both soil corrosivity indexes have advantages and
disadvantages. As example, in AWWA index 40% of the parameters are measure
qualitatively, where the possibilities for error in judgment occur. However the
advantage the AWWA index is less parameter required. It will be more users
friendly and more practical to industries compared to the Nizam classification index.
Nevertheless in the Nizam classification index, nine parameters needed, there are
soil resistivity, moisture content, pH value, chloride content, sulfate content, sulfide
content, soil type, water access and disturbance factor. The advantage of the Nizam
classification index is more parameter involves hence will give better accuracy, and
the value of the parameter is quantitatively measured. This measurement will reduce
the tendency to error. Other advantage of the Nizam classification index are the soil
classification. In AWWA index only have 2 soil conditions, corrosive and non-
corrosive, whereas in Nizam classification index have 4 soil conditions such as very
corrosive, corrosive, mild corrosive and not corrosive.

2.9.5 MIC Corrosion Model

A group of researchers at Shell Petroleum Company, led by Pots et al.,

(2002) developed an approach to assess the MIC risk for carbon steel pipelines. The
risk is calculated based on the details of water chemistry and operation parameters.
The corrosion rate is calculated based on the following equation:

CR (mm/year) = C x Fp (Eq. 2.7)

With F = f1 x f2 x ….fn
Where C is constant (C=2mm/y),
fs are factors for various influencing parameters, and
p is a power law index (0.57).

This semi-empirical model is calculated based on the factor value for each
parameter, such as pH, total dissolved solids (TDS), temperature, sulfate content,
total carbon from fatty acids, nitrogen content, flow velocity, biocide frequency,
 34

pigging frequency and operational history. An overview of the factor values is given
in Table 2.6.

Table 2.6: Overview of factors for various influencing parameters in MIC

(Pots et al, 2002)
Factor value Factor value
Parameters
when true when false
pH between 5 and 9.5 1 0.01
TDS < 60 g/L 1 0.2
If TDS > 60 g/L, do SRB grow 0.2 0.0001
Temperature between 10°C and 45°C 1 0.2
If Temperature > 45°C, do SRB grow 1 0.2
Sulfate > 10 mg/L 1 0.2
Total carbon (C) from fatty acids > 20mg/L 1 0.2
Nitrogen (as utilizable N) > 5 mg/L 1 0.2
C:N ratio < 10 1 0.4
Flow velocity < 1 m/s
Flow velocity = 2 m/s ~0~1
Flow velocity = 2.5 m/s ~0.6
Flow velocity = 3 m/s ~0~1
Debris on bottom of pipeline 2 1
Pigging frequency = none ~1
Pigging frequency = 13 weeks ~0.3
Pigging frequency= 4 weeks ~0.001
Pigging frequency = 1 week ~0.0001
Prolonged oxygen ingress > 50 ppb 5 1
Biocide routinely used 0.2 1
Operational history;
-age pipeline < 0.5 yr 1
-age pipeline > 0.5 yr & downtime = 1 week ~1
-age pipeline > 0.5 yr & downtime = 50 weeks 2

2.10 Microbial Growth Requirements

Micro-organisms can enter a pipeline as early as it has been hydrostat and

remain viable for long period of time. Pipelines may also receive bacteria-
contaminated fluids which then continuously feed contaminant into the pipelines.
Bacterial growth rates and death are greatly influenced by environmental factors.
The microbiology requirement to growth and multiply are different for each type of
bacteria. Each bacterial species has a specific tolerance range of specific
 35

environmental factors. The requirement for bacteria to growth can be divided into
two categories such as physical and chemical. Physical aspects include temperature,
pH, and pressure, while chemical factors such as water, carbon source, nitrogen, and
minerals. The following factors influence bacterial growth:

2.10.1 Presence of Water

Water means life for microorganism because if there isn‟t high enough water
around them, they can‟t transport water across their cell walls. The microorganism
cannot growth without water. All bacteria require free water in order to proliferate.
In crude oil pipelines, water content typically ranges from 0.1% to 1%. MIC in this
condition is primarily considered under stagnant flow condition. Theoretically, if all
water is entrained in the hydrocarbon phase, there would be no threat of MIC (Pots
et al., 2002). However, in the actual flow condition, the possibility of water to wet
the pipe wall or deposits attached to the pipe wall is really high.

2.10.2 Nutritional Requirements

In order to grow and reproduce, cells must process raw materials necessary to
produce membranes, proteins, cell walls, chromosomes and other compounds. The
major elements are carbon, nitrogen, phosphorus and sulfur. From a microbiological
perspective, the three most critical nutrients (C, N and P) must be in optimum ratios
to achieve maximum bacterial activity. Nutrient availability directly influences
microbial activity. When one of the nutrients essential for microbial activity falls
below the threshold, this activity ceases, until more nutrients become available.

Some bacteria can survive and even grow in the complete absence of sulfate;
they simply use an alternative electron acceptor, such as nitrate. This concept has
been utilized to prevent reservoir souring from flood water. However, this form of
metabolism is unlikely to be significant in oil transport pipelines. Apart from sulfate,
 36

other oxidized sulfur species may be utilized, such as thiosulfate or bisulfite. Hence,
if there are sources of these species present in the water, then they may represent an
important increase in the potential for sulfide generation as sulfate level is very low.

2.10.3 Oxygen Level and Redox Potential

Bacteria can generally be classified according to their affinity for oxygen.

Aerobic species require oxygen to carry out their metabolic functions. Meanwhile,
anaerobic species do not live or metabolize in the presence of oxygen. Facultative
bacteria can grow in either environment, although they prefer aerobic conditions.
Microaerophilic bacteria require low concentration of oxygen. Under conditions of
oxygen deficiency or under anaerobic conditions, aerobic facultative and anaerobic
bacteria are able to reduce inorganic compounds by using electron acceptors, such as
nitrate of sulfate.

Oxygen level within the growth medium can be determined by its redox
potential (Eh) measured in millivolts, which reflects its oxidative-reductive state.
Generally, an oxidation state (+Ehvalue) will support aerobic microbial activities,
whereas a reductive state (-Eh value) will encourage anaerobic functions. Microbial
activities forming sessile growths often occur most extensively over the transitional
redox fringe (aerobic -facultative - anaerobic) from -50 mV to +150 mV (Cullimore
D.R., 1999).

2.10.4 Temperature

Generally, an increase in temperature will increase enzyme activity. But if

temperatures get too high, enzyme activity will diminish and the protein (the
enzyme) will denature. On the other hand, lowering temperature will decrease
enzyme activity. At freezing temperatures enzyme activity can stop. At freezing
temperature, water forms ice crystals, hence cells begin to rupture. Every bacterial
 37

species has a specific growth temperature requirement which is largely determined

by the temperature requirements of its enzymes.

Temperature influences microbial growth in a number of ways, ranging from

extremes where low temperatures cause an inhibition of cellular metabolic processes,
to high temperatures that may impact, particularly on the protein constituents of the
cell through thermal denaturalization to cause the death of the cell. Microbes can
survive a wide range of temperature from -10°C to greater than 100°C. Despite this
wide range, most individual species have much more narrow ranges. Most bacteria
that have implications in corrosion, grow best at the temperature of between15°C
and 45°C.

2.10.5 pH

pH is a numeric scale used to specify the acidity or basicity of an aqueous

solution. It is approximately the negative of the logarithm to base 10 of the molar
concentration, measured in units of moles per liter, of hydrogen ions. With MIC-
related micro-organisms, the extracellular polymeric structure in bio-film will tend to
impart a buffering activity that reduces the influence of any pH shifts in the water,
especially when bio-film act in consortium. In general terms, most species grow best
when the water's pH ranges between 6.5 and 9.0. But when the pH level falls below
the prescribed range, the bio-film would kick in to buffer this effect.

2.10.6 Salinity

High concentration of salts is generally considered to have an impact on the

forms of microbial growth. Most bacteria are inhibited at the range of 5% to 8% of
salt concentration (Cullimore, 2000). Stott (2010) reported that SRB are unlikely to
grow if the salinity of water is more than approximately 7% (wt %NaCl). A recent
analysis of soil microbial communities reported that the microbial composition in a
 38

saline soil is influenced as other chemical factors such as temperature or pH

(Canfora et al., 2014). Pereira (Pereira et al., 2012) recently examined the bacterial
and archaeal diversity in saline soils using a meta-analysis approach indicating that
approximately 50% of the archaeal diversity and 25% of the total bacterial diversity
has been recorded from saline soil habitats.

2.10.7 Ferrous Ion Concentration (Fe2+)

It has been pointed out that for the medium with low iron concentration, there
is an adherent iron sulfide film on the steel surface and bio-film accumulation would
follow in the adherent film (Mara and Williams, 1972). When the ferrous ion
concentration in the medium increased to a certain level, the medium reaches super-
saturation very quick and the precipitation of iron sulfate takes place. This process
will either inhibit the protective film formation or break it. Once iron sulfide
particles penetrate the protective film and come in contact with the steel surface, the
corrosion rate increases greatly (King and Wakerley, 1973). The ferrous ion
concentrations also promote formation iron sulphate. Previous research by Booth et
al. in 1968 revealed the importance of iron sulphate (FeS) onto the rate of corrosion
promoted by SRB. The rate of corrosion is significantly higher with addition of FeS
compared to without FeS, providing evidence for the involvement of FeS in cathodic
depolarisation. These mechanisms agreed by other researchers that solid FeS formed
on the metal surface becomes the cathodic site where hydrogen evolution from
electrons and protons occurs more easily than on metal (King and Miller, 1971 and
Widdel, 1992).

2.10.8 Effect of Sulfate Concentration (SO42-)

SRB obtain their energy by reducing sulfates to sulfides while oxidizing

lactate to CO2 and H2O (Hilton and Oleszkiewicz, 1988). Macpherson and Miller
observed increased SRB cell population with the increase of sulfate concentration in
 39

the media (Macpherson and Miller, 1963). Anaerobic pathway depends on one or
more limiting nutrient in the medium, including sulfate.

2.11 Operating Condition Factors Affecting MIC

Apart from microbial growth requirements, operating conditions are also

known to affect the MIC in internal pipelines. The operating conditions are usually
affecting the MIC growth are pressure, flow rate, debris in the pipeline and pigging
activities. These parameters are the main factors in affecting the MIC growth as MIC
risk model developed by Pots et al., (2002). Where the risk is calculated based on the
details of water chemistry and operation parameters

2.11.1 Pressure

Pressure is one of the important factors in influencing the micro-organism to

growth. Osmotic pressure is an important factor that might affects the micro-
organism cells. Osmosis is essential in biological systems because biological
membranes are semi-permeable. Osmosis provides the primary means by which
water is transported into and out of cells. But some researcher highlighted micro-
organisms are relatively simple forms of life and can adapt to radical changes in
pressure (Cullimore, 2000). Therefore, operating pressure might not change in
micro-organisms, but MIC might change due to the formation of bio-films.

2.11.2 Impact of Flow Rate

Flow rate will directly influence the nature of bio-film formation and the rate
of nutrient delivery. As flow rate increases, bio-films become less bulky and more
adherent. However, above a certain threshold, the initiation of bio-film formation is
 40

significantly limited. The transition zone is considered to be between 2 and 3m/s. At

the other extreme, stagnation is often associated with the severest MIC incidents.

2.11.3 Impact of Debris on the Bottom of the Pipeline

In a maintenance scenario, the role of the pipeline pig is to keep the inside of
the pipeline debris-free, remove deposits and monitor the condition of the line. The
pipeline pigging programmes are conducted to make sure that the pipeline is
efficient. The presence of debris exacerbates the MIC because the presence of debris
can create stagnant conditions at the bottom of low velocity lines, where bio-films
can form.

2.11.4 Impact of Pigging Frequency

Pigging can potentially have a significant effect on bio-film and MIC. The
more frequent the pigging operation runs, the less time bio-film has to recover. A
pigging frequency of at least once every two weeks will be very effective in
controlling the formation of bio-film. The extent to which pigging is effective in
controlling bio-film-associated corrosion depends on how well a corrosion process is
established prior to the pigging program. Some of the reasons for running pigs are, to
improving the flow efficiency of the pipeline, removing debris, solid products and
bio-films, and for improved the efficiency of the chemical or biocides treatments.

2.11.5 Impact of Biocide Treatment

Biocides are used to control unwanted microbial activity. If a biocide is used

in a pipeline, the risk of failure due to MIC should be significantly reduced or even
eliminated. Biocides are much more effective in killing planktonic micro-organisms
and when they are used in conjunction with frequent pigging, which disrupts bio-
 41

films from the metal surface. It is essential that the biocide is selected carefully and
the treatment regime is appropriate and applied properly.

2.12 Experimental Design, Mathematical Model and Statistical Analysis

2.12.1 The Concept of Factorial Experiments

In experimental design, factorial experiments are where the design consists of

two or more variables. Such an experiment studies of the effect of each as well as the
interaction between variables variable on the outcomes. Most experiments are
conducted with factorial variables studied at two levels. For example, an experiment
is named 22 (or 2×2), because it contains two stages for each variable, which
ultimately resulted in four factorial points. Experimental design can involve many
independent variables. As an example, the effect of three variables on two levels can
be evaluated in experimental conditions indicated by the corner of a cube.

When there are a lot of factors, more experimental runs are required even
without replication. For example, experiments with 10 factors at two levels produce
210 = 1,024 combinations. At some point, it would be impractical because of high
cost and/or lack of resources. In this case, fractional factorial design can be used.

As with any experiment distribution, experimental runs in factorial

experiments should be carried out at random to reduce bias in experimental results.
Factorial experiments can also be used for more than two levels for each factor.
However, the number of experimental runs required for a three-level (or more) is
more than those for two levels. Therefore, factorial design appears less attractive if
researchers consider more than two levels.
 42

2.12.2 Main and Interactive Effects on Variables

Factorial design with two levels comprise breaking up of each factor at each
level, commonly known as low and high levels. To facilitate the calculation of the
variables, the low and high levels are positioned with a value of -1 and +1,
respectively.

Formulated by Yates (1966), the Yates Analysis is used to exploit the special
structure design to produce estimates of least squares, in order to estimate the main
effects for each variable and all interactions between the variables. Before carrying
out the analysis, the data must be sorted in Yates order. This means that if there are k
factors, the column has a 2k-1 "-" sign, followed by 2k-1 "+" sign. For example, for a
full factorial design with three variables, the design matrix will be:

───
+──
─+─
++─
──+
+─+
─++
+++

Yates analysis produces the following output:

a) Identify factors (from Yates order), for example,

1 = main effect of variable 1
2 = main effect of variable 2
3 = main effect of variable 3
12 = interaction of variables 1 and 2
13 = interaction of variables 1 and 3
23 = interaction of variables 2 and 3
123 = interaction of variables 1, 2 and 3
 43

b) List the most important variable according to the size of the main effects. It is
the least squares estimates of the impact of genetic factors that are ordered
from the largest in magnitude (most effective) to the smallest (least effective).

2.12.3 Linear Model for Factorial Experiment Results

Linear regression is a form of regression analysis in which the dependent

variable is modelled in terms of a linear combination of one or more independent
variables. Linear regression function dependent variable is represented by a straight
line. The results of fitting the data are subjected to statistical analysis.

The data consist of m values taken from the observations (dependent variable
or response), y. The dependent variable has errors. These errors are considered to be
a random variable with an average zero. Related systematic treatment may exist, but
it is within the scope of the regression analysis. Independent variables (variable
qualifier) have no errors. In linear model regression, data model is expressed as:

(Eq. 2.8)

Here, the error ɛi observed, βo (shortcut) and β1 (LOA) are the model
parameters. For a more general model:

(Eq. 2.9)

Here the coefficient Xij is a constant, or a function, of the independent

variables, x. Model specification must be treated as a non-linear regression. It is
assumed that the observation error is not tolerant and has a normal distribution. This
assumption is used when the test subset treats the results of linear regression as a
model for the conditional expectation given by
 44

(Eq. 2.10)

The first objective in the regression analysis is to obtain best fit for the data
by adjusting the model parameters. The criterion of least squares is to get the best fit.
The objective function S is defined as the total revenue squares, ri.

(Eq. 2.11)

For each product (residual), the difference between the observed value and
the value calculated by the mode is given as:

(Eq. 2.12)

The best fit can be obtained when the result of the sum of squares is
minimized. The theory of linear least squares would be resolved by using a
parameter estimator normal equation:

(Eq. 2.13)

Normal equations are written in the matrix form as:

(Eq. 2.14)

When it is not a single matrix XTX (singular), the solution to the normal
equation can be written as:

(Eq. 2.15)
 45

2.12.4 Full Factorial Experiments 23 for Composite Design

In central composite statistical design, the experiments are used in the design
of response surface method to build a quadratic model for the variable response
without using three-level factorial experimental. After conducting experiments,
linear regression is used to obtain the results. Coded variables are commonly used
when producing this design. The design consists of three main sets of experimental
runs:

1. Factorial design of the variables studied, each with two levels.

2. The midpoint set experiments, in which the value of each variable is the
median value used in fraction factorial. This point is usually replicated to
improve the accuracy of experiments.
3. To set the axis point, the same test is run with the midpoint except for one
factor, which will take the second-two above and two levels below the
median for the factorial and typically are outside the range. All of the
variables are different in this way.

The design matrix for central composite experimental design involving k

variables is the middle of the matrix d that consists of three distinct parts in relation
to the three types of experimental runs:

1. The matrix F obtained from factorial experiments. Scaled factor makes its
input in the form of a code of +1 and -1.
2. The matrix C of the midpoint, expressed in coded variables (0, 0, 0, 0), where
k is zero.
3. The matrix E of the axial line of 2k. Each factor is placed in a sequence in
value +α and -α and all the factors are at zero. The value of α is determined
by the user, although in some random state, they may be able to design the
necessary properties. This section will look like:
 46

(Eq. 2.16)

Then d is a vertical matrix:

(Eq. 2.17)

There are many different methods for selecting the useful α. F is the number
of dots caused by factorial design and T = 2k + n is the number of additional points,
where n is the number of halfway point in the design. The most commonly used
methods are as follows:

1. Design the orthogonal:

where (Eq. 2.18)

2. Spinning design:

(Eq. 2.19)

2.12.5 Quadratic Response Surface and Calculation for Variable Phase

Experiments

To evaluate the experimental variables at the point of maximum revenue,

quadratic equations should be written in full conditions, which include the terms bji
 47

and bij where in the calculation of the regression coefficients, they have the same
variables in X. To remove ambiguity, every member of every pair of coefficients can
be replaced with the average given by:

(Eq. 2.20)

which is given by half of the value of the coefficient as given in the above
regression. Complete quadratic equations can be written in matrix notation as:

(Eq. 2.21)

Where, Bi = [b1, b2, b3] (Eq. 2.22)

(Eq. 2.22) (Eq. 2.23)

(Eq. 2.24)

To get the coordinates of a maximum result, partial derivatives of the

response function are set to zero, whereas the maximum value and the second
derivative are checked by consecutive equations that are solved in the first issue for
the coordinates of the maximum result.
 48

Partial derivatives can be written as

(Eq. 2.25)

This may be expressed in matrix notation as:

(Eq. 2.26)

Himmelblau (1970) solved the above equation for X to provide a set of

values xi at the maximum point results as:

(Eq. 2.27)

The theoretical maximum yield can be calculated by entering the value of xi

in the equation Xmax.

2.12.6 Verification Model using Orthogonal Factor

Factorial analysis method can be used to select the most reliable experimental
variables in modelling the corrosion rate. Factor analysis methods enable a wide
range of experimental variables to be described in terms of mutually orthogonal
factor, which are not correlated with each other, but have the same mean and
variance in the standard form of experimental variables. Mutually orthogonal factors
are important factors that may be used to form a linear model, in which the
interaction between factors is not taken into account. The empirical model is built to
describe the results in terms of these mutually orthogonal factors.

The impact of each factor on the result is then determined by removing

certain factors in the model affected by all these factors. Then, the average value of
 49

the squared differences between the actual data with estimates of the resulting model
is compared with the average squared difference between the actual and budget data
using the model that involves all factors using statistical F-test. The factors can then
be classified into categories based on how they affect each of the results. If the
experimental variables contribute only to the factors that are not effective to produce,
it can be concluded that the factors are not relevant to the outcome and they can be
removed from subsequent experiments.

It is assumed that the surface response has a maximum. If the review has no
maximum, it may be reduced sufficiently to a linear model to represent the surface.
If there is not enough curve, then it has a maximum. The linear model is made in
advance. If it does not meet the data and the results of orthogonal factor, then the
non-linear model may be matched with regard to the likely shape of the curve near
the maximum result.

Linear model can be used to explain the dependent variable Y in terms of the
independent variables Xi and regression coefficients ai

(Eq. 2.28)

To put all experimental variables on the same scale, they should be

standardized with an average value of 0 and a variance of 1.0 as follows:

(Eq. 2.29)

Here,
xij is the experimental variable in their units
μi is the average of the variables that are not standardized test which is
defined as:
 50

(Eq. 2.30)

N is the total number of experiments

ѵi is the deviation r.m.s. to xi from the mean μi
wij is the generic for each standardized test wi

There are two conditions that must be met before the above equation can be
used:

1. Experimental data have to be in a normal distribution.

2. There is no correlation between the variables.

Usually, the second condition cannot be fulfilled because of the combined

level of the standard experimental variables is always in a random selection. Then,
any standard experimental variable has an average value of 0 and a variance of 1.0,
but there is still a significant correlation, providing:

(Eq. 2.31)

where Wij and Whj are the standards for two different experimental variables.
The correlation between the two variables can be considered a standard test for all
possible combinations and the results are shown in a matrix table. The correlation
between the experimental variables and values is 1.0. The correlation between the
two variables can either be positive or negative depending on how the variables are
selected for each experiment. Since there is a correlation between the standard
variables, it should be converted into uncorrelated variables. To do this, each of the
different categories should be distributed to a number of factors that are orthogonal,
with each representing a combination of factors that contributes to the same
categories of different variables. The orthogonal factor can be used in the model, in
which the independent variables are independent.
 51

Factor analysis method allows the experimental variables to be expressed in

terms of standard orthogonal factors, with an average and variance equal to the level
of the experimental variables, but there is no correlation between them. Each
standard experimental variable has an average of 0 and variance of 1.0 for
distribution in the experimental stage. By definition, each factor has the same
average of 0. Variance experimental variables are similar to the experimental
variables, with the value of 1.0. There is zero correlation between them. Harman
(1967) has shown that an orthogonal factor level table for all experiments can be
obtained from the tabled variable level of the original experiment by calculating the
value of each factor using the following equation:

(Eq. 2.32)

Where Frj is the generic j for each factor Fr

ηri is the coefficient r eigen-vector related to the eigen-values of the
correlation matrix between the standard experimental variables,
n is the total number of experimental variables,
wij is the generic for each of the standardized wi.

Eigen-values are actually a solution when completing a set of consecutive

equations using matrix techniques. It can be observed that the size of each source
(eigen-values) shows the contribution of source in describing the total variance. The
factors, calculated using the coefficient eigen-vectors associated with the smallest
eigen-values, cannot accurately describe the variance in the standard experimental
variables.
 52

2.12 Summary of Research Gap

In this chapter, numbers of issues are highlighted to signify the importance of

the research; the research gap and the following summary were listed from the
finding in the literature review:

i) SRB has been acknowledged as one of the serious threats to the piping
integrity especially in the oil and gas industry where their piping system can be
categorised as the most strategic asset. Since SRB strain can be originated
from different species with diverse impact towards corrosion severity, the
actual behaviour of these strains towards MIC and pipeline integrity is still
remain equivocal in nature.

ii) Previous researches have shown great efforts in controlling the MIC threats.
Yet majority of the studies mainly focused on marine environment. Research
focusing on the underground SRB attack on buried infrastructure is rarely
found in the literature. Coverage on SRB threat upon underground pipeline is
vital in Malaysia since this asset is as valuable as marine pipelines.

iii) Previous studies on SRB mainly utilised a standard SRB bacteria obtained
from established sources such as ATCC to replicate the performance of actual
SRB strain from the affected site. Attention on the isolation of SRB strain from
the local site is scarcely available. It is of importance to utilise local SRB strain
to study the influence of this strain towards corrosion rate for a more precise
findings.

iv) There is no solid evidence showing that SRB strain obtained from established
source (ATCC7757TM) can well represent the SRB strain isolated from local
environment. Therefore, this study has embarked on detail investigation of the
performance of local SRB strain against ATCC strain towards the progress of
MIC.
 CHAPTER 3

RESEARCH METHODOLOGY

3.1 Introduction

This research is divided into two parts. Part 1 explained the field work where
carbon steel coupons were buried at the selected location. The site location was
selected through maintenance record and recommendations from pipeline operators
where it is found to be susceptible area for SRB activity. Initial survey found that the
selected site fits the requirement. The soil samples and bacteria were collected from
the site for further analysis and study. The bacteria were isolated and identified using
available methods. The details of the methods are discussed in the next chapter. The
analysis of buried coupons was carried out using a statistical analysis approach to
develop the corrosion growth rate model.

Part 2 concentrated on experimental study which consists on isolating SRB,

bacteria culture and growth, OFAT study and Response Surface Methodology
(RSM). The referred SRB was acquired from Sungai Ular site following the initial
investigation. The American Type Culture Collection; with reference number ATCC
7757TM; was also used for comparative study since both SRB strains has similar
DNA structure. The whole view of the research methodology is exemplified in
Figure 3.1.
 54

Part I Part II

SITE STUDY LOCAL SRB ATCC 7757TM

Simulated
Maintenance & SRB Sample Experiment
Inspection Data from from Soil -Weight Loss
Vendor Method
-OCP
Enrich Medium Tafel Slope
-FESEM
Site Determination
Screen SRB OFAT
Experiment
Conduct Field Work

Isolate SRB Simulated

Experiment
Retrieved Every 3
Month Interval
Bacteria
Identification Proposed RSM
Model
Soil Sampling and
Bacteria Sampling
Simulated
Experiment

Weight Loss Method

Correlate between Soil

Parameters

Weight
Statistical Analysis Loss/Corrosion Rate
Model using RSM

Metal Loss Corrosion

Model for Selected Site

Figure 3.1: Flow chart of methodology

 55

3.2 Coupon Preparation

The carbon steel coupon was taken from 36 inch diameter of pipe graded API
5L-X70 obtained from pipeline operator‟s office, Pasir Gudang, Johor. The pipe was
cut into manageable sizes of 12 inch × 12 inch using an oxyfuel cutter. Figure 3.2
shows the cutting process conducted at yard. Cold cutting method was applied in
UTM laboratory to remove the heat-affected zone (HAZ) and to obtain the required
sizes. Figure 3.3 shows the cutting equipment for cold cutting method used at UTM
laboratory. All coupons were cut according to ASTM corrosion testing standard
(ASTM G1-03, 2011). Three sizes of coupons used for this study are shown in Table
3.1.

Table 3.1: 3 sizes of coupons used in this study

No. Coupon Size (mm) Coupon Application

1 10 mm × 10 mm × 7 mm Electrochemical cell testing and

open circuit potential (OCP)

2 10 mm × 30 mm × 7 mm Weight loss method in

anaerobic vials and SEM
analysis

3 80 mm × 60 mm × 7 mm Weight loss method buried at

the selected site

Figure 3.2: Carbon steel pipe cut to small sizes before being sent to UTM for
subsequent cutting process
 56

Figure 3.3: Cutting machine used to remove heat-affected zone (HAZ)

For electrochemical testing, the coupon was connected with a copper wire
using conductive glue, mounted on polyester resin (EpoKwick® Buehler, USA) and
cured for 24 hour. Cured polyester resin was then cut and cleaned from excess
polyester resin to ensure the size is suitable with electrochemical glass cell
mounting. Figure 3.4 shows the schematic diagram and a picture of the mounted
carbon steel coupon known as working electrode. The exposed area of the working
electrode was polished with a series of Si-C papers grade (100, 320, 600 and 800),
recorded and measured (exposed area), followed by ethanol degreasing prior to use.

Figure 3.4: Schematic diagram of carbon steel coupon mounted in polyester resin
for electrochemical experiment
 57

Metal loss experiments were performed in 125 mL anaerobic vials. Since the
size of the mounting of the vials is small (around 1.2 cm2), the size and thickness of
the coupons are precisely monitored. The coupons were polished with 100 grit Si-C
paper, rinsed with ethanol and treated with a sonicator for five minutes to remove all
forms of dirt, grease and small Si-C particles on the coupons‟ surfaces. Clean and
dried coupons were then coated with paint, leaving only the top surface exposed.
Next, the coupons were dried overnight in an oven at 40°C. The exposed area of the
coupons was polished again with a series of Si-C papers grade (320, 600 and 800),
followed by ethanol degreasing. Clean coupons were finally weighed (known as
initial weight, W0), systemically recorded and stored in a desiccator until further use.
Figure 3.5 shows the pictures of the polished and coated carbon steel coupons.

Figure 3.5: Carbon steel coupons used in anaerobic vials (10 mm × 20 mm × 7 mm)

3.3 Chemical Composition and Morphology of API-5LX70

The composition of the carbon steel used in this study was analysed using
energy dispersive spectroscopy (EDS). The result from the chemical composition
analysis is presented in Chapter 4. An optical microscope, Nikon Microphot-FXL
was used to obtain the optical micrograph after the coupon was polished and etched
with 2% of nital reagent (Al-Abbas et al., 2012).
 58

3.4 Weighing and Labelling Process

All coupons were weighed using an analytical balance and preserved in zip
lock plastic bags to avoid contamination and corrosion caused by atmospheric
moisture. Precaution was taken to avoid any contamination or any possible element
that could influence the corrosion mechanism. Coupons were then categorized based
on initial weight (Wo), length (l), width (w), and thickness (t) dimensions. Area of
exposure (A) and time of exposure (T) are also necessary to be recorded and would
be used during the determination of corrosion rate (CR).

3.5 Methodology for Site Study

3.5.1 Site Selection

Site study was performed at chosen site areas to understand the actual
behaviour of corroding carbon steel coupon through direct contacts with real
environment. Site study work can be a lengthy process with tests where it can take a
duration of 18 months to complete. The longer the tests are conducted, the more
precise the results are and more accurate for external corrosion model to be
developed. As stated by Wang et al., (2003), comprehensive research is needed to
improve field database in order to construct a proper model. Site study work
comprised of soil sampling and on-site measurement (soil resistivity, pH, and
moisture content). Moreover, in certain research areas, if groundwater was found
during digging activities; further tests involving groundwater parameters such as
conductivity, temperature and salinity must be measured in-situ. However, due to
time constrains, the 48 coupons used for the study were left buried for only 18
months.
 59

3.5.2 Research Area Selection

Five sites were proposed by the pipeline operator and they were labelled as
Site A, B, C, D and E respectively. These sites were near to gas transmission
pipeline. However out of the five, only one site was selected based on SRB activity
potential. All carbon steel coupons installation was conducted within the right-of-
way (ROW) land of the pipeline operator. The description of the entire proposed site
is shown in Table 3.2.

Table 3.2: Site locations and their corresponding description

Site Location Remarks
Identification
Site A Kerteh, Terengganu Hilly area, the soil texture was
identified as gravel
Site B Kemaman, Terengganu Near to mangrove forest, the soil
texture was identified as silt
Site C Sungai Ular, Cherating, Near to a river and influenced by
Pahang the water tide from the sea, the
soil texture was identified as
loam
Site D Kuantan, Pahang Near to a palm oil plantation, the
soil texture was identified as clay
Site E Segamat, Johor Near to a small river, the soil
texture was identified as sand

Site C consists of three types of pipeline; 6 and 8 inch propane and butane
gas pipelines, as well as 36 inch peninsular gas utilization (PGU) pipeline. Out of the
five proposed sites, Site C was chosen due to its potential SRB activity. One of the
main indicators of potential SRB activity is the location of the site is closer to a river
(swampy area) and it is also supported by the pipeline operator finding. As it
location is near to the river, sea water would flow into the area during high tide and
it can cause the soil to emit strong pungent sulphuric odour (refer to SRB activity).
The operator‟s record also classified this site as corrosive. The soil particles are dark
 60

brown and have strong smell of rotten eggs, which can be associated with hydrogen
sulfide gas. The gas in swamp is always linked to anaerobic respiration process by
sulfide-reducing bacteria.

3.5.3 Installation of Coupons

The holes were drilled using a hand auger machine driven by a petrol engine.
The coupon was placed into the hole at 0.5 meter and 1 meter deep. The space
between the holes was set at two meter apart. The coupons were buried for 18
months, whereby eight coupons were retrieved at every three months to determine
their average metal loss. Each coupon was then properly measured, labelled and
recorded. The dimension of the coupon was approximately at 80 mm long, 60 mm
wide and seven mm thick, with a nominal area of 115.60 cm2, drilled and tapped on
one edge to accommodate string as a holder.

3.5.4 Coupon Retrieval, On-Site Measurement and Soil Sampling

After three months, four holes were excavated to recover eight steel coupons
at five research sites. The excavation was conducted manually using a hoe, a spade, a
shovel and other digging tools. During the excavation, site measurements were also
carried out to determine soil parameters, such as pH, chloride, sulfate, sulphide, soil
resistivity, moisture content, and salinity. These samples were gathered in zip lock
plastic bags and conveyed to the laboratory.

Soil resistivity refers to the resistance toward conduction of electric currents

that affects the rate of corrosion of metal. Robinson (1993) described the relationship
between environmental factors and the corrosive nature of soil, where the soil
resistivity is considered the most important factor to determine soil corrosivity.
According to previous study by Paillet et al., (2010), there are several factors that
correlate to resistivity such as nutrients and salinity (Ismail and El-Shamy, 2009),
 61

water content and preferential direction of water flow (Michot et al., 2003), texture-
related properties (e.g. sand, clay, depth to clay-pans or sand layers) and other
indirectly measured soil properties (e.g. organic matter) (Fedotov et al., 2005).

The Wenner four-electrode method was used to conduct soil resistivity test at
the field work as complied with ASTM G57 (ASTM International, 2012) This
method requires four metal electrodes to be placed with equal distances in a straight
line on the surface of the soil to a depth not exceeding 5% of the minimum distance
of the electrodes. A voltage is impressed between the outer electrodes, causing
current to flow, and the voltage drop between the inner electrodes is measured using
a sensitive voltmeter. The soil resistance can be calculated directly using Ohm‟s Law
(V = IR). The soil resistivity, Re is determined using Equation below;

Re = 2. Π. A. R (Eq. 3.1)

Where;
Re = soil resistivity (in Ω.cm)
A = probe distance (in cm)
R = resistance (direct measurement in Ω)
π = 3.142

In this study, the electrodes was placed perpendicular to the underground

pipeline, rather than placing the electrodes parallel with the pipeline, otherwise
would result in measurements lower than actual resistivity. Figure 3.6 shows the
installation of electrode to measure soil resistivity.
 62

Figure 3.6: The Wenner four-electrode method in soil resistivity measurement

3.5.5 Coupon Cleaning After Retrieval

A total of 48 steel coupons used in the site study were retrieved as specified
by their time intermission as stated in the previous section. The coupons were
conveyed to the laboratory for further examination that is complied with ASTM
G01-90 (ASTM International, 1999). The coupons were cleaned, dried and weighed
to determine the final weight loss (Wi). Mechanical and chemical cleaning was used
to eliminate corrosion on the steel coupons. The cleaning process was repeated
several times and the weight loss after each cleaning was recorded. Mechanical
cleaning such as scraping, scrubbing and brushing were carried out to eliminate
heavy encrusted corrosion. Scrubbing with a non-metallic bristle brush and mild
abrasive distilled water slurry were utilized to eliminate corrosion products.
Precaution must be taken to avoid any removal of base metal. The coupons were
then dipped in 1000 mL of chemical reagent, which was a mixture of 500 mL of HCl
with specific gravity of 1.19, added with 3.5 g of hexamethylenetetramine and
distilled water for the cleaning process.

This chemical reagent was specially invented to eliminate corrosion with

minimal dissolution of any base metal for iron and steel-type materials. The steel
coupons were dipped for 10 minutes at a temperature between 20° and 25° C. Longer
 63

time may be needed for certain cases. The chemical cleaning was repeated several
times until constant or almost constant mass loss was achieved. The steel coupons
were weighed after each cleaning process and recorded.

3.5.6 Calculation of Corrosion Rate According to ASTM

Corrosion rate can be stated as the mass of metal turned into corrosion
products per unit area of surface per unit of time (mass loss) or an increase in
corrosion depth per unit time (penetration rate). The mass loss test is one of several
techniques to calculate the corrosion rate of the metal. A clean coupon then is
measured, weighed (Wo), exposed to the environment for a specific time, detached,
cleaned to eliminate corrosion products and re-weighed (Wi). The average corrosion
rate is then obtained using Equation 3.2 (ASTM G1-03, 2011).

K W
CR  (Eq.3.2)
AT  D

Where:
K = a constant (8.76 ×10 4 for mm/yr) obtained from ASTM G01
W = weight loss in g (W = Wo-Wi)
A = exposed area (cm2)
T = time of exposure (h)
D = metal density in (7.86 g/cm3) for carbon steel X-70

3.6 SRB Bacteria

The lab grown sulfate reducing bacteria (SRB) sample species used was
obtained from the American Type Culture Collection (ATCC), with the standard no.
ATCC 7757TM in a form of freeze-dried culture. The freeze-dried SRB culture was
rehydrated using the recommended method by the ATCC. The SRB was grown in
125 mL anaerobic vials in 100 mL liquid medium. The two-day-old SRB subcultures
 64

were used in all experiments. Other equipment used in the experiment including
vials, medium, syringes, and needles needed to be cleaned and sterilized at a
temperature of 121°C for 15 minutes.

3.6.1 Site SRB Bacteria

Isolated SRB bacteria samples were obtained during maintenance works

conducted at Sungai Ular, Kuantan, Pahang Darul Makmur (based on the record and
conversation with the personnel). The site was selected based on the pipeline
operator‟s pigging record, which showed serious occurrence of internal and external
corrosion in the area. A site visit also revealed the pipeline was buried underground
close to the river and constant overflow with sea water during high tide. The strong
smell of rotten egg (strong sulphuric smell) may be caused by the presence of SRB.
Sterilized syringe and needles were used to transfer the sample to three types of
medium; ATCC 1249TM, Postgate‟s B and Postgate‟s C in anaerobic vials. The vials
were left for incubation for three days at a temperature of 37°C. Once the medium
shows a sign of bacteria growth (medium turned to black), 1 mL of the growth
medium was withdrawn using a sterilized syringe and serially diluted (10 fold) with
9 mL of medium, before the sample was plated in solid ATCC 7757TM medium in a
petri dish. The sealed petri dish was left in an anaerobic glove box to incubate for
two to three days at 37°C. The aforementioned step was repeated for two to three
times until a single colony of SRB can be obtained. Single bacteria colony can be
identified by observing the morphology process resulted on solid medium. Figure 3.7
shows the anaerobic glove box used at the Faculty of Biosciences and Medical
Engineering, UTM.
 65

Figure 3.7: Anaerobic workstation used for isolation and manipulates bacteria under
anaerobic condition.

3.7 Molecular Characterisation of SRB

3.7.1 DNA Extraction

A two-day-old (48 hours) of bacteria cultures were prepared in the ATCC

7757TM medium. 1 mL of SRB culture with approximately 2 × 107 cell /mL were
centrifuged for two minutes at 16,000 × g. The supernatant was then discarded and
the cells were suspended in 480 µL of 50 mM EDTA. An amount of 120 µL of lytic
enzymes were added and incubated in a water bath at 37°C for 60 minutes. The cells
extract was centrifuged again for 2 min at 16,000 × g. The supernatant was removed
and the cells extract mixed gently with 600 µL of Nuclei Lysis solution (DNA
purification kits, Promega Corp., Madison Wis). The cells extraction was incubated
again at 80°C for five minutes before it was cooled to room temperature. Three µL
of RNase solution was added to the extraction and incubated at 37°C for 60 min.
After the incubation, the cells extract were proceed with PCR extraction (section
3.7.2).
 66

3.7.2 PCR Amplification of 16S rRNA Fragment

Genomic DNA (5 µL) was mixed with 2 µL of 10 mM dNTP mix containing

all four deoxyribonucleotides and 2.5 µL of 1 U/µL of temperature-resistant DNA
polymerase, along with 3 µL of 30 pmol of two short synthetic DNA fragments,
which are complementary to DNA sequence. The DNA was mixed with 10 µL of
buffer, 66.5 µL of distilled water and 8 µL of 25 mM MgCl2. The 16s rRNA
fragment from the above extraction were send for sequencing for further analysis
(section 3.7.3 and 3.7.4).

3.7.3 Agarose Gel Electrophoresis

PCR products were electrophoresed and 1% (w/v) of agarose gel in 1× TBE

buffer was used as the running buffer. The gel was electrophoresed at 4.8 v/cm for 1
hr and 45 min. A 100 bp DNA ladder (Promega Corp., Madison Wis.) was used as a
marker to estimate the size of DNA bands. The gel was stained with ethidium
bromide-TBE solution for 20 minutes before being photographed using UV
transilluminator.

3.7.4 rDNA Fragments Sequencing and Analysis

The purified PCR product was sent to First BASE Sdn. Bhd. for
sequencing. The obtained sequences then were aligned using Clustal W version 1.82.
The obtained nucleotide was then compared against related sequences derived from
GenBank database using BLASTN program.
 67

3.8 Media Preparation

Bacteria require a suitable medium to grow and multiple. Therefore, a

selection of suitable SRB medium is required. Three types of medium were used in
this study; ATCC 1249TM, Postgate‟s B and Postgate‟s C. All these mediums were
tested with SRB to determine the most suitable media to promote the growth. ATCC
1249TM medium was selected for the experiment.

3.8.1 ATCC 1249 Medium

ATCC 1249TM broth medium, also known as The Baar‟s medium, is a

recommended medium in ASTM standard test method E979-91 (Standard method
for evaluation of antimicrobial agents in aqueous metal working fluids). Table 3.3
shows the composition of the medium.

Table 3.3: Composition of ATCC 1249TM‟s medium

Components Chemicals and Compositions
Component I MgSO4, 2.0g
Sodium citrate, 5.0g
CaSO4, 1.0g
NH4Cl,1.0g
Distilled water, 400.0ml
Component II K2HPO4, 0.5 g
Distilled water, 200mL
Component III Sodium lactate, 3.5 g
Yeast extract, 1.0 g
Distilled water, 400.0 mL
Component IV Fe(NH4)2(SO4)2, 50 ppm

The chemical form in each component might interact at high temperature

during the autoclave process. Therefore, component I, II and III were prepared and
autoclaved in separate bottles at 121°C for 15 minutes. The medium was left to cool
in laminar flow for 1 hour before they were mixed. Ferrous ammonium sulfate
 68

(Fe(NH4)2(SO4)2) requires filter sterilization as it is heat sensitive and thus cannot be

autoclaved. Sterile syringe and filter (0.45µm) were used to add ammonium sulfate
to the medium. Solid medium was prepared by adding agar (2 % w/v, gelling agent
or agarose) to the liquid medium prior to autoclave.

SRB needs suitable anaerobic condition to grow and this condition was
achieved by bubbling the medium with filtered (0.45 µm) nitrogen for approximately
one hour to remove dissolved oxygen in the medium. Oxygen-free nitrogen was used
to bubble the medium with a gas regulator at 0.5 psi. The temperature of the medium
was increased to between 50°C to 80°C, accelerating the deoxygenated process as
compared to a cooled medium. Since the anaerobic glove box facilities are not yet
available at Faculty of Civil Engineering (FKA), the medium was transferred to
sealed anaerobic vials using a peristaltic pump. Modification was conducted to
Schott bottle cap by fabricating manifold to allow the medium to be transferred and
for nitrogen to bubble without exposing the medium to the atmosphere. Figure 3.8
shows the process of transferring the medium to anaerobic vials under laminar flow.

Figure 3.8: Transferring the medium to anaerobic vials in the laminar flow

3.8.2 Postgate’s C Medium and Postgate’s B Medium

Postgate‟s C medium is commonly used in a diverse range of applications for

growth and enrichment of marine SRB. This medium is used as a selective medium
for SRB screening from indigenous marine organism. Table 3.4 shows the
 69

composition of Postgate‟s C medium. The medium was sterilized and subjected to

anaerobiosis using the aforementioned procedure. Postgate‟s B medium chemical
composition shows in Table 3.5;

Table 3.4: Composition of modified Postgate‟s C medium (Cortas et al. 2012)

Chemical Composition(g/L)
Sodium lactate 6.0
Sodium sulfate 4.5
Ammonium chloride 1.0
Yeast extract 1.0
Potassium phosphate 0.5
Sodium citrate 2H2O 0.3
Calcium chloride 6H2O 0.06
Magnesium sulfate 7H2O 0.06
Iron (II) sulfate 7H2O 0.004

Table 3.5: Composition of modified Postgate‟s B medium (Tanner,1989).

Chemical Composition (g/L)
Lactic acid 2.7
Calcium sulfate H2O 1.3
Ammonium chloride 2.0
Potassium phosphate 1.0
Magnesium sulfate 7H2O 4.0

3.9 Corrosion Testing Using Electrochemical Methods

The participation of micro-organisms in the corrosion process changes the

properties of the interface between the metal and the bulk solution. These
modifications of metal surface conditions resulted in many effects, including
changes from general corrosion to localized corrosion. However, electrochemical
 70

techniques are still useful in explaining the phenomenon of bio-fouling and bio-
corrosion influenced by micro-organisms. All electrochemical measurements are
indirectly based on Faraday‟s law and are able to give instant results. Furthermore,
they are powerful and efficient techniques in investigating corrosion mechanisms.

3.9.1 Open-circuit Potential Measurement (OCP)

The sample (working electrode) and electrode were immersed in a corrosive

liquid medium without any connection to any instrument. The potential term was
assumed as corrosion potential (relative to reference electrode) Ecorr. The corrosion
potential is defined as a potential where the oxidation rate is exactly equal in
magnitude to the reduction rate when measured with an electrometer. The open-
circuit potential (OCP) was measured against a standard saturated calomel electrode
(SCE) immersed in the medium. Working electrode and SCE electrode were
sterilized by rinsing in 70% of alcohol and exposed under UV light prior to immerse
in the SRB inoculated medium. The potential was measured daily using a multimeter
(Kyoritsu digital multimeter). All the mediums used and the electrochemical cell
were autoclaved at 121°C for 15 minutes. Figure 3.9 presents the picture of the OCP
experiment setup in an electrochemical cell.

Figure 3.9: OCP test conducted in an electrochemical cell inoculated with SRB
 71

3.9.2 Tafel Slope Analysis and Corrosion Rate Experiment

An electrochemical experiment was carried out in Faculty of Sciences (FS)

electrochemical lab according to ASTM standard cell (ASTM Designation G3-14,
2014) under anaerobic condition by bubbling nitrogen gas into the electrochemical
cell. Three electrode systems were used in the experiment: with carbon steel as the
working electrode; saturated calomel electrode (SCE) as the reference electrode and
a platinum rod as the counter electrode. Tafel slope and corrosion rate (CR) were
obtained using the Autolab PGSTAT30 system (Eco Chemie B.V., Netherlands) and
the General Purpose Electrochemical System (GPES) software. The determination of
corrosion rate was obtained from the software, which showed the corrosion potential
and the polarization resistance at the corrosion potential. Figure 3.10 show the image
of Autolab PGSTAT30 system in the Faculty of Sciences, UTM.

Figure 3.10 : Autolab PGSTAT 30 with GPES system software.

Polarization resistance, Rp is defined according to Equation 3.3, as the

tangent of the polarization curve at the corrosion potential was under steady-state
polarization conditions using low amplitudes of perturbation.
 72

 dE 
Rp   
 dI ai ,E Ecorr ( Eq. 3.3)

In the charge-transfer reaction, Icorr is associated with Rp and can be

determined through the well-known Stern-Geary equation (Eq3.4) (Stern & Geary,
1957):

a c 1 1
I corr 
2.303(  a   c ) R p A
(Eq. 3.4)

Where:
βa = anodic Tafel slope (V/decade)
βc = cathodic Tafel slope (V/decade)
Icorr = corrosion current density
Rp = polarization resistance (ohm)
A = exposed area (m2)

In this experiment, the exposed area of the coupon‟s contact with the liquid
medium is 1.0 cm2. Once the value of current density was obtained, the corrosion
rate was calculated according to the following equation( Eq. 3.5) (Sun, 2003):

I corr M w
CR 
nF
(Eq. 3.5)

Where:
CR = corrosion rate (mm/yr)
Mw = molecular weight of iron
Icorr = corrosion current density (A/m2)
n = number of electrons transfer during the reaction
F = Faraday‟s constant
ρ = density of iron (kg/m3)
 73

3.10 Analytical Methods

3.10.1 SRB Enumeration

SRB cell enumeration was performed to monitor bacterial growth. Sterile

syringe was used to withdraw the medium from the vials or glass cell before the
medium was precisely diluted in distilled water. Enumeration of bacteria was
counted in the Petroff-Hausser chamber under an optical microscope at 400× (three
times for each sample). The SRB test kit from Bio Sani-Check product #100 was
also used to validate the result (Abdullah et al., (2014).

3.10.2 Sulfate (SO42-) Ions Concentration

Sulfate precipitation with barium was used to measure sulfate concentration

(Kolmert et al., 2000). 1 mL of liquid sample was withdrawn from a vial at a certain
time, followed by centrifuged at 3,000 rpm for two minutes. For each assay, 0.1 mL
of sample was diluted with 0.9 mL of distilled water, and then mixed with 60 mg of
barium chloride and 1 mL of testing reagent. The testing reagent contained 150 g of
NaCl, 100 mL of glycerol/L, 60 mL of concentrated hydrochloric acid, 200 mL of
95% ethanol and the balance is distilled water. A spectrophotometer (Model 340,
Sequoia-Turner Corporation, Mountain View, CA, USA) was used to measure the
optical density of the reaction mixture at the wavelength of 420 nm.

3.10.3 FESEM Observation

Bio-films on the coupon surface were observed using FESEM (model Supra
35VP). Sessile cells in the bio-film were first fixed in 4% (wt) glutaraldehyde for
four hours and then rinsed with a series of ethanol (25%, 50%, 75% and 100%
purities) to dehydrate the bio-films. They were subsequently dried using critical
point dried (CPD) equipment using supercritical CO2 and coated with gold (Au)
 74

prior to the examination of the bio-films under FESEM. Pits observation on the
coupon surface was obtained by removing the bio-films using Clark‟s solution
(ASTM G1-03, 2011) and washed with distilled water before being observed under
FESEM. The image was taken in the area with the largest pits observed.

3.10.4 Ferrous (Fe2+) Ions Concentration

The concentration of Fe2+ ions was determined by spectrophotometer cells

using HACH DR4000U spectrophotometer at the wavelength of 450 nm. Detailed
procedure of Fe2+ ions concentration was described using HACH standard method
#8146.

3.10.5 pH Value

pH value is an important parameter in growing SRB. pH is an indication of

the H+ ion concentration in the solution. For this study the pH value was recorded
using Hanna pH meter. Standard buffer solutions at pH 4 and 10 were used for
calibration, where the pH meter was used as per recommended by the manufacturer.
The pH meter needs to be calibrated regularly to obtain accurate result.

3.11 Weight Loss Techniques

The conventional method for evaluating the corrosion of steel is by

measuring the loss of mass of a metal coupon after a period of exposure to a
corrosive environment. All coupons used in the study were prepared according to the
ASTM standards (ASTM G1-90, 1992; ASTM G 1-72, 1993). The weight loss
measurement was calculated by using the following equation (Eq. 3.6):
 75

Wo  Wa
Weight loss rate (W) 
A (Eq. 3.6)

Where: Wo = initial weight before corrosion (g)

Wa = weight after corrosion (g)
A = surface area exposed (cm2)

After the coupons were exposed to the SRB environment for the designated
period of time, they were cleaned mechanically by scrubbing with a soft brush,
followed by chemical cleaning to remove the bio-films and FeS film, using Clarke‟s
solution (Haynes and Bobian, 1983). The cleaning procedure to remove corrosion
products and the Equation 3.1 was used to calculate the corrosion rate as stated in
ASTM G1-03.

3.11.1 Weight Loss in Anaerobic Vials

Anaerobic vials were used to study the effect of each parameter upon
corrosion rate. Two pieces of coupons were inserted into the vials. Nitrogen gas was
later purged inside the vials for a few minutes before they were capped and crimped
with aluminium seal. The vials with coupons were autoclaved at 121°C for 15
minutes. 100 mL of the designated medium was anaerobically transferred to vials
conducted in laminar flow. A two-day-old cultured SRB was injected into the vials
using sterilized syringe and needle. Meanwhile, no bacteria was added for control
vial. The exposed surface of the coupons inside the vials should be placed face up
for bio-films to grow and give maximum exposure to SRB. The vials were incubated
at the designated temperature for a certain period of time. Figure 3.11 shows the
picture of the SRB inoculated vials and control vials.
 76

Figure 3.11: The differences between inoculated and control vials

3.12 Response Surface Method (RSM)

STATISTICA 8.0 software was utilized for response surface methodology.

The experiment used central composite design (CCD) in developing the corrosion
model due to the ability of the software to analyse up to five levels of factors. For
statistical analysis, the relation between the coded values and actual values is
described in Equation 3.7:

2( X  X high )
xcoded  1
( X high  X low ) (Eq. 3.7)

Where:
xi = dimensionless value of an independent variable
Xi = real value of an independent variable

The predicted corrosion model in the quadratic model in coded equation is as

follows (Yates, 1966):

Y   0    ixi    iixi2    ijxixj

(Eq. 3.8)
 77

Where:
Y = predicted response variables
β0 = offset term
βi = linear effect
βii = squared effect
βij = interaction effect
x = coded levels of independent variables

All experiments were conducted in a random order to avoid systematic bias.

In addition, each experiment was conducted in triplicate. The result from the
experiment was analysed using ANOVA test.

3.12.1 Selecting Factors

Before designing the experiments to be used in the corrosion model, the most
influential factors were identified. This stage was also conducted to screen the
parameters and to identify the range of values of each parameter to be used in the
experiment for obtaining the corrosion model. The selecting factor might come from
OFAT experiment, previous research and also from the literature review

3.12.2 SRB Bacteria Sample

Microbiologically-influenced corrosion consists of aerobic and anaerobic

types of bacteria. Nevertheless, an anaerobic bacteria species such as SRB, received
specific attention by the researchers due to the impact and implication caused by this
species. Table 3.6 shows some of the SRB bacteria that have been used by other
researchers in biocorrosion study using steel coupon.
 78

Table 3.6: Summary of SRB species and used medium in SRB research
Researcher SRB Medium Species
1. Sungur et al. (2007) Postgate‟s C Desulfovibrio sp.

2. Gu et al. (2009a) Modified Baar‟s Desulfovibrio desulfuricans

3. Javaherdashti et al. (2008) Postgate‟s B Isolated and consortia of

SRB
4. Wen et al. (2010) Baar‟s D. desulfuricans subsp.
Aestuarii (ATCC 14563)
5. Sahrani et al. (2008) Modified Postgate Desulfovibrio sp.

6. Abdullah et al. (2014) Modified Baar‟s Desulfovibrio sp. And

isolated SRB.

Therefore, based on the literature listed in the table, Desulfovibrio sp. species
is the most highlighted SRB species in the study of SRB corrosion and Baar‟s
medium is the most suitable medium to grow Desulfovibrio sp. for bio-corrosion
study.

3.12.3 Incubation Period

Incubation period is one of the parameters that needs to be considered in the

study of the exposure of metal coupons to the medium and SRB. Exposure time will
be used in the calculation of corrosion rate for weight loss method. Meanwhile, for
electrochemical method, suitable incubation period is important to evaluate the effect
of SRB on the tested coupons. Table 3.7 shows the inoculation period used by other
researchers to obtain the corrosion rate influenced by SRB.
 79

Table 3.7: Summary of incubation period by other researchers in bio-corrosion

research
Period
Researcher Method
(Days)
1. Gu et al. (2009b) Weight loss method 30

2. Sahrani et al. (2008) Weight loss method & open circuit 14 & 30
potential
3. Eashwar et al. (1993) Weight loss method & open circuit 15 & 25
potential
4. Javaherdashti et al. (2008) Open circuit potential 35

5. Venzlaff et al. (2013) Open circuit potential & otentiodynamic 7 & 28

measurement
6. Abdullah et al. (2014) Weight loss method 15 to 30

Based on the above result, the incubation period of 15 to 30 days was

selected in the inoculation of SRB for API X-70 carbon steel coupon.

3.12.4 Incubation Temperature

Temperature is an important factor when studying bacterial responses. The

optimum incubation temperature will give full potential to the bacteria to grow.
Some SRB bacteria are thermophilic and mesophilic as discussed in the literature.
Table 3.8 presents the works of some researchers on SRB with their incubation
temperature.
 80

Table 3.8: Summary of incubation temperature by other researchers in bio-corrosion

research
Researcher Incubation Temperature (°C)
1. Franca F. P. et al. ( 1999) 30~65°C
2. Sungur I. E. et al. (2007) 37°C
3. Wen J. et al. (2009) 37°C
4. Javaherdashti et al. (1999) 25-40°C
5. Abdullah et al. (2014) 370C

Based on the above literature, the temperature of 37°C was selected as the
central temperature for the study. The effect of temperature on bacterial growth
curve and bio-corrosion is discussed in the next chapter.

3.12.5 Test Matrix for Other Parameters

The test matrix for other parameters is shown in Table 3.9. The selection of
the test parameters is based on the literature review and operating conditions. The
experiments were conducted in anaerobic vials with a limited period of time.
Furthermore, the experiments were conducted differently by executing one-factor-at-
a-time (OFAT) method to obtain the highest corrosion effect for each studied
parameter. Incubation temperature, initial pH value of the medium, content of SO42-
and Fe2+ concentration were the examined parameters. OFAT method was used to
determine the range of values of each parameter decided to be used in RSM method.
The experiments were carried out according to the following test matrix as shown in
Table 3.9.
 81

Table 3.9: Test matrix of OFAT study for SRB

Test Strain ATCC7757TM
Test Medium Baar‟s Medium/ ATCC 1249TM
Coupon Material Carbon steel (API 5L X-70)
Initial Bacteria Number during Inoculation ±1.0~3.0×107
Incubation Duration 15 ~ 60 days
Temperature (0C) 5°C~ 50°C
pH 4.0 ~10.0
Total initial Fe2+ concentration (g/L) 0 ~ 400
Total Sulfate (SO42-)concentration (g/L) 0 ~ 7.5
Flow of the bulk fluid No flow

3.12.6 SRB Corrosion Model using RSM

Based on the result from OFAT method, selected factors and range of the
optimized values further evaluation was conducted using RSM. The selected
variables obtained from OFAT were; pH value, temperature, total initial Fe2+
concentration, and total sulfate concentration. For the SRB corrosion model, the
design was made up of a full 24 factorial with a total of 30 experiments, as per Table
3.10. The star point was set to α of 1.682 from the coded centre point. Each
independent variable was studied at five different coded levels (-α, -1, 0, +1, +α).
 82

Table 3.10: Full factorial experimental design matrix with four variables (coded and
natural)
Temp. SO4-2 Fe2+
Run Standard pH Temp SO4-2 Fe2+ pH
(0C) (g/l) (ppm)
1 28 0 0 0 0 7.5 30 1.5 75
2 8 1 1 1 -1 8.5 40 2.0 50
3 4 1 1 -1 -1 8.5 40 1.0 50
4 19 0 -α 0 0 7.5 10 1.5 75
5 23 0 0 0 -α 7.5 30 1.5 25
6 16 1 1 1 1 8.5 40 2.0 100
7 19 -1 -1 -1 1 6.5 20 1.0 100
8 25 0 0 0 0 7.5 30 1.5 75
9 12 1 1 -1 1 8.5 40 1.0 100
10 21 0 0 -α 0 7.5 30 0.50 75
11 15 -1 1 1 1 6.5 40 2.0 100
12 1 -1 -1 -1 -1 6.5 20 1.0 50
13 18 +α 0 0 0 9.5 30 1.5 75
14 26 0 0 0 0 7.5 30 1.5 75
15 14 1 -1 1 1 8.5 20 2.0 100
16 5 -1 -1 1 -1 6.5 20 2.0 50
17 10 1 -1 -1 1 8.5 20 1.0 100
18 7 -1 1 1 -1 6.5 40 2.0 50
19 3 -1 1 -1 -1 6.5 40 1.0 50
20 22 0 0 α 0 7.5 30 2.5 75
21 11 -1 1 -1 1 6.5 40 1.0 100
22 20 0 +α 0 0 7.5 50 1.5 75
23 29 0 0 0 0 7.5 30 1.5 75
24 2 1 -1 -1 -1 8.5 20 1.0 50
25 6 1 -1 1 -1 8.5 20 2.0 50
26 30 0 0 0 0 7.5 30 1.5 75
27 27 0 0 0 0 7.5 30 1.5 75
28 24 0 0 0 +α 7.5 30 1.5 125
29 13 -1 -1 1 1 6.5 20 2.0 100
30 17 -α 0 0 0 5.5 30 1.5 75

3.13 Concluding Remarks

This chapter presented the methodology of the study in modelling SRB

bacteria in the corrosion of API 5L X-70 carbon steel pipe. Two SRB bacteria strains
were used in the experiments to assess the differences of each SRB strain corrosion
rate. In a natural condition, SRB live in consortia. It is almost impossible to
incorporate all the strains and natural conditions in the modelling, but the most
influential and important parameters have been considered according to OFAT
experiment, as well as from the literature. RSM was used in order to evaluate the
interaction of each parameter against the corrosion rate.
 83

CHAPTER 4

DATA ANALYSIS AND DEVELOPMENT OF SITE CORROSION MODEL

4.1 Introduction

In this chapter, the analysis of weight loss data for carbon steel coupon and
site corrosion parameters for the development of soil corrosion model is presented.
The first part of the chapter covers experimental results of the observed metal loss
data and on-site soil parameters. It is then followed by data screening processes
including normality testing, hypothesis testing and outliers detection. A graph of
metal loss data versus exposure period was plotted to observe the power law form of
deterioration development. Linear correlation analysis (LCA), principal component
analysis (PCA) and multiple linear regressions (MLR) were then carried out to
define the relationship among the examined factors. The important factors related to
corrosion rate, k and corrosion growth pattern, n were then integrated into the power
law model and the grouping of k and n was done based on the best model with the
maximum coefficient of determination (R2).

4.2 Site Selection Description

One site was selected from five proposed sites by the pipeline operator. The
sites were named Site A, B, C, D and E. All the sites are near to the gas transmission
pipeline and the installation of steel coupons were conducted within the Right-Of-
Way (ROW) land of the vendor. The description of the selected site is shown in
Table 4.1.
 84

Table 4.1: Selected site location information (Site C)

Site Code Location Remarks

Site C Sungai Ular, Cherating, Near to a river and influenced

Pahang by the water tide from the sea,
the soil was identified as loam

From the five proposed sites, Site C was chosen. The site was selected as the
location is close to a river (swampy area), influenced by the sea water during high
water tide and has high smell odour of sulphur (refer to SRB activity). The pipeline
operator‟s record also consider this site as highly corrosive and recorded as MIC
suspected area. From the preliminary visit to the site, the soil particles are dark
brown in colour and have strong smell of rotten eggs, which can be associated to
hydrogen sulfide gas. The gas in swampy soil has always been linked to anaerobic
respiration process by sulfate-reducing bacteria. Figure 4.1 show the image of the
carbon steel coupon retrieved from the Sg.Ular site. From the image black colour of
corrosion product and soil is observed. This is an early indication of the presence of
SRB bacteria at Sungai Ular site.
 85

Figure 4.1: Carbon steel coupon retrieved from Sungai Ular site after 3 months

4.3 Preliminary Data Analysis

A number of 48 carbon steel coupons of API 5L X-70 was installed and

buried underground at 0.5 m and 1.0 m depth at selected Site C, or namely Sungai
Ular site. The analysis of the result can be divided into two parts; preliminary
analysis and corrosion model analysis. Preliminary analysis was designed to collect
the information and analyse the significance of the available data on the parameters
of the study. The preliminary analysis was conducted in four stages as follows:

i) Stage 1 – Data was classified according to the depth of the buried coupons.
48 carbon steel coupons were buried at 0.5 m and 1.0m depth.
ii) Stage 2 – Non-parametric test was conducted using T-test to determine
whether the depth of the buried coupons gives statistical significance
between both depths.
 86

iii) Stage 3 – Data was classified according to the central tendency. In this stage,
the data was classified as mean data (avg), median data (med) and
maximum data (max).
iv) Stage 4 – The central tendency was identified. Further analysis was
conducted to select mean data, median data or maximum data for the
corrosion model analysis.

4.4 Experimental Results

Mass loss as a consequence of corrosion development in soil is a dependent

variable controlled by a set of independent variables, which are soil pH (pH), sulfate,
chloride (Cl-), sulfide, moisture content, resistivity and organic content. Table 4.2
present the summary of the observed soil parameters at 1.0 m depth according to the
period of coupons retrieval. While Table 4.3 show the results of soil analysis for
Sungai Ular site after data treatment.

Table 4.2: Results of laboratory analysis of soil for Sungai Ular site

Sulfate Sulfide Organic

Site Time Chloride Moisture Resistivity
No. pH content Content Content
Code (Month) (mg/kg) Content(%) (Ohm.cm)
(mg/kg) (mg/kg) (ppm)
1 C 3 4.1 1495 2384 59 24 94 24505
2 C 6 2.6 1802 67 37 50 82 41011
3 C 9 4.8 2204 5731 21 28 94 55792
4 C 12 3.9 700 48 21 31 104 47228
5 C 15 4.2 3198 408 25 31 622 65211
6 C 18 3.7 1708 1708 39 44 352 49621
 87

Table 4.3: Results of soil analysis for Sungai Ular site after data treatment
Log Sulfide LogRE Log (Oc)
Time Moisture
No. pH LogSulfate (Ch) Content (resistivity) Organic
(Month) Content
(mg/kg) Chloride (mg/kg) (Ohm.cm) Content(ppm)
1 3 4.1 3.175 3.37 59 24 1.973 4.39
2 6 2.6 3.256 1.82 37 50 1.914 4.61
3 9 4.8 3.343 3.75 21 28 1.973 4.74
4 12 3.9 2.845 1.68 21 31 2.017 4.67
5 15 4.2 3.505 2.61 25 31 2.794 4.81
6 18 3.7 3.232 3.23 39 44 2.547 4.69

Based on the result from Table 4.2, throughout the 18 month period of data
collection, the pH value is ranging from 2.6 to 4.8 and 21 mg/kg up to 59 mg/kg for
sulfide content. Huge range was recorded for sulfate content from 700 mg/kg ~ 3198
mg/kg and chloride content from 48 to 5731 mg/kg. The minimum percentage of
moisture content was 24%, while the maximum moisture content recorded was 50 %
due to limited measurement capability of the recording device The resistivity value
of the soil at site is ranging from 82 ~ 622 Ohm.cm and last parameter is organic
content where the value is ranging from 24505~ 65211. By using the AWWA
assessment method, the cumulative score of this site is measured at 13.5 point which
can be classified as corrosive to buried pipe. Furthermore, classification based on the
proposed method by Nazim (2015) shows that 6 out of 7 measured soil parameters
(expect for organic content) are categorized as corrosive to highly corrosive. Table
4.4 summarized the corrosiveness of the site based on assessment method according
to AWWA and Nazim (2015).
 88

Table 4.4: Sungai Ular soil corrosiveness based on AWWA and Nazim (2015) at 3
months of exposure.

Soil Classification Soil Classification

Parameter and measured
Using AWWA index by Nazim (2015)
value
Score Score
pH (4.1) 0 Corrosive Corrosive
Sulfate content (1495 mg/kg) N/A (>10) total Corrosive
Chloride(2384 mg/kg) N/A score is Corrosive
Sulfide (59 mg/kg) 3.5 more than Highly Corrosive
Moisture content (24.0 %) N/A 10, the soil Highly Corrosive
Resistivity (94.0 ohm.cm) 10 in corrosive Highly corrosive
Organic Content (24505) N/A classification N/A
Total Score 13.5 -
Note: N/A – not available

The results of the metal loss (ML) were then used to calculate the corrosion
rate (CR) according to ASTM G1-03 (ASTM International, 2011). The results of
metal loss were classified into three different sets of data, namely median data
(med), average data (avg), and maximum data (max) for two different depths of
installation, which are at 0.5 m and 1.0 m. The calculation of metal loss central
tendency was conducted based on the overall data and maximum data (average high
metal loss volume). The results of soil parameters and metal loss data (median,
average and maximum) are presented in Table 4.2 to Table 4.19. Different types of
corrosion data classification as mentioned previously were used to identify the best
prediction model of metal loss with the least error. The error associated with the
variation of metal loss data such as outliers may affect the quality of prediction.
Hence, it is imperative to summarize the data according to varying methods. The
existence of errors may be due to overestimation or underestimation (extreme data).
The median of metal loss is not affected by extreme data as compared to average.
Since corrosion is a slow process, extreme low rate of corrosion may also affect the
central tendency of metal loss distribution (i.e. average). Therefore, low metal loss
may be removed from the database. To overcome these issues, it is necessary to
calculate the single value of metal loss data for every exact period of exposure using
average and median for overall and maximum data. The results of metal loss and
corrosion rate data at the depths of 0.5 m and 1.0 m obtained from the site after 18
months of exposure to soil environment are shown in Table 4.5 and Table 4.6.
 89

Table 4.5: Metal loss (ML) and corrosion rate (CR) data at 0.5 m depth
Coupon
Time CR
No. Code ML (g)
(Month) (mm/y)
Ref.
1 1A 3 2.3748 0.075
2 2A 3 1.9932 0.063
3 13A 3 6.1113 0.193
4 15A 3 4.4752 0.142
5 9A 6 21.8128 0.461
6 10A 6 16.2682 0.344
7 19A 6 7.0053 0.148
8 24A 6 6.5801 0.139
9 6A 9 28.6429 0.389
10 8A 9 13.3571 0.181
11 20A 9 6.2811 0.085
12 25A 9 4.3334 0.059
13 11A 12 38.5921 0.388
14 12A 12 36.8178 0.370
15 14A 12 10.8761 0.109
16 16A 12 8.5663 0.086
17 5A 15 94.3131 0.720
18 7A 15 34.4821 0.263
19 17A 15 16.2534 0.124
20 18A 15 10.3631 0.079
21 21A 18 12.9732 0.078
22 22A 18 11.4170 0.069
23 3A 18 31.4470 0.190
24 4A 18 41.6791 0.251
 90

Table 4.6: Metal loss (ML) and corrosion rate (CR) data at 1.0 m depth
Coupon Time CR
No. ML (g)
No. (month) (mm/y)
1 1B 3 4.2592 0.135
2 2B 3 3.8644 0.122
3 13B 3 0.8701 0.028
4 15B 3 4.6434 0.147
5 9B 6 5.1649 0.108
6 10B 6 6.8371 0.145
7 19B 6 9.6162 0.203
8 24B 6 4.0273 0.085
9 6B 9 12.0604 0.164
10 8B 9 3.5683 0.048
11 20B 9 15.2801 0.207
12 25B 9 2.2319 0.030
13 11B 12 18.8943 0.190
14 12B 12 12.9546 0.130
15 14B 12 10.9722 0.110
16 16B 12 35.9894 0.361
17 5B 15 19.1297 0.146
18 7B 15 17.9414 0.137
19 17B 15 58.1194 0.444
20 18B 15 13.9384 0.106
21 21B 18 31.1503 0.188
22 22B 18 9.8122 0.059
23 3B 18 19.5103 0.118
24 4B 18 17.4310 0.105

4.5 Data Screening

4.5.1 Normality Test

The tests were carried out at 95% confidence interval and the results are
presented in Table 4.7. Log transformation was used to regulate the range of two
independent variables, which are chloride content and organic content. The corrosion
data would be regarded as normally distributed if the p-value was greater than 0.05
(Sig. value) for S-W.
 91

Table 4.7: Normality test results

Shapiro-Wilk
Remarks
Data Statistic p-value Sig.
p>0.05; cannot reject
ML (avg) at 0.5 m 0.955 0.779 .05 Ho: Sample follows a
normal distribution
p>0.05; cannot reject
ML (avg) at 1.0 m 0.913 0.504 .05 Ho: Sample follows a
normal distribution
p>0.05; cannot reject
ML (med) at 0.5 m 0.893 0.336 .05 Ho: Sample follows a
normal distribution
p>0.05; cannot reject
ML (med) at 1.0 m 0.845 0.145 .05 Ho: Sample follows a
normal distribution
p>0.05; cannot reject
ML (max) at 0.5 m 0.877 0.255 .05 Ho: Sample follows a
normal distribution
p>0.05; cannot reject
ML (max) at 1.0 m 0.936 0.626 .05 Ho: Sample follows a
normal distribution
p<0.05; reject Ho:
MC 0.7 0.006 .05 Sample does not follow
normal distribution
p>0.05; cannot reject
pH 0.840 0.131 .05 Ho: Sample follows a
normal distribution
p<0.05; reject Ho:
Cl- 0.770 0.031 .05 Sample does not follow
normal distribution

4.5.2 Mann – Whitney U Test

The results were interpreted using Mann-Whitney U test probability table at

95% confidence limit compared to the calculated U values for the two samples for
each type of metal loss data. Since the U values calculated for the two samples were
greater than U critical at 95% confidence limit (p>0.05), thus the null hypothesis
(Ho) could not be rejected. Table 4.8 shows the summary of hypothesis testing for
the desired metal loss data. From this result, it is concluded that there was no
statically significant difference between the metal loss data at the depths of 0.5 m
and 1 m. Therefore, further analysis of metal loss data could be carried out by
 92

ignoring the influence of different depths of the buried samples. Table 4.9 and Table
4.10 present the metal loss data (avg, med, max) at the depths of 0.5 m and 1.0 m,
respectively.

Table 4.8: Hypothesis testing for metal loss data at depths 0.5 m and 1.0 m
U critical U calculated p-value Conclusion
p > 0.05 (Accept H0)
288 330 0.392 Therefore, the data has no
influence towards depth

Table 4.9: Metal loss (ML) and corrosion rate (CR) data at 0.5m depth

Period CRavg MLmed CRmed MLmax CRmax

MLavg (g)
(Month) (mm/yr) (g) (mm/yr) (g) (mm/yr)
3 3.739 0.118 3.425 0.108 6.111 0.193
6 12.917 0.273 11.637 0.246 21.813 0.461
9 13.154 0.179 9.819 0.133 28.643 0.389
12 23.713 0.238 23.847 0.239 38.592 0.388
15 38.853 0.297 25.368 0.194 94.313 0.720
18 24.379 0.147 22.210 0.134 41.679 0.251

Table 4.10: Metal loss (ML) and corrosion rate (CR) data at 1.0m depth

Period CRavg MLmed CRmed MLmax CRmax

MLavg (g)
(Month) (mm/yr) (g) (mm/yr) (g) (mm/yr)
3 3.409 0.108 4.062 0.128 4.643 0.147
6 6.411 0.136 6.001 0.127 9.616 0.145
9 8.285 0.112 7.814 0.106 15.280 0.207
12 19.702 0.198 15.924 0.160 36.818 0.370
15 27.282 0.208 18.536 0.142 58.119 0.444
18 19.476 0.117 18.471 0.110 31.150 0.188

4.5.3 Outliers Detection

A proper outlier‟s detection technique was applied to decide whether the

uncertain results need to be retained or rejected. The extreme potential outliers
intrinsic in the metal loss data may be due to the over cleaning works (mechanical
brushing) to remove the soil and rust on the coupons. Meanwhile, the potential
 93

outliers for dependent variables may be due to possible error during field work
measurements. Salinity and chloride contents were not tested for the outlier‟s data,
since the numbers of data are small. Figure 4.2 presents the box plot results for all
soil corrosion parameters and metal loss. The symbols of asterisks and circles in
Figure 4.2 represent the possible outliers that need to be further investigated. The
potential outliers from the box plot were then rechecked using Dixon test and t-test
(Table 4.11). The data found to be outliers from at least 2 of 3 tests were considered
false and had been removed from datasets for further analysis. Three averages, two
maximums and one median of metal loss data were classified as outliers. The bold
values in Table 4.12 represent the values of the outliers before being removed from
the datasets. The removal of the outliers was done since it can affect the accuracy of
the predicted model. The error of reading might came from the portable device used
to measure the data onsite, as example the pH value. The results of metal loss (avg,
med, max) after the removal of outliers are presented in Table 4.13 to Table 4.19.

Figure 4.2: Summary of box plot for metal loss data

 94

Table 4.11 Outliers calculation using Dixon‟s test, Grubbs‟ test and box plot(t-test)
Dixon’s test (t-test)
Outliers Data R22 R22 (critical) p-value Remarks
Detected (observed)

94.313 p-value <

0.05; there is
0.572 0.350 < 0.0001
outliers in the
58.119 data

Grubbs’ test
Outliers Data G G (critical) p-value Remarks
Detected (observed)
p-value <
94.313 0.05; there is
4.567 3.112 < 0.0001
outliers in the
58.119 data
Box Plot (t-test)
Outliers Data Median Kurtosis
Skewness (Pearson) Remarks
Detected (Pearson)
94.313 From the plot,
the circle dot
58.119 11.739 2.436 7.627
showed
outliers
41.679
 95

Table 4.12: Metal loss (ML) and soil parameters (outliers‟ values are bold)
Salinity Moisture
Coupon Time, ML, Resistivity Chloride
(ppt) Content, pH
Code (month) (g) (Ὠ.cm) (mg/kg)
(%)
1A 2.375 N/A 24.0 4.79 94 2384
1B 4.259 N/A 24.0 4.79 94 2384
2A 1.993 N/A 24.0 4.79 94 2384
2B 3.864 N/A 24.0 4.79 94 2384
3
13A 6.111 N/A 24.0 4.79 94 2384
13B 0.870 N/A 24.0 4.79 94 2384
15A 4.475 N/A 24.0 4.79 94 2384
15B 4.643 N/A 24.0 4.79 94 2384
9A 21.813 16.4 50.0 4.36 82 67
9B 5.165 16.4 50.0 4.36 82 67
10A 16.268 16.4 50.0 4.36 82 67
10B 6.837 16.4 50.0 4.36 82 67
6
19A 7.005 16.4 50.0 4.36 82 67
19B 9.616 16.4 50.0 4.36 82 67
24A 6.580 16.4 50.0 4.36 82 67
24B 4.027 16.4 50.0 4.36 82 67
6A 28.643 6.8 28.0 6.0 94 5731
6B 12.060 6.8. 28.0 6.0 94 5731
8A 13.357 6.8 28.0 6.0 94 5731
8B 3.568 6.8 28.0 6.0 94 5731
9
20A 6.281 6.8 28.0 6.0 94 5731
20B 15.280 6.8 28.0 6.0 94 5731
25A 4.333 6.8 28.0 6.0 94 5731
25B 2.232 6.8 28.0 6.0 94 5731
11A 38.592 N/A 31.0 5.63 104 48
11B 18.894 N/A 31.0 5.63 104 48
12A 36.818 N/A 31.0 5.63 104 48
12B 12.954 N/A 31.0 5.63 104 48
12
14A 10.876 N/A 31.0 5.63 104 48
14B 10.972 N/A 31.0 5.63 104 48
16A 8.566 N/A 31.0 5.63 104 48
16B 35.989 N/A 31.0 5.63 104 48
5A 94.313* N/A 31.0 5.87 622 408
5B 19.130 N/A 31.0 5.87 622 408
7A 34.482 N/A 31.0 5.87 622 408
7B 15 17.941 N/A 31.0 5.87 622 408
17A 16.253 N/A 31.0 5.87 622 408
17B 58.119* N/A 31.0 5.87 622 408
18A 10.363 N/A 31.0 5.87 622 408
 96

18B 13.938 N/A 31.0 5.87 622 408

21A 12.973 N/A 44.0 5.79 352 1708
21B 31.15 N/A 44.0 5.79 352 1708
22A 11.417 N/A 44.0 5.79 352 1708
22B 9.812 N/A 44.0 5.79 352 1708
18
3A 31.447 N/A 44.0 5.79 352 1708
3B 19.51 N/A 44.0 5.79 352 1708
4A 41.679 N/A 44.0 5.79 352 1708
4B 17.431 N/A 44.0 5.79 352 1708
Notes: N/A : not detected by portable device due to very low concentration and
limitation of the detection kits.”
* : bold values represent outliers

Table 4.13: Metal loss (avg) after removal of outliers at 0.5m depth

Period CRavg Grubbs’

MLavg (g) Dixon’s Test Box Plot
(month) (mm/yr) Test
3 3.739 0.118
6 12.917 0.273
9 13.154 0.179 No outliers No outliers No outliers
12 23.713 0.238 detected detected detected
15 38.853 0.297
18 24.379 0.147

Table 4.14: Metal loss (med) after removal of outliers at 0.5m depth

Period MLmed CRmed Dixon’s Grubbs

Box Plot
(month) (g) (mm/yr) Test Test
3 3.425 0.108
6 11.637 0.246
No
9 9.819 0.133 No outliers No outliers
outliers
12 23.847 0.239 detected detected
detected
15 25.368 0.194
18 22.210 0.134
 97

Table 4.15: Metal loss (max) after removal of outliers at 0.5 m depth

Period MLmax CRmax Dixon’s Grubbs’ Box

(month) (g) (mm/yr) Test Test Plot
3 6.111 0.193
6 21.813 0.461
No No
9 28.643 0.389
outliers outliers
12 38.592 0.388 detected detected
15 94.313 0.720 Outlier
18 41.679 0.251

Table 4.16: Metal loss (Avg) after removal of outliers at 1.0m depth

Period MLavg CRavg Dixon’s Grubbs’ Box

(month) (g) (mm/yr) Test Test Plot
3 3.409 0.108
6 6.411 0.136
No No No
9 8.285 0.112
outliers outliers outliers
12 19.702 0.198 detected detected detected
15 27.282 0.208
18 19.476 0.117

Table 4.17: Metal loss (med) after removal of outliers at 1.0m depth

Period MLmed CRmed Dixon’s Grubbs’ Box

(month) (g) (mm/yr) Test Test Plot
3 4.062 0.128
6 6.001 0.127
No No
9 7.814 0.106 No outliers
outliers outliers
12 15.924 0.160 detected
detected detected
15 18.536 0.142
18 18.471 0.110

Table 4.18: Metal loss (max) after removal of outliers at 1.0m depth

Period MLmax CRmax Dixon’s Grubbs’

Box Plot
(month) (g) (mm/yr) Test Test
3 4.643 0.147
6 9.616 0.145
No No No
9 15.280 0.207
outliers outliers outliers
12 36.818 0.370 detected detected detected
15 58.119 0.444
18 31.150 0.188
 98

Table 4.19: Metal loss (ML) and corrosion rate (CR) for after removal of outliers
for both depth (0.5m and 1.0 m)
Period ML CR ML CR ML CR
Site
(Month) (mean) (mean) (med) (med) (max) (max)

Sungai 3 3.574 0.113 4.062 0.128 6.111 0.193

Ular

Sungai 6 9.664 0.204 6.921 0.146 21.813 0.461

Ular

Sungai 9 10.719 0.145 9.171 0.124 28.643 0.389

Ular

Sungai 12 21.708 0.218 15.924 0.160 38.592 0.388

Ular

Sungai 15 33.067 0.253 18.536 0.142 94.313 0.720

Ular

Sungai 18 21.927 0.132 18.471 0.111 41.679 0.251

Ular

4.6 Corrosion Growth Pattern

The metal loss as a result of corrosion due to exposure of steel coupons to

underground environment up to 18 months period was analysed in order to
determine the power law pattern of the corrosion growth. Figure 4.3 to Figure 4.5
illustrate the corrosion growth pattern of metal loss (avg, med, max) against
exposure period. Almost all the metal loss data (avg, med, max) are fitted with the
power law pattern with R2 higher than 0.85. The average metal loss data and
maximum value show the power law with convex pattern (n > 1). While metal loss
for median show the power law concave pattern (n < 1). Based on the observation,
the higher n value, will give the higher the corrosion growth. This selected area is
considered as aggressive in regards to it corrosivity level and corrosion accelerate
over time. In the next section, the identification of the influence of soil parameters
towards the magnitude of corrosion (k) and corrosion growth pattern (n) is discussed
in detail.
 99

Figure 4.3: Plot of average metal loss against exposure period for Sungai Ular site

Figure 4.4: Plot of median metal loss against exposure period for Sungai Ular site
 100

Figure 4.5: Plot of maximum metal loss against exposure period for Sungai Ular site

4.7 Linear Correlation Analysis (LCA)

In general, linear correlation analysis (LCA) is utilized to examine the linear

relationship of corrosion parameters between two variables. The main purpose of
correlation analysis is to determine the variables that are closely related to corrosion.
Table 4.20 to Table 4.25 show the correlation coefficient results of Spearman‟s and
Pearson‟s tests between each corrosion parameter. The first observation collected
from the correlation table was the relative degree of influence exerted by each of the
predictor variable on the observed corrosion metal loss (avg, med, max). The
analysis indicates that there was a close correlation exhibited between the metal loss
(avg, med, max) and soil variables such as MC, Cl- and Res for both correlation
methods. From this result, it is possible to extract the key variables related to
corrosion metal loss by linear correlation. Since some of the variables were highly
correlated to each other, for example (Table 4.21), pH and moisture (r =0.843), high
co-linearity could yield large estimated variances and it is difficult to detect the
significant regression coefficients. Therefore, principal component analysis (PCA)
was conducted for more precise extraction of variables.
 101

Table 4.20: Spearman‟s rho correlation of metal loss (med) with soil parameters
Variables MED pH LOGchloride LOGSulfate LOGSulfide LOGresistivity moisture LOGorganic
MED 1 0.143 -0.257 0.371 -0.348 0.899 0.319 0.829
pH 0.143 1 0.600 0.429 -0.406 0.261 -0.783 0.543
-
LOGchloride 0.257 0.600 1 0.314 0.232 -0.116 -0.609 0.143
LOGSulfate 0.371 0.429 0.314 1 -0.203 0.174 0.116 0.714
- -
LOGSulfide 0.348 0.406 0.232 -0.203 1 -0.103 0.015 -0.551
LOGresistivity 0.899 0.261 -0.116 0.174 -0.103 1 0.000 0.667
-
Moisture 0.319 0.783 -0.609 0.116 0.015 0.000 1 0.058
LOGorganic 0.829 0.543 0.143 0.714 -0.551 0.667 0.058 1
Values in bold are different from 0 with a significance level alpha=0.05

101
 102

Table 4.21: Pearson‟s correlation of metal loss (med) with soil parameters
Variables MED pH LOGchloride LOGSulfate LOGSulfide LOGresistivity moisture LOGorganic
MED 1 0.138 -0.190 0.043 -0.493 0.798 0.151 0.747
pH 0.138 1 0.670 0.174 -0.373 0.171 -0.843 0.230
-
LOGchloride 0.190 0.670 1 0.458 0.230 0.110 -0.432 -0.093
LOGSulfate 0.043 0.174 0.458 1 0.036 0.526 0.060 0.375
- -
LOGSulfide 0.493 0.373 0.230 0.036 1 -0.112 0.135 -0.814
LOGresistivity 0.798 0.171 0.110 0.526 -0.112 1 0.068 0.573
-
Moisture 0.151 0.843 -0.432 0.060 0.135 0.068 1 0.176
LOGorganic 0.747 0.230 -0.093 0.375 -0.814 0.573 0.176 1
Values in bold are different from 0 with a significance level alpha=0.05

102
 103

Table 4.22: Spearman‟s rho correlation of metal loss (avg) with soil parameters
Variables AVG pH LOGchloride LOGSulfate LOGSulfide LOGresistivity moisture LOGorganic
AVG 1 0.143 -0.257 0.371 -0.348 0.899 0.319 0.829
pH 0.143 1 0.600 0.429 -0.406 0.261 -0.783 0.543
-
LOGchloride 0.257 0.600 1 0.314 0.232 -0.116 -0.609 0.143
LOGSulfate 0.371 0.429 0.314 1 -0.203 0.174 0.116 0.714
- -
LOGSulfide 0.348 0.406 0.232 -0.203 1 -0.103 0.015 -0.551
LOGresistivity 0.899 0.261 -0.116 0.174 -0.103 1 0.000 0.667
-
Moisture 0.319 0.783 -0.609 0.116 0.015 0.000 1 0.058
LOGorganic 0.829 0.543 0.143 0.714 -0.551 0.667 0.058 1
Values in bold are different from 0 with a significance level alpha=0.05

103
 104

Table 4.23: Pearson‟s correlation of metal loss (avg) with soil parameters
Variables AVG pH LOGchloride LOGSulfate LOGSulfide LOGresistivity moisture LOGorganic
AVG 1 0.128 -0.279 0.208 -0.526 0.849 0.078 0.778
pH 0.128 1 0.670 0.174 -0.373 0.171 -0.843 0.230
-
LOGchloride 0.279 0.670 1 0.458 0.230 0.110 -0.432 -0.093
LOGSulfate 0.208 0.174 0.458 1 0.036 0.526 0.060 0.375
- -
LOGSulfide 0.526 0.373 0.230 0.036 1 -0.112 0.135 -0.814
LOGresistivity 0.849 0.171 0.110 0.526 -0.112 1 0.068 0.573
-
Moisture 0.078 0.843 -0.432 0.060 0.135 0.068 1 0.176
LOGorganic 0.778 0.230 -0.093 0.375 -0.814 0.573 0.176 1
Values in bold are different from 0 with a significance level alpha=0.05

104
 105

Table 4.24: Spearman‟s rho correlation of metal loss (max) with soil parameters
Variables MAX pH LOGchloride LOGSulfate LOGSulfide LOGresistivity moisture LOGorganic
MAX 1 0.143 -0.257 0.371 -0.348 0.899 0.319 0.829
pH 0.143 1 0.600 0.429 -0.406 0.261 -0.783 0.543
-
LOGchloride 0.257 0.600 1 0.314 0.232 -0.116 -0.609 0.143
LOGSulfate 0.371 0.429 0.314 1 -0.203 0.174 0.116 0.714
- -
LOGSulfide 0.348 0.406 0.232 -0.203 1 -0.103 0.015 -0.551
LOGresistivity 0.899 0.261 -0.116 0.174 -0.103 1 0.000 0.667
-
Moisture 0.319 0.783 -0.609 0.116 0.015 0.000 1 0.058
LOGorganic 0.829 0.543 0.143 0.714 -0.551 0.667 0.058 1
Values in bold are different from 0 with a significance level alpha=0.05

105
 106

Table 4.25: Pearson‟s correlation of metal loss (max) with soil parameters
Variables MAX pH LOGchloride LOGSulfate LOGSulfide LOGresistivity moisture LOGorganic
MAX 1 0.204 -0.164 0.471 -0.504 0.867 -0.013 0.786
pH 0.204 1 0.670 0.174 -0.373 0.171 -0.843 0.230
-
LOGchloride 0.164 0.670 1 0.458 0.230 0.110 -0.432 -0.093
LOGSulfate 0.471 0.174 0.458 1 0.036 0.526 0.060 0.375
- -
LOGSulfide 0.504 0.373 0.230 0.036 1 -0.112 0.135 -0.814
LOGresistivity 0.867 0.171 0.110 0.526 -0.112 1 0.068 0.573
- -
Moisture 0.013 0.843 -0.432 0.060 0.135 0.068 1 0.176
LOGorganic 0.786 0.230 -0.093 0.375 -0.814 0.573 0.176 1
Values in bold are different from 0 with a significance level alpha=0.05

106
 107

4.8 Power Law Equation

A mathematical corrosion model can express the relation between the

quantitative of metal loss and the soil corrosion parameter. To achieve this, a
predictive corrosion model over time should be developed. Power law equation is
always used in modelling corrosion growth over time due to the tendency of the
corrosion rate behaviour to decrease over time (Li, 2003). Romanoff (1957)
highlighted that the value of k and n can be varied according to the soil parameters.
Therefore, it is possible to relate the value of k and n with the soil parameters.

P = Ktn (Eq 4.1)

4.9 Principal Component Analysis (PCA)

The main purpose of principal component analysis (PCA) is to extract the

most outstanding key variables correlated with corrosion metal loss (k) and time
factor (n), as can be found in power law equation. Even though the result of LCA
showed that there was a correlation between metal loss and some variables, the high
correlation between the independent variables may be uncertain for the extraction.
Therefore, the second approach of PCA can be used to extract the factors affecting
corrosion to a smaller set of uncorrelated variables more precisely. PCA was
performed on all soil parameters (Table 4.3) with metal loss data (Table 4.19) and
corrosion growth pattern (Figure 4.3 to Figure 4.5). Soil parameters that have a
strong influence on k would affect the metal loss, whereas an influence on n would
contribute to the corrosion growth pattern as time passes. The relationship between
the variables for metal loss (k) and time factor (n) is graphically shown in Figure 4.6
to Figure 4.11.

The two components were extracted based on the eigenvalue>1, which was
considered to be highly influential on metal loss and time function. Component 1
 108

(Comp.1) was selected since the eigenvalue was higher than component 2 (Comp.2).
Higher eigenvalue represents high loading of all variables that retained the maximum
amount of information about the variables. The result for all corrosion metal loss
data shows that Sulfide (S2-) and Organic content (OC) fell under component 1
(Comp.1) for metal loss, k while Chloride (Cl-), pH and moisture content (MC) has
more influence towards time function, n. This result indicates that these five factors
appeared to be the controlling factors on metal loss (k) and corrosion growth pattern
(n). Table 4.26 lists the influence of soil parameters towards metal loss (k) and time
factor (n) for all types of metal loss data.

Figure 4.6: Extracted component of soil variables for metal loss (med data)
Results: k = LogSulfide, LogOrganic
 109

Figure 4.7: Extracted component of soil variables for time factor (med data)
Results: n= pH, LogChloride, moisture content

Figure 4.8: Extracted component of soil variables for metal loss (avg data)
Results: k = LogSulfide, LogOrganic
 110

Figure 4.9: Extracted Component of soil variables for time factor (avg data)
Results: n= pH, LogChloride, moisture content

Figure 4.10: Extracted component of soil variables for metal loss (max data)
Results: k= LogSulfide, LogOrganic
 111

Figure 4.11: Extracted component of soil variables for time factor (max data)
Results: n= pH, LogChloride, moisture content

Table 4.26: k and n of power law model according to average, median and
maximum data
Data Metal Loss (k) Time factor (n)

Average
pH, LogChloride,
LogSulfide,
Median Moisture content
LogOrganic
(MC)
Max

4.10 Multiple Linear Regressions (MLR)

Multiple regression analysis was adopted to study the influence of the

extracted soil parameters with metal loss (k) and corrosion growth pattern (n). The
significant parameters extracted from the PCA results were used in the multiple
regression analysis. The multiple regressions were carried out via the „Enter‟ method
to obtain the linear regression equation for both constant k and n. In order to discover
the best model to predict the corrosion metal loss as a function of time, five
 112

combinations of influential factors (S2-, OC, Cl-, pH, MC) were carried out for k and
n (Table 4.27 to Table 4.30). The coefficient of determination (R2), F-test and t-stat
results from the regression analysis were used to interpret the linear model of k and n
as shown in Table 4.31 to Table 4.33.

The results for R2 showed that the linear equation incorporating S2-, OC, Cl-,
pH, MC parameters yield the highest value of R2 compared to other models. The
result of p-value (<0.05) for F-test indicates that the linear equation is highly
significant to k and n. The contributions of in-situ parameters on k and n can be
estimated by observing the t-stat significant value (p-value) of individual coefficient.
A low p-value (< 0.05) means that the particular parameter is essential in predicting
the corrosion rate. Based on the t-stat results, only a single parameter appeared to
have superior dominance upon metal loss (k) and corrosion growth pattern (n). The
linear equations of k and n for each category of metal loss data (avg, med, max)
obtained from regression analysis were then used to yield the predicted model. The
development of the on-site soil corrosion model is presented in the next section.
 113

Table 4.27: Possible combination of k and n constant

Combination of k and n
n = pH, LogCL, MC
k = LogS, LogORG
n= pH,logCL,MC
k= LogS
n= pH,logCL,MC
k= LogORG
n= pH,LogCL
k= LogS, LogORG
n= pH, MC
k= LogS, LogORG
n= LogCL, MC
k= LogS, LogORG
n= pH
k= LogS, LogORG
n= LogCL
k= LogS, LogORG
n= MC
k= LogS, LogORG
n= pH
k= LogS
n= LogCL
k= LogS
n= MC
k= LogS
n= pH
k= LogORG
n= LogCL
k= LogORG
n= MC
k= LogORG
 114

Table 4.28: Multiple regression analysis for constant k and n (average metal loss
data)
Model Combination of k and n
n = -2.50660+0.64560*pH-
A 0.19646*LOGchloride+0.02975*moisture
k = 167.87356-52.34693*Sulfide-15.41588*LOGorganic
n = -2.50660+0.64560*pH-
B 0.19646*LOGchloride+0.02975*moisture
k = 80.65740-42.01686*LOGSulfide
n = -2.50660+0.64560*pH-
C 0.19646*LOGchloride+0.02975*moisture
k = -151.63677+36.40039*LOGorganic
n = -0.10612+0.23298*pH-0.11160*LOGchloride
D k = 167.87356-52.34693*LOGSulfide-
15.41588*LOGorganic
n = -1.83104+0.40155*pH+0.02203*moisture
E k = 167.87356-52.34693*LOGSulfide-
15.41588*LOGorganic
n = 0.53656+0.01076*LOGchloride-0.00214*moisture
F k = 167.87356-52.34693*LOGSulfide-
15.41588*LOGorganic
n = -0.07264+0.14538*pH
G k = 167.87356-52.34693*LOGSulfide-
15.41588*LOGorganic
n = 0.43243+0.02164*LOGchloride
H k = 167.87356-52.34693*LOGSulfide-
15.41588*LOGorganic
n = 0.57981-0.00254*moisture
I k = 167.87356-52.34693*LOGSulfide-
15.41588*LOGorganic
n = -0.07264+0.14538*pH
J
k = 80.65740-42.01686*LOGSulfide
n = 0.43243+0.02164*LOGchloride
K
k = 80.65740-42.01686*LOGSulfide
n = 0.57981-0.00254*moisture
L
k = 80.65740-42.01686*LOGSulfide
n = -0.07264+0.14538*pH
M
k = -151.63677+36.40039*LOGorganic
n = 0.43243+0.02164*LOGchloride
N
k = -151.63677+36.40039*LOGorganic
n = 0.57981-0.00254*moisture
O
k = -151.63677+36.40039*LOGorganic
 115

Table 4.29: Multiple regression analysis for constant k and n (median metal loss
data)
Model Combination of k and n
n = -2.50660+0.64560*pH-
P 0.19646*LOGchloride+0.02975*moisture
k = 92.18702-31.66593*LOGSulfide-7.01091*LOGorganic
n = -2.50660+0.64560*pH-
Q 0.19646*LOGchloride+0.02975*moisture
k = 52.52241-26.96796*LOGSulfide
n = -2.50660+0.64560*pH-
R 0.19646*LOGchloride+0.02975*moisture
k = -101.09251+24.33400*LOGorganic
n = -0.10612+0.23298*pH-0.11160*LOGchloride
S
k = 92.18702-31.66593*LOGSulfide-7.01091*LOGorganic
n = -1.83104+0.40155*pH+0.02203*moisture
T
k = 92.18702-31.66593*LOGSulfide-7.01091*LOGorganic
n = 0.53656+0.01076*LOGchloride-0.00214*moisture
U
k = 92.18702-31.66593*LOGSulfide-7.01091*LOGorganic
n = -0.07264+0.14538*pH
V
k = 92.18702-31.66593*LOGSulfide-7.01091*LOGorganic
n = 0.43243+0.02164*LOGchloride
W
k = 92.18702-31.66593*LOGSulfide-7.01091*LOGorganic
n = 0.57981-0.00254*moisture
X
k = 92.18702-31.66593*LOGSulfide-7.01091*LOGorganic
n = -0.07264+0.14538*pH
Y
k = 52.52241-26.96796*LOGSulfide
n = 0.43243+0.02164*LOGchloride
Z
k = 52.52241-26.96796*LOGSulfide
n = 0.57981-0.00254*moisture
AA
k = 52.52241-26.96796*LOGSulfide
n = -0.07264+0.14538*pH
AB
k = -101.09251+24.33400*LOGorganic
n = 0.43243+0.02164*LOGchloride
AC
k = -101.09251+24.33400*LOGorganic
n = 0.57981-0.00254*moisture
AD
k = -101.09251+24.33400*LOGorganic
 116

Table 4.30: Multiple regression analysis for constant k and n (maximum metal loss
data)
Model Combination of k and n
n = -2.50660+0.64560*pH-
0.19646*LOGchloride+0.02975*moisture
AE k = 603.64305-164.86611*LOGSulfide-65.97947*LOGorganic
n = 0.53656+0.01076*LOGchloride-0.00214*moisture
AF k = 603.64305-164.86611*LOGSulfide-65.97947*LOGorganic
n = -2.50660+0.64560*pH-
0.19646*LOGchloride+0.02975*moisture
AG k = -402.65132+97.21532*LOGorganic
n = -0.10612+0.23298*pH-0.11160*LOGchloride
AH k = 603.64305-164.86611*LOGSulfide-65.97947*LOGorganic
n = -1.83104+0.40155*pH+0.02203*moisture
AI k = 603.64305-164.86611*LOGSulfide-65.97947*LOGorganic
n = 0.53656+0.01076*LOGchloride-0.00214*moisture
AJ k = 603.64305-164.86611*LOGSulfide-65.97947*LOGorganic
n = -0.07264+0.14538*pH
AK k = 603.64305-164.86611*LOGSulfide-65.97947*LOGorganic
n = 0.43243+0.02164*LOGchloride
AL k = 603.64305-164.86611*LOGSulfide-65.97947*LOGorganic
n = 0.57981-0.00254*moisture
AM k = 603.64305-164.86611*LOGSulfide-65.97947*LOGorganic
n = -0.07264+0.14538*pH
AN k = 230.36069-120.65372*LOGSulfide
n = 0.43243+0.02164*LOGchloride
AO k = 230.36069-120.65372*LOGSulfide
n = 0.57981-0.00254*moisture
AP k = 230.36069-120.65372*LOGSulfide
n = -0.07264+0.14538*pH
AQ k = -402.65132+97.21532*LOGorganic
n = 0.43243+0.02164*LOGchloride
AR k = -402.65132+97.21532*LOGorganic
n = 0.57981-0.00254*moisture
AS k = -402.65132+97.21532*LOGorganic
 117

Table 4.31: Summary of R2 from the proposed model for average metal loss data
MODEL R2 (MLactual vs MLpredicted)
A 0.569
B 0.677
C 0.776
D 0.512
E 0.065
F 0.485
G 0.109
H 0.487
I 0.490
J 0.252
K 0.523
L 0.600
M 0.780
N 0.761
O 0.761

Table 4.32: Summary of R2 from the proposed model for median metal loss data
MODEL R2 (MLactual vs MLpredicted)
P 0.560
Q 0.652
R 0.870
S 0.475
T 0.580
U 0.483
V 0.590
W 0.491
X 0.478
Y 0.593
Z 0.587
AA 0.574
AB 0.850
AC 0.861
AD 0.850
 118

Table 4.33: Summary of R2 from the proposed model for maximum metal loss data
MODEL R2 (MLactual vs MLpredicted)
AE 0.350
AF 0.660
AG 0.360
AH 0.332
AI 0.348
AJ 0.314
AK 0.332
AL 0.313
AM 0.312
AN 0.515
AO 0.011
AP 0.490
AQ 0.691
AR 0.128
AS 0.666

4.11 Development of On-Site Corrosion Model

Based on the above analysis, a site corrosion model was developed. Power
law equation (P = ktn) was utilized in order to find the best model to predict metal
loss in MIC soil environment. There are 45 combinations of linear equation for k and
n for all metal loss data (avg, med and max) as tabulated in Table 4.28-4.30. The
model was chosen based on the value of the coefficient (R2). The R2 with the highest
value near 1.0 was considered as the best predictive model. The results showed that
not all 45 combinations of linear equation were able to predict the actual metal loss.
Some of the R2 values are too low such as 0.011. For all types of data, the R2 values
ranged from 0.011 to 0.87.

The R2 coefficient for average metal loss data ranged from 0.109 to 0.780,
0.475 to 0.870 for median metal loss data and 0.011 to 0.691 for maximum metal
loss data. From all 3 types of data, the median metal loss data showed the best fitting
to the model compared to the other 2 sets of data. The best combination of k and n
that produced the highest coefficient (R2) was found in the median metal loss data
 119

with R2 of 0.870 (Table 4.32). The R2 of 0.870 means that about 87% of the site
corrosion rate is able to be expressed by the model. The result showed that the metal
loss (k) was influenced by the organic content. Meanwhile, the corrosion growth
pattern (n) was primarily influenced by the value pH value, chloride content and
moisture content. Table 4.34 shows the results of the predicted vs. experimental
median metal loss data. A perfect line was plotted to observe how close the predicted
metal loss compared to the actual value of the metal loss from the site experiment
data as show in the Figure 4.12. The metal loss (k) and corrosion growth pattern (n)
can be expressed as a linear equation as follows:

k = -101.09251 + 24.334*LogOrganic (4.1)

n = -2.50660 + 0.64560*pH - 0.19646*LogChloride + 0.02975*moisture (4.2)

Where;
k = the calculated metal loss value
Log Organic = the value of organic content in ppm
n = is the calculated value
pH = is the value of pH
Log Chloride = is the value of chloride in ppm
Moisture = the value of moisture content in %

Table 4.34: Predicted metal loss data from the proposed model compared with
experimental result
n = -2.5066 + Predicted Percentage
Time k = -101.092 + 0.6456*pH - Metal Experimental Error (%)
(year) 24.33*LogOrg 0.19646*LogChl+ Loss result
n
0.02975*moisture (P=kt )
0.25 5.716 0.191 4.387 4.062 7.4%
0.50 11.158 0.301 9.059 6.921 23.5%
0.75 14.411 0.687 11.827 9.171 11.8%
1.00 12.650 0.603 12.650 15.924 11.3%
1.25 16.060 0.614 18.419 18.536 0.63%
1.50 13.172 0.555 16.498 18.471 11.8%
 120

20
18

Actual Metal Loss(g)

16
14
12
10
8
6
4
2
0
0 5 10 15 20
Predicted Metal Loss (g)

Figure 4.12: Experimental metal (Actual) loss vs. predicted metal

loss

4.12 Conclusion

This chapter presents the analysis of metal loss data against the site soil
parameters such as soil‟s pH value, chloride concentration, sulfide content, moisture
content and organic content. The first analysis was successfully conducted to screen
and remove the outlier, which may affect the final result. The second analysis was
carried out to identify the effect of the buried depth towards the metal loss data.
From this, it can be concluded that neither the buried depth of 0.5 m nor 1.0 m has
significantly affected the metal loss data. Power law patterns were observed from the
data of metal loss over time for all types of data (average, median and maximum).
Further analysis has found that the median metal loss data gives the most suitable
result in developing the site corrosion model. PCA analysis was utilized in order to
develop the metal loss model using power law equation. The corrosion of metal loss,
k was found to be related to the organic content, whereas the corrosion growth
pattern was influenced by the pH value, chloride content and moisture content. This
combination will give the best R2 of the model, with the value of R2 of 0.870.
 121

The site is categorized as corrosive based on the classification using AWWA

and Nazim assessment method. The corrosiveness of Sungai Ular site is a combined
effect of many factors such as pH value, chloride concentration, sulfide content,
moisture content, resistivity and organic content. Some of the parameter can be
linked to the SRB activities (i.e: pH, Sulfide, sulfate, organic content) and some are
not (i.e: chloride, resistivity). The result from the model and PCA clearly indicates
that organic content and sulfide; which has direct link to the existence of SRB
underground; are the most influential parameters on metal loss (k) whilst pH has
better governance of metal loss pattern (n). This proves that the high severe pitting
recorded by pigging inspection and high corrosion rate measured on site is highly
likely influenced by SRB. Because the nature of the field work study, it is difficult to
identify the contribution of each parameter individually. Another approach known as
one-factor-at-a-time (OFAT) is required to study individually the influence of
environmental parameters towards bacterial growth and its effect on metal loss.
 122

CHAPTER 5

ANALYSIS OF CORROSION PARAMETERS

5.1 Introduction

This chapter delivers the analysis of laboratory work, identification of the

bacteria, the bacteria growth curve profile, OFAT study, SEM images of bio-films
and corrosion, Tafel slope testing and corrosion analysis. The results from this
section will be utilised to identify the range of pH value, ferum, sulfate and
temperature for modelling the metal loss subject to SRB activities using Response
Surface Method analysis in the Chapter 6.

5.2 Chemical and Microstructure Analysis of Carbon Steel Coupons

A 36 inch diameter pipe was cut to coupon size and the chemical
composition of the coupon was analysed from the original segment of carbon steel
pipes with the specification number API 5L X-70. Table 5.1 shows the chemical
composition of the carbon steel coupon using GDS technique. The details of the
result can be found in Appendix A (Table A-5). The microstructure of the carbon
steel coupon was examined using optical micrograph. The image of the material‟s
microstructure was obtained after polishing and etching with 2% of nital reagent.
Figure 5.1 shows the optical micrograph of the carbon steel coupon API 5L-X70.
The image revealed a mixed ferrite microstructure and dispersion of small carbides
on the coupon.
 123

Table 5.1: Chemical components of carbon steel coupon for API 5L X-70 using
GDS technique
C Mn Si P S Cr Ni Mo V Ti Nb Cu Fe
0.06 1.05 0.27 0.006 0.002 0.02 0.02 0.008 0.05 0.02 0.05 0.24 Bal

Figure 5.1: Microstructure of carbon steel coupon under microscope

5.3 SRB Bacteria

This experiment was conducted to confirm the presence of SRB at Sungai

Ular site as suspected by the pipeline operator. In previous study on Chapter 4, no
solid evidence of the presence of SRB can be made. The previous study can only
give the value on the soil corrosiveness. Therefore this study was conducted to
identify the presence of SRB at Sungai Ular site. Suspected SRB samples on the
coupon surface were collected from the site and then inoculated with the prepared
liquid medium. The bacteria were regrown several times before proceeding to the
next step. In the early step of identification, the presence of SRB was detected based
on visual observation after an incubation period of 7 days in Baar‟s liquid medium.
The medium turned black after an incubation period of 3 to 4 days at 37°C, as shown
in Figure 5.2.
 124

Figure 5.2: Sani SRB detection kit

5.3.1 SRB Isolation

The presence of SRB bacteria was detected based on observation. After 7

days, the medium turned to black. From all 5 selective mixed-cultures isolated, only
3 samples showed the presence of SRB (from SRB kit). All 3 detected Sungai Ular
SRB samples showed rapid growth in liquid medium. The samples also showed
positive result for Sani SRB test kit from Bio Sani-Check product #100 (Sani
CheckTM, Biosan Lab Inc., USA) as show in Figure 5.2. The black precipitate was
indicative of iron sulfide (FeS) as a reduction of sulfate (SO42-) to sulfide (S2-). The
Sungai Ular SRB was grown on solid agar under anaerobic condition. Some of the
surfaces of the agar turned black, making it easy to visualize the SRB colonies
(Figure 5.3). A strong smell of rotten eggs was detected, which indicated the
production of H2S as a result of sulfide-reducing activity by SRB. Microorganism is
frequently found as a mixed culture in their natural habitat. Most cells survived by
adhering to a surface after they are covered with a layer of polysaccharide or
normally known as biofilms. In the biofilms, microorganisms develop their own
 125

micro-environment, in which their own species could resist harsh conditions

(Abdullah at el., 2014). The morphologies of the isolated Sungai Ular SRB revealed
that their features are related to Desulfovibrio species based on the above results.

Figure 5.3: Colony of Sungai Ular SRB grown on agar medium after 7 days of
incubation at 37°C anaerobically

5.4 Characterization of SRB from Sungai Ular

In this part, the SRB sample was further analysed using DNA method. From
this identification, it can be determined if the quality of the sample is good and the
bacteria are successfully isolated.

5.4.1 DNA Amplification and Sequencing

Three strains of SRB bacteria were successfully purified from 5 pools of

mixed SRB screened using the SRB kits. The strains were named SgU 1, SgU 2 and
SgU 3 respectively . DNA extraction was conducted as prescribed in Section 3.7 in
order to obtain the genome DNA for amplification procedure. Figure 5.4 and 5.5
show the 16S rDNA fragment for of purified plasmid DNA and of purified PCR
product respectively. Figures 5.4 and 5.5 show the 16S rDNA fragment for a purified
plasmid DNA and a purified PCR product respectively. Figure 5.4 show genome
 126

DNA was successfully purified from samples SgU1 and SgU2. The purified PCR
product in Figure 5.5 shows the corresponding group of bacteria. Nevertheless, the
purified 16s rDNA fragments was only successfully recovered from SgU1 sample.

Figure 5.4: Agarose gel electrophoresis of purified plasmid DNA for SRB SgU 1
(line 1), SgU 2 (line 2) and SgU3 (line 3)

Figure 5.5: Agarose gel electrophoresis of purified PCR product 16S rDNA for SRB
SgU 1 (line 1), SgU 2 (line 2) and SgU 3 (line 3)
 127

5.4.2 16s rDNA Sequence Analysis

Partial sequence of 16S rDNA fragments was only detected for the Sungai
Ular 1 (SgU1) sample out of all 3 samples submitted with the labels namely Sungai
Ular 1, Sungai Ular 2 and Sungai Ular 3 samples. This might due to the low quality
of the samples during the PCR product extraction process. The PCR product of SgU
1 showed a good quality sequence data. Figure 5.6 shows the 16SrDNA sequence for
Sungai Ular A. Colony morphology of bacteria:

Figure 5.6: Partial 16SrDNA sequence of Sungai Ular 1 strain using BLASTN
analysis
 128

The identification of Sungai Ular 1 strain was confirmed by 16s rDNA partial
sequencing. The closest matches for the strain were Desulfovibrio vulgaris (98%)
and Desulfovibrio (100%). Figure 5.7 shows the phylogenetic tree diagram based on
16SrDNA gene sequences indicating the position of Sungai Ular 1 strain among the
closely related members. The topology shown is a rooted tree obtained with
neighbour-joining algorithm, in which the bootstrap value expresses the percentage
of 1000 replication. The phylogenetic analysis was conducted using MEGA 5.2.

Figure 5.7: Phylogenetic dendrogram based on 16SrDNA gene sequences indicating

the position of Sungai Ular A strain among closely related members

Desulfovibrio sp. strain can be found in many literatures, especially in

research related to SRB and MIC. Boyle et al. (1999) successfully isolated
Desulfovibrio sp. TBP-1 from marine sediments in New York harbour estuary.
Meeta et al. (2010) also successfully isolated Desulfovibrio sp. from the crude oil at
Mumbai offshore. The bacteria is a gram-negative, vibrio-shaped and requires
anaerobic condition, which grows in different carbon source substrates including
lactate, pyruvate, glycerol, ethanol, hydrogen and fumarate with optimal growth
temperatures between 25°C and 40°C (Leu 1999; Madigan, 2009). When the
bacteria have sufficient sulfate as an electron acceptor, it becomes sulfate-reducing
bacteria. These bacteria had been reported to play a significant role in oil field
reservoir souring by generating hydrogen sulfide (Leu et al., 1999).

For Malaysian environment, Desulfovibrio sp. strain has been successfully

isolated by previous researches (Sahrani et al., 2008). This SRB was collected from
the seawater near the harbour in Pasir Gudang, Johor. In the study, VMNI medium
 129

was utilized to grow the SRB. From the isolation, they also found other genus of
SRB species, namely citrobacter. This type of SRB has also been reported to have
the ability to reduce sulfate (Barton, 1995).

5.5 Bacteria Growth Curve

From the previous section (Sec 5.4) the SRB bacteria was successfully
identified. As mentioned in the research problem, the corrosiveness performance of
local strain SRB may be difference with commercially available SRB (available in
the market). Therefore, in this section, the isolated from Sungai Ular SRB was
compare with ATCC7757TM in order to investigate whether there are significantly
different intensities between SRB strains and environmental parameters. In fact, both
bacteria share similar DNA structure. The bacteria growth curve is one of the
important element in study the bio-corrosion. In this study, optimum SRB‟s activity
with the limited amount of medium available need to be measured and analysed. The
incubation period of 7 days up to 60 days was determined by other researchers in the
literature in various MIC study such as weight loss method, open circuit potential
and electrochemical study (refer section 3.12.3). The SRB growth curve was
obtained by utilizing 100 mL of medium in 125 mL anaerobic vials. 2 mL of bacteria
seed (2-3 days old estimated ± 1×107) was added to the vials. Bacterial enumeration
was obtained as mentioned in Chapter 3.

Figure 5.8 illustrates the concentration of sessile bacteria growth incubated at

37°C for the ATCC7757TM and isolated Sg. Ular SRB. The cultures were grown in
anaerobic vials and the enumeration was conducted by withdrawing from the
anaerobic vials. The cell enumeration for both types of SRB showed a similar
increased pattern started from the first day of incubation period until day 14. The
exponential growth phase (1-14 days) was observed in both test cultures. The
maximum value of bacterial enumeration was observed on day 14 with 2.5×108
cell/mL for ATCC7757TM culture, whereas 2.0×108 cell/mL was observed on day 14
and 21 for the isolated Sungai Ular SRB. The number of bacteria concentration
began to decrease significantly thereafter. After 30 days of incubation, the cell
 130

numbers of bacteria were measured at less than 1.0×108 cell/mL for both SRB.
Since the experiments were conducted in batch unit, the bacteria growth decreased
after a certain period due to the increase of toxicity of sulfate (FeS or H2S) towards
SRB metabolism. In the real environment, SRB may not be exposed to sulfate
toxicity due to the open environment and flow of the fluid. The bacterial metabolic
activities may also have developed resistance to toxic agents; hence sustain the
growth of bacteria. The growth of SRB in the limited containing culture medium
showed three stages of SRB growth, namely exponential phase, steady phase and
dead phase.

Figure 5.8: Growth curve of SRB bacteria incubated at 37°C for 60 days in
anaerobic vials

5.6 Open Circuit Potential

OCP is one of the electrochemical studies that can be used to measure the
corrosion potential. Figure 5.9 shows the result of open circuit corrosion potential,
where (Ecorr) is measured again using a standard saturated calomel electrode in
culture solution. Ecorr as a function of time data has shown that SRB growth had
substantially shifted Ecorr to a more positive value. This shift of Ecorr value is known
 131

as ennoblement. The potential shift clearly supports the activity and growth of SRB
in the culture medium by enhancing the redox quality of the medium and accelerates
iron dissolution. The SRB attached on the coupon surface to develop biofilms and
active metabolism and altered the electrochemical process on the coupon surface.
The positive shift of Ecorr as a function of time was observed from Figure 5.9 for
both bacteria. The electrochemical changes associated with a reduction in resistance
of metals are referred as ennoblement. The ennoblement has been attributed by the
bacteria colonization and biofilms formation, which resulted in organometallic
catalysis and acidification of the electrode surface. The ennoblement also promotes
localized corrosion such as pitting corrosion due to the transpassive dissolution of
the passive film (Cetin and Aksu, 2009). Figure 5.9 also revealed the ennoblement
phenomenon whereby the finding is similar with other researchers such as Johnsen
and Bradal (1985), Little and Lee (2007) and Cetin and Aksu (2009).

Figure 5.9: Plots of open circuit potential for carbon steel with Sungai Ular and
ATCC 7757TM SRB
 132

5.7 Corrosion by Isolated SRB

The corrosion rate of the carbon steel coupons inoculated with and without
SRB was calculated as described in the weight loss procedure in chapter 3 is shown
in Table 5.2. The values of corrosion rate for both inoculated medium showed a
similar trend with higher corrosion rate observed compared to the control medium,
which proves the influence of SRB activity in the inoculated medium. Higher
corrosion rate values were observed in both inoculated vials certainly due to H2S/S2
produced by the SRB activity. Iron sulfide precipitated on the surface of the coupons
and formed a passive film. EDS analysis detected iron sulfide in the corrosion
products at the coupons surface for both types of SRB.

Table 5.2: Comparison of corrosion rate for ATCC 7757TM and isolated SRB
(Sungai Ular) inoculated at 37°C for 30 days

ATCC 7757TM Corrosion rate Sungai Ular Corrosion rate

SRB’s Bacteria (mm/yr) SRB’s Bacteria (mm/yr)
Control (without 0.0407 Control (without 0.0546
bacteria) bacteria)
Inoculated 0.2530 Inoculated 0.2017
% of CR between % of CR between
83.91% 72.93%
control vs inoculated control vs inoculated

Figure 5.10 illustrates the polarization curves (PC) for both SRB strains
inoculated in the medium for the incubation period of 7 and 21 days. Corrosion rate
of ATCC7757TM and Sungai Ular SRB strains has increased by 108.0 % and 211.0 %
respectively from 7 days to 21 days of incubation period. From the extrapolation of
the Tafel slope, corrosion current density (Icorr), corrosion rates, anodic (βa) and
cathodic (βc) for SRB in ATCC1249‟s medium were computed and presented in
Table 5.3. At the initial stage of polarization curves testing on day 7, the surface of
the carbon steel coupon might not yet fully covered with thick biofilms and
corrosion products. This could contribute to the low corrosion rate recorded (SRB
activity still did not influence the corrosion rate). Higher corrosion rate was recorded
on day 21 for both SRB strains. This could be attributed by the SRB activity that
 133

produced hydrogen sulfide (H2S) as a secondary metabolite, where H2S can easily be
diluted in the medium to corrode the carbon steel. This is an important proof to point
out that the electrochemical corrosion behaviour of carbon steel is influenced mainly
by the redox quality on the biofilm, in which the redox quality mainly depends on
the SRB metabolism activity.

Figure 5.10: Tafel plot from GPES software for ATCC 7757TM and Sungai Ular
SRB for 7 and 21 days of incubation period

Table 5.3: Corrosion parameters obtained from Tafel plots for ATCC 7757TM and
Sungai Ular strains for 7 and 21 days of exposure

Days/ SRB Icorr Corrosion Rate Βa Βc

(Acm-2) (mmpy) (V/dec) (V/dec)
7 ATCC 7757TM 1.036E-5 0.1204 0.280 0.060
Sungai Ular 9.385E-6 0.1090 0.949 0.095
21 ATCC 7757TM 2.218E-5 0.2500 0.029 0.036
Sungai Ular 5.800E-6 0.3390 0.121 0.21
 134

5.8 Microscopic Observation

Figures 5.11 to Figure 5.14 show the FESEM images of corrosion products
and biofilms developed on the carbon steel coupons exposed to Sungai Ular and
ATCC 7757TM strains for 30 days. Figure 5.11 and Figure 5.12 represent Sungai Ular
SRB strain whereas Figure 5.13 and Figure 5.14 represent ATCC 7757TM SRB strain.
For both images in Figures 5.11 and 5.13 shows a typical rod-shaped Desulfovibrio
sp. SRB cells were observed with approximate size of 1.0-2.0 µm. FESEM
observations were conducted to prove the adhesion of SRB on the carbon steel
coupon and also to verify any contamination to the medium. From the FESEM
images, a layer of biofilms was distinctly observed on the entire coupon surface that
may consist of bacteria cells, extracellular polymeric substance (EPS) and also
corrosion products.

Figure 5.11: FESEM images of the biofilms and SRB incubated at 37°C for 30 days
for isolated Sungai Ular
 135

Figure 5.12: FESEM images of the biofilms incubated at 37°C for 30 days for
isolated Sungai Ular SRB
 136

Figure 5.13: FESEM images of the biofilms and SRB incubated at 37°C for 30 days
for ATCC7757TM SRB
 137

Figure 5.14: FESEM images of the biofilms incubated at 37°C for 30 days for
ATCC7757TM SRB

After the carbon steel samples were cleaned, surface analysis was carried out
again to observe the corrosion morphology of the coupons. Figure 5.15 shows some
of corrosion morphology for carbon steel coupons exposed to ATCC7757TM and
Sungai Ular SRB. The morphology of the corrosion process was determined as
localized and pitting corrosion. Figure 5.16 shows the comparison between the
control coupons (a) with the coupons exposed to SRB (b) after 30 days of
experiment at 37°C. Figure 5.16 (a) shows no existence of pitting corrosion whereas
Figure 5.16 (b) indicates the initiation of pitting corrosion at the entire coupon
surface. Figure 5.17 (a) and (b) shows the biofilm for ATCC7757TM and Sungai Ular
respectively. High density and homogeneous thickness of biofilm could be observed
on the coupon surface at shown in Figure 5.17
 138

Figure 5.15: SEM micrographs of the morphology of the corrosion for a)

ATCC7757TM and b) Sungai Ular SRB

Figure 5.16: SEM images for (a) control coupon and (b) coupon with Sungai Ular
SRB after removal of biofilms for 30 days of inoculation at 37°C

Figure 5.17: SEM images of biofilms for ATCC7757TM (a) and Sungai Ular SRB (b)
inoculated at 37°C observed after 30 days
 139

Figure 5.18: EDS analysis of the corrosion products on the coupon surface after
exposure to SRB for 30 days

Figure 5.18 revealed the presence of the corrosion products, cell spores and
EPS fibres distributed over the coupon. The analysis of the corrosion products on the
coupon surface revealed extensively large S and Fe peaks. An oxygen peak was also
revealed from the analysis due to the exposure to oxygen during the handling
process. An iron sulfide layer was formed on the metal surface where Fe2+ reacted
with hydrogen sulfide produced by SRB metabolism (Xu et al., 2011). Surface
analysis showed pitting corrosion morphologies on the carbon steel coupon
inoculated with isolated SRB. EDS analysis supported the finding with high peak of
Fe and S due to the presence of iron sulfide as shown in the Figure 5.18. Figure 5.19
show the comparison ESD results for ATCC7757TM (a) and Sungai Ular SRB (b).
from the figure similar location of peak were detected for C, S and Fe. While Au was
detected in the in Sungai Ular EDS result. This Au might come from the gold coating
process conducted before the analysis.
 140

Figure 5.19: EDS analysis of biofilms for ATCC7757TM (A) and Sungai Ular SRB
bacteria (B) inoculated at 37°C for 30 days

5.9 Screening the Optimum Parameter Range Using One-Factor-at-a-Time

(OFAT) Method

From the previous section, the comparison between isolated and commercially
available ATCC7757TM SRB strain was conducted. But the interaction between the
conducted parameter upon SRB corrosion is still not inclusive. Further analysis need
to be conducted to compare between this two SRB strains. Therefore the RSM
models need to be developed. Moreover, the identification of appropriate value and
range of the selected parameter need to be conducted. Study on the medium
conditions for SRB was conducted using one-factor- at-a-time (OFAT). This study
was conducted to determine the maximum corrosion rate (CR) from the SRB
cultured in the selected parameter and the appropriate range values. From the OFAT
study, the optimum value for each parameter were determined and later utilized in
the RSM model. The selected parameters involved in OFAT study are incubation
temperature, pH value of the medium, SO4-2 concentration and initial iron (Fe2+)
concentration. Only ATCC7757TM bacteria was used in this study and test matrix to
summary of the OFAT study was illustrated in Table 3.9 in Chapter 3.
 141

5.9.1 Effect of Incubation Temperature on Corrosion Rate in Bio-corrosion

Bacterial growth is very sensitive to incubation temperature. Some SRB

bacteria can tolerate a huge temperature difference while some species require
specific temperature to grow healthily. Most SRB species are mesophiles and they
will grow well in the temperature range between 5°C to 50°C (Javaherdashti et al.,
1999). In order to study the effect of incubation temperature influenced by
ATCC7757TM SRB bacteria, the experiments were carried out at 5°C, 20°C, 30°C,
400C , 500C and 60°C for an incubation period of 30 days. The result of the
experiment is shown in Figure 5.20. While Table 5.4 show the result of OFAT
experiment conducted for Sungai Ular SRB conducted by Ismail (2015).

Figure 5.20: Corrosion rate (mm/yr) of the carbon steel coupons incubated with
ATCC7757TM at 5°C, 20°C, 30°C, 400C, 50 0C and 60°C

Table 5.4: CR of OFAT study for Sungai Ular SRB incubated at different
temperature for 30 days by Ismail (2015).
Temperature 00C 200C 370C 600C
ML(g) 0.11 0.023 0.026 0.023
CR(mm/yr) 0.086 0.178 0.199 0.180
 142

4°C 50°C
20°C 40°C

30°C

Figure 5.21: Tafel plot of OFAT for different incubation temperatures for 14 days

Table 5.5: Summary of Tafel plot of OFAT for different incubation temperatures for
14 days
Parameter Ecorr Icorr βa βc Corrosion
Value (V vs. (µ ACm-2) (V/dec) (V/dec) Rate (mm/yr)
(°C) SCE)
4 -0.610 1.631×10-8 0.034 0.073 0.00019
20 -0.801 5.5×10-7 0.028 0.018 0.00639
30 -0.912 2.13×10-6 0.165 0.037 0.0248
40 -0.853 2.79×10-6 0.039 0.053 0.0325
50 -0.825 1.149×10-6 0.003 0.007 0.0133

Figure 5.21 shows the Tafel plot of OFAT for different incubation
temperature of 14 days. From the extrapolation of the Tafel plot, corrosion potentials
(Ecorr), corrosion current densities (Icorr), corrosion rates, anodic (βa) and cathodic (βc)
of the Tafel slopes were computed and the corresponding values are given in Table
5.5.

The temperature value influenced the corrosion potentials (Ecorr), corrosion

current densities (Icorr) and the parameters fluctuated from 4°C to 50°C. The highest
value of Icorr of 2.79×10-6 was obtained at 40°C and the lowest value of 1.631×10-8
was obtained from the lowest temperature, 4°C. Ecorr shifted slightly to the anodic
direction from -0.610 V vs. SCE for the temperature value of 4°C to -0.825 V vs.
SCE of 50°C. The Tafel anodic was obtained from the regions with the slopes
ranging between 0.034 V/dec and 0.003 V/dec, where for the cathodic regions, the
 143

slope ranging from 0.073 V/dec to 0.007 V/dec. The most suitable temperature
growth for both SRB can be concluded at 370C. At a very low temperature (00C), the
bacteria cannot grow and this affected the CR result. The range of 20 0C to 40 0C for
temperature parameter is considered appropriate for the bacteria to grow and have
significant effect on corrosion rate. Based on CR result from Figure 5.20 the
maximum CR was observed at temperature 300C – 400C from the weight loss
method. This high pattern of CR also has been observed in the Tafel plot experiment
Table 5.4 where the CR was recorded maximum at 400C. This finding also supported
by finding from Ismail, 2015 as show in Table 5.4, where 370C is the maximum CR
recorded for Sungai Ular SRB.

5.9.2 Effect of Medium pH on Corrosion Rate in Bio-corrosion

Figure 5.22 shows image of the anaerobic vials in OFAT study at different
pH values. The images show the colour of the medium turn to black at pH 5.5, 7 and
8.5 only. Figure 5.23 shows the CR result from OFAT study for pH value ranging
between 4.0 -10.0. It is clearly seen that CR for all pH value recorded low for control
sample compared with CR with SRB inoculated. The maximum CR was recorded at
pH 7.0 while the lowest CR was recorded at pH 5.5.

Figure 5.22: Anaerobic vials experiments conducted at different pH

 144

Figure 5.23: OFAT for pH value incubated at 37°C for ATCC7757TM SRB and
control sample

Table 5.6 show the OFAT result for Sungai Ular SRB inoculated at different
pH conducted by Ismail 2015. The maximum CR was recorded at pH was 7.5.
followed by pH 9.5. The Tafel plot study for OFAT at various pH values is also
presented in Figure 5.24. From the extrapolation of the Tafel plot, corrosion
potentials (Ecorr), corrosion current densities (Icorr), corrosion rates, anodic (βa) and
cathodic (βc) Tafel slopes for different pH values incubated at 37°C for 14 days were
computed and the corresponding values are given in Table 5.7.

Table 5.6: CR of OFAT study for Sungai Ular SRB incubated at different pH for 30
days by (Ismail, 2015)

pH 5.5 6.5 7.5 8.5 9.5

ML(g) 0.026 0.028 0.037 0.029 0.035
CR (mm/yr) 0.205 0.218 0.295 0.230 0.280

Based on the data shown in Table 5.7, the pH value influenced the corrosion
potentials (Ecorr), corrosion current densities (Icorr) and the parameters fluctuated due
to the different values of pH. The highest value of Icorr of 4.80×10-5 was obtained at
pH 7.0 and the lowest value was obtained at the highest pH of 10. Ecorr shifted
slightly to the anodic direction from -0.669 V for pH 4 to -0.825 V of pH 5.5 and
then slowly shifted back to the cathodic direction from -0.825 V for pH 5.5 to -0.755
 145

V of pH 8.5. Ecorr shifted drastically to the anodic direction with the lowest value of -
0.586 V for pH 10 and showed that the corrosion energetically favoured higher
values of pH (shifted to cathodic values). The Tafel anodic was obtained from the
regions with the slopes ranging between 0.269 V/dec and 0.023 V/dec, whereas
0.047 V/dec to 0.031 V/dec were the ranges for cathodic region slopes. Based on the
weight lost experiment for ATCC7757TM and finding from Ismail (2015) for Sungai
Ular SRB pH 7.5 was selected as the centre value for that to be used in the RSM
experiment where the suggested optimal value of pH is between 6.5 and 8.5 to gain
maximum CR from the study. This pH value also aligned with the result from Tafel
plot as per Table 5.7.

pH 8.5
pH 4 pH 7
pH 10
pH 5.5

Figure 5.24: Tafel plot of OFAT for different pH values incubated at 37°C for 14
days

Table 5.7: Summary of the Tafel plot of OFAT for different pH values incubated at
37°C for 14 days
Parameter Ecorr Icorr βa βc Corrosion
Value (V vs. (µ ACm-2) (V/dec) (V/dec) Rate (mm/yr)
(pH) SCE)
4 -0.699 V 1.55×10-6 0.269 0.047 0.0180
5.5 -0.825 V 1.15×10-6 0.003 0.007 0.0133
7.0 -0.756 V 4.80×10-5 0.165 0.041 0.0550
8.5 -0.755 V 4.11×10 -5 0.235 0.025 0.0477
10 -0.586 V 1.35×10-8 0.023 0.031 0.00015
 146

5.9.3 Effect of SO42- Concentration on Corrosion Rate in Bio-corrosion

Figure 5.25 show the result of OFAT experiment conducted for

ATCC7757TM for SO42- at 370C. The experiment was conducted with a control
experiment. The control experiment (downward diagonal) show a low CR for all
concentrations compared with the experiment conducted with SRB (solid fill). From
the result, the highest CR was recorded at 2.0 g/l of SO42- concentration followed by
CR from the 1.0g/L as illustrated from the figure. At 3.0 g/l of sulfate concentration,
the CR recorded much lower as compared to both 1.0 and 2.0 g/L concentration.
This pattern was also identified from the Tafel plot result.

Figure 5.25: OFAT for SO42- concentration incubated at 37°C for 30 days
 147

5.0 g/L

4.0 g/L 2.0 g/L

3.0 g/L

Figure 5.26: Tafel plot of OFAT at different initial sulfate concentrations incubated
at 37°C for 14 days

Table 5.8: Summary of the Tafel plot of OFAT for different initial sulfate
concentrations incubated at 37°C for 14 days
Parameter Ecorr Icorr βa βc Corrosion
Value SO42- (V vs. SCE) (µ ACm-2) (V/dec) (V/dec) Rate (mm/yr)
(g/L)
2.0 -0.912 2.13×10-6 0.165 0.037 0.2480
3.0 -0.961 8.18×10-6 0.071 0.042 0.0950
4.0 -0.820 7.293×10-7 0.503 0.038 0.00847
5.0 -0.582 1.51×10-8 0.032 0.033 0.0017

The Tafel plot of OFAT for different initial sulfate concentrations incubated
for 14 days is shown in Figure 5.26. Based on the data shown in Table 5.8, the Ecorr
value increased slightly from -0.912 V (SCE) for the concentration of 2.0 g/L to the
value of -0.582 V (SCE) for 5.0 g/L. The highest value of Icorr of 8.18×10-6 was
obtained at the concentration of 3.0 g/L and the lowest value was obtained from the
highest concentration value of 5.0 g/L. Based on the result from the Figure 5.25, the
maximum CR was obtain at the sulfate concentration at 1.0g/L – 2.0g/L. This result
also show a similar pattern in the Tafel plot experimental result in Table 5.8 where
the highest CR was recorded at sulfate concentration 2.0g/L.
 148

5.9.4 Effect of Fe2+ Concentration on Corrosion Rate in Bio-corrosion

Figure 5.27 show the result of weight loss study for OFAT at various Fe2+
concentration. Based on the result from Figure 5.27 the highest CR obtain at 100ppm
followed by 200ppm of Fe2+ concentration. The lowest CR recorded at the Fe2+
concentration is 0 ppm (control test).

Figure 5.27: OFAT for Fe2+ concentration incubated at 37°C for 30 days

Tafel plot for OFAT at different initial Fe2+ concentrations incubated for 14
days is shown in Figure 5.28. Based on the data shown in Table 5.9, the Ecorr value
decreased from -0.624 V (SCE) for the concentration of 0 ppm to the value of -0.612
V (SCE) for 300 ppm and Ecorr shifted slightly in the cathodic direction. The highest
value of Icorr of 4.69e-5 µACm-2 was obtained at the concentration of 50 ppm and the
lowest value, 1.83e-8 µ ACm-2 was obtained from the concentration of 300 ppm.
 149

300 ppm
O ppm 50 ppm

100 ppm

Figure 5.28: Tafel plot of OFAT at different initial Fe2+ concentrations incubated at
37°C for 14 days

Table 5.9: Summary of the Tafel plot of OFAT at different initial Fe2+
concentrations incubated at 37°C for 14 days
Parameter Ecorr Icorr βa βc Corrosion
Value (V vs. (µ ACm-2) (V/dec) (V/dec) Rate (mm/yr)
(ppm) SCE)
0 -0.624 2.17×10-8 0.043 0.091 0.0025
50 -0.622 4.69×10-5 0.214 0.042 0.1540
100 -0.906 9.41×10-6 0.078 0.058 0.1094
300 -0.612 1.83×10-8 0.039 0.063 0.0021

From the weight loss experiment, the maximum CR was obtained from the 100ppm
of Fe2+ concentration. While result from the Tafel plot experiment show 50ppm
concentration of Fe2+ give the highest CR followed by Fe2+ value of 150ppm.
Therefore the concentration of Fe2+ between 50 ppm to 100 ppm was selected as a
range value that to be used in the RSM modelling.
 150

5.10 Conclusion

This chapter has presented the results from the isolated SRB strain and it
growth curve. It was then compared with growth curve of the pure strain of
ATCC7757TM bacteria. From the growth curve, the suitable incubation period was
recorded at 30 days. The isolated Sungai Ular and ATCC7757TM was then further
analysed using morphology technique in identified the corrosion product and to
images the bio-films. Afterward, analysis of experimental study related to SRB
growth and their influence parameters towards metal was performed using OFAT
approach. This approach was conducted to identify the influence of stipulated
parameters on metal loss over time subject to different SRB strains. The results show
that all the parameters give significant influence toward the corrosion rate subject to
SRB. From the OFAT approach, the appropriate value for each parameter was
successfully identified. Parameter pH indicates that the suitable range for CR should
be between 6.5 and 8.5. In order to obtain the highest CR under SRB influence, the
temperature range is suggested between 20 0C and 40 0C. SO42- concentration was
selected between 1.0 to 2.0 g/L and the Fe2+ parameter from 50 to 100 ppm.
 151

CHAPTER 6

RESPONSE SURFACE METHOD ANALYSIS

6.1 Introduction

In this chapter, statistical analysis was conducted using Statistica 8.0

software for isolated SRB strains from Sungai Ular site and ATCC7757TM. In this
analysis, the range of parameters for the study was designed according to the OFAT
study in the previous chapter. Based on the result of OFAT method, several factors
and corresponding range of the optimized values were selected, and further
evaluation was conducted. The selected variables from OFAT study were pH value,
temperature (°C), SO42- concentration (g/L) and initial iron (Fe2+) concentration
(ppm) obtained using central composite design (CCD). The range and coded level of
the parameters are listed in Table 6.1. CCD factorial design allows the estimation of
the regression between parameters. The response of the experiments is corrosion rate
upon the studied parameters. The design was made up of full 24 factorial as shown in
Table 6.1. The star point was set to alpha (α) of 1.682 from the coded centre point.
Each independent variable was studied at five different coded levels (-α, -1, 0, +1,
+α).
 152

Table 6.1: Experimental range and coded level of independent variables

Range and Level
Factors Symbol
-1 0 +1

pH of the medium (pH) X1 6.5 7.5 8.5

Incubation temperature (°C) X2 20 30 40
SO4-2 concentration (g/L) X3 1.0 1.5 2.0
Initial Fe2+ concentration X4 50 75 100
(ppm)

6.2 Response Surface Methodology for Sungai Ular Isolated SRB

Response surface methodology was employed to determine the interaction

between the studied parameters and their response (corrosion rate). A number of 30
experimental runs were conducted in the study of corrosion rate influenced by SRB
for selected parameters. Each run was conducted at least 3 times to obtain more
accurate results. Figure 6.1 shows the image of the weight loss experiment
conducted in anaerobic vials for 30 days of incubation period. From the experimental
result, the lowest corrosion rate recorded was 1.23 mm/yr, whereas the highest
corrosion rate recorded was 6.21 mm/yr. The results from RSM study are given in
Table 6.2. The design of experiments, actual experimental results and predicted
responses from the model are tabulated in the table. The predicted quadratic model
between the studied parameters with corrosion rate is shown in the predicted
mathematical model as follows:

Y = -43.5365 + 10.3892X1 + 0.3006X2 + 2.505X3 + 0.0492X4 – 0.6342X12

– 0.0056X22 + 0.2798X32 – 0.0003X42 + 0.0004X1X2 – 0.05912X1X3
– 0.0016X1X4 + 0.0484X2X3 + 0.0002X2X4 + 0.0013X3X4
(6.1)
 153

Figure 6.1: Images of the weight loss experiment conducted in anaerobic vials at
different studied parameters

The fitness of the model from the experimental result and predicted values
can be verified using the value of coefficient of determination (R2). Using Statistica
version 8.0 software, the value of R2 was found to be 0.704. This R2 value means
that approximately 30% of the total variations did not fit the proposed model,
whereas 70% out of the total variations fit and was justified by the model. The
obtained R2 value showed that the model is marginally acceptable and reliable.
However, this R2 value is less than the minimum acceptable R2 value of 0.75 (Wan,
et al., 2011). Nevertheless, the obtained R2 value is still not far from the acceptable
R2 value. The lower R2 value might come from the error of the experiment, such as
from the manual weight loss experiment and unsuitable range of parameters. This
experimental response might also be due to microorganism activities, where some
researchers used R2 as low as 0.70 when dealing with microorganism experiments
(Zainol, 2008).
 154

Table 6.2: Experimental design, experimental results and predicted responses using
response surface methodology for Sungai Ular isolated SRB
Coded Values (Variable Values) Actual Predicted
Run A B C D Values Values
(pH) (°C) (g/L) (ppm) (mm/yr) (mm/yr)
1 7.5 30 1.5 75 2.2854 4.2660
2 8.5 40 2.0 50 2.9609 3.5713
3 8.5 40 1.0 50 3.0532 3.2525
4 7.5 10 1.5 75 1.2304 0.9513
5 7.5 30 1.5 25 4.3033 3.5961
6 8.5 40 2.0 100 2.9767 3.5041
7 6.5 20 1.0 100 2.4050 2.1712
8 7.5 30 1.5 75 6.2126 4.2660
9 8.5 40 1.0 100 3.1859 3.1225
10 7.5 30 0.5 75 4.0233 4.0885
11 6.5 40 2.0 100 4.575 4.4075
12 6.5 20 1.0 50 2.397 2.3313
13 9.5 30 1.5 75 2.2044 1.4899
14 7.5 30 1.5 75 4.9087 4.2660
15 8.5 20 2.0 100 1.4365 1.8487
16 6.5 20 2.0 50 2.4243 2.8642
17 8.5 20 1.0 100 2.4659 2.4353
18 6.5 40 2.0 50 3.8217 4.3141
19 6.5 40 1.0 50 2.8486 2.8100
20 7.5 30 2.5 75 5.9065 5.0030
21 6.5 40 1.0 100 2.4499 2.8436
22 7.5 50 1.5 75 3.6475 3.0883
23 7.5 30 1.5 75 4.2542 4.2660
24 8.5 20 1.0 50 2.212 2.7560
25 8.5 20 2.0 50 2.0386 2.1066
26 7.5 30 1.5 75 3.9601 4.2660
27 7.5 30 1.5 75 3.9747 4.2660
28 7.5 30 1.5 125 3.4998 3.3688
29 6.5 20 2.0 100 2.5045 2.7669
30 5.5 30 1.5 75 2.0923 1.9686

To further justify the model, ANOVA test was conducted by analysis of the
variance. The results of the analysis are presented in Table 6.3. This mathematical
model was tested with 5% significance level. From the calculation, it was found that
the value of the calculated F-value was higher compared to the F-value from the
 155

distribution table. This positive value showed that the model was justified and
significant (Fauzi et al., 2013).

Table 6.3: ANOVA summary for CR of Sungai Ular SRB inoculated at selected
parameters

Sum of Degree of Mean F-Value

F-Value
Sources Squares Freedom Square (tabulated)
(calculated)
(SS) (d.f) (MS) (α= 0.05)

Regression (SSR) 29.6829 14 2.1202

Residual (R) 12.4720 15 0.8314 2.550 2.420
Total (SST) 42.1549 29

The correlation between the values from experiments versus predicted values
is presented by the parity plot in Figure 6.2. From this figure, the predicted and
actual values were plotted and it is shown that the model is marginally acceptable,
with R2 = 0.704. Figure 6.3 illustrates the Pareto chart for corrosion rate of SRB
inoculated with isolated Sungai Ular SRB. This chart presents an analysis in
identifying the relative importance of the regression coefficients for the obtained
mathematical model. From the figure, it can be proven that pH value has the largest
effect towards the corrosion rate with a negative value (-3.642).
 156

Observed vs. Predicted Values

4 factors, 1 Blocks, 30 Runs; MS Residual=.8314664
DV: Exp
5.5

5.0

4.5

4.0
Predicted Values

3.5

3.0

2.5

2.0

1.5

1.0

0.5
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0

Observed Values

Figure 6.2: Parity plot for corrosion rate of Sungai Ular isolated SRB inoculated at
selected parameters

Pareto Chart of Standardized Effects; Variable: Exp

4 factors, 1 Blocks, 30 Runs; MS Residual=.8314664
DV: Exp

pH(Q) -3.64246
Temp(Q) -3.22519
(2)Temp(L) 2.87037
1Lby3L -1.29663
(3)g/L(L) 1.22833
ppm(Q) -1.12507
2Lby3L 1.061773
(1)pH(L) -.642942
g/L(Q) .4017782
(4)ppm(L) -.30532
2Lby4L .209218
1Lby4L -.176153
3Lby4L .0688986
1Lby2L .0162582

p=.05

Standardized Effect Estimate (Absolute Value)

Figure 6.3: Pareto chart for corrosion rate of Sungai Ular isolated SRB inoculated at
selected parameters
 157

6.3 Response Surface Plots for Sungai Ular Isolated SRB

The graphical figures from RSM that represent the interactions between
independent variables and corrosion rate are shown in Figure 6.4 to 6.9. Figure 6.4
illustrates the corrosion rate of Sungai Ular isolated SRB as a function of pH and
temperature. The response increased with increasing parameters. Beyond
approximately pH of 7.5 and temperature of 40°C, the response started to decrease
gradually. The corrosion rate of Sungai Ular isolated SRB for different values of pH
and SO42- concentration is presented in Figure 6.5. Conducting experiments at pH
lower than 6.0 or higher than 9.5 resulted in lower response. Meanwhile, there would
be not much difference towards the response when conducting experiments at any
SO42- concentration, as long as the range of pH is between 7.0 and 8.0.

Figure 6.4: Response surface plot as a function of pH and temperature for corrosion
rate of Sungai Ular isolated SRB
 158

Figure 6.5: Response surface plot as a function of pH and SO4-2 concentration for
corrosion rate of Sungai Ular isolated SRB

The influence of pH and initial Fe2+ concentration on the corrosion rate of

Sungai Ular isolated SRB is depicted in Figure 6.6. The response would remain at
approximately 4.0 mm/yr and above when conducting experiments for the values of
pH between 6.0 and 8.5 regardless of the values of initial Fe2+ concentration. As
shown in Figure 6.7 for the plot of the corrosion rate of Sungai Ular isolated SRB as
a function of temperature and SO42- concentration, conducting experiments at
temperatures lower than 15°C would give lower corrosion rate regardless of the
values of SO42- concentration. Hence, at temperatures lower than 15°C would give
lower CR.
 159

Figure 6.6: Response surface plot as a function of pH and initial Fe2+ concentration
for corrosion rate of Sungai Ular isolated SRB

Figure 6.7: Response surface plot as a function of temperature and SO4-2

concentration for corrosion rate of Sungai Ular isolated SRB
 160

Figure 6.8 presents the response surface plot of the corrosion rate of Sungai
Ular isolated SRB as a function of temperature and initial Fe2+ concentration. The
response increased as the temperature increased, reached optimum at approximately
30°C and decreased afterwards. However, there would be not much increase of the
response as initial Fe2+ concentration varies at different temperatures. The corrosion
rate of Sungai Ular isolated SRB as a function of SO4-2 concentration and initial Fe2+
concentration is plotted in Figure 6.9. From the figure, response that is lower than
3.0 mm/yr would only be achieved if experiments are conducted at SO4-2
concentration of lower than 1.4 g/L.

Figure 6.8: Response surface plot as a function of temperature and initial Fe2+
concentration for corrosion rate of Sungai Ular isolated SRB
 161

Figure 6.9: Response surface plot as a function of SO4-2 concentration and initial
Fe2+ concentration for corrosion rate of Sungai Ular isolated SRB

6.4 Response Surface Methodology for ATCC7757TM SRB

Response surface methodology was also employed to determine the

interaction between the studied parameters and their response (CR) for
ATCC7757TM SRB. A total of 30 experimental runs were conducted in the study of
corrosion rate influenced by ATCC7757TM on selected parameters. Each run was
conducted at least 3 times to obtain more accurate result. From the experimental
result, the lowest corrosion rate recorded was 2.1 mm/yr, whereas the highest
corrosion rate recorded was 5.57 mm/yr. The results from RSM study are given in
Table 6.4. The design of experiments, actual experimental results and predicted
responses from the model are tabulated in the table. The predicted quadratic model
between the studied parameters with corrosion rate is shown in the predicted
mathematical model as follows:
 162

Y = -21.5525 + 3.4X1 + 0.6024X2 + 4.9907X3 – 0.0032X4 – 0.2013X12 –

0.0047X22 – 0.1596X32 – 0.0003X42 – 0.0145X1X2 – 0.6003X1X3 +
0.0128X1X4 – 0.0173X2X3 – 0.0019X2X4 + 0.008X3X4
(6.2)

The fitness of the model from the experimental result and predicted values
can be verified using the value of coefficient of determination (R2). Using Statistica
version 8.0 software, the value of R2 was found to be 0.853. This means that 85% of
the total variations fit with the proposed model, whereas the remaining 15% of the
total variations did not fit and were not well explained by the model. The obtained
R2 value of 0.853 showed that the model is acceptable and reliable. This value is
higher than the minimal acceptable R2 value of 0.75 (Wan et al., 2011).
 163

Table 6.4: Experimental designs, experimental results and predicted responses using
RSM for ATCC7757TM

Coded Values (Variable Values) Actual Predicted

Run A B C D Values Values
(pH) (°C) (g/L) (ppm) (mm/yr) (mm/yr)
1 7.5 30 1.5 75 4.337 4.5123
2 8.5 40 2.0 50 3.880 3.6614
3 8.5 40 1.0 50 4.256 4.5423
4 7.5 10 1.5 75 2.100 1.7690
5 7.5 30 1.5 25 3.860 3.7920
6 8.5 40 2.0 100 3.330 3.4412
7 6.5 20 1.0 100 2.110 2.5848
8 7.5 30 1.5 75 5.570 4.5123
9 8.5 40 1.0 100 4.010 3.9212
10 7.5 30 0.5 75 4.505 4.2601
11 6.5 40 2.0 100 3.589 3.6872
12 6.5 20 1.0 50 2.589 2.5743
13 9.5 30 1.5 75 3.775 3.7125
14 7.5 30 1.5 75 5.021 4.5122
15 8.5 20 2.0 100 3.589 3.9862
16 6.5 20 2.0 50 2.890 3.2395
17 8.5 20 1.0 100 4.184 4.1209
18 6.5 40 2.0 50 5.023 5.1865
19 6.5 40 1.0 50 4.999 4.8667
20 7.5 30 2.5 75 4.567 4.4452
21 6.5 40 1.0 100 2.757 2.9667
22 7.5 50 1.5 75 3.552 3.5163
23 7.5 30 1.5 75 4.006 4.5123
24 8.5 20 1.0 50 2.664 2.8314
25 8.5 20 2.0 50 2.405 2.2960
26 7.5 30 1.5 75 4.155 4.5123
27 7.5 30 1.5 75 3.989 4.5123
28 7.5 30 1.5 125 3.885 3.5821
29 6.5 20 2.0 100 3.837 3.6507
30 5.5 30 1.5 75 4.005 3.7015

To further analyse the model, ANOVA test was conducted by analysis of the
variance. The results from the analysis are presented in Table 6.5. This mathematical
 164

model was tested with 5% significance level. From the calculation, it was found that
the value of the calculated F-value (6.247) was higher as compared to the F-value
(2.42) from the distribution table. This positive value shows that the model was
justified and significant (Fauzi et al., 2013).

Table 6.5: ANOVA summary of RSM for ATCC7757TM SRB inoculated at selected
parameters
Sum of Degree of Mean F-Value
F-Value
Sources Squares Freedom Square (tabulated)
(calculated)
(SS) (d.f) (MS) (α= 0.05)

Regression (SSR) 19.0660 14 1.3618

Residual (R) 3.2784 15 0.2185 6.2467 2.420
Total (SST) 22.3444 29

The correlation between the experimental results versus predicted values is

shown by the parity plots in Figure 6.10. From this figure, the predicted and actual
values were outlined and it is shown that the model is fairly acceptable, with R2 =
0.853. Figure 6.11 shows the Pareto chart for corrosion rate inoculated with
ATCC7757TM SRB. This chart presents an analysis in identifying the relative
importance of the regression coefficients for the obtained mathematical model.
From the figure, it can be proven that temperature has the largest effect towards the
corrosion rate with a negative value (-5.236).
 165

Observed vs. Predicted Values

4 factors, 1 Blocks, 30 Runs; MS Residual=.2185615
DV: Corrosion Rate (mm/yr)
6.0

5.5

5.0

4.5
Predicted Values

4.0

3.5

3.0

2.5

2.0

1.5

1.0
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

Observed Values

Figure 6.10: Parity plot for corrosion rate of ATCC7757TM SRB inoculated at
selected parameters
Pareto Chart of Standardized Effects; Variable: Corrosion Rate (mm/yr)
4 factors, 1 Blocks, 30 Runs; MS Residual=.2185615
DV: Corrosion Rate (mm/yr)

Temp (oC)(Q) -5.23629

(2)Temp (oC)(L) 4.577696
2Lby4L -4.08674
1Lby4L 2.735742
1Lby3L -2.56815
2+ -2.31132
Fe (ppm)(Q)
pH(Q) -2.2553
1Lby2L -1.244
3Lby4L .8572624
2Lby3L -.738761
(4)Fe 2+ (ppm)(L) -.549753
(3)SO42- (g/L)(L) .4849582
2- -.44708
SO4 (g/L)(Q)
(1)pH(L) .0290355

p=.05

Standardized Effect Estimate (Absolute Value)

Figure 6.11: Pareto chart for corrosion rate of ATCC7757TM SRB inoculated at
selected parameters
 166

6.5 Response Surface Plots for ATCC7757TM SRB

The graphical figures from RSM that represent the interactions between
independent variables and corrosion rate are shown in Figures 6.12 to 6.17. Figure
6.12 presents the response surface plot of the corrosion rate of ATCC7757 TM SRB as
a function of pH and temperature. It can be seen that lower response could be
achieved by conducting experiments lower than 40°C. Furthermore, there would be
not much difference for the response across different values of pH at any
temperature. According to the response surface plot of corrosion rate of
ATCC7757TM SRB as a function of pH and SO4-2 concentration (Figure 6.13),
conducting experiments at two regions would yield lower corrosion rate, which are
low pH and low SO4-2 concentration, as well as high pH and high SO4-2
concentration.

Figure 6.12: Response surface plot as a function of pH and temperature for

corrosion rate of ATCC7757TM SRB
 167

Figure 6.13: Response surface plot as a function of pH and SO4-2 concentration for
corrosion rate of ATCC7757TM SRB

The influence of pH and initial Fe2+ concentration on the corrosion rate of

ATCC7757TM SRB is presented in Figure 6.14. Increasing both parameters would
gradually yield lower corrosion rate. Moderate values of parameters would give high
response. The response surface plot of corrosion rate of ATCC7757TM SRB as a
function of temperature and SO4-2 concentration is shown in Figure 6.15. The
response increased for increasing temperature. Meanwhile, there would be not much
difference in the response for increasing SO4-2 concentration at any temperature. In
addition, conducting experiments at temperatures lower than 15°C would give lower
corrosion rate.
 168

Figure 6.14: Response surface plot as a function of pH and initial Fe2+

concentration for corrosion rate of ATCC7757TM SRB

Figure 6.15: Response surface plot as a function of temperature and SO4-2

concentration for corrosion rate of ATCC7757TM SRB
 169

Figure 6.16 shows the response surface plot of the corrosion rate of
ATCC7757TM SRB as a function of temperature and initial Fe2+ concentration. The
response increased as both parameters increase. It would be good to conduct
experiments at temperature less than 15°C and initial Fe2+ concentration of less than
80 ppm to obtain low corrosion rate. The response surface plot for corrosion rate of
ATCC7757TM SRB as a function of SO4-2 concentration and initial Fe2+
concentration is shown in Figure 6.17. The response decreased gradually as both
parameters increased. From the plot, conducting experiments at initial Fe2+
concentration higher than 120 ppm would be favourable to produce low corrosion
rate.

Figure 6.16: Response surface plot as a function of temperature and initial Fe2+
concentration for corrosion rate of ATCC7757TM SRB
 170

Figure 6.17: Response surface plot as a function of SO4-2 concentration and initial
Fe2+ concentration for corrosion rate of ATCC7757TM SRB

6.6 Conclusion

From the RSM model study for both SRB strains, the models were successful
developed with R2 0.70 for isolated Sg. Ular SRB and 0.85 for ATCC7757TM. For
Sungai Ular isolated SRB, the most influential parameter towards metal loss is pH
value, whereas incubation temperature was found to be more influential for the
ATCC7757TM. Both of these effects have always been associated with the bacteria
growth activity and their after effect. As a conclusion, the Response Surface
Methodology, involving an experimental design and regression analysis can be an
effective technique in finding the optimum point of CR and their effects on the
selected parameter. Both SRB strains showed different pattern and behaviour in
terms of the most influential parameter and corrosion rate. It seems that Sungai Ular
SRB is more aggressive as depicted by its high corrosion rate as opposed to
ATCC7757TM.
 171

CHAPTER 7

DISCUSSION

7.1 Introduction

This section is intended to elaborate the findings from the preceding chapters
through deep discussions on specific topics related to data collection, empirical
models, comparative study and parameters influence. The intention is to connect
findings from three different methodologies of field work, One-factor-at-a-time
(OFAT) and Response Surface Method (RSM). The content will demonstrate how
both empirical models complement each other in detailing the influence of SRB-
related parameters toward corrosion of buried pipeline at Sungai Ular site.

7.2 Field Work Approach

The 18-month period of field work conducted at Sungai Ular was designed to
produce data so as to model the metal loss under influence of soil corrosion
parameters. Among these parameters are a group of SRB-related parameters that can
be an indicator of possible SRB activities that causing the corrosion. Previous
investigation by pipeline operator has indicated that Sungai Ular was highly
corrosive according to pigging data. This was later affirmed by corrosion rate and
the intensity of parameters recorded at the site for a period of 18-month. Yet, there
 172

was no strong evidence to link the corrosion mechanism with SRB activities.
Further, this research has successfully isolated SRB strain from Sungai Ular proving
the possibilities of MIC mechanism. Nevertheless, field work data also revealed that
other parameters that have no strong relation with SRB such as Chloride and
moisture content may also dominate the high rate of corrosion on site. It is vital to
find the missing link so that high corrosivity of Sungai Ular can be scientifically
proven and associated with SRB activities. PCA and MLR models were then utilised
to find the missing link whereby SRB-related parameters of pH, sulfide and organic
content were found to be influential towards corrosion rate and corrosion growth
pattern.

7.2.1 Soil Corrosion Data

Sungai Ular site was selected based on the recommendation from pipeline
operator. They classified this site as SRB suspected area and very corrosive based on
high metal loss from pigging data record and site observation. Seven types of soil
parameters were measured throughout the 18 months of the site experiment. These
parameters are commonly related to the soil corrosiveness. The measured parameters
are pH, sulfate, chloride, sulfide, moisture content (MC), resistivity (Res) and
organic content (OC). Assessment of the site corrosiveness was conducted using
AWWA and soil corrosivity index as proposed by Nazim (2015). The results are
tabulated in Table 7.1.
 173

Table 7.1: Assessment of soil corrosiveness using AWWA and Nazim (2015)
corrosivity index
Time Score obtain Score obtain according
Parameter Measured Value
(Month) according AWWA Nazim (2015).
pH 4.1 0 Not Influence 3 H. Influence
Sulfate content (mg/kg) 1495 n/a n/a 6.5 Influence
Chloride (mg/kg) 2384 n/a n/a 4.5 Influence
3 Sulfide Content (mg/kg) 59 3.5 Influence 0 H. Influence
Moisture Content (%) 24 1 Influence 4.5 Influence
Resistivity (Ohm.cm) 94 10 H. Influence 0 H. Influence
Organic Content 24505 n/a n/a n/a n/a
pH 2.6 3 Influence 0 H. Influence
Sulfate content (mg/kg) 1802 n/a n/a 5 Influence
Chloride (mg/kg) 67 n/a n/a 10 Not influence
6 Sulfide Content (mg/kg) 37 3.5 Influence 0 H. Influence
Moisture Content (%) 50 1 Influence 5.5 Influence
Resistivity (Ohm.cm) 82 10 H. Influence 0 H. Influence
Organic Content 41011 n/a n/a n/a n/a
pH 4.8 0 Not Influence 3 H. Influence
Sulfate content (mg/kg) 2204 n/a n/a 5 Influence
Chloride (mg/kg) 5731 n/a n/a 2 H. Influence
9 Sulfide Content (mg/kg) 21 3.5 Influence 0 H. Influence
Moisture Content (%) 28 1 Influence 2 H. Influence
Resistivity (Ohm.cm) 94 10 H. Influence 0 H. Influence
Organic Content 55792 n/a n/a n/a n/a
pH 3.9 3 Influence 3 H. Influence
Sulfate content (mg/kg) 700 n/a n/a 6.5 Influence
Chloride (mg/kg) 48 n/a n/a 10 No Influence
12 Sulfide Content (mg/kg) 21 3.5 Influence 0 H. Influence
Moisture Content (%) 31 1 Influence 2 H. Influence
Resistivity (Ohm.cm) 104 10 H. Influence 0 H. Influence
Organic Content 47228 n/a n/a n/a n/a
pH 4.2 0 Not Influence 3 H. Influence
Sulfate content (mg/kg) 3198 n/a n/a 5 Influence
Chloride (mg/kg) 408 n/a n/a 10 No influence
15 Sulfide Content (mg/kg) 25 3.5 Influence 0 H. Influence
Moisture Content (%) 31 1 Influence 2 H. Influence
Resistivity (Ohm.cm) 622 10 H. Influence 0 H. Influence
Organic Content 65211 n/a n/a n/a n/a
pH 3.7 3 Influence 3 H. Influence
Sulfate content (mg/kg) 1708 n/a n/a 5 Influence
Chloride (mg/kg) 1700 n/a n/a 4.5 Influence
18 Sulfide Content (mg/kg) 39 3.5 Influence 0 H. Influence
Moisture Content (%) 44 1 Influence 5.5 Influence
Resistivity (Ohm.cm) 352 10 H. Influence 0 H. Influence
Organic Content 49621 n/a n/a n/a n/a

Soil corrosiveness may be dominated by a single factor or as combination

effect of few soil parameters. As shown in the aforementioned table, some of the soil
parameters such as resistivity and Sulfide content are categorized as highly
influential towards site corrosiveness. All other parameters are categorized to have
 174

minor-to-moderate influence on the site corrosiveness except organic content that is

not covered by both corrosivity indexes. Nevertheless, the result from the site
showed high organic content constantly recorded during all six trips to the site. The
finding from Othman (2015) stated that the organic content more than 23000 mg/kg
is considered corrosive. In theory, SRB utilizes the soil organic matter to carry out
reduction reaction in the soil. Therefore organic content also can be considered as
parameter contributes to the site corrosiveness.

Among all of the soil parameters that were found to be influential towards the
site corrosiveness, some of it such as sulfide content, organic content and pH can be
strongly linked to the presence of SRB. On the other hand, the parameters without
strong and direct connection to SRB such as chloride, moisture content and
resistivity were also a major contributor to the corrosion at Sungai Ular according to
corrosion index. Chloride has been previously reported by Wang et al. (2013) to
have some influence on SRB growth. The author described that suitable amounts of
chloride ions are beneficial to the growth of SRB, and lower concentrations will
inhibit them. Nevertheless, the author also mentioned that when the chloride ions
exceed 0.5wt%, the amounts of SRB remained stable with chloride ion
concentrations increasing, which indicates that the higher addition of chloride ions
have little influence on the SRB growth. Chloride can also be indirectly linked to
SRB through reduction of pH from alkaline to acidic environment. For consistency,
the parameters of pH, sulfide and resistivity are the highly influence parameters with
the most consistent reading throughout 18-month period.

This finding is in line with other researcher that had mentioned about pH
parameter as one of the indication of the SRB presence in that particular
environment. Previous study highlighted the influence of pH towards cell adhesion
of the bacteria (Sheng et al., 2007). SRB is reported active over wide range of pH
from acidic to alkaline. Suitable pH is crucial for bacteria growth as it affects
ionization and therefore the interaction of a myriad of molecular processes. In
addition, pH range plays a main role in the solubility of various substances that
bacteria need (Faletra et al., 2012).
 175

Colwell and Grimes (2000) also highlighted that the high amount of sulfide is
associated with the SRB activity. Theoretically, sulfide was produced from the
reaction of the bacteria consumption (Khur and Vlught, 1934). Roberge (2000)
highlighted that high organic content also is one of the indicator with high
microorganism activity including SRB.

7.2.2 Corrosion Growth Pattern

In this study, the power law equation was decided to be used as the basis to
our soil corrosion growth model. Based on the power law model, the corrosion
growth pattern can be explained by the n value (the exponential factor). The
exponential factor, n < 1 (convex) shows corrosion growth pattern is slowing down
over time. This might come from a number of factors such as formation of passivity
layer by corrosion product that prevents the corrosion progress. In contrast, the n > 1
(concave) indicate that the corrosion growth pattern is accelerating over time
representing extreme cases of metal loss due to reinitiation of corrosion mechanism
after the breakdown of passivity layer. This phenomenon can be linked to SRB
activity. From the field work results, Sungai Ular site was found to have n > 1
whereby SRB related parameters such as Sulfide, pH and organic content was found
highly influential on high corrosivity of the site based on AWWA and corrosivity
index by Nazim (2015).

The site located in swampy, closed to mangrove forest area, experiencing

constant change of water level due to low and high tides and rich with organic
content. This environment is found to be suitable for SRB growth (Parkes, et al.,
1989). The tides can bring the salinity and fresh medium to the bacteria and it also
can wash the corrosion product, hence change the tendency of corrosion pattern to n
> 1. This action might lead to the high corrosion growth rate. High organic content
in soil environment is also one of the features that have a direct relation to the SRB
activity (Wilmott and Jack, 2011). Overall, the results from field work at Sungai
Ular has confirmed the influence of SRB upon high corrosion rate and also reported
 176

severe pitting from pigging inspection. This conclusion can be justified since the
SRB related parameters of pH, sulfide and organic content fell in the corrosive and
highly corrosive categories. Moreover, the physical evidence from direct observation
on site can fortify the aforementioned claim.

7.2.2.1 Parameter Classification using PCA

The metal loss data obtained from the buried coupons were analysed using
principal component analysis (PCA). This technique is widely used in statistics,
signal processing and neural computing (Qiu et al., 2012) to reduce the number of
variables in database and to classify variables. PCA technique is one of the methods
available in analysing the data to find the connection between the data with the
proposed power low model. From the PCA analysis, it showed that the k value is
governed by sulfide and organic content (OC). While Chloride, pH and moisture
content has more influence towards time function, n. This demonstrated good
agreement with previous finding on soil parameter analysis (Table 7.1.), where the
pH, sulfide, chloride and organic content (OC) showed high influence toward the
corrosiveness of the site.

7.2.2.2 Multi Linear Regression Model

Based on the metal loss data, a corrosion model for the selected site was
proposed. The model was developed from Sungai Ular site with the selected soil
parameters from the statistical analysis. This site represents SRB rich-soil area. Prior
to corrosion modelling, the measured parameters were filtered using PCA whereby
five parameters; sulfide, organic content, pH, moisture content and chloride; have
been identified to have more influence on metal loss parameter of power law model,
k and n. To further the analysis, these five parameters were then used to develop the
 177

best multi linear regression model through different combinations. The best
combination of k and n that produced the highest coefficient (R2) was found in the
median metal loss data with R2 of 0.870. This model has organic content as the sole
parameter associated with metal loss, k. Meanwhile, pH, moisture content and
Chloride were found best fitted to the corrosion growth pattern, n. Most of the
aforementioned parameters are highly related to SRB activity. This finding has
confirmed that the site may experience high corrosion rate due to parameters related
to MIC and this statement is in line with the reported severe pitting on the pipeline
buried at Sungai Ular area. However there is no physical evident yet for the presence
of SRB at the site. Therefore it is crucial to carry out further test for the detection of
SRB.

The proposed empirical model can be technically used as a predictive tool to

calculate the potential metal loss over time experienced by buried steel structure
subject to MIC. The model can give an insight to pipeline operator on the level of
site corrosiveness due to SRB presence. Having said that, the model can only
developed using data collected from Sungai Ular site. Therefore, the accuracy of this
model is restricted to the environmental condition and soil structure that is similar to
Sungai Ular site. For a more comprehensive model, additional data must be required
from different sites with diverse soil parameters.

7.3 Laboratory Works

From the previous section, there is a need for physical proof of the presence
of SRB on site, hence laboratory works started with SRB detection using SRB kits,
followed by bacteria isolation and DNA identification. The identified SRB was
further tested with weight loss method, OCP, Tafel plot experiment and FESEM
images to study how the SRB influence the corrosion rate. Afterward, OFAT method
was employed to determine the suitable range of the stipulated parameters so that the
highest corrosion rate subject to optimum bacterial growth can be achieved. The
determined range was then used for RSM model development to find relationship
between tested parameter and metal loss.
 178

7.3.1 Bacteria Identification

The bacteria from the selected site were successfully detected using the SRB
kits. This showed that the collected soils samples from Sungai Ular indeed contained
the SRB bacteria strain. The collected SRB was then identified as Desulfovibrio
Vulgaris trough DNA identification. The ATCC7757TM bacteria are one of the most
commonly used commercially available bacteria for SRB corrosion testing. From the
DNA data, it was found that the ATCC7757TM have a similar DNA with the Sungai
Ular isolated SRB strain. A bacteria growth curve study was conducted for both
strains to determine their active period in the limited medium. The result showed a
similar pattern of active period of growth curve. From the OCP and weight loss
study, the results indicated that corrosion rate for both ATCC7757TM and Sungai
Ular SRB is much higher than the control sample.

7.3.2 Influence of Stipulated Parameters from OFAT Analysis

OFAT study was conducted to determine the suitable range of every single
stipulated parameter in order to simulate the optimum condition for bacterial growth
in corrosive environment. In the initial stage, the range for all four parameters (pH,
Temperature, SO42- and Fe2+) were selected based on literature review (Table 3.9).
The ATCC7757TM bacteria were used in the OFAT study to obtain the appropriate
range to be used in RSM modelling. The results were then compared with OFAT
analysis on Sungai Ular SRB done by Ismail (2015) since both strains share similar
DNA. Results show that the highest corrosion was recorded at 6.5 – 8.5 for pH, 20 –
40 0C for temperature, 1.0 - 2.0 gram/L for SO42- concentration and 50 to 100ppm
for Ferum. This range was then used as centre/ basis in deciding the range for the
RSM because OFAT is not able to determine the level of influence between each
parameter. The selected range was used for both strains based on the fact that both
strains share similar DNA. The selected range covers the recorded value of the
 179

parameters from OFAT analysis for both strains. For the purpose of comparative
study, the modelling of corrosion rate subject to SRB must use similar range so that
the level of influence of the stipulated parameters can be compared for both strains.

7.3.3. Influence of Stipulated Parameters from RSM Model

RSM modelling technique was utilised for both Sg. Ular and ATCC7757 TM
strains to determine the combine effect for all selected parameters towards corrosion
rate. The pH was found as the most influential parameter for Sg. Ular RSM model.
Meanwhile, the ATCC7757TM RSM model was highly influenced by temperature as
opposed to Sungai Ular model. The coefficient, R2 for Sg. Ular model is slightly
lower as compared to ATCC7757TM model. This is probably due the selected range
of parameters which may not be suitable for the Sungai Ular strain bacteria to grow
optimally and produce detrimental effect on corrosion rate. It was found that the
Sungai Ular SRB is more aggressive as compared to ATCC7757TM based on the
corrosion rate obtained in RSM study. This finding shows the importance of using
local SRB strain to estimate the corrosiveness for the site rather than using
commercially available bacterial strain such as ATCC7757TM as reference to assess
the site corrosiveness. This conclusion is drawn on the basis of dissimilarity of
corrosion rate (aggressiveness), influential parameter and correlation coefficient of
RSM model. The utilisation of local strain will give more accurate description of
what is happening underground. Hence, give better understanding on the effect of
SRB upon underground corrosion.

7.4 Relationship Between Field Work, OFAT and RSM Analysis

By referring to the outcome from three different methodologies of field work,

OFAT and RSM approaches, a distinct connection in regards to the performance of
Sungai Ular SRB strain towards high corrosivity at Sungai Ular site can be
constructed. The field work has revealed that pH, Sulfide, moisture content, organic
 180

content and chloride governs the high corrosivity of Sungai Ular by using PCA
analysis. The results were later refined using MLR model whereby the
aforementioned parameters were filtered statistically in the form of empirical model.
The MLR model achieved the best fit by incorporating pH, Chloride, moisture
content and organic content to predict the metal loss. Most of these filtered
parameters are directly related to SRB. The OFAT analysis and RSM model has
affirmed field work results when pH was identified as the most influential
parameters.

The pH can be defined as the strength of hydrogen ion (H+) measurement in

the concentration. The pH was classified as a physical factor and it is always
highlighted as a governing factor on the growth and development of organisms or
biological communities. Metabolism of SRB can be influenced by these physical
factors because microorganisms are sensitive to changes in the environment.
Previous researcher proved that SRB can cause a great drop in pH level due to
hydrogen Sulfide as a by-product of its metabolism (Yuzwa et al., 1991). Generally,
most SRB species grow best at pH ranging from 6.5 to 9.0. Hence, it is necessary to
maintain the pH level within its prescribed control limits to ensure the growth of
SRB.

7.5 Comparison between ATCC7757TM and Sungai Ular SRB Strains

The highest corrosion rate was recorded from isolated Sungai Ular SRB
strain at 6.21 mm/year as compared to the ATCC7757TM strain of 5.57 mm/year, a
difference of 11.4%. The lowest corrosion rate was recorded at 1.23 mm/year and
2.1 mm/year for Sungai Ular and ATCC7757TM strains respectively. According to
the bacteria growth curve as shown in Figure 5.8, the Sungai Ular SRB growth was
better and more consistent as compared to the ATCC7757TM for 60 days. The
bacteria growth in the limited containing culture medium showed three phases of
growth, namely exponential, steady phase and dead phase. The ATCC7757TM strain
achieved maximum growth on the day-14 as opposed to day-21 for Sungai Ular
strain. The ATCC7757TM bacteria numbers start to decrease rapidly from day-14 to
 181

day-42. In contrast, Sungai Ular strain demonstrated a longer steady phase period.
Beginning on day-21 until day-28, that bacteria growth was consistent and steady.
The decline of bacterial numbers (dead period) was only witnessed after day-28 until
day-35. This exponential and steady phase of growth behaviour might contribute
towards the higher corrosion rate recorded by the Sungai Ular strain. Since the
experiments were conducted in batch unit, the bacteria growth decreased after a
certain period due to the increase of toxicity of sulfate (FeS or H2S) towards SRB
metabolism. In the real environment, SRB may not be exposed to Sulfide toxicity
due to the open environment and flow of the fluid. (Cetin and Aksu, 2009).
 182

CHAPTER 8

CONCLUSION

8.1 Introduction

This research has demonstrated a comprehensive and detail investigation on

the influence of Sungai Ular SRB strain on external corrosion of underground
pipeline at Sungai Ular site. The research framework consists of field work and
laboratory experiment to produce two empirical models using MLR and RSM
approaches. The field work involved installation of 48 steel coupon underground for
metal loss measurement to develop the corrosion model. The OFAT approach was
conducted in the lab to determine the range of the stipulated parameters so that
bacteria can grow optimally to influence metal loss in the RSM analysis. These two
models were used to study the influence of environmental parameters towards
underground corrosion subject to local SRB strain. The results from field work are
supported by findings from OFAT and RSM analysis. The DNA analysis was carried
out to identify the exact species of Sungai Ular SRB strain.

8.2 Conclusion

1. Sungai Ular site was found corrosive according to corrosion parameters and
measured metal loss. The 18-month collection of data showed that the site
corrosiveness can be associated with SRB presence. The intensity of soil
parameters were referred to AWWA and corrosion index by Nazim (2015)
 183

whereby most of the parameters with high influence on corrosion are related to
SRB such as pH, Sulfide and organic content.

2. SRB strain was isolated from Sungai Ular soil sample to prove the presence of
SRB at Sungai Ular site that may cause MIC on the underground pipeline.
DNA analysis has confirmed that both strains of Sungai Ular SRB and
ATCC7757TM are from the same species of Desulfovibrio vulgaris. A
comparative study has shown that even though both strains share similar DNA,
the bacteria growth pattern and level of aggressiveness in corrosive
environment is rather different.

3. Open circuit potential (OCP), Tafel slope and weight-loss method (OFAT
approach) has confirmed that pH, sulfate content, ferum content and
temperature plays important roles in the growth of SRB. The optimum value
for each parameter has been successfully identified together with the suitable
range of value for RSM purposes.

4. MLR model has been successfully developed using metal loss data and soil
parameters recorded over 18-month period with a very good correlation value.
The predictive models assisted by PCA analysis has confirmed that the
corrosion at Sungai Ular was highly governed by SRB related parameters such
as organic content, sulfide and pH. Moreover, RSM model for Sungai Ular has
identified pH as the most influential parameter towards metal loss subject to
SRB to affirm the findings from MLR model.

Overall, this research has successfully revealed vital information regarding the
influence of soil parameters upon underground corrosion subject to local strain of
SRB for Sungai Ular site. The findings can give a profound understanding on
underground SRB strain and MIC to pipeline operator in Malaysia. This research
also demonstrated a comprehensive works on the modelling of corrosion subject to
SRB using different techniques which has been proven beneficial to the
identification of influential corrosion parameters.
 184

8.3 Research Contribution

Pipeline operator can now use information from this research to pinpoint the
main factor that lead to high corrosion of the pipeline installation site caused by
SRB. Previous reports and inspection record cannot clearly identify how the
particular site is highly corrosive. This is of importance since soil corrosion is
governed by so many factors as opposed to corrosion in seawater. The presence of
SRB in the soil can change the growth pattern of corrosion which may lead to severe
pitting externally. By referring to the findings from this research, pipeline operator
can now has more confident to identify site with high potential of MIC by measuring
important parameters related to SRB such as pH, organic content and Sulfide. Even
the developed predictive model from the field work can be used to calculate the
potential metal loss due to MIC on their underground assets. This can later be used
as a guideline to design a more efficient repair and maintenance scheme as well as
relocation program of the existing pipeline in suspected SRB zone. This research has
also demonstrated the importance of using local SRB strain to study its effect on
MIC for better results.

8.3 The Recommendations for Future Work

To improve the study, several recommendations are listed as follows:

a) Consider other soil variables such as pipe-to-soil potential of the pipeline

(voltage potential generated between a buried pipe and its surrounding soil)
as this data is easier to obtain by the pipeline operator compared to soil
analysis.

b) Instead of measuring the metal mass loss from the buried coupons, the data
from the pigging record from the pipeline operator can also be used to
develop a soil corrosion model.
 185

c) SRB corrosion currently uses a variety of optical and electron microscopy

methods to obtain the images of the biofilms and the bacteria. For further
study, it will be good if atomic force microscopy (AFM) can be applied. This
method can provide more information about the morphology of SRB and
biofilms.

d) For assessing the corrosion effect due to SRB, it will be more specific if the
corrosion model is developed using the data from the electrochemical cell. In
order to do this, the researcher needs to have a few electrochemical cells to
shorten the time in developing the corrosion model.

e) Another effort for future work is to study the effect of MIC in a continuous
system. Previously, all of MIC studies were conducted in batch units. It will
be great if the study on MIC is conducted in a continuous system where the
fresh medium for SRB is continuously fed to the system.

f) Another recommendation that may be applied in future work is the

application of various types of biocide to reduce the MIC in the system and
reduce the effect from bio-corrosion. In this work, a natural biocide is
considered to have a bright future and new to the researcher.
 186

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 198

APPENDIX A

Table A1: Corrosion Rate of carbon steel coupon X-70 exposed to the soil at Sungai
Ular Site.

Coupon Exposure Corrosion Coupon Exposure Corrosion Rate,

Code No Period Rate, mm/yr Code No Period mm/yr
(Year) (Year)
1A 0.25 0.075 11A 1.0 0.388
1B 0.25 0.135 11B 1.0 0.190
2A 0.25 0.063 12A 1.0 0.370
2B 0.25 0.122 12B 1.0 0.130
13A 0.25 0.193 14A 1.0 0.109
13B 0.25 0.028 14B 1.0 0.110
15A 0.25 0.142 16A 1.0 0.086
15B 0.25 0.147 16B 1.0 0.361
9A 0.5 0.461 5A 1.25 0.720
9B 0.5 0.109 5B 1.25 0.146
10A 0.5 0.344 7A 1.25 0.263
10B 0.5 0.145 7B 1.25 0.137
19A 0.5 0.148 17A 1.25 0.124
19B 0.5 0.203 17B 1.25 0.444
24A 0.5 0.139 18A 1.25 0.079
24B 0.5 0.085 18B 1.25 0.106
6A 0.75 0.389 21A 1.5 0.078
6B 0.75 0.164 21B 1.5 0.188
8A 0.75 0.181 22A 1.5 0.069
8B 0.75 0.048 22B 1.5 0.059
20A 0.75 0.085 3A 1.5 0.190
20B 0.75 0.207 3B 1.5 0.118
25A 0.75 0.059 4A 1.5 0.251
25B 0.75 0.030 4B 1.5 0.105
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Table A2: Metal loss (ML) and corrosion rate (CR) for after removal of outliers for
both depth (0.5m and 1.0 m)

ML CR ML CR ML CR
Site Period
(mean) (mean) (med) (med) (max) (max)
Sungai 0.25 3.574 0.113 4.062 0.128 6.111 0.193
Ular
Sungai 0.50 9.664 0.204 6.921 0.146 21.813 0.461
Ular
Sungai 0.75 10.719 0.145 9.171 0.124 28.643 0.389
Ular
Sungai 1.00 21.708 0.218 15.924 0.160 38.592 0.388
Ular
Sungai 1.25 33.067 0.253 18.536 0.142 94.313 0.720
Ular
Sungai 1.50 21.927 0.132 18.471 0.111 41.679 0.251
Ular

Table A3: Soil chemical properties for Sungai Ular site

Sample Soil Chloride Sulfate Sulfide Organic
No PHC1 content, content, content, Content,
(mg/kg) (mg/kg) (mg/kg) (mg/kg)
1 4.1 2384 1495 59 24505
2 2.6 67 1802 37 41011
3 4.8 5731 2204 21 55792
4 3.9 48 700 21 47228
5 4.2 408 3198 25 65211
6 3.7 1726 1708 39 49621

Table A4: Soil Physical properties for Sungai Ular site

Clay
Moisture Plasticity Particle Size, Soil
Sample No Content,
Content, (%) Index, (mm) Resistivity,
(%) (Ohm.cm)
1 24 11.6 0 0.083 94
2 50 12.7 7 0.055 82
3 28 9 20 0.121 94
4 31 12 15 0.055 104
5 31 14 7 0.074 622
6 44 18 13 0.069 352
 200

Figure A1: Location of the site that the sample were taken

Figure A2: Box plot data for site Sungai Ular for over 1.5 yer exposure period
 201

Figure A3: Site location where the coupon was installed

Figure A4: Site location where the coupon was installed

 202

Table A5: Chemical composition of API 5L X-70 carbon steel pipe by GDS
Technique
 203

APPENDIX B

Tafel slope OFAT experimental result for various incubation temperature

Figure B-1: Tafel slope for OFAT at incubation temperature 4 0C

Figure B-2: Tafel slope for OFAT at incubation temperature 20 0C

 204

Figure B-3: Tafel slope for OFAT at incubation temperature 30 0C

Figure B- 4: Tafel slope for OFAT at incubation temperature 40 0C

 205

Figure B-5: Tafel slope for OFAT at incubation temperature 50 0C

 206

Tafel slope OFAT experimental result for various initial pH value

Figure B-6: Tafel slope for OFAT at pH 4

Figure B-7: Tafel slope for OFAT at pH 5.5

 207

Figure B-8: Tafel slope for OFAT at pH 7

Figure B-9: Tafel slope for OFAT at pH 8.5

 208

Figure B-10: Tafel slope for OFAT at pH 10

 209

Tafel slope OFAT experimental result for various Fe2+ concentrations

Figure B-11: Tafel slope for OFAT at 0 ppm Fe2+ concentration

Figure B-12: Tafel slope for OFAT at 50ppm Fe2+ concentration

 210

Figure B-13: Tafel slope for OFAT at 100ppm Fe2+

Figure B-14: Tafel slope for OFAT at 300ppm Fe2+

 211

Tafel slope OFAT experimental result for various SO4-2 concentration

Figure B-15: Tafel slope for OFAT at 2.0 g/liter of Fe2+ concentration

Figure B-16: Tafel slope for OFAT at 3.0 g/liter of Fe2+ concentration
 212

Figure B-17: Tafel slope for OFAT at 4.0 g/liter of Fe2+ concentration

Figure B-18: Tafel slope for OFAT at 5.0 g/liter of Fe2+ concentration