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

BIOFILM FORMATION BY SULFATE REDUCING BACTERIA AND BIOCORROSION OF METALS IN SEAWATER VU PHUONG THANH (B.ENG., HANOI UNIVERSITY OF TECHNOLOGY) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgements ACKNOWLEDGEMENTS My deep and sincere gratitude goes to my supervisor Professor Ting Yen Peng for allowing me to join his team, for his expertise, kindness, and for his patience. Without his guidance and support during two years of the master course, I might not finish my research work. I would like to thank Dr. Serena Teo for providing facilities for my isolation of new sulfate reducing bacterium from local marine and Professor Hong Liang for allowing me to use the equipment in his lab. I would like to express my recognition and appreciation to our lab officers Ms. Sylvia Wan and Ms. Jamie Sew for their support and assistance in my experiments. I would like to acknowledge the advice and guidance of Ms. Samantha Fam, Dr. Yuan Ze Liang, Mr. Chia Phai Ann and Dr. Rajarathnam Dharmarajan on the operation of AFM, XPS, SEM and HPLC equipment. I also thank Mr. Ng Kim Poi for supplying and fabricating metal coupons for the corrosion study. I have furthermore to thank my lab members for their friendship and encouragement. My special thank goes to my friend, Ms. Pham Van Anh, for her understanding and helpfulness during my time in Singapore. I would like to thank my family members, especially my husband, Nguyen Ngoc Chien, for supporting and encouraging me to pursue this degree. This work received the assistance of Tropical Marine Science Institute (Singapore) National University of Singapore and Data Storage Institute (A-Star). i Table of Contents TABLE OF CONTENTS ACKNOWLEDGEMENTS .......................................................................................... i TABLE OF CONTENTS ............................................................................................. ii SUMMARY ................................................................................................................... v LIST OF TABLES ...................................................................................................... vii LIST OF FIGURES ...................................................................................................viii NOMENCLATURE..................................................................................................... xi CHAPTER I: INTRODUCTION ................................................................................ 1 1.1 Importance of microbiologically influenced corrosion........................................ 3 1.2 Microorganism in biocorrosion ............................................................................. 5 1.3 Objective and Scope of the project........................................................................ 6 CHAPTER II: LITERATURE REVIEW .................................................................. 8 2.1 Fundamental aspects of corrosion......................................................................... 8 2.2 Biofilm formation and proposed mechanisms of MIC ...................................... 10 2.2.1 Biofilm formation process ................................................................................... 10 2.2.2 Biocorrosion mechanisms.................................................................................... 12 2.3 Sulfate reducing bacteria and its influence on the corrosion of metals ........... 15 2.4 Titanium and titanium corrosion ........................................................................ 18 2.5 Continuous systems in biocorrosion study ......................................................... 20 CHAPTER III: MATERIALS AND METHODS.................................................... 23 3.1 MIC study of titanium in continuous and semi-continuous system ................. 23 3.1.1 Metal coupons preparation................................................................................... 23 3.1.2 Bacteria ................................................................................................................ 24 3.1.3 Bacterial culture for MIC studies......................................................................... 25 3.1.3 SEM sample preparation and operation............................................................... 26 3.1.4 AFM sample preparation and operation .............................................................. 27 ii Table of Contents 3.1.5 EIS sample preparation and operation ................................................................. 27 3.1.6 XPS sample preparation and operation................................................................ 28 3.1.7 ICP-MS sample preparation and operation.......................................................... 29 3.2 SRB Isolation and Characterization ................................................................... 30 3.2.1 Isolation................................................................................................................ 30 3.2.2 Morphological characterization ........................................................................... 31 3.2.3 Physiological characterization ............................................................................. 32 3.2.4 HPLC sample preparation and operation............................................................. 33 3.2.5 Phylogenetic classification................................................................................... 34 3.2.6 MIC study of the new isolate ............................................................................... 34 CHAPTER IV: RESULTS AND DISCUSSION...................................................... 36 4.1 Biocorrosion behavior of titanium grade 5 in semi continuous system ........... 36 4.1.1 Experiment conditions ......................................................................................... 36 4.1.2 Biofilm formation and detachment ...................................................................... 39 4.1.3 Pit observation and measurement ........................................................................ 43 4.1.4 Electrochemical study of corrosion behavior ...................................................... 45 4.1.4.1 Modeling of control coupons ...............................................................46 4.1.4.2 Modeling of coupons exposed to Desulfovibrio desulfuricans and Desulfovibrio singaporenus in semi-continuous cultures................................49 4.1.5 ICP analysis for confirmation of bacterial effect on metal dissolution ............... 53 4.1.6 Verification of titanium biocorrosion mechanism using X-ray photoelectron spectroscopy.................................................................................................................. 57 4.1.7 Summary .............................................................................................................. 59 4.2 Biocorrosion behavior of titanium grade 5 in continuous system .................... 60 4.2.1 Experiment conditions ......................................................................................... 61 4.2.2 Biofilm formation and detachment ...................................................................... 62 4.2.3 Pit observation and measurement ........................................................................ 64 4.2.4 EIS data for corrosion behavior and evidence of pitting corrosion ..................... 67 4.2.4.1 Modeling of control coupons ...............................................................67 4.2.4.2 Modeling of coupons exposed to Desulfovibrio desulfuricans continuous culture............................................................................................69 iii Table of Contents 4.2.5 ICP analysis ......................................................................................................... 73 4.2.6 Summary .............................................................................................................. 75 4.3 Isolation and characterization of new SRB from local marine......................... 76 4.3.1 Bacterial cell morphology.................................................................................... 76 4.3.2 Physiological properties....................................................................................... 77 4.3.3 The growth of TA1L in lactate and sulfate containing medium.......................... 80 4.3.4 Phylogenetic properties........................................................................................ 82 4.3.5 MIC study of stainless steel 316, mild steel, aluminum alloy 6064 and titanium grade 5........................................................................................................................... 86 4.3.6 Summary .............................................................................................................. 89 CHAPTER V: CONCLUSION AND RECOMMENDATION .............................. 91 5.1 Conclusion ............................................................................................................. 91 5.2 Recommendation................................................................................................... 92 REFERENCES............................................................................................................ 94 iv Summary SUMMARY The project focuses on titanium and its corrosion behaviors under the influence of sulfate reducing bacteria in seawater. The major focus of the project is a study on the corrosion of titanium in semi-continuous and continuous systems. The isolation of a local marine SRB is also reported. The corrosion behavior of titanium grade 5 in seawater was investigated under the influence of two axenic sulfate reducing bacterial cultures of Desulfovibrio singaporenus (EF178280) and Desulfovibrio desulfuricans (ATCC 27774) in semicontinuous conditions, using electrochemical impedance spectroscopy technique. Observations using scanning electron microscope (SEM) showed that the bacteria quickly colonized on titanium surface and biofilm formation occurred within the first week of exposure. Biofilm detachment was also observed during the period. Pitting corrosion was noted after 3 months, and surface pits were revealed using atomic force microscopy. Analysis using inductively coupled plasma mass spectroscopy (ICP-MS) showed higher concentration of titanium in the bacterial culture medium for both D. singaporenus and D. desulfuricans cultures than in the control without the bacteria. All the data suggested that titanium dissolution occurred in seawater, with the bacteria playing a role in its corrosion. A continuous system based on a reactor provided by Centre of Disease Control (CDC reactor) was established using the sulfate reducing bacterium D. desulfuricans. Bacterial colonization and biofilm formation rapidly occurred as in the semicontinuous system. The biofilm formed in the continuous system was more compact and less patchy, and covered the metal surface with fewer voids. AFM data showed different section profile of pits on titanium surface after 3 months and 4 months in the v Summary continuous culture. On the surface of the control coupon, some holes were also found but it is unlikely that they were corrosion pits because of their small number and shallow depth. EIS was successfully used in modeling the behavior of titaniumbacterium-medium interaction. DC polarization data showed some evidence of biocorrosion. ICP data confirmed the influence of the bacterium in titanium corrosion as bacterial culture consistently showed higher metal concentration than the control in the aqueous system. A novel sulfate reducing bacterium, named TA1L, was isolated from a biofilm on titanium substratum in the local marine environment in Singapore. The cell was Gram negative, rod curve shaped, motile and non-spore forming. The bacterium is halophilic and is capable of using many common electron acceptors as sulfate reducing bacteria. The bacterium also uses sulphite, thiosulphate as electron acceptors. Optimal condition for cell growth was 35oC, pH 7.5 and a salinity of 2.5%. Desulfoviridin test as well as partial 16S-rRNA encoding gene sequence analysis showed that the bacterium is a member of Desulfovibrio genus. Phylogenetic tree indicated that the new isolate is a close relation to Desulfovibrio bizertensis and Desulfovibrio singaporenus. Microbiologically influenced corrosion studies suggested the role of TA1L in initiating and promoting the corrosion of SS316, mild steel, aluminum and titanium grade 5 in seawater. vi List of tables LIST OF TABLES Table 3.1: Modified Barr’s Medium for D. desulfuricans ........................................... 24 Table 3.2: Postgate medium B for D. singaporenus .................................................... 24 Table 3.3: Artificial seawater ....................................................................................... 25 Table 4.1.1 Value of model parameters obtained by fitting the two models [Rs(QbRb)] and [Rs(Qb[Rb(QpRp)])] to EIS data for the control coupons in semi-continuous system. ....................................................................................................................................... 48 Table 4.1.2 Value of fitted parameters for EIS data of D. desulfuricans coupons in semi-continuous system ................................................................................................ 51 Table 4.1.3 Value of fitted parameters for EIS data of D. singaporenus coupons in semi-continuous system ................................................................................................ 51 Table 4.1.4 Titanium concentration (mg/L) in bacterial cultures and controls immersed with 3-month old and 4-month old coupons in semi-continuous conditions. (n denotes the number of samples).................................................................................................. 54 Table 4.2.1 Value of fitting parameters obtained by fitting model [Rs(QbRb)] and model [Rs(Qb[Rb(QpRp)])] to EIS data of control coupons in continuous system. ....... 68 Table 4.2.2 Value of fitted parameters obtained by fitting the two models [Rs(Qb[Rb(QpRp)(QfRf)])] and [Rs(Qb[Rb(QfRf)])] to EIS data of control coupons in semi-continuous system. ............................................................................................... 71 Table 4.2.3 Titanium concentration of bacterial cultures and controls immersed with 3-month old and 4-month old coupons in continuous conditions................................. 74 Table 4.3.1 Morphology and physiological properties of SRB isolated from titanium sample. .......................................................................................................................... 79 Table 4.3.2 Comparison of cell morphology and physiological properties of TA1L and its two close relatives Desulfovibrio bizertensi and Desulfovibrio singaporenus ........ 85 vii List of figures LIST OF FIGURES Figure 1.1 Corrosion cell (Properties of Internal Pipe Corrosion)................................. 9 Figure 2.1 The biofilm life cycle (Biofilm hypertextbook). ........................................ 12 Figure 3.1 CDC reactor system set up (picture from manufacturer) ........................... 26 Figure 4.1.1 Batch growth of ( ) D. singaporenus and ( ) D. desulfuricans in ASW at 25oC under anaerobic condition................................................................................ 38 Figure 4.1.2 pH value and cell concentration of ( ) D. singaporenus and ( ) D. desulfuricans cultures during two 6-day replenishment periods (6th day was chosen as replenishment point). .................................................................................................... 39 Figure 4.1.3 D. desulfuricans biofilm at week 10 (a) before and (b) after replenishment................................................................................................................ 39 Figure 4.1.4 AFM images of (a) D. desulfuricans and (b) D. singaporenus cells and their EPS on titanium surface ....................................................................................... 40 Figure 4.1.5 (1) D. desulfuricans and (2) D. singaporenus biofilm formed on titanium surface after (a) 3 days, (b) 7 days and (c) 14 days ...................................................... 41 Fig. 4.1.6 Metal surfaces with (a1) D. desulfuricans biofilm at week 5; (a2) D. desulfuricans biofilm detachment at week 6; (b1) D. singaporenus biofilm at week 6 and D. singaporenus biofilm detachment at week 7..................................................... 42 Figure 4.1.7 Section profile of pits on (1) D. desulfuricans and (2) D. singaporenus (a) 3-month and (b) 4-month samples by AFM. ................................................................ 44 Figure 4.1.8 Electrical circuits for model (a) [Rs(QbRb)] and model (b) [Rs(Qb[Rb(QpRp)])] for semi-continuous control samples. ........................................... 47 Figure 4.1.9 (a) Nyquist, (b) Bode modulus and (c) Bode phase plots for control coupons and their fitted data with two models [Rs(Qb[Rb(QpRp)])] and [Rs(QbRb)] .... 48 Figure 4.1.10 Electrical circuits for (a) model [Rs(Qb[Rb(QpRp)(QfRf)])] and (b) model [Rs(Qb[Rb(QfRf)])] for semi-continuous D. desulfuricans samples. ............................. 49 Figure 4.1.11 (1) Nyquist and (2) Bode phase plots for (a) D. desulfuricans and (b) D. singaporenus coupons and their fitted data with two models [Rs(Qb[Rb(QfRf)])] and [Rs(Qb[Rb(QpRp)(QfRf)])].............................................................................................. 50 Figure 4.1.12 Cyclic polarization curves of titanium coupons exposed to semicontinuous culture (a) D. desulfuricans, (b) D. singaporenus and (c) seawater after 14 weeks............................................................................................................................. 53 Figure 4.1.13 Comparison of average value of total titanium concentrations in D. desulfuricans and D. singaporenus 3-month samples and controls.............................. 55 viii List of figures Figure 4.1.14 Comparison of average value of total titanium concentrations in D. desulfuricans and D. singaporenus 4-month samples and controls.............................. 55 Figure 4.1.15 Average values of titanium concentration in 3-month and 4-month samples.......................................................................................................................... 57 Figure 4.1.16 XPS spectra of titanium and sulfur on the Ti coupon exposed to D. singaporenus culture after removal of biofilm. ............................................................ 58 Figure 4.2.1 Desulfovibrio desulfuricans growth curve in artificial seawater at 25oC, pH 7, and under ambient conditions ............................................................................. 62 Figure 4.2.2 AFM images of Desulfovibrio desulfuricans colonization and (a) exopolymer substances after 1 week and (b) biofilm after 4 weeks ............................. 62 Figure 4.2.3 SEM images of biofilm on titanium surface under continuous condition after (a) 3 days, (b) 1 week, (c) 2 weeks and (d) 10 weeks. ......................................... 63 Figure 4.2.4 Section profile of pits on (a) 3 month and (b) 4 month continuous coupons ......................................................................................................................... 66 Figure 4.2.5 AFM image of a shallow hole on control coupon after 4 months in continuous system......................................................................................................... 66 Figure 4.2.6 (a) Nyquist, (b) Bode modulus and (c) Bode phase plots for control coupons and their fitted data with model [Rs(QbRb)] and model [Rs(Qb[Rb(QpRp)])] .. 68 Figure 4.2.7 (a) Nyquist, (b) Bode modulus and (c) Bode phase plots for coupons exposed to D. desulfuricans continuous culture and their fitted data with model Rs(Qb[Rb(QfRf)])] and model [Rs(Qb[Rb(QpRp)(QfRf)])] .............................................. 71 Figure 4.2.8 Cyclic polarization curves of titanium coupons exposed to continuous (a) D. desulfuricans culture and (b) control after 14 weeks............................................... 72 Figure 4.2.9 Comparison of titanium concentration in 3rd month and 4th month controls and samples exposed to continuous bacterial culture ..................................... 74 Figure 4.3.1 FESEM images of TA1L cells and biofilm on SS316 coupon after 2 weeks of exposure to seawater...................................................................................... 77 Figure 4.3.2 Growth curve of TA1L in term of ( ) logarithm of cell concentration. ( ) Lactate consumption, ( ) acetate production and (x) pyruvate production during the cell growth. ................................................................................................................... 81 Figure 4.3.3 Partial 16S-rRNA encoding gene of TA1L ............................................. 83 Figure 4.3.4 Phylogenetic tree based on 16S-rRNA gene of new isolate TA1L and other bacteria from Desulfovibrio genus. The scale bar represents 7 nucleotide substitutions per 100 nucleotides. ................................................................................. 84 Figure 4.3.5 Bacterial colonization on (a) SS316, (b) mild steel, (c) aluminum and (d) titanium after 5 days...................................................................................................... 86 ix List of figures Figure 4.3.6 Potentiodynamic scanning curves of (—) control and (—) D. desulfuricans sample of (a) SS316 coupon after 7 days, (b) mild steel coupon after 5 days, (c) Al coupon after 1 day, (d) Cu coupons after 4 weeks and Ti coupon after 3 months........................................................................................................................... 88 Figure 4.3.7 SEM images of pits found on (a) aluminum surface after 5 days, (b) stainless steel after 7 weeks and (c) mild steel after 1 week......................................... 89 x Nomenclature NOMENCLATURE AFM Atomic force microscopy ASW Artificial seawater CBR Centre of Disease Control Biofilm Reactor CDC Centre of Disease Control CPE Constant phase element Epit Breakdown potential (V) Epp Passivation potential (V) Ecorr Corrosion potential (V) EASW Enriched artificial seawater EIS Electrochemical impedance spectroscopy EPS Extracellular polymeric substance HPLC High performance liquid chromatography ICP Inductively coupled plasma ICP-MS Inductively coupled plasma-Mass spectrometry MIC Microbiologically influenced corrosion MPN Most probable number method ppm Part per million OCP Open circuit potential (V) Qb Capacitance of the compact oxide barrier (µF) Qedl Capacitance of the electrical double layer (µF) Qp Capacitance of the porous oxide film (µF) Rb Resistance of the compact oxide barrier (k ) Rct Charge transfer resistance (k ) xi Nomenclature Rp Resistance of the porous oxide film (k ) Rs Solution resistance ( ) n Constant phase angle of CPE (rad) SD Standard deviation SEM Scanning electron microscope SRB Sulfate-reducing bacteria XPS X-ray photoelectron spectroscopy ZCPE Impedance of the constant phase elements ( ) icorr Corrosion current density (A) µmax Specific growth rate (h-1) Yo Constant of CPE (µ -1 n s) xii Introduction CHAPTER I: INTRODUCTION The term microbiologically influenced corrosion (MIC) is usually understood as the deterioration of metal which is promoted and enhanced by the effect of microorganisms. Microorganisms in general, and bacteria in particular, are the main factor in MIC, apart from other requirements such as energy source, carbon source, electron donor, electron acceptor and water (Javaherdashti, 2008). However microorganism not only cause the corrosion of metals but may indeed inhibit this phenomena (Jayaraman et al., 1997; Zuo, 2007), and the term “biofouling” is more generally used. When a metal is exposed to water, the water-borne microorganisms colonize on its surface and form biofilm through a series of steps. Problems which occur when the biofilm builds up are termed biofouling. Microbial fouling not only causes MIC but may also lead to other severe consequences such as product contamination, reduction in a transfer or mechanical blockages. Microorganisms are ubiquitous; as long as the environment contains water, bacteria can form biofilm and biofouling occurs. Metal corrosion is one of the most important aspects of biofouling. Although metal corrosion can occur without microorganism, microbial activities accelerate the reactions rate (Horn et al., 2002). Metabolic products such as organic acids, sulfide, protein and polysaccharides affect the kinetics of cathodic and/or anodic reactions and also modify the properties of the metal surface as well as passive layers which are formed on the metal surface (Beech et al. 2004a). Microbiologically influenced corrosion (MIC) or biocorrosion has long been recognized and is a topic of interest due to the complexities resulting from the involvement of living organisms (Coetser et al. 2005). From the pioneer work of 1 Introduction Wolzogen Kuhr and Van de Flugt in 1934 (Von Wolzogen Kuhr et al. 1934) until the 1960s, only few studies were published. The first stage in the development of our understanding of MIC is the discovery and connection between the presence of bacteria and the corrosion phenomena. During the 1960s and early 1970s, publications were focused on explaining the role of sulfate reducing bacteria (SRB) and the use of some electrochemical techniques for MIC investigation. During this period, the major factors of biocorrosion, bacterial colonization and biofilm settlement, were not widely investigated (Videla, 1991a). The 1980s is considered the booming stage in MIC studies with efforts to establish a mechanism for the corrosion process. Videla and his colleagues (1991b) considered this stage a progress of understanding MIC. Various hypotheses were proposed to interpret the hidden mechanisms of MIC. Understanding of biocorrosion at that point of time created an industrial awareness of this phenomenon. From the 1990’s, many modern applications have been introduced in the detailed investigation of MIC; these include microscopy techniques and electrochemical techniques (Beech, 2004b). Modern microscopy techniques such as atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) reveal the metal surface covered with biofilm, microorganism products such as extracellular polymer substances (EPSs) and corrosion products. Energy Dispersive Xray Analysis (EDX), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and other methods were used in the analysis of corrosion products and the establishment of corrosion mechanisms. Quantitative assessments of MIC including electrochemical techniques have improved, and numerous new approaches such as Electrochemical Impedance Spectroscopy (EIS) and Electrochemical Noise (ECN) have been widely utilized. These electrochemical techniques, in addition to traditional 2 Introduction weight loss measurement, are effective methods for calculating corrosion rates. Other techniques such as Attenuated Total Reflectance/Fourier Transform Infrared Spectroscopy (ATR/FTIR), Inductively Coupled Plasma spectrometry (ICP) and TimeOf-Flight Secondary Ionization Mass Spectrometry (TOF-SIMS) are also applied in this field. Over the years, research has intensified with detailed examination with regard to corrosion materials, environments as well as the microorganisms involved. 1.1 Importance of microbiologically influenced corrosion MIC occurs in all aqueous environments such as fresh water, sea water, and humid soil and in industrial systems. Therefore, its sphere of influence is very extensive, from bridges, buildings and infrastructures on the ground to pipelines and rails underground, from marine ships, harbors and offshore systems to airplanes and aerospace devices. Biocorrosion as well as other types of corrosion attack are responsible for a large number of economic losses. The following cases are some of the negative impact of MIC. MIC reportedly contributes 10- 20% of the total corrosion damage of all metals and building materials out of 4% GNP loss in developed countries (Geesey et al., 2000). In England, it has been estimated that biocorrosion contributes up to 10% of corrosion cases (De Romero et al., 2000). Annually, the direct cost of MIC is estimated at 30-50 billion US dollar. In USA alone, industries spend about 1.2 billion US dollar annually on biocides to mitigate MIC (Flemming, 1996). In the oil and gas industry, biocorrosion accounts for 15-30% of the corrosion cases. In USA alone, overall loss to oil and gas industry due to MIC has been reported to be more than $100 million per annum (Maxwell et al., 2004). Underground corrosion of steel gas or water pipes which cost 0.5 to 2 billion US dollars per year 3 Introduction (Greathouse et al., 1954) is the best known economic disaster engendered by sulfate reducing bacteria (SRB), a group of bacteria involved in MIC. Microorganisms have been held to be the major or minor cause of accidents such as oil spill, building and bridge collapses, etc. MIC-inducing microorganisms are also involved in another significant issue: they cause severe contaminations to industrial products, and their metabolic products excreted poison the reservoirs. The black color of the Black Sea is believed to be the result of the activities of SRB. SRB have also been reported to be responsible for many other environmental problems such as massive fish kills, death of sewer workers by H2S generation, and the formation of poisonous fogs, etc. (Singleton, 1993). In recent years, the use of metal components for human implants has been increasing. In-vivo biocorrosion of these implants by human parasitic and other microorganisms and by body fluids are issues of concern. Singh and Dahotre (2007) reported that corrosion of metallic implants due to the body environment and bacteria cause many problems: it may release undesirable metal ions and/or corrosion products which are non-biocompatible to human body; it may shorten the life of implant device which leads to another costly surgery, and even shorten the patient’s life. Singapore is the third biggest oil refinery centre in the world. The quantity of metals used in infrastructure, including offshore pipelines, and vessels is extremely high. These metal-based systems are highly susceptible to marine biocorrosion. Understanding MIC helps us to protect our systems by applying protection methods such as coating and alloying the metals, and the use of biocides and inhibitors. This approach will significantly reduce the cost of damage to both the material and product. At the same time, the structure integrity of the infrastructure will not be compromised. 4 Introduction Titanium is well known to be a highly resistant material. The term titanium biocorrosion is relatively new as titanium has been shown to be not susceptible to MIC under natural conditions. However, the corrosion of titanium in human fluids as well as in industrial environment has been reported (Yen et al., 1998, Hsu et al., 2004, Rao et al., 2005). Over the years, titanium has been increasingly used. As such, the role of bacteria in the corrosion of titanium over the long term should be investigated. 1.2 Microorganism in biocorrosion Many bacteria have been shown to be associated with the process of biocorrosion since 1960s. According to Beech (2003), sulfate reducing bacteria (SRB), iron/manganese oxidizing bacteria, iron reducing bacteria, sulfur oxidizing bacteria and bacteria which produce organic acids and slime are the dominant types of microorganism involved in MIC in terrestrial and aquatic habitats. These microbes are found in natural biofilm as they all contribute to the complex consortia of biofilm (Beech et al., 2004a). Among others, SRB is not only the first group of interest in biocorrosion study (Videla et al., 1991b) but has also been recognized as the most significant contributors to MIC (Coetser et al., 2005). In the early stage of MIC study, SRB were thought to be the only bacteria causing biocorrosion (Horn et al., 2002). Overtime, however, it is recognized that this group is facilitated in natural biofilm owing to the activities of aerobic bacteria which form reducing conditions as well as nutrient source for SRB. Currently, SRB continues to be the most published group of bacteria in the field of MIC. SRB has been widely investigated in the corrosion of iron-based alloys, aluminum-based alloys, copper-based alloys, etc. The study of bacteria in their natural environment helps us to understand the natural consortia and provides us effective approaches to exploit their advantages as 5 Introduction well as minimize their disadvantages in engineered systems. In MIC studies, SRB as well as other bacteria involved are usually isolated from field sites and construction materials. The isolation and characterization of new bacteria are necessary for the investigation of underlying corrosion mechanisms as there is no universal mechanism that fully explains the phenomenon. In addition, study on the activities of bacteria in their axenic cultures and in mixed cultures is important in understanding the role of an individual bacterium as well as synergistic effects of different bacteria. Many SRB have been isolated from field sites such as Desulfovibrio halophilus sp. nov. from Solar Lake in Sinai (Caumette et al., 1991); Desulfovibrio vietnamensis from oil storage tank in Vietnam (Dang et al., 1996); Desulfotomaculum sp. from oilwater in Turkey (Cetin et al., 2007); Desulfovibrio marinus sp. nov. from marine sediments in Tunisia; etc. In our lab, a new SRB named Desulfovibrio singaporenus was isolated from corroded stainless steel 316 surface in natural seawater. The bacterium has been shown to cause the corrosion of SS316 in the marine environment (Sheng et al., 2007a). Its role in other metals including titanium should be investigated. 1.3 Objective and Scope of the project The project focuses on titanium and its corrosion behavior under the influence of SRB. The SRB biofilm formation on its surface in different conditions is also an aspect of this work. Based on the above objectives, the focus of this work is as follows: (i). Study on the influence of two SRB D. desulfuricans (ATCC 27774) and D. singaporenus on the corrosion of titanium grade 5. The formation of biofilm on metal surface in artificial seawater (ASW) is observed. The morphology of the corrosion and type of attack are revealed. The presence of metal ions and precipitate in the culture media is used to confirm the dissolution of metal under the impact of the bacteria. The nature of corrosion products such as elemental composition and oxidation states is examined. 6 Introduction (ii). Study on influence of Desulfovibrio desulfuricans (ATCC 27774) on the corrosion of titanium in a flow-through system. Under more realistic conditions (i.e. continuous flow and ambient conditions), a CDC reactor system is operated, and the corrosion behavior of titanium is investigated. Biofilm formation and detachment are observed. The morphology of microbial cell, their distribution on the surface and the presence of EPS are also described. Evidence of corrosion pits, morphology and type of corrosion attack are investigated. Due to the diversity of SRB in different water sources and different locations, it is necessary to isolate and investigate the effect of local bacteria on the corrosion of some commonly used metals. Understanding of local bacteria MIC would help to establish more effective metal protection strategy resulting in lower cost of replacement. Hence, another objective of this study is to investigate the biocorrosion of some metals under the influence of a local marine SRB which is isolated from a titanium substratum. The following works are conducted for that purpose. (iii). Isolation of new SRB from natural seawater in St. John’s Island. The SRB is then characterized and classified to confirm its SRB origin. The morphology, physiology and phylogenesis of the cell are investigated. The influence of the new bacterium on the corrosion of different metals is examined in batch cultures, using seawater as the culture medium. This study would contribute to the understanding of titanium biocorrosion which has not been widely investigated. The use of CDC reactor for corrosion study over a long time period would provide more realistic conditions compared to other studies in batch and semi-continuous conditions. The study would also provide an insight into MIC by a SRB isolated locally in laboratory condition. 7 Literature Review CHAPTER II: LITERATURE REVIEW 2.1 Fundamental aspects of corrosion Corrosion is a natural phenomenon. According to ISO 8044 standard, corrosion is defined as a “physicochemical interaction between a metal and its environment which results in changes in the properties of the metal and which may often lead to impairment of the function of the metal, the environment, or the technical system of which these form a part” (Stearns, 2004). In nature, corrosion is the result of a series of redox reactions between a metal-based material and its surrounding medium which bring about the deterioration of the metal or its properties. The reduction of an electron acceptor (often but not always oxygen) located adjacent to the metal surface, in tandem with the oxidation of the metal is the ultimate cause of the metal dissolution. There are many underlying agents that increase the likelihood of corrosion in an aqueous system, such as the dissimilarity of metals or alloys, soil variances, naturally corrosive soils, atmospheric corrosion, environmental contamination and microbial corrosion (Stearns, 2004). However, in order for corrosion to happen, the following components must be present (Barton et al., 2007): + an anode site, + a cathode site, + an electrolyte, + a cathodic reactant, and + an anodic reactant. Corrosion reactions are electrochemical in nature, and take place when there is contact between an oxidizing agent and a reducing agent. The overall corrosion reaction comprises two half reactions as follows: (i) oxidation of metal or anodic reaction: 8 Literature Review Meo å Men+ + ne(ii) reduction of chemical species in contact with the metal or cathodic reaction, for example, the reduction of oxygen: ½ O2 + H2O +2e- å 2OHThe overall reaction is: 2Meo + ½ nO2 + nH2O å 2Men+ + 2nOHThe anodic and cathodic reactions can occur separately at distant locations on the metal surface. When anodic sites are sparse and irregularly distributed on the surface, the attack is localized corrosion in the form of pitting and/or crevice corrosion. On the other hand, when the two half-reactions are close to each other, they form a corrosion cell (Figure 1.1) and may cause evenly distributed corrosion if the corrosion cells are dense (Beech et al., 2007). Figure 1.1 Corrosion cell (Properties of Internal Pipe Corrosion) If a thermodynamic driving force exists and promotes one or both of the halfreactions, corrosion will take place (Mattsson, 1989). The corrosion rate (or rate of metal dissolution) is directly proportional the anodic reaction rate or the magnitude of anodic current. However, the anodic current and the cathodic current must be equal due to the tendency to obtain electrical neutrality. Hence, the corrosion rate depends on the cathodic 9 Literature Review reaction rate or on the surrounding environment. A reductive environment is usually aggressive to the metals. When metals and alloys are exposed to an oxygen-containing environment, or when a passivation process is applied to protect them, a passive film forms on the surface of these materials. The passive layers are usually in the form of metal oxide and consist of not only the major metal but also the alloying metals. For some metals and alloys such as stainless steels and titanium, the passive layers are strong and highly resistant to corrosion. The corrosion of these materials only occurs when this protective layer is damaged by an external factor such as a mechanic force or aggressive chemicals (Beech et al., 2007). An important concept in corrosion study is “polarization”. When a metal is exposed to an aqueous environment, there is an equilibrium between the oxidation of metal atoms to metal ions and the reduction of metal ions to metal atoms: Men+ + ne- Me At the equilibrating state, no electrode reactions occur and the electrode potential is called equilibrium potential. If a current is applied to change the electrode potential and equilibrium no longer exist, we call the process polarization. Polarization curve is a graph of electrode potential when current is altered. Polarization is composed of anodic polarization and cathodic polarization. Understanding the process and its components helps us to comprehend kinetics of the corrosion, calculate the corrosion rate as well as determine a proper strategy for metal protection. 2.2 Biofilm formation and proposed mechanisms of MIC 2.2.1 Biofilm formation process Although MIC has been studied for many years and many mechanisms have been proposed, none of them fully explain the phenomenon of biocorrosion. Nonetheless, it is 10 Literature Review generally accepted that the key to surface alterations and enhancement or inhibition of corrosion is biofilm formation (Videla, 1996). The presence of biofilm on a metal surface often establishes new electrochemical reaction pathways or promotes reactions which are not normally favored in the absence of microorganisms. This may result in the increase of corrosion rates and undesired effects on the performance of metal (Lewandowski et al., 1997). Biofilm formation on metal surface is an accumulation process that starts immediately after metal immersion in an aqueous environment. This process consists of different stages (Figure 2.1). In the first stage, the deposition of inorganic and organic high molecular mass compounds from the aqueous environment alters the electrostatic charges and wetability of the substratum, thus facilitating the colonization of bacteria. In the second stage, microbial growth and extracellular polymeric substance (EPS) production results in biofilm formation. The presence of EPS in aqueous media makes them slimy. Biofilm are thought to contain up to 95% water and this is attributed to the presence of EPS (Guiamet et at, 1999). EPS is believed to protect the biofilm from the attack of external factors such as shear stress, and also improve the nutrient absorbability of biofilm. Subsequently, the biofilm thickening via transport processes and chemical reactions produce the third step of biofilm formation. Biofilm may be thin (10-12µm) or as thick as 100µm which prevents the diffusion of ions between metal surface and bulk fluid (Guiamet et at, 1999). The forth stage, detachment, is the least understood process of biofilm accumulation. Detachment refers to the release of a patch of cells and their associated EPS from the biofilm to the bulk fluid. Hunt et. al. (2004) listed some of the factors that have been suggested to be important in biofilm detachment as matrixdegrading enzymes, microbially generated gas bubbles, nutrient levels and microbial growth status, availability of multivalent cross-linking cations, fluid shear stress, contact 11 Literature Review attrition, quorum-sensing signals, and the activation of a lytic bacteriophage. Among others, accumulation of a metabolic product and the depletion of a metabolic substrate are the most potential factors (Stoodley et al., 2001; Kaplan et al., 2003; Mai-Prochnow et al., 2004; Parsek et al., 2004). Detachment provides inoculum for further biofilm accumulation and this phenomenon has been hypothesized to be a factor in the promotion of MIC (Stewart, 1993). The development and detachment of biofilm are influenced by many factors such as nutrient sources, flow conditions, surface roughness, temperature and pH, etc. (Coetser et al., 2005). Figure 2.1 The biofilm life cycle (Biofilm hypertextbook). 1: Individual cells populate the surface. 2: EPS is produced and biofilm is formed. 3 and 4: biofilm thickens via transport processes and chemical reactions. 5: Biofilm detachment and inoculation of new generation of biofilm. 2.2.2 Biocorrosion mechanisms Videla and his co-workers stated that any biological effect that either facilitates or impedes one of the anodic or cathodic reactions or permanently separates anodic and cathodic sites will increase corrosion (Videla et al., 1991b). Based on this principle, many possible mechanisms of MIC have been proposed. The first mechanism, proposed by Wolzgen Kuhr and Van der Vlugt (1934), was cathodic depolarization. It was suggested that the dissociation of hydrogen from the cathodic site is the rate-limiting step in the corrosion process, and the consumption of hydrogen by SRB depolarizes the cathode and thus accelerates the corrosion. 12 Literature Review 4Fe å 4Fe2+ + 2e- Anodic reaction 8H2O å 8H+ + 8OH- Dissociation of water 8H+ + 8e- å 8H Cathodic reaction SO42- + 8H å S2- +4H2O Cathodic polarization by SRB Fe2+ + S2- å FeS Corrosion product 3Fe2+ + 6OH- å 3Fe(OH)2 Corrosion product The overall reaction is: 4Fe + SO42- + 4H2O å 3Fe(OH)2 + FeS + 2OHHowever, this theory has been rejected in later studies due to the fact that in many experiments, SRB pure cultures gave lower corrosion rate than that at field sites. Some mechanisms such as the formation of occluded areas on the metal surface, fixing the anodic site and underdeposit acid attack were proposed by Pope and Morris (1995) but none of these mechanisms fully explain the phenomena of MIC. According to Hamilton (1985), the role of microorganism in MIC is either to promote the development of electrolytic cell or to enhance the anodic and/or cathodic reactions. Lee and his colleagues (1995) considered MIC as the unavoidable result of the following processes: • Transport and accumulation of materials from the environment to the metal surface. • Microbial and electrochemical transformation processes within the biofilm and the metal surface. Microbial transformation processes affect the interface between the biofilm and the metal. • Detachment of biofilm from the metal surface. This process controls the overall extent of fouling deposit accumulation. 13 Literature Review According to Cloete and Flemming (1997), microorganisms affect the corrosion rate in several manners: • Consumption of oxygen by aerobic organisms develops an oxygen gradient which shifts the metal surface potential and result in the initiation of localized corrosion. • The use of hydrogen by microorganisms via a cathodic reaction depolarizes the cathode which increases the rate of metal loss at the anode. • Microbial degradation of protective coatings. • Microbial degradation of corrosion inhibitors. • Generation of corrosive metabolites. • Metabolic by-products such as H2S precipitate metal ions, thereby producing corrosive products. Coetser and Cloete (2005) listed five factors which are believed to contribute to MIC: • The creation of oxygen concentration cells, • The creation of ion concentration cells, • Activities of iron- and manganese-oxidizing bacteria • Microbiological acid production • Anaerobic conditions promoting the growth of SRB There are many different ways to interpret the phenomena of MIC. However, no model is universally accepted as the mechanism of biocorrosion because the effects of microorganism on corrosion reactions are the result of very complicated interactions (Little et al., 2002). Thus, there is no particular mechanism has been assigned to any instance of biocorrosion. Together with the development and application of modern techniques, the underlying mechanisms of MIC continue to be investigated and currently are better understood. 14 Literature Review 2.3 Sulfate reducing bacteria and its influence on the corrosion of metals Sulfate reducing bacteria (SRB) is a large group of anaerobic microorganisms that play an important role in many biogeochemical processes. They are widely distributed in nature and participate in our lives in a variety of manners. They are considered regular components of many engineered systems (Barton et al., 2007). Their appearance in nature is indicated by blackish (caused by corrosion product FeS) and smelly (due to the generation of H2S) water. Postgate (1984) defined SRB a class of microbes which conducts dissimilarity sulfate reduction. These microorganisms use sulfate the as terminal electron acceptor in their energy metabolism. However, most SRB are flexible; they can use many electron acceptors other than sulfate, such as elemental sulfur (Finster et al., 1998; Bottcher et al., 2005; Haouari et al., 2006), nitrate (Krekeler et al., 1995; Sass et al., 2004), Mn(IV) (Myers et al., 1998; Sass et al., 2004), and Fe(III) (Lovley et al., 2004). Since the dissimilarity sulfate reduction is inhibited under oxic conditions, SRB are usually considered to be strictly anaerobic. Some SRB, however, are reportedly facultative anaerobes, capable of growing under aerobic conditions too (Dannenberg et al., 1992; Lemos et al., 2001; Lobo et al., 2007). The role of SRB in the corrosion of iron and iron-based alloys is well understood (King et al., 1971). According to Hamilton (1994), during the process of metal dissolution and in the absence of oxygen, protons H+ or H2S might serve as electron acceptor at the cathode: 2H+ + 2e- 2H H2 or 2H2S + 2e- 2HS- + H2 15 Literature Review Subsequently, SRB oxidize H2 and lead to the depolarization of the cathode. In combination with their use of sulfate as electron acceptor, S2- is produced and corrosion products are in the form of sulfide: 4H2 + SO42Men+ + S2- 4H2O + S2MeS Guezennec et al. (1991) noted that the application of a current to confer the corrosion resistance on metals can stimulate the growth and activities of SRB to produce H2 at the cathode. Bryant and colleagues (1991) stated that the highest corrosion rates are acquired after a period of bacterial activities, and corrosion products also act as aggressive agents towards the corrosion. Beech (2002) affirmed that even under identical medium, different genera of SRB or even different species of the same genus can modify their ability of affecting the metal deterioration. She also proposed several models of SRB-mediated corrosion and described current hypothesis based primarily on important corrosion reactions. In these models, the depolarizing effect of hydrogenase enzyme, the cathodic depolarization due to biogenically produced iron sulfide and the anodic depolarization due to the formation of corrosion products are considered the key factors. It has been demonstrated that hydrogenase enzyme itself can promote the corrosion of mild steel (Chatelus et al., 1987; Boivin et al., 1990) even when it is in the cell-free extracted form. However, no quantitative assessment of hydrogenase contribution to SRB biocorrosion of metals has been conducted (Beech, 2002). Booth et al. (1968) investigated the cathodic depolarization by adding solid FeS and concluded, based on their assays, that the extent of corrosion was proportional to the added FeS. It has been shown that the biogenically produced FeS has the same corrosion influence as FeS from inorganic reactions (Smith et al., 1975). An understanding of cathodic depolarization and the role of sulphide has given 16 Literature Review rise to an alternative hypothesis – anodic depolarization (Beech, 2002). This model argued that the primary formation of anode is due to the generation of local H+ by SRB activities with ensuing metal dissolution (Bryant et al., 1991; Beech et al., 1995). The localized acidification at the anode due to the reaction: Men+ + HS- å MeS + H+ is the key element which resist the local acidification owing to the action of reducing H2S/HS buffer system (Beech, 2002). Beech also proposed two other theories explaining the corrosion mediated by SRB: the generation of corrosive phosphorus compounds, and the binding of metal ion to extracellular polymeric substances produced by SRB. Phosphorus-containing substances are commonly present in the aqueous environment. These phosphorus compounds are the products of SRB activities and the effect of H2S on phosphates, phosphites and hypophosphites. Metal phosphides are corrosion products of several corrosion reactions. It has been found that the presence of inorganic phosphates can intensify the corrosive activity of hydrogenase (Bryant et al., 1991). Binding of EPS produced by SRB with metal ions such as Fe, Cr, Mo, and Ni has been investigated (Beech et al., 1995). EPS which bind to metal ions can come from biofilm as well as planktonic cell in the environment. The binding occurs when EPS attach to the metal surface and contact with metal ions at the corroding sites. Metals ions are then released into the surrounding environment together with EPS. Therefore, it can be concluded that SRB exopolymers contribute to the corrosion of metals. Although SRB have been extensively investigated and much effort have been directed at understanding the role of these bacteria in MIC, there are still ambiguities which remain to be resolved such as the contribution of their metabolic products, 17 Literature Review exopolymers and enzymes in corrosion reactions. The current trend in SRB research is to examine in detail areas such as enzymatic activities, exopolymers effects and the role of oxygen as the ultimate electron acceptor (Lee et al.; 2004). The development of new biomolecular techniques would assist in understanding how SRB affect the corrosion of different metals and alloys. 2.4 Titanium and titanium corrosion Titanium was first discovered as an impure metal oxide by British mineralogist William Gregor in 1791. It took more than 100 years before Hunter found the first method to isolate the metal in 1910, by heating titanium tetrachloride (TiCl4) with sodium in a steel bomb. Titanium is not a rare metal: it is the forth abundant metal on the Earth. However, titanium and titanium-based products are still expensive due to the fact that titanium is usually found in low concentration and in impure state, and the cost for purifying it is very high (Leyens et al., 2003). During the years that titanium has been commercially available, many alloys have been developed. Titanium is usually combined with aluminum, iron, vanadium, etc. at different proportions to produce light and hard alloys. Each alloy has its own properties and applications in industry and in medicine, and all Ti alloys have excellent corrosion resistance. The superior resistance is result of the formation of a highly stable protective oxide film – TiO2. This film is just 5-20 nm in thickness but it forms quickly when fresh metal is exposed to an oxygen-containing environment (Schultz, 1991). Due to its high resistance to corrosion and low density, more and more titanium and its alloys are replacing other traditional metals such as iron and copper. However, none match the 50% market share that Ti-6Al-4V (Ti grade 5) enjoys (Matthew et al., 1989). Ti-6Al-4V is an - type alloy containing 6% Al and 4% V, and is unique because of its combination of strength and toughness along with excellent corrosion resistance. These 18 Literature Review attractive properties, together with inherent workability, high workshop fabricability, and the production experience and commercial availability has led to reliable and economic usage of Ti-6Al-4V. Thus, Ti-6Al-4V has become the standard alloy against which other alloys must be compared when selecting titanium alloy for specific application. Typical uses of Ti-6Al-4V include aerospace applications, pressure vessels, marine and chemical applications, automotive, aircraft turbines and compressor blades and disc, surgical implants, etc. Titanium usage is expected to increase for advanced gas turbines and airframes (Leyens et al., 2003). As with many other materials, titanium is susceptible to biofouling (Lewis, 1982; Licina, 1988). Microorganism can easily attach and colonize on titanium-based surfaces and form biofilm. However, due to the high resistance of titanium, there appears to be very few reports on the biocorrosion of this metal and its alloys. Schultz (1991) studied the corrosion behavior of titanium under different conditions, and concluded that titanium is resistant to sulfur oxidizing bacteria (SOB), SRB and acid producing bacteria. Little and his colleagues (1992) investigated the impact of SRB and iron/sulfur-oxidizing bacteria on corrosion of titanium and did not observe any MIC. Mansfeld et al. (1992; 1994) concluded that corrosion rate of titanium in natural seawater is very low because of the presence of a tenacious passive oxide layer. Recently, some papers have reported the corrosion of titanium under microbial influence and in biological solution. Hsu et al. (2004) reported that the corrosion resistance of Ti-6Al-4V implant alloy in joint fluid was lower than that of Ti-6Al-4V in serum and urine. This group also found that Ti-6Al-4V implant alloy was significantly corroded in PBS solution at pH 4 and human body temperature or in the same solution at neutral pH and 45oC. 19 Literature Review Rao and his colleagues (2005) studied the pitting corrosion of titanium grade 2 under the influence of an axenic SRB culture. After 90 days of exposure, it was found that SRB induced titanium pitting corrosion with pit size of up to 50µm. From these investigations, it is clear that titanium is also susceptible to MIC with corrosion current density (icorr) in the order of 10-2 to 10-1 µA/cm2. A corrosion density (icorr) of up to 45.93 µA/cm2 was recorded in NaCl solution (Yen et al., 1998). To better understand this phenomenon and to add to the body of knowledge, research should be conducted using modern analytical techniques including electrochemical and microscopy techniques. 2.5 Continuous systems in biocorrosion study Most laboratory studies on MIC were carried out in batch system (Deckena et al., 1992; Beech et al., 1994; Watkins et al., 1995; Sheng et al., 2007a; Nguyen et al., 2008). Under batch condition, the bacterial culture is kept for a duration with no medium replenishment during the period of investigation. Metal samples are immersed in the bacterial culture of interest and are collected after a certain period of time for analysis. This approach is simple and convenient for operation but it is not very realistic. In batch systems, the bacterial culture conditions change over time due to change in pH and chemical composition of the environment as a result of microbial activities, including the generation of metabolites. Marchal et al. (2001) experimented on the growth of Desulfovibrio gabonesis in hermetically closed flasks and found that the growth of this SRB was clearly restricted by the growth conditions. According to Gubner and Beech (1996), the biocorrosion data from batch systems are relatively difficult to reproduce. Besides, such systems do not adequately mimic the natural environment where the microorganisms grow in continuous culture condition. Only few biocorrosion studies have been conducted under continuous conditions. Walker et al. (1991) described a two-stage chemostat system for axenic culture in the 20 Literature Review study of copper corrosion, using low dilution rate in the first stage for microbial growing, while the second stage was operated at higher dilution rate for corrosion study. Angell et al. (1997) established a multiple chemostat system with bacterial consortia to study the synergistic effect of two aerobic bacteria and one SRB on stainless steel 316L electrode. The system was shown to produce a mixed biofilm community where the three bacteria were implicated in the MIC process. Some continuous systems were also designed for the investigation of inhibiting agents for SRB-induced corrosion such as antimicrobial peptides or biofilm and even living bacterial cells (Jayaraman et al., 1999; Örnek et al., 2002; Zuo et al., 2004). In laboratory scale studies, a glass fermenter equipped with temperature controller, pH controller, stirrer, gas and medium suppliers are commonly used in continuous system for both aerobic and anaerobic culture (Weimer et al., 1988; Gubner et al., 1996; Marchal et al., 2001). These systems may be interfaced with electrochemical data acquisition system for in situ measurement of corrosion rate as well as other corrosion parameters. Indeed, apart from more effectively mimicking the real natural environment, continuous systems also have an advantage over batch systems in terms of control over the experimental conditions. However, almost all published work on continuous systems were operated over a short period of time and with a small number of metal samples assembled. This is due to the fact that most of the reactors were designed based on commercially available fermenters which were not designed for corrosion study. Some newly designed reactors have recently been engineered to overcome these disadvantages. CDC Biofilm Reactor (CBR), a product of Centre of Disease Control, is a newly developed system for biofilm study (Honraeta et al., 2005). The reactor incorporates eight coupon holders which can each house three coupons of different materials. Mixing and 21 Literature Review shear is generated by a magnetic stirrer using a magnetic stir plate. The medium is circulated through tubing connected to the reactor by a peristaltic pump. The CBR facilitates the study of biofilms under high shear condition which is common in nature or industry. Some advantages of CDC reactor can be listed as follows: - CBR is a relatively simple system which can be easily assembled, operated and sampled. - CBR, as a continuous flow stirred tank reactor (CFSTR), has all the advantages of a CFSTR such as: (i) Bulk fluid can be assumed as well mixed; (ii) Residence time in the reactor can be adjusted for each bacteria; (iii) Nutrient feed can be specified and adapted for various bacteria or conditions; and (iv) a portion of effluent can be recycled; and can be operated in anaerobic condition. In addition, it also offers additional advantages such as: (i) Coupons holder can be aseptically replaced without any difficulties by blank rods. This permits the removal of samples over time and also retain stable flow pattern. (ii) 24 removable samples in the same reactor facilitates long-term study, and (iii) Consistent shear across all samples. For these reasons, CBR reactors were used in this study in continuous systems for the study of microbiologically influenced corrosion of titanium. The system is maintained for several months for biofilm formation observation and biocorrosion measurement. 22 Materials and Methods CHAPTER III: MATERIALS AND METHODS 3.1 MIC study of titanium in continuous and semi-continuous system 3.1.1 Metal coupons preparation Ti-6Al-4V (titanium grade 5, GoodFellow) was used. The compositions of this alloy include: Al 6%, V 4%, Fe 0.03%, C 0.22%, H 0.1%, N 0.1%, O 0.65%, Ti balance. Metal samples were fabricated as circular coupons of diameter 12.5 mm and thickness 1 mm. The coupons were then manually polished using abrasive papers of grit sizes 400, 800 and 1200 and followed by liquid diamond (Kemet). Kemet 320 manual polishing and lapping machine was used with liquid diamond of sizes 6 µm, 3 µm and 1 µm. Finishing size of liquid diamond, 0.5 µm, enables the metals with mirror surface to be produced. After polishing, the coupons were degreased in acetone in an ultrasonic bath for 15 seconds for 3 cycles. In studying the influence of a newly isolated SRB on the corrosion of different metals, Titanium grade 5 (above mentioned) and the following commonly used metals are utilized: (i) Aluminum alloy 6064 of composition: Si 0.4%, Cu 0.15%, Mg 0.8%, Cr 0.05%, Pb 0.2%, Bi 0.5%, Al balance. (ii) Stainless Steel 316 of composition: Cr 16.5 - 20%, Mn [...]... biocorrosion since 1960s According to Beech (2003), sulfate reducing bacteria (SRB), iron/manganese oxidizing bacteria, iron reducing bacteria, sulfur oxidizing bacteria and bacteria which produce organic acids and slime are the dominant types of microorganism involved in MIC in terrestrial and aquatic habitats These microbes are found in natural biofilm as they all contribute to the complex consortia of biofilm. .. first stage in the development of our understanding of MIC is the discovery and connection between the presence of bacteria and the corrosion phenomena During the 1960s and early 1970s, publications were focused on explaining the role of sulfate reducing bacteria (SRB) and the use of some electrochemical techniques for MIC investigation During this period, the major factors of biocorrosion, bacterial... to any instance of biocorrosion Together with the development and application of modern techniques, the underlying mechanisms of MIC continue to be investigated and currently are better understood 14 Literature Review 2.3 Sulfate reducing bacteria and its influence on the corrosion of metals Sulfate reducing bacteria (SRB) is a large group of anaerobic microorganisms that play an important role in many... surgery, and even shorten the patient’s life Singapore is the third biggest oil refinery centre in the world The quantity of metals used in infrastructure, including offshore pipelines, and vessels is extremely high These metal-based systems are highly susceptible to marine biocorrosion Understanding MIC helps us to protect our systems by applying protection methods such as coating and alloying the metals, ... believed to protect the biofilm from the attack of external factors such as shear stress, and also improve the nutrient absorbability of biofilm Subsequently, the biofilm thickening via transport processes and chemical reactions produce the third step of biofilm formation Biofilm may be thin (10-12µm) or as thick as 100µm which prevents the diffusion of ions between metal surface and bulk fluid (Guiamet... promotion of MIC (Stewart, 1993) The development and detachment of biofilm are influenced by many factors such as nutrient sources, flow conditions, surface roughness, temperature and pH, etc (Coetser et al., 2005) Figure 2.1 The biofilm life cycle (Biofilm hypertextbook) 1: Individual cells populate the surface 2: EPS is produced and biofilm is formed 3 and 4: biofilm thickens via transport processes and. .. known economic disaster engendered by sulfate reducing bacteria (SRB), a group of bacteria involved in MIC Microorganisms have been held to be the major or minor cause of accidents such as oil spill, building and bridge collapses, etc MIC-inducing microorganisms are also involved in another significant issue: they cause severe contaminations to industrial products, and their metabolic products excreted... grade 5 The formation of biofilm on metal surface in artificial seawater (ASW) is observed The morphology of the corrosion and type of attack are revealed The presence of metal ions and precipitate in the culture media is used to confirm the dissolution of metal under the impact of the bacteria The nature of corrosion products such as elemental composition and oxidation states is examined 6 Introduction... using abrasive papers of grit sizes 400, 800 and 1200 and followed by liquid diamond (Kemet) Kemet 320 manual polishing and lapping machine was used with liquid diamond of sizes 6 µm, 3 µm and 1 µm Finishing size of liquid diamond, 0.5 µm, enables the metals with mirror surface to be produced After polishing, the coupons were degreased in acetone in an ultrasonic bath for 15 seconds for 3 cycles In. .. resulting in lower cost of replacement Hence, another objective of this study is to investigate the biocorrosion of some metals under the influence of a local marine SRB which is isolated from a titanium substratum The following works are conducted for that purpose (iii) Isolation of new SRB from natural seawater in St John’s Island The SRB is then characterized and classified to confirm its SRB origin ... process of biocorrosion since 1960s According to Beech (2003), sulfate reducing bacteria (SRB), iron/manganese oxidizing bacteria, iron reducing bacteria, sulfur oxidizing bacteria and bacteria. .. is produced and biofilm is formed and 4: biofilm thickens via transport processes and chemical reactions 5: Biofilm detachment and inoculation of new generation of biofilm 2.2.2 Biocorrosion. .. under the influence of sulfate reducing bacteria in seawater The major focus of the project is a study on the corrosion of titanium in semi-continuous and continuous systems The isolation of a local