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Studies on Breakdown of Passivity of Titanium iofilms covered with in vitro Biofilms B. Anandkumar1, Nanda Gopala Krishna2, R.P. George2*, N. Parvathavarthini2 and U. Kamachi Mudali2 1Department of Biochemistry and Biotechnology, Sourashtra College, Madurai-625004, India 2Corrosion Science and Technology Group, Indira Gandhi Centre for Atomic Research, Kalpakkam-603102, India *Corresponding author: Dr. R.P. George E-mail: firstname.lastname@example.org , Tel: +91 44 27480121, Fax: +91 44 27480301 ABSTRACT This paper presents the influence of pure biofilms of sulfate reducing bacteria (SRB) and heterogeneous biofilms of SRB and manganese oxidizing bacteria (MOB) on the passivity of titanium. Two to eight weeks old SRB biofilm and 12 week old heterogeneous biofilms with SRB and MOB were developed on titanium. Epifluorescence microscopy, scanning electron microscopy, X-ray diffraction and X-Ray Photoelectron spectroscopy were used to characterize the biofilms. Cyclic polarization experiments were conducted in natural sea water with pure and heterogeneous biofilms. These biofilms breakdown the passive film leading to localized corrosion and a probable mechanism of passivity breakdown is proposed. 1 Keywords: Keywords: titanium, polarization, microbiological corrosion, passivity, Sulfate Reducing Bacteria (SRB), Manganese Oxidizing Bacteria (MOB). Introduction Water is one of the most common heat transfer fluids and most of the problems associated with corrosion and deposits are water-related. If long term reliability of heat transfer equipment has to be achieved, the mechanism of microbiologically influenced corrosion (MIC) and fouling has to be understood. It is well known that sulfate reducing bacteria (SRB) are found in marine and freshwater sediments where sulphate is present. They have also been commonly isolated from industrial water systems such as cooling waters and oil fields [1, 2]. They were considered as the major bacterial group involved in MIC . The open cooling systems are particularly suitable for the activities of SRB, because they handle large volumes of water which is maintained at the temperature range of 30 to 60°C with pH of 69, and provide a continuous source of bacteria from make-up water and ambient air. For metabolic activities, SRB utilize sulphate as an electron acceptor and produce H2S [3, 4]. H2S directly attacks metal surfaces and causes corrosion of various metals including carbon steels, stainless steels, and copper alloys . When materials are exposed to cooling waters, aerobic microbes attach and form biofilms. Underneath these biofilms, as the oxygen content decreases, anaerobic SRB flourish and mixed biofilms are formed which leads to more aggressive MIC . Titanium has been selected as the 2 condenser tube material for Prototype fast breeder reactor which is being built at Kalpakkam, Tamilnadu, India. Titanium possesses outstanding corrosion resistance in a wide range of aggressive environments . The reason for the remarkable corrosion resistance of this material is the presence of inert, strongly adherent surface oxide film which forms almost instantly on exposure to moisture, air and many sources of oxygen . With all its technical superiority, titanium has proven to be the choice of heat exchanger material for sea water cooled power plants. However, the inertness of titanium surface and its excellent biocompatibility makes it highly susceptible to biofouling . Not much information is available on the influence of heterogeneous biofilms consisting of aerobic MOB and anaerobic SRB on the passivity of Ti alloys. Earlier studies in the author's laboratory on year long biofouling studies of titanium in seawater have confirmed the presence of aerobic MOB and anaerobic SRB in biofilms . Recently, Rao et al., has reported that titanium becomes susceptible to pitting corrosion by freshwater anaerobic SRB Desulfovibrio sp. . The main objective of this work is to study the influence of a consortium of anaerobic and aerobic bacterial biofilms and pure anaerobic bacterial biofilms on the localized corrosion resistance of titanium in natural seawater. Cyclic potentiodynamic polarization experiments were done for establishing localized corrosion resistance and surface characterization were used for characterizing the passive film before and after forming biofilm. 3 Experimental Material The material used in this investigation is commercially pure (CP) Titanium (Ti) (ASTM Grade 2). The chemical composition of the material is presented in Table 1. Elements Fe O N C Ti Weight % <0.3 0.25 0.03 0.1 Balance Table 1. Chemical composition of CP Grade 2 Titanium Specimen Specimen preparation Ti specimens with two different dimensions were used in the study as given below. (i) Ti specimens with dimensions (20 x 15 x 1.6 mm) were ground with 80 to 1200 grit SiC paper. After grinding, the specimens were cleaned ultrasonically first with soap solution followed by distilled water and air-dried. These specimens were used in biofilms exposure studies, X-Ray Diffraction (XRD) and X-ray Photoelectron Spectroscopy (XPS) analysis. (ii) Ti specimens with dimensions (10 mm x 10 mm x 1.6 mm) were mounted in epoxy resin (araldite) with a brass rod for electrical connection. The details are described elsewhere 4 . This mounted specimen with an exposed area of 1 cm2 was ground to 1200 grit and was used for biofilm formation and for carrying out electrochemical cyclic potentiodynamic and cathodic polarization experiments. Development of biofilm on Titanium specimens Isolation and characterization of microbes SRB was isolated from Titanium coupons exposed to sea water for two weeks in the pumping station of Nuclear Desalination Demonstration Plant located at Kalpakkam, India. Isolation and biochemical characterization of SRB was done as described in Bergey's Manual of Determinative Bacteriology . The Postgate B medium used for isolation contains 10 g Tryptone, 1 g Na2SO4, 1 g Na2SO3, 2.5 g NaCl and 0.5 g Ferric Citrate in one liter of distilled water. Manganese oxidizing bacterium (MOB) was isolated from surfaces of steel scraps in Indira Gandhi Centre for Atomic Research (IGCAR) storage yard and identified as B. flexus in the authors' laboratory , and the same was used in the present study. Molecular identification and phylogenetic analysis of isolated SRB Genomic DNA of the bacterial isolate and amplification of gene encoding small subunit ribosomal RNA was carried out as described elsewhere . The amplified product was purified using GFXTM PCR DNA and Gel Band Purification kit (Amersham Biosciences) 5 and cloned in pTZ57R/T vector according to the manufacturer's instruction (InsT/AcloneTM PCR Product Cloning Kit, MBI Fermentas), and transformants were selected on Luria Bertani medium containing ampicillin (100 g/ml) and X-gal (80 g/ml). DNA sequencing was carried out using ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems). For sequencing reaction, Big Dye Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit (Perkin-Elmer) was used. The sequence obtained was analyzed for its close related sequences using NCBIBLAST search version 2.2.20  and tools of Ribosomal Database Project II Release 10 (http://rdp.cme.msu.edu) for taxonomic hierarchy of the sequences. Phylogenetic analysis was performed with multiple sequence alignments using CLUSTAL X2  with a collection of taxonomically related sequences obtained from the National Center for Biotechnology Information (NCBI) Taxonomy and Homepage (http://www.ncbi.nlm.nih.gov/Taxonomy/ Project-II Release 10 (http://rdp. taxonomyhome.html/) Ribosomal Database cme.msu.edu). Molecular Evolutionary Genetic Anlaysis (MEGA) version 4.1 program  was used to construct phylogenetic tree (neighbor-joining method) and similitude analysis was done with the common 16S rRNA gene regions. 1,000 bootstrap replications were carried out to validate internal branches . MatGAT v. 2.01 software  was used to calculate the similitude percentages among sequences. 6 Exposure Exposure studies in cultures Ti specimens were exposed to two bacterial groups viz., anaerobic SRB and aerobic MOB B. flexus. Semi-continuous SRB culture was maintained by replacing 50 % culture with sterile Postgate Medium B (described above) for every four days on the basis of the SRB growth curve. Maintenance of semi-continuous MOB culture was done as described elsewhere . Biofilms of various duration were formed on the test coupons of Ti by exposing for various durations (two weeks, eight weeks and 12 weeks) in various cultures (SRB and MOB) as follows: (i) (ii) (iii) exposed to SRB for two weeks exposed to SRB for eight weeks exposed to SRB for 10 weeks and MOB for two weeks The SRB and MOB cells present in the biofilms on the exposed specimens were recultured in respective media and their presence and viability were confirmed. Post exposure characterization of biofilms and Titanium surfaces Microscopic observation of biofilms Heterogeneous biofilm of SRB and MOB cells on Ti specimens were stained with 0.1 % Acridine orange stain and examined using Epifluorescence microscope (Nikon E600) for 7 confirming the presence of bacterial groups. Detailed methodology is described elsewhere . Biofilmed Ti specimens were cleaned with sterile water to remove all loose biofilm. The specimens were fixed with 0.25 % Glutaraldehyde at 16°C overnight and then dehydrated with a series of ethanol-water combinations (20 % to 100 %) and then kept in vacuum desiccators. The surface morphological characteristics of the specimens were observed under scanning electron microscope (SEM) (XL30ESEM M/s Philips) at magnification ranging from 100X to 10000X operated at accelerating voltage of 15-30 kV. XRD analysis of the corrosion products Ti coupons exposed to 12 week old heterogeneous biofilms were dried and the XRD pattern of the biofilm was recorded using computer controlled XRD-system (PanAnalytical Xpert) with Cu-K radiation (Ni filtered = 13418 Ao) at the range of 40kV, 20A. The `peak search' and `search match' program built in software (syn master 7935) was used to identify the peak table and ultimately for the identification of XRD peaks. XPS analysis XPS analysis was carried out on unexposed Ti (control) and Ti exposed to heterogeneous biofilm for 12 weeks to understand the influence of biofilm on passive layer of titanium. Ti specimens exposed to bacterial cultures were cleaned in sonicator bath with 8 absolute ethanol for removing all traces of biofilm and surface analysis of the specimens was carried out. Measurements were carried out by X-ray Photoelectron Spectrometer (SPECS make, Germany) using Al K line of the X-ray source at 1486.74eV. The anode was operated at a voltage of 13 kV and source power level at 300 W. Sputter-etch cleaning of specimens was done for 1, 3 and 5 minutes using an Ar ion source operated at 5 kV and 50 A. Spectra were collected using the PHOIBOS 150 MCD-9 analyzer with a resolution of 0.67 eV for 656 kcps at pass energy of 10 eV. Cyclic potentiodynamic polarization studies of Ti covered with bacterial biofilms potentiodynamic biofilms Cyclic potentiodynamic polarization experiments were carried out in sea water as per ASTM standard G61-86  on (i) freshly polished specimens (without any biofilms) and (ii) specimens with two weeks and eight weeks old SRB biofilms and (iii) specimens with heterogeneous biofilm of 10 week exposure in SRB culture followed by two weeks exposure in MOB culture. A computer controlled potentiostat (AUTOLAB PG60, Netherlands) was used to conduct the cyclic potentiodynamic polarization experiments. A three-electrode electrochemical cell was used consisting of test coupon as the working electrode, saturated calomel electrode (SCE) as the reference and a platinum foil as the auxiliary electrode. Sea water from Kalpakkam coast (salinity 35 ppt, chloride 18,981 ppm, sulfate 2650 ppm, pH 8.1)  was used as the electrolyte. Open circuit potential (OCP) was measured after 9 exposing the specimens to the test solution for 10 minutes. Cyclic potentiodynamic polarization was carried out by scanning the electrode potential from OCP anodically at a scan rate of 10 mV/min up to 1000 mV(SCE) and reversing the scan direction. For all experiments, electrode potential Vs current plots were recorded. After every cyclic polarization experiments, the specimens were cleaned ultrasonically and air dried and examined under Leica make Optical microscope for evaluating the nature of corrosion attack. Potentiodynamic cathodic polarization studies In order to understand the influence of 2 week SRB biofilm on the cathodic polarization behavior of CP Ti, cathodic polarization experiments were carried out from OCP to -1200 mV(SCE) at a scan rate of 10 mV/min for the material with and without SRB biofilm. Results and Discussion Characterization of microbes and Biofilms on Titanium 10 The morphological and biochemical characteristics of SRB isolate revealed that it was Gram negative vibrio shaped, non-sporulating, H2S producing, lactate utilizing Desulfovibrio sp. The 16S rRNA sequencing and phylogenetic analysis (Fig. 1) confirmed that the SRB isolate as Desulfovibrio ferrophilus, having 99.6 % similarity with D. ferrophilus of Genbank (AY274449). The 16S rRNA gene sequence of the D. ferrophilus of the present study has been submitted to Genbank under accession number JQ742965. Fig.1. Neighbor-joining tree based on 16S rRNA gene sequences, showing phylogenetic relationships between Desulfovibrio related sequences. Pelobacter acetylenicus was used as the out-group sequence. Numbers at nodes indicate bootstrap values >50% from 1,000 replicates. GenBank accession numbers are given in parentheses. The scale bar indicates sequence divergence 11 Fig. 2 (a) presents the epifluorescence micrograph of the microbial cell of rod shaped Bacillus flexus and Fig. 2 (b) presents that of vibriod shaped Desulfovibrio sp. from the bacterial cultures streaked on glass slides and stained with acridine orange. The distinct morphology of the two bacterial species is clearly brought out in these figures. (2a) 20 um (2b) (b) 100 um Fig. 2 Epifluorescence micrographs of (a) rod shaped sporulating MOB B.flexus and (b) vibrio shaped SRB Desulfovibrio ferrophilus 12 SEM micrographs and epifluorescence micrographs of heterogeneous biofilm of SRB with MOB on Ti specimens are presented in Figs. 3. Microbial cells with dense orange fluorescence of RNA could be observed in epifluorescence micrographs (Fig. 3a). SEM micrographs (Fig. 3b) distinctly showed the presence of vibrioid shaped SRB D. ferrophilus and rod shaped MOB B. flexus on Ti. (3a) (3b) 2 um 13 Fig. 3 (a) Epifluorescence micrographs of heterogeneous biofilm on titanium showing the fluorescing SRB and MOB cells, and (b) SEM micrograph of mixed biofilm on titanium distinctly showing vibrio shaped SRB and rod shaped MOB cells XRD analysis The XRD pattern obtained for Ti specimen exposed to heterogeneous biofilm is presented in Fig. 4. From these data it can be inferred that these compounds namely FeO(OH), Fe2O3, FeS, Fe(OH)2 and hydrated Fe(OH)3 are present on Ti specimen with 12 weeks mixed biofilm. Desulfovibrio ferrophilus is reported  to be capable of reducing sulfate and ferric ions in the growth media leading to the formation of these compounds. Fe2O3 Intensity Fe(OH)3 H2O FeO(OH) FeS FeO(OH) Fe(OH)2 Ti with SRB + MOB 20 30 40 50 Fe2O3 60 2 Theta Fig. 4 X-Ray Diffractogram for Ti with heterogeneous (SRB and MOB) biofilm 14 XPS analysis XPS spectra of the passive film of Ti samples are shown in Figs. 5a to 5l representing the presence of metallic and oxidized Ti peaks and spectra for the passive film of Ti specimen exposed to mixed biofilm. The presence of metallic and oxidized Ti peaks was seen in these Ti specimens (Figs. 5a to 5d). XPS analysis of the passive films of control Ti and bio-film grown Ti samples, sputtered for 1 min and 3 min, reveal that the films are different. The control Ti sample shows that the passive film is TiO2 and with sputtering, lower oxides of Ti are revealed due to the preferential sputtering of Oxygen. This is seen from de-convolution of the high resolution spectra of 2p core level of Ti (Figs. 5a-5d) and 1s core level of O (Figs. 5e-5h). The 2p region of Ti shows the doublet structure of 2p3/2 and 2p1/2 regions distinctly. The de-convolution shows four doublets. The doublet at 454.3 eV & 459.7 eV suggest metallic Ti state, at 455.1 eV and 460.5 eV suggest Ti (+2) state, at 456.4eV and 461.9 eV suggest Ti (+3) state and at 458.9 eV and 464.5 eV suggest Ti (+4) state. The de-convoluted 1s region of O shows three peaks at 530.5 eV, 531.5 eV and 533.0 eV indicating the O-Ti, -OH and adsorbed H2O respectively. Similarly, the biofilm grown Ti sample shows that the passive film is modified slightly that can be seen from the presence of Fe and S along with Ti and O. Within the binding energy range from 705 to 740 eV the presence of Fe2+ peaks (Figs. 5i and 5j) and within the binding energy range from 160 to 166 eV the presence of S2- peaks (Figs. 5k and 5l) was seen on this surface 15 25000 (a) Titanium - Control Ti (1 min sputter) 15000 (b) Titanium - Control Ti (3 min sputter) 20000 Intensity (cps) Intensity (cps) 10000 15000 10000 5000 5000 0 450 452 454 456 458 460 462 464 466 468 470 0 450 452 454 456 458 460 462 464 466 468 470 Binding Energy (eV) 9000 8000 7000 Binding Energy (eV) 8000 (c) Titanium- Biofilm cleaned Ti (1 min sputter) 7000 (d) Titanium- Biofilm cleaned Ti (3 min sputter) Intensity (cps) 6000 5000 4000 3000 2000 1000 450 452 454 456 458 460 462 464 466 468 470 Intensity (cps) Binding Energy (eV) 6000 5000 4000 3000 2000 1000 450 452 454 456 458 460 462 464 466 468 470 Binding Energy (eV) 30000 (e) Oxygen - Control Ti (1 min sputter) 25000 (f) Oxygen - Control Ti (3 min sputter) Intensity (cps) Intensity (cps) 528 530 532 534 536 538 25000 20000 20000 15000 15000 10000 10000 5000 Binding Energy (eV) 528 530 532 534 536 538 Binding energy (eV) 16 20000 (g) Oxygen- Biofilm cleaned Ti (1 min sputter) 16000 16000 (h) Oxygen- Biofilm cleaned Ti (3 min sputter) Intensity (cps) Intensity (cps) 528 530 532 534 536 12000 12000 8000 8000 4000 4000 526 Binding Energy (eV) 526 528 530 532 534 536 Binding energy (eV) 18000 16000 (i) Iron- Biofilm cleaned Ti (1 min sputter) 18000 16000 (j) Iron- Biofilm cleaned Ti (3 min sputter) Intensity (cps) 14000 12000 10000 8000 6000 700 705 710 715 720 725 730 735 740 745 Intensity (cps) 14000 12000 10000 8000 6000 700 705 710 715 720 725 730 735 740 745 Binding Energy (eV) Binding Energy (eV) 2800 (k) Sulfur- Biofilm cleaned Ti (1 min sputter) 2800 (l) Sulfur- Biofilm cleaned Ti (3 min sputter) 2400 Intensity (cps) 2000 Intensity (cps) 160 162 164 166 168 2400 2000 1600 1600 1200 1200 800 158 158 160 162 164 166 168 Binding Energy (eV) Binding Energy (eV) Fig. 5 XPS analysis of Titanium on Control Ti and biofilm cleaned Ti 1 and 3 minute sputtered with Argon (a to d); Oxygen on Control Ti and biofilm cleaned Ti 1 and 3 minute 17 sputtered with Argon (e to h); Iron on biofilm cleaned Ti (i and j); Sulfur on biofilm cleaned Ti (k and l) even after sputter etch cleaning for 5 minutes. Fe is found to be in +2 oxidation state where as S is as FeS. This is seen from the de-convolution of the 2p core levels of the Fe and S. The 2p region of Fe shows the distinct doublet structure. The de-convolution shows three doublets. The doublet at 707.1 eV and 720.1 eV suggest metallic Fe, at 709.4 eV and 722.6 eV suggest Fe (+2) state and at 716.0 eV and 730.2 eV suggest the satellite peak structure of Fe (+2) state. The de-convolution of the 2p region of S shows a single doublet at 161.8 eV and 163.0 eV indicating FeS. With sputtering, the concentration of higher oxidation states of both Fe and Ti reduce and the metallic states increase. potentiodynamic Cyclic potentiodynamic polarization studies Cyclic potentiodynamic polarization curves obtained for clean Ti and Ti under biofilms of SRB and MOB are given in Fig. 6 (a) and 6 (b). 18 Fig. 6 Cyclic anodic potentiodynamic polarization curves for (a) clean Ti and (b) Ti specimens with SRB and mixed biofilms (SRB and MOB) in seawater Electrochemical parameters viz. OCP, breakdown potential, repassivation potential and passive current density were measured from these curves and are presented in the Table 2. S.No Type of period OCP Eb mV(SCE) - Erepass mV(SCE) - ipass A cm-2 0.8 biofilm/exposure mV(SCE) 1. Freshly polished unexposed (no biofilm) - 180 2. 3. 4. With 2W SRB With 8W SRB With 10W SRB + 2W MOB - 80 - 40 - 20 + 740 + 480 -25 8.0 10.0 14.0 80.0 22.0 100.0 19 Table 2. Electrochemical parameters observed from anodic polarization curves with Ti specimens exposed to SRB and MOB biofilms Cyclic polarization curves of freshly polished clean Ti showed very low passive current (0.8 A cm-2) and negative hysterisis  implicating the passivity of Ti and absence of any localized corrosion. Two week old SRB biofilm on Ti increased the passive current to >10 A cm-2 with positive hysterisis showing the initiation of localized corrosion activity. Eight week old SRB biofilm caused one fold increase of passive current density (20 to 80 A cm-2) and 12 week old heterogeneous biofilm of SRB and MOB caused two fold increase of passive current density (40 to 100 A cm-2). After the cyclic polarization experiments, the specimens were examined under optical microscope. The clean Ti did not undergo any localized attack and hence no break down potential can be seen from the cyclic polarization curve. However, 8 weeks old SRB and 12 weeks old mixed biofilm resulted in breakdown of passive film at +740 mV(SCE) and +480 mV(SCE) respectively. Pitting attack can be seen as presented in Fig. 7. 20 Fig. 7. Optical Micrographs of anodically polarized Titanium specimens in seawater (a) Control Ti, (b) Ti with heterogeneous biofilm (arrow indicating localized attack) Cathodic Potentiodynamic Polarization Curves Cathodic potentiodynamic polarization curves (Fig. 8) showed one order magnitude of increased cathodic current on Ti with 2 week SRB biofilm (10-4 A cm-2) compared to clean Ti (10-5 A cm-2). Fig. 8. seawater Cathodic polarization curve for Ti specimens with 2 week SRB biofilm in 21 D. ferrophilus isolated and identified in this present study have been so far reported only in the corrosion of iron . Aerobic biofilms of MOB are reported to cause ennoblement of materials due to manganese deposition [25, 26]. MOB isolate B. flexus used in this study was also reported in corroding SS304 material . However, this is the first study on corrosion on titanium by a mixed biofilm of anaerobic SRB isolate D. ferrophilus and aerobic MOB isolate B. flexus. SRB strains require strict anaerobic condition  for growth and hence the titanium with 2 weeks and 8 weeks patchy biofilms of SRB (Fig. 4a) has oxygen concentration cells. Due to non-availability of oxygen for TiO2 maintenance, thinning of passive film can happen and the XPS results have shown reduction in intensity of titanium and oxygen peaks under biofilms. Pouilleau et al  reported that the passive films composed of an amorphous TiO2 outer layer (10-20 nm thick) and an intermediate TiOx layer, in contact with the TiO2 layer and the metallic substrate. According to these workers, this outer layer is sensitive to environment and its thickness decreases in corrosive environments. The XPS results have confirmed this under SRB biofilms in the present study. For anaerobic respiration SRB use sulphate as terminal electron acceptor and the reduction of the same with hydrogen result in sulphide production . In metal interphases utilization of hydrogen result in cathodic depolarization that can enhance cathodic current [30, 31]. 22 Cathodic polarization curves on titanium with two week SRB biofilm clearly showed enhanced cathodic current that may be due to electron transfer mechanism from metallic surfaces. The same observation has also been discussed by Cordas et al  on the enhanced hydrogen reduction reaction obtained with the SRB biofilms formed in polarized SS surfaces. They observed that the electrodes polarization seems to have a selective effect on the electro catalytic enhancement, with the observation of higher currents towards the hydrogen reduction due to SRB metabolism. The presence of sulfide in biofilms on titanium was confirmed by XRD patterns in the present study. XRD patterns of biofilms on titanium also showed the presence of FeS. The source of ferrous ions is the bacterial medium. Hang  has studied this species of D. ferrophilus in detail and reported that they can also reduce ferric ions (Ferric citrate in the bacterial medium), hence the name D. ferrophilus was proposed. These ferrous ions react with sulfide and form ferrous sulfide. They can also form FeO(OH), Fe2O3, Fe(OH)2 as confirmed by XRD analysis of biofilm. Cyclic potentiodynamic curves showed increased passive current and positive hysterisis for Ti specimens with 2 week and 8 week SRB biofilm indicating localized corrosion initiation by the action of SRB alone on the passive film. According to Moon et al  pitting corrosion occurs under SRB biofilm by accelerated cathodic reaction and oxygen concentration cell. 23 The 12 weeks heterogeneous biofilm on titanium is comprised of 10 weeks SRB biofilm and 2 weeks MOB biofilm. XPS analysis of the surface showing presence of Ti4+, Fe2+ and S2- peaks in the passive films of cleaned biofilmed surface and even after sputter etch cleaning for 5 minutes suggest the modification of the passive film due to biofilm activity even before polarization. Sulfide ions could have interacted directly with the porous TiO2 passive film and helped the entry of Fe2+ ions also. From the cyclic potentiodynamic curves of titanium covered with heterogeneous biofilms, it can be seen that the passive current was varying between 22 to 100 µA cm-2. This is much higher than that observed for 2 weeks and 8 weeks biofilms of SRB. This implies that the localized corrosion attack of titanium covered with heterogeneous biofilm is more severe than that of homogeneous 2 weeks and 8 weeks SRB biofilm. Optical microscopic observation confirmed localized corrosion and pits on Ti. On the surface of the specimens covered with heterogeneous biofilms there is a combination of the oxygen reduction step by aerobic B. flexus in addition to sulfide metabolism by anaerobic SRB. Biomineralization of manganese oxide by B. flexus is already confirmed in authors' earlier studies . This biomineralization of manganese oxide on the titanium surface by MOB can cause ennoblement with aerobic biofilmed areas which in turn create better cathodes. These cathodes accelerate corrosion in the anodic areas covered with anaerobic SRB biofilms. Thus heterogeneous biofilms provided better galvanic couplings that led to increased localized corrosion of titanium. 24 Schematic representation of localized attack of titanium covered with SRB biofilms by thinning of passive films and when covered with mixed biofilms of SRB and MOB by galvanic coupling is shown in Fig. 9. Conclusions The influence of a consortium of anaerobic and aerobic bacterial biofilms and pure anaerobic bacterial biofilms on the localized corrosion resistance of titanium in natural seawater is studied in this investigation. Exposure of titanium covered with bacterial biofilm to seawater resulted in pitting corrosion. The severity of localized attack increased with increase in the age of the biofilm. From the XRD and XPS investigations, it was inferred that anaerobic biofilms of SRB has created oxygen depleted areas where sulphate reduction by SRB led to sulphide ion formation which caused thinning of passive film and pitting attack. 25 Fig. 9. Schematic representation of localized attack of Ti under SRB and heterogeneous biofilms of SRB and MOB 26 In the case of heterogeneous biofilm, MOBs can create additional cathodes due to biomineralisation of Mn and SRBs can create local anodes under the oxygen depleted condition. Thus heterogeneous biofilms provided more severe galvanic coupling which leads to severe pitting attack. From this investigation it is established that although titanium possess excellent localized corrosion resistance in seawater, bacterial biofilms can breakdown the passive film leading to localized corrosion. Acknowledgments Authors sincerely acknowledge Shri. S.C. Chetal, Director, Indira Gandhi Centre for Atomic Research and Dr. T. Jayakumar, Director, MMG, for their keen interest in the study and constant encouragement. Authors also acknowledge the assistance of Shri. Chitta Ranjan Das and Dr. Panneer Selvam for SEM and XRD studies respectively. 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