Direct and Indirect Evidence of the Microbially Induced Pitting Corrosion of Steel Structures in Humid Environments

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Article Direct and Indirect Evidence of the Microbially Induced Pitting Corrosion of Steel Structures in Humid Environments

Jingzhong Zhou 1, Kuoteng Sun 1, Songqiang Huang 1, Xuemin He 1, Zhaowei Hu 2,* and Wenge Li 2,* 1 Liuzhou Bureau of EHV Transmission Company, China Southern Power Grid Co., Ltd., Liuzhou 545006, China; [email protected] (J.Z.); [email protected] (K.S.); [email protected] (S.H.); [email protected] (X.H.) 2 Merchant Marine College, Shanghai Maritime University, Shanghai 201306, China * Correspondence: [email protected] (Z.H.); [email protected] (W.L.)

Received: 9 September 2020; Accepted: 13 October 2020; Published: 15 October 2020

Abstract: Corrosion is a severe problem for steel structures in humid environments. In particular, humidity usually triggers the surface adhesion of microorganisms, leading to microbiologically induced corrosion. This study aims to explore the effect of bacterial biofilm formation on the pitting corrosion of stainless steel. This research uses electrochemical methods to obtain indirect evidence of the pitting corrosion of steel. In addition, in order to obtain direct evidence of the pitting corrosion of stainless steel, field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM) were used to characterize the dimensional morphology of the stainless steel after pitting. It was shown that the bacterial adhesion increased with the pH and temperature, which significantly increased the surface roughness of the stainless steel. Electrochemical analysis revealed that the formation of biofilm greatly destroyed the oxide film of 304 SS and accelerated the corrosion of stainless steel by forming an oxygen concentration battery. SEM and AFM analyses showed cracks and dislocations on the surface of stainless steel underneath the attached bacteria, which suggested a direct role of biofilm in corrosion induction. The results presented here show that the bacterial biofilm formation on the steel surfaces significantly accelerated the corrosion and affected the pitting corrosion process of the steel structure.

Keywords: microbiologically induced corrosion; pitting corrosion; electrochemical testing; steel structure; SEM; AFM

1. Introduction Corrosion has long been a major concern for steel structures. In some harsh environments—for instance, in humid service environments—microorganisms attach easily to steel surfaces and develop highly organized communities known as biofilms [1–3]. Humidity has been one of the major concerns for the maintenance of steel-structured high-voltage transmission line towers in southern China. Biofilm formation is a multi-stage process, with the adhesion of microorganisms such as bacteria as one of the most important steps [4]. Depending upon where they grow, these bacteria can be beneficial to wastewater treatment [5] or troublesome in biomaterial-related infections [6] and engineering equipment fouling and corrosion [7]. In humid environments, the attachment of bacteria will significantly affect the corrosion of materials [8,9], and pitting corrosion is one of the main means of damage [10]. It is not surprising that microbiological effects are of significant concern in failure analysis and prevention. Microbially induced corrosion problems afflict water-handling operations and manufacturing processes in oil and gas production, pipelining, refining, petrochemical synthesis,

Coatings 2020, 10, 983; doi:10.3390/coatings10100983 www.mdpi.com/journal/coatings Coatings 2020, 10, 983 2 of 11 power production, fermentation, waste water treatment, drinking water supply, pulp and paper making, and other industrial sectors. Microbially induced corrosion is also a concern whenever metals are exposed directly to the environment in applications including marine or buried piping, storage tanks, ships, nuclear waste containers, pilings, marine platforms, and so on. Many studies have reported the effects of bacterial attachment on the pitting corrosion of metallic materials in humid environments [11,12]. The causes of pitting corrosion can be divided into four categories: physical factors, chemical factors, biological factors, and metallurgical factors [13]. Corrosive ions such as Cl− and organisms act as chemical and biological factors for the pitting corrosion of metallic materials. Especially for steels [14], their physical and chemical properties are severely affected by fouling organisms attached to their surfaces [15,16]. The role of bacteria in the pitting corrosion of steels has been a concern of researchers. Yin et al. [17] studied the possible mechanism of accelerated pitting corrosion in 303 stainless steel by Vibrio natriegens. It was found that the fixation of nitrogen by this bacterium promoted the pitting corrosion of 303 stainless steel, where the effect of ammonia was negligible. Xu et al. [18] investigated the role of Pseudomonas aeruginosa in the pitting process of 2205 duplex stainless steel. Their results showed that Pseudomonas aeruginosa produced soluble CrO3 on the surface of the duplex stainless steel, which accelerated the dissolution of the passivation film, in turn contributing to pitting corrosion. Moreover, simulation models were also proposed to study crucial factors affecting the corrosion process [19]. However, there is still a lack of knowledge on the mechanisms of the pitting process by fouling microorganisms on steel. Electrochemical methods have been widely used as an indirect measurement technique to study pitting corrosion. For example, Igual Muñoz et al. [20] studied the effect of bromide concentration and the presence of chromate in solution on the pitting behavior of 2205 duplex stainless steel using cyclic polarization curves and impedance measurements. Their results showed that the pitting potential increased with the increase in the lithium bromide concentration in a semi-logarithmic decreasing trend, suggesting that the addition of bromide reduced the pitting corrosion ability of the chromate on the stainless steel. While electrochemical studies can be used to investigate pitting corrosion, these approaches cannot be used to study and identify microscale pit features [21]. Recently, scanning electron microscopy (SEM) has been used to characterize the morphology of single pits and the growth of pits based on the corrosion morphology. Zhu et al. [22] investigated the influence of additives on the pitting process of 2024-T3 aluminum alloy in a neutral sodium chloride solution using SEM. It was found that bis-[3-(triethoxysilyl) Propyl] tetrasulfide inhibited the pitting corrosion of the aluminum alloy. Pidaparti et al. [23] studied the pitting/cracking of nickel-aluminum bronze alloy in a composite corrosive environment comprised of ammonia and artificial seawater. More recently, AFM observation was used to study the characteristic features of the pitting process. Pan et al. [24] conducted an in situ AFM observation of the pitting corrosion behavior of 304 stainless steel and its nanocrystallization thin films in 3.5% NaCl solution, indicating that nanocrystallization changed the geometry and growth mechanism of stable pits. Zhang et al. [25] investigated the pitting corrosion behavior of 304 stainless steel in 3.5% NaCl solution with the in situ AFM method. The results showed that pitting corrosion products played a major role in promoting the growth of pits. Yet, there are limited studies of pitting corrosion that utilize a combined method of electrochemical testing and direct observation (including SEM and AFM) [26,27]. Pitting corrosion is a long process for stainless steel in humid environments, however most researchers have focused on the effect of microorganisms on the pitting of materials within 30 days [8,9,28]. Long-term data of the high-voltage transmission line tower caused by the microbiologically induced corrosion is yet lacking. In this study, we investigated the effects of different environmental factors—including pH, temperature, and culture age (90 days)—which directly affect the extent of the initial adhesion of typical bacteria and bacterial biofilm formation. The indirect evidence of the effect of Bacillus sp. biofilm formation on the pitting of the steel was analyzed using an electrochemical method. Moreover, the pitting morphology of the steel surfaces was observed Coatings 2020, 10, 983 3 of 11 through SEM and AFM images to provide direct evidence of the impact of bacterial biofilm on the pitting corrosion.

2. Materials and Methods A typical bacteria Bacillus sp. strain (MCCC1A00791) was used in this study. The culture medium for the Bacillus sp. was prepared by dissolving 1 g of yeast extract and 5 g of peptone in 1000 mL of artificial sea water (ASW), which modelled the typical humid environments in most places of southern China close to the sea. The bacteria suspension was shaken at 30 ◦C for 24 h. The inoculated medium of 6 Bacillus sp. was prepared with a density of 10 cells/mL by dilution at 30 ◦C under aerobic conditions. 304 stainless steel (304 SS) plates (10 mm 10 mm 2 mm) were typically used as the substrates × × in this study. The substrates were polished to a mirror finish, followed by a brief rinsing with distilled water, and cleaning ultrasonically in acetone. Prior to performing the corrosion testing, the samples were sterilized with 75% ethanol and UV light for 1 h. All the tests were carried out in ASW. Peptone at 3.0 g/L was added to ASW as carbon and energy sources for the bacteria and sterilized at 121 ◦C for 20 min. The culture media were refreshed every 2 days. To investigate the effect of pH on the bacterial attachment, the media were adjusted to a pH of 4.5, 7, and 9. In order to study the effect of the culture temperature, the inoculated media was grown at 4, 20, and 30 ◦C. A field emission scanning electron microscope (FESEM, FEI Quanta FEG 250, Amsterdam, The Netherlands) was used to characterize the morphology of the surface of the 304 SS specimens and identify any unique features that appeared after exposure to the bacterial media. The energy of the accelerator beam used was 5 kV. A confocal laser scanning microscope (CLSM) (Leica TCS SP5 II, Wetzlar, Germany) was utilized to observe the bacterial attachment and obtain images of the biofilm on the 304 SS surfaces. After incubation, the samples were rinsed with distilled water three times to remove the bacteria that did not adhere to the samples, which was followed by fixation in 2.5% glutaraldehyde. Then, the samples were stained with 150 µL of propidium iodide (PI) for 30 min in the dark, followed by three cycles of cleaning with PBS. The Z-stack technique was used to measure the thickness of the biofilm. A Zeiss LSM 700 confocal laser scanning microscope was used to observe the surface topography of the steel samples before and after their immersion in ASW media. To analyze the biofilm and the pits that formed on the surface, AFM (DimensionTM 3100, Digital Instrument Inc., Veeco, Santa Barbara, CA, USA) in air was used in tapping mode (RTESP Silica cantilever, spring constant: 40 N/m). To examine the extent of the biocorrosion of 304 SS, the biofilm on the substrate was removed by ultrasonic cleaning with distilled water for 5 min and with 17% nitric acid for 2 min, followed by rinsing with distilled water. Electrochemical testing was conducted in a three-electrode cell system where a platinum plate and a saturated calomel electrode (SCE) were used as the counter electrode and reference electrode, respectively [28]. The samples with an exposed area of 1 cm2 served as the working electrode. The steel samples were immersed in clean ASW and also in ASW that contained bacteria, and incubated at 30 ◦C in a shaker at 120 rpm. Cyclic potentiodynamic polarization (CPP) and electrochemical impedance spectroscopy (EIS) spectra were obtained by a Solartron Modulab system (2100A, Solartron Electronic Group, Leicester, UK). EIS measurements were achieved by applying an AC signal of 10 mV at a frequency ranging from 100 kHz to 0.01 Hz. The obtained data were then analyzed using ZSimpWin (version 3.30). The CPP curves were acquired by scanning the potential in the forward direction with a scanning rate of 0.5 mV/s from 500 to 900 mV. For the back scan, the potential was reversed to − complete the cycle test. Three samples were tested in each group to ensure reproducibility. To examine the bacterial growth rate and the dissolved oxygen (DO) concentration, a 1.5 mL 8 bacteria suspension (10 Cells/mL) was added to 150 mL of medium and cultured at 30 ◦C in a shaker at 120 rpm. The DO concentration was measured using a logging DO meter (HANNA instruments HI2400, Limena, Italy). Optical density measurements were performed at different time points at 600 nm using a spectrophotometer (SpectraMAX 190, Molecular DevicesTM, San Jose, CA, USA). Coatings 2020, 10, 983 4 of 11 Coatings 2020, 10, x FOR PEER REVIEW 4 of 11

3. Results and Discussion

3.1. Surface Morphology and Bacterial Adhesion In order to obtainobtain the identical surface roughness of the steel in thisthis study, thethe surfacessurfaces of thethe 304 SS were polished to a mirror finish,finish, since surface topography is an important factor aaffectingffecting the bacterial attachment to surfaces prior to biofilmbiofilm formationformation [[29,30].29,30]. Previous studies have shown that the colonizationcolonization of of microorganisms microorganisms on on surfaces surfaces nucleates nucleates on surfaceon surface irregularities irregularities and growsand grows from thesefrom areasthese [areas31,32 ].[31,32]. Figure Figure1 shows 1 CLSMshows imagesCLSM images of bacterial of bacterial biofilm formedbiofilm onformed the smooth on the 304 smooth SS surfaces 304 SS aftersurfaces the diafterfferent the immersiondifferent immersion periods. They periods. show Th theey coverage show the (Figure coverage1a) and (Figure thickness 1a) and (Figure thickness1b) of the(FigureBacillus 1b) of sp. thebiofilms. Bacillus Fromsp. biofilms. the images, From itthe can images, be seen it can that be biofilm seen that thickness biofilm increased thickness overincreased time, andover a time, uniform and distribution a uniform ofdistribution the bacterial of cells the onba thecterial smooth cells surfaceon the wassmoot observed.h surface Paramonova was observed. et al. foundParamonova that, for et microorganismal. found that, for biofilms microorganism less than 120biofilmsµm thick, less than the CLSM120 µm method thick, the reliably CLSM measures method thereliably biofilm measures thickness the [ 33biofilm]. thickness [33].

Figure 1.1. CLSMCLSM imagesimages of ofBacillus Bacillus sp. sp.biofilm biofilm formed formed on theon the steel steel surfaces surfaces after after (a–1) ( 1a–1 day,) 1( a–2day,) 1(a–2 week) 1, andweek, (a–3 and) 1(a–3 month) 1 month and side and view side imagesview images of biofilm of biofilm that showthat show biofilm biofilm thickness thickness after after (b–1 )(b–1 1 day,) 1 (day,b–2) ( 1b–2 week,) 1 week, and ( b–3and) ( 1b–3 month.) 1 month.

The topographies of the smooth surfaces in the presence and absence of biofilmsbiofilms are shown in Figure2 2.. TheThe 304304 SSSS averageaverage surfacesurface roughnessroughness (Ra)(Ra) beforebefore andand afterafter thethe attachmentattachment ofof thethe bacteriabacteria were 0.580.58 µµmm (Figure(Figure2 2a)a) and and 6.30 6.30 µµmm (Figure(Figure2 2b),b), respectively. respectively. These Theseresults resultsshow showthat thatthe thesurface surface roughness increasedincreased significantlysignificantly afterafter thethe biofilmbiofilm formationformation onon thethe smoothsmooth surface.surface.

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Figure 2. SurfaceSurface topography of the steel plates ( a) with and ( b) without Bacillus sp. biofilmbioﬁlm formation.

Bacterial adhesion may be significantly significantly affected affected by the pH of the solution in which the steel substrates are immersed. As As shown shown in in Figure 33a,a, therethere werewere onlyonly aa fewfew bacteriabacteria thatthat adheredadhered toto thethe substrate atat aa pHpH of of 4.5, 4.5, and and few few bacteria bacteria were were observed observed on on the the substrate substrate at a at pH a pH of 7; of however, 7; however, there there was awas dense a dense accumulation accumulation and adhesion and adhesion of the of bacteria the bacteria on the on substrate the substrate at a pH at of a 9. pH These of 9. results These suggestresults thatsuggest the changethat the in change bacterial in bacterial adhesion adhesion at different at pHsdifferent was likelypHs was due likely to the due ionization to the ionization of the functional of the groupsfunctional of Bacillusgroups sp.of ,Bacillus such as sp. carboxyl, such as and carboxyl amino groupsand amino [34]. groups The electrostatic [34]. The forceselectrostatic between forces the Bacillusbetween sp. theand Bacillus the 304 sp. SSand surface the 304 were SS relativelysurface were weak relatively at pH 4.5. weak At aatpH pH of 4.5. 7, theAt a appearance pH of 7, the of negativelyappearance charged of negatively carboxyl charged groups carboxyl increased groups the electrostatic increased force the ofelectrostatic attraction betweenforce of 304attraction SS and Bacillusbetween sp. 304, resulting SS and Bacillus in greater sp. adhesion., resulting Thein greater adhesion adhesion. increased The further adhesion at a increased pH of 9 compared further at to a that pH atof a9 pHcompared of 7 (Figure to that3(a–3)). at a pH In of this 7 study,(Figure except 3(a–3)). for In pH, this it study, was also except found for that pH, the it was temperature also found of that the culturethe temperature solution of aff theects culture the bacterial solution adhesion. affects the In ba dicterialfferent adhesion. seasons andIn different regions, seasons the temperature and regions, of seawaterthe temperature varies greatly, of seawater generally varies varying greatly, from generally1 to 30varyingC[35 ].from To optimize−1 to 30 °C the [35]. culture To optimize temperature the − ◦ forculture bacterial temperature adhesion, for this bacterial study hasadhesion, conducted this bacterialstudy has adhesion conducted tests bacterial at different adhesion temperatures. tests at Fromdifferent Figure temperatures.3b, it is observed From that FiguBacillusre 3b, sp.it producedis observed a biofilm that Bacillus that contained sp. produced only sparse a biofilm clusters that of cellscontained at 4 ◦ Conly (Figure sparse3(b–1)). clusters This of wascells probably at 4 °C (F dueigure to 3(b–1)). the lower This growth was pr rateobably of the due bacteria to the under lower lowgrowth temperatures. rate of the bacteria However, under at both low 20 temperatures and 30 ◦C (Figure. However,3(b–2,b–3)), at both the 20 biofilm and 30 showed°C (Figure a relatively 3(b–2,b– complex3)), the biofilm 3D structure, showed consisting a relatively of complex dense aggregates 3D structure, of Bacillus consisting sp. held of dense together aggregates by an extracellular of Bacillus matrix.sp. held Thus,together the by temperature an extracellular of 30 ◦matrix.C was chosenThus, the for temperature the following ofBacillus 30 °C was sp.-induced chosen for pitting the corrosionfollowing experiments.Bacillus sp.-induced pitting corrosion experiments.

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Figure 3. SEM images of the attached Bacillus sp. inin different diﬀerent ( a) pHs and ( b) at at different diﬀerent temperatures

on the steel steel surface: surface: (a–1 (a–1) pH) pH = 4.5,= 4.5, (a–2 (a–2) pH) pH= 7, =and7, ( anda–3) (pHa–3 )= pH9; (b–1= 9;) at (b–1 4 °C,) at (b–2 4 ◦)C, 20 ( b–2°C, )and 20 (◦b–C, and3) 30 ( b–3°C. ) 30 ◦C.

3.2. Electrochemical Analysis The Nyquist plotsplots forfor the corrosion ofof thethe 304304 SS with and without exposure toto BacillusBacillus sp.sp. during thethe studystudy afterafter 9090 daysdays ofof immersionimmersion areare shownshown inin FigureFigure4 4a.a. InIn thethe presencepresence ofof bacteria, bacteria, thethe charge charge transfer resistance (R ) value was 212.5 kΩ cm2, compared to that of the control in clean ASW of transfer resistance (Rctct) value was 212.5 kΩ·cm2, compared to that of the control in clean ASW of 7355 2 · 2 7355 kΩ2 cm after 90 days. From a previous study [28], the Rct values were 1246 and 2195 kΩ cm2 after kΩ·cm ·after 90 days. From a previous study [28], the Rct values were 1246 and 2195 kΩ·cm· after 7 7 and 30 days of immersion, compared to those of the control in clean ASW of 3733 and 6908 kΩ cm2. and 30 days of immersion, compared to those of the control in clean ASW of 3733 and 6908 kΩ··cm2. By comparing the R data of 316 SS, interestingly, in the presence of bacteria, the R of 304 SS increased By comparing the ctRct data of 316 SS, interestingly, in the presence of bacteria,ct the Rct of 304 SS first and then greatly decreased. The R increased in the presence of bacteria after 30 days of exposure increased first and then greatly decreased.ct The Rct increased in the presence of bacteria after 30 days becauseof exposure biofilm because formed biofilm on the metalformed surface. on the This metal was su likelyrface. due This to thewas fact likely that due the biofilmsto the fact interrupted that the biofilms interrupted the flow of ions and oxygen and the biofilm acted as a protective coating to prevent corrosion. However, extended soaking for up to 90 days greatly reduced the Rct to 212.5

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Coatings 2020, 10, x FOR PEER REVIEW 7 of 11 the flow of ions and oxygen and the biofilm acted as a protective coating to prevent corrosion. However, 2 kextendedΩ·cm2, which soaking was for only up to about 90 days one greatly tenth reducedof that of the thRect sampleto 212.5 after kΩ cm 30 ,days which exposure. was only The about result one · indicatestenth of that that of the sampleoxide film after of 30 days304 SS exposure. was grea Thetly result destroyed, indicates and that the the bacteria oxide film accelerated of 304 SS wasthe corrosiongreatly destroyed, of stainless and steel. the This bacteria could accelerated have been the due corrosion to the biofilm of stainless preventing steel. oxygen This could from have diffusing been todue the to surface the biofilm of stainless preventing steel, oxygen causing from internal diffusing and external to the surface differences of stainless in the steel,oxygen causing concentration internal and externalthereby forming differences an inoxygen the oxygen concentration concentration battery and, which thereby accelerated forming the an oxygencorrosion concentration of stainless steel.battery, Therefore, which accelerated the Rct value the decreased corrosion after of stainless 90 days steel.of expo Therefore,sure. However, the Rct invalue clean decreased ASW solution after (control),90 days of the exposure. Rct value However, increasedin with clean the ASW increasing solution expo (control),sure time, the sinceRct value thin increasedoxide layers with were the formedincreasing by exposurepassivation time, on sincethe 304 thin SS oxidesurface, layers and were these formed oxide layers by passivation protect alloys on the from 304 SSaggressive surface, mediaand these such oxide as chlorides. layers protect In the inoculat alloys fromed solution, aggressive the mediadecrease such in resistance, as chlorides. Rct, Inindicates the inoculated that the oxidesolution, film the on decreasethe surfaces in resistance, is not stableRct in, indicates the presence that theof Bacillus oxide filmsp. bacteria. on the surfaces This was is likely not stable due to in bacteriathe presence destroying of Bacillus the sp.passivationbacteria. film This and was accelera likely dueting to the bacteria pitting destroying of stainless the steel passivation in sensitized film areas,and accelerating such as at thegrain pitting boundaries of stainless where steel elemen in sensitizedts such areas,as chromium such as at (Cr) grain were boundaries dissolved where and consumed,elements such eventually as chromium causing (Cr) stainless were dissolved steel to andfail [36]. consumed, This failure eventually is also causing expected, stainless since steel304 SS to alloysfail [36 are]. This vulnerable failure isto alsopitting expected, in seawater since that 304 SScontains alloys Bacillus are vulnerable sp. Therefore, to pitting the inweakness seawater ofthat the passivecontains filmBacillus at Cr-depleted sp. Therefore, regions the renders weakness the of passive the passive film in film such at regions Cr-depleted less effective regions at renders protecting the passivethe underlying film in suchsteel substrate regions less and e ffresultsective in at film protecting breakdown the underlying and the initiation steel substrate of localized and corrosion results in [37].film breakdownIn this study, and to thefurther initiation investigate of localized the effect corrosion of Bacillus [37]. sp. In adhesion this study, on to the further pitting investigate corrosion the of 304effect SS of by,Bacillus the immersion sp. adhesion time on thewas pitting extended corrosion to 90 ofdays 304 SSand by, cyclic the immersion polarization time was was also extended used to determine90 days and the cyclic change polarization in the corrosi wason also and used pitting to determinemechanism the of changethe metal in surface the corrosion in the presence and pitting of Bacillusmechanism sp. (Figure of the metal 4b). It surface has been in thesuggested presence that of Bacillus the tendency sp. (Figure toward4b). pitting It has been may suggestedbe determined that the by thetendency feature toward of the pitting hysteresis may loop be determined [38]. In the by absence the feature of Bacillus of the hysteresis sp., 304 SS loop has [38 noticeable]. In the absence negative of Bacillushysteresis, sp., 304which SS hassuggests noticeable that negative pitting hysteresis,corrosion whichis not suggestslikely to that occur. pitting However, corrosion the is notpositive likely hysteresisto occur. However, in the presence the positive of bacteria hysteresis indicates in the that presence 304 SS of surfaces bacteria are indicates susceptible that 304 to pitting SS surfaces (Figure are 4b).susceptible to pitting (Figure4b).

Figure 4. (a) Nyquist plotsplots andand ( b(b)) potentiodynamic potentiodynamic polarization polarization curves curves of of the the steel steel samples samples tested tested in the in ASWthe ASW with with (Bacillus (Bacillus sp.) andsp.) withoutand without (sterile) (sterile) the bacteria the bacteria after 90afte daysr 90 ofdays exposure. of exposure. SS: the SS: steel the plates. steel plates. To understand better the effect of Bacillus sp. biofilm formation on the corrosion behavior of 304 SS, the dissolvedTo understand oxygen better (DO2 )the in theeffect solution of Bacillus that containedsp. biofilm bacteria formation was on investigated. the corrosion Figure behavior5 shows of the304 variation in the DO concentration and Bacillus sp. growth during 6 h of incubation. The concentration SS, the dissolved oxygen2 (DO2) in the solution that contained bacteria was investigated. Figure 5 of bacteria in the solution increased with time. The dissolved oxygen was consumed by Bacillus sp. shows the variation in the DO2 concentration and Bacillus sp. growth during 6 h of incubation. The concentrationand the O2 concentration of bacteria in decreased the solution to its increa lowestsed value with (0.03time. mgThe/L) dissolved after approximately oxygen was 600consumed min of exposure. Biofilm inhibits oxygen diffusion towards the metallic surface [39], decreasing the cathodic by Bacillus sp. and the O2 concentration decreased to its lowest value (0.03 mg/L) after approximately 600reaction, min of and exposure. it will cause Biofilm an inhibi inhibitionts oxygen of corrosion diffusion initially. towards It the was me reportedtallic surface that the [39], bacteria decreasing tend theto inhibit cathodic the reaction, corrosion and of stainlessit will cause steel an in inhibition a short period of corrosion [28]. However, initially. as It thewas exposure reported time that wasthe bacteriaextended, tend this to would inhibit form the corrosion an oxygen of concentrationstainless steel battery,in a short resulting period [28]. in the However, accelerated as the corrosion exposure of timestainless was steel. extended, this would form an oxygen concentration battery, resulting in the accelerated corrosion of stainless steel.

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Figure 5. The variation variation in in the the dissolved dissolved oxygen oxygen concentration concentration (mg/L) (mg/L) and and OD OD600nm600nm600nm (the(the(the absorbanceabsorbance absorbance ofof bacterialof bacterial solution solution at 600 at 600 nm) nm) (as (asa growth a growth rate) rate) in thethe in the bacterialbacterial bacterial solutionsolution solution versusversus versus timetime time atat 3030 at °C°C 30 andand◦C and 120120 120rpmrpm rpm speed.speed. speed. DuringDuring During thethe the firstfirst first 600600 600 minmin min ofof of incubationincubation incubation,,, thethe the oxygenoxygen oxygen concentrationsconcentrations concentrations werewere significantly significantlysignificantly decreased andand reachedreached theirtheir lowestlowest valuevalue atat 600600 minmin of of exposure; exposure; meanwhile, meanwhile, the the OD OD600nm600nm600nm valuevalue was was incrementallyincrementally increasedincreaseincreased fromfrom 0.0010.001 toto 0.060.06 andand stayedstayed stable.stable. 3.3. Direct Observation of Bacteria-Induced Pitting Corrosion 3.3. Direct Observation of Bacteria-Inducederia-Induced PittingPitting CorrosionCorrosion Although electrochemical testing can be used to examine pitting, these approaches cannot be Although electrochemical testing can be used toto examineexamine pitting,pitting, thesthesee approachesapproaches cannotcannot bebe used to study microscale pit features [21]. In this study, direct observation, including SEM and AFM, used to study microscale pit features [21]. In this study, direct observation, including SEM and AFM, was also performed to investigate the effect of biofilm on the pitting corrosion. The SEM analysis was also performed to investigate thethe effecteffect ofof biofilmbiofilm onon thethe pipittingtting corrosion.corrosion. TheThe SEMSEM analysisanalysis showed that cracks and dislocations were clearly observed on the metal surface after the removal of showedshowed thatthat crackscracks andand dislocationsdislocations werewere clearlyclearly observedobserved onon thethe metalmetal surfacesurface afterafter thethe removalremoval ofof the biofilm (Figure6a,b). thethe biofilmbiofilm (Figure(Figure 6a,b).6a,b).

Figure 6. SEMSEM imagesimages ofof thethe steelsteel surfacessurfaces (( aa)) beforebefore andand (( bb)) afterafter removingremoving the the biofilm biofilmbiofilm and and corrosion corrosion by-products. These These surfaces surfaces were were initially initially exposedexposed exposed toto to artificialartificial artificial seawaterseawater seawater containingcontaining containing BacillusBacillus sp.sp. forfor for 9090 days.90 days. Afterwards, Afterwards, the the biofilm biofilm and and corrosion corrosion by-products-products by-products werewere were removedremoved removed fromfrom from thethe the surface.surface. surface.

To obtain detailed detailed information information of of the the effect eﬀect of ofBacillusBacillus sp. sp.adhesionadhesionadhesion onon thethe on 304304 the SSSS 304 surface,surface, SS surface, AFMAFM AFMobservation observation was performed was performed in this study. in this AFM study. images AFM of images the 304 of SS the exposed 304 SS for exposed 90 days for to 90 ASW days that to ASWcontainedcontained that containedbacteriabacteria areare bacteria shownshown are inin FigureFigure shown 7.7. in Interestingly,Interestingly, Figure7. Interestingly, thethe AFMAFM imagesimages the AFM beforebefore images andand before afterafter removing andremoving after theremovingthe biofilmbiofilm the fromfrom bioﬁlm thethe from304304 SSSS the surfacesurface 304 SS surfaceshowedshowed showed aa diredirectct a correlation directcorrelation correlation betweenbetween between thethe bacterialbacterial the bacterial cellscells cellsandand underlyingand underlying pits (Figure pits (Figure 7a,b)7 ona,b) the on 304 the SS 304 surface. SS surface. The shape The shapeand size and of size the pits of the on pits the steel on the surface steel aresurface similar are to similar those toof thosethe Bacillus of the sp.Bacillus cell.cell. ThisThis sp. cell.resultresult This suggestssuggests result aasuggests directdirect rolerole a direct ofof Bacillus role ofsp. Bacillus onon thethe pitpit sp. initiationoninitiation the pit onon initiation thethe 304304 SSSS on surface.surface. the 304 AA SS similarsimilar surface. resultresult A similar waswas alsoalso result reportedreported was also forfor DesulfovibrioDesulfovibrio reported for Desulfovibrio (D.)(D.) desulfuricansdesulfuricans (D.) [40].desulfuricans[40]. [40].

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FigureFigure 7.7. AFMAFM topographical topographical images images of of the the steel steel plates plates after after incubation incubation for for 90 90days days in the in the ASW ASW (a) (beforea) before and and (b) ( bafter) after the the removal removal of of the the biofilm biofilm that that demo demonstratesnstrates the the obvious obvious pitting of the steelsteel samplessamples afterafter thethe removalremoval ofof thethe bacterialbacterial biofilm.biofilm. 4. Conclusions 4. Conclusions To simplify the study of the microbiologically induced corrosion of steel structures of high-voltage To simplify the study of the microbiologically induced corrosion of steel structures of high- transmission line towers installed in humid environments, the attachment behaviors of a typical voltage transmission line towers installed in humid environments, the attachment behaviors of a bacterium Bacillus sp. and the subsequent formation of bacterial biofilms on the steel surface were typical bacterium Bacillus sp. and the subsequent formation of bacterial biofilms on the steel surface investigated. The effect of biofilm formation on the pitting corrosion of the steel surface was studied were investigated. The effect of biofilm formation on the pitting corrosion of the steel surface was by indirect and direct methods. The adhesion of bacterium Bacillus sp. to the 304 SS surfaces is studied by indirect and direct methods. The adhesion of bacterium Bacillus sp. to the 304 SS surfaces strongly influenced by physical and chemical parameters, including pH, temperature, and culture is strongly influenced by physical and chemical parameters, including pH, temperature, and culture age. Indirect evidence from electrochemical measurements and direct evidence from SEM and AFM age. Indirect evidence from electrochemical measurements and direct evidence from SEM and AFM indicate that the adhesion of Bacillus sp. to metal surfaces significantly accelerated the corrosion indicate that the adhesion of Bacillus sp. to metal surfaces significantly accelerated the corrosion process of 304 SS after long-term exposure (90 days) to the bacterium. Thus, the initial pitting corrosion process of 304 SS after long-term exposure (90 days) to the bacterium. Thus, the initial pitting influenced by biofilm formation can be obtained using indirect and direct methods. The results can corrosion influenced by biofilm formation can be obtained using indirect and direct methods. The give insight into the understanding and effective control of microbiologically induced corrosion of the results can give insight into the understanding and effective control of microbiologically induced steel structures of high-voltage transmission line towers. corrosion of the steel structures of high-voltage transmission line towers. Author Contributions: Conceptualization, J.Z. and Z.H.; methodology, J.Z. and Z.H.; software, S.H.; validation, J.Z. andAuthor Z.H.; Contributions: formal analysis, Conceptualization, J.Z., K.S., and J.Z. S.H.; and investigation, Z.H.; methodology, J.Z., K.S., J.Z. S.H.,and Z.H.; and software, X.H.; data S.H.; curation, validation, J.Z. andJ.Z. and K.S.; Z.H.; writing—original formal analysis, draft J.Z., K. preparation,S., and S.H.; J.Z.; investigation, writing—review J.Z., K.S., and S.H., editing, and X.H.; Z.H.; data supervision, curation, J.Z. W.L.; and projectK.S.; writing—original administration, draft Z.H.; preparat fundingion, acquisition, J.Z.; writing—review W.L. All authors and haveediting, read Z.H.; and supervision, agreed to the W.L.; published project versionadministration, of the manuscript. Z.H.; funding acquisition, W.L. All authors have read and agreed to the published version of the Funding:manuscript.This research was funded by China Southern Power Grid Co., Ltd. Liuzhou Bureau of EHV Transmission Company, grant number CGYKJXM2017039. Funding: This research was funded by China Southern Power Grid Co., Ltd. Liuzhou Bureau of EHV Acknowledgments:Transmission Company,The authorsgrant number acknowledge CGYKJXM2017039. the Ningbo Institute of Materials Technology and Engineering, CAS for the experiment support, and especially thank Hua Li and Yi Liu for their great help. ConflictsAcknowledgments: of Interest: TheThe authors authors acknowledge declare no conflict the Ningbo of interest. Institute of Materials Technology and Engineering, CAS for the experiment support, and especially thank Hua Li and Yi Liu for their great help. ReferencesConflicts of Interest: The authors declare no conflict of interest.

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