Environment Health Techniques 1168
Johnson Lin and Bafana B. Madida
Research Paper Biofilms affecting progression of mild steel corrosion by Gram positive Bacillus sp. Johnson Lin and Bafana B. Madida School of Life Sciences, University of KwaZulu-Natal Westville, Private Bag X54001, Durban 4000, South Africa
The biodeterioration of metals have detrimental effects on the environment with economic implications. The deterioration of metals is of great concern to industry. In this study, mild steel coupons which were immersed in a medium containing Gram-positive Bacillus spp. and different nutrient sources were compared with the control in sterile deionized water. The weight loss of the coupons in the presence of Bacillus spp. alone was lower than the control and was further reduced when additional carbon sources, especially fructose, were added. The level of metal corrosion was significantly increased in the presence of nitrate with or without bacteria. There was a significant strong correlation between the weight loss and biofilm level (r ¼ 0.64; p < 0.05). The addition of nitrate and Bacillus spp. produced more biofilms on the coupons and resulted in greater weight loss compared to that with Bacillus spp. only under the same conditions. However, Bacillus spp. enriched with carbon sources formed less biofilms and results in lower weight loss compared to that with Bacillus spp. only. The production of biofilm by Bacillus spp. influences the level of metal corrosion under different environmental conditions, thereby, supporting the development of a preventive strategy against corrosion. Keywords: Biofilm / Gram-positive Bacillus / Microbial influenced corrosion / Mild steel coupons / Nutrient supplementation / Weight loss Received: November 20, 2014; accepted: March 1, 2015 DOI 10.1002/jobm.201400886
Introduction For all industries, across all borders, from under developed to highly developed countries, there is a strong concern regarding the factors influencing and affecting the deterioration of metal turbines and pipelines [1]. The biodeterioration of metals does not only have detrimental effects on the environment and population dynamics but also on the economy [2, 3]. The detection of microbial communities in biofilms on corroded metal substrates triggered an interest on the role that bacteria play in biocorrosion [3]. An electrochemical interaction, between the organism and the metallic surface/structure, can promote physicochemical reactions triggering, aiding, and exacerbating the Correspondence: Prof. Johnson Lin, School of Life Sciences, University of KwaZulu-Natal (Westville), Private Bag X54001, Durban 4000, Republic of South Africa E-mail: [email protected] Phone: þ27-31-2607407 Fax: þ27-31-2607809 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
deterioration of the metal leading to metal damage [4, 5]. Biocorrosion contributes to different forms of corrosions namely uniform, pitting, crevice, galvanic, intergranual, dealloying, and stress corrosions [1]. Biofilm structures are not static, but dynamic. Biofilms form immediately after metals are submerged into a medium (e.g., soil, water). Inorganic ions and organic compounds (EPS production) deposited by microorganisms encourage further microbial growth [3, 6, 7] and create localized microenvironments which facilitate the electrochemical process of localized corrosion at the biofilm–metal interface [3, 8]. Their ability to accelerate corrosion is due to bacterial metabolic reactions such as acid production, sulphite production and ammonia production, metal deposition, and most importantly their effects of redox potential on metal corrosion [1]. Fang et al. found that the corrosion patterns appear to match the microbial cluster patterns of the aggregated biofilms [6]. However, no quantitative relationship was found between the degree of increased corrosion and the toxicity of metals/chemicals on
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sulfate-reducing bacteria, or the increased EPS production. EPS presence (biofilms) enables microorganisms to become more resistant to biocides than free living planktonic organisms [9]. Formed biofilms are more resistant to metabolizing inhibiting compounds including aliphatic amines and nitrites. Several factors including availability of nutrients for survival and reduced concentration of toxins encourage biofilm development [10]. Sheng et al. [11] showed that bacterial and metal interactions tend to be reduced when nutrients are present in the solution. Stronger ionic strength in the solution and when the pH of the solution was near the isoelectric point of the bacteria also results in a higher bacteria–metal adhesion forces. Forte Giacobone et al. also showed that Bacillus cereus is capable of promoting corrosion in nutrient poor and nutrient mild conditions [12]. Other researchers suggested that the presence of a nutrient source leads to an increase in the proliferation of bacteria on steel coupons and influences the extent to which corrosion takes place in product pipelines [13, 14]. The form in which corrosion takes place is dependent on environmental factors which impact on the progression of corrosion [15, 16]. In mixed consortia, many different corrosion mechanisms may occur that may even be synergistic in nature. The rates of corrosion obtained during studies using mixed consortia of microbes are considerably higher than with pure cultures [3]. When a material is exposed to a uniform, pure group of micro-organisms, it becomes much easier to detail and explain the mechanisms of corrosion occurring. The aim of this study was to investigate biocorrosion induced by Bacillus spp. on mild steel coupons when supplemented with different nutrient sources. We demonstrate that the ability of Gram-positive Bacillus spp. to form biofilm has a direct impact on the level of mild steel corrosion.
Materials and methods Corroding isolates Gram-positive Bacillus spp. (n ¼ 11) isolated from corroded stainless steel coupons [13] were obtained from the Department of Microbiology, Westville Campus, University of KwaZulu-Natal. A series of sub-culturing on nutrient agar plates was carried out and isolates were maintained in nutrient agar slants for short term storage and stored in 40% glycerol in Microbanks at 80 °C. Characterization and identification of bacteria isolates Characterization of the bacteria isolates was carried out according to Bergy’s Manual of Determinative ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Bacteriology [17]. Characteristics observed include morphological analysis—Gram stain, shape, sporulation, biochemical tests—catalase, IMViC (indole, methyl red, Voges–Proskauer, and citrate tests), urease, nitrate reduction and oxidase tests, carbohydrate metabolism 1 on Analytical Profile Index (API) 50 CH strips (bioMerieux, France) according to manufacturer’s instruction and hydrolysis of starch, casein, and gelatine. This was confirmed by API 50CHB and, thereafter, the sequences were submitted to Bankit (GenBank) to obtain valid accession numbers. Identification was confirmed using 16S rRNA sequencing [18], followed by NCBI Blast comparison software to reveal the identities of the isolates [19] and the MALDI Biotyper (the Peptide and Catalysis Unit, Department of Chemistry, Westville Campus, University of KwaZulu-Natal). The bacterial nucleotide sequences were submitted to the GenBank database under accession numbers JN106407-JN106409, JN106417, JN106418, JN106422, JN106423, and JN106426. Amplifications of Bacillus strains CS2 and CL1 and LS8 using 16S rRNA PCR were not successful. Corrosion experimental set up Determination of corrosion was done using the weight loss method as described by Pillay and Lin [13] and RhylSvendsen [20]. The mild steel coupons (25 25 mm) were 1 manually polished with a Dremel 3000 Variable Speed Rotary Tool, having attached a fine sanding band to it, followed by rinsing the coupons under flowing tap water, dried then degreased in acetone. The weight of each coupon was measured (g), attached to a nylon fishing string and then sterilized. For standardization purposes, Bacillus isolates were grown in 40 ml of nutrient broth overnight at 30 °C in conical centrifuge tubes, followed by centrifugation at 3000 rpm for 10 min at 4 °C to obtain bacterial pellet. The pellet was washed with sterile deionized water twice, followed by centrifugation as described above. The culture inoculums were standardized to an absorbance of 0.80 0.05 at 600 nm in sterilized deionized water (dH2O). One milliliter of standardized culture was used in the following experimental set up to determine the effect of each isolate on corrosion rate in the presence of supplemented nutrients. The experimental sets were prepared according to Rajasekar and Ting [21]. Four pre-treated mild steel coupons were placed in each 250 ml flask each containing 150 ml sterilized dH2O (as the blank control) or in media (as the cell-free control) supplemented with the following individual nutrient sources: Carbon source (50 mg L1—fructose, galactose, or sucrose); MgSO4 (0.5 g L1); Nitrate source (0.5 g L1—NH4NO3 or NaNO3).
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One milliliter of standardized bacterial inoculum was inoculated into each individual set of flasks prepared as above. At the end of the incubation period (30 °C, 100 rpm for a period of 10 days), the coupons were removed for biofilm assay, weight loss determination, and microscopic analysis. The experiments were performed in triplicates. Biofilm assay Biofilm quantification was carried out according to Stepanovic et al. [22] and Adetunji and Isola [23] with modification. Each set (3) of coupons was gently dipped in tap water to remove and wash off any corrosion products. The remaining adherent cells were fixed with a fixative agent (absolute methanol) in staining jars for 15 min, followed by air-drying. Air-dried coupons were then stained with 2% Hucker’s crystal violet for 15 min followed by washing of excess stain under smoothly running tap water. The coupons were then air dried and followed by resolubilization with 15 ml of 33% glacial acetic acid in a sonicator (EUMAX Digital Ultrasonic Cleaner) for 10 min. The absorbance (optical density) of each resolubilization solution was measured at a wavelength of 595 nm (ThermoLabSystems Multiskan RC Microplate Reader coupled with the Ascent Software for MultiScan v2.6). The actual value of biofilm assay for each experimental coupon was obtained by subtracting the absorbance of the blank reading without inoculation. Weight loss determination Following the biofilm assay, the same coupons were soaked in 20% HCl, thereafter, rinsed in deionized water, then allowed to completely dry [20]. The weight loss of mild steel coupons was determined using following equation, representing it as the weight loss per surface area: Weightlosspersurfaceareaðmgcm2 Þ ðW initial Wfinal Þ 1000 ¼ Surfaceareaðcm2 Þ
Microscopic analysis The remaining mild steel coupon after 10-day incubation period was examined at the Electron Microscopy Unit (Westville Campus, University of KwaZulu-Natal) to observe their morphological and topographical characteristics using the Scanning Electron Microscopy (SEM) Leo 1450, or Light Microscope Nikon AZ100. Adhered cells (biofilms) and corroded products were observed according to the method of Abed et al. [24]. Some ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
coupons which had been analyzed (biofilm assay and weight loss) were also analyzed under the microscope. Statistical analysis In correlating the level of biofilm and weight loss of mild steel coupon to nutrients supplemented and present in the environment, the product moment (Pearson’s) correlation coefficient was obtained using the SPSS program version 21 (SPSS, Inc., IL). The significance of the observed results was tested using Student’s t-test to measure the possibility of an existing linear relationship between variables [25]. Probability (significant level) was set at p < 0.05 and p < 0.01.
Results Characterization of bacterial isolates Characterizations and biochemical properties Grampositive Bacillus spp. (n ¼ 11) isolated from corroded stainless steel coupons were further studied. The results are shown in Table 1. All the isolates used in this study were confirmed as Gram-positive Bacillus spp. (Table 1). Sporulation was observed in all isolates with growth at all tested incubation temperatures. Optimal growth was observed in 35 °C for all isolates, with the exception of Isolate CL1 (at 25 °C). All isolates contained oxidase, catalase activities, and hydrolyzed casein as carbon sources with various abilities to use Tween and starch. All isolates except LS4 possessed acid fermentation ability (methyl red positive). The reduction of nitrate to nitrite was positive for all isolates with exception to LS4, SS1, and SS2. All isolates metabolized ribose, glucose, fructose, N-acetylglucosamine, and arbutin as carbon source coupled with acid production. Influence of nutrients on corrosion Effects of nutrients on corrosion of mild steel coupons induced by characterized Bacillus isolates were investigated. Figure 1 represents the corrosion rates of mild steel coupons progressed over a 10-day incubation period with or without nutrients and Gram-positive Bacillus isolates. The corrosion process occurred even without any nutrient supplements or the presence of the bacterial inoculum (108 11.91 mg cm2—the control which were immersed in the sterilized deionized water) indicating other corrosion process than biocorrosion was taking place. An increase in corrosion was observed when the media were supplemented with sodium nitrate (123.47 9.40 mg cm2) compared to the weight loss of the control. There is no significant difference (p > 0.05) in the weight loss of the coupons between the media
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Table 1. Partial biochemical characterization of bacterial isolates. Characteristics cell morphology gram stain shape sporulation growth at 25 °C 35 °C 45 °C biochemical reactions indole test methyl red Voges–Proskauer citrate utilization nitrate reduction starch hydrolysis oxidase test catalase test Tween-80 casein hydrolysis glycerol erythritol D-arabinose L-arabinose ribose D-xylose L-xylose adonitol methyl b-xyloside galactose D-glucose D-fructose mannose sorbose rhamnose dulcitol inositol mannitol sorbitol Characteristics methyl a-D-mannoside methyl a-D-glucoside N-acetylglucosamine amygdalin arbutin aesculin salicin cellobiose maltose lactose melibiose sucrose trehalose inulin melezitose D-Raffinose starch glycogen xylitol b-gentiobiose
CS1
CS2
CL1
CL2
LA2
LS4
LS7
LS8
NL3
SS1
SS 2
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ w þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ w* þ þ w
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ
CS1
CS2
CL1
CL2
LA2
LS4
LS7
LS 8
NL 3
SS 1
SS 2
þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ w
þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ
þ þ þ w w þ – þ þ þ þ –
w w þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ w þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
w þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ w þ þ w þ þ
þ þ þ þ þ þ þ þ w þ þ þ þ þ
(Continued) ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Table 1. (Continued) Characteristics D-turanose D-lyxose D-tagatose D-fucose L-fucose D-arabitol L-arabitol gluconate 2-ketogluconate 5-ketogluconate
CS1
CS2
CL1
CL2
LA2
LS4
LS7
LS 8
NL 3
SS 1
SS 2
þ
þ
þ
– þ
þ
þ þ þ
þ
þ
CS1, SS2: Bacillus pumilus; CS2, CL1: Bacillus sp.; CL2,SS1: Bacillus aerophilus; LS4, NL3: Bacillus megaterium; LS7: Bacillus idriensis; LS8: Bacillus cereus; LA2: Bacillus thuringiensis. * w, weak reaction (no prominent color distinction change).
containing MgSO4 and the control. As shown in Fig. 1, majority of Gram-positive Bacillus spp. managed to mitigate the corrosion process (ranging from 25.65 2.61 to 98.88 31.07 mg cm2) compared to the control in sterilized water only. Further enrichment of nutrients with the bacterial inoculum with the exception of fructose enhanced the corrosion rate compared to the bacteria alone (p < 0.05). The corrosion was mitigated when the coupons were in the medium containing fructose and bacterial inoculum especially for Bacillus pumilus CS1 and Bacillus thuringiensis LA2 (weight loss: 4.52 0.00 and 4.57 0.00 mg cm2, respectively) compared to the bacteria alone (25.65 2.61 and 39.02 10.40 mg cm2, respectively).
Figure 2 highlights some corrosion images of mild steel coupons by MIC in the presence of B. pumilus CS1 with magnesium sulphate (Fig. 2A) and B. thuringiensis LA2 with NaNO3 (Fig. 2B) or carbon source (galactose— Fig. 2C; Bacillus megaterium LS4 with sucrose—Fig. 2D) using the stereo microscope after a 10-day incubation period. The progression of mild steel corrosion in the presence of B. pumilus CS1 with magnesium sulfate could be visualized on the edges of the coupon at the early incubation time and resulted in localized corrosions in the metal surface at the end of the experiment, likely due to MIC (Fig. 2A). Submerging the mild steel coupons in flasks supplemented with nitrate sources especially sodium nitrate and B. thuringiensis LA2 produced crevice
Figure 1. The rate at which corrosion progressed in sterilized deionized water with and without supplementation of nutrient or bacterial inocula. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Figure 2. Stereo microscopic images observed following a 10-day incubation period in deionized, distilled water supplemented with (A) Bacillus pumilus CS1 with Magnesium sulphate; B) Bacillus thuringiensis LA2 with NaNO3; (C) Bacillus thuringiensis LA2 with galactose; (D) Bacillus megaterium LS4 with sucrose.
corrosion (arrows in Fig. 2B). As supported by the weight loss assays, much reduced weight loss of mild steel coupons due to corrosion was also noted in flasks supplemented with galactose and sucrose with Bacillus spp. as shown in Fig. 2C and D, respectively.
Biofilm forming ability and scanning electron microscopic analysis The formation of biofilms on the surface of the coupons was accessed. Figure 3 represents the mean optical density (OD595 nm) from the crystal violet biofilm assay
Figure 3. Mean biofilm forming ability of bacterial isolates involved in mild steel corrosion in the presences and absence of nutrients. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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on corroded mild steel coupons following a 10-day incubation period. The addition of nitrate especially NH4NO3 and Bacillus spp. produced more biofilms on the coupons compared to that with Bacillus spp. only under the same conditions. On the other hand, Bacillus spp. enriched with carbon sources formed less biofilms and compared to that with Bacillus spp. only. It was also found that there are strong correlation between biofilmforming ability of Bacillus isolates that were identified as the same species (r ¼ 0.69; p ¼ 0.29 for two B. pumilus isolates; r ¼ 0.65; p < 0.05 for Bacillus aerophilus isolates, and r ¼ 0.62; p ¼ 0.27 for B. megaterium isolates), but low or medium correlation between different species. Interestingly, Bacillus idriensis (LS7), B. cereus (LS8), and B. thuringiensis (LA2) were capable of developing higher level of biofilms (Fig. 3) with lower corrosion rates (Fig. 1). Figure 4A shows a control SEM image of coupons after autoclave without further treatments. The coupons appeared as a consistently smooth surface with the absence of grains and particles. Figure 4B shows a typical surface area of mild steel coupons following a 10-day incubation period in the medium with ammonium
nitrate but without bacterial inoculation. It is clear that the addition of ammonium nitrate led to deterioration, affecting the integrity of the metal surface of mild steel coupons, but no biofilm was observed. Additionally, cracks on the upper stratum of the mild steel coupon could be visualized in the case with Bacillus sp. and sodium nitrate (Fig. 4C) as well as biofilms attached to the mild steel coupon (Fig. 4C and D). After removal of biofilms, the presence of embedded concave structures with cracks, representing pit morphology, were broadly observed (Fig. 4E and F). The scatter graph of Fig. 5 summarizes the relationship between the biofilm ability of Bacillus spp. and weight loss of the mild steel coupons after a 10-day incubation period. It is clear that the bacterial isolates formed more biofilms on the mild steel coupon in the presence of nitrate than without nitrate while mild steel coupons suffered with more weight loss under the same conditions (triangles inside red circle) compared to those with bacterial isolates alone (black circle). On the other hand, bacterial isolates formed less biofilms in the presence of carbon sources (squares inside green circle) and less weight loss of mild steel coupons was observed.
Figure 4. Scanning electron images of mild steel coupons observed (A) pre-treated, autoclaved before the experiment; and (B) following a 10day incubation period in deionized, distilled water supplemented with ammonium nitrate but without bacterial inoculation; (C) : Bacillus sp. CL1 with sodium nitrate before removal of biofilm; (D) Bacillus megaterium LS4 with glucose before removal of biofilm; (E) Bacillus sp. CS2 with ammonium nitrate after removal of biofilm; (F) Bacillus megaterium LS4 with fructose after removal of biofilm; highlighting some of the products observable following corrosion ultimately leading to deterioration, affecting the integrity of the metal. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Figure 5. Correlation between biofilm formation and weight loss of mild steel coupons under various nutrient conditions.
There is a significant strong correlation between the weight loss and biofilm level (r ¼ 0.64; p < 0.001). The presence of MgSO4 also slightly enhanced the level of weight loss (inside blue circle).
Discussion Pillay and Lin found that environmental conditions play a major role in corrosion processes [26]. In addition, aerobic bacteria isolated from the corroded coupons can play a role in corrosion on mild steel coupons [13]. In this study, the effect of nutrients on corrosion of mild steel coupons by Gram-positive Bacillus isolates from corroded coupons were investigated. The corrosion process occurred after a 10-day incubation in sterilized dH2O only (108 11.91 mg cm2) indicating that other than biocorrosion process(es) was taking place. Increased corrosion due to presence of ammonium nitrate was also reported in the presence of fertilizer solutions in the environment [27]. Other literature and the results in this study (Fig. 1) support that nitrogenous solutions, including ammonium nitrate and sodium nitrate, can have effects on mild steels resulting in having higher weight loss [27, 28]. It is clear in the SEM images (Figs. 4A and B) that enriched with ammonium nitrate only leads to deterioration of mild steel coupons, affecting the ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
integrity of the metal surface of mild steel coupons. Corrosion of metals occurs immediately upon immersion of the metal into a hostile aqueous medium, and involves both an anodic and cathodic reaction. The anodic reaction involves the release of electrons which are transferred to the electron acceptor in the cathodic reaction [29]. The most common electron acceptor is oxygen in neutral and alkaline media. With the presence of nitrogenous compounds, cathodic depolarization could occur leading to the acceleration of corrosion [3]. The progression of mild steel corrosion visualized on the edges of the coupon at the early incubation time can be due to the joining action of mechanical stresses and corrosion (Fig. 2). In the absence of nutrients, majority of Gram-positive Bacillus spp. managed to mitigate the corrosion process (ranging from 25.65 2.61 to 98.88 31.07 mg cm2) especially with Bacillus sp. CS 1 compared to the control in sterilized dH2O only (Fig. 1). The levels of mild steel corrosion by bacterial isolates were much mitigated when supplemented with carbon sources, but were significantly enhanced with the presence of nitrate sources (Figs. 1 and 3). The results in this study demonstrate that with the presence of different nutrient sources, the metabolic pathways of bacterial isolates have been altered resulting in different capacity in the metal corrosion processes. The mechanisms of
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biocorrosion reflect the diverse array of physiological capabilities of microorganisms that can be found within biofilms. Species metabolic specificity within biofilms can have a significant effect on biocorrosion [3]. Respiration by aerobic Bacillus spp. especially in the presence of carbon sources results in the absence of oxygen below the biofilm. This leads to anodic areas due to the localized difference in aeration and the creation of localized corrosion cells. In the absence of electron acceptor (O2), a protective barrier produced by Bacillus sp. resulted in lower weight loss of mild steel coupons as reported by Zuo and co-workers [30]. Rajasekar and Ting [21] found that B. megaterium and Pseudomonas sp. affected the corrosion of stainless steel in the presence of organic and inorganic medium. Gram-positive Bacillus spp. may increase the production of highly corrosive ferric ion, tending to concentrate beneath nodules [14]. In initiating localized biocorrosion, the microorganisms initially seek out any irregularities on the metal surface and initiate biofilm development [31, 32] and anchor themselves with the use of extracellular polymeric substances (EPS). Fang et al. found that increase in concentration of toxic metals and chemicals leads to increase EPS production resulting in an increased degree of mild steel corrosion [6]. Figure 5 demonstrated that the biofilm formation of Gram-positive bacterial isolates on the mild steel coupon and the weight loss of mild steel coupons significantly increased with the presence of nitrate source compared to the coupons in sterilized dH2O. The presence of carbon sources slowed down the biofilm-forming ability of Gram-positive bacteria and the level of mild steel corrosion. Strong correlations of biofilm-forming ability among the isolates that were identified as the same Bacillus species were observed. Our results support the other reports in the literature [33, 34]. This may be explained that manipulation of nutrient supplementation can influence dynamic population changes between planktonic and sessile (biofilm) microorganisms. An increase of metabolically active cells migrated to planktonic state in the medium enriched with carbon sources, rather than remaining dormant in a biofilm on the metal surface leading to a reduction of corrosion [33]. Pitting corrosion is initiated by MIC leading to the localized erosion of iron from the metal coupons and forming pits in the process (Fig. 2). As biofilms have been implicated in biocorrosion, the disintegration of the metal would soon occur, leading to a high presence of irregularities, prominently facilitating the adherence of bacterial cells to the surface as shown in Fig. 4. The corrosion penetrates the mass of the metal, with limited diffusion of ions. This leads to the accumulation of ferric ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
ions which would in turn accelerate corrosion by acting as available oxidizing agents. According to Lewandowski and Beyenal [34], oxygen heterogeneities can be created at the biofilm–metal interface resulting in an increase of mass transport of the corrosion reactants and products such as acids or metabolites. The corrosion metabolites might change the kinetics of the corrosion processes or act as cathodic reactants. It is also possible that corrosion inhibitor(s) has been produced or the production of corrosion inducer (such as electron acceptors) has been reduced due to the supplementation of the nutrients [33]. The role of biofilms in corrosion still remains unclear [9]. Bacteria initiate biofilm formation in response to specific environmental cues, such as nutrient and oxygen availability [35], and produce specific secondary metabolites in the resistivity to biological, chemical, and physical assaults. The biofilm can either play an active role in accelerating or inhibiting the corrosion processes, depending on the environments [21, 35]. Bacillus spp. have been found in water pipelines and galvanized steel and have been shown to be capable of oxidizing manganese and iron as well as producing acid (Table 1) [36] which might subsequently promote corrosion [5]. Zuo et al. reported that a protective barrier produced by B. subtilis WB600 in seawater simulated conditions that led to less weight loss of mild steel coupons [30]. This protective potential was linked to the bacteria’s negative charge which was proposed to have similar roles of cathodic protection through the use of sacrificial anodes. Upon disintegration of the biofilm layer using antibiotics, pitting of mild steel coupons was soon experienced. Little et al. developed two approaches, the use of biofilms and manipulation of environmental conditions, to control of MIC without the use of biocides [33]. The biofilm can form a diffusion barrier against the dissolution of corrosion products. Microorganisms within the biofilm can consume oxygen causing a diminution of the reactant at the metal surface or produce metabolic products as corrosion inhibitor or to prevent the proliferation of corrosion-causing organisms. In this study, B. idriensis (LS7), B. cereus (LS8), and B. thuringiensis (LA2) were capable of developing higher level of biofilms (Fig. 3) with lower corrosion rates (Fig. 1). Species within the same genus can differ significantly in their metal degrading capability. It is these differences within species that may explain findings that experiments conducted under the same environmental conditions and system can vary widely with regard to the corrosion rate [3]. The results emphasize the importance of considering species diversity when investigating the
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corrosion of metals. The study also demonstrates potential tools to develop a preventive strategy against metal corrosion using specific bacterial strains.
[10] Kapellos, G.E., Alexiou, T.A., Payatakes, A.C., 2007. Hierarchical simulator of biofilm growth and dynamics in granular porous materials. Adv. Water Resour., 30, 1648–1667.
Conclusion
[11] Sheng, X., Ting, Y.-P., Pehkonen, S.O., 2008. The influence of ionic strength, nutrients and pH on bacterial adhesion to metals. J. Colloid Interf. Sci., 321, 256–264.
The results of this study highlighted numerous factors which contribute to corrosion, namely the bacteria’s corrosive ability, the organism’s metabolism, the environment, and biofilm forming ability. Without any nutrients, Gram-positive Bacillus spp. retarded the corrosion processes. Supplementation with different nutrients could trigger different metabolic pathways resulting in acceleration or mitigation of the corrosion rates which are strongly correlated to the level of biofilm formation by the microorganisms. The differences in the species present in the biofilm can lead to vastly different metabolic activities within the biofilm. This can explain why corrosion rates may vary with or without bacteria under the same environmental conditions.
References [1] Lane, R.A., 2005. Under the microscope: understanding, detection, and preventing Microbiologically Influenced Corrosion. The AMPTIAC Quarterly, 9(1), 3–8. [2] Beech, I.B., 2004. Corrosion of technical materials in the presence of biofilms—current understanding and state-ofthe art methods of study. Int. Biodeter. Biodegr., 53, 177– 183. [3] Beech, I.B., Sunner, J., 2004. Biocorrosion: towards understanding interactions between biofilms and metals. Curr. Opin. Biotech., 15, 181–186. [4] Cullimore, D.R., 2008. Practical Manual of Groundwater Microbiology. 2nd edn. Sustainable Water Well series, CRC Press, Taylor and Francis Group, Boca Raton, Florida. [5] Rajasekar, A., Babu, T.G., Pandian, K.S., Maruthamuthu, S., et al., 2007. Biodegradation and corrosion behavior of manganese oxidizer Bacillus cereus ACE4 in diesel transporting pipeline. Corros. Sci., 49, 2694–2710. [6] Fang, H.H.P., Xu, L.C., Chan, K.Y., 2002. Effects of toxic metals and chemicals on biofilm and biocorrosion. Water Res., 36, 4709–4716. €, T., Kuok[7] Huttunen-Saarivirta, E., Honkanen, M., Lepisto kala, V.T., et al., 2012. Microbiologically influenced corrosion (MIC) in stainless steel heat exchanger. Appl. Surf. Sci., 258, 6512–6526.
[12] Forte Giacobone, A.F., Rodriguez, S.A., Burkart, A.L., Pizarro, R.A., 2011. Microbiological induced corrosion of AA 6061 nuclear alloy in highly diluted media by Bacillus cereus RE 10. Int. Biodeter. Biodegr., 65, 1161–1168. [13] Pillay, C., Lin, J., 2013. Metal corrosion by aerobic bacteria isolated from stimulated corrosion systems: Effects of additional nitrate sources. Int. Biodeter. Biodegr., 83, 158– 165. doi: 10.1016/j.ibiod.2013.05.013 [14] Rajasekar, A., Balasubramanian, R., Kuma, J.V.M., 2011. Role of hydrocarbon degrading bacteria Serratia marcescens ACE2 and Bacillus cereus ACE4 on corrosion of carbon steel API 5LX. Ind. Eng. Chem. Res., 50, 10041–10046. [15] Ahmad, Z., 2006. Types of Corrosion: Materials and Environments. In Principles of corrosion engineering and corrosion control. Chapter 4. Butterworth-Heinemann, Oxford, 120–270. [16] Vargel, C., Jacques, M., Schmidt, M.P., 2004. Types of Corrosion on Aluminium. Corrosion of Aluminium, Elsevier, Amsterdam, 113–146. [17] Holt, J.G., Kreig, N.R., Sneah, P.H.A., Stanely, J.T., 1994. Bergey’s Manual of Determinative Bacteriology. 9th edn. Williams and Wilkins, Baltimore. [18] Marchesi, J., Sato, R.T., Martin, A.J., Hiam, S.J., et al., 1998. Design and evaluation of bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl. Environ. Microbiol., 64, 795–799. [19] Altschul, S.F., Gish, W., Miller, W., Myers, E.W., et al., 1990. Basic local alignment search tool. Mol. Biol., 215, 403–410. [20] Ryhl-Svendsen, M., 2008. Corrosivity measurements of indoor museum environments using lead coupons as dosimeters. J. Cultural Heritage, 9, 285–293. [21] Rajasekar, A., Ting, Y.P., 2011. Role of inorganic and organic medium in the corrosion behavior of Bacillus megaterium and Pseudomonas sp. in stainless steel SS 304. Ind. Eng. Chem. Res., 50, 12534–12541. [22] Stepanovic, S., Cirkovic, I., Ranin, L., Svabic-Vlahovic, M., 2004. Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface. Lett. Appl. Microbiol., 38, 428–32. [23] Adetunji, V.O., Isola, T.O., 2011. Crystal violet binding assay for assessment of biofilm formation by Listeria monocytogenes and Listeria spp on wood, steel and glass surfaces. Glob. Vet., 6, 6–10.
[8] Landoulsi, J., El Kirat, K., Richard, C., Feron, D., et al., 2008. Enzymatic approach in microbial-influenced corrosion: a review based on stainless steels in natural waters. Environ. Sci. Technol., 42, 2233–2242.
[24] Abed, S.E., Ibnsouda, S.K., Latrache, H., Hamadi, F., 2012. Scanning electron microscopy (SEM) and environmental SEM: suitable tools for study of adhesion stage and biofilm formation, in: Kazmiruk, V. (Ed.), Scanning Electron Microscopy, InTech. pp. 717-730. doi: 10.5772/34990
[9] Videla, H.A., Herrera, L.K., 2005. Microbiologically influenced corrosion: looking to the future. Int. Microbiol., 8, 169–180.
[25] Mukaka, M.M., 2012. Statistics corner: a guide to appropriate use of correlation coefficient in medical research. Malawi Med. J., 24, 69–71.
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[26] Pillay, C., Lin, J., 2014. The impact of additional nitrates in mild steel corrosion in a seawater/sediment system. Corros. Sci., 80, 416–426. [27] Eker, B., Yuksel, E., 2005. Solutions to corrosion caused by agricultural chemicals. Trakia J. Sci., 3, 1–6. [28] Starostin, M., Shter, G.E., Grader, G.S., 2012. Corrosion inhibition of carbon steel in aqueous solution of ammonium nitrate and urea. Mater. Corros., 63, 1–11. [29] Juzeliunas, E., Ramanauskas, R., Lugauskas, A., Leinartas, K. et al., 2006. Influence of wild strain Bacillus mycoideson metals: From corrosion acceleration to environmentally friendly protection. Electrochim. Acta, 51, 6085–6090. [30] Zuo, R., Ornek, D., Syrett, B.C., Green, R.M. et al., 2004. Inhibiting mild steel corrosion from sulfate-reducing bacteria using antimicrobial-producing biofilms in Three-Mile-Island process water. Appl. Microbiol. Biotechnol., 64, 275–83.
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[31] Dunne, Jr., M.W., 2002. Bacterial adhesion: seen any good biofilms lately? Clin. Microbiol. Rev., 15, 155–166. [32] Pope, D.H., Morris, E.A., 1995. Some experiences with microbiologically influenced corrosion of pipelines. Mater. Performance, 34, 23–28. [33] Little, B.J., Lee, J.S., Ray, R.I., 2007. New developments in mitigation of microbiologically influenced corrosion. Defense Technical Information Center Reports (US Department of Defense), 1–7. [34] Lewandowski, Z., Beyenal, H., 2009. Mechanisms of microbially influenced corrosion. Mar. Ind. Biofouling, 4, 35–64. [35] Morikawa, M., 2006. Beneficial biofilm formation by industrial bacteria Bacillus subtilis and related species. J. Biosci. Bioeng., 101, 1–8. [36] Kan, J., Chellamuthu, P., Obraztsova, A., Moore, J.E., Nealson, K.H., 2011. Diverse bacterial groups are associated with corrosive lesions at a Granite Mountain Record Vault (GMRV). J. Appl. Microbiol., 111(2), 329–337.
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Research Paper Biofilms affecting progression of mild steel corrosion by Gram positive Bacillus sp. Johnson Lin and Bafana B. Madida School of Life Sciences, University of KwaZulu-Natal Westville, Private Bag X54001, Durban 4000, South Africa
The biodeterioration of metals have detrimental effects on the environment with economic implications. The deterioration of metals is of great concern to industry. In this study, mild steel coupons which were immersed in a medium containing Gram-positive Bacillus spp. and different nutrient sources were compared with the control in sterile deionized water. The weight loss of the coupons in the presence of Bacillus spp. alone was lower than the control and was further reduced when additional carbon sources, especially fructose, were added. The level of metal corrosion was significantly increased in the presence of nitrate with or without bacteria. There was a significant strong correlation between the weight loss and biofilm level (r ¼ 0.64; p < 0.05). The addition of nitrate and Bacillus spp. produced more biofilms on the coupons and resulted in greater weight loss compared to that with Bacillus spp. only under the same conditions. However, Bacillus spp. enriched with carbon sources formed less biofilms and results in lower weight loss compared to that with Bacillus spp. only. The production of biofilm by Bacillus spp. influences the level of metal corrosion under different environmental conditions, thereby, supporting the development of a preventive strategy against corrosion. Keywords: Biofilm / Gram-positive Bacillus / Microbial influenced corrosion / Mild steel coupons / Nutrient supplementation / Weight loss Received: November 20, 2014; accepted: March 1, 2015 DOI 10.1002/jobm.201400886
Introduction For all industries, across all borders, from under developed to highly developed countries, there is a strong concern regarding the factors influencing and affecting the deterioration of metal turbines and pipelines [1]. The biodeterioration of metals does not only have detrimental effects on the environment and population dynamics but also on the economy [2, 3]. The detection of microbial communities in biofilms on corroded metal substrates triggered an interest on the role that bacteria play in biocorrosion [3]. An electrochemical interaction, between the organism and the metallic surface/structure, can promote physicochemical reactions triggering, aiding, and exacerbating the Correspondence: Prof. Johnson Lin, School of Life Sciences, University of KwaZulu-Natal (Westville), Private Bag X54001, Durban 4000, Republic of South Africa E-mail: [email protected] Phone: þ27-31-2607407 Fax: þ27-31-2607809 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
deterioration of the metal leading to metal damage [4, 5]. Biocorrosion contributes to different forms of corrosions namely uniform, pitting, crevice, galvanic, intergranual, dealloying, and stress corrosions [1]. Biofilm structures are not static, but dynamic. Biofilms form immediately after metals are submerged into a medium (e.g., soil, water). Inorganic ions and organic compounds (EPS production) deposited by microorganisms encourage further microbial growth [3, 6, 7] and create localized microenvironments which facilitate the electrochemical process of localized corrosion at the biofilm–metal interface [3, 8]. Their ability to accelerate corrosion is due to bacterial metabolic reactions such as acid production, sulphite production and ammonia production, metal deposition, and most importantly their effects of redox potential on metal corrosion [1]. Fang et al. found that the corrosion patterns appear to match the microbial cluster patterns of the aggregated biofilms [6]. However, no quantitative relationship was found between the degree of increased corrosion and the toxicity of metals/chemicals on
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sulfate-reducing bacteria, or the increased EPS production. EPS presence (biofilms) enables microorganisms to become more resistant to biocides than free living planktonic organisms [9]. Formed biofilms are more resistant to metabolizing inhibiting compounds including aliphatic amines and nitrites. Several factors including availability of nutrients for survival and reduced concentration of toxins encourage biofilm development [10]. Sheng et al. [11] showed that bacterial and metal interactions tend to be reduced when nutrients are present in the solution. Stronger ionic strength in the solution and when the pH of the solution was near the isoelectric point of the bacteria also results in a higher bacteria–metal adhesion forces. Forte Giacobone et al. also showed that Bacillus cereus is capable of promoting corrosion in nutrient poor and nutrient mild conditions [12]. Other researchers suggested that the presence of a nutrient source leads to an increase in the proliferation of bacteria on steel coupons and influences the extent to which corrosion takes place in product pipelines [13, 14]. The form in which corrosion takes place is dependent on environmental factors which impact on the progression of corrosion [15, 16]. In mixed consortia, many different corrosion mechanisms may occur that may even be synergistic in nature. The rates of corrosion obtained during studies using mixed consortia of microbes are considerably higher than with pure cultures [3]. When a material is exposed to a uniform, pure group of micro-organisms, it becomes much easier to detail and explain the mechanisms of corrosion occurring. The aim of this study was to investigate biocorrosion induced by Bacillus spp. on mild steel coupons when supplemented with different nutrient sources. We demonstrate that the ability of Gram-positive Bacillus spp. to form biofilm has a direct impact on the level of mild steel corrosion.
Materials and methods Corroding isolates Gram-positive Bacillus spp. (n ¼ 11) isolated from corroded stainless steel coupons [13] were obtained from the Department of Microbiology, Westville Campus, University of KwaZulu-Natal. A series of sub-culturing on nutrient agar plates was carried out and isolates were maintained in nutrient agar slants for short term storage and stored in 40% glycerol in Microbanks at 80 °C. Characterization and identification of bacteria isolates Characterization of the bacteria isolates was carried out according to Bergy’s Manual of Determinative ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Bacteriology [17]. Characteristics observed include morphological analysis—Gram stain, shape, sporulation, biochemical tests—catalase, IMViC (indole, methyl red, Voges–Proskauer, and citrate tests), urease, nitrate reduction and oxidase tests, carbohydrate metabolism 1 on Analytical Profile Index (API) 50 CH strips (bioMerieux, France) according to manufacturer’s instruction and hydrolysis of starch, casein, and gelatine. This was confirmed by API 50CHB and, thereafter, the sequences were submitted to Bankit (GenBank) to obtain valid accession numbers. Identification was confirmed using 16S rRNA sequencing [18], followed by NCBI Blast comparison software to reveal the identities of the isolates [19] and the MALDI Biotyper (the Peptide and Catalysis Unit, Department of Chemistry, Westville Campus, University of KwaZulu-Natal). The bacterial nucleotide sequences were submitted to the GenBank database under accession numbers JN106407-JN106409, JN106417, JN106418, JN106422, JN106423, and JN106426. Amplifications of Bacillus strains CS2 and CL1 and LS8 using 16S rRNA PCR were not successful. Corrosion experimental set up Determination of corrosion was done using the weight loss method as described by Pillay and Lin [13] and RhylSvendsen [20]. The mild steel coupons (25 25 mm) were 1 manually polished with a Dremel 3000 Variable Speed Rotary Tool, having attached a fine sanding band to it, followed by rinsing the coupons under flowing tap water, dried then degreased in acetone. The weight of each coupon was measured (g), attached to a nylon fishing string and then sterilized. For standardization purposes, Bacillus isolates were grown in 40 ml of nutrient broth overnight at 30 °C in conical centrifuge tubes, followed by centrifugation at 3000 rpm for 10 min at 4 °C to obtain bacterial pellet. The pellet was washed with sterile deionized water twice, followed by centrifugation as described above. The culture inoculums were standardized to an absorbance of 0.80 0.05 at 600 nm in sterilized deionized water (dH2O). One milliliter of standardized culture was used in the following experimental set up to determine the effect of each isolate on corrosion rate in the presence of supplemented nutrients. The experimental sets were prepared according to Rajasekar and Ting [21]. Four pre-treated mild steel coupons were placed in each 250 ml flask each containing 150 ml sterilized dH2O (as the blank control) or in media (as the cell-free control) supplemented with the following individual nutrient sources: Carbon source (50 mg L1—fructose, galactose, or sucrose); MgSO4 (0.5 g L1); Nitrate source (0.5 g L1—NH4NO3 or NaNO3).
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One milliliter of standardized bacterial inoculum was inoculated into each individual set of flasks prepared as above. At the end of the incubation period (30 °C, 100 rpm for a period of 10 days), the coupons were removed for biofilm assay, weight loss determination, and microscopic analysis. The experiments were performed in triplicates. Biofilm assay Biofilm quantification was carried out according to Stepanovic et al. [22] and Adetunji and Isola [23] with modification. Each set (3) of coupons was gently dipped in tap water to remove and wash off any corrosion products. The remaining adherent cells were fixed with a fixative agent (absolute methanol) in staining jars for 15 min, followed by air-drying. Air-dried coupons were then stained with 2% Hucker’s crystal violet for 15 min followed by washing of excess stain under smoothly running tap water. The coupons were then air dried and followed by resolubilization with 15 ml of 33% glacial acetic acid in a sonicator (EUMAX Digital Ultrasonic Cleaner) for 10 min. The absorbance (optical density) of each resolubilization solution was measured at a wavelength of 595 nm (ThermoLabSystems Multiskan RC Microplate Reader coupled with the Ascent Software for MultiScan v2.6). The actual value of biofilm assay for each experimental coupon was obtained by subtracting the absorbance of the blank reading without inoculation. Weight loss determination Following the biofilm assay, the same coupons were soaked in 20% HCl, thereafter, rinsed in deionized water, then allowed to completely dry [20]. The weight loss of mild steel coupons was determined using following equation, representing it as the weight loss per surface area: Weightlosspersurfaceareaðmgcm2 Þ ðW initial Wfinal Þ 1000 ¼ Surfaceareaðcm2 Þ
Microscopic analysis The remaining mild steel coupon after 10-day incubation period was examined at the Electron Microscopy Unit (Westville Campus, University of KwaZulu-Natal) to observe their morphological and topographical characteristics using the Scanning Electron Microscopy (SEM) Leo 1450, or Light Microscope Nikon AZ100. Adhered cells (biofilms) and corroded products were observed according to the method of Abed et al. [24]. Some ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
coupons which had been analyzed (biofilm assay and weight loss) were also analyzed under the microscope. Statistical analysis In correlating the level of biofilm and weight loss of mild steel coupon to nutrients supplemented and present in the environment, the product moment (Pearson’s) correlation coefficient was obtained using the SPSS program version 21 (SPSS, Inc., IL). The significance of the observed results was tested using Student’s t-test to measure the possibility of an existing linear relationship between variables [25]. Probability (significant level) was set at p < 0.05 and p < 0.01.
Results Characterization of bacterial isolates Characterizations and biochemical properties Grampositive Bacillus spp. (n ¼ 11) isolated from corroded stainless steel coupons were further studied. The results are shown in Table 1. All the isolates used in this study were confirmed as Gram-positive Bacillus spp. (Table 1). Sporulation was observed in all isolates with growth at all tested incubation temperatures. Optimal growth was observed in 35 °C for all isolates, with the exception of Isolate CL1 (at 25 °C). All isolates contained oxidase, catalase activities, and hydrolyzed casein as carbon sources with various abilities to use Tween and starch. All isolates except LS4 possessed acid fermentation ability (methyl red positive). The reduction of nitrate to nitrite was positive for all isolates with exception to LS4, SS1, and SS2. All isolates metabolized ribose, glucose, fructose, N-acetylglucosamine, and arbutin as carbon source coupled with acid production. Influence of nutrients on corrosion Effects of nutrients on corrosion of mild steel coupons induced by characterized Bacillus isolates were investigated. Figure 1 represents the corrosion rates of mild steel coupons progressed over a 10-day incubation period with or without nutrients and Gram-positive Bacillus isolates. The corrosion process occurred even without any nutrient supplements or the presence of the bacterial inoculum (108 11.91 mg cm2—the control which were immersed in the sterilized deionized water) indicating other corrosion process than biocorrosion was taking place. An increase in corrosion was observed when the media were supplemented with sodium nitrate (123.47 9.40 mg cm2) compared to the weight loss of the control. There is no significant difference (p > 0.05) in the weight loss of the coupons between the media
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Table 1. Partial biochemical characterization of bacterial isolates. Characteristics cell morphology gram stain shape sporulation growth at 25 °C 35 °C 45 °C biochemical reactions indole test methyl red Voges–Proskauer citrate utilization nitrate reduction starch hydrolysis oxidase test catalase test Tween-80 casein hydrolysis glycerol erythritol D-arabinose L-arabinose ribose D-xylose L-xylose adonitol methyl b-xyloside galactose D-glucose D-fructose mannose sorbose rhamnose dulcitol inositol mannitol sorbitol Characteristics methyl a-D-mannoside methyl a-D-glucoside N-acetylglucosamine amygdalin arbutin aesculin salicin cellobiose maltose lactose melibiose sucrose trehalose inulin melezitose D-Raffinose starch glycogen xylitol b-gentiobiose
CS1
CS2
CL1
CL2
LA2
LS4
LS7
LS8
NL3
SS1
SS 2
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ rod þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ w þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ w* þ þ w
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ
CS1
CS2
CL1
CL2
LA2
LS4
LS7
LS 8
NL 3
SS 1
SS 2
þ þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ w
þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ
þ þ þ w w þ – þ þ þ þ –
w w þ þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ
þ w þ þ þ
þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ
w þ þ þ þ þ þ þ þ þ þ þ
þ þ þ þ þ þ þ w þ þ w þ þ
þ þ þ þ þ þ þ þ w þ þ þ þ þ
(Continued) ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Table 1. (Continued) Characteristics D-turanose D-lyxose D-tagatose D-fucose L-fucose D-arabitol L-arabitol gluconate 2-ketogluconate 5-ketogluconate
CS1
CS2
CL1
CL2
LA2
LS4
LS7
LS 8
NL 3
SS 1
SS 2
þ
þ
þ
– þ
þ
þ þ þ
þ
þ
CS1, SS2: Bacillus pumilus; CS2, CL1: Bacillus sp.; CL2,SS1: Bacillus aerophilus; LS4, NL3: Bacillus megaterium; LS7: Bacillus idriensis; LS8: Bacillus cereus; LA2: Bacillus thuringiensis. * w, weak reaction (no prominent color distinction change).
containing MgSO4 and the control. As shown in Fig. 1, majority of Gram-positive Bacillus spp. managed to mitigate the corrosion process (ranging from 25.65 2.61 to 98.88 31.07 mg cm2) compared to the control in sterilized water only. Further enrichment of nutrients with the bacterial inoculum with the exception of fructose enhanced the corrosion rate compared to the bacteria alone (p < 0.05). The corrosion was mitigated when the coupons were in the medium containing fructose and bacterial inoculum especially for Bacillus pumilus CS1 and Bacillus thuringiensis LA2 (weight loss: 4.52 0.00 and 4.57 0.00 mg cm2, respectively) compared to the bacteria alone (25.65 2.61 and 39.02 10.40 mg cm2, respectively).
Figure 2 highlights some corrosion images of mild steel coupons by MIC in the presence of B. pumilus CS1 with magnesium sulphate (Fig. 2A) and B. thuringiensis LA2 with NaNO3 (Fig. 2B) or carbon source (galactose— Fig. 2C; Bacillus megaterium LS4 with sucrose—Fig. 2D) using the stereo microscope after a 10-day incubation period. The progression of mild steel corrosion in the presence of B. pumilus CS1 with magnesium sulfate could be visualized on the edges of the coupon at the early incubation time and resulted in localized corrosions in the metal surface at the end of the experiment, likely due to MIC (Fig. 2A). Submerging the mild steel coupons in flasks supplemented with nitrate sources especially sodium nitrate and B. thuringiensis LA2 produced crevice
Figure 1. The rate at which corrosion progressed in sterilized deionized water with and without supplementation of nutrient or bacterial inocula. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Figure 2. Stereo microscopic images observed following a 10-day incubation period in deionized, distilled water supplemented with (A) Bacillus pumilus CS1 with Magnesium sulphate; B) Bacillus thuringiensis LA2 with NaNO3; (C) Bacillus thuringiensis LA2 with galactose; (D) Bacillus megaterium LS4 with sucrose.
corrosion (arrows in Fig. 2B). As supported by the weight loss assays, much reduced weight loss of mild steel coupons due to corrosion was also noted in flasks supplemented with galactose and sucrose with Bacillus spp. as shown in Fig. 2C and D, respectively.
Biofilm forming ability and scanning electron microscopic analysis The formation of biofilms on the surface of the coupons was accessed. Figure 3 represents the mean optical density (OD595 nm) from the crystal violet biofilm assay
Figure 3. Mean biofilm forming ability of bacterial isolates involved in mild steel corrosion in the presences and absence of nutrients. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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on corroded mild steel coupons following a 10-day incubation period. The addition of nitrate especially NH4NO3 and Bacillus spp. produced more biofilms on the coupons compared to that with Bacillus spp. only under the same conditions. On the other hand, Bacillus spp. enriched with carbon sources formed less biofilms and compared to that with Bacillus spp. only. It was also found that there are strong correlation between biofilmforming ability of Bacillus isolates that were identified as the same species (r ¼ 0.69; p ¼ 0.29 for two B. pumilus isolates; r ¼ 0.65; p < 0.05 for Bacillus aerophilus isolates, and r ¼ 0.62; p ¼ 0.27 for B. megaterium isolates), but low or medium correlation between different species. Interestingly, Bacillus idriensis (LS7), B. cereus (LS8), and B. thuringiensis (LA2) were capable of developing higher level of biofilms (Fig. 3) with lower corrosion rates (Fig. 1). Figure 4A shows a control SEM image of coupons after autoclave without further treatments. The coupons appeared as a consistently smooth surface with the absence of grains and particles. Figure 4B shows a typical surface area of mild steel coupons following a 10-day incubation period in the medium with ammonium
nitrate but without bacterial inoculation. It is clear that the addition of ammonium nitrate led to deterioration, affecting the integrity of the metal surface of mild steel coupons, but no biofilm was observed. Additionally, cracks on the upper stratum of the mild steel coupon could be visualized in the case with Bacillus sp. and sodium nitrate (Fig. 4C) as well as biofilms attached to the mild steel coupon (Fig. 4C and D). After removal of biofilms, the presence of embedded concave structures with cracks, representing pit morphology, were broadly observed (Fig. 4E and F). The scatter graph of Fig. 5 summarizes the relationship between the biofilm ability of Bacillus spp. and weight loss of the mild steel coupons after a 10-day incubation period. It is clear that the bacterial isolates formed more biofilms on the mild steel coupon in the presence of nitrate than without nitrate while mild steel coupons suffered with more weight loss under the same conditions (triangles inside red circle) compared to those with bacterial isolates alone (black circle). On the other hand, bacterial isolates formed less biofilms in the presence of carbon sources (squares inside green circle) and less weight loss of mild steel coupons was observed.
Figure 4. Scanning electron images of mild steel coupons observed (A) pre-treated, autoclaved before the experiment; and (B) following a 10day incubation period in deionized, distilled water supplemented with ammonium nitrate but without bacterial inoculation; (C) : Bacillus sp. CL1 with sodium nitrate before removal of biofilm; (D) Bacillus megaterium LS4 with glucose before removal of biofilm; (E) Bacillus sp. CS2 with ammonium nitrate after removal of biofilm; (F) Bacillus megaterium LS4 with fructose after removal of biofilm; highlighting some of the products observable following corrosion ultimately leading to deterioration, affecting the integrity of the metal. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Figure 5. Correlation between biofilm formation and weight loss of mild steel coupons under various nutrient conditions.
There is a significant strong correlation between the weight loss and biofilm level (r ¼ 0.64; p < 0.001). The presence of MgSO4 also slightly enhanced the level of weight loss (inside blue circle).
Discussion Pillay and Lin found that environmental conditions play a major role in corrosion processes [26]. In addition, aerobic bacteria isolated from the corroded coupons can play a role in corrosion on mild steel coupons [13]. In this study, the effect of nutrients on corrosion of mild steel coupons by Gram-positive Bacillus isolates from corroded coupons were investigated. The corrosion process occurred after a 10-day incubation in sterilized dH2O only (108 11.91 mg cm2) indicating that other than biocorrosion process(es) was taking place. Increased corrosion due to presence of ammonium nitrate was also reported in the presence of fertilizer solutions in the environment [27]. Other literature and the results in this study (Fig. 1) support that nitrogenous solutions, including ammonium nitrate and sodium nitrate, can have effects on mild steels resulting in having higher weight loss [27, 28]. It is clear in the SEM images (Figs. 4A and B) that enriched with ammonium nitrate only leads to deterioration of mild steel coupons, affecting the ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
integrity of the metal surface of mild steel coupons. Corrosion of metals occurs immediately upon immersion of the metal into a hostile aqueous medium, and involves both an anodic and cathodic reaction. The anodic reaction involves the release of electrons which are transferred to the electron acceptor in the cathodic reaction [29]. The most common electron acceptor is oxygen in neutral and alkaline media. With the presence of nitrogenous compounds, cathodic depolarization could occur leading to the acceleration of corrosion [3]. The progression of mild steel corrosion visualized on the edges of the coupon at the early incubation time can be due to the joining action of mechanical stresses and corrosion (Fig. 2). In the absence of nutrients, majority of Gram-positive Bacillus spp. managed to mitigate the corrosion process (ranging from 25.65 2.61 to 98.88 31.07 mg cm2) especially with Bacillus sp. CS 1 compared to the control in sterilized dH2O only (Fig. 1). The levels of mild steel corrosion by bacterial isolates were much mitigated when supplemented with carbon sources, but were significantly enhanced with the presence of nitrate sources (Figs. 1 and 3). The results in this study demonstrate that with the presence of different nutrient sources, the metabolic pathways of bacterial isolates have been altered resulting in different capacity in the metal corrosion processes. The mechanisms of
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biocorrosion reflect the diverse array of physiological capabilities of microorganisms that can be found within biofilms. Species metabolic specificity within biofilms can have a significant effect on biocorrosion [3]. Respiration by aerobic Bacillus spp. especially in the presence of carbon sources results in the absence of oxygen below the biofilm. This leads to anodic areas due to the localized difference in aeration and the creation of localized corrosion cells. In the absence of electron acceptor (O2), a protective barrier produced by Bacillus sp. resulted in lower weight loss of mild steel coupons as reported by Zuo and co-workers [30]. Rajasekar and Ting [21] found that B. megaterium and Pseudomonas sp. affected the corrosion of stainless steel in the presence of organic and inorganic medium. Gram-positive Bacillus spp. may increase the production of highly corrosive ferric ion, tending to concentrate beneath nodules [14]. In initiating localized biocorrosion, the microorganisms initially seek out any irregularities on the metal surface and initiate biofilm development [31, 32] and anchor themselves with the use of extracellular polymeric substances (EPS). Fang et al. found that increase in concentration of toxic metals and chemicals leads to increase EPS production resulting in an increased degree of mild steel corrosion [6]. Figure 5 demonstrated that the biofilm formation of Gram-positive bacterial isolates on the mild steel coupon and the weight loss of mild steel coupons significantly increased with the presence of nitrate source compared to the coupons in sterilized dH2O. The presence of carbon sources slowed down the biofilm-forming ability of Gram-positive bacteria and the level of mild steel corrosion. Strong correlations of biofilm-forming ability among the isolates that were identified as the same Bacillus species were observed. Our results support the other reports in the literature [33, 34]. This may be explained that manipulation of nutrient supplementation can influence dynamic population changes between planktonic and sessile (biofilm) microorganisms. An increase of metabolically active cells migrated to planktonic state in the medium enriched with carbon sources, rather than remaining dormant in a biofilm on the metal surface leading to a reduction of corrosion [33]. Pitting corrosion is initiated by MIC leading to the localized erosion of iron from the metal coupons and forming pits in the process (Fig. 2). As biofilms have been implicated in biocorrosion, the disintegration of the metal would soon occur, leading to a high presence of irregularities, prominently facilitating the adherence of bacterial cells to the surface as shown in Fig. 4. The corrosion penetrates the mass of the metal, with limited diffusion of ions. This leads to the accumulation of ferric ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
ions which would in turn accelerate corrosion by acting as available oxidizing agents. According to Lewandowski and Beyenal [34], oxygen heterogeneities can be created at the biofilm–metal interface resulting in an increase of mass transport of the corrosion reactants and products such as acids or metabolites. The corrosion metabolites might change the kinetics of the corrosion processes or act as cathodic reactants. It is also possible that corrosion inhibitor(s) has been produced or the production of corrosion inducer (such as electron acceptors) has been reduced due to the supplementation of the nutrients [33]. The role of biofilms in corrosion still remains unclear [9]. Bacteria initiate biofilm formation in response to specific environmental cues, such as nutrient and oxygen availability [35], and produce specific secondary metabolites in the resistivity to biological, chemical, and physical assaults. The biofilm can either play an active role in accelerating or inhibiting the corrosion processes, depending on the environments [21, 35]. Bacillus spp. have been found in water pipelines and galvanized steel and have been shown to be capable of oxidizing manganese and iron as well as producing acid (Table 1) [36] which might subsequently promote corrosion [5]. Zuo et al. reported that a protective barrier produced by B. subtilis WB600 in seawater simulated conditions that led to less weight loss of mild steel coupons [30]. This protective potential was linked to the bacteria’s negative charge which was proposed to have similar roles of cathodic protection through the use of sacrificial anodes. Upon disintegration of the biofilm layer using antibiotics, pitting of mild steel coupons was soon experienced. Little et al. developed two approaches, the use of biofilms and manipulation of environmental conditions, to control of MIC without the use of biocides [33]. The biofilm can form a diffusion barrier against the dissolution of corrosion products. Microorganisms within the biofilm can consume oxygen causing a diminution of the reactant at the metal surface or produce metabolic products as corrosion inhibitor or to prevent the proliferation of corrosion-causing organisms. In this study, B. idriensis (LS7), B. cereus (LS8), and B. thuringiensis (LA2) were capable of developing higher level of biofilms (Fig. 3) with lower corrosion rates (Fig. 1). Species within the same genus can differ significantly in their metal degrading capability. It is these differences within species that may explain findings that experiments conducted under the same environmental conditions and system can vary widely with regard to the corrosion rate [3]. The results emphasize the importance of considering species diversity when investigating the
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corrosion of metals. The study also demonstrates potential tools to develop a preventive strategy against metal corrosion using specific bacterial strains.
[10] Kapellos, G.E., Alexiou, T.A., Payatakes, A.C., 2007. Hierarchical simulator of biofilm growth and dynamics in granular porous materials. Adv. Water Resour., 30, 1648–1667.
Conclusion
[11] Sheng, X., Ting, Y.-P., Pehkonen, S.O., 2008. The influence of ionic strength, nutrients and pH on bacterial adhesion to metals. J. Colloid Interf. Sci., 321, 256–264.
The results of this study highlighted numerous factors which contribute to corrosion, namely the bacteria’s corrosive ability, the organism’s metabolism, the environment, and biofilm forming ability. Without any nutrients, Gram-positive Bacillus spp. retarded the corrosion processes. Supplementation with different nutrients could trigger different metabolic pathways resulting in acceleration or mitigation of the corrosion rates which are strongly correlated to the level of biofilm formation by the microorganisms. The differences in the species present in the biofilm can lead to vastly different metabolic activities within the biofilm. This can explain why corrosion rates may vary with or without bacteria under the same environmental conditions.
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