Airborne bacteria associated with corrosion of mild

Environ Sci Pollut Res DOI 10.1007/s11356-017-8501-z

RESEARCH ARTICLE

Airborne bacteria associated with corrosion of mild steel 1010 and aluminum alloy 1100 Aruliah Rajasekar 1,2 & Wang Xiao 1 & Manivannan Sethuraman 1,3 & Punniyakotti Parthipan 2 & Punniyakotti Elumalai 2

Received: 20 September 2016 / Accepted: 23 January 2017 # Springer-Verlag Berlin Heidelberg 2017

Abstract A novel approach to measure the contribution of airborne bacteria on corrosion effects of mild steel (MS) and aluminum alloy (AA) as a function of their exposure period, and the atmospheric chemical composition was investigated at an urban industrial coastal site, Singapore. The 16S rRNA and phylogenetic analyses showed that Firmicutes are the predominant bacteria detected in AA and MS samples. The dominant bacterial groups identified were Bacillaceae, Staphylococcaceae, and Paenibacillaceae. The growth and proliferation of these bacteria could be due to the presence of humidity and chemical pollutants in the atmosphere, leading to corrosion. Weight loss showed stronger corrosion resistance of AA (1.37 mg/cm2) than MS (26.13 mg/cm2) over the exposure period of 150 days. The higher corrosion rate could be a result of simultaneous action of pollutants and bacterial exopolysaccharides on the metal surfaces. This study demonstrates the significant involvement of

Responsible editor: Diane Purchase Electronic supplementary material The online version of this article (doi:10.1007/s11356-017-8501-z) contains supplementary material, which is available to authorized users. * Aruliah Rajasekar [email protected]

1

Department of Civil and Environmental Engineering & Minerals, Metals and Materials Technology Centre (M3TC), Faculty of Engineering, National University of Singapore, Block EA, 9 Engineering Drive 1, Singapore 117576, Singapore

2

Present address: Environmental Molecular Microbiology Research Laboratory, Department of Biotechnology, Thiruvalluvar University, Serkkadu, Vellore, Tamil Nadu 632 115, India

3

Present address: Biofouling and Biofilm Processing Section, Water and Steam Chemistry Division, BARC Facilities, Kalpakkam, Tamil Nadu 603 102, India

airborne bacteria on atmospheric corrosion of engineering materials. Keywords Mild steel . Aluminum alloy . Atmospheric corrosion . Biofilm . Microbial community . 16S rRNA analysis

Introduction Microbiologically influenced corrosion (MIC) is an electrochemical progression in which microorganisms initiate, facilitate, or accelerate the corrosion reaction (Beech 2004; Rajasekar et al. 2010, 2011). From previous studies, it is known that microorganisms tend to append themselves to surfaces exposed to the ambient environment to colonize, proliferate, and form a biofilm (Flemming 1996). The biofilm, consisting of microbial cells and their metabolites as well as extracellular polymeric substance (EPS), creates gradients of pH, dissolved oxygen, nutrient contents, temperature and pressure, leading to MIC of metals and alloys (Sarro et al. 2006; Sherar et al. 2011; Narenkumar et al. 2016). The overall economic burden of corrosion amounts to at least 4%–5% of the GNP (Gross National Product), and 20%–25% of this cost has been estimated to be due to the action of microorganisms (Flemming 1996; Koch et al. 2002). Steel and its alloys are the most commonly employed metallic materials for construction of a wide range of equipment and metallic structures deployed in open-air environments due to their low cost and excellent mechanical strength (Brown and Masters 1982; De la Fuente et al. 2011). Most types of steel are exposed to open-air conditions, habitually in exceedingly polluted atmospheres, where corrosion is much more severe than in clean rural environments. Atmospheric corrosion leads to degradation of structures, devices, and products

Environ Sci Pollut Res

exposed to ambient air (Sarro et al. 2006). Hence, the field of atmospheric corrosion has received considerable attention from both scientific and industrial communities. The atmospheric corrosion of metals is primarily influenced by the nature of the environment to which they are exposed and has been widely studied (Qing et al. 2004; Ma et al. 2010; De la Fuente et al. 2011). Metal corrosion is caused by relative humidity (RH), concentrations, and deposition rates of key gaseous air pollutants such as sulfur dioxide (SO2), hydrogen sulfide (H 2S), oxides of nitrogen (NOx), and chlorides (Varotsos et al. 2009; Watkinson and Emmerson 2016; Ramachandran and Srivastava 2016), along with meteorological factors such as temperature, moisture, rainfall, solar radiation, wind velocity, and biological aerosols (Brown and Masters 1982). Airborne bacteria can colonize atmospheric metal surfaces and cause corrosion of the exposed structures. The bacterial polymers, generally polysaccharides, act as adhesives, catching grime and other particulate materials, increasing the disfiguring effects of the biofilm and leading to corrosion of the metal surface. The colonization process commences with autotrophs that require only inorganic materials for growth, followed by heterotrophic organisms (May et al. 1993). Both autotrophs and heterotrophs may accelerate the occurrence of MIC of exposed engineering materials by natural weathering processes and their major microbial activities. The gaseous species mentioned above are also present in the atmosphere and can additionally influence the corrosion rate of exposed materials (Qing et al. 2004; Ma et al. 2010; De la Fuente et al. 2011). However, the role of airborne bacteria on atmospheric corrosion of engineering materials has not been systematically investigated, especially in tropical environments where the prevailing weather conditions are favorable for the growth of microorganisms. The present study evaluates the role of airborne bacteria on atmospheric corrosion and delineates the mechanism of MIC on mild steel (MS) and aluminum alloy (AA) in a tropical marine atmospheric environment. This study focused on the characterization of the microbial biofilm formed on the selected MS and AA using molecular identification (16S rDNA gene) analysis. The morphology of biofilms formed over the metal surfaces was analyzed by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDS), while the rate of corrosion was assessed by electrochemical analysis (polarization and impedance) and weight loss (WL) method.

100 × 150 mm2 (WL analysis) and 10 × 10 mm2 (for SEM analysis) were cut from 1-mm-thick sheets and polished with no. 800 grade emery sheets followed by ultrasonic cleaning in pure acetone, then rinsed with distilled water. In order to evaporate the water on the metal surface completely, the specimen were rinsed with 99.9% ethanol and air dried, and then kept in desiccators until further use (Machuca et al. 2016). After the treatments, each coupon was weighted (initial weight, W0) prior to exposure tests using an analytical balance (the precision is 10 μg). The SEM images for polished control coupons of MS and AA were presented with smooth surface in Fig. S1 (Supplementary material). Bacteriological analysis of exposed mild steel and aluminum alloy samples Exposure site The exposure site was located on the rooftop of a tall building with no visible obstructions at the National University of Singapore (1°18′N; 103°46′E, 67 m above sea level). The site is influenced by vehicular traffic; a busy expressway connecting to the Central Business District runs southeast at a distance of 200 m north from the sampling location and another road runs southward at a distance of 50 m west from the sampling site. Petroleum, petrochemical, and specialty chemical industries are located at a distance of 5–10 km to the southwest. Corrosive pollutants analysis Automated online aerosol/gas analyzer (model ADI 2080, MARGA, Applikon Analytical B. V. Corp., Netherlands) with PM2.5 inlet was used to measure the mass concentrations of major water-soluble aerosol inorganic ions, ammonia, and major inorganic acidic gases at the time resolution of 1 h. The exposure site was further characterized with measurements of metrological parameters (relatively humidity, temperature, wind speed, and rainfall). All the MS and AA specimens were mounted on a test rack, exposed at a 45° angle horizontal to the sky and facing west for 30, 60, 90, 120, and 150 days. At the end of the each exposure periods, both MS and AA coupons were transferred to laboratory for bacterial enumeration, WL, and surface analysis. Enumeration of bacteria

Materials and methods Materials preparation Mild steel (MS 1010) and aluminum alloy (AA 1100) were used as the test materials, and their chemical composition is listed in Table 1. Metal coupons with dimensions of

Samples of biofilm formed on the exposed coupons (MS and AA) were transferred into sterilized 0.9% NaCl solution (30 mL) to enumerate the total viable bacterial count at different exposure periods (30, 60, 90, 120, and 150 days) . The biofilm formed on the coupons was scrapped using a sterilized spatula and was subjected to sonication for 3 min to detach the

Environ Sci Pollut Res Table 1

Chemical compositions of mild steel 1010 and aluminum alloy 1100 (wt.%)

Metals

C

Mn

S

Zn

Ni

Mo

Fe

Al

Cr

Cu

Si

P

Mg

Mild steel 1010

0.01

0.196

0.014

–

0.013

0.015

Balance

–

0.043

–

0.007

0.009

–

Aluminum 1100

–

0.015

–

0.003

–

–

0.518

Balance

0.005

0.061

0.152

–

0.01

biofilm into the solution phase (An 1997; Maruthamuthu et al. 2008). The samples were serially diluted (tenfold) with sterile distilled water followed by pour plate technique for the isolation of aerobic heterotrophic bacteria. The nutrient agar (NA) medium was used to enumerate heterotrophic bacteria. The bacterial counts were expressed as colony-forming units per centimeter (CFU/cm2) of biofilm samples. Morphologically, dissimilar colonies were selected randomly from all plates and isolated colonies were purified and stored at 4 °C until further analysis. The isolated bacterial cultures were subjected to morphological and biochemical characterization by Gram staining, indole test, methyl red test, motility test, citrate test, H2S test, lipid hydrolysis test, carbohydrate fermentation test, Voges-Proskauer test, catalase test, starch test, gelatin test, oxidase test, etc. (Holt et al. 1994). In addition, citrate agar was used to detect the iron-reducing activity of the isolates. Molecular identification of bacteria Bacterial DNA from the isolates was extracted according to Ausubel et al. (1988). DNA from microbial biofilms of MS and AA was PCR amplified with universal bacterial primers for 16S rRNA, 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACGGTACCTTGTTACGACTT-3′) (Teske et al. 2002). The PCR conditions used were as follows: initial temperature was set to 94 °C for 3 min; 30 cycles of 30 s denaturation at 94 °C, annealing for 1 min at 54 °C, and elongation at 72 °C for 2 min; a final elongation was run at 72 °C for 10 min. The PCR products were purified by QIAquick PCR purification kit (MBI Fermentas) as illustrated by the maker and cloned with Qiagen PCR cloning plus kit (MBI Fermentas). T3 and T7 primers were used to sequence the cloned fragments in both directions by Big Dye terminator method. Sequences were checked and assembled using Sequencer 2.0. (PE Applied Biosystems). DNA sequences from each sample were used to obtain the top sequence matches from GenBank using NCBI-BLAST (Altschul et al. 1990) and RDP Seqmatch (http://rdp.cme.msu.edu). Using ClustalX2 (Larkin et al. 2007), multiple sequence alignments were constructed containing 16S rRNA gene sequences with their respective top GenBank matches. The alignments were analyzed using Mega 5.05 (Tamura et al. 2011) to generate distance neighbor joining trees with a nucleotide model (Kimura 1980) and bootstrap values of 1000 (Hillis and Bull 1993). The identicalness percentages between sequences were calculated using MatGAT v. 2.01 software (Campanella et al.

2003). Sequences used in phylogenetic trees were submitted to GenBank (Accession Numbers JX984632 to JX984646). Weight loss measurements For WL measurements, coupons collected from different exposure periods (30, 60, 90, 120, and 150 days) were utilized. The corrosion products of MS and AA metal surfaces were chemically removed by immersion in an appropriate pickling solution (for MS 500 ml HCl + 500 ml distilled water + 3.5 g hexamethylenetetramine (Ma et al. 2010) and for AA 50 mL H3PO4 + 20 g CrO3 + 1 L H2O for 5–10 min at 80 °C (Davis 1993). After removal of the corrosion products from exposed coupons at different time intervals, coupons were rinsed with deionized water, dehydrated with hot air, and weighed to obtain the final weight (W1). The initial weight of the metal coupons before corrosion exposure experiments is W0. The difference between W0 and W1 is the corrosion WL. The procedure was repeated three times for each metal coupons and the average WL was calculated (Rajasekar et al. 2005). Statistical analysis (analysis of variance) was performed on these results. Final mean weights of the coupons in each system were used to calculate the average corrosion rates (CR) as recommended by the American Society for Testing and Materials, using this formula: CR = (K × W) / (A × T × D), where, K = a constant (8.76 × 104), W = mass loss in grams, A = area in cm2, T = exposure time in hours and D = density in grams per cubic centimeter. Surface analysis and corrosion products characterization Each metal coupon was exposed in triplicate at the outdoor atmosphere for 30, 60, 90, 120, and 150 days for WL measurements and corrosion product were collected, further subjected to morphological analysis and biofilm analysis. For surface analysis, both metal coupons were fixed using 3% glutaraldehyde in phosphate buffer saline (PBS) for 4 h, rinsed using PBS, and dried using an ethanol gradient (25%, 50%, 70%, 90%, and 99%) before storage in desiccators. Proceeding to observation, both metal coupons were coated using platinum at a electrical energy of 30 mV for 100 s. A scanning electron microscope (JEOL JSM-5600LV) with 15kV beam electrical energy was applied to imagine the morphology of the biofilm (Harimawan et al. 2011). The surface scenery of both metal coupons and elemental composition was characterized after removal of the corrosion products


using dilute nitric acid for 15 min and subjecting the coupons to scanning electron microscopy-energy-dispersive X-ray (SEM-EDAX) analyses. The elemental composition of biofilm formed on both metal surfaces at different exposure periods was examined by Fourier transform infrared (FTIR) spectroscopy (Bio-Rad Model FTS 135) analysis performed in the reflectance mode with wavenumber between the range of 400 and 4000 cm−1 using FTIR spectrometer prepared with a narrow band of liquid nitrogen-cooled HgCdTe (MCT) detector. Unexposed surface of both metals, MS and AA, was used as a control (Harimawan et al. 2011).

Results and discussion Pollutant analysis and metrological parameters In the study area, the main atmospheric chemical pollutants of concern are sulfur dioxide (SO2), sulfate (SO42−), nitrate (NO3−), and chloride (Cl−) (Table 2). It was observed that the average concentration of Cl−, SO2, NO3, and SO4 levels during the exposure periods were 0.25, 18.98, 1.26, and 3.26 μg/m3 respectively. The average RH was about 76% and the temperature varied during the entire month from 22 °C and 32 °C. It revealed that the atmospheric environment was rich in chemical pollutants due to industrial pollution. (Chemical industry is located near the coastal area is about 1.5 km from the exposure site). The wind speed (WS) and relative humidity (RH) over the exposure period are given in Table 2. A sudden increase in WS and RH was observed in the fifth month of the exposure period, reaching about 2.88 m/s and 78% respectively when compared to other exposure periods. It appears that WS and RH were mainly responsible for the erosion corrosion and increased retention of electrolyte within the rust film, leading to the reverse phenomenon in the corrosion process. The average rainfall at the exposure site was 338 mm with maximum rainfall in November (60 day exposure), leading to a higher WL than that observed after 30 days of exposure. The WL at the exposure period of 150th day was severe but rainfall was Table 2

Bacterial enumeration versus atmospheric exposure time The total viable count of heterotrophic bacteria (HB) on MS coupons was higher after the exposure period of 90 days (21.0 × 10−1 CFU/cm2) when compared to the exposure period of 60 days (8.7 × 10−1 CFU/cm2). The increasing trend of HB counts was observed up to 150th day of exposure (Fig. S2). This suggests that HB were able to colonize the MS coupon and may have accelerated the corrosion process. Bacterial growth was influenced by atmospheric conditions like temperature, RH, and chemical pollutants. RH during the exposure period was observed in the range of 70% to 79%. On 120th day of the exposure period, RH was observed as 78.8%, creating a favorable condition for the proliferation of bacteria on the metal surface. Since the RH was higher in the initial periods 30 and 60 days, the total viable counts obtained were lower when compared to longer exposure period (90, 120, 150 days). It can be explained that the rainfall was higher (507 and 563 mm) during those exposure periods of 30 and 60 days respectively, leading to the washing off of the biofilm formed on the metal surfaces. Hence, the washing effect by rainfall on surface of the metal coupons resulted in lower bacterial counts in the biofilm. The total viable count of HB of AA was higher in the initial exposure period of 30 days (6.75 × 10−1 CFU/cm2), and then counts were considerably lower (4.2 × 10−1 CFU/cm2) at 60th day of exposure period (Fig. S2). The counts of HB were stable after the exposure period of 90 days, and maximum counts were recorded at the end of exposure period of 150 days (16.4 × 10−1 CFU/cm2). Similar to MS, higher rainfall in the initial 2 months of exposure led to lower cell counts. From total viable HB counts, it was revealed that both metal surfaces

Average climatic parameters and pollutant data for the test site, Singapore

Exposure months (2011–2012)

Oct Nov Dec Jan Feb Average

lower at 140 mm of rain. It appears that the dry atmosphere (i.e., less rainfall) favors the metal surface as thick dry corrosion product and leads to the severe pitting corrosion process. This dry atmosphere leads to the dry deposition of environmental pollutants on metal surfaces leading to increased oxidation of metals within the rust film, which tends to be enhanced by high RH in ambient air.

Average temperature (°C) Min

Max

26.1 25.8 22.8 25.6 26.6 25.4

30.8 30.6 31.0 31.3 32.1 31.16

Average relative humidity (RH%)

77.4 79.3 70.1 76.5 78.8 76.4

Average rainfall (mm)

507.20 563.60 320.85 157.73 140.46 337.97

Wind speed (m/s)

0.22 0.20 0.18 0.20 2.88 0.7

Chemical pollutant concentration (μg/m3) SO2

Cl−

NO3

SO4

27.27 20.00 17.17 14.75 15.72 18.98

0.25 0.28 0.25 0.26 0.24 0.25

1.33 1.33 1.16 1.23 1.26 1.26

5.45 4.40 2.33 2.13 2.03 3.26


were prone to bacterial colonization and thus influences the corrosion reaction. Bacterial identification Preliminary identification of bacteria isolated from both metal surfaces (MS and AA) by biochemical tests indicated that the isolates belonged to the genera Bacillus, Staphylococcus, Cohnella, and Paenibacillus. The phenotypic profiles of the bacterial species identified from MS and AA are shown in Table S1a and S1b (supplementary material) respectively. All bacterial strains isolated from MS and AA showed deposits of ferric hydroxide precipitate around the colony, formed as rustred color (citrate agar test). The percentage distribution of general species in MS and AA metal surfaces exposed to the marine environment are presented in Fig. 1. In MS and AA, the percentage of distribution of each genus was about 78% Bacillus sp., 11% Cohnella sp., 11% Staphylococcus sp. and 66% Bacillus sp., 17% Staphylococcus sp., 17% Paenibacillus sp., respectively. Among the airborne heterotrophic bacteria isolated, only the Gram-positive bacteria were identified on both metal surfaces. Among the isolates, the Bacillus genera were more dominant than other isolates in both MS (78%) and AA (66%). The generic distribution of bacterial strains isolated from MS was found to be Bacillus sp. RBM4 and RBM5 (33%) and 17% of each species of B. megaterium RBM1, Paenibacillus sp. RBM2, B. thuringiensis RBM3, and Staphylococcus sp. RBM6. The generic distribution of bacterial strains isolated from AA was found to be Bacillus sp. RBA5 and RBA7 (22%), B. subtilis (22%), and 11% of each species of B. cereus RBA3, B. megaterium RBA8, B. thuringiensis RBA2, Cohnella sp. RBA6, Staphylococcus sp. RBA1. Gram-positive bacteria are more actively involved in biofilm formation on both metal surfaces (Rajasekar et al. 2011). Phylogenetic analysis of bacterial isolates from MS and AA are shown in the neighbor joining trees (Figs. 2 and 3, respectively). Phylogenetic trees for a total of 15 bacterial isolates revealed the presence of major phylum Firmicutes, 90

Aluminium alloy

Percentage, %

80

Mild steel

70 60 50 40 30 20 10 0

Bacillus

Cohnella

Staphylococcus Paenibacillus

Fig. 1 Distribution of bacterial species in biofilm formed on MS and AA metal surface exposed to urban industrial coastal site

having three families: Bacillaceae, Staphylococcaceae, and Paenibacillaceae. Bacillus cereus, B. megaterium, B. subtilis, B. thuringiensis are the predominant species in the family Bacillaceae. Staphylococcus sp. is the only genus detected in the family Staphylococcaceae present in the two trees. High similarities between the sequence of the bacterial isolates and database sequences are given in brackets ranging from 98.5% to 99.6% which confirmed the isolates to species level. The 16S rRNA and phylogenetic analyses show that Firmicutes were the predominant phylum of bacteria detected in AA and MS samples. This result is in accordance with studies that have found the bacterial phylum and mostly Bacillus species in the air over urban, rural, and high alpine locations (Despres et al. 2007). Aerosol formation is one of the causes for the atmospheric corrosion which is mainly influenced by the abundant microbes in the air. The microbial distributions and their metabolism in the atmospheric environment might enhance emission into the atmosphere leading to corrosion of metals and materials exposed to atmosphere. Our study shows the ubiquitous nature of Bacillus-related genera in atmospheric environment correlating with the results and studies of Urbano et al. (2010). Ten bacterial species among the 15 isolates from the two samples are spore formers which have been confirmed with morphological characterization. Urbano et al. (2010) and Onyenwoke et al. (2004) have previously observed the influence of bacterial spore formation by Bacillus related genera in atmospheric corrosion. Weight loss of mild steel 1010 and aluminum alloy 1100 The WL and the corresponding CR of MS are shown in Fig. 4a, with an average WL of 26.13 mg/cm2 and an average CR of 0.001 mm/year following environmental exposure of up to 150 days. After the initial exposure period of 30 days, the WL of MS was about 12.75 mg/cm2 and steadily increased to about 30 mg/cm2 following exposure for 90 days. However, the WL decreased to 24.31 mg/cm2 in the next 30 days, which can be attributed to the thick rust product formed on the metal surface acting as a passive barrier layer. At the end of the exposure period (after 150 days), the WL reached about 42 mg/cm2. It can be explained that Breverse phenomenon^ occurred during the long atmospheric exposure. In general, the atmospheric CR decreased with prolonged exposure time as observed by Fonseca et al. (2004). It has also been reported that atmospheric corrosion rates in marine surroundings with apparent reverse phenomenon showed that the average CR initially enhances, then decreases, and again suddenly increases after a certain period (Fonseca et al. 2004; Qing et al. 2004; Ma et al. 2010). Similar to MS, WL and corresponding CR of AA are shown in Fig. 4b, with an average WL of 1.37 mg/cm2 and average CR about 0.0001 mm/year for 150 days. After the initial exposure period of 30 days, WL of

Environ Sci Pollut Res Fig. 2 Neighbor joining tree for the 16S rDNA sequences belonging to the families Bacillaceae, Paenibacillaceae and Staphylococcaceae (Phylum Firmicutes) of the isolates from mild steel. The identified species have been given in boldface with its sample ids (RBM series 1–6). Sequences taken from databases for analyses have been given with their accession numbers in parentheses. Similarity percentages between sequence of isolate and sequence of closest relative are given in brackets with boldface. Enterococcus aquimarinus was used as a bacterial outgroup. Bootstrap values having >50% are shown in the main nodes after the analyses of 1000 replicates; the scale bar represents the expected number of substitutions averaged over all sites analyzed

AA was about the 0.25 mg/cm2 and consistently increased with respect to the exposure period. The WL was lower for AA when compared to MS through the exposure periods. In terms of CR, it was observed that after the initial exposure period, the CR was high. The CR then gradually decreased and remained low with the prolonged exposure period (Fig. 4a). The CR was within a range of 0.001 mm/year for MS, and this observation was consistent with those reported by Ma et al. (2010). The initial higher CR was mainly due to the availability of the Bfresh^ metal surface to the atmospheric

Fig. 3 Neighbor joining tree for the 16S rDNA sequences belonging to the families Bacillaceae, Paenibacillaceae, and Staphylococcaceae (Phylum Firmicutes) of the isolates from aluminum alloy. The identified species have been given in boldface with its sample ids (RBA series 1–9). Sequences taken from databases for analyses have been given with their accession numbers in parentheses. Similarity percentages between sequence of isolate and sequence of closest relative are given in brackets with boldface. Lactobacillus alvi was used as a bacterial outgroup. Bootstrap values having >50% are shown in the main nodes after the analyses of 1000 replicates; the scale bar represents the expected number of substitutions averaged over all sites analyzed



Fig. 5 Surface appearance of a mild steel and b aluminum alloy exposed to the atmospheric corrosion (AC) at test sites

layer with severe corrosion; (2) long exposure period, exfoliation of the layer due to interaction with the environment pollutant leads to the slowdown of the corrosion process; (3) extended exposure period, removal of thick corrosion product due to the physicochemical parameters leads to enhance the fresh surface for corrosion reaction and causes severe corrosion on the metal surface. Surface/morphology analysis of the corrosion product formed on mild steel and aluminum alloy Fig. 4 Weight loss data: a mild steel, b aluminum alloy versus exposure time in the test site

environment, creating favorable conditions for metal reactions to occur due to environmental parameters like physical, chemical, and biological corrosive factors. However, after this Bhoneymoon corrosion period,^ there was formation of a protective layer of corrosion products on the metal surfaces. This led to the limited contact of the corrosive species to the metal surface, thus decreasing the CR. This was suggestive that corrosion was mostly reliant on the rate of incessant removal of corrosion products, or the exfoliation of the protective layer on the metal surface (Fonseca et al. 2004; Natesan et al. 2006). It was also reported by De la Fuente et al. (2011) along with the explanation of the phenomenon in terms of the structure of the protective layer formed on the metal surface. It was noticed that initially, severe corrosion was followed by the formation of a thick corrosion product layer on long exposure period and thus led to limit the entry of corrosive species to the metal surface and caused the CR to slow down (Fonseca et al. 2004; De la Fuente et al. 2011). In the present investigation, an overall increasing trend in mass loss indicated that continuous corrosion process occurred on the MS metal surface and this trend was also observed by De la Fuente et al. (2011). Hence, the current observation on atmospheric corrosion of MS metals consists of three concurrent corrosion processes: (1) initial exposure period, formation of the corrosion product

Surface appearances of MS and AA exposed for various days at the test sites are shown in Fig. 5a, b. At the exposure period of 30 days, MS was observed as light brown in color (corrosion product) with some pitting attack (Fig. 5a). When the exposure period was increased, the MS surface was dark brown in color. This could be due to the severe corrosion product formed on the MS surface at the test site, and a similar trend was observed for increased exposure period. Figure 5b shows the surface appearance of AA at 30 days of exposure period, and it was noticed that a few white discrete corrosion products formed on the surface. The white corrosion product was observed at all exposure periods. The surface appearance of the two exposed metals over the exposure periods revealed that the test site environment was very prone to corrosion. The surface morphology of MS was varied significantly between different exposure periods as shown in Fig. 6. The morphology of the oxide layer formed on MS has been observed by several groups (Antunes et al. 2003; Raman et al. 1987, 1989; De la Fuente et al. 2011). The phases most Fig. 6 Surface morphological structures found in mild steel corrosion products formed in urban industrial exposure site; SEM micrographs shows a, a1 30 and b, b1 60 days–fine plates (Bflowery^ structures) typical of lepidocrocite, c, c1 90 and d, d1 120 days–globular (Bcotton balls^) structures typical of goethite, e, e1 150 days–Brosette^ morphologies typical of akaganeite. Note: a1–e1 are a magnified view of the respective exposure periods



frequently observed as lepidocrocite appears as small crystalline globules (sandy crystals) or as fine plates (flowery structures) (Fig. 6 a1, b1); goethite appears as globular structures known as cotton balls (semi-crystalline goethite) (Fig. 6 c1, d1) or even as acicular structures (crystalline goethite); akaganeite appears with cotton ball and rosette morphologies (Sagoe-Crentsil and Glasser 1993) (Fig. 6 e1) and d-FeOOH shows a distorted plate-like morphology (Sagoe-Crentsil and Glasser 1993). Figure 6 a1 shows an irregular, cracked, and non-protective oxide layer (open structure) which allows the easy access of corrosive microorganisms to the metallic surface; the usual condition in atmospheres of highly aggressive environment. In comparison, dense oxide film (closed structures) favors protection of the metallic substrate. Due to the higher rate of chloride deposition in marine atmospheres, greater degree of flaking was observed; loosely adherent flaky rust favored layer breakdown (spallation, detachment) and the initiation of fresh attack (Fig. 6a, c, e). The rust layer on the skyward side of the panels was usually thinner and finer than on the rougher ground ward side (Fig. 6 d1). This roughness was due to the large rust particles and easy spallation of large flaky rust (Raman et al. 1989). The surface morphological characteristics of the exposed MS samples after the removal of corrosion product are shown in Fig. 7. During the initial period (30th day), uniform corrosion with severe pit was observed (Fig. 7 a1) whereas with increased exposure period the pitting corrosion became more severe (Fig. 7b–e). Severe pitting attacks were noted, with a pit size of more than 50 μm in diameter over the surface of steel exposed to 150th day (Fig. 7 d1). Figure 8 shows the typical SEM surface appearances of AA coupons after a series of exposure times. The images show the significant evolution of surface morphology of the samples at various times (Fig. 8a–e). After 1 month of field exposure, the surface of AA became slightly rougher and darker than the original sample (Fig. 8 b1). Cracks formed in an insect shape and were distributed over the surface. The maximum diameter of the cracks was about 10 μm. After 3 months of exposure, the cracks obviously increased in quantity. Few cracks appeared to sparkle, indicating that there were possible corrosion products formed in the vicinity of or under the crack (Fig. 8 d1). After exposure of 5 months, the AA showed larger cracks and pit and more corrosion products on the surface of the coupon (Fig. 8 e1). With prolonged exposure times, the number of pits was increased and gradually appeared as a honeycomb distribution over the surface of AA. Figure 9 shows that the corrosion attack was initiated within 30 days of exposure and the pits were severe with respect to the exposure period. Significantly, the pit size was higher than about 30 μm at the exposure period of 90 days (Fig. 9 b1) when compared to 30 days (Fig. 9 a1). The severe corrosion pits (40 μm) were observed at the exposure period of 150 days

(Fig. 9 d1). This suggested that the AA metal was very prone to a pitting type of corrosion rather than uniform corrosion in an urban industrial atmospheric environment. After the exposure period, the corrosion products that formed on MS and AA surfaces were removed by a pickling solution and the surface morphology and EDAX are presented in Fig. S3 (supplementary material). As presented in Fig. S3a, iron was present as a major component on MS surface and increasing the exposure period increased the intensity of iron, as noted by very low intensity at 30 days of exposure period compared with longer exposure periods at 90 and 150 days that had higher intensity peaks (Fig. S3b and Fig. S3c). Trace amounts of aluminum, chloride, and zirconium were also present on the MS surface. Similar to MS, SEM-EDAX observation of AA showed aluminum, chloride, and zirconium oxides (Fig. S3d–f) as major corrosion products. Increases in the exposure period also increased the concentration of corrosion products. The presence of chloride on both metal surfaces may favor the pitting type of corrosion. It was clear that the formation of insulating amorphous oxide film on the AA surface had little solubility within air and aqueous solutions between the pH range of 4 to 8.6. Common corrosion product aluminum sulfate was observed on the AA surfaces. It indicates that the adsorption of gaseous pollutant SO2 in the air reacts with AA surface and formed aluminum sulfate (Barton 1976; Graedel 1989) as observed in the EDAX data. Figure 10 shows the FTIR spectrum of MS and AA metal samples exposed at different exposure periods at an urban industrial coastal site. The peak at 3380 cm−1 revealed the presence of NH stretch for NH 2 group. The peak at 1720 cm−1 indicated the existence of SH. Other peaks at 1440 and 1353 cm−1 specified that presence of CH def for CH3 group. One more peak at 1019 cm−1 showed the occurrence of C=O stretch. In addition, the presence C-Cl was seen at 558 cm−1. The intensity of the FTIR peak for MS at the exposure period of 90 days was higher (Fig. 10a); this may be due to the high accumulation of bacterial metabolites i.e., EPS. Moreover, at the AA surface, stronger intensity of the SO4 asymmetric stretching peak around 1107 cm−1 at all exposure periods (Fig. 10b) indicated an increase in basic aluminum sulfate in the corrosion product layer (Contreras et al. 2006; Yalfani et al. 2007; Dan et al. 2011). It can be explained that airborne bacteria were able to colonize on both metal surface and formed a thicker biofilm. Hypothesis of atmospheric microbial influenced corrosion (AMIC) In the present study, the presence of acid-forming bacteria, B. cereus and Staphylococcus sp., in the samples suggested that these bacteria may play a key role in the corrosion of both MS and AA. Staphylococcus sp., which was detected in the biofilm sample collected from both

Environ Sci Pollut Res Fig. 7 SEM images of the coupon surfaces after removal of the corrosion products: a, b, c and d images of MS sample exposed for 30, 90, 120, and 150 days, respectively; a1, b1, c1, and d1 images of MS samples show higher magnification of respective exposure periods

metal surfaces, are often present in soil, bioaerosols, and water. They are able to fix nitrogen under anaerobic or micro-aerobic conditions (Holt et al. 1994). These bacteria generate nitric acid and/or nitrates that may contribute to metal corrosion (Zhu et al. 2003; Rajasekar et al. 2010). Bacillus thuringiensis RBM3 and B. megaterium RBM1 have minimal nutritional requirements and are often present in aquatic/atmospheric environments that are loaded with organic contaminants such as inorganic nutrient, gasoline, and solvents (Zhu et al. 2003). In addition, Bacillus megaterium RBA8 and B. megaterium RBM1 contribute to biofilm formation by producing exopolysaccharides and facilitating the attachment on metal surfaces. The

metabolites of EPS produced on metal surfaces create a potential difference on metal surface and thus enhance corrosion (Machuca et al. 2016). Cohnella sp. RBA6 is a Gram-positive, endospore-forming, motile, and rodshaped bacterial strain ubiquitously present in the environment (Kim et al. 2011). It is capable of producing spore during the unfavorable condition for growth. It was previously shown that this bacterium forms a biofilm with a corrosion layer on the metal surface (Machuca et al. 2016), helping the bacteria further proliferate on metal surfaces. The results obtained on the durability of MS and AA were interpreted as a function of exposure time, environmental pollution levels, and total bacterial counts.



Fig. 8

Surface morphological structures found in aluminum alloy corrosion products formed in urban industrial exposure site; SEM micrographs a, b, c, d and e shows sample for 30, 60, 90, 120, and 150 days respectively; a1, b1, c1, d1, and e1 images of AA samples show higher magnification of respective periods

Fig. 9 SEM images of the coupon surfaces after removal of the corrosion products: a, b, c, and d images of AA sample exposed for 30, 90, 120, and 150 days, respectively; a1, b1, c1, and d1 images of AA samples show higher magnification of respective exposure periods

The chemical pollutants are deposited on the metal surface and act as nutrients for the heterotrophic bacteria. Besides, RH aids in bacterial proliferation on the metal surface and forms an intact biofilm. Staphylococcus sp. utilizes the SO4 and converts it into sulfuric acid. It creates the low pH environment on the metal surface and thus accelerates corrosion. Fe oxidizers


Fig. 10 FTIR spectrum of biofilm formed on the a MS and b AA metal surfaces

Fig. 11 Schematic representation of the atmospheric corrosion mechanism in MS and AA metal surface in presence of environmental pollutants and microorganism

like Bacillus sp. oxidize Fe2+ to Fe3+ and form ferric oxides as corrosion products (reaction 3) as clearly observed by the SEM-EDAX analysis. It can be claimed that airborne bacteria persuade the corrosion of steel. All isolated bacterial strains are catalase and oxidase positive. Jones (1986) suggested that the insoluble materials are a combination of ferric oxide (Fe2O3) and ferric hydroxide (Fe (OH)3), which can be referred to as FeO (OH)n and explained that ferric ions are unlikely to precipitate completely, especially in the acidic crevice regions. As a consequence, ferric ions present in the biofilm serve as highly oxidizing species and thus may accelerate corrosion. The presence of chloride in the atmospheric pollutant indicates (Table 2) that the likelihood of corrosion occurs on the metal surface. It is well known that chloride is a corrosive agent. Moreover, due to the presence of chloride ions on the metal surface, the iron-oxidizing bacteria may be directly involved in the formation of ferric chloride (FeCl3), which is an exceptionally corrosive material that can concentrate under corrosion nodules (Jones and Amy 2000). Chloride ions are capable of drift into a fracture location by neutralizing the increased charge via anodic dissolution and then combining with the oxidized products of ferrous and aluminum ions, as evident from SEM-EDAX analysis (Fig. S3 in supplementary material). Staphylococcus sp. and Bacillus sp. are facultative anaerobes, and biochemical tests indicate the presence of catalase and cytochrome oxidase. Staphylococcus and Bacillus sp. have a peroxidase enzyme, which produces hydrogen peroxide during the metabolism. Furthermore, it generates catalase that conquers the toxic nature of hydrogen peroxide, breaking


it down into water and oxygen (Borenstein 1988; Busalmen et al. 2002). It can also be assumed that Staphylococcus sp. and Bacillus sp. favor the Fenton reaction (Bentiss et al. 2000), (Eqs. 1 and 2) by oxidizing ferrous iron, important for the generation of hydroxyl radicals that can damage biological macromolecules (Touati 2000). . 1 2O þ2Hþ þ 2e → H2 O ð1Þ 2

Fe þH2 O2 → Fe3þ þ OHþOH‐ 2þ

ð2Þ

It is inferred that the formation of Fe3+ combines with OH ions and exopolymeric substances (organic matter) to form an iron-organic complex as a corrosion product. Hydrogen and carbon are consumed by airborne bacteria from chemical pollutants deposited on the metal surface. The oxygen from peroxide and H+ molecules from the organic carbon layer (i.e., chemical deposited on the metal surface) combines with Fe2+ to produce Fe3+. Since these bacteria are capable of living in low-pH environments, Fe3+ formation is encouraged by peroxide production and corrosion according to Eq. 2. Airborne isolates further convert ferric ion to ferric oxides by inclusion of oxygen from the bacterial metabolism (Eq. 3), and finally the ferric organic complex may be formed as a corrosion product on the metal surface, as observed in the FTIR (Fig. 11) and SEM-EDAX (Fig. S3 in supplementary material) analysis. . Fe2þ þ1 4O þH‐ → Fe3þ þH2 O2 ð3Þ 2

The overall biocorrosion mechanism of MS and AA metal surface can be described as follows (Schematic diagram in Fig. 11). Airborne isolates are facultative anaerobes; initial biochemical characterization specified the existence of catalase and cytochrome oxidase. Dominant species Staphylococcus and Bacillus have a peroxidase enzyme (Eq. 4), which produces hydrogen peroxide, which is further broken down by the catalase enzyme into water and oxygen (Eq. 5). Superoxide dismutase O2 þ2H2 O þ 2e‐ → H2 O2

ð4Þ

Catalase . H2 O2 → H2 O þ 1 2O

2

ð5Þ

Since the ferric/aluminum present in the metals have high affinity for oxygen, it obtains oxygen from product acquired through hydrogen peroxide and encourages the formation of ferric/manganese oxides and speeds up the corrosion reaction. It can be elucidated that since electrons are needed continuously for bacterial metabolic activity, it converts the Fe2+ to Fe3+, Al2+ to Al3+, and forms ferric oxides (Fe2O3) and

aluminum oxides (Al2O3) by continuous addition of oxygen. Besides, the rich source of chemical pollutants at the test site acted as a nutrient source for the bacteria and led to the proliferation of bacteria in the biofilm.

Conclusions The influence of airborne microbial biofilm on AMIC of MS and AA exposed to urban industrial coastal site, Singapore, has been examined in detail using WL method, bacterial analysis (16S rDNA gene analysis), FTIR, and SEM-EDAX. The 16S rRNA and phylogenetic analyses showed that Firmicutes are the predominant bacterial phylum detected in aluminum alloy and mild steel samples. The Bacillus sp. and Staphylococcus were identified as dominant airborne bacteria and capable of accelerating corrosion of mild steel and aluminum alloy. It is concluded that the acceleration of corrosion was due to the activity of bacterial metabolism, which was encouraged by chemical pollutants. FTIR study confirmed the presence of C=O stretch, C-Cl and NH2 group, owing to the bacterial EPS. Corrosion products like ferric oxides, aluminum oxides, aluminum sulfate were noticed in the SEMEDAX analysis and revealed an influence on corrosion as iron oxidizer and acid producer. Higher CR of mild steel was noticed when compared to aluminum alloy due to the simultaneous action of pollutants with airborne bacterial metabolism and thus leading to severe AMIC. This study shows the role of airborne bacteria on microbial influenced corrosion of MS and AA. Acknowledgements The authors are thankful to the two anonymous reviewers for improving the earlier version of our manuscript.

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