How Often Should Microbial Contamination Be Detected in Aircraft Fuel Systems? An Experimental Test of Aluminum Alloy Corrosion Induced by Sulfate-Reducing Bacteria part 1

[Audio] materials Article How Often Should Microbial Contamination Be Detected in Aircraft Fuel Systems? An Experimental Test of Aluminum Alloy Corrosion Induced by Sulfate-Reducing Bacteria Bochao Lu 1,2,†, Yimeng Zhang 1,*,†, Ding Guo 1, Yan Li 3, Ruiyong Zhang 1 , Ning Cui 2,* and Jizhou Duan 1 1 Key Laboratory of Advanced Marine Materials, Key Laboratory of Marine Environmental Corrosion and Biofouling, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; [email protected] (B L ); [email protected] (D G ); [email protected] (R Z ); [email protected] (J D ) 2 School of Mechanical and Automotive Engineering, Qingdao University of Technology, Qingdao 266520, China 3 Qingdao Campus of Naval Aviation University, Qingdao 266041, China; [email protected] * Correspondence: [email protected] (Y Z ); [email protected] (N C ) † These authors contributed equally to this work. Citation: Lu, B.; Zhang, Y.; Guo, D.; Li, Y.; Zhang, R.; Cui, N.; Duan, J How Often Should Microbial Contamination Be Detected in Aircraft Fuel Systems? An Experimental Test of Aluminum Abstract: Microbial contamination in aircraft fuel-containing systems poses significant threats to flight safety and operational integrity as a result of microbiologically influenced corrosion (M-I-C--). Regular monitoring for microbial contamination in these fuel systems is essential for mitigating M-I-C risks. However, the frequency of monitoring remains a challenge due to the complex environmental conditions encountered in fuel systems. To investigate the impact of environmental variables such as water content, oxygen levels, and temperature on the M-I-C of aluminum alloy in aircraft fuel systems, orthogonal experiments with various combinations of these variables were conducted in the presence of sulfate-reducing bacteria. Among these variables, water content in the fuel oil demonstrated the most substantial influence on the corrosion rate of aluminum alloys, surpassing the effects of oxygen and temperature. Notably, the corrosion rate of aluminum alloys was the highest in an environment characterized by a 1:1 water/oil ratio, 0% oxygen, and a temperature of 35 C Within this challenging environment, conducive to accelerated corrosion, changes in the corrosion behavior of aluminum alloys over time were analyzed to identify the time point at which M-I-C intensified. Observations revealed a marked increase in the depth and width of corrosion pits, as well as in the corrosion weight-loss rate, starting from the 7th day. These findings offer valuable insights for determining the optimal frequency of microbial contamination detection in aircraft fuel systems. Alloy Corrosion Induced by Sulfate-Reducing Bacteria. Materials Keywords: Desulfovibrio; biocorrosion; aluminum alloy; fuel oil; corrosive environments 2024, 17, 3523. doi 10.3390/ma17143523 Academic Editor: Helena Otmaˇci´c ´Curkovi´c 1. Introduction Received: 3 June 2024 Revised: 10 July 2024 Accepted: 13 July 2024 Published: 16 July 2024 Copyright: © 2024 by the authors. Licensee M-D-P-I-, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Corrosion has emerged as a significant impediment to economic development, environmental preservation, and safety, resulting in a substantial 3.34% loss in China’s Gross Domestic Product (G-D-P--) [1]. Metallic materials in aircraft structures are susceptible to corrosion, accounting for 23.6% of the total maintenance costs of the total sustainment costs for U-S Air Force aviation and missiles in the fiscal year of 2018 [2]. Among the wide range of corrosion forms, microbiologically influenced corrosion (M-I-C--) is metal corrosion associated with the action of microorganisms present in the corrosion system. It is one of the significant contributors to the corrosion of man-made infrastructure and equipment in natural environments, especially in marine environments with high humidity, temperature, and salinity, as well as other harsh and extreme conditions [3]. Estimates suggest that losses attributable to M-I-C could constitute as much as 20% of total corrosion-related losses, with corrosion rates reaching up to 4 millimeters per year on metal materials [4]. Microbial contamination.

[Audio] Materials 2024, 17, 3523 2 of 18 the aircraft, leads to serious consequences ranging from filter and injector fouling to engine malfunctions and accelerated equipment corrosion [5]. The microbial tests to monitor the microbial contamination in fuel and fuel systems were only commenced in 2002 following safety incidents attributed to MIC [6]. In such fuel-containing systems, hydrocarbons with C10–C18 carbon chains in fuel were shown to be degraded and serve as the carbon sources to support microbial growth. Additionally, nitrogen and inorganic salts from both fuel and non-metallic materials, such as nitrile rubber and polyurethane foam in the fuel tank, are also utilized for microbial growth and multiplication [7]. The fuel tank of an aircraft will inevitably contain water that is condensed from the fuel itself and the outside air during the process of transportation, daily use, and maintenance of the aircraft, supporting microbial growth [8]. The concentration of oxygen in the headspace of fuel systems can also significantly impact microbiologically influenced corrosion M-I-C--. Furthermore, aircraft experience fluctuating temperatures throughout their flights. When conditions such as temperature, water presence, oxygen levels, nutrients, and other factors align to support microbial growth, a rapid proliferation of microorganisms occurs, leading to corrosion in the fuel tank and escalating maintenance and repair expenses [9–11]. Therefore, the assessment of microbial contamination in fuel-containing systems must consider the influence of the surrounding environment, including varying humidity, oxygen levels, and temperature, on microbial growth [12]. The contaminative microorganisms in aircraft fuel and fuel systems mainly include fungi and bacteria. Filamentous fungi with hydrocarbon-degrading ability, including Aspergillus, Cladosporium, and Penicillium, can be a major problem that causes corrosion by producing a variety of acids. In addition, facultative bacteria, such as Bacillus, and anaerobes, such as sulfate-reducing bacteria (S-R-B--), are possible threats [13]. Among diverse contamination-causing microorganisms in maritime aircraft, S-R-B--, with a sulfate-respiration metabolism, are the most threatened microbial populations to the corrosion of aviation fuel systems [14–17]. The corrosion mechanism of S-R-B has been studied extensively, including currently proposed chemical-MIC through an excreted chemical agent and electrical-MIC through direct electron uptake [18–22]. Aluminum alloys are widely used in aircraft as the skin, wing beam, partition frame, and integral fuel tank panel because of their good performance in terms of high corrosion resistance, high strength, and low density [23]. The dense oxide passivation film (mainly Al2O3) is formed on the surface of aluminum alloys to resist corrosion. Research on microbiologically influenced corrosion (M-I-C--) in aviation fuel systems has predominantly focused on studies influenced by sulfate-reducing bacteria (S-R-B--). The key finding from these studies suggests that S-R-B accelerate aluminum corrosion by disrupting the protective passivation film through the action of biogenic sulfide [24–28], while some research pointed out that the biofilms formed by S-R-B can also reduce corrosion by protecting the aluminum alloy from acidic corrosive substances [29]. The corrosion behavior of S-R-B is likely influenced by the surrounding environments within aviation fuel systems, including factors like water content, oxygen levels, and temperature [30], whereas comprehensive studies in this area are currently lacking. To reduce the threat of microbial corrosion of aluminum alloy-made materials in the fuel systems of aircraft, it is necessary to detect and monitor microbial contamination of fuel periodically. However, a better understanding of the risk of microbial corrosion in the aircraft fuel system, as well as the growth features and corrosion behavior of corrosioncausing microorganisms under different environmental factors, is still lacking. There are no systematic and uniform detection standards for microbial detection in marine aircraft, especially how often the microbial contamination should be monitored. To select the environment in which the M-I-C of aluminum alloys is accelerated the most,.

[Audio] Materials 2024, 17, 3523 3 of 18 factors and the number of experiments required, and (2) systematically studying the effects of multiple factors on experimental outcomes, facilitating comparison of the impact of different factors. As the multi-environmental factors were considered in the present study, the orthogonal experiment is an effective way to access M-I-C in these complex environments. The orthogonal experimental design here combined three environmental factors (water content, oxygen content, and temperature) with various gradient concentrations separated into nine different groups. The influence of environmental factors on microorganisms was clarified, and the combination of environmental factors with the greatest impact on M-I-C was screened out through this orthogonal experiment. Secondly, under such a combination of environmental factors that highly accelerate corrosion, a 14-day time immersion experiment was carried out to study the effects of a corrosion-causing S-R-B strain on aluminum alloy corrosion at different growth stages. Subsequently, under the identified combination of environmental factors that greatly accelerated corrosion, a 14-day immersion experiment was conducted to investigate the effects of a corrosion-causing sulfate-reducing bacteria (S-R-B--) strain on aluminum alloy corrosion at different growth stages. It was observed that corrosion of the aluminum alloy accelerated notably from the 7th day, coinciding with a significant deterioration in fuel quality and heightened activity of S-R-B on the alloy surfaces. These findings offer valuable theoretical insights for establishing the detection cycle of contaminated microorganisms in maritime aircraft fuel systems. 2. Materials and Methods 2.1. Materials and Microbes A commonly used aluminum alloy in aircraft manufacturing, 7B04 aluminum alloy, was chosen as the representative material in the present study. The chemical composition of 7B04 was as follows (wt %): Al 91.2, Si 0.10, Fe 0.05, Cu 1.40, Mn 0.2, Mg 1.80, Cr 0.10, Ni 0.10, Zn 5.0, and Ti 0.05. Firstly, the aluminum alloy plates were cut into 10 × 10 × 3 millimeters3 square coupons using a wire cutting machine and polished to 2000 grit with SiC papers step by step. Secondly, the coupons were washed with anhydrous ethanol and sonicated for 20 minutes at room temperature to remove the impurities attached to the surface. Finally, the coupons were dried in N2 atmosphere and placed in an anaerobic glove box. The electrochemical working electrode has a working area of 1 centimeters2, and the rest of the electrode is sealed with polytetrafluoroethylene. All coupons were placed under UV light for 40 minutes and then transferred to modified P-G-C medium. The UV lamp is a 750 watt BBS-SDC model, and the distance between the UV lamp and the aluminum alloy coupon is 500 millimeters. A marine sulfate-reducing bacteria, D bizertensis, which was isolated from the corroded metals, was cultivated using a modified Postgate’s Medium C [31,32]. The modified Postgate’s Medium C contained 0.5 grams KH2PO4, 1 grams NH4Cl, 0.06 grams CaCl2·6H2O, 0.06 grams MgSO4·7H2O, 6 mL 70% sodium lactate, 1 grams yeast extract, and 0.3 grams sodium citrate in 1 liters aged seawater from a Qingdao offshore area. Prior to testing, the medium pH was adjusted to 7.2 ± 0.1. L-cysteine was added to remove residual oxygen from the medium after N2 was passed through the medium for 30 minutes. The rubber stopper, culture medium, aeration needle, and cotton are sterilized in an autoclave at 121 C for 20 minutes. 2.2. Orthogonal Experiments of Various Environmental Factors In practical cases, the contaminated microbes in aircraft systems usually grow in various and complicated environments with diverse humidity, oxygen, and temperature levels. To determine the extent of environmental factors that affected M-I-C process, a statistical orthogonal experiment was set.

[Audio] Materials 2024, 17, 3523 4 of 18 immersion experiment contained 50 mL of medium and was inoculated with 0.5 mL of S-R-B seeds; the 500 mL wide-mouth flask with stopper used for the electrochemical experiment contained 450 mL of medium and was inoculated with 4.5 mL of S-R-B seeds. The aluminum alloy coupons were put into these bottles, which were put into incubators with different temperatures, as shown in Table 1. The corrosion rate of these coupons, as shown by pit depth and average weight loss tests under the 9 conditions, was tested after 14 days of incubation, as described in the following part. Table 1. Nine groups of orthogonal experiments under different environmental conditions. Group Water Content Oxygen Content Temperature ( C) 1 20% 10% 15 2 20% 20% 35 3 20% 0% 25 4 50% 20% 15 5 50% 10% 25 6 50% 0% 35 7 90% 10% 35 8 90% 0% 15 9 90% 20% 25 2.3. Corrosion Behavior of Aluminum Alloy Influenced by D bizertensis under Harshest Conditions After choosing the most corrosive conditions in which the aluminum alloy had the highest corrosion rate, corrosion behavior tests over time were performed to determine the time point at which the corrosion rate obviously increased under the influence of D bizertensis. The tests of electrochemical behavior, surface morphology, mineralogy, and cell growth were performed as described below. The OD values were observed using a spectrophotometer with an absorbance of 600 nanometers to characterize the planktonic cell count. The initial inoculate concentration of bacterial cells was 108 cells/mL. Sessile cells were rinsed using P-B-S to observe the counts (deionized water 1 liters, KH2PO4 0.27 grams, Na2HPO4 1.42 grams, KCl 0.2 grams, NaCl 8 g). Observed and counted in a light microscope at 400× magnification using a blood cell counter. 2.4. Biofilm and Corrosion Product Film Characterizations The coupons were removed from the medium and immersed in glutaraldehyde solution for 2 hours to immobilize the bacteria. Stage-by-stage dehydration using anhydrous ethanol (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% (v/v)), followed by blowdrying of the coupons under N2. Scanning electron microscopy (S-E-M--) (Zeiss, Oberkochen, Germany) was used to observe the corrosion product films on aluminum alloy surfaces. The distribution of live/dead bacteria on the surface of the coupon was observed using fluorescent microscope (F-M---) (Olympus, Tokyo, Japan) after the following steps. Firstly, rinse the surface of the specimen with P-B-S to remove the planktonic bacteria on the surface. Secondly, use a blotting paper to absorb the P-B-S solution on the surface. Finally, drop the stain evenly onto the surface of the specimen. Staining was carried out under dark conditions for 20 minutes for fluorescence observation. X-ray diffraction (X-R-D--) (Rigaku, Akishima-shi, Japan) and energy-dispersive spectroscopy (E-D-S--) (INCAx, Oxford, UK) are used to research the chemical composition of corrosion product films on alloy coupons after collecting corrosion products as the following steps: open the anaerobic vial in a medical clean bench and remove the aluminum alloy coupons using forceps, then rinse the specimen in deionized water followed by anhydrous ethanol. Finally, the specimens were blown dry with N2 and sealed into 5 mL centrifuge tubes. M-D-I Jade software (version 6.0) was utilized to analyze data from X-R-D characterization..

[Audio] Materials 2024, 17, 3523 5 of 18 2.5. Weight Loss and Pit Morphology The weight loss of coupons is calculated by Equation (1): vcorr = m0 − m1 A (1) The weight loss rate of coupons is calculated by Equation (2) [33]: vcorr = (m0 − m1) × K A × t × ρ (2) The pits on the surface of the coupons were observed using confocal laser scanning microscope (C-L-S-M-). Aluminum alloy coupons removed from the medium were rinsed with anhydrous ethanol and blown dry with N2. The coupons were immersed in H-N-O-3 solution for 5 minutes to remove corrosion products firmly adhered to the surface based on GB/T 16545-2015 [34]. The Gaussian fit of the collected pitting data was performed. 2.6. Electrochemical Measurements A series of electrochemical measurements of aluminum alloys in the culture medium were performed using an Gamry electrochemical workstation (Gamry, PA, USA). Electrochemical testing uses a three-electrode system: aluminum alloy as the working electrode, platinum electrode as the counter electrode, and saturated calomel electrode as the reference electrode. The 4.5 mL of D bizertensis was inserted into 450 mL of medium. The electrochemical impedance spectroscopy (E-I-S--) has a frequency range of 105 Hz to 10−2 Hz with an amplitude of 10 millivolts. Potentiodynamic polarization ranged from −0.5 volts to plus 1 volts with respect to the open circuit potential (O-C-P--), with a scan rate of 0.000167 V/s. 3. Results 3.1. Statistical Orthogonal Experiments under Various Environmental Conditions Figure 1 shows the S-E-M images of corrosion products covered by aluminum alloy coupons after the 14 d incubation in the fuel-containing media with different environmental conditions. It showed that the bacteria in fuel oil grew slowly and accumulated little corrosion products under the environmental conditions of Group 1, Group 2, and Group 3. However, the bacteria in fuel oil grew rapidly, and the thick corrosion product layer appeared on the aluminum alloy substrate after the 14 d incubation in Group 4, Group 5, and Group 6. The depth and width of pits in the fuel-containing media after the 14 d incubation with different environmental conditions are shown in Figure 2. The pit depth profiles showed that the deepest pits of aluminum alloy coupons in Group 6 (water/fuel ratio of 1:1, obligately anaerobic (0%) and 35 C) were 27.8 µm, which was higher than that of other groups. This indicates that D bizertensis induced the most severe pitting corrosion under the environmental conditions of Group 6. Figure 3 ampere shows the weight loss of aluminum alloy coupons with different environmental conditions after the 14 d incubation. The corresponding corrosion rates of Group 1, Group 2, and Group 3 were 4.4 ± 0.8 milligrams/cm2, 4.9 ± 1.4 milligrams/cm2, and 5.8 ± 1.3 milligrams/cm2, respectively. These three groups had no obvious difference. The corrosion rate for Group 6 was 46.6 ± 6.1 milligrams/cm2, which was 10.5-fold, 9.5-fold, and 8.1-fold higher than that of Group 1, Group 2, and Group 3, respectively. The results of the weight loss test are in accordance with the pit corrosion. The extent of the effect of three individual environmental factors on corrosion was also analyzed (Figure 3B–D). The corrosion weight loss increased significantly along with increasing water concentration when the water concentration was below 50% but decreased significantly along with increasing water concentration when it was below 50% (Figure 3B). When the oxygen content was in the range of 0–10%, the corrosion decreased significantly with the increase in the oxygen content. When the oxygen content was 10–20%, the.

[Audio] g y g g Materials 2024, 17, 3523 6 of 18 (Figure 3B). When the oxygen content was in the range of 0%-10%, the corrosion decreased significantly with the increase in the oxygen content. When the oxygen content was 10%20%, the corrosion did not decrease significantly. The effect of temperature on the whole corrosion process is not significant. Furthermore, according to the orthogonal experiment, we used the R-value to express the degree of influence of three environmental factors on corrosion. A larger R-value means that environmental factors have a greater influence on corrosion. The corrosion index indicated by the R-value of the aluminum alloy coupons were 44.8, 2.63, and 6.8, respectively, showing the same result. Thus, water content in fuel oil has the greatest impact on the M-I-C of aluminum alloy among the three environmental factors. In conclusion, D bizertensis influenced the corrosion of aluminum alloy the most uncorrosion did not decrease significantly. The effect of temperature on the whole corrosion process is not significant. Furthermore, according to the orthogonal experiment, we used the R-value to express the degree of influence of three environmental factors on corrosion. A larger R-value means that environmental factors have a greater influence on corrosion. The corrosion index indicated by the R-value of the aluminum alloy coupons were 44.8, 2.63, and 6.8, respectively, showing the same result. Thus, water content in fuel oil has the greatest impact on the M-I-C of aluminum alloy among the three environmental factors. der Group 6 conditions, namely with a water/fuel ratio of 1:1, obligately anaerobic (0% oxygen), and a temperature of 35 degrees celsius. Water concentration influenced the M-I-C of aluminum alloy the most among the three environmental factors in fuel-containing systems. The following experiments were performed in the Group 6 environment. Materials 2024, 17, x For peer review 7 of 20 Figure 1. S-E-M images of the corrosion products on aluminum alloy coupons after the 14 d incubation with different environmental conditions: (A) and (a) for Group 1; (B) and (b) for Group 2; (C) and (c) for Group 3; (D) and (d) for Group 4; (E) and (e) for Group 5; (F) and (f) for Group 6; (G) and (g) for Group 7; (H) and (h) for Group 8; (I) and (i) for Group 9 in the fuel-containing media. Figure 1. S-E-M images of the corrosion products on aluminum alloy coupons after the 14 d incubation with different environmental conditions: (A,a) for Group 1; (B,b) for Group 2; (C,c) for Group 3; (D,d) for Group 4; (E,e) for Group 5; (F,f) for Group 6; (G,g) for Group 7; (H,h) for Group 8; (I,i) for Group 9 in the fuel-containing media. Figure 2. The C-L-S-M images of coupons after the 14 d incubation with different environmental conditions: (a) for Group 1; (b) for Group 2; (c) for Group 3; (d) for Group 4; (e) for Group 5; (f) for Group 6; (g) for Group 7; (h) for Group 8; (i) for Group 9 in the fuel-containing media. Figure 2. The C-L-S-M images of coupons after the 14 d incubation with different environmental conditions: (a) for Group 1; (b) for Group 2; (c) for Group 3; (d) for Group 4; (e) for Group 5; (f) for Group 6; (g) for Group 7; (h) for Group 8; (i) for Group 9 in the fuel-containing media..

[Audio] Figure 2. The C-L-S-M images of coupons after the 14 d incubation with different environmental conditions: (a) for Group 1; (b) for Group 2; (c) for Group 3; (d) for Group 4; (e) for Group 5; (f) for Group 6; (g) for Group 7; (h) for Group 8; (i) for Group 9 in the fuel-containing media. Materials 2024, 17, 3523 7 of 18 Figure 3. Weight loss of aluminum alloy coupons in the fuel-containing media with different environmental conditions after the 14 d incubation (A), and the influence of water content (B), oxygen content (C), and temperature (D) on weight loss of aluminum alloy coupons. Figure 3. Weight loss of aluminum alloy coupons in the fuel-containing media with different environmental conditions after the 14 d incubation (A), and the influence of water content (B), oxygen content (C), and temperature (D) on weight loss of aluminum alloy coupons. In conclusion, D bizertensis influenced the corrosion of aluminum alloy the most under Group 6 conditions, namely with a water/fuel ratio of 1:1, obligately anaerobic (0% oxygen), and a temperature of 35 C Water concentration influenced the M-I-C of aluminum alloy the most among the three environmental factors in fuel-containing systems. The following experiments were performed in the Group 6 environment. 3.2. Bacterial Growth and Biofilm Characterization The growth curves of sessile and planktonic D bizertensis during the 14 d incubation on aluminum alloy coupons in the D bizertensis media with different immersion times are shown in Figure 4. The growth curves of D bizertensis can be divided into three phases: linear, stable, and decay. D bizertensis showed an exponential growth stage in the first 6 days, when cells grew rapidly, and the planktonic cell counts reached about 108 cells/mL on the 7th day. From the 7th day, cell growth slows down and enters a stagnant period. Figure 5 shows the S-E-M images of D bizertensis and corrosion products on aluminum alloy coupons during the 14 d incubation in the D bizertensis media with different immersion times. It showed that more and more D bizertensis cells attached to surfaces over time and attended to form biofilms after 14 days. To indicate the activity of the attached D bizertensis, the live and dead cells on the surfaces of aluminum alloy coupons were stained using multiple fluorescence staining (Figure 6). Red and green areas denote dead and live D bizertensis cells, respectively. Live D bizertensis cells increased over time from the first day to the seventh day (Figure 6A–C). After the seventh day, the total cells reached a high number, while dead cells also began to.

[Audio] To indicate the activity of the attached D bizertensis, the live and dead cells on the Materials 2024, 17, 3523 8 of 18 surfaces of aluminum alloy coupons were stained using multiple fluorescence staining (Figure 6). Red and green areas denote dead and live D bizertensis cells, respectively. Live D bizertensis cells increased over time from the first day to the seventh day (Figure 6 ampere, Figure 6B, Figure 6C). After the seventh day, the total cells reached a high number, while dead cells also began to increase over time (Figure 6D). This indicates high activities of attached cells from the 7th day, consistent with the results of sessile cell counts (Figure 4B) and biofilm observations (Figure 5). increase over time (Figure 6D). This indicates high activities of attached cells from the 7th day, consistent with the results of sessile cell counts (Figure 4B) and biofilm observations (Figure 5). Materials 2024, 17, x For peer review 9 of 20 Figure 4. D bizertensis planktonic cell count (A) during the 14 d incubation and sessile cell count (B) on aluminum alloy coupon surfaces with different immersion times. Figure 4. D bizertensis planktonic cell count (A) during the 14 d incubation and sessile cell count (B) on aluminum alloy coupon surfaces with different immersion times. Figure 5. S-E-M images of the D bizertensis biofilm and the corrosion products on aluminum alloy coupons during the 14 d incubation with different immersion times: (A) and (a) for 1 d; (B) and (b) for 3 d; (C) and (c) for 7 d; (D) and (d) for 14 d in the D bizertensis media. Figure 5. S-E-M images of the D bizertensis biofilm and the corrosion products on aluminum alloy coupons during the 14 d incubation with different immersion times: (A) and (a) for 1 d; (B) and (b) for 3 d; (C) and (c) for 7 d; (D) and (d) for 14 d in the D bizertensis media..

[Audio] Materials 2024, 17, 3523 9 of 18 Materials 2024, 17, x For peer review 10 of 20 Figure 6. FM images of D bizertensis biofilm on aluminum alloy coupons during the 14 d incubation: (A) for 1 d; (B) for 3 d; (C) for 7 d; (D) for 14 d in the D bizertensis media. Figure 6. FM images of D bizertensis biofilm on aluminum alloy coupons during the 14 d incubation: (A) for 1 d; (B) for 3 d; (C) for 7 d; (D) for 14 d in the D bizertensis media. 3.3. Composition of Corrosion Products Figure 7 illustrates the X-R-D patterns of the corrosion products on aluminum alloy 3.3. Composition of Corrosion Products coupons during the 14 d incubation in the D bizertensis media. The characteristic peaks of Al2O3 and Al can be observed in the pre-corrosion immersion period. With increasing immersion time, the characteristic peaks of Al2(S-O-4--)3 can be clearly observed. Materials 2024, 17, x For peer review 11 of 20 Figure 7 illustrates the X-R-D patterns of the corrosion products on aluminum alloy coupons during the 14 d incubation in the D bizertensis media. The characteristic peaks of Al2O3 and Al can be observed in the pre-corrosion immersion period. With increasing immersion time, the characteristic peaks of Al2(S-O-4--)3 can be clearly observed. Figure 7. X-R-D pattern of the corrosion products on aluminum alloy coupons during the 14 d incubation in the D bizertensis media with different immersion times. Figure 7. X-R-D pattern of the corrosion products on aluminum alloy coupons during the 14 d incubation in the D bizertensis media with different immersion times. 3.4. The Pitting Corrosion 3.4. The Pitting Corrosion The pit morphology of corroded coupons during the 14 d incubation in the D bizertensis media was observed (Figure 8). The number, diameter, and depth of the pits induced by D bizertensis increased remarkably over time. The maximum pit depth was 5.1 µm, 9.8 µm, 23.1 µm, and 33.4 µm detected at 1 d, 3 d, 7 d, and 14 d, respectively. The pit morphology of corroded coupons during the 14 d incubation in the D bizertensis media was observed (Figure 8). The number, diameter, and depth of the pits induced by D bizertensis increased remarkably over time. The maximum pit depth was 5.1 µm, 9.8 µm, 23.1 µm, and 33.4 µm detected at 1 d, 3 d, 7 d, and 14 d, respectively..

[Audio] 3.4. The Pitting Corrosion The pit morphology of corroded coupons during the 14 d incubation in the D bizerMaterials 2024, 17, 3523 10 of 18 tensis media was observed (Figure 8). The number, diameter, and depth of the pits induced by D bizertensis increased remarkably over time. The maximum pit depth was 5.1 µm, 9.8 µm, 23.1 µm, and 33.4 µm detected at 1 d, 3 d, 7 d, and 14 d, respectively. Figure 8. The C-L-S-M images (A–D), the maximum depth of corrosion pits (a–d) and the statistical analysis of pits (a’–d’) during the 14 d incubation: (A) and (a,a’) for 1 d; (B) and (b,b’) for 3 d; (C) and (c,c’) for 7 d; (D) and (d,d’) for 14 d in the D bizertensis media. Figure 8. The C-L-S-M images (A–D), the maximum depth of corrosion pits (a–d) and the statistical analysis of pits (a’–d’) during the 14 d incubation: (A) and (a,a’) for 1 d; (B) and (b,b’) for 3 d; (C) and (c,c’) for 7 d; (D) and (d,d’) for 14 d in the D bizertensis media. Materials 2024, 17, x For peer review 12 of 20 3.5. Weight Loss 3.5. Weight Loss Figure 9 shows the weight loss of aluminum alloy coupons during the 14 d incubation in the D bizertensis media with different immersion times. The ExpDec1 model was used Figure 9 shows the weight loss of aluminum alloy coupons during the 14 d incubation in the D bizertensis media with different immersion times. The ExpDec1 model was used to fit the corrosion weight loss. The corrosion rate of the alloy coupons increased remarkably over immersion times. The weight losses of the aluminum alloy coupons were 1.3 ± 0.2 milligrams/cm2 (0.26 µm/year), 5.1 ± 1.3 milligrams/cm2 (1.42 µm/year), 15.7 ± 2.6 milligrams/cm2 to fit the corrosion weight loss. The corrosion rate of the alloy coupons increased remarkably over immersion times. The weight losses of the aluminum alloy coupons were 1.3 ± 0.2 milligrams/cm2 (0.26 µm/year), 5.1 ± 1.3 milligrams/cm2 (1.42 µm/year), 15.7 ± 2.6 milligrams/cm2 (1.87 µm/year), and 43.1 ± 9.9 milligrams/cm2 (3.05 µm/year) detected at 1 d, 3 d, 7 d and 14 d, respectively. Corrosion rate curves at 1 d, 3 d, 7 d, and 14 d were fitted tangentially in the D bizertensis media, and the corresponding tangent values were −0.41, −0.52, −0.83, and −0.15, respectively. It can be clearly seen that the corrosion accelerated significantly from the 7th day. In the sterile control system, no significant corrosion loss was observed in the aluminum alloy coupons. (1.87 µm/year), and 43.1 ± 9.9 milligrams/cm2 (3.05 µm/year) detected at 1 d, 3 d, 7 d and 14 d, respectively. Corrosion rate curves at 1 d, 3 d, 7 d, and 14 d were fitted tangentially in the D bizertensis media, and the corresponding tangent values were −0.41, −0.52, −0.83, and −0.15, respectively. It can be clearly seen that the corrosion accelerated significantly from the 7th day. In the sterile control system, no significant corrosion loss was observed in the aluminum alloy coupons. Figure 9. Weight loss and corrosion rate of aluminum alloy coupons during the 14 d incubation in the D bizertensis media (A) and sterile culture media (B) with different immersion times. Figure 9. Weight loss and corrosion rate of aluminum alloy coupons during the 14 d incubation in the D bizertensis media (A) and sterile culture media (B) with different immersion times..

[Audio] Materials 2024, 17, 3523 11 of 18 Materials 2024, 17, x For peer review 13 of 20 3.6. Electrochemical Measurements of the aluminum alloy coupons was 3.7 µA/cm2, 9.8 µA/cm2, 51.6 µA/cm2, and 69.8 µA/cm2 detected at 1 d, 3 d, 7 d and 14 d, respectively. The corrosion current density obviously increased on the 7th day, which was nearly two orders of magnitude higher than that on the 1st day and the 3rd day. The O-C-P variation of aluminum alloy coupons during the 14 d incubation in the D bizertensis media with different immersion times was analyzed (Figure 10). As the immersion time increases, the O-C-P gradually shifts negatively to −900 millivolts, implying that the presence of D bizertensis leads to a significant increase in the corrosion activity of aluminum alloy coupons. In the sterile control system, O-C-P values did not fluctuate significantly. Figure 10. The O-C-P (A) and pH value (B) of aluminum alloy coupons during the 14 d incubation in the D bizertensis media with different immersion times. Figure 10. The O-C-P (A) and pH value (B) of aluminum alloy coupons during the 14 d incubation in the D bizertensis media with different immersion times. The E-I-S behavior of aluminum alloy coupons during the 14 d incubation in the D bizertensis media with different immersion times was further analyzed (Figure 11). In the Nyquist plots, the radius of the semicircle obviously increased with time in the initial 9d in the D bizertensis medium. For 9–15 days, the radius of the semicircle was basically unchanged. The radius of the semicircle changed little in a sterile medium. The impedance values decreased over immersion time in the Bode plots, indicating much higher corrosion rates. The impedance values changed little in the sterile medium. Figure 12 illustrates the fitting of E-I-S data using two equivalent circuit models. In the equivalent circuit models, corresponding to Rs, Rf, Rct, Qdl, and Qf, the means were the solution, film, charge transfer resistances, the double electric layer, and the capacitances, respectively. The fitted data for the above circuit system are demonstrated in Tables 2 and 3. By fitting the data, it can be seen that the charge transfer resistance in the sterile control group was significantly higher than that in the D bizertensis media. This is because the dense passivation film formed on the surface of the aluminum alloy prevents corrosion. At the beginning of the immersion, the Rct decreased by an order of magnitude, which is attributed to the accelerated corrosion of the aluminum alloy by the H-2-S produced by the metabolism of S-R-B-. Table 2. Electrochemical parameters fitted from the electrochemical impedance data of aluminum coupons in the sterile culture media. Time RS (Ω cm2) Qf × 10−5 (Ω−1 centimeters2 Sn) Rf (Ω cm2) Qdl × 10−5 (Ω−1 centimeters2 Sn) Rct (KΩ cm2) 1 12.51 ± 0.89 3.89 ± 0.69 73.21 ± 16.08 5.84 ± 1.20 14.98 ± 2.38 3 10.77 ± 0.61 1.49 ± 2.52 33.62 ± 5.58 6.06 ± 1.42 19.66 ± 3.52 5 12.72 ± 0.82 1.86 ± 1.60 50.66 ± 5.10 3.72 ± 2.18 26.82 ± 1.51 7 11.32 ± 1.49 2.99 ± 0.95 59.85 ± 2.91 4.27 ± 0.38 16.92 ± 2.82 9 16.92 ± 0.41 5.58 ± 3.86 57.27 ± 4.10 4.92 ± 1.52 15.87 ± 4.14 11 18.44 ± 2.02 2.67 ± 1.40 22.69 ± 6.10 4.74 ± 1.14 23.12 ± 1.58 13 11.08 ± 0.18 4.09 ± 0.21 26.12 ± 2.92 9.20 ± 1.04 16.69 ± 1.73.