Characterization of the rust layers of Q345 steel after immersion in the South China Sea for 5.5 years
The rust layers generated on carbon steel (Fig. 1) were analyzed to get a better understanding of corrosion mechanisms by SEM (Fig. 2), XRD (Fig. 3a), µ-Raman spectroscopy (Fig. 3b), IR (Fig. 3c) and XPS (Fig. 3d and Fig. 4).
Analysis of the composition of IL
A large number of spherical and lamellar corrosion products, as well as some irregular block corrosion product, were found in the inner rust layer. It was worth mentioning that rod-liked bacteria were also detected in Fig. 2a. As shown in Fig. 3a, the crystalline diffraction peaks of magnetite (Fe3O4), green rust and iron sulfide showed up in IL by XRD analysis. Typical Raman spectra of the IL was shown in Fig. 3b. In dark-green regions of this layer, magnetite with characteristic Raman peaks at 550 cm− 1 and 670 cm− 1 were detected40,41. The weak peak at 207 cm− 1 might come from nanocrystalline mackinawite42,43. And the peaks at 430 cm− 1 and 489 cm− 1 might be corresponded to green rust44, but previous studies also showed a competitive relationship between the formation of magnetite and green rust45.
Figure 3c showed FT-IR analyze results. The strongest absorbance at 1140 cm− 1 could be attributed to sulfide. Absorbance at 796 cm− 1 indicated Fe-OH stretch. Peaks at 1652 and 1422 cm− 1 corresponded to asymmetric and symmetric stretching of carboxylate in the side chain, respectively. Besides, the O-H stretching vibrations of hydroxyl groups were found to have a large band at 3324 cm− 1 10,46.
Figure 3d and Fig. 4 showed the high resolution XPS spectra for these three rust layers. Basically, the carbon spectra of C1s contained three contributions: a C-C peak at 284.72 eV, a C-N peak at 286.37 eV, and a C = O peak at 288.5 eV. The O1s spectrum could be curve-fitted with six peaks (Fig. 4a2). The peaks at 529.7 and 531.0 eV were corresponded to Fe(Ⅱ)-O and Fe(Ⅲ)-O, respectively. And the peaks at 530.75, 531.44, 533.14, 533.18 eV could be attributed to H-O, SO42−, C = O, and NO3−, respectively. It was found that Fe2p3/2 iron spectra revealed the following four contributions: an iron peak (Fe0) at 707.94 eV, a divalent oxide peak (Fe2+) at 709.95 eV, a trivalent oxide peak (Fe3+) at 711.07 eV, and an iron sulfide peak at 712.74 eV in IL (Fig. 4a3). As to the S2p3/2 binding energy (Fig. 3a4), sulfur speciation was almost dominated by sulfate at 168.58 eV and sulfide at 163.7 eV. There was only a smaller component of FeS at 161.52 eV10,47−49. The XRD and Raman results corroborated the XPS findings for all main phases components.
These results illustrated that the main corrosion products of IL were magnetite, and a small amount of green rust and iron sulfide was also detected.
Analysis of the composition of ML
In ML, acicular-like and some flake-like structures showed up, and little spherical-like products were also detected (Figs. 2c and 2d). More corrosion product phases showed up in ML than those in IL. A large number of maghemite (γ-Fe2O3) were found to be present according to XRD patterns50. There were also diffraction peaks of magnetite and mackinawite, which was similar to the IL composition.
The Raman spectra peaks (Fig. 3b) from a black area in ML were clearly visible at 266 cm− 1, 307 cm− 1, 400 cm− 1, 487 cm− 1 and 717 cm− 1. The peaks at 717 cm− 1 and 487 cm− 1 were assigned to maghemite51–53, which was regarded as the dominant part in this layer. A strong peak at 266 cm− 1 could belong to mackinawite54. There were two weak peaks at 307 cm− 1 and 400 cm− 1, possibly corresponded to magnetite and akaganeite (β-FeOOH), respectively. Akaganeite, a kind of marine atmospheric corrosion products, might form due to the post-processing (ie. separation or drying process) in lab, which was difficult to avoid45.
The FT-IR results (Fig. 3c) of ML were similar to that of IL. However, the absorption peak at 1140 cm− 1 was weak, indicating less sulfide content in this layer. For XPS fitting results (Fig. 4c), no peaks of Fe0 were found in ML but iron oxyhydroxide at 712.31 eV (Fe2p3/2) and 531.07 eV (S2p3/2) were detected.
As a result, the main corrosion products formed in ML were determined as maghemite, while other components mackinawite and magnetite were detected as well. Due to the continual structural modification during the long-term seawater immersion, relatively stable corrosion products would like to formed in ML rather than IL. Yang et al.55 also reported similar structures in marine environments, supporting these results.
Analysis of the composition of OL
Non-crystalline structures along with fouling organisms were discovered in OL (Fig. 2e). For instance, the smooth, roughly oval-shaped objects were identified as diatoms (Fig. 2f).
Numerous phases were identified via XRD analysis (Fig. 3a). The main corrosion products of OL were Fe(Ⅲ)-oxyhydroxides, including goethite (α-FeOOH), lepidocrocite (γ-FeOOH), and a small amount of maghemite. Highly crystalized quartz (SiO2), halite and clays from shattered shells also showed up, in accord with the SEM results.
The Raman spectrum (Fig. 3b) displayed a strong peak at 240 cm− 1, which was regard as the characteristic of γ-FeOOH56,57. The strong peak at 490 cm− 1 and comparatively weak peak at 301 cm− 1 was related to α-FeOOH58, Similar with ML, the peak at 400 cm− 1 could be attributed to β-FeOOH. In addition, a ghost peak at 171 cm− 1 appeared due to 532 nm laser3.
FT-IR results of OL showed the strongest absorbance at 1485 cm− 1 from C-C stretch, which resulted from the attachment of small fouling organisms. The strong band corresponded to C-H stretching vibrations was also detected at 864 cm− 1, which indicated certain organic compounds in this layer. The XPS results of Fe2p and S2p in OL (Fig. 4c) were consistent with ML. Due to the low N content (as shown in Table S1), OL showed no C-N peak, but a C-Cl peak at 288.71 eV appeared. For O1s, the unique SiO2 spectrum was also detected at 532.88 eV.
Thus, Fe(Ⅲ)-oxyhydroxides were the main corrosion products together with a small amount of magnetite. Besides, large amounts of fouling organisms were also found due to the direct contact with sea water.
According to Fig. 3d and Table 1, from IL to OL, the content of nitrogen decreased but the content of chlorine increased. In OL, silicon (Si) was found while the iron content was relatively low, which might attribute to the existence of fouling organisms such as coral and diatom. Among these three layers, IL showed the lowest oxygen content, resulted from the diffusion and consumption of dissolved oxygen by aerobic microorganisms.
Results fitting these XPS narrow scan spectroscopies were given in Fig. 4 and Table 2. It was found that the ratio of divalent iron oxide Fe(II) decreased from IL to OL, while Fe(III) increased. The low oxygen content led to low iron oxidation degree in IL. Besides, the lowest sulfate content and the highest sulfide content showed up in IL. The sulfate and sulfide contents of ML and OL were close. Moreover, the presence of iron sulfide existed only in IL, probably resulting from SRB activities as previous studies repoted45.
Table 1
Substance content in three rust layers obtained by wide-scan XPS fitting (%).
|
C1s
|
O1s
|
Fe2p
|
S2p
|
N1s
|
Cl2p
|
Si2p
|
IL
|
38.86
|
47.11
|
6.11
|
2.40
|
2.83
|
2.70
|
-
|
ML
|
26.83
|
58.75
|
6.65
|
1.67
|
1.02
|
5.09
|
-
|
OL
|
29.58
|
55.08
|
4.52
|
2.26
|
-
|
7.47
|
1.10
|
Table 2
Substance content in each valence state by narrow-scan XPS fitting (%).
|
Fe(Ⅱ)/Fe(Total) (%)
|
Fe(Ⅲ)/Fe(Total) (%)
|
Sulfate/S(Total) (%)
|
Sulfide/S(Total) (%)
|
IL
|
57.33
|
32.88
|
44.06
|
39.97
|
ML
|
25.26
|
74.74
|
97.71
|
2.29
|
OL
|
21.09
|
78.91
|
96.90
|
3.10
|
Microbial community analysis of three rust layers
Microorganisms at the class level and genus level were studied to identify the representative taxa that responded positively to metal corrosion. The common and unique OUTs in the three rust layers were displayed and analyzed in the Venn diagram (Fig. S4). IL, ML, and OL shared unique 199 OTUs (13% of total OTUs), 106 OTUs (7% of total OTUs), and 366 OTUs (25% of total OTUs), respectively. According to the rarefaction curves (Fig. S3), 16S rRNA gene sequences in this study, which were generated from corrosion samples, could represent the whole bacterial population.
Figure 5a showed that the prokaryotic phyla comprised 91.57% of the overall community in these rust layer samples, and there were more than 11 phyla detected in these samples. Proteobacteria was the most common phylum in ML and OL, accounting for 19.49–52.67% of all phyla, however, Chloroflexi was the most common phylum in IL (33.60%).
More than 65 genera were identified (Fig. S1). Desulfovibrio and Desulfobacter were the most common bacterial genera in the top 100, according to the hierarchical clustering heat map (Fig. S2). Desulfovibrio was the most abundant genera, accounting for 1.63, 37.43, and 6.43% of the total number of genera in IL, ML, and OL, respectively (Fig. 5b and Table 3). While Desulfobacter was the secondly abundant phylum, accounting for 0.55, 6.69, and 0.64% in IL, ML, and OL, respectively. The average abundance study revealed that several other genera were well-represented (e.g., Gammaproteobacteria and Ralstonia). In all rust layers, anaerobic SRB became the dominant bacteria, and the abundance of SRB in different rust layers was quite different.
Distinct rust layers had different microbial population compositions, based on the weighted pair-group method with an arithmetic mean (UPGMA) compared (Fig. S6). The alpha diversity analysis could be used to determine the quantity and diversity of the microbial population in each rust layer, and Table 3 showed the microbial populations of the different rust layers significantly different. The microbial communities in seawater were more diverse and complex than those in the rust layer. For ML, the diversity of microorganisms was the lowest. The community abundance and diversity of IL and OL were similar. A similar trend was also seen in the NMDS plots of Bray-Curtis distances (Fig. S5) that were based on OTUs.
The functionality of the microbial community in all rust layers was also analyzed. The high dsrA and dsrB (0.32%) genes in ML indicated that dissimilatory sulfate and sulfite reduction and oxidation reactions were mainly reacted, which were much higher than that in IL (0.0097%) and OL (0.0057%). Sulfate reductase gene (dsrAB) was an important gene in the anaerobic sulfate metabolism process of SRB and has been used as a biomarker to detect and monitor the diversity and abundance of SRB in target samples. In these three rust layers, Sat genes had been detected, which could be related to assimilatory sulfate reduction.
SRB and other microorganisms were crucial to forming corrosion products in the rust layers. Iron shlfide preponderance in the IL layer showed a high level of SRB activity. Castaneda et al.59 studies showed SRB metabolic activity resulted in the generation of FeS. The formation process of green rust might also be related to the aerobic bacteria in IL. Duan et al.35 also showed similar findings. The content of divalent and trivalent iron oxides in ML and OL were similar. Due to the presence of typical IOB (Pseudomonadales) and IRB (Shewanellaceae) in these two rust layers (as show in Fig. S2), the conversion between different iron oxides was balanced. However, the abundance of this type of microorganisms in the rust layer community was very low. Meanwhile, corrosion products also showed great influence on the abundance and diversity of bacteria. For ML, due to the large amount of FeOOH that acted as the electron acceptors for SRB60, the abundance of Desulfovibrio revealed the highest abundance but lowest diversity among all rust layers. The OL had the most bacterial diversity, which might be due to the close contact with seawater.
Table 3
Relative abundance of bacterial 16S rRNA gene sequences obtained from rust and seawater samples at the genus level (%).
Genera
|
OL
|
ML
|
IL
|
SW
|
Desulfovibrio
|
6.43
|
37.43
|
1.63
|
0.54
|
Desulfobacter
|
0.64
|
6.69
|
0.55
|
0.53
|
Methyloceanibacter
|
1.06
|
1.59
|
0.13
|
0.24
|
Woeseia
|
1.69
|
0.21
|
0.29
|
3.58
|
Filomicrobium
|
1.42
|
0.65
|
0.21
|
0.54
|
Gammaproteobacteria
|
0.86
|
0.11
|
0.64
|
1.78
|
Brevinema
|
0.87
|
0.53
|
0
|
0
|
Ralstonia
|
0.66
|
0.4
|
1.35
|
1.73
|
Constrictibacter
|
0.43
|
0.03
|
0.59
|
0.04
|
Ilumatobacter
|
0.25
|
0.09
|
0.24
|
2.97
|
Others
|
85.69
|
52.27
|
94.37
|
88.05
|
Laboratory study on D. bizertensis SY-1 separated from the rust layers
Isolation and cultivation of D. bizertensis SY-1
When the rust layer was inoculated into an aerobic solid agar culture medium, SRB was separated and concentrated. For the first two days after inoculation in PGC medium containing lactate as a carbon source, the culture solution became black. It revealed that iron sulfide was formed when iron oxide in rust reacted with hydrogen sulfide created by SRB. After 7 days of incubation at 30 oC, the color of the culture become darker (as show in Fig. 6d). On solid agar plates, black and round colonies with a diameter of 3-5 mm appeared, showing SRB growth in the medium (as show in Fig. 6c).
16S rDNA sequence is not only conservative but also highly variable, so it is widely used in bacterial taxonomy16,61. This SRB strain was recognized as Desulfovibrio bizertensis (98% similarity) and Desulfovibrio singaporenus (98% similarity) by 16S rDNA gene sequencing (Fig. 6g). According to the phylogenetic relationship, this strain of bacteria was named as Desulfovibrio bizertensis SY-1.
A series of physiological metabolism test data showed that this strain belonged to SRB (as show in Fig. 7). Desulfovibrio was also the dominant genera in all rust layer (as show in Table 3). The TEM microstructure images of SY-1 strain was presented in Figs. 6a and 6b, and the length of this cell was about 2-4 mm.
The surface morphologies of carbon steel exposed in the SY-1 strain for 15 days were shown in Figs. 5e and 5f. With a net-like structure, the biofilm's homogeneity and looseness were apparent. More crowded cells and polysaccharides with a defined structure and a smooth surface were identified in Fig. 5f. It also produced an iron sulfide corrosion product layer with visible spherical granules.
H2S generation by SRB, particularly those in the Desulfovibrio genus, was a significant factor in the MIC. H2S would alter the environment's pH, resulting in the creation of FeS. D. caledoniensis62, D. vulgaris63, D. vietnamensis64 and other Desulfovibrio strains isolated from rust layers were shown to have severe corrosion abilities, as demonstrated by laboratory tests. In addition, the ability of some SRB species to degrade alkanes and aromatic hydrocarbons utilizing Fe3+ as the ultimate electron acceptor was a characteristic that was significant to metal corrosion37,38,65.
Growth process of D. bizertensis SY-1 strain
To analyze the growth curve of the planktonic SY-1 strain, the OD600 value of the culture medium was evaluated. Figure 7a represented the typical three-stage curve, and the first five days revealed exponential development. The culture media became rather turbid due to the fast expansion of cells and its metabolites. The number of SY-1 cells reached the maximum value on the 5th day, and the cell concentration was about 108 cells/mL by the direct microscope counting. After then, the cell growth slowed down and to a standstill.
Figure 7b showed the pH values of SRB media were nearby neutral. During 15 days, SRB culture media increased sharply in initial 9 days, but dropped in the last 6 days. Sulfate reduction by SRB in the normal metabolism used H+, which was obtained through organic carbon oxidation. In most cases, the total procedure resulted in no net change in scalar or vectorial proton concentration. Zhou et al.66 reported similar findings. SO42− values were also determined in Fig. 7c. In the SY-1 strain medium, the change in SO42− levels mirrored the change in OD600. Its concentration was rapidly consumed by the fast development of planktonic SY-1 cells in the first three days, after which the SO42− concentration stabilized.
Above all, SY-1 strain was a strictly anaerobic bacterium. This strain grew rapidly to reach the highest value in the initial three days, while the sulfate in the culture solution was rapidly consumed. Meanwhile, the pH in the culture medium remained neutral throughout the process. Wang et al. had similar findings, indicating that H2S would not contribute much to the corrosion of carbon steel at neutral pH67.
Corrosion rate tests on D. bizertensis SY-1
To evaluate the corrosion capacity of SY-1 strain, the corrosion behavior of the Q235 coupons induced by SY-1 and a typical well-known corrosive SRB, Desulfovibrio caledoniensis35, were tested via electrochemical tests, weight loss tests and pit depth measurements for compare.
Electrochemical test results
The MIC process is a bio-electrochemical process25, so the electrochemical measurements and analyses would reveal the corrosion influence by the SY-1 strain. LPR measurements were performed on the coupons in sterile, SY-1 strain, and D. caledoniensis inoculated culture medium after various periods of time (Fig. 8a). The Rp value in the sterile media coupons increased in first three days, indicating that the corrosion rate reduced with time. Similarly, Qiang et al.68 previously also found this. After three days, the Rp value started to drop, indicating the corrosion rate had increased. However, its decreased continuously in both types of SRB medium for 15 days immersion, indicating the increase of the corrosion rate. Although the FeS layers might inhibit carbon steel corrosion, the SRB biofilm had a considerably greater corrosion-promoting impact, which might result in fast carbon steel degradation. A comparison of these two strains LPR results showed that the Rp values in the SY-1 strain media were much lower than those in the D. caledoniensis medium.
After 15 days of exposure, Fig. 8b showed the potentiodynamic polarization curves in various mediums. The corrosion current density of the SY-1 strain (22.6 µA/cm2) was nearly seven times higher than that of sterile media (2.9 µA/cm2) and almost three times higher than that of D. caledoniensis culture medium (6.5 µA/cm2). The presence of SY-1 strains considerably increased the corrosion rate of carbon steel, according to these findings.
Corrosion rate and pit depth
To exactly determine the corrosion capacity of SY-1 strain, the weight loss of the coupons was measured. Some typical SRB corrosion rates were also given in Fig. 8c to help compared the SY-1 strain's corrosion rate. Although there were differences in the composition of the medium for different SRBs, the weight loss tests were similar, and the comparison of the corrosion ability of different SRB also had certain value. The weight loss of the Q235 steel in the SY-1 medium after 15 days exposure was 13.561 ± 2.89 mg/cm2, and its corrosion rate was four times than that in sterile cultured medium. Compared with other SRB corrosion data63,69−72, the carbon steel coupons in the SY-1 strain medium had a larger corrosion weight loss than other SRB, and the corrosion rate of SY-1 strain was more than one third higher than that of the more corrosive D. desulfuricans.
After separating the corrosion products, Fig. 8d showed the pitting morphologies of carbon steel under different culture conditions. During the 15 days of anaerobic and sterile conditions exposure, no apparent corrosion pits formed on the coupon surfaces. Different SRB showed different corrosion forms. Within 15 days exposure, the carbon steel coupons in the D. caledoniensis medium had uniform corrosion morphologies. However, in the SY-1 strain medium, the coupon surface displayed substantially more severe localized corrosion morphologies. Figure 8d showed that the SY-1 biofilms developed the highest depths of the pits was 43.769 µm, which was approximately four times higher than that in D. caledoniensis media (10.027 µm). These results demonstrated that the SY-1 strain dramatically aggravated localized corrosion.
Schematic of the rust layer structure
Electrochemical reactions between atoms on the coupon surface and ions/atoms in seawater would cause corrosion, hence the microstructure of the corrosion layers could be changed over time with the long-time seawater immersion and corrosion microorganisms22,39,73.
For Q345 carbon steel immersed for 5.5-year in the South China Sea, Specific corrosion products gradually evolved following the proposed corrosion mechanism (Fig. 9). Three distinct layers could be discovered: an inner rust layer, a thick intermediate layer, and an outer rust-biofilm layer. The rust layers and the microorganisms living in them strongly affected each other. Microorganisms revealed a substantial impact in the process of rust layer formation, and rust layers also showed major effects on the variety of microbial communities.
When initially immersed in seawater, carbon steel suffered from severe corrosive environment to form primal corrosion products. Meanwhile, marine microorganisms also adhered onto the steel surface. The dissolved oxygen species might react with the surface iron atoms, with reaction (1) occurring at the anode and reaction (2) occurring at the cathode.
Fe → Fe2+ + 2e− (1)
O2 + 2H2O + 4e− → 4OH− (2)
It could take place via Reactions (3):
2Fe + O2 + 2H2O → 2Fe(OH)++ 2OH− (3)
Because Fe(OH)+ ions were unstable, it could further combine with O2 to generate FeOOH film55:
4Fe(OH)+ + 4OH− + O2 → 4β-FeOOH + 2H2O; or (4)
4Fe(OH)+ + 4OH− + O2 → 4γ-FeOOH + 2H2O (5)
Green rust would form near the steel layer42,74:
5Fe(OH)2 + Fe2+ + 1/2O2 + 9H2O + SO42− → GR (6)
Subsequently, the dehydration reaction γ-FeOOH → γ-Fe2O3 occurred to form a stable iron oxide55. These substances were detected in XRD and Raman spectroscopy in ML. The excess positive charge and acidity were increased by Fe2+ hydrolysis, which was formed in anodic areas on the steel/rust contact. According to XPS research, the extra positive charge at the steel/rust interface attracted Cl− and SO42− ions from seawater, resulting in greater chlorinity in the rust layers (Table 1). The high sulfate content might facilitate the formation of green rust near the steel surface.
γ-FeOOH would be also reduced to Fe3O4 by Fe2+ through the following reaction75:
2γ-FeOOH + Fe2+ → Fe3O4 + 2H+ (7)
Meanwhile, Fe3O4 could also be directly formed from metals in an oxygen-deficient environment45.
3Fe + 4H2O → Fe3O4 + 4H2 (8)
Therefore, Fe3O4 was abundantly present in the IL, and was also detected in the ML and OL.
Microorganisms also played an important role on the formation process of corrosion products. Microorganisms in seawater would utilize these minerals to support their own survival and metabolism as the rust layer increased. The formation of iron oxides and the consumption of oxygen by aerobic bacteria in the rust layer may support SRB growth and metabolism as well. As shown in Fig. 5 and Table 3, anaerobic SRB became the dominant bacteria in all rust layers. As reported, SRB always utilized sulfate as the terminal electron acceptor, eventually converted it to HS− 76.
SO42− + 9H+ + 8e− → HS− + 4H2O (9)
2FeOOH + 3HS− + 3H+ → 2FeS + S + 4H2O (10)
HS− would react with Fe2+ produced by metal dissolution to form FeS. On one hand, the HS− generated by SRB would diffuse into the seawater. On the other hand, it would accumulate on steel surface, which was ascribed to the metal surface acting as an anode (1). The IL exhibited the highest HS− content among these layers, while the HS− content in ML and OL were relatively lower than that in IL, as shown in Table 2.
Near steel surface, IL was a steel-surface-connected electronic conductor, which mainly composed of magnetite and small amount of iron sulfide. Some SRBs might obtain electrons directly from the steel surface through DET34. Zhang et al.18 found that the dominant community in the inner rust layer of carbon steel was mainly Desulfovibiro, which could directly transfer electrons. Electroactive SRBs could also sustain their long survival by utilizing the Fe3O4/FeS particle network attached to the metal surface45. Meanwhile, H2 produced during Fe3O4 formation (8) might also be utilized by SRB or hydrogen-consuming microorganisms. Carbon steel could be served as a cathodic zone, while SRB acted as the cathode region. So, although the IL was basically considered as an anaerobic environment, carbon steel corrosion would still be aggravated. Fe2+ and H+ generated in IL would diffuse to ML, which would further promote the corrosion of ML and increase the thickness of ML.
After long-time immersion, previous studies77–79 found that ferric oxide could act as an electron acceptor and easily be reduced by microbial metabolism in an anaerobic environment. Wei et al.60 also reported SRB could use not only sulfate ions but also ferric hydroxide as electron acceptors to acquire energies for their growth. FeOOH might be converted into Fe3O4 under the action of SRB. In our research, ML contained a large amount of γ-Fe2O3, which might be attributed to the γ-FeOOH dehydration reaction and the consumption of FeOOH by SRB. XRD and Raman results also indicated a small amount of magnetite and mackinamite in the ML (Fig. 3).
For OL, a large number of fouling organisms, such as diatoms and corals, colonized due to direct contact with seawater (Figs. 2e and 2f)). Marine biofouling was a complex natural phenomenon. Marine organisms in seawater would colonize and grow on the surface of the material to complete their life cycle and form a fouling biological community adapting to the environment. Under the influence of these biological communities, the iron oxide would further be oxidized and fell off. Under the influence of these complex microbial community in marine environment, a relatively stable three rust layers were formed.
Meanwhile, aerobic bacteria such as Filomicrobium and Ruegeria were also the dominant microorganisms in all rust layers. They also played key roles in the formation of corrosion products, which could form anoxic and acidic environment in the rust layer and produce extracellular polymeric substance (EPS). For example, Methyloceanibacter was the dominant aerobic microorganism in all rust layers, and this organism was a methylotroph consuming methanol, methylamine, trimethylamine, and a range of other multi-carbon chemicals80.
Therefore, IL and ML were formed under the influence of SRB. Previous studies found8,45 that under actual seawater immersion, the black corrosion products in the IL were mainly magnetite and green rust, and the orange-brown corrosion products in the OL were mainly Fe(Ⅲ)-oxyhydroxides and a small amount of maghemite. In this work, the IL layer was mainly composed of magnetite as well as slight iron sulfide and green rust, so the electroactive SRB might directly obtain electrons from the iron surface and utilize the Fe3O4/FeS particle network to survive persistently. ML was mainly γ-Fe2O3 as well as little magnetite and mackinamite because of the action of FeOOH dehydration reaction and SRB.
SRB was the dominant bacteria in all rust layers, but the proportion was quite different. SRB was most abundant in ML (up to 44%) due to the diffusion of seawater and the nutrients produced by biofouling in OL, which supported sufficient electron donors and acceptors for SRB. SRB accounted for 6% in OL, less than that in ML. Zhang et al. 18 also found that the dominant bacteria in the OL of carbon steel were SRB that could produce spores by high-throughput sequencing.
SRB exhibited the least content (only 2%) in IL, resulting from the lack of energy substances (which consumed by ML SRB) even in an appropriate anaerobic environment, As shown in Table 2.
Overall, the interaction between microorganisms and the rust layer strongly affected each other. With the formation of iron minerals, microorganisms would use these minerals to promote their own growth and metabolism. Under the influence of EPS secreted by some key microorganisms, specific corrosion products, such as iron sulfide, were also induced to form in rust layers.