Biochemical and microbiological characterization of a thermotolerant bacterial consortium involved in the corrosion of Aluminum Alloy 7075

doi:10.21203/rs.3.rs-2371670/v1

Microorganisms can play a significant role in material corrosion, with bacterial biofilms as major participants in microbially influenced corrosion (MIC). The exact mechanisms by which this takes place are poorly understood, resulting in a scarcity of information regarding MIC detection and prevention. In this work, a consortium of moderately thermophilic bacteria isolated from a biofilm growing over aluminum alloy 7075 was characterized. Its effect over the alloy was evaluated on a 40-day period using Electron Microscopy, demonstrating acceleration of corrosion in comparison to the abiotic control. The bacterial consortium was biochemically and microbiologically characterized as an as an attempt to elucidate factors contributing to corrosion. Molecular analysis revealed that the consortium consisted mainly of members of the Bacillusgenus, with lower abundance of other genera such as Thermoanaerobacterium, Anoxybacillus and Paenibacillus. The EPS polysaccharide presented mainly mannose, galactose, rhamnose and ribose. Our observations suggest that the acidification of the culture media resulting from bacterial metabolism acted as the main contributor to corrosion, hinting at an unspecific mechanism. The consortium was not sulfate-reducing, but it was found to produce hydrogen, which could also be a compounding factor for corrosion.

Biofilm

corrosion

microbial consortium

polysaccharides

AA7075

Metal alloys are used in a wide variety of applications, ranging from the building trade and manufacturing of industrial equipment to military gear and electronics. A downside of their use is their thermodynamic instability, reflected by the spontaneous oxidation these materials undergo in the process known as corrosion (Farag et al., 2020), affecting the conductivity, rigidity, and mechanical properties of the materials. This results in the eventual need for maintenance and replacement of the affected goods, which has a significant impact in many sectors of the economy. Yearly, worldwide expenses associated with the prevention of corrosion damage and replacement of affected materials have been calculated to be around US$ 4 trillion. (Li et al., 2015).

Corrosion has been observed to be the result of both biotic and abiotic factors. Microbially induced corrosion (MIC) or biocorrosion, refers to the acceleration of the corrosion process by the direct or indirect activity of microorganisms (Narenkumar et al., 2019). Environmental bacteria are the most common organisms associated with accelerating corrosion. It has been established that the formation of bacterial biofilms over the surface of metal alloys is a determining factor in the acceleration of corrosion (Videla, 2002). Many bacterial species have been reported to induce corrosion, and rich bacterial communities can be found in places where corrosion occurs (Little et al., 2020a). Furthermore, it is believed that mixed populations of bacteria, where some contribute directly while others help in maintaining an ideal environment for the community, are the most corrosively damaging (Loto, 2017).

The formation of extracellular polymeric substances (EPS), comprising proteins, polysaccharides and in minor proportion also nucleotides and lipids, seems to be a major contributing factor to MIC. Biofilm formation is the main mechanism of microbial surface colonization, and EPS plays a central role in adhesion and survival of the inhabiting bacterial community. Polysaccharides are a major contributor to the resistance of biofilms, as they anchor microorganisms and form gel matrices that maintain their structural integrity (Beech and Sunner, 2004). The glycosidic content of EPS has been established for common biofilm forming pathogens (Bales et al., 2013), but little is known about the composition and structure of the EPS produced by environmental thermophilic bacteria capable of inducing corrosion.

Furthermore, specific mechanisms involved in corrosive damage caused by bacteria are not entirely clear to date. A significant body of research supports the hypothesis that MIC is driven by electron transfer from carbon steel to bacteria. Some microorganisms have been reported to use elemental iron as an electron donor to derive reducing power for microbial respiration in oligotrophic conditions, which are common in places where MIC occurs (Little et al., 2020b). Accordingly, MIC has been generally studied under minimal media in laboratory-controlled conditions. However, the relevance of MIC in carbon-rich environments, like wastewater treatment plants, sewerage systems, metal pipes in carbon-rich soils and oil pipes, is less understood and mostly attributed to sulfate reduction, which occurs under anaerobic conditions. The observation that metal pipes are also corroded in oxygen and carbon rich environments, where thermodynamic constraints favor the development of aerobic metabolisms rather than sulfate reduction, suggest that other mechanisms are also involved in MIC.

This work studied the biofilm formed by a thermotolerant bacterial consortium isolated from the corroded aluminum alloy (AA7075-T6) surface of an airplane engine, and its effect over corrosion in a carbon rich environment. The sample was obtained in Antarctica, however, the bacterial consortium showed to be thermotolerant in nature, capable of growing at 50°C, likely due to the temperatures reached by the engine while functioning. Similar to the environmental biofilm, this bacterial consortium retained its capability to induce corrosive damage under laboratory conditions. The compositions of its bacterial community, the carbohydrates found in the EPS it secretes, some of the enzymes it produces and the effect its growth had on the pH of the culture media were determined in an attempt to elucidate factors driving corrosion in carbon rich environments.

2.2 Bacterial cultures and growth conditions

The consortium used to produce the biofilm was obtained from a sample taken from a bacterial biofilm growing on the outer surface of a functional DHC-6 Twin Otter airplane engine showing signs of corrosion. The sample was immediately used to inoculate an enriched medium in situ. The medium used for laboratory growth consisted of 1.2 g of NH₄Cl, 0,5 g de NaCl, 0.3 g of KH₂PO₄, 2 g of MgSO₄·7H2O, 3,5 g of MgCl, 0.3 g of CaCl₂, 0,35 g of Kcl, 0,06 g of NaBr, 0,015 g of NaBr, 0,02 g of KI, 0,006 g of Sodium Citrate, 0,02 g of SrCl₂, 5 g of yeast extract, 5 g of maltose, 5 g of tryptone, and trace amounts of other metal salts. The inoculated medium was incubated for 70 h at 50°C and pH 6.8. This pH and temperature were selected based on the values registered at the time of sampling using litmus paper (Macherey-Nagel) and a type K/J Digi-Sense temperature meter (Cole-Parmer) respectively. Microscope slides were added to the culture vessel to stimulate formation of biofilm over their surface. For subsequent analysis, the planktonic phase of the culture was separated from the EPS forming biofilm. This was achieved collecting the supernatant of the culture containing flasks, which contains the planktonic phase of the microbial community. In parallel, the dense EPS as well as the biofilm forming in the surface of the microscope slides were collected by scraping and analyzed separately to study the biofilm composition. For pH measurements, 50 ml consortium cultures were incubated for 40 days at 50°C using the conditions described above. Three ml aliquots were taken after 1, 7, 14, 21 and 40 days of incubation. The pH of the media was registered at the time of sampling using a pH211 microprocessor pH-meter (Hanna Instruments).

2.3 Corrosion assays

Sterile 1 x 1 cm aluminum alloy 7075 coupons were submerged in either uninoculated sterile culture media (sterile controls) or sterile media inoculated with the corrosive bacterial consortium, and then incubated at 50°C for 14 or 40 days, under aerobic conditions. After incubation, samples were visually inspected for signs of oxidation and photographs were taken. Experiments were carried out in duplicates. In a parallel set of experiments, identical coupons were submerged for 40 days in uninoculated sterile media with pH adjusted to 4.0, to evaluate the contribution of pH to the corrosion of the alloy.

2.4 Bacterial community composition.

After 36 hours of incubation, DNA was extracted from consortium cultures using a modified phenol-chloroform extraction procedure (Amenabar et al., 2018). Briefly, cultures were centrifuged (14,000 × g, 20 min, 4°C) to obtain biomass for DNA extractions. Cell pellets were re-suspended in 150 mM Tris-HCl buffer (pH 7.5) containing 15 mM EDTA (pH 8.0) and 1 mg ml^− 1 of lysozyme and incubated at 37°C for 20 min. Following incubation, proteinase K (200 µg ml^− 1 final concentration) and sodium dodecyl sulfate (1% final concentration) were added to the extract and incubated at 50°C for 20 min. After incubation, a lysis buffer containing guanidine thiocyanate (1.8 M final concentration) and sarcosyl (0.23% final concentration) was added and the mixture was incubated at 50°C for 20 min. Cellular debris were removed by centrifugation (9,000 x g for 20 min.). DNA was purified from the clarified phase, precipitated, and resuspended in molecular grade water as previously described (Amenabar et al., 2018). The concentration of the resuspended DNA was determined using a Genova Nano spectrophotometer (Jenway).

The extracted DNA was subjected to PCR amplification of 16S rRNA genes according to previously described protocols (Amenabar and Boyd, 2018) using archaeal primers 344F (5’-ACGGGGYGCAGCAGGCGCGA-3’) and 915R (5’-GTGCTCCCCCGCCAATTCCT-3’), and bacterial primers 1100F (5’-YAACGAGCGCAACCC-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). An annealing temperature of 55°C or 61°C was used for bacterial or archaeal primers, respectively. PCR amplification was verified by running the sample on 1.5% agarose gel stained with SYBR gold (Invitrogen). Archaeal amplicons were not detected in any of the DNA extracts. Negative DNA extraction blanks were negative in PCR screens. Bacterial 16S rRNA gene amplicons were purified using previously published methods (Monsalves et al., 2020) and were submitted to MrDNA (Shallowater, TX) for barcoding and sequencing. PCR amplicons were sequenced via multiplexed, paired-end Illumina MiSeq tag sequencing. Post sequence processing was performed by MrDNA (Shallowater, TX).

2.5 Purification of the carbohydrates present in the biofilm.

After growing for 36 hours, consortium bacterial cultures were centrifuged to separate the EPS producing biofilm from the planktonic phase (4,000 x g for 10 min). The biofilm was treated with formaldehyde before further processing, to avoid cellular rupture. The formaldehyde-biofilm mixture was incubated in a shaker (100 rpm) for 1 h at room temperature. Bacteria were separated from the biofilm by the addition of 4 ml NaOH 1 M to each 10 ml of biofilm. The resulting solution was incubated at room temperature, while shaking at 100 rpm, for 3 h. Suspended cells were then centrifuged (16,800 x g) for 1 h at 4°C. The supernatant with the soluble EPS was filtered using a 0.2 mm filter and dialyzed for 24 h against distilled water using a 12–14 kDa molecular weight cut-off membrane at 25°C. EPS components were selectively precipitated in successive steps. Trichloroacetic acid (TCA) was added to a final concentration of 20% w/v to the extracted EPS solutions and was left at 20°C to precipitate proteins and nucleic acids. The solution was then centrifuged (16,800 x g) for 1 h at 4°C. 95% propanol (2 volumes) was added to the resulting supernatant. The mixture was incubated at 20°C for 24 h to separate the polysaccharides from the lipids. The solution was then centrifuged (16,800 x g) for 1 h at 4°C and the exopolysaccharide pellet was resuspended and dialyzed (12–14 kDa membrane) against distilled water for 24 h at 4°C to remove low molecular weight impurities and leftover TCA and then lyophilized overnight. Fractions were tested for the presence of carbohydrates by the phenol-sulfuric acid method of Dubois et al., (1956).

2.6 Glycosyl Analysis

Glycosyl composition analysis was performed by a third party at the Complex Carbohydrates Research Center, Georgia (CCRC). Two polysaccharide samples (PSI and PSII), obtained from the EPS collected by the method described above, were sent for analysis. These samples were obtained from two independent cultures obtained from media inoculated from the same original consortium culture.

Glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) methyl glycoside derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis.

For determination of glycosyl linkages, the sample was permethylated, reduced, re-permethylated, depolymerized, reduced, and acetylated and the resultant partially methylated alditol acetates (PMAAs) were analyzed by GC/MS, as described by Heiss et al., (2009). PMAAs analysis was done on an Agilent 7890A GC interfaced to a 5975C MSD; separation was performed on a Supelco 2331 fused silica capillary column (30 m 0.25 mm ID).

2.7 Sulfate reduction Activity

Sulfate reduction activity of cultures was evaluated in terms of production of total sulfide (S²⁻; proxy for SO₄²- reduction), using methods previously described (Amenabar et al., 2017). The presence of dissolved sulfide was determined with the methylene blue reduction method (Fogo and Popowsky 1949). This method allows for the indirect determination of sulfide concentration by monitoring the increase in absorbance at 670 nm that results from the formation of methylene blue when samples containing sulfide are treated with N,N-dimethyl-p-phenylenediamine oxalate and ferric chloride. Cultures were incubated at 50°C for 14 days using gauze and cotton stoppers, and then the presence of sulfide was determined using the methylene blue reduction method. In a second set of experiments, cultures were incubated for 3 days using gauze and cotton stoppers and then these stoppers were replaced by rubber gas-tight stoppers to generate a microaerobic-anaerobic environment due to the microbial consumption of oxygen. This was done with the intent of stimulating sulfate reduction in microaerobic-anaerobic conditions.

2.8 H₂ determination

Hydrogen gas production was evaluated on the same cultures where gas-tight stoppers where used for sulfate reduction determination (see above). The concentration of H₂ was determined via gas chromatography using a YL6100 gas chromatograph (Young Lin Instrument Co) with an injector temperature of 200°C and an oven temperature of 35°C. Gas was separated on a 30m × 0.53mm Carboxen 1010 PLOT fused silica capillary column (Supelco) using ultrapure N₂ as a carrier gas. H₂ was measured on a thermal conductivity detector (TCD) at a temperature of 230°C.

2.9 Enzymatic assays

Peroxidase activity was detected using a reaction mixture containing 980 µl of MSM medium, 10 µl of enzyme extract, 5 µl of 30% hydrogen peroxide (H₂O₂) aqueous solution and 5 µl of guaiacol (Sigma-Aldrich). Tetrahydroguaiacol formation was measured at an absorbance of 470 nm for 1 min. Assays were performed at 25°C and 50°C.

Catalase activity was measured by registering the decrease in absorbance at 240 nm corresponding to the decomposition of H₂O₂. One hundred µl of enzyme extract was added to potassium phosphate buffer 50 mM pH 7.0, containing 10 mM H₂O₂, and the reaction were allowed to run for 5 minutes. Assays were performed at 25°C and 50°C.

2.10 Microscopic Imaging

The pitting of the coupons was observed under a scanning electron microscope (JEOL, model JSM-6010LA), with a beam voltage of 14–15 kV. The AA7075 coupons exposed to the conditions described above were prepared for analysis by fixing cells using a 2.5% glutaraldehyde solution at 50°C for 45 min. Afterwards, samples were dehydrated using ethanol at increasing concentration (25, 50 and 75%) for 15 min each and then dried at room temperature.

3.1 Microbial growth and corrosion

Samples taken from a surface of aluminum alloy 7075 showing visible corrosion damage were inoculated in an enriched media and incubated at 50°C resulting in microbial growth. With the aim of assessing the corrosive potential of the microorganisms present in the samples, aluminum alloy 7075 coupons were exposed for 40 days in inoculated media. This resulted in clear signs of oxidation after 40 days and no visible oxidation in the uninoculated sterile control (Figure 1). After 14 days of incubation there was already a noticeable difference with the sterile controls (Figure 1A and 1C).

Variations in the pH of culture media as a result of bacterial growth were also monitored. As can be observed in table 1, bacterial growth had an acidifying effect on the culture media. Freshly inoculated media had an initial pH of 6.6. Just a day later, a marked decrease in pH was observed. The pH continued decreasing on the following 40 days of incubation, although at a slower rate and stabilizing around pH 4. In the case of the uninoculated sterile controls, an acidification of the media was also observed but at a much slower rate and with a linear behavior, confirming that the decrease in pH observed in consortium cultures was the result of bacterial activity and not only due to abiotic reactions.

3.2 Biochemical characterization

In order to address the potential influence of sulfate reducing bacteria, a group commonly associated with biocorrosion, on the oxidation of aluminum alloy 7075, the production of total sulfide was evaluated in the cultures. However, no sulfate reduction activity or any type of sulfide producing activity was observed, neither when the consortium was incubated using gauze and cotton stoppers, nor under microaerobic conditions. H₂ production was also evaluated, as hydrogen adsorption over alloys has been related to corrosion (Biezma, 2001). The consortium under study showed hydrogen gas production when grown under microaerobic conditions.

Enzymatic assays were performed for the detection of peroxidase and catalase activity, since both enzymes have been reported to contribute to corrosion (Karn et al., 2017; Landoulsi et al., 2008). No peroxidase activity was detected neither in the extracellular fraction, nor in the crude extract. Catalase activity was only detectable in the extracellular supernatant when the assay was performed at 50°C. No intracellular catalase was detectable in our assays, suggesting that catalase is rapidly excreted from the bacterial cells.

3.3 Microbial community analysis

Total DNA was independently extracted from both, the planktonic fraction (P) and the EPS-forming biofilm (BF) precipitated at the bottom of the flask. 16S rRNA genes were amplified from both samples and sequenced. From the sequences obtained a reconstruction of the microbial community present in the culture was proposed.
Bacterial 16S rRNA gene amplicons were detected in both P and BF. The composition of the microbial community present in both P and BF (Table 2) indicates a similar structure at the genus level. The diversity of genera observed in P and BF was composed mainly by members of the Bacillus genus (around 94% in both samples), followed by Thermoanaerobacterium (4.5% and 3.8%, respectively). A minor percentage of the bacteria present were classified as Anoxybacillus (0.9% and 0.2%, respectively) and Paenibacillus (0.4% and 1%). Although differences of the microbial diversity at the genus level were negligible between P and BF, most abundant OTUs of each sample were related to different unclassified Bacillus species.

3.4 Glycosyl composition analysis

The type and number of glycosyl residues present in two samples (PSI and PSII) of the polysaccharide fraction of the studied consortium were determined. As expected, both samples showed similar proportions of the same monosaccharide residues (Table 3). The samples contained mostly mannose, galactose and rhamnose. Other glycosides were present in small amounts. The EPS also showed the presence of ribose, which is considered uncommon in bacterial biofilms (Lembre et al., 2012).

Regarding the analysis of glycosyl linkages (Table 4), it was surprising to find the presence of xylopyranosyl residues, considering that the monosaccharide analysis showed no presence of xylose, but this could be explained by the low proportion in which it was found. Another unexpected result was the presence of a significant percentage of an unidentifiable peak, which does not correspond to any known monosaccharide linkage and is listed as “unknown” in table 4. This typically indicates the presence of an unreduced monosaccharide. This could be the result of the chemical modification of a glycosyl residue, which could block the reduction step of the analysis pipeline. Other than that, results are consistent with the glycosyl composition analysis and fall within expected.

3.5 Effect of pH over aluminum alloy 7075

Since it was determined that consortium growth resulted in the acidification of the culture media, reaching a final pH ~ 4 (Table 1), the contribution of protons to corrosion was evaluated. Coupons incubated for 40 days in sterile uninoculated media at 50°C and pH 4.0 behaved similarly to the ones submerged in consortium cultures. At 14 days, corrosion damage was already apparent, and by day 40 the amount of visible damage was comparable to the coupons incubated in inoculated media (Figure 2).

To further elucidate the correlation between bacterial growth, acidic pH and corrosion over the surface of AA7075, SEM images of the coupons subjected to the corrosion assays were obtained (Figure 3). Images obtained at 30X magnification confirmed the trend observed upon visual inspection. Coupons exposed to sterile media showed little signs of pitting after 40 days (Figure 3a, I & II). Coupons incubated with media inoculated with the corrosive consortium, however, showed clear signs of localized modification of the surface after only 14 days, with a thick deposition becoming apparent after 40 days (Figure 3a, III & IV). When the coupons were exposed to uninoculated sterile media adjusted to pH 4, scattered pitting was observable after 40 days (Figure 3a, V & VI).

SEM images obtained at larger maximization values further stablish the differences between the coupons subjected to each condition (Figure 3b). On the surface of coupons incubated in sterile media, only crystal-like structures were visible, presumably salt deposited from the culture media (Figure 3b, I & II). In the case of coupons exposed to consortium cultures, bacteria can be seen attached to the surface of the alloy at 14 days, developing into a thick biofilm after 40 days (Figure 3b, III & IV). Coupons exposed to pH 4 showed an irregular surface with no signs of any kind of deposition (Figure 3b, V & VI).

The consortium obtained from inoculating the original sample in rich media showed a notorious influence on the corrosion process of the metal alloys exposed to it, which can be seen as soon as 14 days after exposure. This acceleration of corrosion, in comparison to the abiotic control, was accompanied by an acidification of the media. This was most likely the result of the accumulation of metabolites or metabolic end-products in the extracellular environment of the consortium. Furthermore, when the effect of low pH was tested on coupons incubated in acidic sterile uninoculated media, clear signs of corrosion were visible in the aluminum alloy, supporting the idea of corrosion resulting from the acidic pH produced as a consequence of the metabolic activity of bacteria and the different metabolites they produce and release, including organic acids. A higher concentration of protons in the surface of the metal alloy primes the material for oxidation and degradation, as the redox potential is inversely correlated to the pH at which electron exchange takes place. As evidenced by the results here obtained, pH 4.0 by itself is sufficient to induce corrosion on aluminum alloy 7075. When SEM was used to analyze the corroded coupons, a localized damage was observed on the AA7075 exposed to consortium cultures, with pitting on the places where bacterial biofilm was forming. The coupons exposed to media adjusted to pH 4 showed scattered pitting in various sites on the surface of the alloy. This suggests that the lowering of pH is in itself sufficient for the corrosion of the alloy, although the presence of the biofilm still induces localized damage where it grows. Recent investigations suggest that accumulation of acidic metabolites in bacterial EPS are an important contributing factor for corrosion (Wang et al., 2020).

Microbial community analyses of the planktonic and biofilm consortium cultures showed no major difference at the genus level, in the bacterial community present in the planktonic fraction and the biofilm of the consortium cultures, showing a great predominance of Bacillus. It should be noted, however, that the community composition here presented is unlikely to be an exact representation of the community present in the original sample site, as the rich media provided to the microorganisms with all certainty had an effect over their ecological dynamics. Still, the community recovered on the enrichment media retained its capacity to induce corrosion, as shown in the corrosion assays against AA7075, and the phenomenon occurred even when carbon sources were available and abundant for the bacteria.

In general, it is difficult to correlate a certain genus to metal corrosion, as it has been previously shown that some bacteria, such as those from the Bacillus genus, can either accelerate or inhibit metal corrosion (Juzeliūnas et al., 2006; Karn et al., 2017). Some of the genera detected in our analysis of the consortiium have been reported to be thermophilic in nature, including Thermoanaerobacterium and Anoxybacillus. A very recent work reports the presence of bacteria from the genus Thermoanaerobacterium in the microbial community growing at corrosion sites in oil plant pipelines (Nasser et al., 2021). This genus has also been shown to be able to corrode metal surfaces (Lan et al., 2012). The identification of an Anoxybacillus in the context of biocorrosion is unusual, and to the best of our knowledge rarely reported before. The 16S rRNA sequence of the one present in the consortium here studied has less than 97% identity with its closest match on the database used, which could mean that it is an uncharacterized species. The expanding list of bacteria involved in corrosion suggests that this characteristic is widely distributed, and not limited to certain genera (Dou et al., 2021).

The determination of the composition and structure of the EPS secreted by bacteria is relevant to understanding and remediating biocorrosion, as it is the main mechanism of adherence of bacteria and what makes biofilms so recalcitrant. Here we report seven different monosaccharides conforming the EPS and the glycosyl linkages that connect them. In theory, one could use this information to develop an enzymatic method for dismantling the EPS, specifically targeting the linkages found. The studied EPS showed high percentage of 3,4-Man and 4-Man residues (Table 3), which theoretically indicates the EPS could be dismantled using Mannan endo-1,4-beta-mannosidase enzymes (EC 3.2.1.78). Ideally, the three-dimensional structure of the polysaccharides involved should be determined for the design of useful enzymatic treatments, being NMR a powerful tool for the analysis of polysaccharide structures (Yao et al., 2021). A commonly discussed disadvantage of enzymatic treatments is their expensive nature, which makes large-scale application unfeasible in infrastructure (Little et al., 2020a). However, they can be useful for localized treatment of specific surfaces affected by corrosion, induced by microbial colonizers.

An aspect of these results to discuss is the ribose found in the polysaccharides of the biofilm. In some cases, D-ribose has been shown to inhibit the formation of biofilms (Lee et al., 2014). Alternatively, the presence of this glycosyl residue could imply RNA remnants in the sample, because it is not commonly found in bacterial biofilms. Nonetheless, these samples were treated with TCA to eliminate nucleic acids and proteins, and no deoxyribose or peptides were found in GC/MS, indicating that the TCA precipitation was probably successful.

In the glycosyl linkage analysis, ribose appears as terminal ribofuranosyl residue and 2-linked ribofuranosyl residues. This does not correspond to the ribosyl residues expected to be found in RNA, where ribose is linked to the nitrogenous bases at carbon 1, and to phosphate at carbon 5. Still, the presence of an unidentified peak in GC/MS analysis of the reduced polysaccharides could be attempted to be explained as resulting from the presence of nucleotide. But if this was the case, it would be possible to distinguish between the four different types of nucleotides in the chromatogram. All this considered, it seems likely that ribose is, in this case, a structural component of the biofilm herein studied. This, albeit unusual, is not without precedent, as ribose can be found in the biofilm formed by some species (Dominguez et al., 2018, Lembre et al., 2012).

The mechanisms by which thermophilic bacteria participate in corrosion are poorly understood. A bulk of research suggests that MIC is caused by the production of metabolic byproducts (Kip et al., 2015; Lahme et al., 2019). The acidification of the media reported here, as discussed above, could support this hypothesis. In the case of SRB, hydrogen sulfide production is an obvious candidate for explaining the influence on corrosion. In the present study, no sulfate reduction activity was measurable in the consortium cultures, ruling out the presence of active SRB in the bacterial community isolated from our initial sample. This result was expected, as in the 16S rRNA gene community analysis no genera associated to SRB were found. The acidification observed could be the result of the production of organic acids by bacteria, or active release of protons to the media, as a consequence of metabolic activity. The involvement of organic acids in bacterial influenced corrosion has been previously discussed in the literature (Salvarezza et al., 2007).

Interestingly, the cosortium under study was also found to produce hydrogen gas (H₂). This fact is also relevant to corrosion, as biological activity has been linked to hydrogen embrittlement, a phenomenon caused by adsoprtion of the gas over metallic surfaces, directly damaging the material and causing desagregation of grains (Biezma, 2001; Quej-Ake et al., 2020). The mechanisms through which H₂ is capable of damaging aluminum alloys has recently been proposed (Zhao et al., 2022). Although hydrogen production usually occurs under anaerobic conditions, it is known that in biofilm forming bacterial communties, anaerobic microenvironments are formed at the inner layers of the biofilm, in contact with the surface over which it is formed. Hydrogen production by this consortium is likely one of the compounding factors in its capability of inducing corrosion.

Proteins, including enzymes, are present in the extracellular environment of biofilms. Among them, redox enzymes are relevant in the context of biocorrosion, as they can produce the accumulation of oxidizing agents, which increase the corrosion potential in the surface of affected materials. The presence of catalase activity was found in the supernatant of the consortium cultures herein studied. Catalase has been reported to play a role in corrosion in electrochemistry studies using immobilized enzymes (Landousli et al., 2008). Although further work must be done to stablish a causal link between catalase secretion by bacteria and corrosion in a situation involving living cells, it must be noted that the involvement of catalase has been reported in different works regarding MIC (Atalah et al., 2019; Baeza et al., 2012). As more studies evaluate the secretion of redox enzymes by corrosion inducing microorganisms, a clearer picture of the mechanisms involved will emerge.

This work aimed to broadly characterize the biofilm produced by a thermotolerant pro-corrosive consortium at the level of microbial community, EPS polysaccharide content and enzymes produced in carbon rich conditions. Many questions remain open about the mechanisms involved in MIC, such as whether there are specific mechanisms conducive to corrosion damage, or if the presence of associated microorganisms over a surface is enough, and corrosion is just a consequence of their normal metabolic activity (Little et al., 2020a). There is growing evidence that the ability to induce corrosion is found in bacteria of numerous genera, and no trend in terms of the EPS structure has been found. Such characterizations are still valuable, as they can open the doors to the development of specific treatments against recalcitrant biofilms and corrosion prevention methodologies.

With the findings here reported it can be concluded that the bacterial consortium studied exerted an induction of corrosion over AA7075, visible after a period of 14 days using SEM. It produced catalase, an enzyme that has been linked to MIC, did not display sulfate reduction activity, and produced an EPS which polysaccharide content consisted mainly of mannose, galactose, rhamnose and ribose. Bacterial growth also resulted in acidification of the media. The exposure of AA7075 coupons to the pH reached suggested that this is at least one of the factors driving the induction of corrosion. Hydrogen production was also detected, and could be related to corrosive effect observed. Taken together, this results support the hypothesis that MIC can result from the normal metabolic activity of microorganisms that are able to colonize susceptible surfaces, and does not necessarily imply specific biochemical mechanisms, or even specific bacterial genera.

Acknowledgements

This work was supported by the AFOSR under project FA9550-19-1-0234.

We would like to thank Caitlin Bojanowski for her support and feedback.

Disclosure statement

No potential conflict of interest was reported by the authors.

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Table 1: Registered changes in the pH of bacterial cultures in time. Cultures were kept at 50°C for 40 days and pH was measured at the stated intervals. Shown pH values are the means obtained from four independent experiments. As a control, sterile media was also incubated for 40 days at the same temperature.

Intervals:

t₀

1 day

7 days

14 days

21 days

40 days

Measured pH (Consortium):

6.60

(+/- 0.00)

4.41

(+/- 0.05)

4.11

(+/- 0.05)

3.97

(+/- 0.02)

4.13

(+/-0.02)

3.95

(+/- 0.12)

Measured pH (Sterile Control):

6.80

(+/- 0.00)

6.70

(+/- 0.14)

6.07

(+/- 0.00)

5.86

(+/- 0.05)

5.69

(+/- 0.01)

5.46

(+/- 0.04)

Table 2.- Microbial composition at the genus level of the planktonic phase of the consortium and the Biofilm-forming bacteria.

Bacterial Genera	Relative abundance (%)
Bacterial Genera	Planktonic phase	Biofilm
Bacillus	94.23	94.89
Paenibacillus	0.4	1.06
Anoxybacillus	0.88	0.24
Thermoanaerobacterium	4.5	3.81

Table 2: Estimated amounts and mole percentage of each detected monosaccharide in each sample

	PS I		PS II
Glycosyl residue	Mass (μg)	Mol %	Mass (μg)	Mol %
Ribose (Rib)	6.0	18.2	7.2	23.3
Rhamnose (Rha)	5.5	15.3	5.2	15.5
Mannose (Man)	11.0	27.7	9.2	25.0
Galactose (Gal)	11.6	29.1	10.6	28.7
Glucose (Glc)	3.6	9.1	2.6	7.0
N-Acetyl Glucosamine (GlcNAc)	0.3	0.5	0.2	0.4
Sum:	38.0	99.9%	34.9	99.9%

Table 3: Relative percentage of each detected monosaccharide in analyzed samples.

Glycosyl residue	PS I	PS II
Terminal Ribofuranosyl residue (t-Ribf)	0.4	0.4
Terminal Rhamnopyranosyl residue (t-Rha)	0.2	0.2
2 linked Ribofuranosyl residue (2-Ribf)	0.6	0.5
Terminal Mannopyranosyl residue (t-Man)	0.2	0.3
4 linked Rhamnopyranosyl residue (4-Rha)	11.3	11.2
Terminal Glucopyranosyl residue (t-Glc)	2.8	1.9
Terminal Galactopyranosyl residue (t-Gal)	0.2	0.2
4 linked Xylopyranosyl residue (4-Xyl)	0.9	0.7
3 linked Glucopyranosyl residue (3-Glc)	1.2	1.0
2 linked Mannopyranosyl residue (2-Man)	0.8	0.1
3 linked Mannopyranosyl residue (3-Man)	1.0	1.6
2 linked Glucopyranosyl residue (2-Glc)	0.6	0.4
3 linked Galactopyranosyl residue (3-Gal)	12.5	16.4
4 linked Mannopyranosyl residue (4-Man)	12.7	14.1
6 linked Glucopyranosyl residue (6-Glc)	1.0	0.8
4 linked Galactopyranosyl residue (4-Gal)	0.3	0.3
4 linked Glucopyranosyl residue (4-Glc)	12.6	10.1
6 linked Galactofuranosyl residue (6-Galf)	8.2	8.4
3,4 linked Mannopyranosyl residue (3,4-Man)	13.9	13.5
3,4 linked Galactopyranosyl residue (3,4-Gal)	0.6	0.4
3,6 linked Glucopyranosyl residue (3,6-Glc)	0.3	0.2
2,6 linked Mannopyranosyl residue (2,6-Man)	0.5	0.4
4,6 linked Glucopyranosyl residue (4,6-Glc)	2.7	2.4
4 linked N-acetyl Glucosamine (4-GlcNAc)	1.4	1.9
Unknown	13.0	12.4

No competing interests reported.

Biochemical and microbiological characterization of a thermotolerant bacterial consortium involved in the corrosion of Aluminum Alloy 7075

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Methods

2.2 Bacterial cultures and growth conditions

2.3 Corrosion assays

2.4 Bacterial community composition.

2.5 Purification of the carbohydrates present in the biofilm.

2.6 Glycosyl Analysis

2.7 Sulfate reduction Activity

2.8 H₂ determination

2.9 Enzymatic assays

2.10 Microscopic Imaging

3. Results

4 Discussion

5. Conclusions

Declarations

References

Tables

Additional Declarations

Status:

Journal Publication

Version 1

Biochemical and microbiological characterization of a thermotolerant bacterial consortium involved in the corrosion of Aluminum Alloy 7075

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Methods

2.2 Bacterial cultures and growth conditions

2.3 Corrosion assays

2.4 Bacterial community composition.

2.5 Purification of the carbohydrates present in the biofilm.

2.6 Glycosyl Analysis

2.7 Sulfate reduction Activity

2.8 H2 determination

2.9 Enzymatic assays

2.10 Microscopic Imaging

3. Results

4 Discussion

5. Conclusions

Declarations

References

Tables

Additional Declarations

Status:

Journal Publication

Version 1

2.8 H₂ determination