Assessment of the corrosivity of AISI 1020 steel through microbiological analyzes and the mass loss technique in a clayey soil

This search aims to evaluate the corrosivity of AISI 1020 steel in clayey soil through microbiological analyzes and the mass loss technique. Through the results obtained, according to the identification methodology of the Bergey manual, the presence of two microorganisms was verified, Acidithiobacillus thiooxidans and ferrooxidans , responsible for the biocorrosion process , in addition to filamentous fungi. The presence of these bacteria does not generate a classification for the soil, however, it is known that they can accelerate the corrosion process when in contact with a metallic structure. Regarding the mass loss rate, a criterion used to simulate the assessment of corrosivity in pipes, NACE Standard RP-07-75 was adopted, which defined the intensity of the corrosive process, obtaining as a result for the sterilized sample a value considered low, since it was free of microbial activities and any other contaminating factor, but for the sample without sterilization, the soil was classified as having severe potential. Therefore, this research sought to correlate the characteristics of the soil representative of the Amazon with a process of corrosion of buried pipes due to the presence of microorganisms, which would correspond to a microbiological corrosion. Although, in many cases, there is suspicion of the accuracy of corrosion monitoring techniques, mass loss and microbiological identification techniques were used, which had a positive result in relation to the microbiological one.


Introduction
For Gentle (2022) Corrosion can be defined as the deterioration or even destruction of a material, usually metallic, by chemical or electrochemical action of the environment, due or not to mechanical efforts.
Several methods have been proposed to study and monitor corrosion processes, such as mass loss tests (rate of mass loss), analysis of aqueous extract (microbiological analysis), detection of galvanic current, measurement of electrical resistance and electrochemical measurements, among others. others (Macdonald et al., 1998;Ferreira, 2005;Rodrigues, 2006;Da Silva, 2007).
The investigation of the soil corrosivity rate is of great relevance for several activities directly associated with the useful life of materials, equipment and structures, especially vital structures such as electrical energy transmission towers, which must, mandatorily, maintain their physical integrity (Labegalini et al., 1992;Sharma & Kumar, 2021).
With the increasing number of reservoirs and buried pipes, the study of soil as a corrosive agent is of great importance, since its capacity for deterioration can represent serious economic and environmental problems over the years.
The wear process in buried reservoirs and pipes is due to physical-chemical and microbiological characteristics and some superficial factors, which generally cause high costs for a variety of sectors, such as the industrial sector (Gomes, 2001;Ert hal et al., 2017).
Carbon steel is often used in the manufacture of pipelines for use in the petroleum industry. Buried pipelines traverse a variety of soils, textures, depths, and in some cases, ions increase and accelerate soil corrosivity. Thus, the useful life of the tubes depends on the thickness of the metal, the exposed area and the maintenance/repair techniques employed (Oguzie et al., 2004;Wang et al., 2020).
It is important to emphasize that microbiological corrosion can also occur, in which colonies of microorganisms chemically modify the environment, releasing products of their metabolism such as acids that accelerate the corrosive process, in addition to the formation of films adhered to the metallic surface, which promote corrosion by aeration. differential (Gentil, 2012;Gentil, 2022;EMBRAPA, 2018).
According to Oliveira (2010), there are several types of bacteria that are fully involved in the soil corrosion process, such as: iron precipitating bacteria, acid-producing bacteria, exopolysaccharide-producing bacteria (Pseudomonas aeruginosa) and sulfate-reducing bacteria (SRB). Research, Society andDevelopment, v. 12, n. 6, e1112641752, 2023 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v12i6.41752 3 Another way to assess corrosivity is through the mass loss rate, which consists of monitoring the behavior of the metallic sample as a function of time, determining the mass loss per unit surface. The test provides objective values of soil aggressiveness for cases of generalized corrosion (Trabanelli et al., 1972;Erthal et al., 2017). It is the simplest and most widely used corrosion assessment method.
In this context, the present work aimed to study the corrosivity process in samples of AISI 1020 steel, similar to oil and gas pipelines, promoted by soils in the state of Amazonas, by the technique of mass loss/corrosion rate, by microbiological analyzes of the aqueous extract of soils and the identification of active bacteria in the process of corrosivity of this type of soil.

Soil Sample Collection
Soil samples were collected at the following locations: province Urucu-Coari oil company (geographical coordinates:   Each soil sample was collected in a maximum radius of 1 m from the helical metallic stake n at a depth of 0.5 to 1 m, with the aid of an auger in the amount ~1 kg, transferred to a plastic bag, sealed with adhesive tape and coded. Samples A1 (oil receiver), A2 (GLP), A3 (oil shipment) and A4 (GLP) were taken to the laboratory for drying, sieving and maceration, for further analysis.

Test Body Samples (AISI 1020 Steel)
The AISI 1020 steel samples, in the cylindrical format of 2.54 cm in diameter and 3 cm in height, were preserved and kept in a dry environment. This type of steel consists of carbon steel alloy with chemical properties similar to metallic tubes used in the oil industry. Research, Society and Development, v. 12, n. 6, e1112641752, 2023 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v12i6.41752

Microbiological Characterization
Aqueous extracts of collected soils were used to obtain bacterial samples according to the procedure described by Ferreira (2005).
Approximately 500 g of soil sample was transferred to a plastic tray, then this sample was manually disaggregated and left to dry in ambient air for 5 days. With the help of tweezers any leaves, branches and roots present were removed from the sample, then it was sieved through a 2.5 mm mesh sieve, the passing material was macerated in an agate mortar/pistil. Finally, the soil aqueous extract was prepared to determine the concentration of soluble species that could interfere with the corrosive process of the soil. Among these, the concentrations of sulfates, chlorides, potassium, sodium and calcium were of interest.
The preparation of the aqueous extract basically consisted of a solution composed of soil and water in the proportion of 1:100 , which was kept under agitation for 24 hours, enough time for the dissolution of the ions to occur. A 1 mL aliquot of the sample was inoculated into a nutrient medium composed of tryptose , lactose, bile salts, monopotassium phosphate , dipotassium phosphate , sodium chloride and distilled water, followed by incubation for a period of 48 hours in a bacteriological oven at 35° W.
This temperature was maintained for the selection of mesophilic bacteria (most bacteria prefer growth temperatures of 37 °C). After this period, the cultures of this sample were prepared in an acid medium ( ), being 30 mL of sulfuric acid at 0.5 mol L -1 , 10 mL of nutrient medium, 60 mL of distilled water, which was incubated for 24 h at 35 °C.
The final identification was made using a specific broth for the bacilli. Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans , respectively, in which the two media were distributed in test tubes and inoculated with 1 mL of the concentrate obtained from the nutrient medium and incubated in a bacteriological oven at 35 °C for 3 to 5 days. For a positive result, the solution should be cloudy, indicating the presence of microorganisms.
After this period, the cultures of this sample were prepared in an acid medium ( ) and incubated for 36 hours at 35 °C.
For the first identification of bacteria, the manual by Bergey (1994) was used, initially pre-selecting two possible organisms, Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans , both of which are Gram-negative and that could grow in these parameters and temperature where the experiments were carried out.
For the samples collected in December 2018 (Samples A3 and A4), the procedure adapted from Videla (2002) was used. Then, they were transferred to Petri dishes and distributed in bacteriological ovens, at temperatures of 25, 30 and 35 ºC, for an average period of 3 to 5 days. After this period, noticing the appearance of formed colonies, these bacteria were isolated, in the respective culture medium and , in order to preserve and favor the growth of a single type of bacteria, being able to identify it. Identification was performed as described by Holt (1994), where Gram staining and chemical tests, temperature, motility, oxidase, catalase and nitrate reduction were performed.

Mass Loss Rate
The determination of the mass loss (difference between the initial and final masses) allows calculating the corrosion rate and, therefore, evaluating the intensity of the corrosive process and estimating the wear of the metallic material in a given environment (Castro, 2013;Gentil, 2022).
The specimens were treated, and the masses were measured before the beginning of the experiments. The treatment consisted of sanding in an Arotec polishing machine, with sandpaper nº 80, 120, 200, 400, 600, 800 and 1200, for a better visualization of the corrosion. Mass measurements were taken daily on an analytical scale with 0.0001g precision, during a period of 30 days to calculate the corrosion rate, which were subjected to situations that simulate a steel tube or a pipeline Research, Society andDevelopment, v. 12, n. 6, e1112641752, 2023 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v12i6.41752 5 buried in the ground. The three forms of evaluation and treatment of steel in soil samples were carried out in accordance with ASTM G1-90 (2003): • Experiment 1 with fresh soil + 1020 steel; • Experiment 2 containing aqueous soil extract + 1020 steel (cleaned with isopropyl alcohol); • Experiment 3 with aqueous soil extract + 1020 steel.
The estimate of the corrosion rate was made with the results of the mass losses, from Equation 1: (1) where is the constant of proportionality tabulated (mm year -1 ) equal to ; is the difference between the masses, before and after exposure, in g; is the exposed area of the metallic sample, cm²; is the exposure time, in hours, and is the density, g cm −3 (carbon steel, 7.86 g cm −3 ).

Microbiological Analysis
Microbiological analyzes were performed in the following steps: • Step 1: Enrichment that consisted of sowing each sample in Petri dishes, with agar and salts, with the objective of verifying the bacterial colonies that grew in this medium, under pre-established conditions of temperature.

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Step 2: Isolation on new Petri dishes, each colony identified in Step 1 was streaked.

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Step 3 -Identification was performed according to the parameters of the Bergey manual (1994), using Gram stain and chemical tests, , temperature, motility, nitrate reduction, oxidase and catalase.

Temperature and
Microbiological analyzes were performed in the following steps: • Step 1: Enrichment that consisted of sowing each sample in Petri dishes, with agar and salts, with the objective of verifying the bacterial colonies that grew in this medium, under pre-established conditions of temperature.

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Step 2: Isolation on new Petri dishes, each colony identified in Step 1 was streaked.

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Step 3 -Identification was performed according to the parameters of the Bergey manual (1994) (2013) observed the first signs of formation of bacterial colonies between 3 and 4 days, at a temperature of 31 ± 0.5 °C, using bromine-cresol green and phenol red, for a total period of 10 days.
Through the results in Table 2, it is clear that there was a moderate growth for pH 4.0 and 5.0 at a temperature of 35 o C, thus confirming the presence of these bacterial colonies.

Gram stain
After the isolation of the cultivated bacterial colonies, the Gram staining test was performed to verify the shape, cell arrangement and coloration, identified through the result in Gram positive and Gram negative, illustrated in Figure 2. The results confirm what Bergey (1994) denotes, which shows that Gram-positive bacteria are classified by the color they acquire after applying a chemical process called Gram staining. Gram-positive bacteria stained blue, in the case of samples A1 and A2, while the other bacteria-stained red.

Motility
To verify the locomotion capacity of the microorganism, the samples were distributed in four test tubes and incubated in semisolid medium for 24 hours. It is also possible to identify motility when the culture medium becomes cloudy, as shown in Figure 3 (Houry et al., 2018). Source: Authors.

Nitrate Reduction
The bacteria were added to the nitrate medium (broth). Incubation was carried out for 5 days at 37°C. After this period, the tests were carried out in two steps: the first, adding 1 mL of solution A (sulfanilic acid) and the second, adding solution B (αnaphthyl -amine). The two-phase test is necessary because some bacteria convert nitrate to nitrite, and nitrite to nitrogen at high speed, as a result, there is no color change; and when powdered zinc was added, it reacted with the nitrate present. Source: Authors.

Catalase
To verify the presence of catalase in the selected bacteria, a slide was divided into four parts ( Figure 5). A bacterial concentrate was placed in each part, adding 1 mL of hydrogen peroxide.

Oxidase
It is a qualitative procedure and serves to determine the presence or absence of cytochrome C oxidase activity in bacteria. Used as a test to differentiate between anaerobic and facultative aerobic Gram-negative bacteria. This activity depends on the presence of an intracellular cytochrome oxidase system that catalyzes the oxidation of cytochrome C through molecular oxygen, which, in turn, will function as a receptor in the body's electron transport system (Steel, 1961). Source: Authors.

Scanning Electron Microscopy (SEM)
In order to characterize the occurrence of microbiological corrosion by indicating the presence of Acidithiobacillus bacteria thiooxidans and Acidithiobacillus ferrooxidans, the specimen that was submitted to experiment 1 was used. Figure 7 shows the images of the AISI 1020 steel sample before and after exposure to the aqueous extract formed with the clayey soil. Research, Society andDevelopment, v. 12, n. 6, e1112641752, 2023 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v12i6.41752 9 The metallic specimens were previously weighed before the beginning of the experiments.
The SEM images can be seen in Figure 8.

Corrosion rate
The test was performed according to Ferreira's procedure (2005), the corrosion rate results are shown in Table 3. For the evaluation criterion of corrosivity in gas pipelines, the NACE Standard RP-07-75 was adopted, according to Table 4. As for the sample without sterilization, the results can be seen in Table 5.

Microbiological analysis Temperature and
Bergey 's manual (1994), it is stated that the ideal for the growth of both species is 2.0 to 4.0, with temperatures in the ranges of 25 to 30 °C and 30 to 35 °C, for Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans, respectively.
In this test, a significant growth of bacterial colonies was observed at , at a temperature of 35 ± 2.0 ºC, over a period of 5 days (120 h) as shown in Table 2. Figure 2 shows the presence of Gram negative bacteria, in the form of rods (bacilli) and arranged in diplobacilli.

Gram stain
Jensen and Webb (1994), however, report that Acidithiobacillus can also occur alone or as streptobacillus.

Motility
In this test, it was possible to visualize that the bacteria grew along the incubation line, indicating the ability to move, as shown in figure 3. It is also possible to identify motility when the culture medium becomes cloudy.

Nitrate reduction
The bacteria selected for the test in the samples were not able to reduce nitrate to nitrite in vitro, as shown in Figure 4, which represents the four samples, this result is in line with the Bergey manual (1994).
Catalase Bergey (1994) was used, whose expected result for these types of bacteria was a negative result. As shown in Figure   5, no bubble formation or effervescence was observed.

Scanning Electron Microscopy (SEM)
By means of the SEM images, a continuity in the corrosion pattern can be observed, with the accumulation of biological material on the steel surface, which can be seen in Figure 8. These images show the surface of the partially polished AISI 1020 steel, with appearance grooves caused by polishing the metal part. Comparing it with the images from experiment 2, it is possible to visualize the surface with a spongy appearance formed by the deterioration promoted by microorganisms.

Corrosion rate
For the evaluation criterion of corrosivity in pipelines, the NACE RP-07-75 Standard was adopted, which defines the intensity of the corrosive process, according to Table 4. It was observed that the result for the sterilized sample was considered low, a since it was free of microbial activity and any other contaminating factor.
As for the sample without sterilization, the soil was classified as having a severe potential, which can be seen in Table 5. There was a significant difference in the corrosion rate in sample 1 in relation to samples 2 and 3, since it refers to a sample with clayey soil in natura and the others are samples with aqueous soil extract.

Conclusion
According to the identification methodology of the Bergey manual, two microorganisms were found, Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans , responsible for the biocorrosion process . The presence of these bacteria does not generate a classification for the soil, however, it is known that they can accelerate the corrosion process when in contact with a metallic structure.
As a process of corrosion of buried pipes due to the presence of microorganisms, which would correspond to microbiological corrosion. Although, in many cases, there is suspicion of the accuracy of corrosion monitoring techniques, mass loss and microbiological identification techniques were used, which had a positive result in relation to microbiology, in addition to emphasizing the idea that this monitoring of the corrosion rate it must be done with complementary techniques, which was the case with the chemical and mineralogical characterization techniques of the samples used.