Preparation of the specimens, bacteria, and culture medium
The B. cereus Mc-1 bacteria isolated in a previous study was grown in a nutrient broth medium up to absorbance of 1.0 at a wavelength of 600 nm. After satisfactory growth, the culture was centrifuged at 8,000 g, then the supernatant was discarded and the precipitate was washed with Phosphate Buffered Saline (PBS) (Merck, Germany). The centrifugation and washing processes were repeated twice. Then, the cell pellet was used for inoculation in 3 ml of broth medium for growth of iron precipitating bacteria Ammonia Ferric Citrate medium (CFA), which was designed based on the chemical composition of 0.5 g/l (NH4) 2SO4, 0.2 g/l CaCl2.6 H2O, 0.5 g/l MgSO4.7 H2O, 0.5 g/l NO3- and 10.0 g/l Ammonia Ferric Citrate, 0.5 g/l K2HPO4, and the pH adjusted for 6.6 using 6N HCl. After 48 hours the culture was centrifuged, the supernatant discarded and the precipitate was used to inoculate for the corrosion experiment (described below).
Coupon preparation and experimental setup
Three different metal alloys were employed. ASI-1020 carbon steel, which has a chemical composition of C = 16%, Mn = 0.63%, P = 1.2%, S = 3.1%, Si = 1.2%, Cu = 1%, Cr = 3% and Ni = 1%; 316L stainless steel with maximum chemical composition of 0.030% C, 2.00% Mn, 0.75% Si, 0.045% P, 0.030% S, from 16.00 to 18.00% Cr, from 10.00 to 14.00% Ni, from 2.00% to 3.00% Mo, and copper Cu = 99.9%, Sb = 0.002%, Bi = 0.001%, As = 0.002%, Fe = 0.005%, Pb = 0.005%, and S = 0.005%. The carbon steel coupons were cut to 3 cm2, whereas the 301L stainless steel and copper coupons were cut to 4 cm2. They were exposed to absolute ethanol to degrease the surface, washed with acetone to remove organic matter, dried in an oven 70°C for 30 minutes, and kept in a desiccator. After the coupons were sterilized by autoclaving and then were cooled, identified, and weighed, to evaluate the weight loss by corrosion.
The determination of corrosion was done using the treated coupons placed in the bottom of the Erlenmeyer glass of 250 ml, and the growth medium was the CFA medium without the Ammonia Ferric Citrate compound (CFA.Ico-). Initially, a pre-inoculum was prepared from an isolated colony of B. cereus Mc-1 strain in CFA medium for 72 hours under the same conditions described above. After the growth reached the optical density of 0.50 at a wavelength of 600 nm, 100 µl were withdrawn and used to inoculate the CFA.Ico- medium with the coupons. Were analyzed the mass loss by microbial corrosion after seven and 15 days of exposition to CFA.Ico-. All analysis was performed in triplicate. In order to analyze the influence of other nutrients in the corrosion rate by Mc-1 strain, additional coupons assays were performed followed by the same parameters described above with supplements of the individual compounds: 2.0 gL-1 NaCl and 2.0 gL-1 of NO3-.
Weight loss determination
After each determined period for analysis, three coupons from each system were retrieved for the measure of mass loss and corrosion rate. The rust deposited was scraped, and the coupons were immediately immersed in acid pickling (15% HCl) to remove all surface corrosion products according to ASTM G1 (Standard Practice for Preparing, Cleaning, and Evaluating Corrosion Test Specimens) (ASTM G1-03 2017). The acid reaction was stopped by the application of thiourea solution for 5 seconds, next the coupons were washed with distilled water, and then the reaction was neutralized with 10% NaOH for 5 seconds, and finally immersed in acetone for the same period. To determine the corrosion rate during the experimental period of incubation in all conditions, the weight-loss evaluation method was employed. The results of weight-loss measurement were utilized to calculate the value of corrosion rates (CR) following the equation:
CR= (W x 365 x 1000)/(D x A x T),
where the W is the decrease in metal weight during the time period analyzed, K is the constant (3650), D the metal density in g/cm3, A the coupon area (mm2), and T the exposure time in days (NACE 2005). In order to evaluate the significant differences in the corrosion rates, an analytical analysis t test was employed.