Coupon finishes were selected based on the roughness attainable in standard machining operations under traditional (i.e., milling) and non-traditional processes (i.e., superfinishing) (Grzesik et al., 2010). Pure 101 copper (Cu) (ThyssenKrupp batch# 91V3461, Cu >99.99%wt. with Oxygen <0.0005%wt. and other trace elements) was purchased from Online Metals (Seattle, WA). Coupons were cut from a bar to the dimensions of 10 × 10 × 2mm using electrical discharge machining (EDM) process to minimize defects such as mechanical deformation that may later influence the corrosion behavior. Coupons were then ground and polished on both sides to four different surface finishes: 400 US grit, 600 US grit, 800 US grit and 3-micron diamond suspension polishing using metallographic procedures with an Allied High Tech MetPrep 3 Grinder/Polisher equipped with a PH3 power head. During the surface grinding and polishing processes, copper mechanically deformed primarily through dislocation motion because of its face-centered cubic (FCC) crystal structure characterized by a high number of slip planes. The deformation associated with each surface finish may have contributed to the development of dislocation’ “forests”. However, previous research on FCC metals demonstrated that, over a wide range of strains, the interaction between forest and mobile dislocations only marginally increased the forest density (Csanádi et al., 2011). For this reason, the degree of deformation introduced by the preparation process was expected not to influence the corrosion behavior of the copper coupons.

Coupons without any surface modification (“as received”) were also used for comparison. To remove any polishing residue, ultrasonic cleaning was performed in a two-step process with 100% acetone (Fisher Chemical Lot#187993) and 100% methanol (Fisher Chemical Lot#217743) for 10 min each. Prior to abiotic and biotic exposures, all coupons were prepared in a biosafety cabinet by dipping in ethanol solution (70% v/v) prepared with pure ethyl alcohol (Sigma-Aldrich Lot # SHBL 3646) and sterile nanopure water. This step was followed by exposure to UV light for 30 min for each side of the sample during drying in a biosafety cabinet (Ha and Ha, 2010; Guridi et al., 2019; Raeiszadeh and Adeli, 2020).

The microorganism selected for this study Desulfovibrio alaskensis G20 was originally isolated from a soured oil reservoir (Feio et al., 2004) and recently reclassified to Oleidesulfovibrio alaskensis G20 (Waite et al., 2020). Oleidesulfovibrio alaskensis G20 has been extensively studied due to its role in MIC and its versatile metabolic capabilities (Wikieł et al., 2014; Nair et al., 2015; Chilkoor et al., 2018; Krantz et al., 2019; Mehta-Kolte et al., 2019; Singh et al., 2022), therefore making this strain a model organism for studying corrosion of metals by SRB.

The anoxic bacterial growth medium (LS4D) was prepared with 10.39 g/L PIPES buffer disodium salt (Sigma-Aldrich #P3768), 1.63 g/L MgCl₂·6H₂O (Research Products International M24000, 1.07 g/L NH₄Cl, 0.09 g/L (Sigma-Aldrich #A9434). CaCl₂·2H₂O, 0.64 mg/L (Research Products International C36200) as previously described (Mukhopadhyay et al., 2006). The LS4D growth medium was designed to minimize precipitate formation, maximize growth, and allow for optical density OD600 measurements of growth under sulfate-reducing environments and has been used under numerous growth conditions (Klonowska et al., 2008; Borglin et al., 2009; Holman et al., 2009; Zhou et al., 2015). The pH of the medium was adjusted to 7.2 using a 2 M solution of HCl and 14 mL was anaerobically dispensed into each 25 mL Balch tube. The tubes were flushed with N₂/CO₂ (80%/20%), sealed with a butyl-rubber stopper, crimped with an aluminum cap, and autoclaved. A total volume of 0.24 mL sterilized pre-mix solution containing 0.01 mL of Thauers vitamins, 0.19 mL of trace minerals, and 0.03 mL of 1M potassium phosphate solution was added to each tube after sterilization. The experiments were conducted under an electron donor and acceptor balanced condition that uses 60 mM lactate:30 mM sulfate as previously described (Franco et al., 2018; Krantz et al., 2019). Resazurin (0.1% v/v) was also added to the medium solution to serve as a general indicator of the oxidative-reduction potential (ORP) (Clark et al., 2006; Krantz et al., 2019).

For both abiotic and biotic exposures, three coupons for each surface finish were anaerobically placed in sterile growth medium in Balch tubes using a biosafety cabinet with sparging of sterile N₂ gas. The tubes were then sealed with a sterile rubber stopper and crimped with an aluminum seal cap. Blank coupon tubes were co-incubated to verify sterility during coupon processing. For biotic copper coupon exposures, O. alaskensis G20 was cultivated in previously prepared anaerobic tubes with LS4D medium for 3 days at 30°C and 0.1–0.2 mL of the culture was inoculated into each prepared tube containing the medium with one copper coupon. The optical density (600 nm) was measured using a Thermo Scientific, Genesys 10S UV-VIS Spectrophotometer as previously reported (Caffrey and Voordouw, 2010; Gao et al., 2016; Dong et al., 2023). For abiotic exposures, copper coupons of the as received and of each surface finish were also anaerobically exposed to sterile LS4D media in 25 mL Balch tubes to assess if any corrosion or surface deterioration occurred over the test period.

After 7 days of exposure, aluminum seal caps and stoppers were removed from the abiotic and biotic tubes inside a fume hood and the coupons were immediately placed in vacuumed tubes for further analysis after rinsing with sterile, anoxic medium. A ZEISS SUPRA 55VP FE-SEM microscope (Carl Zeiss Microimaging, GmbH, Gottingen) was used for the enumeration of SRB sessile cells. Representative triplicates of FE-SEM images of biotically exposed copper coupons were selected for each surface finish. Three 10 × 10 µm areas were randomly chosen per each image for the quantitative evaluation of sessile SRB cell by direct surface counts. The recorded values were then extrapolated to obtain the cell density for 100 mm² corresponding to the coupons’ total surface area. The abiotic control coupons (prepared the same way but not inoculated with O. alaskensis G20) did not demonstrate any microbial presence therefore no interfering species were introduced during the preparation and exposure processes. Removal of O. alaskensis G20 surface-associated cells and of any corrosion product resulting from the biotic exposures was performed by ultrasonic cleaning with hydrochloric acid (HCl) (Fisher Chemical Lot#179019) for 1 min as (ASTM G1-03, 2017)e1, Standard Practice for Preparing, Cleaning, and Evaluating Corrosion Test Specimens, 2017). FE-SEM analyses and weight loss measurements were performed to confirm that this procedure did not introduce any weight change or additional surface damage.

Prior to abiotic and biotic exposures the as received and all prepared copper coupons were analyzed using a Filmetrics Profilm 3D Optical Profilometer to evaluate the surface topography and the distribution of asperities, defined by the peaks’ height and valleys’ depth. Three area roughness parameters were measured, namely, the arithmetical mean height (Sa), the root mean square height (Sq) and the maximum valley depth (Sv). Samples did not need any further preparation and white light beam setting was used for all profilometer analysis. The HCl cleaning process described previously was performed prior to FE-SEM imaging following biotic exposure while no additional steps were taken for abiotically exposed samples.

To assess the distribution, thickness, and morphology of the formed mineral layers, analysis of metallographically prepared cross sections was also performed for all exposed samples. Polishing was conducted up to 1 micron diamond suspension using Allied High Tech MULTIPREP Polishing system. EDX (Princeton Gamma-Tech, Inc, Rocky Hill, NJ) was used to evaluate the pit depth using the X-ray attenuation methodology (Avci et al., 2015). To characterize the developed mineral layer, grazing incidence X-Ray diffraction GI-XRD analysis at a grazing incidence angle of 0.8° was performed using Bruker Nano Analytics D8 Advance XRD on all coupons after the biotic exposure experiments and prior to HCl cleaning. Statistical analysis of corrosion rates and pit depth from the biotic exposure experiments was carried out using MiniTab 21.4 (MiniTab, LLC). Statistical significance was determined via Tukey simultaneous tests for differences of means to determine the effects of each surface finish.

The corrosion rates for all surface finishes were calculated from the weight loss measurement, as per ASTM Standard G1-03, using Equation 3 (ASTM G1-03, 2017)e1, Standard Practice for Preparing, Cleaning, and Evaluating Corrosion Test Specimens, 2017). Weight loss measurements were performed by weighing the coupons before biotic exposure and after HCl cleaning as described in Section 2.3. C o r r o s i o n   R a t e m m y e a r = 8.76 × 10 4 × M a s s   l o s s g A r e a   c m 2 × T i m e   o f   e x p o s u r e   h o u r s × D e n s i t y g c m 3 (3)

RPS was used to comprehensively quantify corrosion accounting for any weight loss due to uniform corrosion. Despite being recognized as the “MIC signature”, if only pitting corrosion was considered, the overall corrosion severity would be underestimated. Pit depth would in fact be measured from the corroded surface instead of the uncorroded one. Hence, both pit depth and weight loss should be used to properly describe corrosion severity. RPS defined as the ratio of pit growth rate to uniform corrosion rate based on specific weight loss was calculated from Equation 4 (Dou et al., 2018) R P S = a v e r a g e   m a x i m u m   p i t   d e p t h   c m x   m e t a l   d e n s i t y   g c m 3 a v e r a g e   s p e c i f i c   w e i g h t   l o s s   g c m 2 (4)

The calculated RPS values were then used to reflect the relative importance of pitting corrosion to uniform corrosion as follows:

- RPS ≫ 1, pitting corrosion is much more severe than uniform corrosion

The 3D topography and corresponding distribution of asperities for all prepared surfaces before biotic exposures, is shown in Figures 1A–E while the Sa, Sq and Sv values are summarized in Table 1The same decreasing trend was observed for all the measured roughness parameters. The as-received samples were characterized by random distribution of surface features most likely related to the manufacturing process of the copper bars (Figure 1A). A homogeneous distribution of unidirectional grinding marks with asperities decreasing in size with increasing US grit values (Figure 1B′-1D′) was observed for the 400, 600 and 800 US grit (Figures 1B–D) and was supported by the measured values of Sv and Sq (Table 1). The 3 μm diamond suspension polished samples had a uniform distribution of asperities in the range 0.03–0.07 μm (Figure 1E′). For surfaces finished with the 800 US grit and 3 μm diamond suspensions the roughness values were below the average cell size of O. alaskensis G20.

The O. alaskensis G20 planktonic growth in the presence of the copper coupons with different surface finishes over the exposure period is presented in Figure 2. The lack of significant difference in the planktonic growth of O. alaskensis G20 between cultures cultivated with and without copper coupons and the abundant presence of sessile cells (Figures 3A–E) indicated that, irrespective of surface finish, the levels of Cu(I) ions released by copper coupons were not toxic to either planktonic or sessile O. alaskensis G20 cells.

FE-SEM imaging of copper coupons after 1-week of abiotic exposure to sterile medium, revealed unaltered surfaces (Supplementary Figure S1) with topographies like those observed using 3D optical profilometry prior to biotic exposures (Figures 1A–E). Surfaces of the exposed as-received copper coupons demonstrated the presence of dense crystalline deposits and numerous associated SRB cells (Figure 3A). The 400 US grit finished coupons were characterized by a random distribution of individual crystals varying in size (Figure 3B). For the 600 US grit, 800 US grit and 3 μm diamond suspension finished surfaces, the morphology of individual crystals regions was similar to that seen on the 400 US grit finish along with dense mineral regions with a morphology comparable to the as-received samples (Figures 3C–E). Smaller size crystals were detected in both regions for the 3 μm diamond suspension surface finish, when compared to crystal sizes measured on surfaces with higher roughness values (Figure 3E). Spectra obtained with GI-XRD demonstrated the prevalence of copper (I) sulfide (Cu₂S, chalcocite) minerals in all biodeposits, irrespective of the surface finish (Figure 4).

Irrespective of the surface finish, FE-SEM cross-sectional imaging of biodeposits demonstrated a continuous mineral layer spreading over the copper surface, decorated with scattered individual copper (I) sulfide crystals with a size varying from 3 μm to 7 μm. (Supplementary Figure S2). The thickness of the layer decreased with the surface roughness, measuring approximately 3 μm, 2 μm, 1.6 μm, 1.2 μm, 0.5 μm on the as received, 400, 600, 800 US grit SiC paper and 3 μm diamond suspension polished surfaces, respectively.

Using the FE-SEM enumeration procedure, sessile SRB cell counts (cells/cm²) of 2.96 ± 0.31 × 10⁷, 2.40 ± 0.27 × 10⁷, 2.57 ± 0.42 × 10⁷, 3.07 ± 0.42 × 10⁷, 2.53 ± 0.21 × 10⁷ for the as received, 400 US grit, 600 US grit, 800 US grit finished coupons and 3 μm diamond suspension polished coupons respectively was recorded (Supplementary Table S1). Based on the Tukey simultaneous test for differences of means (Supplementary Table S2) among the sessile cell counts, it was determined that the surface roughness did not have a statistically significant effect.

FE-SEM imaging of copper surfaces following the removal of biodeposits revealed uniform corrosion along with pitting attack (Figure 5). The size and distribution of pits varied with the surface finish. Nesting pits were observed for the as received and the 400 US grit finished samples with external pit diameter ranging from 3 μm to 6 μm while the internal pit diameter value was estimated to be between 1 μm and 3 μm (Figures 5A, B). For the 600 US grit and the 800 US grit finished samples, single pits with a diameter between 1 μm and 2 μm size were observed over the entire surface (Figures 5C, D). No specific trend was noted in the pit distribution. Intergranular corrosion was observed for the 3 μm diamond suspension polished surfaces; a scarce localized attack was found with pits of a diameter <1 μm being randomly distributed on the grains (Figure 5E).

Table 2 summarizes the corrosion rates calculated for the 400 US grit, 600 US grit, 800 US grit finished coupons and 3 μm diamond suspension polished coupons. The number of samples tested per each surface finish is N, the “mean” is the average value of measured corrosion rate, “StDev” is the standard deviation and “95% CI” is the 95% confidence interval recorded for the as-received coupons and for each surface finish. The results indicated that a decrease in the roughness caused a decline in the overall copper corrosion rates. A reduction of 48%, 52%, 62% and 75% in corrosion rate for the 400 US grit, 600 US grit, 800 US grit finished coupons and 3 μm diamond suspension polished coupons respectively were recorded, when compared to as-received coupons. However, the Tukey simultaneous test for differences of means (Supplementary Table S3) using the corrosion rates values determined that the surface roughness Sa, did not have a statistically significant effect until it decreased from 2.71 μm (as-received samples) to 0.006 μm (3 μm diamond suspension polished samples) which corresponds to a highly finished surface (e.g., superfinishing).

No weight loss was observed for abiotic 7-day exposure in sterile medium subsequently the corrosion rates could not be calculated.

The pit depth evaluation was carried out using the X-ray attenuation methodology in EDX spectra as previously described (Avci et al., 2015). Ten pits were randomly selected and characterized for each surface finish. A minimum of three measurements were conducted on the same pit to minimize uncertainties introduced by the X-ray production statistics. A reduction in the pit depth of 53%, 75%, 58% and 68% was recorded for the 400 US grit, 600 US grit, 800 US grit finished coupons and 3 μm diamond suspension polished samples respectively, when compared to as-received coupons (Table 3). The Tukey simultaneous test for differences of means performed on the measured pit depths, revealed that the pit depth decrease for each surface finish was statistically significant when compared to the as-received coupons. However, no specific pit depth trend was found among the surface finishes (Supplementary Table S4).

Table 4 shows the RPS values calculated for the 400 US grit, 600 US grit, 800 US grit finished coupons and 3 μm diamond suspension polished coupons. It was observed that, regardless of the surface finish all values were lower than 1, supporting the relative severity of uniform corrosion.