The consortium of sulfate reducing bacteria (sulfate reducing microorganisms) used in this study was collected with an in-line inspection tool used for internal inspection in pipelines (the so-called smart ‘PIGS”). The sample consortium was inoculated in ATCC1250 modified Baar's medium for Sulfate Reducers with NaCl (2.5 wt.%) [ATTC®]. The pH value was adjusted to 7.2 under de-aeration by applying nitrogen to the electrolyte. The medium was autoclaved at 120°C for 15 min. The SRM consortium was incubated at 37°C.

The Zn-rich epoxy primers and multi-walled carbon nanotubes were supplied by Tesla NanoCoatings company. The epoxy resin (Tesla P1150ASAS) was mixed with CNTs at 1 and 2 wt.%. After the CNTs were completely dispersed in the epoxy resin through mechanical stirring according to a previously method (Cubides and Castaneda, 2016), Zn particles at 70 wt.% were slowly added while stirring the solution, and a hardener (Tesla P1150BSAS) was then poured in to a final weight ratio of 2:1 (epoxy resin-hardener). The 70 wt. % was used based on the previous work (Cubides and Castaneda, 2016) where is was demonstrated the Zn content was the most balanced for cathodic protection or sacrificial conditions. In addition, Zn-rich epoxy primer without CNTs was also used in this study. The coatings were air-sprayed onto UNS1008 steel plates. This procedure was performed previously (Cubides and Castaneda, 2016). Table 1 shows the different sample characteristics.

A three-electrode electrochemical cell was used for the experimental characterization. The electrochemical cells were autoclaved. The working electrode was the multifunctional coating sample. The counter electrode was a platinum mesh, and the reference electrode was a saturated calomel electrode (SCE). The working electrodes and the reference and auxiliary electrode were sterilized with 70% ethanol and acetone and set under UV light and laminar flow. They were placed on a 25 mL electrochemical cell containing culture medium under N₂ gas injection before each experiment. It was conducted at 25°C. OCP was performed on prior EIS measurements during a period of 10 min. EIS was performed in a frequency range of 100 KHz to 10 mHz with a ±10 mV peak-to-peak amplitude vs. the open circuit potential (OCP). All the tests were carried out in a potentionstat/galvanostat (VSP-300; Bio-Logic Science Instruments), using the software EC-Lab® V.10.32. All electrochemical experiments were performed in duplicate to ensure reproducibility of the electrochemical behavior.

Once the electrochemical test was completed, the samples were rinsed with PBS 1X (8.0 g of NaCl, 0.2 g of KCl, 1.4 g of Na₂HPO₄·2H₂O, and 0.2 g of KH₂PO₄ per liter, pH = 7.2). Subsequently, the samples were fixed with glutaraldehyde 2.5% w/v in PBS for 2 h, with washing every 30 min. All fixation was conducted at room temperature. After fixation, the cells were washed twice in PBS and then re-suspended in sterilized ultrapure water to avoid salt crystallization during the drying process. The samples were dried for 24 h in a LABCONO Freeszone 6 lyophilizer. Finally, the samples were sputtered with silver particles and observed under a JSM-7000F/JOEL microscope through energy-dispersive spectroscopy (EDS).

The ZnR coating system shows the potential evolution influenced by different conditions and interfacial configurations such as the epoxy physical barrier, the galvanic phenomenon between zinc and steel, the physicochemical barrier of corrosion products, the concentration of Fe and Zn near the particle/coating or substrate/coating interfaces and inside the coating, amongst other factors, as described by Cubides (Cubides and Castaneda, 2016). The cathodic protection of steel has been established at ≤ −0.90 V vs. Cu/CuSO₄ (−0.826 vs. SCE) under anaerobic conditions (Schwerdtfeger, 1958; Guan et al., 2016; Liu and Cheng, 2017). This level is drawn as a dash line in Figure 1. At higher (more positive) potentials, barrier protection tends to be the dominant control mechanism, which may be associated with the epoxy-polymer and the corrosion products of Zn and/or Fe. The OCP monitoring of the AISI/SAE 1008 steel substrate and the multifunctional coatings under biotic conditions is shown in Figure 1. The OCP for the bare steel is more positive (passive region or protective layer region based on thermodynamics) than the cathodic potential criteria because the galvanic effect results in a negative potential value, in which the steel faces immune thermodynamic conditions. The OCP for the bare steel 1008 was determined in a test where the sample was kept in the culture medium in bacterial consortium conditions, and the steel potential was found to be influenced by the biofilm and corrosion products formed at the surface of the substrate. Classic work by Castaneda (Castaneda and Benetton, 2008) characterized mild steel in seawater in the presence of SRB, which resulted in positive potentials due to the combination of corrosion products and biofilm. Figure 1 shows the initiation and growth stages for the biofilm formation. At this respect, Castaneda marked 14 days for the initiation to biofilm growth transition, similar to this condition, where the OCP increases the magnitude following 2 weeks of exposure.

The Zn-rich primer (70 wt.% Zn) exposed to the bacteria consortium shows a potential oscillating within the barrier protection zone over the exposure time. This means that the Zn particles are randomly distributed in the epoxy matrix with a connectivity between them that is insufficient to trigger the galvanic effect formation. There is a transient state during the first 10 days of exposure, and during this time, the potential values are in the barrier zone. This behavior is associated with water uptake in the coating and the formation of Zn products during the initial days of exposure. Finally, the potential is around the OCP of 1008 carbon steel in biotic conditions, but both experimental results are above the corrosion protection located in the barrier protection zone, and barrier zone dominance can result from the Zn-Fe corrosion products and/or biofilm formation to produce a blocking effect for the water uptake process.

At the early stage, the Zn-1CNT potential is located close to the cathodic protection zone for the first day. Following the negative potentials, a transient stage occurs during the next 10 days within the barrier protection area. The initial conditions could be the response of the bioelectrolyte wetting over the Zn particles and due to the interconnectivity among Zn particles and steel arising from the influence of MWCNTs or the cathodic protection effect; the potential between 4 and 10 days may be the result of Zn and/or Fe corrosion products acting as a barrier or passive particle state, which switches the system to prevail in more positive potentials. These latter compounds fill the porous surfaces within the coating structure, and the potential subsequently increases at day 2. The interaction between consortium/coating subsequently changes the quantity and morphology of the products, and the potential decreases at day 10. Eventually, the potentials reach ~ −0.680V vs. SCE in the barrier zone, 100 mV more negative than the ZnR sample. Subsequently, the potential remains stable at close to −0.680V vs. SCE. This latter effect could be due to the influence of the biofilm at the surface, in which the zinc ions inhibit the formation of the biofilm layer at initial conditions (Babich and Stotzky, 1978; Bong et al., 2010; Billanger et al., 2015; Tong et al., 2015), but the ions are depleted and the biofilm starts to grow over time to become more homogeneous and distributed compared with the 2CNT, and the magnitude of the potential becomes more negative than the ZnR sample to produce a less dominant barrier effect with the addition of 1CNT.

OCP for the Zn-2CNT sample are more negative than the threshold for the cathodic protection magnitude during the first 2 days. This behavior is associated with a greater MWCNTs concentration, resulting in better interconnection between the Zn/Zn electrical connection particles due to the CNT/Zn/CNT interconnectivity. This latter finding is confirmed in the cross-section SEM images. These potentials are more negative than the Zn-1CNT results. A transient potential state exists between days 3 and 10 of exposure. After 10 days, there are two transient states during the barrier protection zone and close to the cathodic protection threshold, one between 10 and 15 days and the other between 16 and 24 days. Finally, the potential keeps fluctuating in the barrier protection zone and reaches lower values than the OCP of carbon steel in biotic conditions. The potentials reveal the dominance of cathodic protection for this sample in different stages. Particle activation was promoted by the distribution of CNTs within the coating, and the products formed in the coating are not sufficiently compact to stop bioelectrolyte uptake within the coating, with the unstable biofilm leading to lower potential magnitudes. This may result from greater electrochemical activity among particle/coating interface. This latter result is evidence of a certain grade of galvanic effect, i.e., the system is at a lower potential than the value found for steel without coating due to reaction with the Zn particles. The presence of two increments may be due to the creation of products that fill the pores in the coating, with the decrease resulting from the particle layer and an interaction forming with an incipient heterogeneous biofilm. That is, zinc ion formation inhibits biofilm growth and leads to various areas of biofilm depletion, thus affecting the morphology of these compounds over time and altering the distribution of the biofilm at the surface of the coating.

Figure 2 shows the complex and phase angle diagrams for Bare steel and Zinc Rich Epoxy multifunctional coatings in SRM consortium environment.

The Nyquist representation for the AISI 1008 bare steel sample exposed to the biotic electrolyte is shown in Figure 2A. The results show a loop with a large diameter characteristic of a capacitance and charge transfer process. The phase angle representation is illustrated in Figure 2B. The medium frequency response is associated with capacitance (double layer) at the surface, whereas the response at low frequency is associated with the electrochemical reactions at the steel/electrolyte interface. On day 5, this system represents biofilm formation, and the corrosion products form an inorganic and organic mixing layer. At low frequencies, the magnitude of the semicircle's diameter is associated with the substrate/mixing layer/electrolyte interface. The magnitude for the biotic conditions results in a loop intercepting the locus point on the order of 12,000 Ω cm², whereas at 10 days, an increase in the loop diameter occurs, which may be associated with the accumulation of a mixing layer formed at the surface. At the surface, the number of active sites decreases, and the Rct increases, as previously described by Castaneda and Benetton (2008), with mild steel exposed to an SRM electrolyte during the first 2 weeks. At day 15, the impedance magnitudes begin to decrease, reflecting an activation mechanism, and an increase in the number of active sites occurs at the metallic substrate/mixing layer interface. On day 20, the impedance lowers the magnitude for this sample, an effect associated with the active sites, owing to the charge transfer process. After 25 days, the contribution of two potential interfaces is revealed by two maximum points in the phase angle representation. At days 25, and 29, the active sites remain constant, owing to the maintenance of the impedance at low magnitudes and the formation of two separate contributions at the interface layers, owing to the stability and physical presence of a mixing layer on the surface, as a result of precipitation of corrosion products and presumed biofilm formation. This finding corroborates the OCP biofilm transition from initiation to growth stage. The EC representation for this system can be described by Figures 3A,B; the first days include an activation capacitance RC analog, and after 25 days, two time constants are represented by two constant phase elements.

Figure 7 shows the cross-section SEM images obtained for the multifunctional coatings after 29 days of immersion in the SRM bioelectrolyte. These images corroborate the findings from the OCP data and the impedance analysis. For Zn-1CNT and Zn-2CNT coatings, it was possible to detect and demonstrate the presence and interconnectivity of Zn particles with carbon nanotubes at the interface of carbon steel, the coating matrix itself and the EPS/corrosion product layer. The biofilm layer can be considered to provide greater extension for the ZnR coating because, as described above, the CNT content leads to cytotoxicity toward the main bacterial and fungal constituents of the SRM consortium (Upadhyayula and Gadhamshetty, 2010). The increase in CNT content promotes the formation of Zn ions, thus resulting in a cytotoxic environment. The images in Figure 7 reveal biofilm patch formation instead of a continuous layer at the electrode surface. Therefore, the presence of biofilm is not favored when CNTs are present.

The presence of CNTs, as evident in Figure 7, was associated with an excellent influence on conductivity at the inner section of the coating according to the impedance analysis. Lack of uniform layer is ascribable to the dissolution of zinc cations, which are presumably cytotoxic to the microorganisms. In turn, zinc ions could be also responsible for the formation of corrosion products such as ZnO and ZnCl₂, also the presence of these corrosion products also became evident according to the EDS elemental analysis, this latter illustrated in Figure 8.

Figures 8a–c show top-view SEM images of the three multifunctional coatings after 29 days of immersion in bioelectrolyte. For ZnR in Figure 8a, a continuous layer of extracellular polymeric substances is observed covering the surface, which becomes less evident in the presence of CNT particles. Figure 8b illustrates the initial formation and lack of persistence of a layer of biofilm that shows some heterogeneous and residual over time. This finding is attributable to the inhibition of mature biofilm formation by the CNTs influence along with a poisoning mechanism by the cytotoxic effect of Zn soluble products (Babich and Stotzky, 1978; Bong et al., 2010) At this respect, CNTs are considered to have adverse effects on biofilm growth at some levels according to the investigations of Upadhyayula and Gadhamshetty (2010). When a double content of CNTs is present, a greater porosity can be appreciated as shown in Figure 8c, which is mainly attributed to the lack of continuous biofilm layer caused by an increased level of zinc corrosion products and the double content of carbon nanotubes that alter the development and formation of a mature biofilm.

Also, Figures 8a–c show the EDS analysis demonstrating that the ZnR coating has a higher content of organic compounds, such as carbon, sulfur, nitrogen, and phosphorous (C, S, N, and P) due to presence of a more mature biofilm, which supports the OCP data and impedance results. For the Zn-1CNT and Zn-2CNT coatings, a higher content of Zn and O is evident, and a lower level of organic compounds is found. Higher Zn levels indicate the formation of zinc corrosion products over the coating surface. In addition, the Zn-1CNT and Zn-2CNT coatings exhibit higher Zn and O content, thus further indicating that these coatings inhibit the formation of extracellular polymeric substances (Babich and Stotzky, 1978; Bong et al., 2010; Billanger et al., 2015; Tong et al., 2015), and therefore disturb biofilm growth to a greater extent due to the toxicity of the high levels of CNTs (Upadhyayula and Gadhamshetty, 2010).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.