*2.4. Long-Term Corrosion Experiments*

Initially, nine plastic reagent bottles (500 mL) were placed in 500 mL co-culture medium and autoclaved for 30 min. The coupons of X70 pipeline steel (18.7 mm × 7.8 mm × 1 mm) were provided by School of Materials Science and Engineering, USTB. Three experimental groups were set up, and the inocula are shown in Table 1. The samples were sealed and placed at room temperature for 3, 6 and 9 months to observe the corrosion and corrosion inhibition over a long period of time.

**Table 1.** The inocula volumes of the three experimental groups.


#### **3. Results and Discussion**

*3.1. Identification of P. stutzeri Strain*

3.1.1. Observation of Colonies

After the collected soil samples were cultured in liquid and enriched several times with strains, the samples were isolated and purified by the spread plate method. The growth rate of colonies in the autotrophic medium was slow, and small beige or white colonies appeared on the plates after about 5 days of incubation. After 7 days of incubation, there was a significant difference between each soil sample plate's surface and internal colony morphology. The small yellowish translucent needle-tip colonies on the surface of the plates were *P. stutzeri*.

The single colonies of *P. stutzeri* were inoculated in the sterilized medium for more than three generations of enrichment. The acceleration voltage of SEM test was set to 3 kV and the emission current was set to 100 µA. Then, the purified strains were subjected to Gram staining and SEM observation, as shown in Figure 1. *P*. *stutzeri* stained dark red, indicating that they were Gram-negative bacteria, and the cells were short rods with a length between 0.5 and 1 µm. The Gram staining experiment also showed that the purified strains were relatively pure, and no other miscellaneous bacteria were mixed in.

## 3.1.2. Identification of the Denitrification Capacity of *P. stutzeri*

After incubation with the Giltay medium, *P. stutzeri* could be observed for its discoloration reaction. *P. stutzeri* can turn the color of Giltay liquid medium blue-green as they are autotrophic denitrifying bacteria. Under anaerobic conditions, *P. stutzeri* can produce nitrogen, so air bubbles (N2) can be trapped in the Duchenne tubules. Without the presence of the denitrifying bacteria *P. stutzeri*, the Giltay medium was still dark green, but no air bubbles appeared in the Duchenne tubules. The results of the experiment are shown in Figure 2. *P. stutzeri* bacteria discolored the Giltay medium, and thus had a denitrification ability that could be used for subsequent experiments.

**Figure 1.** *P. stutzeri* colony (**a**); *P. stutzeri* liquid culture (**b**); *P. stutzeri* Gram staining (**c**); *P. stutzeri*  SEM (**d**). **Figure 1.** *P. stutzeri* colony (**a**); *P. stutzeri* liquid culture (**b**); *P. stutzeri* Gram staining (**c**); *P. stutzeri* SEM (**d**).

**Figure 2.** Identification of denitrifying bacteria using Giltay medium. (**a**) The blank Giltay medium; (**b**) medium for inoculation with *P. stutzeri*; (**c**) local amplification of (**b**). **Figure 2.** Identification of denitrifying bacteria using Giltay medium. (**a**) The blank Giltay medium; (**b**) medium for inoculation with *P. stutzeri*; (**c**) local amplification of (**b**).

3.1.3. Identification by 16S rDNA Sequencing

*3.2. Growth Characteristics of P. stutzeri and SRB*

3.2.1. Effect of Temperature on the Growth of the Strain

*stutzeri*.

3.1.3. Identification by 16S rDNA Sequencing The sequencing of the strains was conducted by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Then, the sequencing results were spliced with ContigExpress and the faulty parts at both ends were removed. Next, the spliced sequences were compared in the NCBI database (blast.ncbi.nlm.nih.gov) with the standard strains' rRNA type The sequencing of the strains was conducted by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Then, the sequencing results were spliced with ContigExpress and the faulty parts at both ends were removed. Next, the spliced sequences were compared in the NCBI database (blast.ncbi.nlm.nih.gov) with the standard strains' rRNA type

strains/16S\_ribosomal\_RNA database. After that, the species with the highest homology

Activated SRB and *P. stutzeri* were inoculated into liquid medium at pH 7 and incubated at a temperature ranging from 10 to 50 °C. After 7 days, the OD<sup>600</sup> values of the

bacterial broths were measured and the results are shown in Figure 3.

strains/16S\_ribosomal\_RNA database. After that, the species with the highest homology was selected and an evolutionary tree was constructed to confirm that the strain was *P. stutzeri*.

#### *3.2. Growth Characteristics of P. stutzeri and SRB*

#### 3.2.1. Effect of Temperature on the Growth of the Strain

Activated SRB and *P. stutzeri* were inoculated into liquid medium at pH 7 and incubated at a temperature ranging from 10 to 50 ◦C. After 7 days, the OD<sup>600</sup> values of the bacterial broths were measured and the results are shown in Figure 3. *Materials* **2023**, *16*, x FOR PEER REVIEW 7 of 19

**Figure 3.** Effect of temperature on strain growth. **Figure 3.** Effect of temperature on strain growth.

From Figure 3, it can be seen that the isolated and purified strains of SRB and *P. stutzeri* maintained high bacterial concentrations in the range of 25–40 °C. The absorbance of both SRB and *P. stutzeri* strains reached the maximum at around 30 °C. In this experiment, the optimum growth temperature of both SRB and *P. stutzeri* was around 30 °C. Thus, temperature affected the growth of SRB and *P. stutzeri* to a similar extent. From Figure 3, it can be seen that the isolated and purified strains of SRB and *P. stutzeri* maintained high bacterial concentrations in the range of 25–40 ◦C. The absorbance of both SRB and *P. stutzeri* strains reached the maximum at around 30 ◦C. In this experiment, the optimum growth temperature of both SRB and *P. stutzeri* was around 30 ◦C. Thus, temperature affected the growth of SRB and *P. stutzeri* to a similar extent.

#### 3.2.2. Effect of pH on the Growth of the Strain

3.2.2. Effect of pH on the Growth of the Strain After the activation, SRB and *P. stutzeri* were respectively inoculated into a liquid medium (the incubation temperature was 30 °C) with pH ranging from 1 to 13 to determine the effect of pH on the growth of SRB and *P. stutzeri*. OD600 values of the bacterial solution were measured after 7 days, and the results are shown in Figure 4. After the activation, SRB and *P. stutzeri* were respectively inoculated into a liquid medium (the incubation temperature was 30 ◦C) with pH ranging from 1 to 13 to determine the effect of pH on the growth of SRB and *P. stutzeri*. OD<sup>600</sup> values of the bacterial solution were measured after 7 days, and the results are shown in Figure 4.

**Figure 4.** Effect of pH on strain growth. **Figure 4.** Effect of pH on strain growth.

As shown in Figure 4, the maximum concentration of both SRB and *P. stutzeri* was reached at pH 7. The experiment showed that both SRB and *P. stutzeri* were able to grow in a wide pH range from 5 to 8, and the optimum growth pH was about 7. Thus, the effect of environmental pH on the growth of SRB and *P. stutzeri* was similar. The above experiments provide a feasible basis for the coexistence of SRB and *P. stutzeri* in the same growth environment. As shown in Figure 4, the maximum concentration of both SRB and *P. stutzeri* was reached at pH 7. The experiment showed that both SRB and *P. stutzeri* were able to grow in a wide pH range from 5 to 8, and the optimum growth pH was about 7. Thus, the effect of environmental pH on the growth of SRB and *P. stutzeri* was similar. The above experiments provide a feasible basis for the coexistence of SRB and *P. stutzeri* in the same growth environment.

#### 3.2.3. Graphical Growth Curves of *P. stutzeri*, SRB and Co-Culture of *P. stutzeri* + SRB 3.2.3. Graphical Growth Curves of *P. stutzeri*, SRB and Co-Culture of *P. stutzeri* + SRB

First, it was necessary to separately prepare the media of *P. stutzeri*, SRB, and mixed medium (mixed in the ratio of 1:1). Then, *P. stutzeri*, SRB and SRB + *P. stutzeri* mixture were inoculated into the above media at a 1/100 ratio and incubated in blue-capped culture flasks under anaerobic conditions at 30 °C constant temperature. After two days, samples were taken every 24 h and analyzed for OD<sup>600</sup> values. Based on the results, the growth curves of SRB, *P. stutzeri* and co-culture of *P. stutzeri* + SRB were plotted, and the results are shown in Figure 5. First, it was necessary to separately prepare the media of *P. stutzeri*, SRB, and mixed medium (mixed in the ratio of 1:1). Then, *P. stutzeri*, SRB and SRB + *P. stutzeri* mixture were inoculated into the above media at a 1/100 ratio and incubated in blue-capped culture flasks under anaerobic conditions at 30 ◦C constant temperature. After two days, samples were taken every 24 h and analyzed for OD<sup>600</sup> values. Based on the results, the growth curves of SRB, *P. stutzeri* and co-culture of *P. stutzeri* + SRB were plotted, and the results are shown in Figure 5.

From Figure 5, it can be seen that SRB reached the logarithmic growth phase on the third day and entered the stable phase after 8 days. However, after 12 days, SRB gradually began to enter the decay phase due to nutrient depletion, which changed the optimal growth environment of SRB. In contrast, *P. stutzeri* entered the stable phase after 5 days and slowly started to enter the decay phase after 11 days. As a result, it can be concluded that the growth cycles of SRB and *P. stutzeri* are consistent.

When SRB was co-cultured with *P. stutzeri*, the growth of SRB was not inhibited by *P. stutzeri* in the first 2 days; the concentrations of the mixed bacteria on the third and fourth days were greater than those of the two bacteria in separate cultures; after the fifth day, the concentrations of the two bacteria in co-culture were higher than those of *P. stutzeri*, but lower than those of SRB in separate cultures. It can be tentatively assumed that the growth of SRB was inhibited by *P. stutzeri* when the two bacteria were co-cultured together.

This is likely because when the two bacteria were co-cultured in the same medium, they grew competitively as the medium substrate was consumed over a longer incubation time. Namely, possible factors inhibiting the growth of SRB by *P. stutzeri* include their substrate utilization or their secretions. *Materials* **2023**, *16*, x FOR PEER REVIEW 9 of 19

**Figure 5.** Growth curves of SRB, *P. stutzeri* and co-culture of *P. stutzeri* and SRB. **Figure 5.** Growth curves of SRB, *P. stutzeri* and co-culture of *P. stutzeri* and SRB.

#### From Figure 5, it can be seen that SRB reached the logarithmic growth phase on the *3.3. Corrosion Inhibition of X70 Pipeline Steel*

#### third day and entered the stable phase after 8 days. However, after 12 days, SRB gradually 3.3.1. Effect of Temperature on the Inhibition of SRB Corrosion by *P. stutzeri*

began to enter the decay phase due to nutrient depletion, which changed the optimal growth environment of SRB. In contrast, *P. stutzeri* entered the stable phase after 5 days With the temperature change, there was a fairly obvious change in the weight loss of the samples, and the results are shown in Figure 6.

and slowly started to enter the decay phase after 11 days. As a result, it can be concluded that the growth cycles of SRB and *P. stutzeri* are consistent. When SRB was co-cultured with *P. stutzeri*, the growth of SRB was not inhibited by *P. stutzeri* in the first 2 days; the concentrations of the mixed bacteria on the third and fourth days were greater than those of the two bacteria in separate cultures; after the fifth day, the concentrations of the two bacteria in co-culture were higher than those of *P. stutzeri*, but lower than those of SRB in separate cultures. It can be tentatively assumed that the growth of SRB was inhibited by *P. stutzeri* when the two bacteria were co-cultured together. This is likely because when the two bacteria were co-cultured in the same medium, they grew competitively as the medium substrate was consumed over a longer incubation time. Namely, possible factors inhibiting the growth of SRB by *P. stutzeri* include their substrate utilization or their secretions. *3.3. Corrosion Inhibition of X70 Pipeline Steel* 3.3.1. Effect of Temperature on the Inhibition of SRB Corrosion by *P. stutzeri* With the temperature change, there was a fairly obvious change in the weight loss of The weight loss was relatively low when the temperature was between 10 ◦C and 20 ◦C. With the increase in temperature, the weight loss increased significantly. The corrosion of the steel sheets was more severe in the system in which only SRB was inoculated at 30 ◦C, while at 50 ◦C severe corrosion was noted in all three systems. From the analysis, the main factors were identified: most of the microorganisms had a suitable growth temperature of 20–35 ◦C—the higher the temperature, the stronger the microbial activity. If the external temperature were reduced or increased, the growth of the microorganisms would be affected to a certain extent. At temperatures lower than 20 ◦C, the metabolic activity of microorganisms (e.g., related enzymes) would be inhibited; at temperatures as low as 10 ◦C, the growth rate of microorganisms would be significantly reduced and they would basically be in a dormant state, making the corrosion of X70 steel relatively weak. The corrosion under these conditions would mainly be based on electrochemical corrosion, and since the temperature is low, the electrochemical corrosion would also be relatively low. With the increase in temperature, the activity of bacteria increased. Since the weight loss of steel sheets in the medium only inoculated with SRB was more apparent between 30 and 40 ◦C, it can be concluded that SRB grew faster and were more active.

the samples, and the results are shown in Figure 6.

**Figure 6.** Weight loss curves (7 days) of X70 steel under different temperature conditions. **Figure 6.** Weight loss curves (7 days) of X70 steel under different temperature conditions.

The weight loss was relatively low when the temperature was between 10 °C and 20 °C. With the increase in temperature, the weight loss increased significantly. The corrosion of the steel sheets was more severe in the system in which only SRB was inoculated at 30 °C, while at 50 °C severe corrosion was noted in all three systems. From the analysis, the main factors were identified: most of the microorganisms had a suitable growth temperature of 20–35 °C—the higher the temperature, the stronger the microbial activity. If the external temperature were reduced or increased, the growth of the microorganisms would be affected to a certain extent. At temperatures lower than 20 °C, the metabolic activity of microorganisms (e.g., related enzymes) would be inhibited; at temperatures as low as 10 °C, the growth rate of microorganisms would be significantly reduced and they would basically be in a dormant state, making the corrosion of X70 steel relatively weak. The corrosion under these conditions would mainly be based on electrochemical corrosion, Meanwhile, there was a slight trend of increased weight loss of steel sheets in the blank medium, mainly due to electrochemical corrosion. In the system inoculated with *P. stutzeri* + SRB, the weight loss of steel sheets was greatly reduced compared with the system inoculated with SRB only, indicating that *P. stutzeri* reduced the corrosion of X70 steel by SRB. When the temperature reached 40 ◦C, the growth of bacteria and the activity of enzymes would be somewhat inhibited by the high temperature. Hence, the corrosion induced by SRB was weakened, and the electrochemical corrosion was enhanced. Moreover, when the temperature reached 50 ◦C, as the temperature was far too high, the activity of bacteria was reduced by the influence of temperature. However, this high temperature promoted electrochemical corrosion, which caused more severe corrosion and a greater loss of weight. Thus, it can be concluded that *P. stutzeri* may have a good inhibitory effect on the corrosion of steel sheets caused by SRB in a wide temperature range.

#### and since the temperature is low, the electrochemical corrosion would also be relatively low. With the increase in temperature, the activity of bacteria increased. Since the weight 3.3.2. Effect of Time on the Inhibition of SRB Corrosion by *P. stutzeri*

loss of steel sheets in the medium only inoculated with SRB was more apparent between 30 and 40 °C, it can be concluded that SRB grew faster and were more active. There was a gradual weight loss per area of the steel sheets as the incubation time increased (the incubation temperature was 30 ◦C), as shown in Figure 7.

Meanwhile, there was a slight trend of increased weight loss of steel sheets in the blank medium, mainly due to electrochemical corrosion. In the system inoculated with *P. stutzeri* + SRB, the weight loss of steel sheets was greatly reduced compared with the system inoculated with SRB only, indicating that *P. stutzeri* reduced the corrosion of X70 steel by SRB. When the temperature reached 40 °C, the growth of bacteria and the activity of enzymes would be somewhat inhibited by the high temperature. Hence, the corrosion induced by SRB was weakened, and the electrochemical corrosion was enhanced. Moreover, when the temperature reached 50 °C, as the temperature was far too high, the activity of bacteria was reduced by the influence of temperature. However, this high temperature promoted electrochemical corrosion, which caused more severe corrosion and a greater The corrosion loss of the steel sheets inoculated with SRB was much greater than that observed in the other two tested systems. The 30-day loss was 8.57 g/m<sup>2</sup> , measured from the steel sheets in the blank group, which was mainly due to electrochemical corrosion caused by certain salts in the incubation medium. The corrosion rate of X70 steel in the test system inoculated with SRB alone was greater, which reached 15.67 g/m<sup>2</sup> at 10 days, 19.26 g/m<sup>2</sup> at 15 days, 21.87 g/m<sup>2</sup> at 20 days and 22.57 g/m<sup>2</sup> at 30 days. The growth curve of SRB bacteria showed four typical phases: retardation phase (0–2 days), log phase (3–6 days), stabilization phase (7–11 days) and decay phase (>12 days). In the range of 0–5 days, SRB were in the activation period after inoculation, and the number of active bacteria was small. Therefore, the corrosion was mainly caused by chemical corrosion; in days 5–10, the number of SRB increased drastically, and the corrosion enhanced. Notably,

after 7 days, the number of strains reached the maximum, the number of active bacteria was high, both enzyme activation and metabolism were at the peak, and the corrosion also intensified. Although the number of bacteria remained at a high state in the period of 10–15 days, the weight loss was lower than that in the 5–10 days period, probably because the growth of high quantities of bacteria led to the formation of biofilms on the surface of the steel sheets, which slowed the rate of increase of corrosion. Furthermore, at 20–30 days, corrosion was significantly delayed due to the large consumption of nutrients at this stage, while the toxic metabolites accumulated in large quantities, leading to an increase in SRB mortality and a decrease in viable bacteria. At the same time, the chemical corrosion also slowed because inorganic substances were consumed during their growth. In that case, it can also play a role in mitigating the corrosion on the surface layer as the corrosion product film became thicker and its combination with the microbial film was denser, which led to the corrosion into a slower stage after 15 days. The weight loss of steel sheets in the medium inoculated with SRB + *P. stutzeri* was 11.52 g/m<sup>2</sup> after 30 d immersion experiment. During the 0–30 days period, the weight loss in the mixed bacteria culture was significantly lower than that of SRB alone; between 15 and 30 days, the weight loss was basically at a stable stage. It was assumed that in the co-culture of *P. stutzeri* + SRB, the presence of *P. stutzeri* influenced the growth of SRB and slowed the corrosion of steel sheets by SRB, while on the other hand, the bacteria, extracellular polymers and corrosion products were mixed to generate a dense biofilm, which played a protective role on the steel sheets. Thus, it can be noted that *P. stutzeri* has a better protective effect on the corrosion of steel sheets caused by SRB with the extension of time. *Materials* **2023**, *16*, x FOR PEER REVIEW 11 of 19 loss of weight. Thus, it can be concluded that *P. stutzeri* may have a good inhibitory effect on the corrosion of steel sheets caused by SRB in a wide temperature range. 3.3.2. Effect of Time on the Inhibition of SRB Corrosion by *P. stutzeri* There was a gradual weight loss per area of the steel sheets as the incubation time increased (the incubation temperature was 30 °C), as shown in Figure 7.

**Figure 7.** Weight loss curves of X70 steel under different test times. **Figure 7.** Weight loss curves of X70 steel under different test times.

The corrosion loss of the steel sheets inoculated with SRB was much greater than that 3.3.3. Effect of Initial pH on the Inhibition of SRB Corrosion by *P. stutzeri*

observed in the other two tested systems. The 30-day loss was 8.57 g/m<sup>2</sup> , measured from the steel sheets in the blank group, which was mainly due to electrochemical corrosion caused by certain salts in the incubation medium. The corrosion rate of X70 steel in the test system inoculated with SRB alone was greater, which reached 15.67 g/m<sup>2</sup> at 10 days, 19.26 g/m<sup>2</sup> at 15 days, 21.87 g/m<sup>2</sup> at 20 days and 22.57 g/m<sup>2</sup> at 30 days. The growth curve The rules of electrochemical corrosion of steel affected by pH were as follows: when pH < 4, the corrosion of carbon steel was severe, mainly caused by hydrogen evolution corrosion; at pH 5–13, the corrosion was slower, mainly caused by oxygen absorption corrosion. From Figure 8, it can be seen that when pH 3, steel corrosion was more serious. At this stage, the role of microorganisms was not prominent; the bacteria in the acidic medium

of SRB bacteria showed four typical phases: retardation phase (0–2 days), log phase (3–6 days), stabilization phase (7–11 days) and decay phase (>12 days). In the range of 0–5 days,

the number of SRB increased drastically, and the corrosion enhanced. Notably, after 7 days, the number of strains reached the maximum, the number of active bacteria was high, both enzyme activation and metabolism were at the peak, and the corrosion also intensified. Although the number of bacteria remained at a high state in the period of 10–15 days, the weight loss was lower than that in the 5–10 days period, probably because the growth of high quantities of bacteria led to the formation of biofilms on the surface of the steel sheets, which slowed the rate of increase of corrosion. Furthermore, at 20–30 days, corrosion was significantly delayed due to the large consumption of nutrients at this stage, while the toxic metabolites accumulated in large quantities, leading to an increase in SRB mortality and a decrease in viable bacteria. At the same time, the chemical corrosion also slowed because inorganic substances were consumed during their growth. In that case, it

grew slowly in general, so it the main corrosion mechanism was chemical hydrogen precipitation corrosion due to acidic conditions, and the rate of X70 corrosion was higher. With increased pH, electrochemical corrosion weakened, and microbial corrosion increased. At pH 6–8, conditions were more suitable for microbial growth, and SRB and chemistry caused the corrosion. At pH 7, the corrosion caused by SRB was more severe, and the weight loss reached 39.31 g/m<sup>2</sup> , but the weight loss in the combined system of *P. stutzeri* + SRB was weakened to 12.33 g/m<sup>2</sup> ; meanwhile, in the blank medium system, the weight loss was 9.89 g/m<sup>2</sup> , which was mainly due to chemical oxygen absorption corrosion. Discounting the electrochemical corrosion, the addition of *P. stutzeri* resulted in an 83% reduction in weight loss. Therefore, *P. stutzeri* provided better protection against steel sheet corrosion at pH 7. At pH > 9, the corrosion was relatively weaker because both chemical and microbial corrosion were slowed down under alkaline conditions. Overall, the growth of microorganisms was somewhat restricted in alkaline environments, enzyme activity was reduced, and there was a relative decrease in both corrosion and corrosion resistance. Li et al. also showed that in an alkaline environment, a thick oxide film was generated on the surface layer of pipeline steel, which acted as a passivation layer and slowed the corrosion rate [35]. Therefore, the protection of *P. stutzeri* against SRB corrosion was better in the pH 6–8 environment. *Materials* **2023**, *16*, x FOR PEER REVIEW 13 of 19

**Figure 8.** Weight loss curves of X70 steel under different pH. **Figure 8.** Weight loss curves of X70 steel under different pH.

3.3.4. Analysis of Elements in Corrosion Products 3.3.4. Analysis of Elements in Corrosion Products

From the analysis of corrosion products (Figure 9), it can be seen that the corrosion products in the sterile environment were mainly with inorganic compounds, such as iron oxides, and the P elements were mainly derived from K2HPO<sup>4</sup> in the medium. The content of Fe and S in the corrosion products inoculated with SRB was significantly higher than that in the sterile environment and in the environment with *P. stutzeri*; presumably, the corrosion products mainly contained sulfides and iron oxides. In the environment with mixed bacteria, the corrosion products mainly contained C, O, Fe and S, but the content From the analysis of corrosion products (Figure 9), it can be seen that the corrosion products in the sterile environment were mainly with inorganic compounds, such as iron oxides, and the P elements were mainly derived from K2HPO<sup>4</sup> in the medium. The content of Fe and S in the corrosion products inoculated with SRB was significantly higher than that in the sterile environment and in the environment with *P. stutzeri*; presumably, the corrosion products mainly contained sulfides and iron oxides. In the environment with mixed bacteria, the corrosion products mainly contained C, O, Fe and S, but the content of S

of S was significantly lower than that of corrosion products inoculated with SRB. The oxidation of sulfatide induced by *P. stutzeri* hindered the accumulation of corrosive sulfide,

was significantly lower than that of corrosion products inoculated with SRB. The oxidation of sulfatide induced by *P. stutzeri* hindered the accumulation of corrosive sulfide, and the SRB-involved corrosion can be weakened thereby. *Materials* **2023**, *16*, x FOR PEER REVIEW 14 of 19

**Figure 9.** Element content of corrosion products on steel coupon surface.

**Figure 9.** Element content of corrosion products on steel coupon surface. 3.3.5. Tafel Curve

3.3.5. Tafel Curve The polarization curve test is damaging, and hence it is also known as a disposable test, where irreversible damage is caused to the sample surface during the measurement. Because the Tafel polarization can damage the sample surface, which can effectively exclude interference, the relationship between the polarization current and electrode poten-The polarization curve test is damaging, and hence it is also known as a disposable test, where irreversible damage is caused to the sample surface during the measurement. Because the Tafel polarization can damage the sample surface, which can effectively exclude interference, the relationship between the polarization current and electrode potential of the respective anodic and cathodic reactions can be observed separately, rendering the polarization curves capable of revealing the mechanism of electrochemical reactions and their kinetic characteristics in depth.

tial of the respective anodic and cathodic reactions can be observed separately, rendering the polarization curves capable of revealing the mechanism of electrochemical reactions and their kinetic characteristics in depth. The polarization curves of X70 steel samples in SRB and SRB + *P. stutzeri* solutions from 0 to 14 days of inoculation were measured with activated strains. In addition, the corrosion current density (icorr) was obtained after fitting using extrapolation software (Origin 2018), and the variation trend is shown in Figure 10.

The polarization curves of X70 steel samples in SRB and SRB + *P. stutzeri* solutions

from 0 to 14 days of inoculation were measured with activated strains. In addition, the corrosion current density (icorr) was obtained after fitting using extrapolation soft-

ware(Origin 2018), and the variation trend is shown in Figure 10.

**Figure 10.** (**a**) The potentiodynamic polarization curves of SRB; (**b**) the potentiodynamic polarization curves of SRB + *P. stutzeri*; (**c**) the corrosion current density. **Figure 10.** (**a**) The potentiodynamic polarization curves of SRB; (**b**) the potentiodynamic polarization curves of SRB + *P. stutzeri*; (**c**) the corrosion current density.

Figure 10a,b are the corrosion polarization curves for SRB and SRB + *P. stutzeri* samples, respectively, and Figure 10c shows the corrosion current density in both media. As can be seen from Figure 10, the corrosion current density in both media was the same at the beginning of the experiment, and with the extension of the incubation time, the corrosion current density of X70 steel in the media inoculated with SRB only reached the maximum at 7 days. At this stage, the growth rate of SRB was faster and the number of strains increased, while corrosion intensified. In the next 7 days, there was a decrease in corrosion current density compared to the seventh day owing to the formation of corrosion products on the surface, which had a protective effect on the specimens. As can be seen from the test results of 14 days of inoculation, after 2 days, the corrosion current density of X70 steel in the mixed medium of SRB + *P. stutzeri* was always lower than that of the medium inoculated with SRB alone, which indicated that the addition of *P. stutzeri* had a certain inhibitory effect on the corrosion caused by SRB. In brief, the corrosion current density generally showed a trend of first increasing and then decreasing, which means the corrosion was initially enhanced and then weakened. can be seen from Figure 10, the corrosion current density in both media was the same at the beginning of the experiment, and with the extension of the incubation time, the corrosion current density of X70 steel in the media inoculated with SRB only reached the maximum at 7 days. At this stage, the growth rate of SRB was faster and the number of strains increased, while corrosion intensified. In the next 7 days, there was a decrease in corrosion current density compared to the seventh day owing to the formation of corrosion products on the surface, which had a protective effect on the specimens. As can be seen from the test results of 14 days of inoculation, after 2 days, the corrosion current density of X70 steel in the mixed medium of SRB + *P. stutzeri* was always lower than that of the medium inoculated with SRB alone, which indicated that the addition of *P. stutzeri* had a certain inhibitory effect on the corrosion caused by SRB. In brief, the corrosion current density generally showed a trend of first increasing and then decreasing, which means the corrosion was initially enhanced and then weakened.

Figure 10a,b are the corrosion polarization curves for SRB and SRB + *P. stutzeri* samples, respectively, and Figure 10c shows the corrosion current density in both media. As

#### 3.3.6. Long-Term Exposure Weight Loss Test 3.3.6. Long-Term Exposure Weight Loss Test

*Materials* **2023**, *16*, x FOR PEER REVIEW 16 of 19

The weight losses of X70 steel sheets exposed to media for 3, 6 and 9 months are shown in Figure 11. The weight losses of X70 steel sheets exposed to media for 3, 6 and 9 months are shown in Figure 11.

**Figure 11.** Weight losses of steel coupons from long-term exposure (3, 6, 9 months). **Figure 11.** Weight losses of steel coupons from long-term exposure (3, 6, 9 months).

The weight loss of the steel sheets in all systems gradually increased with time, which indicates that the degree of corrosion varied in different environments. Still, as shown in Figure 11, the media inoculated with SRB alone showed more weight loss and more severe The weight loss of the steel sheets in all systems gradually increased with time, which indicates that the degree of corrosion varied in different environments. Still, as shown in Figure 11, the media inoculated with SRB alone showed more weight loss and more severe corrosion. The weight of specimens in the blank group lost slightly more than

corrosion. The weight of specimens in the blank group lost slightly more than those inoculated with SRB + *P. stutzeri* because the blank medium had a higher electrolyte concenthose inoculated with SRB + *P. stutzeri* because the blank medium had a higher electrolyte concentration than the solution inoculated with SRB + *P. stutzeri*, where bacteria grew and consumed part of the electrolytes. This is consistent with the results of the short-time corrosion weight loss experiments described above. In addition, the weight loss of steel sheets in the media inoculated with SRB + *P. stutzeri* was significantly lower than that in the system inoculated with SRB, which suggests that *P. stutzeri* had a better effect on preventing and controlling SRB-induced corrosion on steel sheets.

#### **4. Conclusions**

(1) A strain of *P. stutzeri* was obtained by isolation and purification. The strain is a Gram-negative bacterium with short rod-shaped cells, about 0.5–1 µm in length, and white transparent colonies, which could discolor Giltay medium and produce gas, showing that it has denitrification ability.

(2) The sequencing results of 16SrDNA also verified the properties of the *P. stutzeri* strain: its growth conditions were similar to those of SRB, with an optimum culture temperature of about 30 ◦C and an optimum growth pH of about 7. It entered the stabilization phase after 5 days and started to enter the decay phase slowly after 11 days. The growth cycle was the same as that of SRB, and the two bacteria can co-exist in the common growth environment.

(3) The results of the weight loss tests showed that *P. stutzeri* could inhibit the corrosion of X70 steel caused by SRB at 20–40 ◦C and pH 6–8. The electrochemical results showed that SRB promoted the corrosion of X70 steel, and the corrosion of X70 steel was inhibited in the *P. stutzeri* + SRB media. In addition, the corrosion current density of X70 steel in the media containing mixed bacteria was less than that of the environment inoculated with SRB alone, and the EDS results showed that the elemental S content was significantly lower than that of the corrosion products inoculated with SRB. The results of the weight loss tests also showed that *P. stutzeri* had a better inhibitory effect on the corrosion of X70 steel caused by SRB in the medium at 3, 6 and 9 months.

**Author Contributions:** Conceptualization, L.Q. and A.G.; methodology, L.Q. and D.Z.; software, Z.L. (Zhipeng Liu) and S.Z.; validation, Y.S., Z.L. (Ziyi Liu) and S.Z.; data curation, L.Q.; supervision, A.G.; project administration, A.G.; funding acquisition, A.G. and L.Q. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Chinese National Natural Science Foundation (No. 51701016).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This work was financially supported by the Chinese National Natural Science Foundation (No. 51701016).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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