*2.2. Growth Characteristics Experiment*

The growth of bacteria has a greater impact on corrosion and anti-corrosion, and the corrosion effect of the strain is more evident at higher concentrations. For this experiment, the optical density at 600 nm (OD600) values of the experimental strains under different

conditions of time, temperature and pH were studied by using a spectrophotometer to determine the growth characteristics of the strains.

1. Effect of temperature on the growth of SRB and *P. stutzeri*

SRB and *P. stutzeri* strains were inoculated in a liquid medium and incubated anaerobically at 10 ◦C, 20 ◦C, 30 ◦C, 40 ◦C and 50 ◦C for 7 days. The OD<sup>600</sup> values at each temperature were measured to determine the optimal growth temperature of the strains.

2. Effect of pH on the growth of SRB and *P. stutzeri*

The SRB and *P. stutzeri* strains were inoculated equally in a liquid medium at pH 1.0, 3.0, 5.0, 7.0, 9.0, 11.0 and 13.0 for 7 days. The OD<sup>600</sup> values were measured at each pH to determine the optimal growth pH of the strains.

3. Graphical growth curves of *P. stutzeri*, SRB and co-culture of *P. stutzeri* + SRB

The media of *P. stutzeri* and SRB were prepared separately, as well as the mixed medium mixed at a ratio of 1:1. *P. stutzeri*, SRB, as well as the mixture of SRB and *P. stutzeri* were inoculated in a 1/100 ratio separately and incubated anaerobically in blue-capped culture flasks. Then the OD<sup>600</sup> values were measured by sampling every 24 h.

All experiments were implemented in three replicates and results were based on the mean after three replicates with a standard deviation range of 0.5–5.0%.

#### *2.3. Exposure Experiments*

#### 2.3.1. EDS and Weight Loss Measurement

Before soaking the specimens, holes were punched at the top middle, and each side was sanded to mirror-smooth using SiC sandpaper from 400 to 800, 1000 and 2000 mesh. Then, the specimens were washed with deionized water and anhydrous ethanol, blown dry and weighed and irradiated with UV light for at least 30 min. The steel sheets were suspended vertically inside the frosted jars with waterproof wires. The surface of the liquid was covered with liquid paraffin. After nitrogen was flushed into the bottle, the gap between the stopper and the bottle body was sealed with the sealing film to isolate the air. The experiments were divided into blank, SRB and SRB + *P. stutzeri*, with different control of incubation time, temperature, pH etc. All experiments were done three times in parallel. When the immersion time was reached, the steel sheets were removed and the surface was descaled according to the Chinese Standard "Corrosion of metals and alloys-Removal of corrosion products from corrosion test specimens" GB/T16545-2015.

The rust remover comprised 500 mL hydrochloric acid (ρ = 1.19 g/mL), 500 mL deionized water and 3.5 g of hexamethylene tetramine, well-mixed. After rust removal, samples were rinsed with deionized water and anhydrous ethanol, then dried and weighed. The inhibition effect of *P. stutzeri* bacteria on the corrosion caused by SRB under each condition was analyzed by the weight loss of steel sheets. The additional specimens were removed after soaking for 7, 14 and 21 days, before scanning X-ray energy-dispersive spectrometer (EDS, from Oxford Instruments, Abingdon, UK). For analysis of corrosion products, the specimens were pretreated by soaking in a phosphate-buffered saline solution containing 2.5% (*v/v*) glutaraldehyde for 8 h to immobilize cells. The specimens were then dehydrated using a serial dilution of ethanol (30%, 50%, 70%, 90% *v/v*), each for 15 min, except the final step for 30 min [31].

#### 2.3.2. Electrochemical Measurements

The steel sheets were polished to smooth grade by grade, soldered with copper conductor, then put into the mold and sealed with epoxy resin, with ethylenediamine as the curing agent. Then, the samples were reserved with a working surface of 10 mm × 10 mm, polished with sandpaper in steps to 2000 mesh before use, and then polished with w 2.5 and w 0.5 diamond grinding paste in turn, rinsed with deionized water, cleaned with anhydrous ethanol, sterilized under UV for 30 min and set aside. A three-electrode system was used for the electrochemical tests, with X70 steel specimen as the working electrode,

platinum sheet as the auxiliary electrode, and Saturated Calomel Electrode (SCE) as the reference electrode, using a Chi760e electrochemical workstation. Specimens were divided into two experimental groups using the co-culture medium—one group was inoculated with SRB only and the other group was inoculated with SRB + *P. stutzeri*. For every 100 mL of medium, 10 mL of bacterial solution was inoculated. The test scan potential range was ±0.5 V vs. OCP, and the potential scan rate was 2 mV·s −1 . The polarization curve (Tafel curve) of the steel sheet was measured. It is worth noting that potential scan rate has an essential role in minimizing the effects of distortion in Tafel slopes and corrosion current density analyses, as previously reported [32–34]. However, based on these reports, the adopted 2 mV/s has no deleterious effects on those Tafel extrapolations [32,33] to determine the corrosion current densities (icorr) of the examined samples.
