**1. Introduction**

Metal corrosion is a spontaneous chemical, electrochemical or biological process that destroys metal and its physical and chemical properties [1]. It affects global security in areas as diverse as oil and gas transportation, offshore engineering equipment and water treatment [2–4]. Among all kinds of metal corrosion, microbially induced corrosion (MIC) accounts for about 20% of the total corrosion [5], and can cause economic losses of tens of billions of dollars every year [6]. Many large accidents have been directly or indirectly caused by MIC, such as the fire on the offshore drilling platform of Mexican oil giant PEMEX in 2015 [7], the Prudhoe Bay oil spill on the North Slope of Alaska [8] and a large explosion caused by a natural gas pipeline leak in Carlsbad, New Mexico [9]. Many types of microorganisms are involved in MIC, which can be divided into aerobic, anaerobic and facultative, according to different requirements for oxygen. These microorganisms generally form biofilms on the surface of metal materials simultaneously. Aerobic and facultative bacteria can consume O<sup>2</sup> in the system during their growth and provide local anaerobic growth conditions for anaerobic bacteria while growing [10]. Common corrosive microorganisms include bacteria, archaea and fungi, such as sulfate-reducing bacteria (SRB) [11], sulfate-reducing archaea (SRA) [12], nitrate-reducing bacteria (NRB) [13], methanogenic bacteria [14], acid-producing bacteria (APB) [15], iron-reducing bacteria (IRB) [16], ironoxidizing bacteria (IOB) [17], sulfur-oxidizing bacteria (SOB) [18], manganese-oxidizing bacteria (MOB) [19], a variety fungi [20] etc. Because sulfate is widely distributed in many systems, such as seawater, brackish water and agricultural runoff water [21], SRB have been extensively studied for decades as major pathogenic microorganisms in microbial

**Citation:** Qiu, L.; Zhao, D.; Zheng, S.; Gong, A.; Liu, Z.; Su, Y.; Liu, Z. Inhibition Effect of *Pseudomonas stutzeri* on the Corrosion of X70 Pipeline Steel Caused by Sulfate-Reducing Bacteria. *Materials* **2023**, *16*, 2896. https:// doi.org/10.3390/ma16072896

Academic Editor: Jose M. Bastidas

Received: 7 March 2023 Revised: 30 March 2023 Accepted: 31 March 2023 Published: 5 April 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

systems. Traditional anti-corrosion measures such as coating, surface treatment and corrosion inhibitors can control most corrosion problems, but inevitably bring environmental problems because of the accumulated release of toxic substances [6,22,23].

Microbiologically influenced corrosion inhibition (MICI) is caused by microorganisms' direct or indirect actions [6]. Compared with traditional anti-corrosion methods, MICI is characterized by low cost, environmental friendliness as well as moderate application and maintenance requirements. Therefore, it has ecological and economic significance and will become the development direction of the next generation of preservative technology. However, due to the diversity of microorganisms and the complexity of their metabolic processes influenced by environmental factors, MICI is still facing the challenge of practical application. Therefore, the research on MICI in China and abroad is still in the initial stage. In addition, some problems remain to be solved, such as single species and a lack of systematic understanding of the preservative mechanism.

*Pseudomonas stutzeri* is a Gram-negative facultative anaerobic bacterium with strong denitrification activity and high denitrification capacity. It is widely used in biological denitrification [24–27]. Liu et al. showed that the inhibition of corrosion of deposit-covered X80 pipeline steel was due to *P. stutzeri* in seawater containing CO<sup>2</sup> [28]. Fu et al. studied the effects of *P. stutzeri* on the biocorrosion of X80 pipeline steel for different nitrate and nitrite concentrations [29]. In this work, a strain of *P. stutzeri* with SRB corrosion resistance was isolated and purified from the soil by enrichment culture, and the anti-corrosion behavior of the bacterium against X70 steel in the presence of SRB was investigated. This provides a theoretical basis for the further development of new green, low-cost and long-term novel SRB corrosion inhibition technologies.

## **2. Materials and Methods**

## *2.1. Isolation and Identification of P. stutzeri*

The liquid culture medium used for *P. stutzeri* was as follows (g/L) [25,30]: K2HPO<sup>4</sup> (2.0), NH4Cl (0.5), Na2S2O3·5H2O (5.0), MgSO4·7H2O (0.6), FeSO4·7H2O (0.01), KNO<sup>3</sup> (2.0), NaHCO<sup>3</sup> (1.0), NaKC4H4O<sup>6</sup> (20.0) and pH 1.0~7.2. Before inoculation, the liquid medium was sterilized by autoclaving at 120 ◦C for 20 min. Then, 1.5–2% agar was added to the solid medium.

The soil sample was weighed to 10 g, inoculated into a conical flask containing 100 mL liquid medium, and shaken for 10 min so that the soil sample was well-mixed and incubated at a constant temperature of 30 ◦C. After the upper layer of liquid was turbid, 5 mL of the upper layer of clear liquid was inoculated into 50 mL of the liquid medium in the sterile table, mixed well and continued to incubate at a constant temperature. When the bacterial fluid became turbid again, 0.2, 0.5, 1.0, 1.5 and 2.0 mL of bacterial fluid were taken to the sterile table, quickly added to sterilized Petri dishes and poured into sterilized warm solid media (the temperature was below 60 ◦C). The media and the bacterial solutions were mixed well by gentle shaking. After cooling and solidifying, the plates were inverted into the constant-temperature incubator at 30 ◦C for observation. After 7 days of incubation, scattered single colonies appeared on the surface and inside of the plates, and single colonies were separately picked and inoculated onto solid plates to continue the culture of the purified strains.

The single purified colonies were inoculated into sterilized test tubes with 10 mL of liquid medium and incubated at 30 ◦C. Once the bacterial solutions were turbid, the strains were preserved and set aside.

The identification of the strains was determined by microscopic observation, Giltay medium denitrification ability identification and 16S rDNA sequencing.
