Abstract

   Previous studies have demonstrated that some methanogens can directly
   accept electrons from Fe(0), leading to metal corrosion under strict
   anaerobic conditions. However, there are few reports on the research of
   anaerobic iron corrosion by some substances that coexist with
   methanogens, such as syntrophic bacteria and activated carbon, which is
   widely distributed in environments. Therefore, in this study, a
   corrosion system consisting of Methanosarcina barkeri, stainless steel,
   and granular activated carbon (GAC), as well as a corrosion system with
   Geobacter metallireducens, was constructed. The aim was to explore the
   mechanism of stainless steel corrosion under the metabolic action of M.
   barkeri. It was found that the GAC and G. metallireducens can
   accelerate the extracellular electron transfer between M. barkeri and
   stainless steel, thereby accelerating corrosion, and this
   intensification mechanism may be related to the mtmC, mtbC, and fwdC
   genes. By understanding these mechanisms, not only can a theoretical
   basis be provided for the protection against metal corrosion, but it
   can also promote environmental protection and safe production.

   Keywords: stainless steel, corrosion, extracellular electron transfer,
   granular activated carbon, Methanosarcina, Geobacter

1. Introduction

   Wear and corrosion resistance are fundamental requirements for
   materials subjected to aggressive environments. Stainless steels are
   notable due to their combination of excellent mechanical properties and
   superior corrosion resistance [[36]1]. They are widely employed in
   construction, food industry, chemical industry, medical equipment,
   aerospace, energy, and other fields. The damaging effect of corrosion
   is supported by estimates of the damage caused, which account for
   approximately 10–15% of the global direct metal production losses.
   However, the actual damages are far greater because to the direct
   losses must be added the indirect losses determined by loss of
   production, loss of capacity, and contamination of products [[37]2]. In
   recent years, with the increasing complexity of the industrial
   environment, the corrosion resistance of traditional materials is
   facing greater challenges. Furthermore, the anaerobic corrosion of iron
   structures is expensive to repair.

   It has been known for over 100 years that the presence of anaerobic
   respiratory microorganisms can accelerate iron corrosion [[38]3].
   Microorganisms can corrode either through metabolites or by forming
   biofilms on metal surfaces. The microbial metabolic activities in the
   biofilms will change the pH value of the local environment and thus
   affect the corrosion efficiency [[39]4]. Thus, it is indisputable that
   the microbial corrosion or bio-corrosion plays a predominant role, with
   considerable economic losses. Despite many microbial species being
   described as corrosive, certain groups, such as sulfate-reducing
   bacteria [[40]5] and nitrate-reducing bacteria, acetogenic bacteria,
   and methanogenic archaea, are the most commonly implicated [[41]6].
   Tang [[42]3] has reported that Geobacter sulfurreducens strain ACL can
   extract electrons from Fe(0) by hydrogenase and C-type cytochrome on
   the outer surface. Previous studies have inferred that methanogenic
   microorganisms accelerate the corrosion of iron-containing metals.
   Holmes [[43]7] report that Methanosarcina acetivorans, which also
   belong to the genus Methanosarcina, obtain electrons from metals to
   produce methane, which enhances the possibility of M. barkeri corroding
   stainless steel.

   Methanosarcina barkeri is also a methanogenic archaea widely
   distributed in the environment, a very versatile methanogen using
   hydrogenotrophic, methylotrophic, and acetoclastic methanogenesis
   [[44]8]. In recent years, M. barkeri has gradually become one of the
   hot spots of research due to its potential in biodegradation and energy
   recovery. However, M. barkeri not only plays an important role in
   anaerobic digestion wastewater treatment [[45]9] and biogas upgrading
   [[46]10] but also takes Fe(0) as a potential electron donor in some
   extreme environments [[47]11], causing metal corrosion, especially in
   anaerobic environments. In these environments, microorganisms may
   affect the stability of metal surface films by producing acids,
   producing gases (such as methane, hydrogen), and excreting other
   metabolic byproducts. It has been reported, for example, that
   planktonic cells of M. fulgenii can form biofilms on iron films and
   improve the biological corrosion activity during anaerobic iron
   corrosion, thus accelerating the corrosion process of metals [[48]12].
   In addition, as an anaerobic microorganism, the growth and metabolic
   activities of M. barkeri often reduce the oxygen concentration in the
   local environment, so that the microorganisms directly absorb electrons
   from metal iron through extracellular electron transfer to increase the
   electrochemical reaction rate of corrosion [[49]13]. In recent years,
   it has been found that in the environment where M. barkeri exists, the
   corrosion rate of stainless steel and other metal materials increases
   significantly [[50]14].

   With the aggravation of environmental pollution, activated carbon has
   been applied to pollutant treatment in an increasing number of
   industries, including the treatment of dairy wastewater [[51]15], the
   removal of pesticides in the atmosphere [[52]16], and the removal of
   pollutants in surface water [[53]17]. This has led to the widespread
   presence of carbon particles in the ecological environment. Granular
   activated carbon (GAC) is a highly porous carbon material with a wide
   surface area and excellent adsorption properties. These properties make
   it an effective material for a variety of applications, including water
   treatment [[54]18] and electrochemical reactions [[55]19]. In anaerobic
   digestion of wastewater, GAC is often used to promote electron transfer
   and thus increase the methane production rate due to its excellent
   electrical conductivity [[56]20]. This indicates that the conductive
   properties of GAC may promote electron transfer between microbial cells
   and metal surfaces, affecting the corrosion of materials such as
   stainless steel caused by microorganisms. A study has shown that
   Geobacter and Methanosarcina often coexist in a variety of anaerobic
   environments, including sediments, hot springs, animal gastrointestinal
   tracts, and rice fields [[57]21,[58]22,[59]23]. Geobacter
   metallireducens can cooperate with Methanosarcina barkeri to enhance
   the metabolic activities of the two microorganisms and promote the
   production of metabolites [[60]24]. Thus, it is hypothesized that the
   interaction between G. metallireducens and M. barkeri can also
   accelerate the corrosion of stainless steel.

   These phenomena have attracted extensive attention from researchers.
   Previous studies have demonstrated that certain microorganisms capable
   of extracellular electron transfer (G. metallireducens and M.
   acetivorans) can corrode stainless steel [[61]7,[62]25]. Meanwhile, GAC
   and syntrophy (G. metallireducens) in co-culture systems have been
   shown to accelerate extracellular electron transfer [[63]24,[64]26].
   However, due to the slow corrosion progression and long experimental
   duration, few studies on M. barkeri mediated corrosion have been
   reported, and the effects of GAC and syntrophy on corrosion remain
   unclear. Therefore, it is important to study some substances that
   coexist with methanogens, such as syntrophic bacteria and GAC. On the
   premise of having proven that GAC and G. metallireducens can promote
   electron transfer, this experiment hypothesizes that GAC and syntrophic
   bacteria (G. metallireducens) can accelerate the corrosion of stainless
   steel by M. barkeri. Based on previous studies on the corrosion of
   stainless steel by M. acetivorans and the corrosion of Fe⁰ by G.
   sulfurreducens [[65]7,[66]27], the experimental scheme of this study
   was designed.

2. Materials and Methods

2.1. Bacterial Strains and Growth Conditions

   Methanosarcina barkeri and Geobacter metallireducens, purchased from
   DSMZ (Braunschweig, German), were routinely cultured in serum vials
   under strict anaerobic conditions using the N[2]:CO[2] (80:20) mixed
   gas adopted in DSMZ’s official culture medium [[67]24]. The vials were
   sealed with butyl rubber stoppers and aluminum rings. M. barkeri was
   cultured in DSM120 medium at a constant temperature of 37 °C [[68]24].
   The formulation of DSM120 medium is as follows: 0.5 mM Na[2]S, 1 mM
   cysteine, 0.002 g/L CaCl[2]·2H[2]O, 1 g/L NaCl, and 2 g/L NaHCO[3]. G.
   metallireducens was cultured in ferric citrate (FC) medium at a
   constant temperature of 30 °C. The formulation of the FC medium
   [[69]28] is as follows: NaHCO[3], 2.5 g/L; CaCl[2]·2H[2]O, 0.1g/L; KCl,
   0.1 g/L; NH[4]Cl, 1.5 g/L; NaH[2]PO[4]·H[2]O, 0.6 g/L; as well as
   vitamin and trace element mixtures. Unless otherwise specified, acetate
   (6.8 g/L) was used as the carbon source. The gas phase was N[2]-CO[2]
   (80:20). The co-culture of M. barkeri and G. metallireducens was
   carried out under anaerobic conditions at a constant temperature of 30
   °C, using DSM120 medium, but instead of sodium acetate as the electron
   donor, ethanol was used [[70]29].

2.2. Construction of the Culture System of Strains, Stainless Steel and GAC

   The model of the stainless steel is 316 L, with the specifications of
   10 mm × 10 mm × 2 mm, and the addition amount is 10 pieces per bottle.
   According to Liu’s experiment, when the granular activated carbon (GAC)
   with a particle size of 8–20 mesh is added at an amount of 25 g/L, the
   electron transfer efficiency between G. metallireducens/G.
   sulfurreducens co-cultures is optimal at 25 g/L [[71]26]. Therefore, 25
   g/L of GAC was selected for addition in this experiment. The stainless
   steel (SS) and GAC were immersed in acetone and then placed in an
   ultrasonic cleaner and soaked at room temperature for 30 min. The
   acetone was replaced with ethanol and then placed in the ultrasonic
   cleaner for 20 min. This step was repeated 2–3 times. After that, they
   were washed with clean water, air-dried, and placed in a desiccator.
   The surface of the SS was polished with three different grades of
   sandpaper (220, 600, and 1200 mesh), and each side was continuously
   polished for 60 s. Then it was soaked in 70% ethanol for 30 min and
   placed in a petri dish. The ethanol was poured out, and the petri dish
   was put into an ultraviolet cabinet for 30–45 min. During this process,
   the petri dish was turned over 2–3 times for sterilization [[72]25].

   During the construction of the culture system of Methanosarcina,
   Geobacter, GAC, and stainless steel, the stainless steel and GAC should
   first be placed in an aerated and anaerobic empty vial. After sealing,
   the vial is sterilized. Once it has cooled down after sterilization,
   the sterilized medium is injected into the vial using a syringe. In
   order to ensure that the environment in the anaerobic vial is under
   strict anaerobic conditions, the oxygen scavenger should be allowed to
   function fully as much as possible. The time of inoculation should be
   at least 2 h or more after the injection time of the oxygen scavenger.
   Three parallels are set for each group. Methanosarcina and Geobacter
   should be inoculated during the logarithmic phase of the cultivation
   process, and the inoculation amount is 20% of the content of the
   medium. All data obtained from the experiments were derived from three
   parallel groups and subjected to significance analysis using a t-test.

2.3. Analytical and Detection Methods

2.3.1. Detection Method of Ions in Solution

   The determination of Fe^2+ in the solution adopts the ferrozine
   spectrophotometric method. The directions for this method are as
   follows: Take 1 mL of the sample to be tested, add 4 mL of 0.5 N HCl,
   and dissolve it for 15 min. Then, take 1 mL of the acid dissolution
   solution and add 4 mL of the ferrozine reagent. After color development
   for 15 min, measure the absorbance at 562 nm.

2.3.2. Detection Method of Ions in Granular Activated Carbon

   After the experiment is completed, take out all the granular activated
   carbon in the anaerobic vial, soak it in 50 mL (close to the volume of
   the experimental system) of 1 M hydrochloric acid solution for 2–3 h,
   and keep stirring or slightly shaking to ensure that the acid solution
   can fully dissolve the iron ions in the activated carbon, obtaining the
   iron–ion eluent of GAC. The ferrozine spectrophotometric method is used
   for measurement.

2.3.3. Determination of the Concentration of Substances in Solution

   With a syringe, draw 1 mL of the sample from the anaerobic vial and
   filter it (using a 0.22 μm filter), then determine the concentration of
   the substrate sodium acetate or ethanol. The acetic acid in the
   solution is determined by a liquid chromatograph (SCION LC6000,
   Beijing, China) [[73]30], with a mobile phase of 5 mM H[2]SO[4]. The
   ethanol is determined by a gas chromatograph (Agilent, GC7890, Beijing,
   China).

2.3.4. Testing Method for Gas Components and Contents

   Use a 1 mL syringe to sample and determine the gas products generated
   by the stainless-steel corrosion system. The content of the gas product
   CH[4] is determined by a gas chromatograph (Tian Mei, GC7900,
   Zhongshan, China). The testing method follows the operation
   instructions of the GC7900.

2.4. Surface Analysis Method

   A scanning electron microscope (SEM) is a commonly used surface
   analysis method for metals. This instrument can conduct microscopic
   analysis of the surface morphology of materials, thereby obtaining
   information such as physical morphology and the morphology of microbial
   films. Before sample detection, pretreatment is required. The sample is
   gently rinsed with 0.1 M phosphate-buffered saline (pH = 7.2). After
   cleaning, the sample is placed in a 2.5% glutaraldehyde solution and
   fixed at 4 °C for 12 h. Gradient dehydration is performed with 50%,
   70%, 80%, and 90% ethanol, successively [[74]25]. Each time, it is
   allowed to stand for 10–15 min and then centrifuged at 3000 rpm to
   remove the upper layer liquid. It is dehydrated with absolute ethanol
   three times. After dehydration, the sample is placed in a freeze dryer
   and dried for 12 h and then taken out when it is ready for monitoring.
   The pretreated sample is pasted onto a copper platform with conductive
   adhesive, and a layer of carbon is plated on the sample surface. It is
   then observed under a scanning electron microscope. SEM–EDS point
   scanning is used to measure the content of each element at the required
   points.

2.5. Detection Method for Electrochemical Indicators

   An electrochemical device with a single-chamber three-electrode system
   was constructed. The working electrode was a stainless steel electrode,
   the counter electrode was a graphite plate, and the reference electrode
   was a KCl electrode. The solution in the device was the 120 media,
   which is the culture medium for M. barkeri. At room temperature, a
   scanning test was carried out in the range of −1.0 V to 0.2 V at a
   scanning rate of 10 mV/s. After obtaining the data, a cyclic
   voltammogram was plotted with the potential as the abscissa and the
   current as the ordinate.

2.6. Transcriptomics

   Cells were harvested from the 50 mL anaerobic M. barkeri corrosion
   systems containing GAC and G. metallireducens, which had been
   cultivated for 60 days. Stainless steel was used as the electron donor,
   and the cells were grown as described above. The cells were centrifuged
   at 8000 rpm for 20 min at 4 °C, immediately frozen in liquid nitrogen
   thereafter, and stored at −80 °C [[75]31]. RNA was extracted from the
   samples using the e.zn.a.^® Soil RNAMidi Kit (Omega Bio-Tek, Norcross,
   GA, USA). The concentration and purity of RNA were detected by
   NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA), and the
   integrity of RNA was examined by 1% agarose gel electrophoresis. The
   RIN (RNA integrity number) value was determined using the RNA 6000 Nano
   Kit on an Agilent 2100 (Beijing, China). Subsequent steps, including
   library construction, high-throughput sequencing, and data
   preprocessing, were completed by Majorbio Bio-Pharm Technology Co.,
   Ltd. (Shanghai, China). The gene expression levels were presented as
   TPM (transcripts per million). Differential expression analysis was
   performed using the DESeq2 software, which employs the negative
   binomial distribution model and generalized linear model (GLM) as its
   core statistical methods, to identify genes with significantly
   differential expression across sample groups. Subsequently, Gene
   Ontology (GO) functional enrichment analysis and KEGG (Kyoto
   Encyclopedia of Genes and Genomes, Kyoto, Japan) pathway enrichment
   analysis were carried out on these genes to explore their potential
   biological functions and the signaling pathways they participated in,
   thus comprehensively elucidating the characteristics of transcriptome
   changes and their biological significance.

3. Results and Discussion

3.1. GAC Can Accelerate the Corrosion of Stainless Steel by Methanosarcina
barkeri

   It is known that M. barkeri can produce methane by obtaining electrons
   through direct electron transfer (DET) [[76]32]. As a carbon-based
   material with a large surface area and high electrical conductivity,
   GAC is often added to anaerobic biogas digesters to promote methane
   production [[77]33]. In this experiment, a control group consisting of
   GAC and stainless steel without cells was set up, with 1 mM NaAc as the
   electron donor to facilitate electron acquisition by M. barkeri from
   stainless steel. A system consisting of M. barkeri, GAC, and stainless
   steel was constructed to explore the enhancement effect of GAC on the
   corrosion of stainless steel by M. barkeri. Methane content, the most
   direct indicator of M. barkeri growth, was first measured, with the
   results shown in [78]Figure 1A. M. barkeri obtained electrons from
   stainless steel to produce methane, and the methane content reached 1.6
   ± 1.08 mM. It is worth noting that in the presence of GAC, the methane
   production of M. barkeri reached 8.22 ± 0.57 mM. When 1 mM NaAc was
   provided as the sole substrate, theoretically 1 mM methane could be
   produced (Equation (1)) [[79]7]. However, the experimental results
   showed that the methane content in the two experimental groups reached
   8 mM or 10 mM, indicating that stainless steel (SS) was utilized by M.
   barkeri as an additional electron donor for CO[2] methanation.
   Additionally, SS does not produce hydrogen non-biologically with water;
   therefore, M. barkeri likely acquires electrons from SS via direct
   electron transfer (DET). This confirmed the possibility that M. barkeri
   in this growth state could attach to stainless steel and carry out
   electron transfer, and GAC likely accelerated electron transfer between
   the microbe and metal, increasing methane production.
   CH[3]COONa + H[2]O → CH[4] + CO[2] + NaOH (1)

Figure 1.

   [80]Figure 1
   [81]Open in a new tab

   Contents of CH[4] (A) and Fe(II) (B) changing with time in different
   experimental groups and the cyclic voltammogram (C) during the
   corrosion of stainless steel by M. barkeri.

   Meanwhile, in order to better and more intuitively evaluate the
   corrosion effect, we measured the Fe(II) in the culture medium, and the
   results are shown in [82]Figure 1B. It can be seen that in the presence
   of M. barkeri, the concentration of Fe(II) increased, which shows that
   M. barkeri can obtain electrons from stainless steel to maintain its
   own metabolism. This key conclusion is highly consistent with the
   research finding that Methanosarcina vacuolata, which also belongs to
   type I methanogens, can obtain electrons by using stainless steel
   [[83]34], further consolidating the theoretical basis for the electron
   transfer between microorganisms and metallic materials. It is
   particularly noteworthy that after introducing GAC into the anaerobic
   corrosion system, its methane production reached 5.14 times that of the
   control group. This data intuitively show the significant accelerating
   effect of GAC on the process of M. barkeri obtaining electrons from
   stainless steel, thereby strengthening the corrosion effect of
   microorganisms on stainless steel. This result coincides with the
   theoretical speculation put forward by Wang that GAC may enhance the
   generation pathway of electron shuttles and promote the intracellular
   and extracellular electron transfer processes [[84]35].

   However, the Fe(II) concentration in the GAC-supplemented experimental
   group was lower than that in the group without GAC (p < 0.05). This is
   hypothesized to result from the adsorption capacity of GAC. According
   to the experimental data in [85]Figure 2A, the total iron contents in
   the “M. barkeri + SS” experimental group and the “M. barkeri + SS +
   GAC” experimental group on the 52nd day were measured to be 42.49 ±
   1.24 mg/L and 37.05 ± 1.83 mg/L, respectively. In addition, the total
   iron content in the GAC eluent was 31.60 ± 1.02 mg/L. This proves the
   speculation that a portion of the irons was adsorbed due to its
   inherent adsorption properties. Concurrently, the cumulative total iron
   content in the GAC group (68.65 mg/L) was significantly higher than
   that in the GAC-free control group (42.49 ± 1.24 mg/L) (p < 0.05). This
   result, from another aspect, proves that the addition of GAC can
   effectively accelerate the corrosion of stainless steel by M. barkeri.

Figure 2.

   [86]Figure 2
   [87]Open in a new tab

   Iron content, (A) total iron of each liquid. Among them, “MB + SS” and
   “MB + SS + GAC” represent the total iron content of the bacterial
   liquid in the anaerobic vial. The GAC eluent is the liquid obtained by
   eluting the GAC in “MB + SS + GAC”; (B) iron element proportion near
   the bacterial cells obtained by SEM–EDS in each experimental group, and
   the scanning object is indicated in the brackets.

   To further prove the speculation, cyclic voltammetry (CV) scans were
   performed on the anaerobic corrosion system at the end of the
   experimental period, with results depicted in [88]Figure 1C. It can be
   seen that the area enclosed by the scanning curve of the “M. barkeri +
   SS + GAC” group is larger than that of the “M. barkeri + SS” group,
   indicating that a larger number of M. barkeri adhered to the surface of
   the electrode, and its capacitance is greater than that of the
   experimental group without the addition of GAC. It is worth noting that
   there is a relatively obvious oxidation peak appearing at around −0.38
   V in the “M. barkeri + SS + GAC” group, while no obvious oxidation
   peaks or reduction peaks are in the other one, indicating that the
   stainless steel underwent an oxidation reaction under the action of M.
   barkeri, losing electrons and changing into Fe(II) or Fe(III) in the
   solution. Meanwhile, a reduction peak appeared at around −0.28 V,
   indicating that a reaction occurred on the electrode in which M.
   barkeri obtained electrons and reduced CO[2] to CH[4].

   It can be observed from the scanning electron microscope images
   ([89]Figure 3) that M. barkeri adhered to the surfaces of the stainless
   steel in both groups, and the cells were in direct contact with the
   surfaces, without observing the accumulation between cells. Therefore,
   it can be reasonably assumed that there is a direct electron transfer
   between the metal and the microorganisms [[90]7]. The surfaces of the
   stainless steel in the images show corrosion signs to varying degrees
   (surface roughness, local pits, or corrosion spots). For example, it
   can be clearly seen that the stainless steel (B) with the added GAC
   system has more severe corrosion cracks and corrosion pits than that
   (A) without the GAC system. The content of iron element near the
   bacterial cells is shown in [91]Figure 2B. The atomic content diagrams
   and comparison tables of other experimental groups are shown in
   [92]Figure S1 and Table S1. When GAC was present, the weight percent of
   Cr and Fe were both lower than those in the other group (p < 0.05),
   indicating that the metal oxide film on the surface of the stainless
   steel in the “M. barkeri + SS + GAC” group is more severely damaged,
   with more iron element loss and more serious corrosion. Meanwhile, the
   M. barkeri cells attached to the GAC and the iron weight percent of
   0.15% once again prove that GAC accelerates the corrosion of stainless
   steel by M. barkeri through electron transfer.

Figure 3.

   [93]Figure 3
   [94]Open in a new tab

   Scanning electron microscope images of different experimental groups.
   ((A): the stainless steel in the “M. barkeri + SS” group, and the arrow
   points to M. barkeri; (B): the stainless steel in the “M. barkeri + SS
   + GAC” group, and the arrow points to M. barkeri; (C): the GAC in the
   “M. barkeri + SS + GAC” group, and the arrow points to M. barkeri; (D):
   the stainless steel in the “G. metallireducens + M. barkeri + SS”
   experimental group, and the arrow points to M. barkeri and G.
   metallireducens).

3.2. Mechanism Analysis of GAC Accelerating the Corrosion of Stainless Steel
by Methanosarcina barkeri

   In order to further understand the mechanism by which GAC accelerates
   the electron transfer between M. barkeri and stainless steel, the
   strains in the GAC-supplemented and GAC-free experimental groups were
   subjected to transcriptome analysis to identify differentially
   expressed genes. Some genes with a difference in gene expression levels
   of more than 1.8 times between the two experimental groups were
   selected, and the data shown in [95]Table 1 were obtained. The other
   highly expressed genes and the specific values of each experimental
   group are shown in [96]Table S2. As shown in [97]Table 1, the gene
   MSBRM_2245, which is related to the editing of annexin, exhibits a
   relatively large expression difference. This indicates that electrons
   are directly transferred through the cell membrane of M. barkeri, which
   belongs to the category of direct electron transfer. In addition, the
   expression difference of mtmC, which encodes methyltransferase-like
   proteins, is also relatively high. The difference in the expression
   levels of this gene between the “M. barkeri + SS + GAC” group and the
   “M. barkeri + SS” group is 7.276 times (p = 0.034). Stephen A. Burke
   described how mtmC is an important gene for catalyzing methylation,
   which establishes a family of cobalamin-binding proteins involved in
   methylotrophic methanogenesis [[98]36]. At the same time, there are two
   other genes related to methanogenesis, cofD and mfnE, with relatively
   large fold changes (FC) in expression, which are 2.122 and 1.819,
   respectively. This explains why the methane production in the group
   with GAC addition was higher than that in the other groups. In
   addition, genes such as fabG encoding 3-oxoacyl-[acyl-carrier protein]
   reductase, aor encoding ferredoxin oxidoreductase, and mtsB encoding
   coenzyme M methyltransferase corrinoid protein have been upregulated to
   varying degrees. Illumina sequence reads have been submitted to the SRA
   NCBI database under BioProjects PRJNA1241488. [99]Figure 4 classifies
   the differentially expressed genes into five categories: cellular
   processes, human diseases, genetic information processing,
   environmental information processing, and metabolism. It can be seen
   that the largest number of differentially expressed genes are related
   to the energy metabolism (including methane metabolism) pathway,
   followed by carbohydrate metabolism. This indicates that GAC can indeed
   enhance the activity of M. barkeri and at the same time increase the
   expression levels of some genes to accelerate the electron transfer
   between M. barkeri and stainless steel. This is most likely to
   accelerate this process by increasing the expression of genes related
   to the methane metabolism pathway, and the gene most closely associated
   with this is mtmC. The KEGG enrichment rates of each gene are shown in
   [100]Figure S2.

Table 1.

   Selected genes with relatively high differential upregulation of M.
   barkeri genes in the “M. barkeri + SS + GAC” experimental group and the
   “M. barkeri + SS” experimental group.
   Gene ID KO Name Gene Description Pathway Definition FC ^a (MB-GAC/MB)
   Log[2]FC ^b (MB-GAC/MB)
   MSBRM_3095 ------ DUF378 domain-containing protein ------ 23.949 4.582
   MSBRM_3339 ------ hypothetical protein ------ 24.252 4.600
   MSBRM_2922 ------ STARP antigen ------ 2.462 1.300
   MSBRM_2359 ------ hypothetical protein ------ 12.126 3.600
   MSBRM_1961 ------ hypothetical protein ------ 14.551 3.863
   MSBRM_2245 ------ putative membrane-associated protein ------ 13.338
   3.738
   MSBRM_1851 mtmC Monomethylamine methyltransferase corrinoid protein
   Methane metabolism 7.276 2.863
   MSBRM_2105 fabG 3-oxoacyl-[acyl-carrier protein] reductase Biotin
   metabolism
   Fatty acid biosynthesis 3.638 1.863
   MSBRM_0533 cofD 2-phospho-L-lactate transferase Methane metabolism
   2.122 1.085
   MSBRM_1439 aor Tungsten containing aldehyde:ferredoxin oxidoreductase
   Pentose phosphate pathway 1.819 0.863
   MSBRM_1541 mtsB Methylthiol:coenzyme M methyltransferase corrinoid
   protein Sulfur metabolism 1.819 0.863
   MSBRM_1197 mfnE delta 1-pyrroline-5-carboxylate synthetase Methane
   metabolism 1.819 0.863
   MSBRM_1079 cbiQ Transmembrane component CbiQ of energizing module of
   cobalt ECF transporter ABC transporters 1.819 0.863
   [101]Open in a new tab

   ^a FC (stress/control): The fold change of this gene between the two
   samples; ^b Log[2]FC (stress/control): the base-2 logarithm of the fold
   change of this gene between the two samples; the control represents the
   control group.

Figure 4.

   [102]Figure 4
   [103]Open in a new tab

   KEGG pathway classification statistics chart of differentially
   expressed genes between the “M. barkeri + SS + GAC” group and the “M.
   barkeri + SS” group.

3.3. Syntrophy Can Accelerate the Corrosion of Stainless Steel by
Methanosarcina barkeri

   M. barkeri can syntrophically coexist with G. metallireducens,
   accelerating the electron transfer rate and generating methane
   [[104]24]. In this experiment, stainless steel with no cells was used
   as the control, and in order to minimize the influence of oxygen anions
   in nutrients on inhibiting local corrosion [[105]37] and make the
   experimental groups comparable, stainless steel and 1 mM ethanol or 1
   mM sodium acetate were selected as electron donors to construct a
   co-culture system of M. barkeri and stainless steel and a co-culture
   system of M. barkeri, G. metallireducens, and stainless steel to
   explore the enhancement effect of syntrophic metabolism on the
   corrosion of stainless steel by M. barkeri. The changes in the contents
   of CH[4] and Fe(II) during the experiment are shown in [106]Figure 5.
   The co-culture system of M. barkeri, G. metallireducens, and stainless
   steel entered the logarithmic growth phase relatively quickly and
   reached a stable state around the 30th day. During this period, the
   maximum value of methane production reached 10.67 ± 0.09 mM
   ([107]Figure 5A), which was higher than the 1.5 mM methane
   theoretically produced by 1 mM ethanol and 5.13-fold that of the group
   with M. barkeri and stainless steel (p < 0.05). Since the main
   component of the FC medium used for culturing G. metallireducens is
   ferric citrate, a large amount of Fe(II) will be carried during
   inoculation. In order to eliminate this influence, when measuring the
   Fe(II) concentration, the part in the inoculum of G. metallireducens
   was deducted, and the curve shown in [108]Figure 5B was obtained. It
   can also be seen from [109]Figure 5 that the content of Fe(II) in the
   experimental group of “M. barkeri + G. metallireducens + SS” was
   significantly higher than that in the experimental group of “M. barkeri
   + SS” (p < 0.05), indicating that it obtained more electrons from
   stainless steel, which proves that syntrophic symbiotic metabolism can
   accelerate the corrosion of stainless steel by M. barkeri. This is
   consistent with Nardy’s description that metal corrosion is affected by
   complex processes such as different electrochemical reactions carried
   out by different microorganisms [[110]38].

Figure 5.

   [111]Figure 5
   [112]Open in a new tab

   Production of CH[4] (A) and Fe(II) (B) changing with time in different
   experimental groups and the cyclic voltammogram (C) during the
   corrosion of stainless steel by M. barkeri.

   As shown in [113]Figure 5C, the area enclosed by the cyclic
   voltammogram obtained from the “M. barkeri + SS” experimental group is
   relatively small, indicating that the capacitance of this system is in
   a relatively small state. In contrast, the area enclosed by the curve
   obtained from the “M. barkeri + G. metallireducens + SS” experimental
   group is significantly larger than that of the previous experimental
   group (p < 0.05), suggesting that more M. barkeri adhered to the
   surface of the electrode, and the capacitance of the system increased
   accordingly. In addition, there are no obvious oxidation peaks and
   reduction peaks in the “M. barkeri + SS” experimental group. However,
   in the “M. barkeri + G. metallireducens + SS” system, a reduction peak
   appears at around −0.66 V, which is similar to the reduction peak in
   the methane production of the microbial electrolysis cell experimented
   on by Yufang Wei [[114]39]. This indicates that at this potential, M.
   barkeri obtained electrons from the stainless steel on the electrode
   and carried out the reaction of reducing CO[2] to generate CH[4].
   However, the potential corresponding to the reduction peak in the
   experimental group with the addition of GAC is −0.28 V, which is
   different from the potential of the reduction peak in the experimental
   group with the addition of G. metallireducens. This may be because GAC
   and G. metallireducens have different overpotentials. At the same time,
   the different corrosion degrees of the stainless steel between the two
   groups will also lead to different overpotentials. Meanwhile,
   Nelabhotla and Dinamarca [[115]40] pointed out that transferring a
   certain number of electrons at a lower potential is more efficient than
   transferring the same number of electrons at a higher potential, which
   may also be the reason why the methane production in the experimental
   group with the addition of G. metallireducens is higher than that in
   the experimental group with the addition of GAC. In conclusion, the
   enlarged capacitance and the existence of the reduction peak both prove
   that in the presence of G. metallireducens, M. barkeri is more likely
   to adhere to the stainless steel electrode, transfer electrons with it,
   and carry out the reaction of reducing CO[2] to generate CH[4], thus
   accelerating the corrosion of the stainless steel.

   The corresponding strains of G. metallireducens and M. barkeri were
   also captured within the corrosion pits of the stainless steel in the
   experimental group with the addition of G. metallireducens ([116]Figure
   3D), and the two strains were attached to each other. Meanwhile, the Fe
   weight percentage in the experimental group with the addition of G.
   metallireducens was 70.26%, which was lower than that in the “M.
   barkeri + SS” group. This indicates that G. metallireducens indeed
   participated in and accelerated the corrosion of stainless steel by M.
   barkeri. Since the iron content of the corroded stainless steel is
   similar to that in the experimental group with the addition of GAC, it
   is speculated that the acceleration rates of the two are similar, but
   the specific situation still needs to be verified by experiments.

3.4. Mechanism Analysis of Syntrophy Accelerating the Corrosion of Stainless
Steel by Methanosarcina barkeri

   Similarly, in order to understand the mechanism by which G.
   metallireducens accelerates the corrosion of stainless steel by M.
   barkeri, transcriptome analysis was still carried out on M. barkeri
   after the experiment was completed. Some genes in the “M. barkeri + G.
   metallireducens + SS” experimental group whose gene expressions were
   upregulated were compared with those in the “M. barkeri + SS”
   experimental group, and the difference multiples of gene expression
   levels greater than 2.5 were selected, and the gene table shown in
   [117]Table 2 was obtained. The other upregulated genes and specific
   values are shown in [118]Table S3. As shown in [119]Table 2, among the
   upregulated genes, apart from some genes encoding hypothetical proteins
   with relatively large upregulation multiples, fwdC encoding furan
   dehydrogenase and mtbC encoding methyltransferase-like proteins, both
   belonging to the methane metabolic pathway, show relatively large fold
   differences, which are 12.816 and 2.563, respectively. Furan
   dehydrogenase was initially regarded as an intermediate in CO-based
   methanogenesis [[120]41]. By catalyzing the conversion of formate to
   carbon monoxide, fwdC indirectly provides electrons for the
   methane-generating process. M. barkeri utilizes these electrons along
   with hydrogen or carbon dioxide to produce methane. Therefore, the fwdC
   gene promotes the methane-generating process by enhancing the
   conversion of formate. It has been newly discovered that CO[2] can be
   captured as a formyl group branched on the amine moiety of the
   methanofuran cofactor, which also represents hydrogenotrophic
   methanogenesis [[121]42]. However, both M. barkeri and G.
   metallireducens use H[2] as a mediator without producing hydrogen, and
   316 L stainless steel abiotically produces little or no H[2] [[122]43].
   Thus, there is almost no hydrogenotrophic methane production in this
   system. mtbC is recognized as a methyltransferase gene that initiates
   methanogenesis from methylamine [[123]44]. However, the research of S.
   L. Tsola found that mtbC can also participate in the anaerobic
   conversion of dimethyl sulfide to methane, challenging the view that
   “substrate-specific methyltransferases are used for methylotrophic
   methanogenesis” [[124]45]. Therefore, mtbC is indeed likely to
   participate in the process of converting carbon dioxide to methane. On
   the other hand, after classifying the differentially expressed genes
   according to KEGG pathways in the same way, [125]Figure 6 was obtained,
   and the KEGG enrichment rates of each gene are shown in [126]Figure S3.
   As can be seen from [127]Figure 6, most of the differentially expressed
   genes belong to the energy metabolism (including methane metabolism)
   pathway. These indicate that when the syntrophic bacterium G.
   metallireducens is co-cultured with M. barkeri, different from the
   genes affected by GAC, it will lead to the high expression of mtbC and
   fwdC to improve the ability of M. barkeri to utilize the electrons of
   stainless steel to complete its own metabolism and produce methane.

Table 2.

   Selected genes with relatively high differential upregulation of M.
   barkeri genes in the “M. barkeri + G. metallireducens + SS”
   experimental group compared with the “M. barkeri + SS” experimental
   group.
   Gene ID KO
   Name Gene Description Pathway Definition FC(MB_GM/MB)
   Log[2]FC(MB_GM/MB)
   MSBRM_0413 ------ hypothetical protein ------ 44.909 5.489
   MSBRM_1961 ------ hypothetical protein ------ 35.886 5.165
   MSBRM_3095 ------ DUF378 domain-containing protein ------ 10.894 3.445
   MSBRM_0718 ------ collagen triple helix repeat domain protein ------
   5.554 2.473
   MSBRM_2952 ------ hypothetical protein ------ 19.968 4.320
   MSBRM_2105 fabG 3-oxoacyl-[acyl-carrier protein] reductase Biotin
   metabolism
   Fatty acid biosynthesis 6.408 2.680
   MSBRM_1954 trpB Tryptophan synthase beta chain Phenylalanine, tyrosine,
   and tryptophan biosynthesis 2.563 1.358
   MSBRM_1237 puuE 4-aminobutyrate aminotransferase Alanine, aspartate,
   and glutamate metabolism 2.563 1.358
   MSBRM_0462 mtbC Dimethylamine methyltransferase corrinoid protein
   Methane metabolism 2.563 1.358
   MSBRM_0475 ipdC Pyruvate decarboxylase Tryptophan metabolism 2.563
   1.358
   MSBRM_0157 pylC Pyrrolysine synthetase Lysine biosynthesis 2.563 1.358
   MSBRM_2496 ------ 8-oxoguanine DNA glycosylase Base excision repair
   2.563 1.358
   MSBRM_0641 fwdC Formylmethanofuran dehydrogenase Methane metabolism
   12.816 3.680
   [128]Open in a new tab

Figure 6.

   [129]Figure 6
   [130]Open in a new tab

   KEGG pathway classification statistics chart of differentially
   expressed genes between the “M. barkeri + G. metallireducens + SS”
   group and the “M. barkeri + SS” group.

4. Conclusions

   This study demonstrated that GAC and the syntrophy G. metallireducens
   can accelerate the corrosion of stainless steel by M. barkeri. Both of
   them promote the conversion of CO[2] into CH[4] through genes encoding
   methyltransferases. The acceleration in the GAC system mainly increases
   the expression level of methane metabolism gene mtmC, and that in the
   G. metallireducens system mainly increases the expression level of mtbC
   and fwdC. This study investigates the effects of the two on the
   corrosion of M. barkeri on stainless steel, filling a gap in this field
   and providing theoretical guidance for microbial anti-corrosion, which
   is of practical significance. However, the effects of GAC and G.
   metallireducens dosage on corrosion, as well as the synergistic
   mechanisms of mtmC, fwdC, and other functional genes in the complex
   stainless steel system, remain unclear.

Supplementary Materials

   The following supporting information can be downloaded a:
   [131]https://www.mdpi.com/article/10.3390/microorganisms13061278/s1:
   Figure S1: Atomic contents near M. barkeri in each experimental group;
   Figure S2: KEGG enrichment analysis (MB-GAC vs. MB); Figure S3: KEGG
   enrichment analysis (MB-GM vs. MB); Table S1: Contents of each atom
   near M. barkeri (the scanning object is indicated in the brackets);
   Table S2: Genes with relatively high differential upregulation of M.
   barkeri genes in the “M. barkeri + SS + GAC” experimental group and the
   “M. barkeri + SS” experimental group; Table S3: Genes with relatively
   high differential upregulation of M. barkeri genes in the “M. barkeri +
   G. metallireducens + SS” experimental group compared with the “M.
   barkeri + SS” experimental group.
   [132]microorganisms-13-01278-s001.zip^ (910.3KB, zip)

Author Contributions

   Conceptualization, Y.D.; methodology, C.Z. and H.D.; software, C.Z. and
   H.L.; validation, S.H.; formal analysis, C.Z.; investigation, H.Z.;
   resources, W.C.; data curation, C.Z.; writing—original draft
   preparation, C.Z.; writing—review and editing, C.Z.; supervision, Y.D.;
   project administration, Y.D. All authors have read and agreed to the
   published version of the manuscript.

Institutional Review Board Statement

   Not applicable.

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The original sequence data were submitted to the Sequence Read Archive
   (SRA) NCBI database under BioProjects PRJNA1241488.

Conflicts of Interest

   The authors declare no conflicts of interest.

Funding Statement

   This research was funded by the National Natural Science Foundation of
   China (No. 52270023, No. 52470026), China High-Level Talent Support
   Program-Youth Project, China Baowu Low Carbon Metallurgy Innovation
   Foundation (No. BWLCF202214), the 5·5 Engineering Research & Innovation
   Team Project of Beijing Forestry University (No: BLRC2023B04).

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References