The effect and mechanism of a microbial agent used for corrosion control in circulating cooling water

Abstract

Corrosion control is vital for the safe operation of a circulating cooling water system. The biological method is a novel treatment method performed by adding a microbial agent to achieve corrosion control. In this study, microbial agents, including Bacillus cereus, Pseudomonas, Bacillus subtilis, and Thiobacillus denitrificans, were selected to investigate the anti-corrosion effect and mechanism of the biological method. The results indicated that the anti-corrosion efficiency of the microbial agent on Q235B carbon steel ranged from 8.06–9.02%, while that for 316L stainless steel was 62.80%. The anti-corrosion mechanisms of functional bacteria were also revealed, which included (a) the formation of a biofilm to isolate oxygen and corrosive substances, (b) the inhibition of a cathodic reduction reaction and reduction of corrosion current, and (c) the inhibition of sulfide corrosion.

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Water impact

Corrosion control is vital for the safe operation of a circulating cooling water system. Biological treatment is a novel, efficient, and environmentally friendly method used for corrosion control achieved through adding a microbial agent. Our study provides a deeper understanding of the roles of functional bacteria, allowing for the advanced development and application of the microbial agent used for water treatment.

1. Introduction

Carbon steel and stainless steel are commonly used metal materials for pipelines and heat exchangers in circulating cooling water systems used in power plants. Under the action of oxygen and aggressive ions in circulating cooling water, carbon steel, and stainless steel may undergo oxygen consumption corrosion, pitting corrosion, and other corrosion behaviors.¹ Corrosion could cause condenser tube perforation, leakage of circulating cooling water to the thermal system, and other adverse effects.² Therefore, processes that foster the anti-corrosion of metals are of great significance to the safe operation of circulating cooling water systems.

Adding a chemical corrosion inhibitor is a common method for circulating cooling water treatment.³ Organic phosphonates, chromate, and molybdate are common corrosion inhibitors, which are usually used in conjunction with scale inhibitors. Chemical methods have good anti-corrosion and anti-scaling effects.^4,5 However, the extensive use of chemical agents may cause problems, such as deterioration of water quality. Related studies have shown that the application of corrosion and scale inhibitors led to the total phosphorus content of circulating cooling water exceeding 3 mg L⁻¹.⁶ This could increase the risk of the water quality exceeding effluent standards. Therefore, the development of an environmentally friendly method for corrosion control has become a research priority in circulating cooling water treatment.

The biological method is used for circulating cooling water treatment by means of adding a specific microbial agent and utilizing the synergistic effect of functional bacteria metabolism.⁷ Chen et al.⁸ used compound microbial preparation (CMP) with nitrobacteria, Bacillus subtilis (B. subtilis), photosynthetic bacteria, and Thiobacillus denitrificans (T. denitrificans, TDN), to treat circulating cooling water. The results showed that CMP had good corrosion and scale inhibition effect, and the corrosion inhibition rate on Q235 carbon steel reached 99.69%. Hu et al.^9,10 evaluated the corrosion inhibition behavior of Bacillus cereus (B. cereus) on Q235 carbon steel in simulated cooling water, whose anti-corrosion efficiency was 73.88%. The results showed that the addition of B. cereus increased the charge transfer resistance of the metal and reduced the corrosion current density, allowing it to attach to the surface of Q235 carbon steel to form a tight biofilm.

Some research studied the anti-corrosion effect of microbial secretions. Li et al.¹¹ studied soluble extracellular polymeric substances (s-EPS) secreted by B. cereus as a new biological agent with anti-corrosion and anti-scaling effects. The results showed that the corrosion rate of s-EPS (40 mg L⁻¹) on 316L stainless steel in an artificial seawater medium reached 91.16% in 30 d. Jayaraman et al.¹² studied the corrosion inhibition of aluminum 2024 using the formation of biofilms by B. subtilis WB600 and Bacillus licheniformis, respectively. The results indicated that the polyaspartate and γ-polyglutamate, which originated from biofilms, slowed down the pitting corrosion. The carboxylic acid group could be capable of chelating or coupling aluminum ions or aluminum oxides that were present at the metal–solution interface via hydrogen bonds, dipole–dipole, and coulombic interactions. Gunasekaran et al.¹³ found that the corrosion rate of carbon steel significantly decreased due to the formation of a surface biofilm in a phosphate-buffered basal salt solution containing Pseudomonas cichorii. The FTIR results proved that the biofilm protects the metal due to the ferrous ions and EPS that formed the Fe-EPS organo-metal complex layer. However, differences exist in the anti-corrosion effect of biofilm formed by microbial functional bacteria on different metals. The process of biofilm formation by functional bacteria and the interaction between biofilm and metal have not yet been clearly recognized. Therefore, it is important to clarify the anti-corrosion mechanism of functional bacteria for engineering the application of microbial agents.

In this paper, Q235B carbon steel and 316L stainless steel were selected to study the anti-corrosion effect of microbial agents and reveal the mechanism of functional bacteria. The corrosion rates of metal coupons with and without the microbial agent were compared by a rotary coupon test. A corrosion electrochemistry test monitored the attachment process of microorganisms onto the metal surface and calculated the anti-corrosion effect of the microbial agent. A biofilm observation test observed the formation process of the biofilm. Our study provides a deeper understanding of the anti-corrosion mechanism of functional bacteria, allowing for the advanced development and application of the microbial agent used for corrosion control in circulating cooling water.

2. Materials and methods

2.1 Materials pretreatment

In this study, a dry powder of the microbial agent was purchased from Yuanneng Chemical Co., Ltd. (Tieling, China). The microbial agent was composed of B. cereus, Pseudomonas, B. subtilis, and T. denitrificans, and the proportion was 2 : 2 : 4 : 1. Moreover, carbohydrates, used as an assistant agent, were used to meet the needs for microbial growth, which included saccharose, arabinose and glucose. The microbial agent of dry powder was pretreated to activate the microorganism, which was mixed with carbohydrates and normal saline. The mixed liquid was heated in a water bath at 36 °C for 24 h. The liquid microbial agent was prepared and used for future experiments. The effective microbial content was varied in the range of 10⁹–10¹⁰ CFU L⁻¹. Other chemicals were of analytical grade throughout the experiments. The circulating cooling water of a power plant was used as the test water, and the water quality depended on temperature and other factors. The water quality index is shown in Table S1.†

2.2 Rotary coupon test

The aim of the rotary coupon test is to study the anti-corrosion effect of the microbial agent on Q235B carbon steel and 316L stainless steel. The Q235B and 316L coupons should be pretreated according to the Chinese Standard GB/T 18175-2014.¹⁴ The coupons were installed in the beaker of the rotary hanging-piece corrosion device. The circulating cooling water was used as a test solution. In one group, a liquid microbial agent was added with a concentration of 50 mL L⁻¹ and carbohydrate daily with a concentration of 75 mg L⁻¹. The other group did not add the microbial agent. The solution was kept at a constant temperature of 36 °C for 30 d, and the rotation speed was set to 100 r min⁻¹. Three parallel samples were tested in each group. The corrosion rate (v_a) and anti-corrosion efficiency (η_c) of the microbial agent could be calculated according to eqn (1) and (2).

3650 is the conversion factor; m₀ and m₁ are the weights of the coupon before and after corrosion (g); A is the surface area of the coupon (cm²); t is the corrosion time (d); ρ is the density of the material coupon (g cm⁻³); v_a1 and v_a0 are the average corrosion rate with and without the microbial agent (mm a⁻¹), respectively.

The coupon with attachment after corrosion was dried under natural conditions. The surface attachment on the coupon was detected by energy dispersive spectroscopy (Aztec Energy EDS, Oxford Instruments) for the elemental component and analyzed by XPert Pro X-ray diffraction (XRD, Malvern Panalytical) and K-Alpha X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific) for the material component. The coupon was further pickled according to the Chinese Standard GB/T 18175-2014.¹⁵ The pickling of the coupon was conducted to remove the corrosion product and surface attachment. The coupons after corrosion and after pickling were observed by MIRA 3 field emission scanning electron microscopy (SEM, Tescan) for microscopic morphology.

2.3 Corrosion electrochemistry test

The conventional three-electrode apparatus measured the open circuit potential (OCP), electrochemical impedance spectroscopy (EIS), and potentiodynamic polarization. The working electrode material used Q235B carbon steel and 316L stainless steel. The working electrode had a working area of 0.5 cm² and a material density of 7.85 g cm⁻³ (Q235B carbon steel) and 8 g cm⁻³ (316L stainless steel). Before the test, the working electrode was mechanically polished and washed with deionized water. The reference electrode used a saturated calomel electrode (SCE) and was placed in an L-shaped salt bridge with the saturated KCl solution. The auxiliary electrode was a 10 mm × 10 mm platinum sheet electrode.

The working electrode was installed in the beaker of the rotary hanging-piece corrosion device, and the working surface was immersed in solution. The circulating cooling water was used as a test solution. In one group, a liquid microbial agent was added with a concentration of 50 mL L⁻¹ and carbohydrate daily with a concentration of 75 mg L⁻¹. The other group did not add an agent. The solution was kept at a constant temperature of 36 °C for 30 d, and the rotation speed was set to 100 r min⁻¹. The working electrode was removed at regular intervals for the electrochemistry test. The test solution was taken out from the beaker as a test electrolyte. All electrochemical tests were performed at the electrochemical workstation.

The OCP was the electrode potential measured when the metal was not carrying current. The EIS test was performed at a stable OCP and a sinusoidal signal of 10 mV was applied to the electrode. The frequency range was 0.1–10⁵ Hz. The OCP and EIS tests were non-destructive, which allowed for the continuous monitoring of the influence of microorganisms on the electrode and the analysis of the corrosion behavior of the electrode. The potentiodynamic polarization method was scanned at the potential range of −0.5 V to +0.5 V (vs. OCP) with a scan rate of 1 mV s⁻¹. Potentiodynamic polarization was a destructive test and only measured at the end of the test. The anti-corrosion efficiency (η_c) could be calculated according to corrosion current density (i_corr), and the equation was as follows:

where i_corr1 and i_corr0 are the corrosion current densities (A cm⁻²) of the electrode with and without the microbial agent after corrosion.

2.4 Biofilm observation test

A biofilm observation test was performed with the intent of observing the biofilm formation process. The 316L stainless steel coupon was selected and pretreated in the same way as the rotary coupon test. The 316L coupon was installed in the beaker of the rotary hanging-piece corrosion device. The circulating cooling water was used as a test solution. A liquid microbial agent with a concentration of 50 mL L⁻¹ was added to the solution, and carbohydrate with a concentration of 75 mg L⁻¹ was added daily. The solution was kept at 36 °C for 30 d, and the speed was set to 100 r min⁻¹.

The coupon-attached biofilm was removed from the beaker every 5 d, and soaked in a phosphate buffer solution containing 3% glutaraldehyde for 4 h to immobilize the biofilm. Then the coupon was dehydrated successively with an ethanol gradient: 25%, 50%, 75%, 90%, and 100% for 10 min.^15,16 The coupon-attached biofilm was observed by SEM and optical surface profilometer (OSP). The organic component of the biofilm was analyzed by Fourier transform infrared spectroscopy (FTIR), where a diamond crystal was used as the reflection accessory.

3. Results and discussion

3.1 Coupon corrosion characteristic

Table S2† shows the corrosion rate of Q235B and 316L coupons after 30 d. The average corrosion rate of Q235B without microbial agent was 0.7261 ± 0.0167 mm a⁻¹, which was slightly higher than that with microbial agent (0.6606 ± 0.0098 mm a⁻¹). The anti-corrosion efficiency of the microbial agent on Q235B carbon steel was 9.02%. However, the corrosion rate of Q235B carbon steel with and without microbial agent exceeded the limit value of the Chinese Standard GB/T 50050-2017.¹⁷ Q235B carbon steel corroded under the water quality condition of circulating cooling water. The average corrosion rates of 316L with and without microbial agent were 0.0005 ± 0.0002 and 0.0006 ± 0.0001 mm a⁻¹, respectively. The corrosion rate of 316L stainless steel with and without the microbial agent was not different and was much lower than the limit value of 0.005 mm a⁻¹. The 316L stainless steel did not incur the obvious corrosion behavior under the circulating cooling water.

Fig. 1 shows the appearance image and SEM image of Q235B and 316L coupons after corrosion and pickling. There was no significant difference between the surface of the Q235B coupon with and without microbial agent after corrosion. The Q235B coupon generated a corrosion product layer that covered the whole surface of the coupon. The scattered attachment appeared on the 316L coupon surface without the microbial agent after corrosion. This could be a particle impurity originating from the circulating cooling water, which remained on the metal surface. A layer of film appeared on the 316L coupon surface with microbial agent after corrosion because the functional bacteria could attach to the metal surface and form a biofilm layer. The surface of the Q235B coupon after pickling with and without microbial agent was uneven and had no metallic luster, but the surface had no obvious pit. The corrosion process of Q235B carbon steel with and without microbial agent in circulating cooling water was uniform corrosion, rather than pitting corrosion. The surface of the 316L coupon after pickling with and without microbial agent was flat and had metallic luster. The 316L stainless steel with and without microbial agent in circulating cooling water had no obvious corrosion behavior.

Fig. 1 Appearance image and SEM image of the coupon under different conditions. (A) After corrosion; (B) after pickling; (a) Q235B without microbial agent; (b) Q235B with microbial agent; (c) 316L without microbial agent; (d) 316L with microbial agent.

The XRD images of surface attachment on the Q235B and 316L coupons are shown in Fig. 2. The diffraction peaks of Fe₃O₄ magnetite crystal (PDF Card No. 99-0073) appeared in attachment of Q235B coupon with and without microbial agent, corresponding to 30.43°(220), 35.43°(311), 43.05°(400), 56.94°(511), and 62.52°(440), respectively. The results indicate that Q235B carbon steel underwent oxygen consumption corrosion and generated the corrosion products of Fe₃O₄. The diffraction peaks of Cr_0.19Fe_0.7Ni_0.11 austenite crystal (PDF Card No. 33-0397) appeared in the attachment of 316L coupon with and without microbial agent, corresponding to 43.58°(111), 50.79°(200), and 74.68°(220), respectively. In fact, a Cr_0.19Fe_0.7Ni_0.11 austenite crystal was the base of 316L stainless steel rather than an attachment.

Fig. 2 XRD images of attachment under different conditions. (a) Q235B without microbial agent; (b) Q235B with microbial agent; (c) 316L without microbial agent; (d) 316L with microbial agent; △Fe₃O₄ crystals; ○Cr_0.19Fe_0.7Ni_0.11 crystals.

The elemental component for the surface attachment of Q235B and 316L coupons by EDS analysis is shown in Table 1. Four elements of C, O, S, and Fe were present in the attachment of the Q235B coupon after corrosion. The C, O, and Fe were derived from the Fe₃O₄ corrosion product, which was confirmed by the XRD results. The element, S, was detected because Q235B carbon steel may have generated a small amount of FeS corrosion product. Sulfate-reducing bacteria (SRB) is a common corrosive microorganism in circulating cooling water. It could consume cathodic hydrogen through an enzyme called hydrogenase to generate H₂S. The accumulation of corrosive H₂S on metal leads to metal corrosion, which generates the FeS corrosion product.^18,19 The growth environment of TDN of functional bacteria is similar to SRB. TDN could convert the corrosive H₂S into non-corrosive SO₄²⁻, thereby reducing the corrosion of sulfide to metals.²⁰ EDS results show that the content of S for Q235B attachment decreased after adding the microbial agent. This proves that the proportion of FeS in the corrosion product decreased and functional bacteria had the effect of inhibiting sulfide corrosion.

Table 1 Element component of attachment under different conditions

Element	Without microbial agent		With microbial agent
	Quality percentage/%	Atomic percentage/%	Quality percentage/%	Atomic percentage/%
(a) Q235B coupon
C	3.65	9.83	3.90	10.10
O	23.65	47.87	26.12	50.83
S	0.62	0.50	0.18	0.14
Fe	72.08	41.80	69.81	38.93
(b) 316L coupon
C	4.21	16.39	33.54	61.57
N	—	—	0.57	0.84
O	0.94	2.74	11.02	15.16
P	—	—	1.22	0.84
Si	0.43	0.72	0.22	0.16
S	0.58	0.85	0.97	0.66
Cr	17.05	15.34	9.63	4.08
Mn	1.04	0.88	0.33	0.12
Fe	66.2	55.46	37.16	14.61
Ni	9.55	7.61	5.33	1.97

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Eight elements, C, O, Si, S, Cr, Mn, Fe, and Ni, were present in the attachment of 316L coupon without microbial agent after corrosion, which was mainly derived from the 316L stainless steel base. After adding the microbial agent, the extra elements, N and P, were detected in the attachment. Furthermore, the content of C and O significantly increased. This may be due to the formation of biofilm on the surface of 316L stainless steel by functional bacteria. The main components are microbial flora and an extracellular polymeric substance (EPS) secreted by functional bacteria (B. cereus, Pseudomonas, and B. subtilis). An EPS is mainly composed of polysaccharides, proteins, and small amounts of phospholipids, humic acids, and nucleic acids.²¹ Therefore, the content proportion of C, N, O, and P is higher in the attachment of 316L coupon with microbial agent.

The material proportion of Fe for the surface attachment of Q235B coupon by XPS analysis is shown in Fig. 3. The characteristic peaks of Fe₃O₄ and FeS appeared at 710.8 eV and 713.6 eV, respectively.^22–24 Therefore, the main components of the corrosion product for Q235B carbon steel are Fe₃O₄ and FeS. In addition, the proportion of FeS in the corrosion product with the microbial agent was 7.27%, which was lower than that without the microbial agent (16.64%). The functional bacteria could inhibit the production of FeS, which has an anti-corrosion effect on Q235B carbon steel. This is consistent with the conclusion of the EDS analysis.

Fig. 3 High resolution Fe 2p_3/2 spectra for the attachment of Q235B coupon under different conditions. (a) Without microbial agent; (b) with microbial agent.

Furthermore, the proportion of Fe₃O₄ in corrosion products was much higher than that of FeS. Therefore, oxygen consumption corrosion is the main type of corrosion for Q235B carbon steel in circulating cooling water, rather than sulfide corrosion. Although the functional bacteria have the capacity to inhibit the corrosion of sulfide, the anti-corrosion efficiency on Q235B carbon steel is low. The functional bacteria could not inhibit the occurrence of oxygen consumption corrosion.

3.2 Corrosion electrochemistry

Fig. 4 shows the change of OCP for the Q235B electrode and 316L electrode with and without microbial agent. The results show that the Q235B electrode had a positive shift of OCP with and without microbial agent. Q235B carbon steel could be corroded under the water quality condition of circulating cooling water and the functional bacteria has difficulty forming biofilm on the metal surface. The influence of the corrosion process for OCP is larger than the attachment process of microorganisms. The change of OCP for the Q235B electrode is mainly caused by the formation of the corrosion product.

Fig. 4 Change of OCP for different electrodes. (a) Q235B without microbial agent; (b) Q235B with microbial agent; (c) 316L without microbial agent; (d) 316L with microbial agent.

There was a significant difference between the change of OCP for the 316L electrode with and without microbial agent. The OCP of the 316L electrode without microbial agent remained unchanged, while that with microbial agent was positively shifted. This positive shift of OCP indicates that the microorganisms have attached to the metal surface, and the shift value is positively related to the number of microorganisms attached to the electrode surface.²⁵ The change of OCP for the 316L electrode is mainly caused by the attachment process of functional bacteria rather than the corrosion process. The OCP remained unchanged for 0–10 d, and the functional bacteria slowly attached to the electrode. After 10 d, the positive shift in OCP was obvious as the amount of functional bacteria increased and biofilm gradually formed on the electrode surface. For the 316L stainless steel electrode, the functional bacteria could attach to the metal surface and form biofilm.

Fig. 5 shows the Nyquist diagram of the Q235B electrode and the 316L electrode with and without microbial agent at different times. There was no significant difference between the Nyquist diagram of the Q235B electrode with and without microbial agent. The extra semicircle occurred in the high-frequency region of the 316L electrode after adding a microbial agent, which was caused by functional bacteria forming a biofilm on the electrode surface. The corresponding EIS data were fitted using the equivalent circuit, as shown in Fig. 5. The one-time constant was used to represent the process of oxide film formation on the electrode of Q235B without microbial agent, Q235B with microbial agent, and 316L without microbial agent. The two-time constant was used to represent the process of double film on the electrode of 316L with microbial agent. The outer layer was biofilm and the inner layer was oxide film. CPE was usually used instead of the capacitor element when the data deviated from ideal capacitance behavior due to the non-uniformity of the scale layer. In the equivalent circuit, R_s represents the solution resistance, R_ct represents the charge transfer resistance, CPE_dl represents the constant phase element of the metal/medium double layer, R_f represents the biofilm resistance, and CPE_f represents the constant phase element of the biofilm layer.

Fig. 5 Nyquist diagram and the equivalent circuit of different electrodes. (a) Q235B without microbial agent; (b) Q235B with microbial agent; (c) 316L without microbial agent; (d) 316L with microbial agent.

According to the equivalent circuit, the impedance data were fitted in Zview software and the relevant parameters are shown in Table 2. The R_s value of the solution changed slightly and was in the range of 32.79–79.16 Ω cm². The R_ct value of the Q235B electrode with and without microbial agent shows a trend of increasing first before slightly decreasing because the oxide film of the corrosion product gradually forms on the Q235B electrode during the initial 0–15 d. As a consequence, the corrosion is inhibited. However, this oxide film is unstable, and its protective effect of metal gradually weakens with time.

Table 2 Impedance parameters of different electrodes

Time/(d)	R s/(Ω cm^2)	CPEdl/(F cm^−2)	n dl	R ct/(Ω cm^2)
(a) Q235B without microbial agent
5	32.79	4.22 × 10^−6	0.99	70.95
10	34.50	1.67 × 10^−6	0.99	161.98
15	39.02	1.58 × 10^−6	0.99	195.98
20	38.26	1.72 × 10^−6	0.98	194.55
25	38.68	1.56 × 10^−6	0.99	175.46
30	35.52	1.62 × 10^−6	0.99	164.09
(b) Q235B with microbial agent
5	60.87	1.37 × 10^−6	0.99	207.93
10	76.83	8.85 × 10^−7	0.97	379.28
15	69.42	9.67 × 10^−7	0.97	357.58
20	67.29	1.01 × 10^−6	0.97	343.03
25	64.53	1.02 × 10^−6	0.97	333.40
30	66.01	9.62 × 10^−7	0.98	331.15
(c) 316L without microbial agent
5	52.23	2.57 × 10^−5	0.88	9.62 × 10^5
10	54.55	2.25 × 10^−5	0.86	4.50 × 10^6
15	58.22	2.49 × 10^−5	0.85	3.53 × 10^6
20	55.18	2.43 × 10^−5	0.85	2.27 × 10^6
25	79.16	2.43 × 10^−5	0.85	2.10 × 10^6
30	73.43	2.31 × 10^−5	0.86	2.89 × 10^6

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Time/(d)	R s/(Ω cm^2)	CPEf/(F cm^−2)	n f	R f/(Ω cm^2)	CPEdl/(F cm^−2)	n dl	R ct/(Ω cm^2)
(d) 316L with microbial agent
5	71.60	8.92 × 10^−6	0.93	194.14	2.60 × 10^−5	0.90	1.63 × 10^6
10	74.02	1.51 × 10^−6	0.92	468.40	2.45 × 10^−5	0.86	7.51 × 10^6
15	69.84	1.52 × 10^−6	0.92	464.74	2.44 × 10^−5	0.88	7.76 × 10^6
20	74.44	1.51 × 10^−6	0.92	470.47	2.52 × 10^−5	0.88	7.18 × 10^6
25	72.12	1.55 × 10^−6	0.92	455.63	2.46 × 10^−5	0.85	7.24 × 10^6
30	76.57	1.41 × 10^−6	0.93	526.48	2.46 × 10^−5	0.85	7.44 × 10^6

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The R_ct value of the 316L electrode without microbial agent increased before slightly decreasing, which was consistent with the changing trend of the Q235B electrode. For 316L, the electrode with microbial agent, the CPE_f value decreased from 8.92 × 10⁻⁶ to 1.41 × 10⁻⁶ F cm⁻², and the R_f value increased from 194.14 to 526.48 Ω cm² after 30 d. The increase in the R_f value indicates that the integrity of the biofilm gradually increased. Furthermore, some studies have shown that the CPE_f value is inversely proportional to the film thickness.^15,18,26 Therefore, the functional bacteria could attach to the 316L electrode surface and form the biofilm, which is consistent with the result of the OCP change. The R_ct value of the 316L electrode with microbial agent reached a maximum of 7.76 × 10⁶ Ω cm² at 15 d and thereafter remained unchanged. Therefore, the biofilm can maintain a long and stable protective effect on 316L stainless steel. In addition, the R_ct value of the 316L electrode with microbial agent is higher than that without microbial agent, indicating that the formation of biofilm inhibits the charge transfer and diffusion process of oxygen molecules. The functional bacteria have an anti-corrosion effect on 316L stainless steel.

The biofilm formation of functional bacteria on the metal surface is one of the important factors for anti-corrosion. The biofilm could isolate the metal from oxygen molecules and inhibit the cathodic reduction reaction; thereby, the biofilm acts as a physical barrier layer that prevents the corrosive substances in the circulating cooling water from coming in contact with the metal and inhibits the metal from being damaged by corrosive substances.^27–29 Moreover, the type of metals and microorganisms influence the biofilm formation process. The results show that biofilm could only be formed on 316L stainless steel but experienced difficulty forming on Q235B carbon steel. When Q235B carbon steel comes in contact with the circulating cooling water, oxygen consumption corrosion will rapidly occur and generate the corrosion products of Fe₃O₄. The process of biofilm formation is relatively slow. The surface of Q235B carbon steel is covered by corrosion products before the attachment of functional bacteria. Therefore, the functional bacteria could not inhibit oxygen consumption corrosion by biofilm formation, and the anti-corrosion efficiency on Q235B carbon steel is low.

The new Q235B and 316L electrode and the electrode after 30 d of test were used for potentiodynamic polarization. Fig. S1† shows the potentiodynamic polarization curve of different electrodes. The corrosion current density (i_corr), corrosion potential (E_corr), anode Tafel slope (β_a), and cathode Tafel slope (β_c) could be calculated by fitting the Tafel lines. The fitting parameters are shown in Table 3. The i_corr value of Q235B electrode with and without microbial agent after 30 d were 5.70 × 10⁻⁵ and 6.20 × 10⁻⁵ A cm⁻², respectively. The anti-corrosion efficiency of the microbial agent on Q235B carbon steel was 8.06%, close to the rotary coupon test. The microbial agent had an anti-corrosion effect on Q235B carbon steel. However, the i_corr value of the electrode after 30 d was higher than that of a new electrode. Q235B carbon steel was corroded in the circulating cooling water with and without microbial agent.

Table 3 Fitting parameters of potentiodynamic polarization curve for different electrodes

Type		i corr/(A cm^−2)	E corr/(V vs. SCE)	β a/(mV dec^−1)	−βc/(mV dec^−1)
Q235B electrode	New electrode at 0 d	7.21 × 10^−6	−0.63	182.31	578.95
	Without microbial agent after 30 d	6.20 × 10^−5	−0.50	376.29	329.18
	With microbial agent after 30 d	5.70 × 10^−5	−0.42	405.98	318.29
316L electrode	New electrode at 0 d	1.70 × 10^−7	−0.09	848.04	134.81
	Without microbial agent after 30 d	1.89 × 10^−7	−0.08	1115.20	135.66
	With microbial agent after 30 d	7.03 × 10^−8	0.19	509.62	87.48

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The i_corr values of the 316L electrode with and without microbial agent after 30 d were 7.03 × 10⁻⁸ and 1.89 × 10⁻⁷ A cm⁻², respectively. In addition, the i_corr value of the 316L electrode after adding a microbial agent decreased compared to that with the new electrode. The microbial agent had an anti-corrosion effect on 316L stainless steel, and the anti-corrosion efficiency was 62.80%. This is because the formation of biofilm inhibits the diffusion process of oxygen molecules, resulting in a decrease in the limiting diffusion current density (i_L) of oxygen. Since the corrosion is controlled by the diffusion process of oxygen, the i_corr is equal to i_L. The results show that the i_corr value of the electrode with microbial agent is lower, which proves the above conclusion.

3.3 Biofilm formation process

Fig. 6(a–g) show the SEM images of the 316L coupon surface at different times under 1000× magnification. The results show that the attachment on the coupon surface increased with time. The functional bacteria could attach to the coupon surface and gradually secrete EPS to form a biofilm. The sporadic and dispersed attachment appeared on the coupon surface at 5 d, which was the attachment process of functional bacteria. Then, the functional bacteria continued to reproduce and gradually gathered to form colonies and produce a large amount of EPS. The complete biofilm layer gradually covered the coupon surface after 20 d. Fig. 6(h) shows the SEM images of biofilm under 20 000× magnification. The “mushroom-like” biological structure was observed, and the microorganisms cross-linked to form clusters, consistent with the biofilm morphology observed in relevant studies.^15,30,31 SEM images show that the biofilm was composed of multi-layer microbial flora, and the secreted EPS closely linked each microbial flora.

Fig. 6 SEM images of the coupon surface at different times. (a) 0 d; (b) 5 d; (c) 10 d; (d) 15 d; (e) 20 d; (f) 25 d; (g) 30 d; (h) 30 d-high magnification.

The 3D topography images were obtained by using OSP to observe the biofilm formed on the coupon surface at different times, as shown in Fig. 7. The coupon surface at 20 d was smooth and had no obvious fluctuation. Some convex regions appeared on the coupon surface with time, which reflected the continuous attachment of functional bacteria. The convex regions gradually increased, and the surface height of the coupon gradually increased. The attached functional bacteria could form a biofilm layer and gradually cover the coupon surface. To quantitatively describe the thickness of biofilm, the height distribution in the observation region was statistically analyzed. Table S3† shows the surface roughness parameters of the coupon at different times, including the surface arithmetical average height (S_a) and the surface root-mean-square height (S_q). The results show that the S_a and S_q values increased in 0–20 d, the surface roughness of the coupon increased, and the thickness of the biofilm increased. The values of S_a and S_q remained unchanged after 20 d. At 20 d, the functional bacteria formed a dense and complete biofilm layer structure on the surface of the 316L coupon, which is consistent with the SEM results. It could be inferred from the roughness parameters that the thickness of the biofilm layer is about 1 μm. The dense biofilm layer formed by functional bacteria could effectively prevent the corrosive substances in the circulating cooling water from making contact with the metal, thus achieving an anti-corrosion effect.

Fig. 7 3D topography images of the coupon surface at different times. (a) 0 d; (b) 5 d; (c) 10 d; (d) 15 d; (e) 20 d; (f) 25 d; (g) 30 d.

Fig. S2† shows the FTIR spectrum of biofilm on the coupon surface after 30 d. The results show that the characteristic absorption peaks of the N–H amino group and O–H hydroxyl group appeared at 3286 cm⁻¹, C=O bond appeared at 1644 cm⁻¹, NH₂ bond appeared at 1539 cm⁻¹, the amine appeared at 1026 cm⁻¹, and carboxylic acid group appeared at 1411 cm⁻¹. These were typical characteristic peaks of a protein secondary structure.^32–34 The vibration band of C–O appeared at 1261 and 1082 cm⁻¹, which was typical of the characteristic peak of a polysaccharide.³⁵ In addition, the characteristic absorption peaks caused by the C–H bond stretching vibration appeared at 2958 and 798 cm⁻¹. The results indicate that the components of biofilm were protein and polysaccharide, which was EPS secreted by functional bacteria.

3.4 Anti-corrosion mechanism analysis

The above results show that adding a microbial agent to circulating cooling water could control corrosion. As shown in Fig. 8, the functional bacteria could establish an anti-corrosion effect in the following three ways.

Fig. 8 Schematic diagram of the anti-corrosion mechanism for functional bacteria. (a) Biofilm formation; (b) corrosion current reduction; (c) sulfide corrosion inhibition.

(a) Biofilm formation. Functional bacteria (B. cereus, Pseudomonas and B. subtilis) could form biofilm on the metal surface, thereby inhibiting corrosion. The biofilm could only be formed on 316L stainless steel, but it had difficulty forming on Q235B carbon steel. The biofilm could isolate the metal from an oxygen molecule and inhibit the cathodic reduction reaction. The biofilm acting as a physical barrier layer could prevent the corrosive substances in circulating cooling water from making contact with the metal and prevent the metal from being damaged by corrosive substances. Relevant studies have reported the behavior of corrosion inhibition by the biofilm. Qu et al.³⁰ studied the corrosive behavior of a cold rolled steel electrode in artificial seawater containing B. subtilis C2. The EIS results show that the R_ct value gradually decreased at the beginning of the test because the lactic acid produced by B. subtilis C2 accelerated the corrosion. The R_ct value increased after 16 h, and a dense biofilm protective layer was formed on the surface of the cold rolled steel, which inhibited corrosion.

The positive shift in the OCP value and the increase of the R_f value prove that biofilm formed on the 316L electrode after adding a microbial agent. The R_ct value of the 316L electrode with the microbial agent is higher than that without microbial agent. This indicates that the formation of biofilm isolates the oxygen molecules and inhibits the consumption corrosion of metal. The SEM images and 3D topography images illustrate that the functional bacteria could attach to the 316L coupon surface and secrete EPS to form biofilm. A dense and complete biofilm layer covers the 316L coupon surface after 20 d. The thickness of biofilm is about 1 μm and the main components of the biofilm are protein and polysaccharide.

Moreover, related studies have shown that not all EPS produced by microorganisms results in a protective effect on metal. Stadler et al.³⁶ found that EPS produced by Desulfovibrio alaskensis had a corrosion inhibition effect on pure iron and carbon steel under anaerobic conditions; however, EPS produced by Desulfovibrio vulgaris and Desulfovibrio indonesiensis did not protect metals. Therefore, it is of great significance to select functional bacteria that could produce EPS that do protect metal. In our study, the B. cereus, Pseudomonas, and B. subtilis were used as functional bacteria, proving that they could form biofilm and produce an anti-corrosion effect on 316L stainless steel.

Suma et al.³⁷ reported that the protective action of Pseudomonas putida RSS on a mild steel surface was enhanced by biofilm formation. The results indicate that no trace of corrosion was present even after 12 months of immersion, with a negligible corrosion rate of 3.01 × 10⁻² mmpy. However, this property of biofilm producing/biopassivating did not appear in our study. The Q235B carbon steel is corroded and covered by corrosion products before the attachment of functional bacteria. The biofilm secreted by functional bacteria is difficult to form on Q235B carbon steel.

(b) Corrosion current reduction. The functional bacteria could attach to the metal surface and consume oxygen molecules through respiration. The formation of biofilm inhibits the diffusion process of oxygen and the cathodic reduction reaction. The results show that the i_corr value of the 316L electrode with microbial agent is lower than that without microbial agent. The presence of functional bacteria reduces the corrosion current and inhibits metal corrosion. Dubiel et al.³⁸ found that the presence of the Shewanella oneidensis strain MR-1 decreased the i_L value of 316 stainless steel. The microbial respiration caused a decrease in oxygen concentration. Therefore, the attached functional bacteria could consume oxygen molecules on the metal surface through respiration and inhibit the cathodic reduction reaction.

(c) Sulfide corrosion inhibition. SRB is a common corrosive microorganism in circulating cooling water, and the growth environment for TDN of functional bacteria is similar to that of SRB. TDN could convert the corrosive sulfide produced by SRB into a non-corrosive sulfate, thereby inhibiting the corrosion of sulfide to metal. The EDS and XPS analyses show that the proportion of FeS corrosion product produced by Q235B carbon steel decreases after adding a microbial agent. It proves that functional bacteria have the effect of inhibiting sulfide corrosion. Yuan et al.³⁹ studied the corrosion of X70 carbon steel when TDN and SRB coexisted and SRB existed alone and found that TDN in a neutral or neutral alkaline environment weakened the corrosion of carbon steel. TDN could consume the corrosive sulfide produced by SRB and inhibit the corrosion of SRB to carbon steel. This was consistent with the conclusion of our study. However, oxygen consumption corrosion is the main type of corrosion for Q235B carbon steel in circulating cooling water, rather than sulfide corrosion. Although the functional bacteria can inhibit the corrosion of sulfide, the anti-corrosion efficiency on Q235B carbon steel is low.

4. Conclusion

This study aimed to evaluate the anti-corrosion effect of a microbial agent and reveal the mechanism of functional bacteria. The results showed that the anti-corrosion efficiency of a microbial agent on Q235B carbon steel was 8.06–9.02% and 62.80% on 316L stainless steel. The anti-corrosion mechanism of functional bacteria included biofilm formation, corrosion current reduction, and sulfide corrosion inhibition.

The results show that functional bacteria could form biofilm on the metal surface, thereby inhibiting corrosion. However, biofilm could only be formed on 316L stainless steel, and it had difficulty forming on Q235B carbon steel. The positive shift of the OCP value and the increase in the R_f value proved the formation of biofilm on the 316L electrode after adding a microbial agent. The SEM images and 3D topography images show that a dense and complete biofilm layer covered the 316L coupon surface after 20 d. The thickness of biofilm was about 1 μm and the main components of biofilm were protein and polysaccharide. The change in the R_ct value of the 316L electrode after adding a microbial agent indicated that the formation of biofilm isolated the oxygen molecule and corrosive substances, which inhibited the metal corrosion.

The attached functional bacteria and biofilm on metal surfaces could consume oxygen molecules through respiration and inhibit the diffusion process of oxygen. The i_corr value of the 316L electrode with microbial agent was lower, which proved that functional bacteria could inhibit the cathodic reduction reaction and reduce the corrosion current.

The TDN of functional bacteria could convert the corrosive sulfide produced by SRB into a non-corrosive sulfate, thereby inhibiting the corrosion of sulfide to metal. The analyses of EDS and XPS showed that the proportion of FeS corrosion products produced by Q235B carbon steel decreased after adding a microbial agent. Although the functional bacteria had the effect of sulfide corrosion inhibition, the anti-corrosion efficiency on Q235B carbon steel was low. The functional bacteria could not inhibit oxygen consumption corrosion because the biofilm experienced difficulty forming on Q235B carbon steel.

Author contributions

Yu Wang: methodology, writing – original draft, writing – review & editing; Hongfeng Liao: methodology, resources; Li Gan: validation, resources; Zhengxiu Liu: data curation, resources; Ziqiang Tang: visualization, supervision; Xiaoran Zhao: writing – review & editing; Yubin Zeng: supervision, writing – review & editing; Chunsong Ye: supervision, writing – review & editing.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Beijing Jingneng Energy Technology Research Co., Ltd.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3ew00629h

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