Effect of pH regulation by sulfate-reducing bacteria on corrosion behaviour of duplex stainless steel 2205 in acidic artificial seawater

Sulfate-reducing bacteria (SRB) can regulate environmental pH because of their metabolism. Because local acidification results in pitting corrosion, the potential capacity of pH regulation by SRB would have important consequences for electrochemical aspects of the bio-corrosion process. This study focused on identifying the effect of pH on the corrosion of duplex stainless steel 2205 in a nutrient-rich artificial seawater medium containing SRB species, Desulfovibrio vulgaris. Duplex stainless steel samples were exposed to the medium for 13 days at 37°C at pH ranging from 4.0 to 7.4. The open-circuit potential value, sulfide level, pH and number of bacteria in the medium were recorded daily. Electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization were used to study the properties of the biofilms at the end of the experiments and the corrosion behaviour of the material. Inductively coupled plasma mass spectrometry was used to measure the concentration of cations Fe, Ni, Mo, Mn, Cr in the experimental solution after 13 days. Scanning electron microscopy and energy-dispersive X-ray spectroscopy (EDX) were used for surface analysis. The results showed the pH changed from acidic values set at the beginning of the experiment to approximately pH 7.5 after 5 days owing to bacterial metabolism. After 13 days, the highest iron concentration was in the solution that was initially at pH 4 accompanied by pitting on the stainless steel. Sulfur was present on all specimens but with more sulfur at pH 4 in the EDX spectra. EIS showed the film resistance of the specimen at pH 4 was much lower than at pH 7.4 which suggests the corrosion resistance of the stainless steel was better at higher pH. The results of this study suggest that the corrosion process for the first few days exposure at low pH was driven by pH in solution rather than by bacteria. The increasing pH during the course of the experiment slowed down the corrosion process of materials originally at low pH. The nature and mechanism of SRB attack on duplex stainless steel at different acidic environments are discussed.

Although the SRB used in this study seems to be a popular corrosive strain, the experimental results hardly mentioned the corrosive effect caused by this strain, but rather emphasized the corrosion inhibitory effect.
Based on the above, ①In the experiments using the same strain, there are some results showing the reproduction of MIC even in neutral pH culture conditions. What is the difference between these results and yours? ②In order to discuss the effects of corrosion under acidic conditions, it should be preferable to set the same pH abiotic experimental condition as a control. Could you tell me the reason why? I would like you to clarify your views and position regarding these. In addition, clarify the novelty and purpose of research more than in the introduction At the same time, I would like it reflected in the title of the paper. Based on these, I would like to request that the research novelty and purpose be made clearer in the introduction part. Similarly, research content should be reflected more clearly in the title of the paper.
Some items pointed out are specifically described below.
(1) Some keywords such as SRB or anaerobic should be included in the title.
(4) P1, l.41: Does Sulfide mean iron sulfide? (5) P1, l.48: In this study, while the suppression of corrosion by biofilm produced by SRB is discussed, there is few mentions about the corrosion phenomenon assisted by SRB. Usually it is easy to find the corrosion phenomenon assisted by SRB (may be same strain) and occurred in neutral pH condition, in some literature. Please see also (8). The strain ATCC 7757 has been treated as a popular corrosive bacterium in many bio-corrosion studies. However, your studies emphasized the corrosive effect of pH on the material, rather than the bacterial corrosive ability. Please explain your view on these. (9) P.5, l.56: Did you measure biofilm thickness? (10) P.9, Figure 1, 2, P.10, Figure 4, 5 ：The letters, legend, and plot are small and unclear. (11) P.11. Figure 6: The circuit model is unclear and unreadable. We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below.
Dear Dr Tran Thi Thuy: Title: Effect of pH regulation by microbes on corrosion behaviour of duplex stainless steel 2205 in acidic artificial seawater environment Manuscript ID: RSOS-200639 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. ********************************************** RSC Associate Editor: Comments to the Author: Quality assurance and control must be clearly mentioned in the Materials and Methods section.

RSC Subject Editor:
Comments to the Author: (There are no comments.) ********************************************** Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) This paper is focusing on identifying the effect of pH on the corrosion of duplex stainless steel 2205 in a nutrient rich artificial seawater medium containing Sulphate reducing bacteria species, namely Desulfovibrio vulgaris. The manuscript contains original results and deserves to be published after minor revision. Here are my comments and observations: 1. Page 3, last line: The title "Analytical methods" does not reflect the content of the section. Maybe something is missing… 2. Page 4, lines 49-52: I suggest moving the phrase "In this paper, all the solution samples changed to pH 7.5 so the term pH 4, pH 5, pH 6, pH 7.4 refers to the initial pH of the samples…" to the previous section where pH level is discussed. 3. Figure 3. The parameter on the x axis should be "Time" expressed in days, like in Figure 1, 2 and 4. 4. Page 5, line 27. "QCPE is the CPE parameter" is not clear. Q is the pseudo-capacitance corresponding to CPE. 5. Page 6. For clarity reasons, I suggest writing the chemical reactions in the same line with the corresponding expressions of e. (The notation "E" is maybe better than "e") 6. Table 1. E should be expressed in mV vs. Ag/AgCl electrode; Icorr should be expressed with the same number of decimals (3 are sufficient). 7. Figure 5. The error bars are not visible; please write on the x-axis "pH" instead "ph" 8. Figure 6. The impedance spectra should be orthonormal (same values on both axes). If the lines represent the curves simulated with the equivalent electrical circuit, this should be mentioned in the legend of the figure. 9. The conclusions should be more detailed.

Reviewer: 2
Comments to the Author(s) It seems to be a meaningful result of MIC research, I would like you to consider the following some points.
Although the SRB used in this study seems to be a popular corrosive strain, the experimental results hardly mentioned the corrosive effect caused by this strain, but rather emphasized the corrosion inhibitory effect.
Based on the above, ①In the experiments using the same strain, there are some results showing the reproduction of MIC even in neutral pH culture conditions. What is the difference between these results and yours? ②In order to discuss the effects of corrosion under acidic conditions, it should be preferable to set the same pH abiotic experimental condition as a control. Could you tell me the reason why? I would like you to clarify your views and position regarding these. In addition, clarify the novelty and purpose of research more than in the introduction At the same time, I would like it reflected in the title of the paper. Based on these, I would like to request that the research novelty and purpose be made clearer in the introduction part. Similarly, research content should be reflected more clearly in the title of the paper.
Some items pointed out are specifically described below.
(1) Some keywords such as SRB or anaerobic should be included in the title.
(4) P1, l.41: Does Sulfide mean iron sulfide? (5) P1, l.48: In this study, while the suppression of corrosion by biofilm produced by SRB is discussed, there is few mentions about the corrosion phenomenon assisted by SRB. Usually it is easy to find the corrosion phenomenon assisted by SRB (may be same strain) and occurred in neutral pH condition, in some literature. Please see also (8).

RSOS-200639.R1 (Revision)
Review form: Reviewer 1 Is the manuscript scientifically sound in its present form? Yes

Comments to the Author(s)
The answers of the authors to the reviewers' comments are satisfactory. Consequently, I recommend the publication of the manuscript in its present form.

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
This paper is an important contribution and I recommend that it be accepted for publication. However, the numbering notation in Figure 8 is small and difficult to decipher. I would like to request a clearer letter.

Decision letter (RSOS-200639.R1)
We hope you are keeping well at this difficult and unusual time. We continue to value your support of the journal in these challenging circumstances. If Royal Society Open Science can assist you at all, please don't hesitate to let us know at the email address below. ******** RSC Associate Editor: Comments to the Author: Your paper has been recommended for publication. However, please be aware that Reviewer 2 has made a minor but important comment that must be attended before during proofreading.

RSC Subject Editor:
Comments to the Author: (There are no comments.) ********* Reviewer(s)' Comments to Author: Reviewer: 1 Comments to the Author(s) The answers of the authors to the reviewers' comments are satisfactory. Consequently, I recommend the publication of the manuscript in its present form.

Reviewer: 2
Comments to the Author(s) This paper is an important contribution and I recommend that it be accepted for publication. However, the numbering notation in Figure 8 is small and difficult to decipher. I would like to request a clearer letter. ***************************

RSC Associate Editor:
We would like to thank you for your constructive comments on the manuscript. We have made changes based on your comments as indicated below in our responses to your comments: Quality assurance and control must be clearly mentioned in the Materials and Methods section.

Response:
A new section has been added to the Materials and Methods section.

Quality Assurance and Control
All the experiments were conducted in the University molecular lab is a certified PC2 lab which undergoes yearly inspection by the Institutional Biosafety Committee. The ICPMS tests are calibrated in the lab that follows documented analytical protocols based on USEPA methodology and other published methodology and includes extensive Quality Control analyses with every sample batch. The lab subscribes to QUASIMEME (Quality Assurance of Information for Marine Environmental Monitoring in Europe) which is a subscription based international quality assurance program for marine environmental monitoring, and the lab participates actively since 1997. Laboratory performance studies include four rounds per annum of 'blind' analysis of seawater, sediment and biota samples provided by QUASIMEME. Submitted data is statistically assessed to provide an external quality assurance for chemical measurements in the marine environment. The lab's performance in QUASIMEME is consistently of very high quality. The potentiostat was calibrated using internal and external dummy cells provided by the instrument manufacturer. The pH meter was calibrated using standard buffer solutions. Hach DR 3000 Colorimeter was calibrated using standard samples and reagents provided by Hach. The JEOL SEM-EDX was calibrated and serviced regularly by a JEOL technician.
The term constant phase element (CPE) was introduced to the model. It presents the deviation from true capacitive behaviour. CPE usually substitutes the capacitance in ECs because of the inhomogeneous conditions (e.g. electrode roughness, coating, and distribution of reaction rate). Its admittance and impedance were defined by following equations respectively: Where Y0 is the magnitude of the CPE, j is the imaginary number, is the angular frequency and n is CPE power index (n < 1).
5. Page 6. For clarity reasons, I suggest writing the chemical reactions in the same line with the corresponding expressions of e. (The notation "E" is maybe better than "e")

Response:
The notation has been changed from "e" to "E" in the manuscript as shown below:  Response: This has been corrected in the manuscript as shown below: 7. Figure 5. The error bars are not visible; please write on the x-axis "pH" instead "ph"

Response:
The size of the figure has been increased to make the error bars visible. We have also corrected the term "ph" in Fig. 5.
8. Figure 6. The impedance spectra should be orthonormal (same values on both axes). If the lines represent the curves simulated with the equivalent electrical circuit, this should be mentioned in the legend of the figure.

Response:
We have changed both axes to the same values and mentioned the equivalent electrical circuit in the caption.

The conclusions should be more detailed.
Response: More details have been added in the conclusions section as shown below: "The effect of pH on corrosion behaviour of duplex stainless steel in acidic seawater environment was evaluated by observing pH level, dissolved sulphide level, OCP, bacteria population, EIS, potentiodynamic polarization, SEM-EDX and ICPMS. The main conclusions obtained from this work are presented below: -The results showed the environmental pH was changed by SRB after around 5 days -The highest iron concentration was at pH 4 and this was 3 times higher than at pH 7.4 indicating increased release of iron due to corrosion at lower pH -EIS results showed the film resistance of the specimen at pH 4 was much lower than at pH 7.4 which suggests the corrosion resistance of the duplex stainless steel was better at pH 7.4 than at pH 4.
-Corrosion current density was higher at first time immersion and reduced with time of exposure due to SRB activities. This suggests that SRB pH regulation activities could slow down the corrosion processes of duplex stainless steel in very low pH environments." We would like to thank you for your constructive comments on the manuscript. We have made changes based on your comments as indicated below in our responses to your comments.
It seems to be a meaningful result of MIC research, I would like you to consider the following some points.
Although the SRB used in this study seems to be a popular corrosive strain, the experimental results hardly mentioned the corrosive effect caused by this strain, but rather emphasized the corrosion inhibitory effect.

Response:
The SRB species, Desulfovibrio vulgaris, was chosen because is a well-known corrosive strain. Our study's focus was on the impact of starting pH conditions on the MIC and thus the overall effect on corrosion is considered including inhibitory effect and pitting corrosion. Based on the above, ①In the experiments using the same strain, there are some results showing the reproduction of MIC even in neutral pH culture conditions. What is the difference between these results and yours?

Response:
Our results support previous results in neutral pH condition [1-4] and we have discussed the neutral pH condition in the context of the culture in low pH environment. We have made this clearer in the introduction at final paragraph as shown below: "Previous report has indicated that environmental pH has significant effect on corrosion of materials not only in abiotic environment [11][12][13][14][15], but also in microbial environment [16][17][18]. However, the recent literature has focussed mostly on MIC behaviour of duplex stainless steel at a neutral pH [19][20][21][22]. Corrosion on stainless steel caused by SRB in different pH environments has received less attention. The effect of pH on the corrosion rate of carbon steel in SRB medium was studied in previous literature [17]. But parameters important in understanding MIC mechanisms were not recorded, such as the change in pH during time and the growth of bacteria. Several previous studies show that some Desulfovibrio species activities are inhibited at pH below 5 while some other Desulfovibrio species have ability to grow well even at very low pH environment [1,4]. In this study, the parameters including changes in OCP, pH, bacteria concentration, dissolved sulphide concentration were measured. The objective of this research was to conduct a comprehensive study on the corrosion behaviour of duplex stainless steel in different pH environments containing SRB." ②In order to discuss the effects of corrosion under acidic conditions, it should be preferable to set the same pH abiotic experimental condition as a control. Could you tell me the reason why?
Response: There are two reasons for not conducting the experiments under abiotic conditions. Firstly, the study emphasises the comparison of the corrosion behaviour between acidic environments and thus relative differences between them can be analysed without the need for abiotic experiments. Secondly, there are several previous studies on corrosion of stainless steel in abiotic environment under acidic condition which can be used for discussing the results of this study.
In the manuscript, we have referred to these studies [5][6][7][8][9]. I would like you to clarify your views and position regarding these. In addition, clarify the novelty and purpose of research more than in the introduction At the same time, I would like it reflected in the title of the paper. Based on these, I would like to request that the research novelty and purpose be made clearer in the introduction part.

Response:
We have added sentences in the introduction to make the research novelty and purpose clearer in the final paragraph of Introduction section as shown above in ①.