Active pathways of anaerobic methane oxidization in deep-sea cold seeps of the South China Sea

ABSTRACT Anaerobic oxidation of methane (AOM) is critical for controlling methane emissions from deep-sea cold seeps. Microbial groups associated with different AOM processes have been briefly investigated, but the AOM activities associated with the utilization of different electron acceptors in the deep-sea methane seeps remain largely unknown. Here, surface sediments (0–4 cm) from two cold seeps (Site F/Haima) and the Xisha trough in the South China Sea (SCS) were incubated to measure AOM potentials driven by nitrite, nitrate, and sulfate as different electron acceptors with isotopically labeled methane gas (13CH4), and the population shifts of microbial communities along the incubation were investigated by high-throughput sequencing and quantitative polymerase chain reaction based on both the 16S rRNA gene and functional genes. Nitrite and nitrate were preferentially used prior to sulfate during the incubation, and nitrate-/nitrite-dependent anaerobic methane oxidation (Nr-/N-DAMO) contributed more to the methane flux in both the cold seeps and the trough. The highest rates of DAMO and sulfate-dependent anaerobic methane oxidation (SAMO) were detected at Site F, consistent with the transcript abundance of mcrA and dsrB genes during the incubation, and might be explained by the in situ active functional groups. The former process might be explained by Campylobacteria, which could couple sulfide oxidation and nitrate consumption to further participate in the N-DAMO process, while the latter process might be due to the higher proportions of Desulfobacterota. This study elucidates the diversity and potential activities of major microbial groups in the DAMO and SAMO processes in the deep-sea cold seeps using isotopic tracing incubations and provides direct evidence for the occurrence of Nr-/N-DAMO as a previously overlooked microbial methane sink in the deep-sea hydrate-bearing sediments in the SCS. IMPORTANCE Cold seeps occur in continental margins worldwide and are deep-sea oases. Anaerobic oxidation of methane is an important microbial process in the cold seeps and plays an important role in regulating methane content. This study elucidates the diversity and potential activities of major microbial groups in dependent anaerobic methane oxidation and sulfate-dependent anaerobic methane oxidation processes and provides direct evidence for the occurrence of nitrate-/nitrite-dependent anaerobic methane oxidation (Nr-/N-DAMO) as a previously overlooked microbial methane sink in the hydrate-bearing sediments of the South China Sea. This study provides direct evidence for occurrence of Nr-/N-DAMO as an important methane sink in the deep-sea cold seeps.

My main concern about this manuscript is the description of the potential AOM rate.I would suggest that the authors should reorganize both parts of 3.1 and 4.1, and the quality of the figures needs to be improved.line 246-247, It's hard for me to come to that conclusion that "the production of 13CO2 in all sediments amended with different electron acceptors was always higher in the cold seeps than that in the trough during".As shown in Fig. 2 (SQ261,SQ82 and SQ81) that the highest observed values of 13CO2 of "13CH4" is higher than those of the groups with nitrate and nitrite.And according to the authors' statement on the calculation of the potential AOM rate, the potential AOM rate of "13CH4" in samples SQ261, SQ82 and SQ81 should also be higher than those of the experimental groups with added nitrate and nitrite.However Fig. 3 shows the opposite result.It is not surprising that the experimental group "13CH4" without any added electron acceptors showed a potential AOM rate during incubation, since some of the substances originally contained in the sediment samples, such as humic acid, can act as electron acceptors for AOM.
Reviewer #3 (Comments for the Author): 1.Line 145: For the determination of 13CO2 from 13CH4, a blank control (which was added with equal of 12CH4) should be designed.2.Line 220: please provide a reference for the determination method or a brief procedure for the determination.3.The statistical analysis of the data should be described in detail.4.Line 225:Please provide a link to the web page where the data is deposited.

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Dear editor and reviewers,
We are sincerely grateful for your comments and modified throughout the text accordingly.Please see below the details responds.
Reviewer #1 (Comments for the Author): My main concern about this manuscript is the description of the potential AOM rate.I would suggest that the authors should reorganize both parts of 3.1 and 4.1, and the quality of the figures needs to be improved.As shown in Fig. 2 (SQ261, SQ82 and SQ81) that the highest observed values of 13CO2 of "13CH4" is higher than those of the groups with nitrate and nitrite.And according to the authors' statement on the calculation of the potential AOM rate, the potential AOM rate of "13CH4" in samples SQ261, SQ82 and SQ81 should also be higher than those of the experimental groups with added nitrate and nitrite.However Fig. 3 shows the opposite result.

Response
Response: In Fig. 2, 13 CO 2 production in slurries of sediment amended with 13 CH 4 and different electron acceptors during 14-day incubations was shown.
While in Fig. 3 potential AOM rate in different samples and regions was shown.
The potential AOM rate calculation was based on the 13 CO 2 production from D3 to D7 of cultivation.Because the production of 13 CO 2 increased rapidly from D3 to D7 during the incubation with various electron acceptors addition, and obvious consumption of nitrite appeared after 3-day incubation (Fig. 4A).And the potential AOM rate of " 13 CH 4 " in sample SQ261 was higher than that of the experimental groups with added nitrate.The potential AOM rate of " 13 CH 4 " in sample SQ81 was higher than that of the experimental groups with added nitrite and sulfate.The potential AOM rate of " 13 CH 4 " in sample SQ82 was lower than that of the experimental groups with added nitrate, nitrite and sulfate.

According to above information, Figs. 2 and 3 in fact showed consistent patterns.
It is not surprising that the experimental group "13CH4" without any added electron acceptors showed a potential AOM rate during incubation, since some of the substances originally contained in the sediment samples, such as humic acid, can act as electron acceptors for AOM.
Response: Yes, the reviewer is correct.We admit that some of the substances originally contained in the sediment samples (such as humic acid, iron, manganese and etc.) may act as electron acceptors for AOM.
Reviewer #3 (Comments for the Author): 1 .Line 145: For the determination of 13 CO 2 from 13 CH 4 , a blank control (which was added with equal of 12 CH 4 ) should be designed.
Response: This study focused on the microbial communities and rates of SAMO and DAMO processes, and to elucidate the utilization of different electron acceptors (nitrite, nitrate and sulfate).The types of electron acceptors were independent variable, so " 13 CH 4 " was used as the control group, while " 12 CH 4 " control group was not set up.This is consistent with the previous studies (Shen et al., 2016; Chen et al. 2021).In addition, the 13 CO 2 produced in the original background would be present in each treatment group (different electron acceptors) as well, thus should have no effect on the trend in each treatment group.

Refs:
Shen, L. D., Hu, B. L., Liu, S., Chai, X. P., He, Z. F., Ren, H. X., Liu, Y., Geng, S., Wang, W., Tang, J. L., Wang, Y. M., Lou, L. P., Xu, X. Y., Zheng, P., 2016.Anaerobic methane oxidation coupled to nitrite reduction can be a potential methane sink in 3. The statistical analysis of the data should be described in detail.Thank you for your great efforts in revising the manuscript according to the comments of two reviewers.I found your revision is satisfying.Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.

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Sincerely, Jianjun Wang Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred : According to the reviewer's suggestion, the description of the potential AOM rate has been re-organized in parts of 3.2 and 4.1 of the revised manuscript.In addition, the figures were compressed with reduced visualization in the previous version.All the figures have been checked and improved with higher resolution in the revised manuscript.line 246-247, It's hard for me to come to that conclusion that "the production of 13CO2 in all sediments amended with different electron acceptors was always higher in the cold seeps than that in the trough during".Response: The average production of 13 CO 2 in the Haima cold seeps (SQ56, SQ58, SQ81) and Site F cold seeps (SQ63, SQ261, SQ264) after14-day incubation was 3.69 nmol g -1 [wet sediment] and 6.20 nmol g -1 [wet sediment], respectively.While in the trough (SQ82, SQ84, SQ87), it was 3.53 nmol g -1 [wet sediment].Based on the above results, we come to this conclusion.The above information was added in lines 256-259 of the revised manuscript.

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Thanks for the suggestion.The statistical analysis of the data has been described in detail in the line 228-236 of the revised manuscript.4. Line 225:Please provide a link to the web page where the data is deposited.Response: The link to the web page where the data was deposited has been added in the line 239 of the revised manuscript.Thanks for the suggestion.jing Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, Sanya No.28 Luhuitou road Sanya China Re: Spectrum02505-23R1 (Active pathways of anaerobic methane oxidization in deep-sea cold seeps of the South China Sea) Dear Prof. hongmei jing: