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This review incorporates comments and contributions from Femi Arogundade, Aneth David, Chen Yang, Teena Bajaj, Ahmad Bashiru, Randa Salah Gomaa Mahmoud, Gliday Yuka, and Adamolekun Emmanuel. The review was synthesized by Femi Arogundade.

Summary of the Study: 

The study focuses on the development and validation of a MicH-specific immunoassay to detect and monitor microbial-influenced corrosion (MIC) caused by certain methanogenic archaea. The assay demonstrates strong specificity for the MicH protein, a key component of the ‘MIC hydrogenase’ involved in corrosion, with minimal cross-reactivity with sulfate-reducing bacteria (SRB) or non-corrosive methanogens. The MicH protein and micH gene were detected in both biofilms and planktonic cells, broadening its potential application for field monitoring using water samples.

Positive Aspects of the Study:

• The study integrates laboratory and field conditions, enhancing its relevance and robustness

• The identification of both the micH gene and MicH protein in the Methanobacterium-like strain IM1 offer molecular insights into the mechanisms of corrosion caused by archaeon.

• The study employs qPCR for micH gene analysis, providing an effective method for quantifying microbial populations associated with corrosion processes. This approach is complemented by ATP quantification and gas chromatography for measuring methane production.

• The study employs recombinant antibodies to target the MicH protein linked to MIC, showing strong potential for precise MIC detection.

• The detection of MicH in both biofilms and planktonic cells broadens its practical application for MIC monitoring in challenging field conditions where biofilm samples are hard to collect.

Major Comments (Validity or Strength of the Methodology, Experiments, and Analyses, Strength of the Conclusions)

• In the development of recombinant antibodies, the statement regarding the selection of peptide sequences for the development of polyclonal antibodies ('For the initial development of polyclonal antibodies, two peptide sequences were chosen from the protein sequence of the target gene micH of the corrosive methanogen M. maripaludis strain OS7...') requires further clarification. The rationale for selecting these specific peptide sequences should be explained in the main text, including details on their location and conservation within the MicH protein. Additionally, the justification for choosing keyhole limpet hemocyanin (KLH) as the carrier protein over other common carriers should be provided. A figure showing the location of the selected peptides in the protein structure would also be helpful for visual clarity.

• The process of calculating the concentration from 20 pg/ml to 0.3 fg should be explained in detail.

• The method for calculating the signal-to-noise (S/N) ratio should be described.

• The basis for calculating 3 × 10^3 protein molecules should be shown.

• Validation of protein stability during sample processing needs to be addressed.

• The authors should discuss previously used assays or techniques and explain how their approach overcomes limitations and why it is better.

• The study could benefit from clarifying potential limitations of antibody use in complex biofilm matrices, where cross-reactivity and signal interference may reduce specificity.

• The variability of MicH expression across different methanogenic strains should be discussed to provide insight into the biomarker's applicability in diverse microbial populations.

• The conclusion should mention the need for further statistical analysis or confirmatory studies to verify the assay’s performance in varied field conditions.

Minor Comments (Clarifications to Statements in the Text, Interpretation of the Results, Presentation of the Data/Figures)

• Clarify whether the synthetic brine composition was specifically adjusted to a certain pH or salinity. While referencing earlier work is fine, key chemical details should be briefly pointed out to ensure clarity for readers unfamiliar with reference 5.

• Clarify how the anaerobic conditions were maintained during the collection of the produced water (e.g., was the water collected in an anaerobic chamber, or were the bottles purged with N2/CO2 immediately after collection to maintain anoxic conditions?).

• Provide clarification on how pipeline solids (pigging debris) and produced water were stored during transport to the lab.

• It is unclear how many replicates were used for enrichment cultures or corrosion bottle tests.

• The statement “After arrival in the destination lab, the cultures were incubated at 32°C for at least two weeks before being used as inoculum for corrosion bottle tests” is unclear. Why not provide a more precise duration or exact number of days the cultures were incubated?

• The location and conservation of the selected peptides within the MicH protein should be described.

• A figure showing the location of selected peptides in the protein structure would be helpful.

• Figure 2 - The 20 pg/ml value is not clearly identified in the figure.

• A table showing S/N ratios at different concentrations would be valuable.

• The statement, 'Protein extract of Methanobacterium-like strain IM1 obtained from cultures that inflicted significant corrosion of 0.3 mm/yr, showed a strong signal in the Western blot,' requires a more quantitative definition of 'strong' to improve the clarity of the result. Additionally, protein extraction efficiency validation data should be provided, results from cells at different growth phases should be included, and validation of protein stability during sample processing needs to be addressed.

• “The results confirm that the MicH-specific immunoassay selectively detects the MicH protein while showing low cross-reactivity with proteins extracted from non-corrosive methanogenic strain S2 and SRB pure cultures (Fig. 3 and Fig. S3A)". However, the definition of 'low cross-reactivity' should be quantified for clarity. Additionally, it would strengthen the study to include more negative control strains, perform cross-reactivity testing with other hydrogenases, and provide complete Western blot images, including molecular weight markers.

• “The ATP results indicated that biofilms have formed on steel surfaces to a varying degree, with generally higher numbers detected on corroded steel surfaces (+MIC; 278 – 71,299 pg/cm²) compared to less corroded ones (-MIC; 6 – 1,762 pg/cm²; Fig. 4B)”. A correlation analysis between ATP and MicH protein levels should be included to better understand their relationship. Additionally, given the wide range of ATP values, logarithmic scaling should be considered for data presentation. The large variation in ATP levels also needs further explanation, and the reliability of ATP as a biofilm formation indicator should be discussed.

• The authors should briefly discuss previously used assays or techniques and explain how this approach overcomes limitations and why it is better.

• The quantification of results could add value to the discussion section.

• A more thorough comparison of the current results with those from recent studies should be included.

Comments on Reporting (Information on the Statistical Analyses or Availability of Data)

• The statistical analysis for protein concentration detection appears adequate, though it would benefit from more detailed error margins in measurements.

• A dedicated statistical methods section, detailing the tests used and providing p-values for key comparisons, particularly between corrosive and non-corrosive samples, would strengthen the findings.

• A table showing S/N ratios at different concentrations would be valuable.

• Results from cells at different growth phases should be included.

• Protein extraction efficiency validation data should be provided.

• The authors should provide information on data availability to support replication or further research, particularly regarding the antibody development protocols.

Suggestions for future studies

• Future research could explore the potential of integrating this immunoassay with rapid field testing kits, such as lateral flow devices, to enhance practicality for on-site corrosion monitoring.

• Expanding the assay to target additional MIC-related biomarkers would provide a more comprehensive diagnostic tool.

• More studies are needed to ensure the reproducibility of the immunoassay results across different research laboratories and field sites.

• Given that MicH is linked to corrosion via hydrogenase activity, further studies are needed to understand how MicH interacts with the steel surface during corrosion processes. 

Competing interests

The authors declare that they have no competing interests.