Beyond Cultivation: Combining Culture-Dependent and Culture-Independent Techniques to Identify Bacteria Involved in Paint Spoilage

Round 1

Reviewer 1 Report

In the manuscript “Beyond cultivation: combining culture-dependent and culture-independent techniques to identify Bacteria involved in paint spoilage” the authors combined traditional culture-dependent techniques with subsequent sequencing, quantitative qPCR and a serial quantification method (most probable number; MPN) to detect paint degrading Bacteria in general and sulfate-reducing bacteria (SRB) in particular. This manuscript is well organized, and the drawn conclusions are coherent with the obtained results. The references should be updated to include more recent studies.

 Lines 25: The keywords should be arranged alphabetically.

Lines 53 – 58: The authors should be better explain their predictions and hypothesis.

Lines 47 – 48: I think that you should add this more recent and interesting reference to support your sentence: “Furthermore, bacteria embedded in biofilms are more resistant to desiccation and treatment with biocides”. I would like to suggest:

Bosso, L., and Cristinzio, G. (2014). A comprehensive overview of bacteria and fungi used for pentachlorophenol biodegradation. Reviews in Environmental Science and Bio/Technology, 13, 387-427.

Lines 101 – 105: I think that you should add this more recent and interesting reference to support your methods: “DNA was extracted using the NucleoSpin® Microbial DNA Kit (Macherey-Nagel, Germany) according to the manufacturer's instructions. The integrity of the DNA was determined on a 1.0% agarose gel and the quantity and purity of the extracted DNA was evaluated via UV-vis spectrophotometry with NanoDrop 2000cTM (PeqLab, Germany)”. I would like to suggest:

Bosso, L., Lacatena, F., Varlese, R., Nocerino, S., Cristinzio, G., & Russo, D. (2017). Plant pathogens but not antagonists change in soil fungal communities across a land abandonment gradient in a Mediterranean landscape. Acta Oecologica, 78, 1-6.

 

Lines 261 – 277: Please, expand the conclusion including also future research.

Moderate editing of English language

Author Response

Review report:

In the manuscript “Beyond cultivation: combining culture-dependent and culture-independent techniques to identify Bacteria involved in paint spoilage” the authors combined traditional culture-dependent techniques with subsequent sequencing, quantitative qPCR and a serial quantification method (most probable number; MPN) to detect paint degrading Bacteria in general and sulfate-reducing bacteria (SRB) in particular. This manuscript is well organized, and the drawn conclusions are coherent with the obtained results. The references should be updated to include more recent studies.

 

The authors would like to thank the reviewer for the valuable input. All suggestions have been incorporated in the revised manuscript. All comments were answered step by step (highlighted in bold letters) with listed changes and corresponding line numbers (associated with the clean version) and a revised manuscript version including “track changes” were uploaded.

We would be very pleased if the revised version of our manuscript now comes up to your expectations and is suitable for publication in Coatings.

We appreciate your time and look forward to your response.

• Lines 25: The keywords should be arranged alphabetically.

We changed the keywords accordingly.

• Lines 53 – 58: The authors should be better explain their predictions and hypothesis.

Thank you very much for this helpful comment. We added more information regarding our hypothesis and predictions (lines 66ff.):

Our hypothesis tested was that culture-independent techniques will not only broaden our knowledge on important key players associated with paint spoilage as they can capture yet undetected, non-culturable and anaerobic microorganisms but also expand the repertoire of current detection techniques by rapid and reproducible methods particularly regarding difficult matrices such as paint.

• Lines 47 – 48: I think that you should add this more recent and interesting reference to support your sentence: “Furthermore, bacteria embedded in biofilms are more resistant to desiccation and treatment with biocides”. I would like to suggest: Bosso, L., and Cristinzio, G. (2014). A comprehensive overview of bacteria and fungi used for pentachlorophenol biodegradation. Reviews in Environmental Science and Bio/Technology, 13, 387-427.

Thanks to the reviewer. We incorporated the suggested literature in the revised version of the manuscript (lines 49ff.):

In this context, Bosso and Cristinzio [9] studied the tolerance of bacteria and fungi to pentachlorophenol, which is present in various paints as antifouling agent and were able to observe various strategies like exclusion from the cell, conversion into a non-toxic compound or by using it as the sole carbon source. These microorganisms comprised (amongst others) well-known paint degraders including aerobic and anaerobic species of the genera Bacillus, Pseudomonas, and Desulfitobacterium as well as Penicillium, Phanerochaete and Rhizopus [9].

• Lines 101 – 105: I think that you should add this more recent and interesting reference to support your methods: “DNA was extracted using the NucleoSpin® Microbial DNA Kit (Macherey-Nagel, Germany) according to the manufacturer's instructions. The integrity of the DNA was determined on a 1.0% agarose gel and the quantity and purity of the extracted DNA was evaluated via UV-vis spectrophotometry with NanoDrop 2000cTM (PeqLab, Germany)”. I would like to suggest: Bosso, L., Lacatena, F., Varlese, R., Nocerino, S., Cristinzio, G., & Russo, D. (2017). Plant pathogens but not antagonists change in soil fungal communities across a land abandonment gradient in a Mediterranean landscape. Acta Oecologica, 78, 1-6.

Thank you for the interesting reference, we supplemented the Material and Methods section with the suggested literature to support our methods (please refer to lines 95-96).

• Lines 261 – 277: Please, expand the conclusion including also future research.

Thank you for this constructive feedback. The content of the conclusions has undergone expansion regarding future research (lines 284ff.):

As paint spoilage is still considered a “black box” with essential information about preventing biodeterioration still being missed, future investigations should also focus on other bacterial groups like iron bacteria as well as fungi by using culture-independent approaches such as targeted amplicon sequencing and metagenomics along with Fourier-transform infrared spectroscopy (FTIR) analyses that can help to elucidate major structural changes or modifications of contaminated water-based as well as oil-based paint, particularly interesting during storage.

 

Reviewer 2 Report

The main question addressed by the research is detection of paint degrading Bacteria in general and sulfate-reducing bacteria in particular by the determination of colony forming units approach.

The topic is original and relevant to the field. So far there is a lack of combination of culture-dependent and culture-independent physico-chemical techniques applied within the same study. This study, therefore, fills this gap, as it extensively utilizes both these approaches. The work uses the state-of-the art sample preparation techniques and the combination of complementary research tools.

As a result of bridging this gap, and compared to already conducted works in the field, this study offers quite comprehensive knowledge that has closer relevance to its application for the production of paints meeting the modern standards.

Despite the fact that the methodology used gives quite complete and self-consistence novel information, it can, nevertheless, be improved if the results obtained were added by the molecular level information on how the molecular content (fragments) of the paints changes upon their degradation obtained, for instance, by FTIR.

Conclusions are totally consistent with the evidence presented and they do address the main issues of the work. They are self-consistent and allow to understand the main message of the study.

Referencing is appropriate both in terms of the completeness of the coverage of the topic, and in taking into account the current state-of-the art in this area of research.

In general, given the scientific quality of the work, clearness and accuracy of its presentation, compliance with the subject of the journal, it is suitable for publication in Coatings in its present form.

Author Response

Revision report:

The main question addressed by the research is detection of paint degrading Bacteria in general and sulfate-reducing bacteria in particular by the determination of colony forming units approach.

The topic is original and relevant to the field. So far there is a lack of combination of culture-dependent and culture-independent physico-chemical techniques applied within the same study. This study, therefore, fills this gap, as it extensively utilizes both these approaches. The work uses the state-of-the art sample preparation techniques and the combination of complementary research tools.

As a result of bridging this gap, and compared to already conducted works in the field, this study offers quite comprehensive knowledge that has closer relevance to its application for the production of paints meeting the modern standards.

Despite the fact that the methodology used gives quite complete and self-consistence novel information, it can, nevertheless, be improved if the results obtained were added by the molecular level information on how the molecular content (fragments) of the paints changes upon their degradation obtained, for instance, by FTIR.

Conclusions are totally consistent with the evidence presented and they do address the main issues of the work. They are self-consistent and allow to understand the main message of the study.

Referencing is appropriate both in terms of the completeness of the coverage of the topic, and in taking into account the current state-of-the art in this area of research.

In general, given the scientific quality of the work, clearness and accuracy of its presentation, compliance with the subject of the journal, it is suitable for publication in Coatings in its present form.

 

The authors would like to thank the reviewer for the valuable feedback and time.

We agree with the reviewer that FTIR analyses would add additional information regarding changes and/or modifications of spoiled paints, particularly during storage and in comparison, with uncontaminated paints. We supplemented the Conclusion section dealing with future research (as also suggested by other reviewers) and incorporated FTIR analyses (lines 284ff.):

As paint spoilage is still considered a “black box” with essential information about preventing biodeterioration still being missed, future investigations should also focus on other bacterial groups like iron bacteria as well as fungi by using culture-independent approaches such as targeted amplicon sequencing and metagenomics along with Fourier-transform infrared spectroscopy (FTIR) analyses that can help to elucidate major structural changes or modifications of contaminated water-based as well as oil-based paint, particularly interesting during storage.

Reviewer 3 Report

The approach taken by the authors is great and what makes it even more interesting is application of MPN/CFU with qPCR. The research As the authors have already mentioned, it is worth to develop the MMM tests using other culture-independent methods such as NGS. 

I would like to suggest to also examine other bacterial groups such as iron bacteria. It will also be good if you consider in your future works the possibility of microbial contamination of oil-based paints.

Lines195 to 209 are repetitive and needs to be adjusted and modified. Please explain what is exactly meant by "recalcitranting VOC" in line 209.

Author Response

Revision report:

The approach taken by the authors is great and what makes it even more interesting is application of MPN/CFU with qPCR. The research As the authors have already mentioned, it is worth to develop the MMM tests using other culture-independent methods such as NGS. 

I would like to suggest to also examine other bacterial groups such as iron bacteria. It will also be good if you consider in your future works the possibility of microbial contamination of oil-based paints.

The authors thank the reviewer for the helpful comments and time! We agree with the reviewer that other bacterial groups such as iron bacteria as well as the analysis of oil-based paints is highly interesting and we keep this valuable information in mind. Furthermore, we supplemented the Conclusion section dealing with future research (as also suggested by other reviewers) and added the investigation of iron bacteria and oil-based paints as well as NGS analyses to this section (lines 284ff.):

As paint spoilage is still considered a “black box” with essential information about preventing biodeterioration still being missed, future investigations should also focus on other bacterial groups like iron bacteria as well as fungi by using culture-independent approaches such as targeted amplicon sequencing and metagenomics along with Fourier-transform infrared spectroscopy (FTIR) analyses that can help to elucidate major structural changes or modifications of contaminated water-based as well as oil-based paint, particularly interesting during storage.

• Lines 195 to 209 are repetitive and needs to be adjusted and modified. Please explain what is exactly meant by "recalcitranting VOC" in line 209.

We would also like to thank the reviewer for the accurate reading of the manuscript. We deleted lines 218-224 (associated with the version containing track changes) as they were indeed repetitive. Also, “recalcitrating VOCs” was better explained in the revised version of the manuscript (lines 213ff.):

Besides, Pseudomonas spp. are able to attack cellulosic derivates next to phenolic resins present in binders and -stable and difficult to degrade- volatile organic compounds (VOCs) such as toluene used as paint thinners.

Reviewer 4 Report

Comments to the Author

This manuscript investigated the combination of culture-dependent techniques with subsequent sequencing, quantitative qPCR, and a serial quantification method to detect paint-degrading bacteria and sulfate-reducing bacteria. The authors’ findings are interesting and worthy of publication after consideration of the following:

line 14 replaces “Bacteria” with “bacteria”

line 37 “(as well as fungi): please remove brackets

lines 73-74 nutrient agar: instead of writing the component of the medium write the company and country of the medium.

lines 75-76 “Morphologically different colonies were aseptically picked from the Petri dishes, amplified via PCR using the Red Taq DNA Polymerase Mastermix”: before amplification, DNA extraction must be performed. please write the kits that were used in DNA extraction.

 

lines 89-90: please write the company and country of the components

Minor editing of English language required

Author Response

Revision report:

This manuscript investigated the combination of culture-dependent techniques with subsequent sequencing, quantitative qPCR, and a serial quantification method to detect paint-degrading bacteria and sulfate-reducing bacteria.

The authors would like to thank the reviewer for the valuable input. As far as possible all suggestions have been incorporated in the revised manuscript. All comments were answered step by step (highlighted in bold letters) with listed changes and corresponding line numbers (associated with the clean version) and a revised manuscript version including “track changes” were uploaded.

We would be very pleased if the revised version of our manuscript now comes up to your expectations and is suitable for publication in Coatings.

We appreciate your time and look forward to your response.

• line 14 replaces “Bacteria” with “bacteria”

Was changed accordingly and corrected throughout the manuscript.

• line 37 “(as well as fungi): please remove brackets

The brackets were removed.

• lines 73-74 nutrient agar: instead of writing the component of the medium write the company and country of the medium.

Was changed accordingly.

• lines 75-76 “Morphologically different colonies were aseptically picked from the Petri dishes, amplified via PCR using the Red Taq DNA Polymerase Mastermix”: before amplification, DNA extraction must be performed. please write the kits that were used in DNA extraction.

Thank you for this comment. We did not perform DNA extraction as we performed Colony PCR with pure cultures. Therefore, a single colony on the agar plates was picked, which then served as template during PCR amplification. Furthermore, a prolonged initial denaturation step was included in the protocol (5 mins at 95 °C) to disrupt the cells. For a better clarification, we added some information regarding Colony PCR to the revised version of the manuscript (lines 86ff.):

Morphologically different colonies were aseptically picked from the Petri dishes and isolated, which then served as template during PCR amplification using the Red Taq DNA Polymerase Mastermix (2x, VWR, USA) and the universal bacterial primer pair 27f and 1492r (Eurofins Genomics, Germany). PCR conditions included an initial denaturation step at 95 °C for 5 min (the prolonged denaturation step intended to disrupt the cells), followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s, and elongation at 72 °C for 45 s. A final elongation step at 72 °C for 10 min completed the protocol.

• lines 89-90: please write the company and country of the components

Was changed accordingly.

Reviewer 5 Report

Dear authors,

congratulations on a very nicely written manuscript. I have no comments that would require revisions, I therefore accept your article as it is.

Author Response

Review report:

Dear authors,

congratulations on a very nicely written manuscript. I have no comments that would require revisions, I therefore accept your article as it is.

The authors would like to thank the reviewer for the valuable feedback and time.