Application of GRAS Compounds for the Control of Mould Growth on Scots Pine Sapwood Surfaces: Multivariate Modelling of Mould Grade

Round 1

Reviewer 1 Report

See attachement.

Comments for author File: Comments.pdf

Author Response

COMMENTS FROM REVIEWER #1:

Title: GRAS Compounds Application to Control Mould Growth on the Surface of Dried Scots Pine Sapwood: Multivariate Modelling of Mould Growth

 

Three ‘non-biocidal’ compounds/treatments were applied on wood to test their ability to inhibit or resist mould growth. One compound were found to more effective than the others. A multivariate model/PLS was developed to capture the importance of and reason for coverage of each of the fungal species on the mould grade.

There is a great need to seek out sustainable surface treatments that can be used on wood to enhance the properties in question – such as here: resistance or control of mould growth. This study tries to add to this picture, but it of fails in several ways.

 

Thank you very much for pointing the importance of the search for sustainable surface treatments and therefore the need for research we have done. We understand that some points of view of reviewer differ from ours, but we greatly appreciate for reviewer time and effort that will improve our manuscript significantly.

 

• I do not know what the end application of the treatments are. I get the feeling that the end applications is outdoor use – but this is not clearly mentioned. SIOO is an outdoor product. But the authors introduce indoor and health issues in the introduction – why?

We thank the reviewer for bringing this application in front, but in our study, the main point is a GRAS nature of treatments that might be useful in outdoor and indoor applications. However, we added the clarification to that question in Line 37-42 of the manuscript.

• The experimental set-up seems quite incidental and immature.

o Why are these two test set-up chosen (direct and indirect)?

A good comment. However, in the previous work that we cited in the current manuscript, we have seen the differences in different test set-ups for mould growth assessment. The reference for the paper is below. We found that those tests give different results regarding different fungi growth on the surface. Therefore, in the current manuscript, we would like to address those differences.

Myronycheva, O.; Sehlstedt-Persson, M.; Karlsson, O.; Sandberg, D. Growth of Mold and Rot Fungi on Copper-impregnated Scots Pine Sapwood: Influence of Planing Depth and Inoculation Pattern. BioResources 2018, 13, 8787-8801–8801.

o In what way will these set-ups be the best to get the results fitting to the end-application? Why are the following test mould fungi chosen: A.p., P.c. and P.v.?

According to the statement of Dr Jos Houbraken from Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, the Aspergillus sp., Penicillium sp. and Paecilomyces sp. are most important Ascomycete genera in food and indoor environment. Those fungi isolated as dominant from the simultaneous test done by Margot Sehlstedt-Persson and therefore we decided to check those fungi performance with selected treatments also.

o If the material is intended for outdoor use – blue stain fungi would be more natural to choose.

A good comment. We mentioned both applications and stressed the GRAS origin that might be universal. According to many studies the Aspergillus, Penicillium spp. are most economically important and deleterious, with food spoilage, mycotoxin production and biodeterioration heading the list and Paecilomyces spp. under attention last years due it similar relevance to human activities. We choose those species because we have seen their dominance in the simultaneous tests and decided to test their interactions on the wooden surface. All fungi from the same order Eurotiales and all cosmopolitans. We want to see if we can model the growth of selected fungi and therefore to develop a method available to predict fungal growth under GRAS surface treatments.

 

o The inoculation method should be described in the paper.

Thanks to this comment, the missing information is added in this section.

▪ Need information about sterilisation steps of the material and the test chambers

Added Line 106

▪ How old where the dishes for both the direct and indirect method?

Added Line 131-132

▪ What was the concentration of the spores?

Added Line 123

▪ Why was the incubation T/RH the first three days from the proceeding 25 day?

Added Line 138-139.

▪ How was the rating scale made up? Inspired of other standards? Why could you not use an existing standard?

Thank you for your comment. We added an additional grade and percentage of growth that we missed to show in the table in Line 143-146.

▪ Was it sterile conditions in the incubation chambers throughout the test period?

Added Line 133-134.

• The result part (especially line 154-264) is long and very badly structured – the read do not have the chance to follow and understand the results. Table 5 is not helping much. A considerable job has to be done to tidy and structure this part. For the modelling part – the authors has to reduce the number of figures – several of the are overlapping. Remember – only the most important result can be presented in a figure. The modelling approach do not give a lot of information that can be used to explain the results.

In order to address the concern of the reviewer, we structured the result part Line 172-267. Based on the comments provided by the reviewer, we could not find strong evidence for the judgement about Table 5 but we structured that part, please see Line 270. We agreed that some figures in the modelling part overload the content. Therefore we removed figures 4 and 5, and corrected manuscript respectively see Line 289-290. We, therefore, insist on keeping the modelling part of the paper because that approach was used the first time and shows the contribution of multivariate modelling for the mould tests improvement.

 

• English editing is very much needed. The authors even mixes the words ‘inoculation’ and ‘incubation’. And more accurate wording is needed in throughout the manuscript..

Thank you for the comment. We used Grammarly app for the proofreading and accurate wording checked.

 

• The whole paper needs to be better structured, have a clearer aim, followed up with more precise test setup that targets these aims, and a correct enhancing of the results that can contribute to the aim and research questions for a proper discussion.

I also lack references to many important studies/works performed mould growth/blue stain on coated and uncoated wood (different wood substrates). The authors should absolutely consider to read/review/include mould growth articles of the following authors; Stig Bardage, Antje Gellerich, Hannu Viitanen, Lone Ross Gobakken, Solrun Karlsen Lie (and probably more).

We addressed the concern of the reviewer and significantly improved our manuscript by structuring and rewriting. However, it is not easy to describe highly variable data like in our study, and we demonstrated that instead of a complicated description of univariate analysis results, the multivariate approach is more useful in the description and modelling of mould test that makes a step forward in the understanding of biodegradation phenomenon.

Regarding the references, we strongly consider the effort on mould growth of articles stated by reviewer authors, but they do not refer to fungal interactions and their differences. Moreover, more attention has done to the standard test in the material evaluation and moisture that we stated in Line 68.

 

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript presents an original work supported by a sound methodology and clearly presented results. The reviewer would suggest that the section discussing the methods used could be improved in order to increase clarity. More precisely, in lines 146-150, it would be beneficial for the reader to have a short description of each method followed by the reason why they are selected in this context. Moreover, the reviewer suggests to include a paragraph/section in the discussion section where the implications and relevance of the study are more evident for the industry. The latter has been shorty introduced in the conclusion section.

More specific minor comments:

Line 92 :  A reference is required for the method used.

Line 130: A reference is required.

140: Fig 3 Typo, sex=six?

Line 18-170: Reformulate sentence to provide clarity.

Author Response

COMMENTS FROM REVIEWER #2:

The manuscript presents an original work supported by a sound methodology and clearly presented results. The reviewer would suggest that the section discussing the methods used could be improved in order to increase clarity. More precisely, in lines 146-150, it would be beneficial for the reader to have a short description of each method followed by the reason why they are selected in this context. Moreover, the reviewer suggests to include a paragraph/section in the discussion section where the implications and relevance of the study are more evident for the industry. The latter has been shorty introduced in the conclusion section.

Thanks for this excellent comment. We agree with the reviewer and are aware of the issue. We are working in this direction. A sentence in section 5 (page 13 line 366-367) has been modified to address this point.

 

More specific minor comments:

Line 92 : A reference is required for the method used.

Thank for your attention. It is Line 100 now, and the reference added.

 

Line 130: A reference is required.

The reference added see Line 147.

 

140: Fig 3 Typo, sex=six?

Thank for your attention. Changed, see Line 160

 

Line 168-170: Reformulate sentence to provide clarity.

We reformulated the whole part. Please, see Line 192-194

 

Author Response File: Author Response.docx

Reviewer 3 Report

Material and methods, treatment of specimens, page 3/ 4:

Have you used an additional end grain sealer, or have you treated all sides of the specimens with the GRAS compounds? Please specify.

Material and methods, Mould test and assessment, page 4:

Why have you used these fungi? Standardised fungi or typical in Swedish environment or ubiquitous fungi? Please add a short explanation.

Material and methods, Mould test and assessment, page 4:

Why have you used two different inoculation methods? Please explain shortly.

Table 2, Rating 2: Conidiophores*, please explain * or delete

Material and methods, Mould test and assessment, page 5: ImageJ, which software version was used? Please add a short information.

Results, Table 4, page 6:

Moisture content below FSP? You explained differences of mould growth between bark and pith side because of the nutrient content, particularly in case of Aspergillus. But the moisture content was also below FSP or just around FSP after test. Maybe you have also an influence because of moisture gradients within the sample? Have you determined the moisture content only for the whole sample or have you also measured the moisture content in different areas of the sample?

Furthermore, has the silicon treatment also an influence on wood moisture content particulary in the surface area of the sample? That could be also an influence beside the effect of a physical barrier.

Author Response

COMMENTS FROM REVIEWER #3:

We appreciate the reviewer’s understanding of the work and comments on the manuscript. We have considered all the comments, including the minor ones, as shown by our responses below.

 

Material and methods, treatment of specimens, page 3/ 4:

Have you used an additional end grain sealer, or have you treated all sides of the specimens with the GRAS compounds? Please specify.

No end sealing used — this information added to the section (Line 123-124).

 

Material and methods, Mould test and assessment, page 4:

Why have you used these fungi? Standardised fungi or typical in Swedish environment or ubiquitous fungi? Please add a short explanation.

Thank you for the suggestion. The explanation added in Line 136-137.

 

Material and methods, Mould test and assessment, page 4:

Why have you used two different inoculation methods? Please explain shortly.

Good comment. We added the explanation in the Line 138-140.

 

Table 2, Rating 2: Conidiophores*, please explain * or delete

Deleted.

 

Material and methods, Mould test and assessment, page 5: ImageJ, which software version was used? Please add a short information.

The explanation added in Line 158.

 

Results, Table 4, page 6:

Moisture content below FSP? You explained differences of mould growth between bark and pith side because of the nutrient content, particularly in case of Aspergillus. But the moisture content was also below FSP or just around FSP after test. Maybe you have also an influence because of moisture gradients within the sample? Have you determined the moisture content only for the whole sample or have you also measured the moisture content in different areas of the sample?

Furthermore, has the silicon treatment also an influence on wood moisture content particulary in the surface area of the sample? That could be also an influence beside the effect of a physical barrier.

Yes. The moisture content of our samples in the test was over FSP, and we mentioned that in the discussion section (344-347). We measured moisture content in the whole sample but not a moisture gradient in different areas of the specimens. We want to have an excess of water in our samples in order to maximum promote fungal growth.

We have a concern regarding silicon treatment. In our latest tests, we checked the Phase 1 of that treatment. The average moisture content was extremely high compared to control samples. The question of uniform distribution of treatment by Phase 2 also raised. We can see from field tests that in some areas the fungal attack by Cladosporum sp. exist. So the physical barrier is a question and more study ongoing in order to find the reason for the fungal inhibition.

 

Author Response File: Author Response.docx