Laboratory Investigations of Mold Growth on Transverse and Longitudinal Wood Surfaces
Round 1
Reviewer 1 Report
This study aims at quantifying and comparing mold growth in pine and spruce wood along three directions (transverse, tangential and radial) using commercial sapwood samples. Since wood is hygroscopic and is considered a valuable engineering material the study of the effects of fungal degradation are of great interest for general society (health issues and consumer’s needs) and is now gaining more attention with regards to the economic and environmental impacts (circular economy as well as green economy targets). However, the major concerns are related to the unclear scientific objectives of the study and the overall lack of scientific soundness (structure and terminology issues) of the ms. The scientific objectives should be clarified in introduction and afterwards the title, abstract, discussion and conclusions should be improved accordingly. It could be important a collaboration with researchers showing background on wood sciences, namely on structure and properties of lignocellulosic materials. Overall, the study and efforts to help improve and/or validate current mold models are acknowledged.
Unfortunately, it was somehow difficult to follow the all the manuscript and its scientific content. The manuscript should be rewritten improving English namely avoiding repeated and/or misplaced information, including the information related to the sampling only in Materials and Methods section, i.e., removing it from the discussion (for example). The mention in the Discussion section related to the observations (results) should not be repeated exactly as it was in the Results section. The terminology according to each scientific domain (microbiology, civil engineering and wood anatomy and wood science) should be more accurate. It is urgent to change “principal anatomical directions” from the title and along the ms. Wood is an anisotropic material and by simplification according to the standard procedures the wood structure and anatomy is studied along three directions: transverse, tangential and radial. The wood characterization is respectively studied in transverse, tangential and radial sections, or surfaces. Please check some wood technology literature and which would be the objective of the study. The scientific names for tree and mold species should be written in italics with the authority (when cited for the first time in the text). There is no need to refer “pine samples” and “spruce samples” so often. The “first microscopic signs”, “earliest macroscopic mold growth”, etc are of common use within these experiments?
It is urgent to address the interest and importance of using pine and spruce wood, including the scientific names of the studied samples. Is it Pine sylvestris and Picea abies? Which are the most traded pine and spruce species in Czech Republic? The same explanations are needed for the use of only sapwood. This aspect needs more attention and should be discussed later considering the results obtained since no heartwood samples were used to be compared. In fact, the authors refer in Conclusions that “It is also necessary to choose between sapwood and heartwood and define the surface quality of the samples.” However, this is not a conclusion since no heartwood was used in the study, and theoretically the wood durability between heartwood and sapwood is found elsewhere in literature (e.g. https://doi.org/10.1016/j.ibiod.2020.105082). this is mentioned for the explanation is missing for Is there any data/literature for the sapwood width for each species according to the wood production forestry national management (or diameter of tree when harvested). Is there any studies for the proportion of heartwood and sapwood for these studied species?
The description of “Materials and Methods” could be considerably improved. Please rewritten the section including all the information related to sampling here i.e., removing it from the discussion (lines 216 to 226). It is critical to explain the wood directions and surface planes studied as well as to clarify the dimensions of the samples in each direction and change the designation of square since the dimension is 20 x 20 x 3 mm cubic. Are the sampling units correct? Clarify the dimension on each direction. Check all the microscopy related information such as the type of microscope, stereo or compound microscope, and used magnifications. The 100x and 400x does not seem entirely correct and in 2.2 section there is a mention to the use of 10x and 40x. Insert information regarding the monitorization and recording of the RH values during the experiment to ensure the stable conditions as highlighted in the title and in the conclusions: “The moisture state of the samples before and during experiments should be carefully controlled”.
It was somehow difficult to follow the text, figures, and tables. The Results section does not seem organized with regards to the wood different sections/surfaces (that would be one of main parameters to be the studied). The Figures should be placed according the first mention in the text. However, it seems that this was not verified before the ms submission. Please check also if the observations/sentences in text are related to the graphs or microscopic images. It is not understandable, for example the maximum mold coverage was observed on day 39 for PX-95 as it is showed in the graph, i.e., was determined. Thus, it should be described after the citation of the Figure 8 (the graph). The number of the Figures showing the microscopic observations are differing between the wood species and RH conditions and it is not quite understandable the used criteria. It would be interesting to relate the mold growth coverage rating with the aesthetic rating (microscopic images), inserting a), b) for all the Figure captions relating the figures with the mold growth stages. Insert the scale bars in Figures as well. Consider also to place the Tables from the 87% RH and 97 RH of the same wood species together to make easier the comparison and reading.
The discussion should be improved according to the previous recommendations. Overall, the results seem consistent with literature regarding water requirements on surface for mold growth (e.g., ref 13), i.e., the Penicillium spp. and Aspergillus spp. are primary colonisers and Alternaria spp. second colonisers. Even if in the Abstract it is mentioned “The dominant mold in the experiment was Aspergillus niger”, this is poorly discussed in ms. Moreover, this Aspergillus species is also known as the most common responsible for fruit decay, for example. The influence of the type of wood cut (quarter sawn or flat sawn boards,) could be also discussed or mentioned during the manuscript. Since pine wood is more susceptible to mould growth than spruce wood as found in literature it could be useful to relate the main differences of wood anatomy and chemical composition. For example, with regards to tissues proportions and the content of starch, cellulose, and lignin. The use of samples codes should be avoided and the repetition of the sentences from the results section. The mention for the parameters studied are few, with exception for line 242. It would be important to know if the authors have any suggestion for the differences mentioned in line 259.
The conclusions should be improved as mentioned before as well as shortened. The last three paragraphs should be removed.
Please revise the text carefully taking also into consideration the following specific comments.
Specific comments:
Abstract
1) The short abstract should be improved. Where all the parameters studied? By “occurrence in a structure” it means “building structure”. It should be considered that it could be interpreted as wood structure.
Introdution
1) There are some misunderstanding sentences, for example: “Wood is affected by a number of loads, of which weathering, chemicals, and biological agents are the most important for its durability”. Is it wood in general or wood durability that is mostly influenced by biological agents, and other factors such weathering and chemical composition?
2) An explanation or references are needed to understand why the changing climatic conditions are one of the main reasons of mold incidence increase. It is too vague since the general idea is related to drought situations caused by climatic changes.
3) Use examples of the structural elements used in buildings (?) to clarify the reader about the type of building structures and interest on the study.
4) Avoid acronyms in Introduction such as RH.
5) References are needed in lines 46 and 47.
6) “Simple carbohydrates” and “low-molecular-weight carbohydrates”: unify terminology and add examples.
7) “the quality of surfaces” please specify. It too vague.
8) Explain what do you mean by “is the surface of dried wood” vs. “spring harvested wood”.
9) Lines 57 to 62, clarify which wood species are being referred to and when it growth is related to mold growth or wood growth.
10) Check if citation are showing the number of reference near the author name (e.g. Segers)
11) Line 63: Which published studies?
12) Line 64 insert the species names
13) Lines 68 to 74 deal with issues related to laboratory and mold growth determination methods. However not once is referred the used standards used in other studies and in the present study it is also missing that information. Why did the authors choose not to follow the standards? If the authors followed some standard, besides the work ref 16 and ref 17, it should be inserted also. Rewrite the paragraph.
14) Rewrite the aims as mentioned before.
Materials and methods
1) The 2.1 is missing.
2) Insert reference for the common mold occurrence
3) “another criteria” which was the first, rewrite
4) Insert the authority in the scientific names of the used molds
5) Insert the reference of the institute in line 89
6) “passasing in the laboratory” – what does this means?
7) Line 97 insert the type of microscope used and model.
8) Consider changing 2.3. Samples to wood samples
9) Introduce the abbreviations when first mentioned in the text “S” for spruce, “P” for pine, etc
10) Clarify the origin of the sample see above comment for sampling misplaced in discussion
11) Insert the model of the planning machine
12) The sampling is quite confusing, rewrite
13) The “nomenclature” and “characteristic photos” are not accurate term to be used here
14) Clarify the number of values obtained by each sample (PT-75, etc)
15) See above comments on magnification
16) Insert details related to the labelling of the samples. Was there a container for each sample?
17) clarify in 2.4.3 section how was the determination of the mold growth/coverage rating (what corresponds to 10%, etc) and obviousness of mold growth (schematic draws)
18) check “quality of the quality of the sporangia”
19) Line 134 reference for “our previous work” is missing
Results
1) Line 136 “the results of mold growth observations” – rephrase
2) Line 136 “for all three” – idem
3) Line 138 “see Figuee 3” – idem
4) The use of both styles: 75th and day 75 makes more difficult to follow the results
5) Replace “Microscopic images” for “Microscopic observations”
6) Consider changing “placed in XX% RH” along the manuscript
7) Line 192, clarify: “The macroscopic mold coverage had not changed until the end of the experiment”
8) Line 193: clarify the sentence to make sure if it was observed or not: “Alternaria alternata was not observed 193 to grow until the end of the experiment” (the point at the end of the sentence is also missing)
9) The title “3.3. Comparison of results” could be improved
10) Line 197 to 199 should be placed in Table title
11) Line 208 – see comment above for line 193
Discussion
1) Remove the lines 211 to 216 for the introduction.
2) Why the use of “pure wood” term?
3) Specify always if the genus and genera is related to the tree species or mold species
Figures
Figures captions should be improved inserting the necessary information as mentioned previously. Figure 1 - The scheme of could be more appealing.
Figure 2 – The photography of the samples is not showing the 3D dimension, “surface” is repeated on the 1st column, the label can be shown in parentheses within the 1st column. The caption is incomplete. Do not use “anatomical directions” terms
Figures 3, 5, 6, 7, 9, 10, 11, 13, 14, 15, 17, 18 and 19 – Insert a more complete caption, using a) and b), etc describing the microscopic observations on each surface as well as relating with the growth stage shown in the graphs (to easier to relate the day of the stage). Check all magnifications and insert the scale bars in each image.
Figures 4, 8, 12 and 16 – insert the information for the y axis (MR) and units (rating?) description in the title
Tables
Table 1 – Avoid “nomenclature”, Insert a more complete title. Units are missing for RH.
Table 2 – Insert a line to separate the index proposed by the authors and the the index used by the authors of ref 16. Avoid the percentage repetition in column at the right. Consider using one more column to show the mold index (rating numbers) also used in ref 16. “Grow” is misspelled. Use “mold” or “mould”.
Others:
Correct the abbreviation “sp.” to “spp.”
Check the affiliations, it seems there are only two different affiliations. Also check the author’s names it seems there is some misspelling.
References related with the fungal degrading process in wood that might be useful:
Eaton, R A and Hale, M. D. C., 'Wood. Decay, pests and protection' (Chapman & Hall, London, 1993).
Dickinson, D J and Levy, J F 'Timber and Forest Products', Encyclopedia of Microbiology, (4) (1992) 231-242.
Tsoumis, G.T. Science and Technology of Wood : Structure, Properties, Utilization; Van Nostrand Reinhold: New York, 1991; ISBN 0-442-23985-8
Wheeler, E.A. Inside Wood—AWeb Resource for Hardwood Anatomy. IAWA J. 2011, 32, 199–211.
Butterfield, B.G.; Meylan, B. Three-Dimensional Structure of Wood: An Ultrastructural Approach.; 1980
Author Response
Thank you for your review, it was very inspiring. We have completely rewritten the article and incorporated all your comments. We have added a figure to the methods and materials section that shows the sample preparation. And we have explained more about magnification during observation and when taking images.
Reviewer 2 Report
The paper presents the results of research on the influence of anatomical directions of pine and spruce wood on the growth of selected mold fungi. I consider the selection of test fungi species justified, although there are no typical cellulolytic fungi, e.g. T. viride or Ch. globosum.
Alternaria alternata belongs to the weak xerophilic fungi, so it was predicted that it would not grow at a humidity (RH) below 85%.
The direction of the research is important, but there are some methodological errors that should not occur when designing this type of research. First of all, large methodological doubts are raised by the lack of proper knowledge about the wood used for the tests (wood purchased in the store):
- whether the wood was free from defects (blue stain, knots, resin)?
- how it was identified that the spruce wood samples intended for testing were free of heartwood (spruce is a wood with an undyed heartwood). The spruce heartwood is undyed, while the heartwood may not have fungus growth. How can you be sure that the spruce wood used does not contain heartwood?
- what part of the tree was the wood taken for testing (this has an impact on the density of the wood and thus on the growth of fungi)? What was the density of the wood? What was the coefficient of variation within the wood density?
-what was the grain of the wood (it is known that pine can be variable-grained, and this can have a great impact on the ability of fungi to grow on a given anatomical cross-section)?
- how many repetitions were made for one fungi species and one anatomical direction. It is impossible to conclude which wood anatomy is conducive to fungal growth without well-planned research statistics. Are there any statistically significant differences between the examined anatomical sections?
Were the test specimens (tangential, radial and transverse specimens) cut from one plank of wood?
What was the proportion of late and early wood? The different anatomy of the early and late wood coils has an impact on the growth of fungi. This is an important feature for drawing appropriate conclusions.
The discussion of the results should be supported by a thorough literature analysis taking into account the influence of fungal growth depending on the anatomical structure of the wood (the share of late-early wood, the type of anatomical cross-section, the share of, for example, crumb cells in a given cross-section, in which easily accessible nutrients are stored).
The list of references is slightly different than the requirements in the instructions for authors.
Other data requiring:
Figure 19 should be before chapter 3.3
Line 151 - replace “whereas” with “whereas”
Line 173 should be changed from 87% to 95%
Line 174 of the genus names should be written in italics
Figure 12 shows the test results for the humidity of spruce samples - 87%, while the text - line 179 gives 95% humidity
Line: 183-184 - please put the names of species and genera in italics
Line 194 - at the end of the sentence, please put the sign "."
Table 4 should contain the same markings as Table 3 "RH [%]", "87", "95"
All names of fungi species and types should be in italics in the text (line: 185, 224, 228, 229, 232, 235, 267)
In references to literature in lines: 231, 239, 244, 245, 251, 256, 258, 264, the dates of publication should be removed.
Following the revision of the "Results and discussion" section, the "Conclusions" section should be corrected.
Author Response
Thank you for your review. We have completely rewritten the article to make it more readable and have incorporated all your comments. We have added the figure of the sample preparation and additional information regarding sampling. We have revised the methods and materials section, the discussion, and the conclusion.
.
Round 2
Reviewer 1 Report
All the improvements made have been acknowledged, namely in the introduction and discussion. However, some arrangements are still necessary.
I not sure if I agree with the present title “The effect of anatomical sections on mold growth on wood under stable relative humidity”, it might be misleading: “the effect “term might not be the most accurate to be used here; one of the goals of the study seems to be the comparison of the results with the results obtained in other experiments which is not expressed in title and it might suggest that only relative humidity was stable, while temperature or other factors were not controlled. Moreover “wood under stable relative humidity” might be interpreted as wood surface humidity, for instance, which was not studied. I would suggest some changes such as “Mold growth contrast between different wood anatomical surfaces and related experimental approaches”, “Mold growth occurrence on transverse and longitudinal wood surfaces and comparison between experimental approaches”, etc.
With regards to the presentation of the results it should be improved to support the conclusions, since photographs of the mold growth in the different wood surfaces are now missing. The authors should consider inserting the most relevant photographs in the results section to support the study (discussion and conclusions) while other photographs could be inserted as Supplementary Material.
It would be important to state the selection for these species even if it is only because are the most common woods used in standard mold test methods. Moreover, the lack of explanation for the reasons to choose ref 12 and ref 24 for comparison is missing.
Since the authors claim for some future improvements, it could be interesting to discuss which should be the wood anatomical surface used for the common standardized methodology based on the results? Is it related to the main commercial wood board types (quarter sawn boards – radial surfaces, flat-sawn – tangential surfaces)? There is no reference for the extractive content of the wood along the ms even if it is one of the main causes for the variation of wood durability between heartwood and sapwood.
The “spp.”should not be in italics.
Please check the following specific comments:
Abstract
Line 14-15: “spp.” should not be presented in italics
Line 18: “was studied by regular microscopic and macroscopic observations.” Studied by observations? Evaluated, monitored, recorded?
Line 21: Why refer here the number of the studies used for the results comparison?
Line 23: What do you mean by “scientific” here? Does it make sense to compare with non-scientific studies?
Line 24: “was developed and followed”? or should be?
Introduction
Line 32: “moisture state” or “moisture condition”?
Line 33: “sufficient”? can be removed
Line 36: “natural limits”? Is it “natural durability limits”?
Line 55: “appearance” or “presence”?
Line 57-58: “In addition to environmental conditions, several other aspects influence mold growth”. Change to “In addition to environmental conditions, wood species and wood surface quality and treatment. For example, pine…” or and add: “such as wood species and wood surface treatment” followed by the examples as presented now. Nevertheless, the mention to the difference of mold growth on sapwood and heartwood should also be underlined here and is missing. This is one the most important wood characteristics when selecting timber for building structures.
Line 64-65: change “containing pure” to “showing the”
Line 67: “moments” or “stages”?
Line 70-71: Please refer specifically to the aim of the study and delete “Finally, conclusions are drawn” is not usual at all to mention within the ms.
M&M
Line 79: “observation” or “monitorization”?
Line 84-85: Botanical authority (when cited for the first time in the text) is missing i.e., Pinus sylvestris L. and Picea abies (L.) Karst, known by the common names of the Scots pine and Norway spruce respectively.
Line 85: remove “see”
Line 87: “The wood without any biocidal treatment.” There is something missing here.
Line 94: use “showing” instead of “with”
Line 96: use “typical” instead of “characteristic”
Line 97: “upper”? Why? Was “upper” used because it would correspond the upper surface as placed in the experiment? Please do not forget that the same anatomical surface is shown twice in the specimen (in the upper and above surface according to the given explanation), i.e., to a “upper square surface (20 × 20 mm2)” always correspond an “above square surface (20 × 20 mm2)”. A figure showing the position of the wood specimen during the experiment would be very helpful. Nevertheless, avoid “upper” or clarify.
Line 98: use “wood species” instead of “type of wood”
Line 101-102: The growth ring width was not recorded (at least it was not mentioned) and from the Figure 1 it seems that growth rings are very narrow within the sapwood region (where the selection was made), i.e., the earlywood (bright area of each growth ring) and latewood (the dark area of each growth ring) will be very narrow too. In Figure 2 the pencil thickness used to identify the specimen orientation seems thicker than the latewood width. Careful must be made. So, if the authors are sure, change to: “The tangential surfaces were composed only of earlywood”.
Line 114-115: The authority in the scientific names of the used molds is still missing
Line 115-116: Delete the information already given (“These fungal genera were obtained from the Czech Collection of Microorganisms). Rewrite to include only the mention for “the number in the brackets is their access number.”
Line 125: Insert the microscope model (this is important since stereomicroscope – used for macroscopic observation- is different from a microscope – used for microscopic observations). The “x” should be in normal text.
Line 126: Was “CFU” mentioned before? If not it should be presented.
Line 131: Present here “RH” for the relative humidity
Line 147-148: Change to “Mold growth was observed under a microscope (Motic, BA 410E Epi) with a magnification of 40× and 100×”.
Line 156: “MI” is not presented before. Does it go for Modl Index? If so, rewrite lines 154-163 accordingly.
Line 162: The authors refer that “The degrees of mold index are illustrated in Table 3”. However, there is no mention for the used wood species, i.e., pine and spruce. The photographs are from which wood species and why only one is shown here. Moreover the description of the wood surface in each photograph is missing and should be inserted. This should be corrected, and the selection explained since differences were described for the two used species. The other wood species, not shown in Table 3 should be presented in Supplementary Material for example.
Results
“3.1. Mold growth on wood specimens” and “3.2. Response times” subsections are not easily understandable. The results are related to growth mold development and should be presented as one subsection. Perhaps the authors would like to present the variation of the growth between mold species and then the mold growth development by wood species and wood surfaces. Please consider inserting the Figures with regards the mold growth variation between the different studied wood surfaces. The last version of the ms detailed it well, i.e, the differences observed in the transverse, radial and tangential surfaces which seems to be removed now. However, this is one of the main aims and some photomicrographs are relevant.
Line 174: Remove (“The mold growth curves are presented as the average for each subset of specimens”) and insert in Figure 3 caption.
Line 185: “transverse spruce specimens”
Line 187: “specimens with tangential surfaces”? All the specimens have the tangential surfaces. However according to the study only some have square tangential surfaces. Do the authors mean that “the fastest growth was always on the tangential surfaces of the specimens of both wood species (PT and ST)?
Line 189: “The lower maximum mold index was reached on spruce specimens”. In which surface?
Line 191: why the use of “PT” if the idea is to refer the transverse surface?
Line 198: Remove “see”
In the subsection “3.3. Comparison to other mold growth studies” there is no description at all of the results (graph analysis).
Discussion
Line 210: “The experimental results showed, consistent with previous findings”? something is wrong in this sentence.
Line 220: “for its life”? Is this necessary?
Line 221: “anatomical sections of wood” or the “main anatomical surfaces of the wood”
Line 222: Was the maximum delay in mold growth found on the transversal surface of spruce samples? Clarify.
Line 222: “maximum mold index was reached the fastest on tangential surface of both wood species”? Do the authors mean that the maximum mold index as well as the fastest (how many days vs. mean days) was reached in the tangential surface of both wood species? Clarify.
Line 225: This is good point. However the main differences between the composition of earlywood and latewood regarding the nutrients availability needs for the mold species should be addressed. Insert reference too.
Line 231: “reasonable agreement”? and Line 238: “relatively good agreement” ? This not so consensual.
Line 239: “low difference” – please insert the value in parenthesis
Line 242-244: ok but what are “small changes in boundary conditions”? Clarify?
Line 249: “Potential factors are discussed”? Should it be “These potential factors”? It seems related to the previous sentence but needs to be clarified. Ref 26 is incomplete.
Conclusions
Line 260-266: are not concluding remarks of the study and should be deleted from this section
Line 272-273: please see related previous comments
Line 277: why “boundary”? Was this explained before in the ms?
Figures and Tables
Figure 1: The “preconditioning (equalization of the moisture)” also applies to the storage and stabilization of the samples for the three studied conditions and UV treatment? If so, mention it in Line 77 (equalization of the moisture under …)
Figure 2: Insert the designation of each code; SP (spruce tangential), etc. Change to “Red dashed lines separate heartwood and sapwood.” If possible, avoid the pencil sketches (except those used to identify the specimen orientation) in the Figure 2, this does not give an accurate idea of the work at all. An illustrative figure might also be given.
Figure 3: Insert “MI” in parenthesis in the caption. It seems that lines are made by hand.
Figure 4: Use the name of the authors also in the graphs instead of “data [12]” and “data [24]” to avoid misinterpretation. Use tangential and radial surfaces instead of “RT” (this is a new code) in the caption.
Table 1: “view from top on the surface of wood specimens”. Which is the “top”? This is not explained in the text. Use the same expression presented in M&M: “square surface”. In the first line of the table, insert the letter code for each anatomical surface in parenthesis: Tangential surface (T), etc. In the second line of the table, insert only the wood species name and the letter code in parenthesis: Spruce (S), etc.
Table 2: Avoid “Mold Index” and “sub-index” uniform to “Index” and “subindex”. Some lines sin “Description” column is empty – check it (merge the lines or introduce the description if it is the case)
Table 3: Should this be a Table or a Figure? Change to “The scale of mold index and subindex (according to Table 2) exemplified by microscopic and macroscopic photographs.”
Table 4: Replace “wood” by “Species” in the first column; remove parenthesis of “(in brackets)” and “finally” form the table title. Insert the explanation of “MR”, “M” and “MI” in the caption.
Author Response
Thank you for your assistance, our response is attached.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The Authors of the research largely responded to the comments and supplemented the content of the publication. Missing data has been corrected. however, some corrections should be made in the final paragraphs of the publication.
In the conclusions, the Authors of the research write that they have noticed significant differences between the results of the conducted experiment and the results of other authors. In order to determine the significance of the differences, a statistical analysis should be performed at this point, which would allow to determine whether the differences are statistically significant.
Author Response
Thank you for your assistance, our response is attached.
Author Response File: Author Response.docx
