Fungal Community Analysis and Biodeterioration of Waterlogged Wooden Lacquerware from the Nanhai No. 1 Shipwreck

Round 1

Reviewer 1 Report

The manuscript by Yin Jia and co-workers is focused on fungal colonization of waterlogged Wooden Lacquerware from the Nanhai No. 1 Shipwreck. This is a relevant and interesting topic, and the presented work seems to me useful to improve the knowledge about the conservation of this peculiar artefacts; however, some revisions should be necessary.

In particular, I suggest that the authors should be better clarify some point of sampling methods and of experimental design: how many replicates they have for each types of samples (sterile cotton swab, water, enzymatic tests)? It is not clear if there are a congruent number of replicates of tests carried out, and if statistical analysis have been performed.

The introduction section can be improved: a focus on knowledge of biodegradation of wooden Lacquerware is lacking.

The title “Fungal Community Analysis and Biodeterioration of Waterlogged Wooden Lacquerware from the Nanhai No. 1 Shipwreck” is a bit inaccurate: authors do not asses the “Biodeterioration” of the objects: they assess the “potential Biodeterioration” with tests on the presence of ligninolytic and cellulolytic enzymes.

The manuscript in its current form is also difficult to follow due to because of an inappropriate English style; a revision by a native speaker is strongly recommended.

A list of comments is subsequently reported to detail some points of the text which should/may be re-checked to improve the clarity of the contents (and some “typos”).

 

Abstract

lines 20-21: “on the two water samples (NHI935 and NHI58) storing the lacquerware and colonies on the surface of lacquerware.” is not clear: in this sections is not necessary to write this detail of information, for the reader “NHI935” and “NHI58” are unclear. This is a material for the M&M section.

lines 22-23: “And then, the Potato Dextrose Agar (PDA) plates were used to isolate and identify the fungi.” also the detail of the cultural medium is not necessary for the abstract section.

Introduction

line 35 =bamboo,wood = insert space between the comma and the term “wood”

line 36 = surface.Rising = insert space before the term “Rising”

line 44 = can authors provide a reference for Chinese lacquerware techniques?

line 45 = “Large quantities of lacquerware have been excavated at different in China” = it seems to me that a word is lacking: “different” what? Maybe “different sites”?

line 47 = “Unearthed wood lacquers were usually situated in water to prevent its deformation” = this sentence it is not clear.

line 49-50 = Studies uncover that different kinds of degradation and microbial successions occur in waterlogged wooded objects = this sentence it is not clear.

line 53-55 = “Typical fungi inhabitants of waterlogged wooden objects are species of soft rot fungi belonging to the group of Ascomycetes and Deuteromycota such as species in genus Cladosporium, Acremonium, Fusarium, and Chaetomium, among others[5,6].” = Cladosporium, Acremonium, Fusarium, and Chaetomium species are really widespread in several habitats (soil, museums, but also frequently detected in the airborne particulates), they are not exclusive to the group that causes soft rot. 

lines 56-57 “large percentage of the world’s cultural heritage artifacts have been severely and irreversibly damaged and degraded by microorganisms[7,8]” in the reference section authors of paper 7 are incorrectly reported as “Katja, S.; Flavia, P.J.E.M.” they are: Katja (first name) Sterflinger (family name), and Flavia (first name) Pinzari (family name).

lines 57-66 = For example, a study about the biodeterioration of the Mausoleum of the Dingtao King isolated Hypochnicium sp. WY- DT1, a strain that withstand low temperatures while degrading cellulose and ligin[9]. Purple spots appeared in seven different parchment manuscripts from museums, libraries and archives in Italy were observed to enrich halophilic and halotolerant microorganisms under the salty environment, which can promote the occurrence of the purple spots[10]. Another investigation examined the potential microfloral inhabitants of an epoxy resin statue exposed to outdoor conditions in Devin. The results indicated that the differentiate microbial community present on the statue surface was composed of algal phtobionts, rock-inhabiting members, Cyanobacteria, black yeasts and several plant-associated fungi[11]. = this paraph sound as “cherry picking”: a few papers, about very different sunstrata are discussed. This is not usefull for the reader: can authors find a similar number of papers focused on Chinese lacquerware, laquer, wood biodeterioration and discuss only these?

Line 59 = ligin = a type for “lignin”?

Line 66 = microbial diseases = “desease” is term used for humans, animals, rarely for crops, but for objects sounds inappropriate,maybe “microbial degradations”?

Line 69-71 = since only by understanding the biological physiological properties of organisms can we control the potential harm they cause[12]. = this sentence is not clear

Lines 72-80 = This paragraph is to move at the M&M section (and maybe authors can add in the Supplementary Materials a figure with a others materials such as map and a plan of the museum)

Lines 87-89 = In this research, the overall microbiological analyses were to identify the fungi present the wood lacquers extracted from Nanhai No.1 and to reveal fungal communities thriving in water samples of wood lacquers. = Maybe “were conducted to identify the fungi present on the wood lacquers extracted from Nanhai No.1 ”

line 97 purifition =purification

2. Materials and Methods

2.1. Sample collection

lines 91-93 = Lacquerware surface samples collection using non-invasive sampling with PDA medium (Figure 1). The petri dishes were opened and the lacquerware surfaces were streak-inoculated on the agar with sterile cotton bud. Then these petri dishes were incubated in the laboratory for futher isolation. = This procedure in not clear TO ME, routinely a sterile swab is rubbed on the surface of the object and then inoculated in a Petri dishes with a culture medium in a laminar flow cabinet; in this study petri dishes with PDA have been opened in the laminar flow cabinet or in the museum? Is “ sampling with PDA medium” or “sampling with a sterile swab and then inoculated in a Petri dishes with PDA medium”? “futher isolation” is a type for “further”, but from what?

Also how many samples? One for each object, two in total? Please better clarify. And what criteria did the authors use to choose the objects to be sampled? Random? Objects that showed traces of colonization?

Line 94 = 50 mL sterile centrifuge tubes were used to collect water sample = how many samples? One for each object, two in total? Please better clarify.

2.2. Microbial isolation, cultivation and identification

Line 102 = According to the growth of microorganisms, = means according to the common cultural laboratory practices?

Lines 103-104 “After several times of isolation and inoculation, pure colonies were obtained.” = maybe “Fungi were isolated and pure cultures obtained after subsampling”?

Line 104 = purified strains = pure strains

Line 114-117 The sequences were analyzed by the National Center for Biotechnology Information (NCBI) BLAST program = means “The sequences were used as queries in BLAST searches”?

2.4. Activity of carboxymethyl cellulase (CMCase) production by fungi

line 126 = Fungal spores = of pure isolates strains illustrated at 2.2 paragraph? Please, specify

line 135 = The blank control group = it is not clear this control? Another strain?

2.5. Biocide susceptibility of fungal strains

line 148 = Preventol ® D 7, P 91, BIT 20 N and Euxyl ® K100 = a table for the tested biocides with a list of manufacturing companies, active agents and concentrations usually used in restoration programs? Why the concentration used is 0,5% for all the biocides? 

A an exhaustive description of the experiment is missing (number of thesis, replicates, positive and negative controls).

Results

3.2. Isolation of dominant fungi

line 175 = fungi isolated and purified from the surface = purified from surface? fungi isolated from the surface is more clear

caption of figure 3 = scale bars are missing in all the images!

3.3. Activity of CMCase production by F. solani NK-NH1 and P. chrysogenum NK-NH3

The number of samples for each activity test is unclear (there are bars in the graphs, but the number of replicates is not written). Figure 4 graphs have low quality...

3.4. Efficiency of biocide products against targeted strains

lines 205-206 = “F. solani NH-NH1 and P. chrysogenum NK-NH3 were studied. Four biocides were able to inhibitthe fungal growth” = were studied? Means “were used for the tests”? (a space is missing between “inhibit” and “the”).

Lines 206-207= applied at 0.5% concentration, which is much lower than the concentration (2%) recommended by manufacturers. = conditions in tests are really different from those of real restoration interventions. Authors should keep this in mind.

Efficacy of different biocides is reported without a number: authors have statistics to support these findings about different effectiveness?

line 219 “The results were shown in Figure 5.” = No: the figure 5 is only “a representative” image of results of the tests; in the M&M section authors wrote: “After incubating at 28°C for 4 days, the inhibition zones around the discs was measured.” They don't have performed statistical analyses of these measures? Can they provide a graph from the statistical analysis?

4. Discussion

4.1. Fungal communities

line 228 = in this article = in the present article

line 229 = Meanwhile = Moreover ?

Line 249 = cellulaese = cellulases

line 276 = mivrobial = microbial

lines 279-284 = Benzalkonium chloride and isothiazolinones are different active agents, and authors may discuss this.

line 288 = In the museum, the fungal contamination of lacquerware is low. = (lines 67-68) Moreover, Nanhai No.1 shipwreck is suffering an outbreak of a white filamentous fungus. It is not clear how many objects have been colonised by fungi? how quickly?

Line 289 = usde =used

I agree: replace the water and consider water to avoid fungal colonization and physical innovative approaches can be adopted to avoid biocide application, but a monitoring program is really important; fungal growth can be very fast.

5. Conclusions

line 295 We studied the fungal biodeterioration = potential biodeterioration

lines 301-302 The fungal isolates, F. solani NK-NH1 and P. chrysogenum NK-NH3, may not be the directly responsible for the current biodeterioration. = what the authors mean by “the current biodeterioration” they have found damages on constituent materials? The presence of biological colonisation does not always cause damage to materials.

Author Response

Thanks for the suggestions and we have made revisions. Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Line 103: 20-30C is a large temperature range. Did the incubation temperature vary over time or did you incubate plates at different temperatures?

 

Line 123-124: need more details on what was done to determine taxonomy. MAG assembly? ITS gene extraction.

 

Line 125: Please reference a published method for the CMCase methods

 

Line 128-129: Is is not clear why these different incubation conditions/variables were chosen. Please describe in the results section why these are relevant.

 

Line 134: 3,5-dinitrosalicylic acid (mispelled)

 

Line 157-158: there are 5 isolates. how can all 5 be closely related to only two strains?

 

Line 171: 0.71%, and 0.69%, respectively.

 

Figure 2: is relative abundance shown as a %? This should be properly labeled on the y-axis. Why do you think OTUs for Penicillium spp. were not found? This is not discussed in your results or in the discussion. Could there be intrinsic biases in both methods that result in differential community composition? Especially, since you were able to isolate a Penicillium strain from the liquid samples (but not detected in the liquid-derived sequences).

 

Figure 3: These names do not match those in Table 1. Please add additional labeling to this figure to indicate which isolate (1-5) these images refer to in Table 1. And preferably in the same order of 1-5 as listed in Table 1. If these are characterized as the same species, can you speculate why all the colony types look different? Were they cultured under the same conditions?

 

Figure 4E. This a really low pH for a maximum activity. Is this relevant to the in situ conditions? What is the pH of the storage liquids? Do the different pH optima for both strains indicate that one is more active than the other in potential biodeterioration on lacquer surfaces? Please discuss these differences between these strains in your evaluation of the results.

 

Line 227-230: the methods used in comparing 2016 and 2017 data are derived from different methods. Greater care should be made in evaluating these results.

 

Line 242: Cadophora is not shown in Table 1. Was this isolated in the current study? As written, this is unclear and potentially misleading.

 

Line 247-250: P. chrysogenum was not detected in water samples...do you hypothesize that this is due to low abundance and just easy to culture on plates? Why do you think it was not detected in your illumina sequencing?

 

Line 276: microbial (mispelled)

 

Line 293: first (mispelled)

 

Line 305: I see no data indicating that FeS was evaluated. This part of the discussion is relevant, however I do not see any indication from your data or hypotheses that FeS is of concern. Please expand and perhaps add some content to the introduction that prefaces the main compounds or chemicals that present a danger to preservation of the lacquerware.

Author Response

Thanks for the suggestions and we have made revisons. Please see the attachment for details.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I have carefully looked over the materials the Authors provided and have found my questions, comments, and suggestions have been adequately addressed.