Environmental Factors Influence Lichen Colonization and the Biodeterioration of Brick Carvings on Roof Ridges of Historic Buildings in Luoyang, China

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The revised manuscript extended the Discussion and Conclusion sections of the original manuscript. 

The overall quality of the investigation has been improved, although not all of the previously suggested revisions have been considered, e.g. for Fig.4 (bar plots’ scale and circles’ size) or for a comment about possible biodeterioration scenarios linked to climate change.

Author Response

Response to Reviewer #1 Comments

We are very grateful to your valuable comments and suggestions for our manuscript to Sustainability (Sustainability-3541462). In response to your comments and suggestions, we reply as follows:

Question: The overall quality of the investigation has been improved, although not all of the previously suggested revisions have been considered, e.g. for Fig.4 (bar plots’ scale and circles’ size) or for a comment about possible biodeterioration scenarios linked to climate change.

Response: We really appreciate your advice. Based on your suggestions, we have refined the visualization of circle sizes in Figure 4 to better represent the proportion of microorganisms, thereby enhancing the overall readability. Additionally, the results section added comments on possible biodegradation scenarios related to climate change.

In the revised manuscript, please see Figure 4 and Results section on line 273-276.

We acknowledge your comments and constructive suggestions very much, which are valuable in improving the quality of our manuscript. We hope that the changes having been made to the manuscript meet to your satisfaction. 

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

See attached file 

Comments for author File: Comments.pdf

Author Response

Response to Reviewer #2 Comments 

We are very grateful to your critical comments and thoughtful suggestions for our manuscript to Sustainability (Sustainability-3541462). Here are our responses to your comment.

Question: As I mentioned in the first report: Overall, 17 bacterial species and 10 fungal genera were identified within the lichen populations (Table 3). The authors just present a theoretical result for 17 bacterial species and 10 fungal. Biological work must be added in detail in the present work (this is the main concepts of this study). Table 3. Microbial community of lichen on roof ridge brick carvings in the microclimate zone of the Luoyang region (Just theoretical data)

Response: We sincerely appreciate your rigorous scientific research attitude. As you correctly pointed out, biological work must be elaborated in detail within the current study, which represents the core concept of this research. Below, we provide a detailed explanation of our biological work:

Firstly, the biological samples consist of lichen samples collected from brick carvings of historical buildings located in various counties within the Luoyang area.  All samples are preserved in our laboratory at -80°C. Currently, isolation culture and antimicrobial susceptibility testing are ongoing.

Secondly, bacterial and fungal genomic DNA was extracted from different samples using CTAB. PCR was employed to amplify the V3-V4 region of the bacterial 16S rRNA gene and the ITS region of the fungal 18S rRNA gene. These variable regions are critical for analyzing microbial community composition and diversity. Based on gene sequence similarity (>97%), they were classified into the same species and grouped into operational taxonomic units (OTUs). The sequences were subsequently searched in the NCBI GenBank database, and fungi and bacteria with high homology were selected. This method is widely used for microbial diversity identification [1,2], and we have adopted this approach as well.

Thirdly, PCR-based identification of lichen microbial communities has been extensively utilized [3-6], and we have adopted this approach as well.

Therefore, the 17 bacterial species and 10 fungal species identified through these biological methods in the lichen population on roof ridge brick carvings in the microclimate area of the Luoyang region (Table 3) represent genuine results obtained from our experiments.

The methods used for sample collection and identification are described comprehensively in the "Materials and Methods" section of the manuscript (lines 186-263).

[1] Gardes M., Brun T. D. ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Molecular Ecology, 1993, 2 (2): 113–118.

[2] Heuer H., Krsek, M., Baker, P., Smalla K., Wellington E.M. Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel electrophoretic separation in denaturing gradients. App. Env. Microbiol. 1997, 63(8), 3233-3241

[3] Asako K., LI X.S., Osuga Y., Kawashima A., Gu J.D., Nasu M., and Katayama Yoko. Bacterial communities in pigmented biofilms formed on the sandstone bas-relief walls of the Bayon Temple, Angkor Thom, Cambodia. Microbes Environ. 2013, 28(4), 422–431

[4] Johannes T., Waldherr M., Ortbauer M., Graf A., Sipek B., Dubravka J.S., Sterflinger., Piñar G. Pretty in pink? Complementary strategies for analysing pink biofilms on historical buildings. Sci. Total Enviro. 2023, 904, 166737.

[5] Suchy H., Zalar P. and Macedo M.F. Microbial diversity of biodeteriorated limestone cultural heritage assets identified using molecular approaches—A literature review. Appl. Sci. 2024, 14, 7429.

[6] Xiong W.X, Tao Y., Wang P.P., Wu K.T. and Chen L.Z. Impact of environmental factors on the formation and development of biological soil crusts in lime concrete materials of building racades. Appl. Sci. 2022, 12, 2974.

 "Materials and Methods" section of the manuscript (lines 186-263):

“2.2. Visual evaluation and sample collection

In March and December 2023, we conducted three visual assessments of Qing Dynasty target buildings located in downtown Luoyang and nine surrounding counties. The external evaluations encompassed the conditions surrounding each building as well as any visible signs of moisture. For the brick carvings on roof ridges, our visual assessment included an examination of the ridge structure, plaster condition, and the state of the brick carvings on the roof ridges themselves. Additionally, a macro-inspection was performed to assess the extent of lichen contamination.Samples comprising biofilm, guano, and building materials were collected from the brick carvings on the roof ridges of historic buildings across each district. The biofilm was meticulously harvested from the surfaces of sculptures using a sterile blade, and small fragments of building materials exhibiting signs of biological colonization were extracted with a sterile chisel. Samples were taken from three distinct locations on each building and subsequently combined into a single sample for analysis. The collected lichen samples were placed into clean sealing bags, with their respective quantities and collection sites documented (Table 1). All biological samples were then stored at -80°C. Lichen-covered brick carving material was systematically sliced into 0.5-cm sections from its surface before being stored at -20°C.

2.3. DNA Extraction and polymerase chain reaction (PCR)

The improved cetyl trimethyl ammonium bromide (CTAB) method was employed for the extraction of bacterial and fungal DNA. Approximately 0.5 g of dry lichen or bird dung samples was taken from the ultra-low temperature water tank, and 10 mL liquid nitrogen was added to rapidly grind them into a powder. Building materials were also crushed into a fine powder. The powdered building material samples were soaked in 1.0 mL of sterile water for 30 min, followed by centrifugation at 3,000 g for 5 min. The supernatant was then subjected to further centrifugation at 12,000 g for an additional 5 min to collect the precipitate. Subsequently, 1.0 mL of 2×CTAB (Real-time (Beijing) Biotechnology Co., LTD.) was added to the lichen, guano powder, and sediment from the building materials. This mixture was preheated at 65°C for 20 min and transferred to a 1.5-mL centrifuge tube before being incubated in a water bath at this temperature for 30 min. Subsequently, it was centrifuged at 12,000 g at 25°C for 10 min, and the resulting supernatant was carefully transferred into a new 1.5-mL centrifuge tube. We then added 5 μL of RNase A solution (10 mg/mL) and allowed fermentation to occur at 37°C for 20 min. An equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was then introduced; after gentle mixing, it underwent centrifugation at room temperature at 12,000 g for 10 min, with subsequent transfer of the supernatant into another new tube containing an equal volume of chloroform: isoamyl alcohol (24:1). After thorough mixing and another round of centrifugation under the conditions described above, we transferred the supernatant into a new 1.5-mL centrifuge tube, added 0.6 times the volume of cold isopropyl alcohol, gently mixed it, and left it for 30 min at -20℃. After centrifugation at 14,000 g at 4℃ for 10 min, we discarded the supernatant, added 1 mL of 70% ethanol for rinsing and precipitation, centrifugated the mixture at 14,000 g at 4℃ for 3 min, added 50 μL of TE buffer to the precipitation to dissolve DNA, and stored it at -20℃.

Bacterial, fungal, and cyanobacterial biodiversity in the samples was measured by polymerase chain reaction (PCR). To this end, the V3–V4 region of bacterial and cyanobacterial 16S rRNA genes and fungal 18S rRNA gene ITS regions were amplified. Fungal 18S rRNA and bacterial 16S rRNA genes were selected to study the composition and distribution of microbial communities. Fungal 18S rRNA gene ITS regions have high variability and polymorphism among different fungal species, which makes them an important tool for fungal classification and identification. The highly variable region of the 16S rRNA gene V3–V4 region is an important region for the analysis of microbial community composition and diversity [23, 24]. The primers were designed based on the consensus sequences of the bacterial 16S rRNA gene, the cyanobacterial 16S rRNA gene, and the fungal 18S rRNA gene. Table 2 shows the primer sets used.

The PCR reactions were conducted using the TaKaRa PCR Amplification Kit (TakaRa Bio Inc., Otsu, Japan) following the manufacturer's instructions. The PCR reactions were carried out in 25-µL reaction mixtures containing 40 pmol of each primer, 20 ng of template DNA, 10 × PCR Buffer 2.5 µL, and TaKaRa Taq 1 µL (5 U/ μL), brought to a volume of 25 µL with ultra-pure water.

The reaction conditions for bacterial DNA amplification are as follows: predenaturation at 95℃ for 5 min, denaturation at 95℃ for 45 s, annealing at 58℃ for 45 s, extension at 72℃ for 100 s, a total of 33 cycles, extension at 72℃ for 10 min. The fungal and cyanobacterial DNA amplification reaction conditions were similar to those for bacterial DNA, except that the annealing temperatures for fungi and cyanobacteria were 55℃ and 56℃, respectively.

2.4. Nucleotide sequence analysis

The PCR products were purified from 1% agarose gel. The purified samples were then submitted to TakaRa Biotechnology (Dalian) Co., Ltd. (Dalian, China) for nucleotide sequencing. The sequencing data obtained were processed and corrected using the BioEdit V7.0.9.0 software, and a local database was established. Sequences with more than 97% similarity were classified as the same species and grouped into operational taxonomic units (OTUs). We searched the sequences in the GenBank database of NCBI and selected lichenous fungi, bacteria, and the reference sequences with high homology. The detection frequencies for bacteria and fungi in each sample were calculated. Through cluster analysis, the distribution patterns of microbial colonies in lichens across different microclimate zones in Luoyang were examined.”

We hope our explanation will be satisfactory to you.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Dear Authors, 

The topic is very interesting and important not only for your region but also for every heritage regions world wide.

I have some minor comments that may facilitate the readability of your manuscript.

1) The Introduction section:

a) It is better to split the introduction section into two section; 1) introduction and 2) background.

b) You should state clearly in the introduction section the research problem and research aim.

c) Kindly, provide a brief paragraph describing the following sections.

2) In the Literature review:

a) Consider illustrate the limitations of the previous studies that inspire you to perform this research.

b) Consider use more recent references as some of them are seems in the past ten years.

3) In the Results and discussion section:

a) In my opinion, Kindly consider adding a table that comprises 1) the type of each bacteria and fungi, 2) a picture of the effect of corresponding bacteria or fundi on the building surface, 3) the effects of these bacteria or fungi on the surface, 4) the factors influence the the growth of these bacteria, and 5) the methods to fight or confront the growth of these types of fungi or bacteria 

 

Author Response

Response to Reviewer #3 Comments

Thank you very much for your valuable comments and suggestions on our Sustainability (Sustainability-3541462). With regard to your comments and suggestions, we would like to reply as follows:

Question 1:  The Introduction section:

a) It is better to split the introduction section into two sections; 1) introduction and 2) background.

b) You should state clearly in the introduction section the research problem and research aim.

c) Kindly, provide a brief paragraph describing the following sections.

Response: We sincerely appreciate your valuable advice. To more clearly articulate our research question and purpose, a detailed description has been added to the preface. In the revised manuscript, please refer to lines 119-146 of the introduction.

Your suggestion to divide the introduction into two distinct sections—introduction and background—is greatly appreciated as it enhances clarity. In the methods section, we provided an in-depth description of the natural environment of Luoyang. In the introduction, we briefly outlined the issue of biological pollution affecting the brick carvings of historical buildings in Luoyang. Although concise, this description ensures a smooth and natural transition following the literature review, thereby maintaining logical coherence.

In the revised manuscript, please see Introduction on line 119-137 and Materials and Methods on line 149-185.

Question 2:  In the Literature review:

a) Consider illustrate the limitations of the previous studies that inspire you to perform this research.

b) Consider use more recent references as some of them are seems in the past ten years.

Response: We extend our gratitude for your valuable suggestions. In the literature review section, we have refined the limitations of prior research that motivated us to conduct this study. In the revised manuscript, please see Introduction on line 108-118.

In the process of conducting this research, we extensively reviewed a substantial amount of literature. Given the scarcity of studies on lichen growth on brick carvings of historical buildings, particularly over the past five years, relevant literature remains limited. This further underscores the necessity and urgency of our research.

Question 3: In the Results and discussion section:

a) In my opinion, kindly consider adding a table that comprises 1) the type of each bacterium and fungi, 2) a picture of the effect of corresponding bacteria or fundi on the building surface, 3) the effects of these bacteria or fungi on the surface, 4) the factors influence the growth of these bacteria, and 5) the methods to fight or confront the growth of these types of fungi or bacteria

Response: Thank you for your valuable advice. As you pointed out, a table presents the following information: 1) the type of each bacterium and fungi, 2) a picture of the effect of corresponding bacteria or fundi on the building surface, 3) the effects of these bacteria or fungi on the surface, 4) the factors influence the growth of these bacteria, and 5) the methods to fight or confront the growth of these types of fungi or bacteria. This approach synthesizes disparate results into a coherent format, thereby enhancing readability. However, due to the complexity of the content, it is challenging to consolidate all this information into a single table without compromising clarity. Consequently, we opted to list the identified bacterial and fungal types separately in Table 3 while providing corresponding images in Figure 2 that depict the effects of lichen-forming bacteria or fungi on building surfaces. This arrangement not only ensures clarity in presenting test results but also ensures the quality of the picture performance. The discussion section offers an in-depth analysis of how these microorganisms affect building surfaces as well as the factors that influence their growth. With regard to methods for inhibiting proliferation, we believe that targeted approaches will become increasingly significant in our future research on biological antibacterial strategies.

In the revised manuscript, please see Results Table 3 and Figure 2 as well as Discussion on line 343-431.

We acknowledge your comments and constructive suggestions very much, which are valuable in improving the quality of our manuscript. We hope that the changes having been made to the manuscript meet to your satisfaction. 

Round 2

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Biological work is considered the core concept of this manuscript.

I'm still not convinced that the lab work is not added in the present work.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Report:

Article title:	Environmental Factors Influence Lichen Colonization and the Biodeterioration of Brick Carvings on Roof Ridges of Historic Buildings in Luoyang, China

 

 My comments are as follows:

Abstract:

In the abstract section, the authors stated, "However, respective studies on brick carvings atop historic buildings remain insufficient." This statement may be inaccurate as numerous studies have been conducted in this area, including but not limited to the following:

1. Favero-Longo, E.,  Matteucci, E.,  , Castelli, D., Iacomussi, P., Martire, L., Ruggiero, M. G., Segimiro, A., An ecological investigation on lichens and other lithobionts colonizing rock art in Valle Camonica (UNESCO WHS n. 94) addresses preventive conservation strategies, The Lichenologist, Vol. 55, 2023, pp. 409–422.
2. Zabihi, M., Sohrabi, M., Favero-Longo, S. E., Nortaghani, A., Lichen colonization and associated biodeterioration processes on ancient bricks of the Gonbad-e Qā tower, UNESCO World Heritage Site, Iran, Research Square, 2024, pp. 1-19.
3. SEAWARD, M.R.D. GIACOBINI, C., Lichen-induced biodeterioration of italian monuments, frescoes and other archaeological materials, Studia Geobotanica, 8: 3-11, 1988, pp. 3-11.

  Materials and methods:

• I thank the authors very much for their description of the research area and its natural environment. They provided all the details, especially regarding the fluctuation in temperature and humidity between January and July, which had an impact on the deterioration process of lichen and associated microorganisms (bacteria and fungi).

Results:

Visual assessment

• The authors stated, "Lichen populations proliferated across roof surfaces, resulting in the expansion and cracking of roof plaster as well as damage to brick carvings located on both the northern and southern sides of these roofs." I believe that the cracks in the roof plaster and damage to brick carvings are not solely caused by lichen populations, but there may be other factors contributing to these aspects of deterioration.

Analysis of microbial diversity in lichen samples collected from brick roofs of historical buildings in Luoyang city

• The authors focused on isolating and identification of microorganisms (bacteria and fungi) associated with lichen. To understand the contribution of these organisms to the decay process, a study on the enzymatic activity of identified bacteria and fungi is necessary.
• Enzymatic activity must be followed by a statistical study of the extracted results.

Discussion

• In the discussion section, the authors mentioned that Actinomycetes were isolated, but they were not mentioned in the results section.
• The authors did not explain the mechanism of the damage that occurred between lichen populations and the accompanying living organisms of the lichen, as well as the surface of the plaster and brick carvings. They will not be able to explain the extent of the damage without conducting a study on the enzymatic activity of the isolated microorganisms.
• In order to provide a thorough interpretation and explanation of the mechanism of deterioration caused by lichen populations and associated bacteria and fungi, it would be beneficial for the authors to study other physical and chemical properties of plaster and brick carvings.

Conclusion

The conclusion did not add any new information to what was found in the other references related to the same topic that were mentioned above.

Recommendation:

I recommend rejecting this manuscript for publication in Sustainability.

Reviewer 2 Report

Comments and Suggestions for Authors

See attached file 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

See attached file 

Reviewer 3 Report

Comments and Suggestions for Authors

The paper presents a study about the microbial communities of lichens on the roofs of historic buildings in Luoyang and surrounding areas in China. The investigation combined visual assessment and molecular biological analyses to evaluate the influence of local climatic conditions and the characteristics of brick carving on the variability of lichen colonization and their biodeterioration effect.

The manuscript is well structured and clearly presented, therefore I suggest only minor revisions to enhance the quality of the study. 

The following comments may be considered:

• discussions can be enhanced through a more explicit and punctual comparison between the analysed samples based and the other case studies already referenced to contextualize the topic;
• fig.4 may be improved in its readability unifying the scale of the bar plots and adjusting the circles’ size based on their number of microbial communities.

Finally, to strengthen the value of the investigation, it would be useful to provide practical advice to manage/limit/prevent roof colonization in Luoyang and in other historic sites with comparable conditions (if applicable). Moreover, it would be interesting to try to reason about possible biodeterioration scenarios linked to the expected climate change.