Harmful Risk of Bioaerosol Pollution at Major Indoor Sites of a Summer Campus in Guilin City

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors investigated the harmful risk of indoor bioaerosols from some areas on the campus. They found that the total number of bacteria (mostly microbes) detected in the classroom during no lesson in the morning (33% of samples) and in the canteen during meal time (55% of samples) was greater than the World Health Organization's recommended value (1000 CFU/m³). The study shows data on controlling indoor bioaerosol pollution. This study is within the scope of the journal and the necessary topic, but I recommend posing the following questions and comments to enhance the manuscript's quality:

1- In the title of the study, the word “indoor” is repeated twice; please revise.

2- More papers should be evaluated, and the literature review section should be improved to highlight the novelty of the study.

3-      Give more information about the material and devices used in the study.

4-      It is better to add table 1 to the “material and methods” section.

5-      The subtitle “3.5. Figure and Tables” is unnecessary. It is better to distribute the figures according to the paper’s structure.

6-      Explain “DOOP, CAOP, CAUP, etc.” term in the first used paragraph. Prepare and add the nomenclature table.

7-      There is no comment for Fig. 6. Explain each figure and table in the manuscript.

8- In line 265, there is a comment for Fig. 7c. However, comments should be added for other graphs (Fig. 7a, b, d, e, f).

9-      In Fig. 6, add a, b, c, and d for the different charts.

 

10-  Enhance the conclusions part, highlighting the novel findings.

Comments on the Quality of English Language

The manuscript's English language quality is sufficient for understanding, yet the authors should review it to eliminate grammar mistakes and typos before publishing.

Author Response

*/1- In the title of the study, the word “indoor” is repeated twice; please revise./* *Response:*Thank you for pointing out this error, we have removed the duplicate word “indoor “in the title section, please review again. */2 - More papers should be evaluated, and the literature review section should be improved to highlight the novelty of the study./* *Response:*Thank you very much for your proposed changes, and we recognize the shortcomings of this section, we have re-summarized and more relevant literature in the introduction section and emphasized the novelty of this study: focusing on the differences in bioaerosols in different indoor environments in different time periods, and, unlike previous studies, we have also compared the community composition of bioaerosols of different particle sizes. The specifics of our modifications and additions are as follows: *Lines 52-75: *Educational buildings have received more attention due to the specificity and sensitivity of their occupants, and many studies have been reported worldwide on the risk assessment of indoor bioaerosol exposures on campuses, in which bioaerosols from indoor campus sites such as cafeterias, classrooms, dormitories, gymnasiums, and libraries are collected and analyzed more frequently [18-26]. The risk of high concentrations of bioaerosols and the presence of pathogenic bacteria in indoor spaces on campuses, especially in public indoor spaces with high levels of activity, has been reported in many reports [27,28]. Still, systematic comparisons of bioaerosols in indoor spaces on different campuses and at different times of day in the same indoor space are lacking in the current study, finding indoor locations on campus that are at higher risk for bioaerosol exposure and further identifying when this risk is located may help us to do a better job of prevention. In addition, many studies have focused on the particle size distribution characteristics of indoor bioaerosols to assess their inhalation risk [29-32], but more have analyzed them only in terms of concentration, and whether there are differences in the community structure of microorganisms in bioaerosols of different particle sizes has not yet received sufficient attention. Therefore, we monitored the concentrations and analyzed the community compo-sition of bacterial, fungal, and actinomycete aerosols in three indoor sites during dif-ferent periods, namely, classroom, dormitory, and canteen, on a university campus in Guilin, China, focusing on the differences of bioaerosols in different indoor environ-ments and during different periods in the same indoor environment, unlike previous studies, we also compared the community composition of bioaerosols with different particle sizes. This study aims to provide basic data for risk assessment and prevention of indoor bioaerosol hazards on campus and try to provide new ideas for analyzing and studying bioaerosols. */3- Give more information about the material and devices used in the study./* *Response:*Thank you for pointing out this deficiency in our manuscript, and we have added information on the material and devices used in 2.2 Sampling and Analytical Methods;The specifics of our addition are as follows: *Lines 100-101:*Bioaerosol samples were collected using a six-stage Andersen (FA-1, Qingdao Polytron China) impactor with retention diameters of 7.0, 4.7, 3.3, 2.1, 1.1, and 0.65 µm and an airflow rate of 28.3 L/min. *Lines 105-115:*Culturable bacteria are collected on nutrient agar dishes (Huankai Microbial, Guangdong, China), culturable fungi are tested on sand agar dishes(Huankai Microbial, Guangdong, China), and culturable actinomycetes are tested on modified Koch's 1 medium dishes(Huankai Microbial, Guangdong, China). Bacterial samples were incubated at 37 ℃for 2 days, fungal samples were incubated at 28℃for 3 days, and actinomycetes were incubated at 28℃for 7 days. The concentration of living microorganisms was counted as the number of colony-forming units in the volume of air (CFU/m^{^3} ). Quartz filter membranes (80 mm diameter, 0.3 µm pore size,Xingya Shanghai, China) were used to collect samples of bioaerosol particulate matter, which were stored in a refrigerator at -20℃after collection and analyzed for microbial community characterization on the particulate matter by high-throughput sequencing. */4- It is better to add table 1 to the “material and methods” section./* *Response:*Thank you very much for your comments, table 1 has been adjusted according to your suggestions to the “material and methods” section. */5- The subtitle “3.5. Figure and Tables” is unnecessary. It is better to distribute the figures according to the paper’s structure./* *Response:*Thank you very much for your comments, subtitle “3.5. Figure and Tables” has been removed, figures and tables have been removed, and figures and tables have been relocated to their corresponding positions in the text. */6-Explain “DOOP, CAOP, CAUP, etc.” term in the first used paragraph. Prepare and add the nomenclature table./* *Response:*Thank you very much for your comments, We explain the meaning of the terms “DOOP, CAOP, CAUP, etc.” when they first appear in Table 2 and add “the nomenclature table” UP-the periods of few personnel activities OP-the periods of personnel activities CFU-colony-forming units IAQ-indoor air quality WHO -the World Health Organization CLUP-the classroom during periods of few personnel activities; CLOP-the classroom during periods of personnel activities; DOUP-the dormitory during periods of few personnel activities; DOOP-the dormitory during periods of personnel activities; CAUP-the canteen during periods of few personnel activities; CAOP-the canteen during periods of personnel activities.*//* */7-There is no comment for Fig. 6. Explain each figure and table in the manuscript./* *Response:*Thank you for pointing out this deficiency, We have corrected the comment about Figure 6 as follows: *Lines 294-295:*The distribution of α-diversity indices (Chao 1, Shannon, Simpson, and PD) for bioaerosols of different particle sizes is shown in Fig6. */8- In line 265, there is a comment for Fig. 7c. However, comments should be added for other graphs (Fig. 7a, b, d, e, f)./* *Response:*Thank you very much for your comments , The comments were added for(Fig. 7a, b, d, e, f). The details are as follows: *Lines 304-311:*The relative abundance of Micrococcaceae on bacterial aerosols with particle sizes ranging from 3.3-7.0 μm was significantly (/p/=0.015) higher than that on bacterial aerosols in the other particle size ranges in the classroom during UP (Fig 7a). Microorganisms originating from soil and water environments such as Micrococcaceae [57,58] are mostly concentrated on bioaerosols with particle sizes larger than 3.3 μm (Fig 7a, b), whereas microorganisms associated with humans, such as Muribaculaceae, Lachnospiraceae, and Bacteroidaceae [59-61] have high abundance in all particle size rangesthis was more pronounced in dormitory and canteen (Fig 7c, d, e, f).*//* */9-  In Fig. 6, add a, b, c, and d for the different charts./* *Response:*Thanks for your suggestion, we add a, b, c, and d for the different charts in fig 6 according to your suggestion. *Line 314: * Fig 6 Folded distribution of the four α-diversity indices (Chao 1, Shannon, Simpson, and PD) on particulate matter of different sizes. (a) Chao 1 indices; (b) Simpson indices; (c) Shannon indices; (d) PD indices */10-  Enhance the conclusions part, highlighting the novel findings./* *Response:*Thank you for your suggestions on our conclusion section, we enhance the conclusions part, highlighting the novel findings. The details are as follows: *Lines 344-365:*We studied the bioaerosol characteristics of dormitory, classroom and canteen during different periods on a university campus. Bacteria were the predominant microorganisms in all three indoor sites, and there was a risk of total bacterial counts greater than 1000 CFU/m^{^3} in the classroom’s morning and during meal times in the canteen.The percentage of respirable bioaerosols (<3.3 μm) was greater than 50% in all three indoor sites.Indoor human activity significantly increases their airborne bacterial aerosol concentrations, and good ventilation reduces the impact of human activities on bacterial aerosol concentrations, but ventilation of the room should be avoided on mornings when outdoor bioaerosol concentrations are high. Analysis in terms of community composition, ventilated indoor places can change the community structure of indoor bioaerosols to some extent, but this effect is relatively limited because human-related microorganisms, such as Muribaculaceae, Lachnospiraceae, and Bacteroidaceaealways have a high contribution to indoor bioaerosols if there is current human activity indoors. The community structure of bioaerosols with different particle sizes showed some differences: four α-diversity indices (Chao 1, Shannon, Simpson, and PD) for bioaerosols of 2.1-3.3 μm were consistently high at all three sampling sites; Micrococcaceae had high relative abundance only for bioaerosols with particle sizes > 3.3 μm. Micrococcaceae and Enterobacteriaceae were conditionally pathogenic bacteria present at all sampling sites, with Micrococcaceae in higher relative abundance in the classroom and the relative abundance of Enterobacteriaceae having unusually high values occurring in the dormitory. The overall assessment is that all three campus sampling sites are at risk from bioaerosol hazards and that precautionary measures are needed based on the specific causes contributing to the risk. We tried our best to improve the manuscript and made some changes marked in blue in the revised manuscript which will not influence the paper's framework. We appreciate for Editors/Reviewers’ warm work earnestly, and hope the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In the reviewed article Authors describe the results of research concerning microbial quality of indoor air and bioaerosols from the canteen, classroom, and dormitory on a university campus in Guilin, China. The reviewed manuscript is well-written, relevant for the field and presented in a well-structured manner. The cited references are mostly recent publications (67% within the last 5 years) and they are relevant to the subject of the article. The figures and tables are prepared appropriately, they properly show the data and are easy to interpret and understand. I must admit, I find the results obtained by the Authors in classrooms very important - obviously open windows and air exchange between indoor and outdoor air have much bigger effect on the number of microorganisms in air than expected. Most literature data indicate that humans are the biggest and the most important 'microbial source' for the air, which was not confirmed here.

However, chapter 2 (Materials and methods) requires some improvement:

1.      How many samples were really taken? Summer months seems to indicate that there were three analyses, each taking three days, but in line 69 there is information “particulate matter sampling was conducted on one day in July”. It is very hard to draw any conclusions based on analysis made only one day.

2. Were the windows open in classrooms all day or only during the classes?

3.      In lines 88-90 authors state “ Quartz filter membranes (80 mm diameter, 0.3 μm pore size) were used to collect samples of bioaerosol particulate matter, which were stored in a refrigerator at -20℃ after collection and analyzed for microbial community characterization on the particulate matter by high-throughput sequencing.” What sequencing method was used? How were the sample prepared? How was it analysed (methods, programmes)?

4. Also, please check the article for the proper formatting (Latin names should be in italics; please see lines 186-211, abstract etc.).

Author Response

Dear Editor, Dear reviewers

This is a revised response to manuscript ID:atmosphere-3031635. We express our gratitude for the professional review work conducted on our article. In response to your insightful suggestions, we have implemented extensive revisions to our initial draft. The detailed corrections are outlined below. We sincerely appreciate the efforts of the editor and all reviewers for providing valuable feedback that has significantly enhanced the quality of our manuscript.

Reviewer comments are presented in italicized font, and specific concerns have been numbered. Our responses are provided in normal font, with changes/additions to the manuscript highlighted in blue text.

Respond to Reviewer #2’s comments

1. How many samples were really taken? Summer months seems to indicate that there were three analyses, each taking three days, but in line 69 there is information “particulate matter sampling was conducted on one day in July”. It is very hard to draw any conclusions based on analysis made only one day.

Response: First of all, the comments you made are still very informative and I would like to express my deep gratitude, but I am also sorry that the particulate samples for our bioaerosol community composition analysis were collected in one day, on the other hand, we collected and performed the community analysis on different six particle size range particulates separately, and the three sites for two periods contained 36 samples that were collected by the glass fiber filter membrane. The number of samples in each group was six, and our conclusions are all based on the analysis of six samples in each group, and the conclusions have credibility. We really hope you can understand.

 

2. Were the windows open in classrooms all day or only during the classes?

Response: Thank you for raising this issue, which we have corrected and explained in the manuscript as follows.

Lines 166-168: the windows and doors of the classrooms are open throughout the day and the fans are turned on to assist in the ventilation of the classrooms during class hours.

 

3. In lines 88-90 authors state “ Quartz filter membranes (80 mm diameter, 0.3 μm pore size) were used to collect samples of bioaerosol particulate matter, which were stored in a refrigerator at -20℃ after collection and analyzed for microbial community characterization on the particulate matter by high-throughput sequencing.” What sequencing method was used? How were the sample prepared? How was it analysed (methods, programmes)?

Response: Thanks to your comments, we added specific processes and methods regarding high-throughput sequencing as described below:

Line 115-133: Sequencing Methods:

(1) Sample DNA purification. Sample gDNA purification was performed using the Zymo Research BIOMICS DNA Microprep Kit (Cat# D4301). gDNA integrity was detected using 0.8 % agarose electrophoresis, followed by nucleic acid concentration detection (PicoGreen dye method) using a Tecan F200.

(2) PCR amplification. According to the sequenced region, specific primers with index sequences were synthesized to amplify the 16S rDNA V4 region of the samples, and the amplification primer sequences were as follows: Primer5'-3': 515F (5'-GTGYCAGCMGCCGCGGTAA-3') and 806R (5'- GGAC-TACHVGGGTWTCTAAT-3')

(3) PCR product detection, purification and quantification. PCR products were mixed with a 6-fold sampling buffer, followed by electrophoretic detection of the tar-get fragments using a 2 % agarose gel. Samples that passed the assay were taken for recovery of the destination bands, and the recovery was performed using Zymoclean Gel Recovery Kit (D4008); quantification was performed using Qubit@ 2.0 Fluorometer (Thermo Scientific); and finally, equimolar amounts were mixed.

(4) Library construction. Library construction was performed using NEBNext Ul-tra II DNA Library Prep Kit for Illumina (NEB#E7645L) from NEW ENGLAND Bi-oLabs.

(5) High-throughput sequencing. PE250 sequencing was used, and the sequencing kit was Hiseq Rapid SBS Kit v2 (FC-402-4023 500 Cycle) for Illumina.

 

4. Also, please check the article for the proper formatting (Latin names should be in italics; please see lines 186-211, abstract etc.).

Response: Thanks to your suggestion, we have corrected the names of bacteria at the genus and species level in the manuscript to italics in accordance with the International Code of Bacterial Nomenclature.

 

 

We tried our best to improve the manuscript and made some changes marked in blue in the revised manuscript which will not influence the paper's framework. We appreciate for Editors/Reviewers’ warm work earnestly, and hope the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript can be accepted with its revised version.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for sending me the revised version of your manuscript and for the very detailed explanations to my comments. All my comments were replied in a very professional manner. I have no further comments.