Biodegradation of Low-Density Polyethylene by Native Aspergillus Strains Isolated from Plastic-Contaminated Soil

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In general, the text seems to have many AI modifications, it should be revised. Remove words like notable, emergence and conversely. English should be improved.

Repeated ideas in the abstract.

Introduction

Too long, ideas should be more concise.

Many semicolons are used where comma should be used.

Some ideas are two long, divide them.

Line 85: in vitro, should be in italics

Materials

I don’t understand the idea in line 135: In the surroundings, solid waste is thrown into them plastics of different types.

Line 215: systems can be prepared not performed.

Figure 3, Species should be in italics

Line 329, figure number is wrong

Table 1, is not necessary to include the control, just say no weight loss was detected. If the data is in the table there is no need to repeat in the text.

Figure 6. Homogenize graphs size.

Discussion

Line 382 should be in past tense.

Rewrite line 467: The emergence of a band at 1650 cm-1 is attributed to carbonyl group formation, indicates structural changes under fungal treatment [71].

Comments on the Quality of English Language

Some ideas are unclear. Please revise english. 

Author Response

Thank you for your thoughtful feedback. Please find our responses to your comments below:

In general, the text seems to have many AI modifications, it should be revised. Remove words like notable, emergence and conversely. English should be improved.

Repeated ideas in the abstract.

Response OK. It was corrected.

Introduction

Too long, ideas should be more concise.

Many semicolons are used where comma should be used.

Some ideas are two long, divide them.

Line 85: in vitro, should be in italics

Response Thank you for pointing that out. Those details have been corrected.

Materials

I don’t understand the idea in line 135: In the surroundings, solid waste is thrown into them plastics of different types.

Response It was corrected.

Line 215: systems can be prepared not performed.

Response It was corrected.

Figure 3, Species should be in italics

Response: Response It was corrected.

Line 329, figure number is wrong

Response It was corrected.

Table 1, is not necessary to include the control, just say no weight loss was detected. If the data is in the table there is no need to repeat in the text.

Response It was corrected.

Figure 6. Homogenize graphs size.

Response We appreciate the comment regarding the standardization of image sizes. We have attempted to adjust each image so that its content is not distorted and there is no excess white space, while maintaining visual consistency with the structure of the manuscript. However, we are open to making additional adjustments if the editorial committee considers a more uniform format to be necessary.

Discussion

Line 382 should be in past tense.

Response It was corrected

Rewrite line 467: The emergence of a band at 1650 cm-1 is attributed to carbonyl group formation, indicates structural changes under fungal treatment [71].

 Response It was rewritten.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. Typographical / Citation Error：On page 3, line 86–87: “fungi… due to the various enzymes they produce [20, 21, 2].” → Reference “2” is repeated incorrectly.
2. Inconsistent Figure Numbering：On page 9, the text refers to “Figure 65” when showing pH variation, but it should be “Figure 5.”
3. Formatting Error: The article has missing metadata placeholders (e.g., “Academic Editor: Firstname Last-name”; “Citation: To be added by editorial staff”) that should have been completed before submission.
4. Calculation Clarity Issue: The half-life (t½) results (Table 1) are presented, but the method description does not clearly state how replicates were treated when calculating mean ± SD for k and t½.
5. Grammatical Error: In the discussion (line 507–509): “Additionally. The extended half-life…” → “Additionally, the extended half-life…” (incorrect punctuation).
6. The introduction highlights plastic waste problems well, but why did the authors not compare the biodegradation potential of fungi versus bacteria directly, since both are mentioned as effective degraders?
7. Pu et al. (2018; doi.org/10.1371/journal.pone.0190236) studied Aspergillus niger, which is also one of the strains in the LDPE biodegradation study, it demonstrates the metabolic versatility of niger and could be cited in the Introduction or Discussion to support its biotechnological potential.
8. The impact of thermal degradation on microbial biodegradation should be discussed in a background comparison such as tang et al explained(doi.org/10.1016/j.polymdegradstab.2025.111205).
9. The study used PEG-400 as a model substrate for preliminary enzymatic activity. Could this choice overestimate fungal potential since PEG is much more biodegradable than LDPE?
10. The reported LDPE weight loss (max ~4.25% in 50 days) is relatively low compared to literature (some >30% in 90 days). What specific factors in experimental design may have limited degradation efficiency here?
11. The FTIR results suggest oxidative and enzymatic activity (lipases). Why did the authors not directly assay lipase or peroxidase activity in the culture supernatants to strengthen the mechanism claims?
12. Given the very long half-lives reported (up to ~1757 days), how do the authors justify the practical feasibility of applying these fungi in real-world plastic waste management?

 

Author Response

Thank you for your thoughtful feedback. Please find our responses to your comments below.

Typographical / Citation Error：On page 3, line 86–87: “fungi… due to the various enzymes they produce [20, 21, 2].” → Reference “2” is repeated incorrectly.

Response Thank you for your comment. The error has been corrected.

 

Inconsistent Figure Numbering：On page 9, the text refers to “Figure 65” when showing pH variation, but it should be “Figure 5.”

Response It was corrected.

 

Formatting Error: The article has missing metadata placeholders (e.g., “Academic Editor: Firstname Last-name”; “Citation: To be added by editorial staff”) that should have been completed before submission.

Response Thank you for your comment. In accordance with the instructions for the authors, the editor field and citations are completed by the editorial team after the acceptance. We have kept the placeholders according to the official template.

 

Calculation Clarity Issue: The half-life (t½) results (Table 1) are presented, but the method description does not clearly state how replicates were treated when calculating mean ± SD for k and t½.

Response Thank you for your comment. It was added (lines 244-246).

 

Grammatical Error: In the discussion (line 507–509): “Additionally. The extended half-life…” → “Additionally, the extended half-life…” (incorrect punctuation).

Response It was corrected.

 

The introduction highlights plastic waste problems well, but why did the authors not compare the biodegradation potential of fungi versus bacteria directly, since both are mentioned as effective degraders?

Response We appreciate the reviewer’s comment. While it is well established that both fungi and bacteria possess the ability to degrade plastics, this study focused exclusively on fungal strains. This decision was based on the remarkable metabolic versatility of fungi, their capacity to secrete potent extracellular enzymes, and their adaptability to diverse and even extreme environmental conditions. These traits make fungi particularly promising candidates for biotechnological applications in real-world ecological contexts. Although a direct comparison with bacterial strains was not included in this phase, we acknowledge its relevance and consider it a valuable direction for future research.

 

Pu et al. (2018; doi.org/10.1371/journal.pone.0190236) studied Aspergillus niger, which is also one of the strains in the LDPE biodegradation study, it demonstrates the metabolic versatility of niger and could be cited in the Introduction or Discussion to support its biotechnological potential.

Response Thank you for the suggested. The studied was added in the Introduction (line 110 -112).

 

The impact of thermal degradation on microbial biodegradation should be discussed in a background comparison such as tang et al explained(doi.org/10.1016/j.polymdegradstab.2025.111205).

Response I appreciate the suggestion regarding the work of Tang et al. (2025). Although the present study does not consider thermal degradation processes, I recognize that this approach can provide valuable insights for a comprehensive understanding of plastic transformation. We will consider incorporating it into future research exploring synergies between physicochemical pretreatments and fungal biodegradation.

 

The study used PEG-400 as a model substrate for preliminary enzymatic activity. Could this choice overestimate fungal potential since PEG is much more biodegradable than LDPE?

Response We appreciate the observation. Indeed, PEG-400 is more biodegradable than LDPE, and its use at this stage was solely for the purpose of demonstrating the preliminary enzymatic capacity of the strains. The intention is not to directly extrapolate these results to the behavior of LDPE, but rather to guide the selection of strains with degradative potential, which were then evaluated directly on LDPE under controlled conditions. Therefore, we have included a brief clarification in lines 182-185.

 

 

 

The reported LDPE weight loss (max ~4.25% in 50 days) is relatively low compared to literature (some >30% in 90 days). What specific factors in experimental design may have limited degradation efficiency here?

Response Thank you for your observation. Indeed, our results show lower values, which can be attributed to certain factors such as the evaluation time, the use of separate fungal strains, and the absence of oxidative pretreatment, as mentioned in other studies. These points were mentioned in the final paragraph of the discussion, under limitations. However, we believe that our results are important and relevant in the field, as they provide valuable evidence on the biodegradation potential of individual fungal strains against LDPE, a polymer with high environmental persistence. By using controlled conditions and no oxidative pretreatments, a useful baseline is established for future research to optimize degradation through microbial consortia or combined strategies.

 

The FTIR results suggest oxidative and enzymatic activity (lipases). Why did the authors not directly assay lipase or peroxidase activity in the culture supernatants to strengthen the mechanism claims?

Response Thank you for the observation. We could include the analysis you mentioned in future work. For this project, however, we are basing our findings on previous research.

 

Given the very long half-lives reported (up to ~1757 days), how do the authors justify the practical feasibility of applying these fungi in real-world plastic waste management?

Response As previously noted in the limitations section, the long half-life of LDPE poses a significant challenge to the practical implementation of fungal biodegradation strategies, especially under natural conditions. While the analyses performed offer a preliminary approximation of the degradative potential of the strains, the incorporation of enzymes such as laccase could strengthen this capacity. In this regard, the study provides a strategic basis for the future development of more efficient biotechnological approaches.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

First of all, thank you very much for the opportunity to read and review of very interesting manuscript entitled Biodegradation of Low-Density Polyethylene by Native Aspergillus Strains Isolated from Plastic-Contaminated Soil. The manuscript focuses on the capability of different soil strains and species of Aspergillus fungi to degrade the low-density poliethylene in experimental conditions. The topic that have been taken up by authors is crucial and relevant, due to the role of microfungi in degradation of toxic and waste substances occuring in natural environment. Therefore, it is necessary to search for new strains/species od high effective fungal and bacterial strains with abbilities to biodegrade substances such as low-density poliethylene, and to optimize conditions of their culture to improve their activity.

The work is, in general, prepared correctly, nevertheless the following is necessary before it is accepted for publication:

1. Minor linguistic correction of the manuscript.
2. Additional literature to the "Introduction Section", according to the posted notes.
3. Detailed revisions in the "Materials and Methods Section".
4. In-depth tracking of the results discussed in "Results Section" and analysis of their significance.
5. Rearrangement of tables and figures explanations according to the posted notes.
6. Rearrangement of the "Discussion Section", its enrichment with the results of own research and literature data according to posted notes. 
7. Detailed revision of "References Section", especially in terms of DOI adresses.

In addition, I have provided below a detailed description of the inaccuracies noted, along with comments that I would like the authors to address:

 

Abstract Section

• Lines 22-23: I suggest not repeating the information regarding the names of the three strains of the genus Aspergillus isolated from plastic-contaminated soil, as well as their origin. This is a repetition of the information provided earlier (lines 16-19).

 

Introduction Section

• Line 41: I suggest giving values in SI units, i.e., instead of using the term “tons,” I suggest using “Mg.”
• Line 46: Do the authors have information on the percentage increase in plastic in municipal solid waste over recent years? If so, I suggest including it in the Introduction Section.
• Lines 47-48: I suggest giving values in SI units, i.e., instead of using the term “tons,” I suggest using “Mg.”
• Line 60: I suggest giving values in SI units, i.e., instead of using the term “tons,” I suggest using “Mg.”
• Line 63: Instead of “/m³,” I suggest giving this value as “m^-3,” without the “/” sign.
• Line 76: I recommend that the authors supplement the information on specific environmental factors determining the microbiological biodegradation of plastic.
• Line 79: I suggest that the authors supplement the information with specific examples of enzymes involved in the biodegradation of plastic and its mechanism.
• Line 80: I ask the authors to explain why biofilm formation is so important in the context of plastic biodegradation?
• Line 85: I suggest that the authors enrich the section with specific examples of bacteria and fungi species that are most commonly used in the biodegradation of plastic.
• Lines 100-102: I recommend that the authors provide information on which of the listed fungi are currently of greatest practical importance.
• Line 113: I suggest that the authors verify whether a period should be written after the abbreviation “et al,” because in my opinion it should be there.
• Line 115: I suggest that the authors verify whether a period should be written after the abbreviation “et al,” because in my opinion it should be there.

 

Materials and Methods Section

• Line 132: I suggest shortening the title of subsection 2.1. and writing it as “Sampling Site.”
• Line 147: I suggest changing the title of Figure 1, starting with “Location of the sampling,” without specifying the platform used (Google Maps) in the first place.
• Line 149: I suggest that the authors change the title of subsection 2.2. to “Plastic contaminated soil characteristics.”
• Line 151: I would like the authors to explain why soil samples were taken from a depth of 3-5 cm? What was the reason for this, and what was the system for selecting sampling sites? Was it a fully randomized system? From how many sites exactly was the soil taken?
• Lines 149-154: I ask the authors to supplement the information regarding the exact characteristics of the tested soil, as well as its systematic classification. What type of soil was analyzed? Was the classification performed according to WRB or FAO?
• Line 157: I suggest changing the units from ml to cm³. Please also provide information on the preparation of soil samples for analysis, including sieving through a sieve with an appropriate mesh size.
• Line 159: I suggest changing the units from ml to cm³.
• Line 160: I would ask the authors to provide information on why the incubation lasted 7 days. Did the fungi not start to appear around day 5?
• Line 161: I would like to ask the authors whether all colonies meeting these criteria were transplanted? If not all of them, what was the selection system used to choose those that were to be used for further analysis?
• Lines 161-162: I suggest that the authors provide information on what specific macroscopic characteristics were taken into account when characterizing the strains.
• Line 162: I suggest using the term “slide cultures” instead of “microculture” because it is a more specific term. I also ask the authors to provide information on the microscopic characteristics that were characterized for the studied strains. Did the authors use any systematic keys? If so, which ones?
• Lines 165-166: I recommend that the authors supplement the information on which enzymes were specifically detected and the exact composition of the solid media used.
• Lines 173-175: I suggest changing the units from “g/L” to “g dm^-3” and providing the chemical composition of the medium using chemical formulas instead of colloquial names. In my opinion, this will improve the readability of this part of the "Materials and Methods" Section.
• Line 176: I ask the authors to provide detailed information on the conditions under which the sterilization of microbiological media took place.
• Line 186: I suggest that the authors verify whether a period should be added after the abbreviation “et al,” as in my opinion it should be there.
• Line 187: I recommend that the authors supplement the information on the exact course of the DNA extraction process.
• Line 189: I recommend that the authors supplement the information on the exact course of the amplicon purification process.
• Line 205: I ask the authors to provide information on how and under what conditions the process of draining the strips took place.
• Line 213: I suggest changing the notation of units from “mL^-1” to “cm^-3.” Additionally, in the complete notation concerning spore suspension, it is not necessary to use the “×” symbols.
• Lines 216-217: I suggest changing the notation of units from ml to cm³.
• Line 219: I ask the authors to explain why the entire process took as long as 50 days. Is this the optimal period?
• Line 221: I suggest changing the title of subsection 2.6.1. to “Aspergillus strains growth on LDPE strips.”
• Lines 222-225: I would ask the authors to explain exactly what this “qualitative analysis” consisted of.
• Lines 227-228: I suggest changing the units from ml to cm³.
• Lines 266-267: I would like to ask the authors to explain what guided them in selecting statistical tests for normality. Why did the authors decide to use the Kolmogorov-Smirnov and Shapiro-Wilk tests, rather than the Shapiro-Wilk test alone?

 

Results Section

• Line 273:  I believe that the subscript for strain H6C is unnecessary.
• Line 275: I suggest that the authors describe the colors of the outer (upper) part of the colony collectively as “obverse of the colony” instead of “conidial heads.”
• Line 276: I suggest that the authors describe the colors of the lower part of the colony collectively as “reverse of the colony” instead of “back of the medium.”
• Line 277: I would like to ask the authors whether they performed periodic measurements of the diameter of the colonies of the tested strains? If so, where the authors mention differences in the growth rate of the analyzed strains, please provide accurate numerical data relating to these differences.
• Line 279: I recommend using the term “slide cultures” instead of “microcultures.”
• Line 285: I suggest that the authors refrain from providing raw data on halo diameter. The most important information is the ratio of halo to colony diameter, which directly indicates the intensity of enzymatic activity. Therefore, I recommend that the authors recalculate the results and present them as the so-called enzymatic index.
• Line 296: Incorrect citation of Figure. Instead of Figure 4, the authors should, in my opinion, cite Figure 3.
• Lines 300-302: I recommend that the authors shorten the title of Figure 3 and limit it to only the first sentence of the current title. I suggest placing the rest of the current text under Figure 3 as “Explanations.”
• Lines 303-304: In my opinion, the first sentence is unnecessary because it is a direct repetition of the title of Figure 4. I suggest that the authors begin the fragment by describing what can be seen in the cited Figure 4.
• Lines 309-310: I suggest that the authors change the title of Figure 4 to “Aspergillus strains growth on LDPE strips after 50 days of incubation: control (a), strain H1C (b), strain H3C (c), strain H6C (d)”. Alternatively, molecularly confirmed species names can be used here.
• Line 314: Please provide information on whether the decrease in pH recorded by the authors was statistically significant.
• Line 316: Please provide information on whether the increase in pH recorded by the authors was statistically significant.
• Lines 318-328: In the case of Figure 5:
  - I suggest changing the title of the Y-axis to “pH values of culture media.”
  - For the X-axis, I suggest omitting the term “day” for each experiment date, as the units “days” are already on the X-axis.
  - I recommend increasing the font size throughout Figure 5 to improve readability.
• Lines 329-330: Incorrect numbering of Figure. Instead of Figure 65, it should remain Figure 5. I suggest placing the last sentence of the current title of Figure 5 directly below Figure 5 as “Explanations.”
• Lines 331-333: I ask the authors to consider removing the first sentence describing what is in Table 1 and starting the fragment with a discussion of the results, citing the indicated table.
• Line 333: I suggest that the authors standardize the way strain names are written in the indicated fragment and throughout the manuscript, whether they are written with a subscript number or without. Where the authors indicate the significance of changes, I suggest specifying the significance level “α” or the probability “p” for which this significance was demonstrated.
• Lines 334-341: I recommend that the authors refer once or twice in the indicated fragment to Table 1, to which the discussed results refer.
• Line 344: In the case of Table 1:
  - For all columns, I recommend giving their units in parentheses after the column title.
  - I suggest standardizing the number of decimal places for the numerical values given in the entire table, and if the authors decide otherwise, at least within the data columns.
  - Place the text below the table as “Explanations.
• Line 347: I suggest that the authors supplement the information on the significance or lack thereof for the discussed increase.
• Line 350: I suggest that the authors supplement the information on the significance or lack thereof for the discussed decrease.
• Line 356: I ask the authors to provide information on whether the indicated strain differed significantly from the others in terms of the analyzed parameter.
• Lines 361-363: I suggest moving the indicated fragment explaining the analyzed phenomenon to the “Discussion” Section.
• Lines 364-366: In the case of Figure 6:
  - I suggest enlarging the font for all components of the Figure to improve its readability.
• Lines 370-376: In the case of the indicated fragment, I suggest quoting Figure 7 again, to which the discussed results refer.
• Line 378: Incorrect numbering of the figure. I think it should be numbered Figure 7 here.

 

Discussion Section

• Lines 382-398: I suggest that the authors consider removing references to specific Figures in the section devoted to the discussion of results, as these references appeared in the “Results” section. This impairs the readability of the chapter and is not recommended for the “Discussion” section.
• Line 399: If the authors wish to cite a specific figure, I suggest using the term “Figure 3” in parentheses without any references such as “see.” However, I suggest omitting references to Tables and Figures entirely.
• Lines 404-417: I suggest that the authors enrich the indicated fragment with literature data and the results of other authors to which the results presented in this work will be referred.
• Line 418: I suggest that the authors completely omit references to Figures in the “Discussion” section.
• Line 425: I suggest that the authors completely omit references to Figures in the “Discussion” section.
• Line 434: If the authors wish to cite a specific Table, I suggest using the term “Table 1” in parentheses without any references such as “see.” However, I suggest omitting all references to Tables and Figures.
• Line 450: I suggest that the authors completely abandon references to Figures in the “Discussion” section.
• Line 462: I suggest that the authors completely omit references to Figures in the “Discussion” section.
• Lines 467-473: I suggest that the authors enrich the indicated fragment with more results from their own research.
• Line 475: I suggest that the authors completely abandon references to Figures in the “Discussion” section.
• Line 481: I suggest that the authors completely abandon references to Figures in the “Discussion” section.
• Lines 482-505: I suggest that the authors increase the number of results from other authors with whom the data included in this section will be discussed.

 

References Section

• Lines 555-697: I suggest that the authors review the accuracy of the citation system for the works included in the References section. Additionally, I suggest adding doi addresses for all works where possible. When providing a doi address, I recommend using the full address, i.e., https://doi.org/NUMBER.

 

Best regards,
Reviewer

Author Response

Thank you for your thoughtful feedback. Please find our responses to your comments below:

Abstract Section

Lines 22-23: I suggest not repeating the information regarding the names of the three strains of the genus Aspergillus isolated from plastic-contaminated soil, as well as their origin. This is a repetition of the information provided earlier (lines 16-19).

Response It was corrected.

 

Introduction Section

Line 41: I suggest giving values in SI units, i.e., instead of using the term “tons,” I suggest using “Mg.”

Response Ok. It was changed.

Line 46: Do the authors have information on the percentage increase in plastic in municipal solid waste over recent years? If so, I suggest including it in the Introduction Section.

Response We appreciate the suggestion to include percentage data on the increase of plastic in municipal solid waste. While we agree on the relevance of such information, we have opted to maintain the current structure of the Introduction in order to preserve the coherence of the proposed approach.

 

Lines 47-48: I suggest giving values in SI units, i.e., instead of using the term “tons,” I suggest using “Mg.”

Response Ok. It was changed.

 

Line 60: I suggest giving values in SI units, i.e., instead of using the term “tons,” I suggest using “Mg.”

Response Ok. It was changed.

 

Line 63: Instead of “/m³,” I suggest giving this value as “m^-3,” without the “/” sign.

Response Ok. It was changed.

 

Line 76: I recommend that the authors supplement the information on specific environmental factors determining the microbiological biodegradation of plastic.

Response Ok. It was added.

 

Line 79: I suggest that the authors supplement the information with specific examples of enzymes involved in the biodegradation of plastic and its mechanism.

Response We appreciate the suggestion to include specific examples of enzymes and mechanisms involved in the biodegradation of plastics. Indeed, several studies have documented the role of extracellular enzymes such as cutinases, lipases, esterases, and laccases, which catalyze the breakdown of polymer bonds through hydrolysis or oxidation, facilitating the conversion of plastics into low molecular weight compounds. These products are subsequently assimilated by microorganisms and mineralized into CO₂, H₂O, or CH₄, depending on environmental conditions (Srikanth et al., 2022; Kotova et al., 2021). We have supplemented the text in the corresponding section to reflect this information without altering the overall structure of the manuscript.

 

Line 80: I ask the authors to explain why biofilm formation is so important in the context of plastic biodegradation?

Response Ok. A brief description was added.

 

Line 85: I suggest that the authors enrich the section with specific examples of bacteria and fungi species that are most commonly used in the biodegradation of plastic.

Response We appreciate your suggestion to include specific examples of microbial species. To enrich the section without losing conciseness, we have incorporated a representative selection of bacteria.

 

Lines 100-102: I recommend that the authors provide information on which of the listed fungi are currently of greatest practical importance.

Response Agradecemos su recomendación sobre destacar los hongos de mayor importancia práctica. En la versión actual del manuscrito ya se incluyen especies fúngicas ampliamente estudiadas en biodegradación de plásticos, como Aspergillus spp., Phanerochaete chrysosporium, Cladosporium cladosporioides y Pleurotus spp., las cuales han demostrado eficacia en la degradación de polímeros como LDPE, PET y PVC. Además, se señala que la clase Eurotiomycetes concentra el mayor número de especies con esta capacidad. Consideramos que esta información ya responde a su sugerencia, evitando redundancias en la sección.

 

Line 113: I suggest that the authors verify whether a period should be written after the abbreviation “et al,” because in my opinion it should be there.

Response It was added.

 

Line 115: I suggest that the authors verify whether a period should be written after the abbreviation “et al,” because in my opinion it should be there.

Response It was added.

 

 

Materials and Methods Section

Line 132: I suggest shortening the title of subsection 2.1. and writing it as “Sampling Site.”

Response Ok. It was changed.

 

Line 147: I suggest changing the title of Figure 1, starting with “Location of the sampling,” without specifying the platform used (Google Maps) in the first place.

Response Ok. It was modified.

 

Line 149: I suggest that the authors change the title of subsection 2.2. to “Plastic contaminated soil characteristics.”

Response We appreciate the suggestion regarding the change of title for subsection 2.2. However, we believe that the current title, “Sampling of LDPE Residues from Contaminated Soil,” more accurately reflects the content of the section, which focuses on the sample collection procedure rather than soil characterization.

 

Line 151: I would like the authors to explain why soil samples were taken from a depth of 3-5 cm? What was the reason for this, and what was the system for selecting sampling sites? Was it a fully randomized system? From how many sites exactly was the soil taken?

Response. Soil samples were collected at a depth of between 3 and 5 cm, focusing on the aerobic surface layer, where microbial activity is highest. Sampling sites were selected based on visible accumulation of plastic waste and human activity.

 

Lines 149-154: I ask the authors to supplement the information regarding the exact characteristics of the tested soil, as well as its systematic classification. What type of soil was analyzed? Was the classification performed according to WRB or FAO?

Response: We appreciate your comment regarding soil characterization. However, the objective of this study does not include soil analysis or systematic soil classification, as this is not a central variable in the experimental design or in the interpretation of the results. The focus has been on analyzing the potential of three strains of Aspergillus to biodegrade plastic under controlled conditions, so it was not considered relevant to incorporate such characterization.

 

Line 157: I suggest changing the units from ml to cm³. Please also provide information on the preparation of soil samples for analysis, including sieving through a sieve with an appropriate mesh size.

Response: We appreciate your comment regarding the unit of volume. In this manuscript, mL has been used for consistency with the language commonly used in microbiological protocols. Since mL and cm³ are equivalent, we believe that the use of mL does not affect the clarity or interpretation of the results. However, if the editorial committee deems it necessary, we are willing to make the adjustment. On the other hand, we appreciate your suggestion regarding the preparation of soil samples. In this study, soil was used exclusively as an environmental source for the isolation of fungi with LDPE degradation potential, without its physical or chemical properties constituting a variable for analysis. Therefore, it was not considered necessary to apply sieving procedures or perform granulometric classification.

 

Line 159: I suggest changing the units from ml to cm³.

Response: It has already been explained.

 

Line 160: I would ask the authors to provide information on why the incubation lasted 7 days. Did the fungi not start to appear around day 5?

Response: Although some fungal colonies may begin to develop by day 5, the incubation period was extended to seven days to ensure complete colony growth, facilitate morphological differentiation, and enable more accurate selection of Aspergillus strains.

 

Line 161: I would like to ask the authors whether all colonies meeting these criteria were transplanted? If not all of them, what was the selection system used to choose those that were to be used for further analysis?

Response: Six colonies with different characteristics were observed, three of which had characteristics typical of Aspergillus in terms of spore texture and color, and were corroborated by molecular identification. The others corresponded to different species, which were not part of the study's objective, which was to work only with Aspergillus.

 

Lines 161-162: I suggest that the authors provide information on what specific macroscopic characteristics were taken into account when characterizing the strains.

Response: The suggestion was included (line 164-165)

Line 162: I suggest using the term “slide cultures” instead of “microculture” because it is a more specific term. I also ask the authors to provide information on the microscopic characteristics that were characterized for the studied strains. Did the authors use any systematic keys? If so, which ones?

Response: The suggested changes were made and the microscopic characteristics concerning Aspergillus were written. However, we did not use taxonomic keys since the species were identified molecularly.

 

Lines 165-166: I recommend that the authors supplement the information on which enzymes were specifically detected and the exact composition of the solid media used.

Response: We appreciate the suggestion. In this study we did not seek to identify specific enzymes, but to perform a preliminary evaluation of the extracellular enzymatic potential of Aspergillus strains in solid media with model substrates. PEG-400 was used as a hydrophobic compound to simulate the properties of LDPE, and 0.1 % (v/v) vegetable oil was used to detect lipolytic activity. The objective was to explore whether the fungal strains present complementary enzymatic machinery that could contribute to plastic degradation processes. This was indicated in lines 170 - 172.

 

Lines 173-175: I suggest changing the units from “g/L” to “g dm^-3” and providing the chemical composition of the medium using chemical formulas instead of colloquial names. In my opinion, this will improve the readability of this part of the "Materials and Methods" Section.

Response: We appreciate your suggestion regarding the notation of units and the presentation of the components of the culture medium. Regarding units, “g/L” has been used for consistency with the usual technical language in microbiology, where this notation is widely recognized and accepted. However, if the editorial committee considers the use of “g·dm⁻³” preferable, we are willing to make the adjustment. Regarding the composition of the medium, we appreciate your recommendation and have replaced the colloquial names with the corresponding chemical formulas.

Line 176: I ask the authors to provide detailed information on the conditions under which the sterilization of microbiological media took place.

Response: We appreciate your suggestion. All microbiological media were sterilized by autoclaving at 121 °C for 15 minutes, following standard procedures widely adopted in microbiological practice. As this is a routine step, its description was kept concise in the manuscript.

 

Line 186: I suggest that the authors verify whether a period should be added after the abbreviation “et al,” as in my opinion it should be there.

Response: It was verified.

 

Line 187: I recommend that the authors supplement the information on the exact course of the DNA extraction process.

Line 189: I recommend that the authors supplement the information on the exact course of the amplicon purification process.

Response to line 187 and 189: We appreciate your comments. Both the DNA extraction and amplicon purification procedures were carried out by an external specialized laboratory as part of a contracted service. The methodological description included in the manuscript reflects the technical information provided by the laboratory, including the reagents, protocols, and conditions reported in the corresponding documentation.

 

Line 205: I ask the authors to provide information on how and under what conditions the process of draining the strips took place.

Response: We appreciate your comment. It was changed for: “After immersion, the strips were aseptically dried for ten minutes to remove excess oil, gently blotted with sterile absorbent paper, and handled inside a biosafety cabinet to maintain sterility”.

 

Line 213: I suggest changing the notation of units from “mL^-1” to “cm^-3.” Additionally, in the complete notation concerning spore suspension, it is not necessary to use the “×” symbols.

Response: The “×” symbols was removed.

 

Lines 216-217: I suggest changing the notation of units from ml to cm³.

Response: It has already been explained.

 

Line 219: I ask the authors to explain why the entire process took as long as 50 days. Is this the optimal period?

Response: We appreciate your comment regarding the total duration of the process. The 50-day period was defined as an experimental criterion, considering that the systems were developed in tubes with limited volumes, which restricted the possibility of performing intermediate evaluations without compromising the sample. This period allowed us to obtain representative results on the degradative activity of the isolated strains, with a view to future scaling up of the system under more controlled conditions and with greater sample availability. We believe that this approach provides a solid basis for the design of subsequent trials in expanded systems.

 

Line 221: I suggest changing the title of subsection 2.6.1. to “Aspergillus strains growth on LDPE strips.”

Response: It was changed.

 

Lines 222-225: I would ask the authors to explain exactly what this “qualitative analysis” consisted of.

Response: We appreciate your interest in the qualitative analysis section. In this study, the term “qualitative analysis” refers to the observation of visible physical changes in the LDPE polymer during the incubation process with fungal strains. These changes included alterations in the texture, coloration, opacity, and surface fragmentation of the plastic, evaluated through visual inspection and photographic recording. Although not quantified using instrumental techniques at this stage, these observations allowed us to identify preliminary signs of biodegradation that warrant further detailed evaluation.

 

Lines 227-228: I suggest changing the units from ml to cm³.

Response: It has already been explained.

 

Lines 266-267: I would like to ask the authors to explain what guided them in selecting statistical tests for normality. Why did the authors decide to use the Kolmogorov-Smirnov and Shapiro-Wilk tests, rather than the Shapiro-Wilk test alone?

Response: Thank you for pointing that out. Only the Shapiro Wilks test was used; there was an error in writing Kolmogorov Smirnov.

 

Results Section

Line 273:  I believe that the subscript for strain H6C is unnecessary.

Response: It was corrected.

Line 275: I suggest that the authors describe the colors of the outer (upper) part of the colony collectively as “obverse of the colony” instead of “conidial heads.”

Response: It was corrected.

 

Line 276: I suggest that the authors describe the colors of the lower part of the colony collectively as “reverse of the colony” instead of “back of the medium.”

Response: It was corrected.

 

Line 277: I would like to ask the authors whether they performed periodic measurements of the diameter of the colonies of the tested strains? If so, where the authors mention differences in the growth rate of the analyzed strains, please provide accurate numerical data relating to these differences.

Response: Measurement was not performed, since we do not consider it as part of the objective; it could be complementary data that could be taken into account in future studies. We appreciate the suggestion.

Line 279: I recommend using the term “slide cultures” instead of “microcultures.”

Response: It was corrected.

 

Line 285: I suggest that the authors refrain from providing raw data on halo diameter. The most important information is the ratio of halo to colony diameter, which directly indicates the intensity of enzymatic activity. Therefore, I recommend that the authors recalculate the results and present them as the so-called enzymatic index.

Response: We sincerely thank the reviewer for this valuable suggestion. However, in our study the enzymatic activity was not evaluated through direct colony growth on agar medium, but rather by applying extracellular supernatants into wells. Therefore, there was no colony diameter to normalize the halo size, and the enzymatic index (ratio halo/colony) could not be calculated in this experimental setup. In this type of well diffusion assay, the absolute halo diameter represents the direct indicator of extracellular enzymatic activity. We have carefully revised the Methods section to ensure this clarification is evident and have emphasized it in the Results to avoid any possible confusion.

 

Line 296: Incorrect citation of Figure. Instead of Figure 4, the authors should, in my opinion, cite Figure 3.

Response: ok. It was corrected.

 

Lines 300-302: I recommend that the authors shorten the title of Figure 3 and limit it to only the first sentence of the current title. I suggest placing the rest of the current text under Figure 3 as “Explanations.”

Response: ok. It was corrected.

 

Lines 303-304: In my opinion, the first sentence is unnecessary because it is a direct repetition of the title of Figure 4. I suggest that the authors begin the fragment by describing what can be seen in the cited Figure 4.

Response: ok. It was corrected.

 

Lines 309-310: I suggest that the authors change the title of Figure 4 to “Aspergillus strains growth on LDPE strips after 50 days of incubation: control (a), strain H1C (b), strain H3C (c), strain H6C (d)”. Alternatively, molecularly confirmed species names can be used here.

Response: ok. It was corrected.

 

Line 314: Please provide information on whether the decrease in pH recorded by the authors was statistically significant.

Response: The Tukey test was performed to observe statistical differences (line 324-325).

 

Line 316: Please provide information on whether the increase in pH recorded by the authors was statistically significant.

Response: The Tukey test was performed to observe statistical differences (line 324-325).

 

Lines 318-328: In the case of Figure 5:
- I suggest changing the title of the Y-axis to “pH values of culture media.”
- For the X-axis, I suggest omitting the term “day” for each experiment date, as the units “days” are already on the X-axis.
- I recommend increasing the font size throughout Figure 5 to improve readability.

Response: We appreciate the reviewer’s suggestions. The X-axis of Figure 5 has been adjusted by removing the word “Day” from each time point, as the unit is clearly indicated. Regarding the Y-axis title and font size, we consider that the figure retains a clear and readable structure that adequately supports the interpretation of the results, and therefore no further modifications were made

 

Lines 329-330: Incorrect numbering of Figure. Instead of Figure 65, it should remain Figure 5. I suggest placing the last sentence of the current title of Figure 5 directly below Figure 5 as “Explanations.”

Response: ok. It was corrected.

 

Lines 331-333: I ask the authors to consider removing the first sentence describing what is in Table 1 and starting the fragment with a discussion of the results, citing the indicated table.

Response: ok. It was corrected.

 

Line 333: I suggest that the authors standardize the way strain names are written in the indicated fragment and throughout the manuscript, whether they are written with a subscript number or without. Where the authors indicate the significance of changes, I suggest specifying the significance level “α” or the probability “p” for which this significance was demonstrated.

Response: ok. Thank you for the suggested

 

Lines 334-341: I recommend that the authors refer once or twice in the indicated fragment to Table 1, to which the discussed results refer.

Response: ok. Thank you for the suggested

 

Line 344: In the case of Table 1:
- For all columns, I recommend giving their units in parentheses after the column title.
- I suggest standardizing the number of decimal places for the numerical values given in the entire table, and if the authors decide otherwise, at least within the data columns.
- Place the text below the table as “Explanations.

Response: We appreciate the reviewer’s suggestions. Table 1 was prepared following editorial criteria previously accepted by the journal, and we believe its current format provides a clear and coherent presentation of the results. Therefore, we have chosen to retain the structure as presented.

Line 347: I suggest that the authors supplement the information on the significance or lack thereof for the discussed increase.

Response: We appreciate the reviewer’s suggestion. The ICO and DBI values shown in Figure 6c are interpreted as descriptive indicators of LDPE oxidation and structural degradation, derived from FTIR spectral analysis. At this stage of the study, no inferential statistical analysis was applied to these indices, as only one spectral measurement per sample was obtained. Nevertheless, the observed differences between treatments reflect consistent trends with weight loss and pH variation results, supporting the biodegradative activity of the evaluated strains.

 

Line 350: I suggest that the authors supplement the information on the significance or lack thereof for the discussed decrease.

Response: Thank you for the observation. The significance of the decrease in –CH band intensity is already addressed in the discussion section, where it is interpreted as evidence of LDPE backbone decomposition and linked to the oxidative and enzymatic activity of the evaluated fungal strains.

 

Line 356: I ask the authors to provide information on whether the indicated strain differed significantly from the others in terms of the analyzed parameter.

Response: We appreciate the observation. However, at this stage of the study, each strain was evaluated with a single repetition, which limits the possibility of applying inferential statistical analyses to determine significant differences between groups. Therefore, the values presented should be interpreted as preliminary trends that suggest variability between strains in the ICO and DBI indices, but do not allow definitive conclusions to be drawn about significant differences. We plan to increase the number of replicates in future stages to strengthen the statistical validity of the findings.

 

Lines 361-363: I suggest moving the indicated fragment explaining the analyzed phenomenon to the “Discussion” Section.

Response: ok. It was corrected.

 

Lines 364-366: In the case of Figure 6:
- I suggest enlarging the font for all components of the Figure to improve its readability.

Response: ok. It was corrected.

 

Lines 370-376: In the case of the indicated fragment, I suggest quoting Figure 7 again, to which the discussed results refer.

Response: ok. It was corrected.

 

Line 378: Incorrect numbering of the figure. I think it should be numbered Figure 7 here.

Response: ok. It was corrected.

 

Discussion Section

Lines 382-398: I suggest that the authors consider removing references to specific Figures in the section devoted to the discussion of results, as these references appeared in the “Results” section. This impairs the readability of the chapter and is not recommended for the “Discussion” section.

Response: We appreciate the suggestion. The discussion section has been reviewed, and references to figures that were redundant or already addressed in the results section have been removed. However, we have retained those mentions we consider necessary to reinforce the interpretation of findings and to facilitate the connection between results and analysis.

 

Line 399: If the authors wish to cite a specific figure, I suggest using the term “Figure 3” in parentheses without any references such as “see.” However, I suggest omitting references to Tables and Figures entirely.

Response: Ok. It was corrected.

 

Lines 404-417: I suggest that the authors enrich the indicated fragment with literature data and the results of other authors to which the results presented in this work will be referred.

Response: We appreciate the suggestion. Although molecular characterization of the strains was performed, this section served a complementary role and was not the main focus of the study. Therefore, it was presented concisely, without extensive comparisons to other works. We consider that the level of detail provided is sufficient to support the identification of the strains used in the biodegradation assays.

 

Line 418: I suggest that the authors completely omit references to Figures in the “Discussion” section.

Response: Ok. It was corrected.

 

 

Line 425: I suggest that the authors completely omit references to Figures in the “Discussion” section.

Response: Ok. It was corrected.

 

 

Line 434: If the authors wish to cite a specific Table, I suggest using the term “Table 1” in parentheses without any references such as “see.” However, I suggest omitting all references to Tables and Figures.

Response: Ok. It was corrected.

 

Line 450: I suggest that the authors completely abandon references to Figures in the “Discussion” section.

Response: Ok. It was corrected.

 

 

Line 462: I suggest that the authors completely omit references to Figures in the “Discussion” section.

Response: Ok. It was corrected.

 

Lines 467-473: I suggest that the authors enrich the indicated fragment with more results from their own research.

Response: It was added in line 514 – 517.

 

Line 475: I suggest that the authors completely abandon references to Figures in the “Discussion” section.

Response: Ok. It was corrected.

 

 

Line 481: I suggest that the authors completely abandon references to Figures in the “Discussion” section.

Response: Ok. It was corrected.

 

Lines 482-505: I suggest that the authors increase the number of results from other authors with whom the data included in this section will be discussed.

Response: We appreciate the suggestion. In the indicated section (lines 482–505), we have prioritized a focused discussion of the morphological findings supported by key references. We believe that further expansion may compromise the overall balance of the manuscript, so we have maintained a moderate length that highlights the results without exceeding the intended scope of the discussion.

 

 

References Section

Lines 555-697: I suggest that the authors review the accuracy of the citation system for the works included in the References section. Additionally, I suggest adding doi addresses for all works where possible. When providing a doi address, I recommend using the full address, i.e., https://doi.org/NUMBER.

Response: Thank you for the suggested.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the modifications. I have some concerns in the methodology of section 2.6. This is a central part of the work but is unclear. For this degradation what does the in vitro system has? is just a LDPE strip submerged in what? a salt medium?, inoculated with what amount of spores? incubated for how long at what conditions (temperatur, light, agitation?

Author Response

Thank you for the modifications. I have some concerns in the methodology of section 2.6. This is a central part of the work but is unclear. For this degradation what does the in vitro system has? is just a LDPE strip submerged in what? a salt medium?, inoculated with what amount of spores? incubated for how long at what conditions (temperatur, light, agitation?

Response: Thanks for your suggestion. It has been modified for clarity.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Accept

Author Response

We appreciate your suggestions. 

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you very much for the chance to read and review the revised version of your manuscript  entitled Biodegradation of Low-Density Polyethylene by Native Aspergillus Strains Isolated from Plastic-Contaminated Soil. The authors addressed all the comments and suggestions I made. They did not agree with all of them, however, they satisfactorily explained the reason for their decision. The manuscript sent for re-review was significantly improved.

In addition, I have provided below a detailed description of the editorial inaccuracies noted:

 

Introduction Section

• Line 72: I suggest that the authors standardize the notation of units to particles × m^-3 as in other cases.
• Line 137: It seems to me that there is no space between the words “by” and “the.” I ask the authors to verify this.

 

Materials and Methods Section

• Line 240: Nevertheless, I suggest that the authors change the way units are written from mL to cm³.
• Line 242: Nevertheless, I suggest that the authors change the way units are written from mL to cm³.
• Line 250: I suggest that the authors standardize the title of subsection 2.3.2 by capitalizing all words, as in the titles of other subsections.
• Lines 258-259: Nevertheless, I suggest that the authors change the units from g/L to g dm^-3.
• Lines 283: Nevertheless, I suggest that the authors change the notation of units from g/L to g dm^-3.
• Line 304: I suggest that the authors standardize the title of subsection 2.5 by capitalizing all words, as in the titles of other subsections.
• Line 321: Nevertheless, I suggest that the authors change the notation of units to spores cm^-3.
• Line 328: I suggest that the authors standardize the title of subsection 2.6 by capitalizing all words, as in the titles of other subsections.
• Line 343: I suggest that the authors standardize the title of subsection 2.6.1 by capitalizing all words, as in the titles of other subsections.
• Line 349-350: Nevertheless, I suggest that the authors change the notation of units from mL to cm³.

 

Results Section

• Line 417: I suggest that the authors standardize the way strain numbers are written, in the indicated place and throughout the manuscript, i.e., use or do not use subscripts.

 

Discussion Section

• Lines 584-604: Nevertheless, I suggest that the authors refrain from referring to figures in tables in the indicated fragment and the entire chapter devoted to discussion, but I leave this to their discretion and that of the editors.

 

References Section

• Lines 824-1000: Nevertheless, I suggest that authors provide the full address when giving doi numbers, i.e., https://doi.org/NUMBER.

 

Best regards,
Reviewer

Author Response

We appreciate your suggestions. 

Introduction Section

• Line 72: I suggest that the authors standardize the notation of units to particles × m^-3 as in other cases.

Response: It was corrected.

• Line 137: It seems to me that there is no space between the words “by” and “the.” I ask the authors to verify this.

Response: It was corrected (line 97).

 Materials and Methods Section

• Line 240: Nevertheless, I suggest that the authors change the way units are written from mL to cm³.

Response: We appreciate the suggestion once again. However, we believe that mL is a unit that we have been using, which has been accepted in publications both in mdpi journals and others.

 

• Line 242: Nevertheless, I suggest that the authors change the way units are written from mL to cm³.

Response: We appreciate the suggestion once again. Our response remains the same as before.

 

• Line 250: I suggest that the authors standardize the title of subsection 2.3.2 by capitalizing all words, as in the titles of other subsections.

Response: It was corrected.

 

• Lines 258-259: Nevertheless, I suggest that the authors change the units from g/L to g dm^-3.

Response: Our response remains the same as before.

 

• Lines 283: Nevertheless, I suggest that the authors change the notation of units from g/L to g dm^-3.

Response: Our response remains the same as before.

 

• Line 304: I suggest that the authors standardize the title of subsection 2.5 by capitalizing all words, as in the titles of other subsections.

Response: It was corrected.

 

• Line 321: Nevertheless, I suggest that the authors change the notation of units to spores cm^-3.

Response: Our response remains the same as before.

 

• Line 328: I suggest that the authors standardize the title of subsection 2.6 by capitalizing all words, as in the titles of other subsections.

Response: It was corrected.

 

• Line 343: I suggest that the authors standardize the title of subsection 2.6.1 by capitalizing all words, as in the titles of other subsections.

Response: It was corrected.

 

• Line 349-350: Nevertheless, I suggest that the authors change the notation of units from mL to cm³.

Response: Our response remains the same as before.

 

                                                           Results Section

• Line 417: I suggest that the authors standardize the way strain numbers are written, in the indicated place and throughout the manuscript, i.e., use or do not use subscripts.

• Response: It was corrected. It was standardized by mentioning the species with the strain number (i.e. niger H1C).

Discussion Section

• Lines 584-604: Nevertheless, I suggest that the authors refrain from referring to figures in tables in the indicated fragment and the entire chapter devoted to discussion, but I leave this to their discretion and that of the editors.

Response: The figures and table 1 were removed from the text in the discussion.

References Section

• Lines 824-1000: Nevertheless, I suggest that authors provide the full address when giving doi numbers, i.e., https://doi.org/NUMBER.

Response: It was added.

 

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

-

Author Response

Thanks