Material Conversion, Microbial Community Composition, and Metabolic Functional Succession During Algal Sludge Composting

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study provides insufficient novelty and methodological rigor, warranting rejection. Key criticisms include:

1. The findings on community succession and functional roles largely confirm existing knowledge from composting other substrates, failing to demonstrate what is unique about "algal sludge" composting. You may present relevant rebuttals to validate your claim.
2. The claim that microbial diversity "did not show any significant response" to composting phases directly contradicts the well-established literature and is biologically implausible given the extreme temperature shifts reported.
3. Proposing Geobacillus (a well-known thermophile) as a "potential biomarker" for a specific phase is trivial and adds no novel insight, as its presence in the thermophilic phase is entirely expected.
4. Attributing specific functions (e.g., N-transformation to bacteria, humification to fungi) based purely on correlation (structural equation model: SEM analysis) without metagenomic or metatranscriptomic data is speculative and not causally proven.
5. I observed mix type citation style in your introduction part. Some are numbered while some are in Zhang et al. (2021) form.
6. The Figures are very poor resolution. It is mandatory to provide better images.
   
   Additionally,

   1. In the introduction part, beside the poor literature review, there are mixed types of citations. Very few of the references are relevant.
   2. The similarity also seems quite high from the ithenticate report.
   3. I am not convinced with the scientific approach of this article. Nothing new has been observed in this paper. 

Author Response

Thank you for your review and comments on our manuscript. While we respect your overall assessment, we believe that our study provides sufficient novelty and methodological rigor. We would like to take this opportunity to address each of your concerns in detail and clarify the contributions of our research. Please allow us to respond to your comments point by point. Relevant changes have been color-coded in blue in the revised manuscript.

Comments:

1.The findings on community succession and functional roles largely confirm existing knowledge from composting other substrates, failing to demonstrate what is unique about "algal sludge" composting. You may present relevant rebuttals to validate your claim.

Response: Thanks for your comments. We sincerely appreciate the reviewer’s comments and the opportunity to clarify the novelty of our study.

Composting is a cost-effective technology for the treatment of organic solid wastes and resource recovery. During this process, complex organic matter is converted into stable humus through microbial metabolic activity, thereby achieving harmless disposal of pollutants. The composting process involves dynamic microbial succession, with bacteria and fungi representing the major microbial groups. However, most existing studies primarily examine structural changes and phase-specific functional characteristics of microbial communities independently, while research on the interaction mechanisms between bacterial and fungal communities remains limited. Our study emphasizes the systematic analysis of the dynamic evolution of different microbial groups and the elucidation of their interactions, which is of great theoretical and practical importance for optimizing composting. To highlight this point, we reviewed microbial community succession and functional shifts in the Introduction and framed the investigation of bacterial–fungal interactions as a central focus of our work.

We selected algal sludge as the composting substrate. Although microbial community composition differs across composting substrates, the analytical framework we developed for studying community interactions can serve as a reference for composting optimization with other types of raw materials. While we did not explicitly emphasize the uniqueness of algal sludge composting in the Introduction, its distinctive characteristics are clearly reflected in the physicochemical parameters described in our manuscript. Specifically:

Algal sludge typically has a very low C/N ratio (~5 on a dry-weight basis), much lower than that of manure or food waste. In practice, algal blooms occur mainly in summer, and collected algal sludge is difficult to store due to rapid decay and potential release of microcystins, which can cause secondary contamination. Thus, algal sludge requires faster and lower-cost treatment. Although bulking agents (e.g., straw, rice hulls, spent mushroom substrate) can raise the C/N ratio and reduce moisture, their addition decreases the proportion of sludge treated, increases economic burden (Han et al., 2014), and may reduce the nutrient value of the final compost product due to their nutrient deficiency (Li et al., 2022). Therefore, in practical operations, algal sludge is often composted under relatively low C/N conditions to achieve rapid treatment. Our previous research has demonstrated that safe and high-quality algal sludge compost can be obtained under low C/N ratios (Wu et al., 2025). In this study, we used algal sludge with mushroom residue at a C/N ratio of 10–15 (Fig. 1d, e). It is worth noting that the typical C/N ratio of most composting feedstocks generally falls within the range of 20–30, further highlighting the uniqueness of this composting.

Rapid temperature rises and high peak temperature. Another distinctive feature is the rapid increase in temperature during the composting process. In our study, the temperature reached 75 °C within only 2 days and maintained a prolonged thermophilic phase (Fig. 1a). Such dynamics are markedly different from those observed in composting of manures, food waste or other organic solid wastes. Since microbial growth and succession are strongly influenced by temperature, this rapid and intense thermophilic stage is a unique factor shaping microbial community dynamics in algal sludge composting.

Taken together, these features differentiate algal sludge composting from other commonly studied substrates and justify the novelty of our investigation into its microbial community interactions.

Han, W., Clarke, W., Pratt, S., 2014. Composting of waste algae: A review. Waste Management 34, 1148–1155. https://doi.org/10.1016/j.wasman.2014.01.019

Li, D., Manu, M.K., Varjani, S., Wong, J.W.C., 2022. Mitigation of NH3 and N2O emissions during food waste digestate composting at C/N ratio 15 using zeolite amendment. Bioresource Technology 359, 127465. https://doi.org/10.1016/j.biortech.2022.127465

Wu, H., Wang, C., Zhou, J., Cong, H., Gao, Y., Cai, W., Feng, S., Zhang, C., 2025. Feedstock optimization with low carbon to nitrogen ratio during algal sludge aerobic composting: Quality and gaseous emissions. Bioresour. Technol. 416, 131811. https://doi.org/10.1016/j.biortech.2024.131811

 

2.The claim that microbial diversity "did not show any significant response" to composting phases directly contradicts the well-established literature and is biologically implausible given the extreme temperature shifts reported.

Response: Thank you for this comment. We respectfully disagree with the reviewer’s interpretation and would like to provide clarification and supporting evidence. In our experiment, the bacterial and fungal communities did not show statistically significant differences in diversity across composting phases. This does not imply that diversity remained completely unchanged, but rather that the observed differences were not significant according to statistical tests. For example, the Shannon and Evenness indices of the bacterial communities showed a slight increase during the cooling phase (Figure 2a). Similar nonsignificant patterns of microbial diversity have also been frequently reported in previous composting studies using different raw materials.

During composting, physicochemical parameters change significantly, and these shifts are accompanied by pronounced changes in microbial community composition. However, microbial diversity itself does not necessarily exhibit significant variation across phases. This can be explained by functional redundancy within microbial communities, where different taxa perform similar ecological roles, and by the dynamic replacement of dominant populations. Thus, even though the taxonomic composition undergoes substantial changes, the overall diversity indices may remain relatively stable. This pattern has also been reported in previous studies. For instance, (Sun et al., 2022) and (Qian et al., 2022) investigated bacterial and fungal communities during composting of different substrates and found that microbial diversity across composting phases did not necessarily show significant differences. These findings further support the validity of our observations.

Qian, X., Bi, X., Xu, Y., Yang, Z., Wei, T., Xi, M., Li, J., Chen, L., Li, H., Sun, S., 2022. Variation in community structure and network characteristics of spent mushroom substrate (SMS) compost microbiota driven by time and environmental conditions. Bioresource Technology 364, 127915. https://doi.org/10.1016/j.biortech.2022.127915

Sun, P., Liu, B., Ahmed, I., Yang, J., Zhang, B., 2022. Composting effect and antibiotic removal under a new temperature control strategy. Waste Management 153, 89–98. https://doi.org/10.1016/j.wasman.2022.08.025

Regarding the reviewer’s concern about the biological plausibility of the high temperatures observed during the thermophilic phase, we emphasize that the primary reason for the rapid and elevated temperature rise lies in the properties of the composting substrate itself. The maximum temperature observed in our study was 75 °C. Although this value is higher than the temperatures commonly reported in composting, it is not unusual according to recent studies. For example, (Hernández-Lara et al., 2022) reported composting of agricultural residues exceeding 75 °C, (Sun et al., 2022) Sun et al. (2022) documented peak temperatures up to 78 °C during co-composting of cow manure, food waste, and cornstalk, and (Chang et al., 2021) observed a maximum temperature of 77.6 °C during composting of rice, corn, and tomato straws. Together, these reports support the plausibility of our findings.

To further improve clarity for readers, we have supplemented the legend of the reactor schematic with additional information about the system (Figure S1): “The bioreactor constructed from 10 mm thick polyvinyl chloride material, with an effective volume of >100 L (Φ60 cm × height 60 cm).” While the PVC structure provides some insulation, we believe the intrinsic characteristics of the algal sludge substrate were the dominant factor driving the rapid temperature increase.

Chang, H., Zhu, X., Wu, J., Guo, D., Zhang, L., Feng, Y., 2021. Dynamics of microbial diversity during the composting of agricultural straw. Journal of Integrative Agriculture 20, 1121–1136. https://doi.org/10.1016/S2095-3119(20)63341-X

Rodríguez, F.J., Fernández, J.A., Egea-Gilabert, C., Pascual, J.A., 2022. Bacterial and fungal community dynamics during different stages of agro-industrial waste composting and its relationship with compost suppressiveness. Science of The Total Environment 805, 150330. https://doi.org/10.1016/j.scitotenv.2021.150330

Sun, P., Liu, B., Ahmed, I., Yang, J., Zhang, B., 2022. Composting effect and antibiotic removal under a new temperature control strategy. Waste Management 153, 89–98. https://doi.org/10.1016/j.wasman.2022.08.025

 

3. Proposing Geobacillus (a well-known thermophile) as a "potential biomarker" for a specific phase is trivial and adds no novel insight, as its presence in the thermophilic phase is entirely expected.

Response: Thank you for your comment. While the composition and succession of microbial communities during composting are strongly influenced by the raw materials and environmental conditions, certain broad patterns are common across different composting processes. For example, as the composting process transitions from the thermophilic phase to the cooling phase, the relative abundance of thermophilic microorganisms, such as Geobacillus, is expected to decrease. This similarity in microbial succession across various composting materials is one of the reasons why optimization strategies in composting can be mutually informative.

Previous studies have primarily focused on identifying indicator species based on the relative abundance of specific microorganisms at different phases. Our study, however, emphasizes the interaction between bacterial and fungal communities. In selecting potential biomarker species, we took this into consideration. We first employed random forest analysis to identify microorganisms whose relative abundance changed significantly during composting, ultimately selecting 48 species. This step follows the general approach used in existing research. However, building on this, we applied network analysis to identify key species that play central roles in the microbial network. By combining the results from random forest and network analysis, we selected the intersection of the two, which included one fungal species and two bacterial species as potential biomarkers.

Thus, while the identification of Geobacillus as a biomarker in the thermophilic phase may seem expected, it is important to note that our approach, which integrates both random forest and network analysis, offers a novel and practical method for identifying biomarkers. This method is not only applicable to Geobacillus, but also reveals other microorganisms that contribute valuable insights. Although microbial community compositions differ between various composting substrates, our methodology—considering both bacterial and fungal interactions—provides a useful framework for identifying microbial biomarkers in the composting of other materials.

 

4. Attributing specific functions (e.g., N-transformation to bacteria, humification to fungi) based purely on correlation (structural equation model: SEM analysis) without metagenomic or metatranscriptomic data is speculative and not causally proven.

Response: Thank you for your comment. We appreciate the reviewer’s concerns regarding the use of correlation-based methods for attributing specific functions to microbial groups without metagenomic or metatranscriptomic data. While we acknowledge that more in-depth sequencing techniques such as metagenomics and metatranscriptomics can provide valuable insights into specific functional genes and their activity, these approaches focus more on gene-level identification and are not necessarily aimed at predicting overall community functions. Additionally, metagenomic approaches often come with significantly higher costs and greater complexity in data interpretation.

High-throughput amplicon sequencing, on the other hand, is currently the most commonly used method for microbial community studies, including those focused on environments such as aquatic systems, soils, and human-controlled processes like composting and wastewater treatment. Amplicon sequencing is well-suited for analyzing the microbial community structure at the genus level and above, providing valuable information on species composition and diversity. With the continuous improvement of functional prediction databases such as FAPROTAX, FUNGuild, and PICRUSt2, amplicon sequencing can also yield reliable predictions of microbial functions. In contrast, metagenomics sequencing focuses primarily on identifying key functional genes and their activity, offering a more granular understanding of microbial functions at the gene level. While this approach can provide more precise insights into gene functions, it also comes with substantially higher costs and is not always necessary for studying community-level functional potential.

In our study, we focused on the diversity, composition, and interactions of bacterial and fungal communities at the genus level and above, and predicted their potential functions. It is important to note that our research did not aim to analyze individual functional genes, but rather to predict the potential functions of microbial groups based on their taxonomic profiles. The methods we used are fully supported by the sequencing data and are widely used in microbial community research, including composting studies.

Furthermore, we used the term “potential functions” in the manuscript, which accurately reflects the scope of our study and does not overstate our findings. Composting is widely recognized as a cost-effective and efficient method for treating organic waste, including algal sludge. Pursuing deeper sequencing depth without considering the associated costs would make the optimization of composting processes prohibitively expensive, especially for general optimization studies where high costs are not justifiable. Amplicon sequencing, therefore, provides an effective and affordable solution for our research objectives.

We believe our approach, utilizing well-established methodologies for microbial community analysis and functional prediction, is both appropriate and sufficient for addressing the research questions posed in our study.

 

5. I observed mix type citation style in your introduction part. Some are numbered while some are in Zhang et al. (2021) form.

Response: Thank you for your comment. Regarding your concern about the literature review and the relevance of the citations, we acknowledge that there were formatting issues due to Zotero when switching citation styles, which we have since manually corrected. We apologize for any confusion this may have caused.

 

6. The Figures are very poor resolution. It is mandatory to provide better images.

Response: Thank you for your comment regarding the figure resolution. We would like to clarify that the figures in the original submission were provided as vector graphics, which are generally of high quality and suitable for publication. However, it appears that during the submission process, the journal automatically converted these vector images when formatting the manuscript, which resulted in a loss of image quality upon enlargement.

We believe that this conversion may have contributed to the issue of image clarity you raised. We have now replaced the figures in the revised manuscript with the original vector images, which retain their high resolution and clarity. We trust that these updated figures will address your concerns about image quality.

 

7. The similarity also seems quite high from the ithenticate report.

Response: Thank you for your comment. Regarding the high similarity index, we found that it was primarily due to the "Materials and Methods" section. To address this, we have thoroughly revised manuscript. We believe these changes now meet the journal's requirements.

 

8. In the introduction part, beside the poor literature review, there are mixed types of citations. Very few of the references are relevant.

Response: Thank you for your comment. We believe that all the cited studies directly relate to our work, either by addressing similar composting processes, microbial community dynamics, or specific techniques and methodologies used in the field of composting research. These references are integral to establishing the background and context of our study. We understand that literature relevance may be subjective, we trust that the references we have included appropriately support the points made in the manuscript.

 

9. I am not convinced with the scientific approach of this article. Nothing new has been observed in this paper.

Response: Thank you for your comment. We understand your concern regarding the novelty of our study. However, we respectfully believe that we have already addressed the scientific approach and the contributions of our work in previous responses. Our study provides new insights into the microbial community dynamics during the composting of algal sludge, particularly with regard to the interactions between bacterial and fungal communities, which is a topic that has been less explored in the existing literature. The methodology we employed offers a novel approach to identifying potential biomarkers and understanding microbial interactions. 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript, written by Zhou et al., is a Material Conversion, Microbial Community Composition and Metabolic Functional Succession During Algal Sludge Composting. Some specific comments should be added for more clarity regarding the manuscript:

1. Please add study background conditions and the problem to be solved in the abstract and the introduction. How does your paper contribute to the advancement of knowledge?
2. The authors measured the physicochemical parameters including temperature, the oxygen (O2) concentration, total organic carbon (TOC) content, total nitrogen (TN), and germination index (GI). How were it influenced each other as relationship between physicochemical characteristics and microorganisms during composting, then how about pH parameter during the aerobic composting of algal sludge?
3. Please add more details about FAPROTAX and FUNGuild analysis that the authors used used to further evaluate the potential functions of the bacterial and fungal communities during the composting process.
4. The conclusion should be improved with clear quantitative findings.

Author Response

Thank you for your constructive comments. We have carefully considered your suggestions and have made the necessary revisions to the manuscript. Specifically, we have added additional data and provided further clarification of our methods in the relevant sections to strengthen and complete the manuscript. We believe these changes address the points you raised and enhance the overall quality of the work. Relevant changes have been color-coded in blue in the revised manuscript.

 

1.The manuscript, written by Zhou et al., is a Material Conversion, Microbial Community Composition and Metabolic Functional Succession During Algal Sludge Composting. Some specific comments should be added for more clarity regarding the manuscript:

Please add study background conditions and the problem to be solved in the abstract and the introduction. How does your paper contribute to the advancement of knowledge?

Response: Thank you for this suggestion. In the abstract, we have already provided the study background and identified the problem to be solved in the first sentence, and described the research method in the second sentence. The sentence “Although bacterial and fungal communities are both important for organic matter degradation and humification during composting, their composition, functional relationships, abiotic compost properties and succession patterns remain unclear”. Given the limited space of the abstract and the typical length of background information in scientific abstracts, we consider this level of detail to be appropriate. However, to emphasize the core focus of our study, we have revised the wording in the first sentence by replacing “functional relationships” with “interactions”.

Regarding the introduction section, please allow me to clarify it first, and then explain the additions I made based on your suggestions. In the first paragraph, we introduce the fundamental concepts of the composting process to highlight the significance of microbial community studies. In the second paragraph, we review the existing literature on the dominant microbial groups during composting, specifically bacteria and fungi, and their role in the process. However, we point out that the interactions between bacterial and fungal communities remain insufficiently explored. The sentence, “Most of the current studies focus on the respective structural changes and phase-specific functional characteristics of different microbial communities, while the study of the interaction mechanism between fungal and bacterial communities is still relatively insufficient. Therefore, the systematic analysis of the dynamic evolution of different microbial communities during the composting process and the elucidation of the interactions between fungal and bacterial communities are of great theoretical and practical importance for optimizing the composting process”. This clarifies the research problem and underscores the significance of our study.

In the third paragraph, we explain that microbial communities vary significantly across different composting feedstocks. Therefore, we provide the research background for our chosen feedstock—algal sludge. We discuss the characteristics of algal sludge and review the current state of research on algal sludge composting. Despite some studies exploring process parameters and microbial community structure in algal sludge composting, the interactions between bacterial and fungal communities remain insufficiently understood, highlighting the need for our study. This gap in knowledge justifies the need for our study, as reflected in the sentence: “Although existing studies have preliminarily revealed the effects of process parameters and microbial community structure in algal sludge composting, the interactions between bacterial and fungal communities and their specific roles in this process are still not well understood.”

Finally, in the fourth paragraph, we outline the main objectives of our study, which are to analyze changes in physicochemical properties during the composting process, investigate the succession of bacterial and fungal structure and function during algal composting, and examine the relationship between microbial communities and physicochemical properties during composting. To further clarify our contribution, we have added the following sentence: “The methodological framework presented here may serve as a valuable reference for microbial optimization in composting of other materials.”

 

2.The authors measured the physicochemical parameters including temperature, the oxygen (O2) concentration, total organic carbon (TOC) content, total nitrogen (TN), and germination index (GI). How were it influenced each other as relationship between physicochemical characteristics and microorganisms during composting, then how about pH parameter during the aerobic composting of algal sludge?

Response: Thank you for your comment.

Since our study primarily focuses on the interactions between bacterial and fungal communities, we chose to use a structural equation model (SEM) to analyze the relationships between microbial communities and physicochemical parameters. This approach allows us to integrate bacterial communities, fungal communities and key physicochemical parameters, such as temperature (T), total nitrogen (TN), humification index (HA/FA), and germination index (GI), which reflect microbial activity, compost quality, and the degree of compost maturity and safety. The complete analysis results are in Chapter 3.5 of the manuscript, and the main conclusions include: The analysis indicated that temperature exerted a significant and direct effect on microbial communities (p < 0.001). Specifically, a positive correlation was observed between temperature and fungal communities (p < 0.001), while a negative correlation was found between temperature and bacterial communities (p < 0.001), suggesting a potential complementary relationship between them. This finding implies that temperature may play a role in regulating microbial community succession, which could, in turn, influence nitro-gen transformation and humification processes, contributing to the overall maturity of the compost. The bacterial communities may have a significant effect on the loss of TN and the humification process. In contrast, the fungal community showed a significant negative correlation with HA/FA (p = 0.012) and had no significant direct effect on nitrogen loss (p = 0.947). Compared with the original manuscript, we have made a few modifications to the description of the SEM results in the revised manuscript to ensure the rigor of the language.

Regarding the pH parameter, we measure it continuously throughout the compost process because pH is the basic parameter that can reflect whether the compost process is steadily proceeding. Typically, pH increases during the initial stages of composting due to the release of ammonia from NH₄⁺ or alkaline amino acids. Later, as organic acids accumulate and microbial activity or nitrification decreases, pH tends to decrease. Mature compost typically shows a mildly alkaline pH. Therefore, generally speaking, the pH changes and temperature during the composting process are significantly related. The pH changes in our study followed this typical pattern, which is consistent with existing literature. The physicochemical parameters presented in the literature can well reflect the nitrogen loss and humification process during composting. So, did not specifically discuss it in the manuscript. Since you raised this question, we have provided the pH data here for your reference (Please see the attachment "responses to comments_2.docx", the picture cannot be displayed here).

3.Please add more details about FAPROTAX and FUNGuild analysis that the authors used used to further evaluate the potential functions of the bacterial and fungal communities during the composting process.

Response: Thank you for your comment. FAPROTAX and FUNGuild are functional prediction databases used to assess the potential functions of bacterial and fungal communities based on high-throughput sequencing data. These tools compare annotated ASV/OTU results with the respective databases to predict microbial functions. Both databases are well-documented, with the authors providing detailed installation and usage instructions. Additionally, several researchers have enhanced or developed alternative analysis scripts in different programming languages, which have been made available on GitHub. As the usage process primarily involves executing the provided code, we did not elaborate on the analysis details in the manuscript.

In response to your suggestion, we have now included the relevant GitHub links to the analysis code for both FAPROTAX and FUNGuild in the manuscript for readers who may wish to explore the methods further. The revised text is as follows: “Bacterial functional characteristics were assessed using the FAPROTAX database (https://github.com/yongxinliu/EasyMicrobiome). Fungal functions were predicted using the FUNGuild database based on the ITS data (https://github.com/UMNFuN/FUNGuild)”.

 

4.The conclusion should be improved with clear quantitative findings.

Response: Thank you for your comment. Our conclusions are entirely based on the physicochemical and microbial community data we measured. In sections 3.1 and 3.2, we have provided a detailed description of these measured data. Since the core focus of our study is the interaction between bacterial and fungal communities during composting, the manuscript primarily discusses and analyzes these data using statistical methods. Therefore, due to the typically concise nature of the conclusion, fundamental physicochemical and microbial changes are not presented in the conclusion. Instead, the conclusion mainly highlights the results of statistical analysis. We have carefully considered your comments and our key findings, and in the revised manuscript, we have included the quantitative relative abundance of microbial communities and the significance of statistical tests (P value) to help readers better understand our conclusions.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors responses are satisfactory and backed with scientific evidences from recent and relevant literature and necessary changes have been made in manuscript. most of my comments are answered with detailed discussion which I personally appreciate.

on the basis of satisfactory answers, I suggest the editor to accept the article. 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been revised and reorganized based on the reviewer.