Combined Use of Biochar and Microbial Agents Can Promote Lignocellulosic Degradation Microbial Community Optimization during Composting of Submerged Plants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments for the manuscript sections that must be considered by the Authors:

Abstract: needs to be revised for clearer understanding; do not use abbreviations without explanation;  either generalize the data and provide rounded/estimated values or provide specific values clearly referring to specific treatment conditions; indicate the novelty and the aim of this study.

Introduction:
L46-47: what is the reduction and harmless principle; why is it in quotation mark; please define and provide a reference.

L68: does the authors typically conduct the mentioned research? Or other authors?

Please clearly underline the novelty of this study in the introduction section.

Materials and methods:

L92-104: please provide specific names and information on the producer and purity for the materials as well as basic information on the apparatus and equipment used (model, producer; adding this information is necessary for the whole section when the use of any equipment is mentioned).

Table 1: please correct the editing of the table.

L109: what is a suitable proportion?

L149: please add information on sample preparation for the measurements.

L138; L191: the Authors mention EC value (probably electric conductivity), but without explaining the abbreviation. Please provide the explanation to each symbol You use in the text of the manuscript in the place where the given term is used for the first time; this refers to all terms and abbreviations throughout the manuscript.

L314-326: please unify the font style according to the Journal’s template.

Microbial composition: please use an italic font where necessary (microbial names); e.g. see lines L389 and L398 and correct the text uniformly.

L465: no information of RDA analysis (what is it and how was it completed) was mentioned in the materials and methods; please add a necessary information.

L517-522: please include the necessary editing for the terms and text.

Section 4: I suggest to change the title to “Summary” or “Conclusions”; please indicate clearly the optimal/the best process conditions for compositing the submerged plants; please indicate future research directions and needs in this field.

Language and editing revision is needed for the text of the manuscript; e.g. L61 (acting as a optimize ??); L385: might be due to…; please put attention to little language and typic errors; please put correct numeration of pages.

Comments on the Quality of English Language

I suggest to revise the English language used in the text of the manuscript. In general, the language is clear and acceptable, but there are some fragments that need to be revised (few specific comments are given in the suggestions for manuscript improvement). Please correct the spelling mistakes, check whether all the sentences are gramatically correct.

Author Response

Respond to Reviewer

We greatly appreciate the reviewers’ valuable comments and suggestions on manuscript entitled “Combined use of biochar and microbial agent can promote lignocellulosic degradation microbial community optimization during submerged plants composting” fermentation-2785960). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion were marked in the Revised-Manuscript. The main corrections in the paper and the responds to the editors and reviewers' comments are as flowing:

 

Reviewer 1's comments:

We greatly appreciate the Reviewer 1’s positive review of the manuscript and valuable suggestions.

Abstract: needs to be revised for clearer understanding; do not use abbreviations without explanation; either generalize the data and provide rounded/estimated values or provide specific values clearly referring to specific treatment conditions; indicate the novelty and the aim of this study.

Response: As Reviewer suggested that we have rewritten the Abstract.

Abstract: Aerobic composting is one of the methods for the resource utilization of submerged plant residues. This study investigated the effects of biochar, wetland sediments and microbial agents added individually or combinedly on humification process, lignocellulose degradation and microbial communities during Ceratophyllum demersum and Potamogeton wrightii composting. The results showed that the addition of wetland sediment and biochar were found to significantly elevate the composting temperature, humification of compost products. The average content of lignin in wetland sediment and/or biochar treatments were 12.2%~13.5%, higher than the control group (10.9%~11.45%). Comparing with the organic matter (19.4%) and total nitrogen concentration (35.3%) of compost treated with complex microbial agent treatments, the homemade microbial agents significantly increased the values by 22.1% and 41.0%, respectively. By comparing the differences in microbial communities among different treatments, the sediments and homemade agents demonstrated greater increases in activity and diversity of lignocellulose degradation-related microbes, especially for Truepera, Actinomarinale. Humus component and temperature were the most critical parameters influencing the changes of bacterial community changes. Based on these results, combination of biochar and homemade agents was promising additives for the effective composting strategy, and sediment was identified as a potential control of bacterial diversity in wetland plant compost.

 

Introduction:

L46-47: what is the reduction and harmless principle; why is it in quotation mark; please define and provide a reference.

Response: As reviewer suggested we have changed "reduction and harmless" to reduction and harmless, Aerobic composting is one of the effective technologies of solid waste recycling treatment, and has been widely applied in the reduction and harmless treatment of waste [3,4].

And added reference [4] Han, S.Q.; Li, J.L.; Zhou, Q.; Liu, G.F.; Wang, T. Harmless disposal and resource utilization of wastes from the lake in China: De-watering, composting and safety evaluation of fertilizer. Algal Res. 2019, 43,101623. https://doi.org/10.1016/j.algal.2019.101623

 

L68: does the authors typically conduct the mentioned research? Or other authors?

Please clearly underline the novelty of this study in the introduction section.

Response: As reviewer suggested we have emphasized the novelty of the study in the introduction.

Finally, extensive researches are typically conducted to explore the use of various additives for optimizing the composting process using different raw materials [19]. The in-fluence of different additives and microbial agents on the composition of microbial communities has emerged as hot research topics [7,20-22]. Nevertheless, investigation into the transformation contribution of microorganisms to various submerged plant components remains unclear during composting under different microbial agent amendment strategies. Therefore, we innovatively proposed the effects of various microbial inoculation strategies on the co-composting process of submerged plant lignocellulose residues and animal manure.

 

Materials and methods:

L92-104: please provide specific names and information on the producer and purity for the materials as well as basic information on the apparatus and equipment used (model, producer; adding this information is necessary for the whole section when the use of any equipment is mentioned).

Response: According to the suggestion we have added names and information on the producer and purity for the materials:

The submerged plants (Ceratophyllum demersum and Potamogeton wrightii) and wetland surface sediment for composting experiment were obtained from Baiyangdian Lake in August 2022. Dry chicken manure (purchased from Nanjing Easy Recycling Agricultural Technology Co., Ltd) and urea (Cangzhou Zhengyuan Chemical Fertilizer Co., Ltd.)were used to adjust compost carbon-nitrogen ratio (C/N), while the biochar was straw biochar, purchased from Pingdingshan Tannuo Environmental Protection Material Co., Ltd. The complex microbial agents (group C) were purchased from Nongkang Storage Trading Co., Ltd, which contained lactic acid bacteria, yeast, the homemade microbial agent (group H) contains bifidobacteria, lactobacillus, Bacillus and other microorganisms.

.

 

Table 1: please correct the editing of the table.

Response: We have corrected the Table 1:

Table 1. Primary properties of raw materials in composts.

Material	TOC （%）	TN （%）	C/N	cellulose （%）	hemicellulose （%）	lignin （%）
Submerged plant	84.62±5.26	1.33±0.09	63.62	15.8±0.43	29.2±0.76	8.90±0.46
Dry chicken dung	63.11±3.23	3.62±0.22	17.43	-	-	-
Wetland sediment	39.64±2.17	2.63±0.19	15.13	-	-	-
Compost material	76.36±4.35	2.69±0.18	28.39	7.62±0.26	15.9±0.87	4.88±0.39

Note: Data are reported as Mean±SD with n=3. TOC, total organic carbon; TN, total nitrogen; -, not detected due to low content. Compost materials consisted of submerged plants mixed with dry chicken manure in a 7:3 ratio, the submerged plants were Ceratophyllum demersum and Potamogeton wrightii mixed 1:1.

 

L109: what is a suitable proportion?

Response: As reviewer suggested we have added the proportion of submerged plant and chicken manure compost:

Note: TOC, total organic carbon; TN, total nitrogen; -, not detected due to low content. The sub-merged plants are Ceratophyllum demersum and Potamogeton wrightii 1:1 mixed. Compost mate-rials consist of submerged plants mixed with dry chicken manure in a 7:3 ratio.

 

L149: please add information on sample preparation for the measurements.

Response: As reviewer suggested we have added information on sample preparation.

The freeze-dried compost sample and potassium bromide (KBr) in a ratio of 1:100, then ground and mixed in an agate mortar under an infrared lamp, and sample powder compressed to form a disc. The Fourier conversion infrared spectroscopy analyzer (FTIR, Perkin Elmer Frontier) determines the main functional groups involved in the compost reaction, with a wave number range ranging from 4,000 to 400 cm-1. A scanning electron microscope (SEM, Czech TESCAN MIRA LMS) was used for appearance shooting and energy spectrum scanning to observe the composting surface morphology and determine the elemental structure. The samples were previously metallized with a 10 nm layer of gold.

 

L138; L191: the Authors mention EC value (probably electric conductivity), but without explaining the abbreviation. Please provide the explanation to each symbol You use in the text of the manuscript in the place where the given term is used for the first time; this refers to all terms and abbreviations throughout the manuscript.

Response: We have checked the whole manuscript, and explanation was added before the abbreviation.

The pH and electric conductivity (EC) values were determined by a pH/EC instrument.

The experimental procedure for cultivating the homemade microbial agent in this study involved dissolving and evenly mixing 2.5 g effective microorganism (EM) bacteria and 500 g of brown sugar in a plastic bucket containing 9 L of distilled water.

Total organic carbon (TOC) was determined by the external heating method [23]. Total nitrogen (TN) was determined by Kjeldahl method [24].

 

L314-326: please unify the font style according to the Journal’s template.

Microbial composition: please use an italic font where necessary (microbial names); e.g. see lines L389 and L398 and correct the text uniformly.

Response: We have changed the font style, and used the italic font for microbial names for whole manuscript.

 

L465: no information of RDA analysis (what is it and how was it completed) was mentioned in the materials and methods; please add a necessary information.

Response: As reviewer suggested, we have added the information in 2.6. Statistical analysis: Redundancy analysis (RDA) was performed using CANOCO 5.0.

2.6. Statistical analysis

Experimental results were determined by triplicate replicates. The final results were shown as the average with standard deviations. The arrangement, statistical analysis and chart drawing of the experimental data were performed in Excel 2016 (Microsoft Inc., Redmond, WDC, USA), SPSS 26.0 (SPSS Inc., Chicago, IL, USA) and Origin 2021b (Origin Lab Inc., Northampton, MA, USA) software, respectively. Analysis of variance (ANOVA) was used to evaluate the effects of different treatments on compost properties. One-way ANOVA was used for sample data and the differences were considered statistically significant at p<0.05 using Tukey’s test. Redundancy analysis (RDA) was performed using CANOCO 5.0.

 

 

L517-522: please include the necessary editing for the terms and text.

Response: As Reviewer suggested that we have re-edited the text:

The Spearman Correlation Heatmap in Figure 8a was showed the investigation of the impact of major bacteria on lignocellulose degradation, indicating a positive correlation between g__Pseudoxanthomonas,g__unclassified_f__Planococcaceae, g_unclassified_c_Bacilli with cellulose and hemicellulose during temrmophilic stage, this finding was consistent with the previous study results on the microbial characteristics [62]. Furthermore, Figure 8b illustrated the gradual degradation of hemicellulose and lignin in the anaphase of composting, and the g_unclassified_c_Bacilli, g_Saccharomonospora exhibited a stimulative effect on the degradation process. Indeed, the degradation necessitates the interaction of multiple microorganisms and formation of symbiotic relationships to collectively facilitate the degradation of organic matter [76].

 

Section 4: I suggest to change the title to “Summary” or “Conclusions”; please indicate clearly the optimal/the best process conditions for compositing the submerged plants; please indicate future research directions and needs in this field.

Response: As Reviewer suggested that we have rewritten the “Conclusions”.

4. Conclusions

The results of this study showed that biochar and sediment as compost conditioner can improve the humus, TN content and GI at the end of composting. The promotion effect of compost maturation in the biochar treatment group was more obvious than the sediment treatment group, whereas sediment treatments exhibited greater microbial diversity. Bacillus, Lysinebacillus, Brevibacillus were the dominant bacteria for lignocellulose degradation, meanwhile Truepera,Actinomarinale, Sphaerobacter as important roles for the degradation of lignocellulose. In the co-occurrence network, the key groups that significantly promote lignocellulose degradation are Firmicutes and Actinobacteria, the structure and function of microbial co-occurrence network are dominated by low abundance species and environmental factors affecting lignocellulose degradation. Based on the relationship between exogenous additives with humic substances and lignocellulose, an optimized strategy of biochar-homemade microbial agent co-inoculation was proposed to improve compost maturity and adjust the microbial community according to the functional needs during the composting process. This study provides new ideas for the resource utilization and harmless treatment of submerged plants.

 

Language and editing revision is needed for the text of the manuscript; e.g. L61 (acting as a optimize ??); L385: might be due to…; please put attention to little language and typic errors; please put correct numeration of pages.

Response: We have made correction according to the Reviewer’s comments, and the language has been carefully revised by native speaker for the whole manuscript. And Language and editing revision were as following:

L61 (acting as a optimize ??)… we have revised:

Microbial agents, due to their capacity to accelerate decomposition and other specialized functions, could serve as an excellent supplement for optimizing the composting process.

L385: might be due to…we have revised:

Compared with the control, the relative abundance of Proteobacteria significantly increased in the biochar and sediment treatments, which could be attributed to the high organic matter content in sediment and the simulating impact of biochar on aeration in the pile, thus providing Proteobacteria with suitable environmental condition and nutrients [47].

 

I suggest to revise the English language used in the text of the manuscript. In general, the language is clear and acceptable, but there are some fragments that need to be revised (few specific comments are given in the suggestions for manuscript improvement). Please correct the spelling mistakes, check whether all the sentences are gramatically correct.

Response: We have made correction according to the Reviewer’s comments. We have revised the language of the whole manuscript and corrected grammatical errors.

 

We tried our best to improve the manuscript and made some changes in the manuscript. We marked the changes in red in Revised Manuscript. We earnestly appreciate for Editors/Reviewers’ warm work, and hope that the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Reviewer 2 Report

Comments and Suggestions for Authors

 

The article "Combined use of biochar and microbial agent can promote lignocellulosic degradation microbial community optimization during submerged plants composting" presented by Wang et al deals with the use of biochar and microbial agent to promote the degradation of submerged plants during composting.

Although the authors have correctly applied a clear methodological approach and analyzed numerous parameters, especially microbiological ones, the article is not convincing. The first problem is the lack of standard deviation in the results. If the values in Figure 1 are labelled with error bars, we don't know what they mean: are the authors trying to represent the uncertainties of the measurement methods or the standard deviation of experiments conducted in triplicate, which is never mentioned in the text?

Secondly, the authors mention degradation or modification rates throughout the text, but on the one hand it is not possible to find these figures from the figures, and on the other how to explain the number of decimal places given for each value, for example 25.51 and 21.68 % on line 210. And finally, in the true sense of the word, these are not rates but yields.

Globally speaking, the authors put forward numerous explanations to interpret their results, but these are not objectively clear. For example, in line 182, the variations in pH are very few and not differentiated between conditions, yet the authors invoke mechanisms to explain these inconspicuous differences.

Summary tables should, for example, include the main results from Figure 1.

Finally, the general discussion and conclusion are also surprising. In line 460 the authors mention that the HA/FA and GI parameters are the most important, but the problem is that these parameters are not defined in the text. In the conclusion at line 534 the authors mention that biochar and sediment accelerate heating and extend the high temperature period but this fact is not mentioned or shown in the text.

Details:

- the values in Table 1 should be given with their uncertainties.

- line 201 TOC is not a substrate but a measure of carbon

Author Response

Respond to Reviewer

We greatly appreciate the reviewers’ valuable comments and suggestions on manuscript entitled “Combined use of biochar and microbial agent can promote lignocellulosic degradation microbial community optimization during submerged plants composting” fermentation-2785960). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion were marked in the Revised-Manuscript. The main corrections in the paper and the responds to the editors and reviewers' comments are as flowing:

 The article "Combined use of biochar and microbial agent can promote lignocellulosic degradation microbial community optimization during submerged plants composting" presented by Wang et al deals with the use of biochar and microbial agent to promote the degradation of submerged plants during composting.

Although the authors have correctly applied a clear methodological approach and analyzed numerous parameters, especially microbiological ones, the article is not convincing. The first problem is the lack of standard deviation in the results. If the values in Figure 1 are labelled with error bars, we don't know what they mean: are the authors trying to represent the uncertainties of the measurement methods or the standard deviation of experiments conducted in triplicate, which is never mentioned in the text?

Response: As suggested, we have added the supplementary explanation of standard deviation. Firstly, in the part of material and method, it was explained that the sampling was mixed samples, and the physicochemical property indexes and microorganism of each sample were tested in triplicate (n=3); secondly, the error bars were written in the title of the figure to represent standard deviation. Specific revisions are as follows:

2.2. Composting design and sampling

The mixed samples were divided into two parts, one sample was air-dried for determinations of physicochemical properties, the other was stored at room temperature to determine the seed germination index. And the fresh samples from days 0, 12 and36 (8 treatments and each replicated thrice) were the stored at -80℃ to determine the genetic diversity of the microbial community, sub-samples were taken from five sampling points and mixed into a composite sample. The samples for microbial analyses labelled as treatment-d0, -d12 and -d36, representing the prophase, metaphase and anaphase, respectively.

2.6. Statistical analysis

Experimental results were determined by triplicate replicates. The final results were shown as the average with standard deviations. The arrangement, statistical analysis and chart drawing of the experimental data were performed in Excel 2016 (Microsoft Inc., Redmond, WDC, USA), SPSS 26.0 (SPSS Inc., Chicago, IL, USA) and Origin 2021b (Origin Lab Inc., Northampton, MA, USA) software, respectively. Analysis of variance (ANOVA) was used to evaluate the effects of different treatments on compost properties. One-way ANOVA was used for sample data and the differences were considered statistically significant at p<0.05 using Tukey’s test. Redundancy analysis (RDA) was performed using CANOCO 5.0.

Figure 1. Changes in EC, pH, Total Organic Carbon (TOC), Total nitrogen (TN), C/N, hemicellulose, cellulose, and lignin during composting in different treatments. Error bars represent standard deviations (SDs) for each test day (n = 3)

 

Secondly, the authors mention degradation or modification rates throughout the text, but on the one hand it is not possible to find these figures from the figures, and on the other how to explain the number of decimal places given for each value, for example 25.51 and 21.68 % on line 210. And finally, in the true sense of the word, these are not rates but yields.

Response: According to the suggestion, we have rewritten the changes of compost materials and compost maturity condition through TOC and TN content analysis. Specific revisions are as follows:

Total organic carbon (TOC) represents the organic carbon content in the compost sample and serves as a source of carbon for microbial metabolism, which exhibited a gradual decrease trend in the composting process [29]. Generally, solid organic matter forms dissolved organic matter (DOM) that is more easily utilized by microorganisms through the metabolic activities, and the small molecule organic matter reorganizes to form humus with stable structure [30]. During the initial 0-12 days of composting, the TOC in all samples decreased significantly, possibly resulting in the volatilization and rapid loss of carbon in the form of gas due to the vigorous microbial activity (Figure 1c). However, the TOC contents showed significant differences due to the different organic matter degradation and humification process with various conditioners (Table S1). Higher TOC degradation degree was observed in the SB-H and SB-C treatments, and the average content of TOC in the late composting period were 58.20% and 60.33%, respectively, indicating that the synergy of wetland sediment and biochar additives was more conducive to the decomposition of TOC. Additionally, the loss of TOC content in the biochar treatment was greater than that of wetland sediment. At the end of composting, the mass fraction of TOC was above 45%, which met the standard of organic fertilizer.

The composting process had also been accompanied by nitrogen loss for the decomposition of organic matter, but the nitrogen continued to undergo ammonification, nitrification and denitrification, as well as the condensation of solid matter, the mass fraction of TN in the pile volume generally showed an increasing trend. When the composting completed, the TN content in each treatment ranged from 3.37%~3.86% (Figure 1d), the combination of sediment and biochar significantly increased the nitrogen content due to the abundant nitrogen source provided by sediments and reduction of nitrogen loss facilitated by biochar. The carbon to nitrogen (C/N) ratio is another important indicator to judge compost maturity. When the C/N<20, the composting can be considered to be decomposed [31]. At the end of composting for each treatment, C/N was between 15.08 to 19.39 for reaching the maturity standard (Figure 1e).

As shown in Figure 1f and Table S1, the hemicellulose content in composts also had a fluctuating decreasing trend. In the early 6d of composting, the average hemicellulose concentration decreased from 7.38%~7.91% to 4.21%~5.55%, because of the hemicellulose can be the preferred carbon source as the easily degradable organic matter. Hemicellulose in aquatic plants is the most easily degradable part due to the relatively simple structural characteristics, and its degradation directly affects the composting process and humification degree of submerged plants [32]. For the 24~36 d, microbial agent supplementation accelerated the degradation of hemicellulose, and the hemicellulose content decreased slightly in the late compost period. At the end of composting, the hemicellulose in both wetland sediment and biochar addition treatments showed a lower concentration of hemicellulose, and the change of hemicellulose in biochar treatment groups was significantly obvious than that in sludge treatment group (p<0.05). The variation of cellulose content in different compost treatments showed a similar trend as hemicellulose. After 36 d of composting, the average content of cellulose in each treatment was basically stable between 11.4% and 13.2%. The results showed that the average content of cellulose for control group was lower than that in other treatments, which was in contrast to hemicellulose treatment, likely due to the synergistic effect of microbial agent with wetland sediment or biochar could not promote the decomposition of cellulase. In addition, compared with hemicellulose and cellulose, lignin has more complex structure, and the molecular structure of lignin is more difficult to be degraded by microorganisms. The microbial degradation process of lignin mainly occurs in the high temperature and cooling decay stage. At the end of composting, the average content of lignin in each treatment was ranged from 4.04%~4.35%. Biochar and wetland sediment played an important role in lignin degradation, for the lignin in SB-H treatment (4.04%) was significantly lower than that of control treatments (CK-C 4.33% and CK-H 4.35%).This could be attributed to the structural properties of biochar were beneficial to the survival of microorganisms, as well as the wetland sediment had a richer microbial community and quantity, producing more enzymes conducive to lignin decomposition [16,19].

 

Globally speaking, the authors put forward numerous explanations to interpret their results, but these are not objectively clear. For example, in line 182, the variations in pH are very few and not differentiated between conditions, yet the authors invoke mechanisms to explain these inconspicuous differences.

Response: According to the suggestion, we have rewritten the compost pH changes and analysis. Specific revisions are as follows:

Dynamic profiles of the pH for different treatments were similar (Figure 1b). The stable value of pH was 7.5–8.0 in the late stage of composting, which meets the necessary conditions for compost maturity [26]. The initial pH of all treatments showed an increasing trend at the reaction process of 0~6 d, mainly because the microorganism degraded the nitrogenous organic matter and produced a large amount of NH₄⁺-N under the high temperature. In the later stage, the pH value was relatively stable and ultimately between 7.4 and 7.9. The main reason is that the porosity of the stack decreased with the decomposition of composting materials, a large amount of organic matter transformed into organic acids of small molecules and gradually accumulated, at the same time, and the carbon dioxide released by microbial respiration also leads to a decrease in pH value [27]. This result was consistent with previous findings of biomass waste compost [15].

 

Summary tables should, for example, include the main results from Figure 1.

Response: According to the suggestion, we have added the summary tables in the supplementary materials, please see Table S1.

 

Finally, the general discussion and conclusion are also surprising. In line 460 the authors mention that the HA/FA and GI parameters are the most important, but the problem is that these parameters are not defined in the text. In the conclusion at line 534 the authors mention that biochar and sediment accelerate heating and extend the high temperature period but this fact is not mentioned or shown in the text.

Response: HA/FA and GI characteristics were investigated and analysed in Supplementary DATA. As reviewer suggested we have added some analysis for this result in 3.1. Composting properties. Specific revisions are as follows:

For the other important indexes to evaluate the quality of compost, the HA contents showed an overall increasing trend, which was basically stable at 4.82% to 5.86% in each treatment. Among them, the HA in the SB-C treatment group ranged from 3.16% at the beginning to 5.73% at the end of composting, which was significantly higher than that in S-C treatment group (47.00%). Due to the relatively small molecular weight and simple structural characteristics, FA in the raw material was greatly decomposed by microorganisms. The decomposition of FA under the microbial action and the rate of disintegration or polymerization into HA affected the dynamic change of FA content [10]. At the end of the composting, the FA content of each treatment was 2.41% to 2.66%. It is generally believed that when HA/FA is greater than 1.9, the conversion of HA and FA tends to be stable. The HA/FA of each treatment showed an increasing trend, and the HA/FA value ranged from 1.9 to 2.4 at the end of this compost experiment. The treatment groups with the highest humification were SB-H and SB-C, indicating that the addition of wetland sediment, biochar and microbial agent could promote humification and polymerization of submerged plant composting, while the microporous biochar structure provided more space for microbial growth and reproduction [15]. Wetland sediment could significantly increase diversity and abundance of microorganisms, as well as the homemade microbial agent promoted the degradation of refractory compounds. Therefore, synergistic effect between additives effectively promoted the formation of HA and FA degradation, improved heap humification degree [3].

Germination index (GI) is the important biological indicator for evaluation of the maturity and biotoxicity of compost. The GI > 80% can be suggested as an indicator of the disappearance of phytotoxicity, and GI > 100% is considered to have a positive effect on plant seed development [8]. After 36 days of composting, the seed germination index increased significantly to 104.00%-117.00%. Similarly, the combination of wetland sediment and biochar had the greatest promoting effect in seed germination.

 

(a)	(b)
(c)	(d)

Figure 2. Changes of HA, FA, HA/FA and seed germination index (a~d) during composting in different treatments. Error bars represent standard deviations (SDs) for each test day (n = 3)

 

Details:

- the values in Table 1 should be given with their uncertainties.

Response: As suggested that we have corrected the table 1.

Table 1. Primary properties of raw materials in composts.

Material	TOC （%）	TN （%）	C/N	cellulose （%）	hemicellulose （%）	lignin （%）
Submerged plant	84.62±5.26	1.33±0.09	63.62	15.8±0.43	29.2±0.76	8.90±0.46
Dry chicken dung	63.11±3.23	3.62±0.22	17.43	-	-	-
Wetland sediment	39.64±2.17	2.63±0.19	15.13	-	-	-
Compost material	76.36±4.35	2.69±0.18	28.39	7.62±0.26	15.9±0.87	4.88±0.39

Note: Data are reported as Mean±SD with n=3. TOC, total organic carbon; TN, total nitrogen; -, not detected due to low content. The submerged plants are Ceratophyllum demersum and Potamogeton wrightii 1:1 mixed. Compost materials consist of submerged plants mixed with dry chicken manure in a 7:3 ratio.

 

 

- line 201 TOC is not a substrate but a measure of carbon

Response: As suggested that we have corrected the sentence.

Total organic carbon (TOC) represents the organic carbon content in the compost sample and serves as a source of carbon for microbial metabolism, which exhibited a gradual decrease trend in the composting process

 

We tried our best to improve the manuscript and made some changes in the manuscript. We marked the changes in red in Revised Manuscript. We earnestly appreciate for Editors/Reviewers’ warm work, and hope that the correction will meet with approval. Once again, thank you very much for your comments and suggestions.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The reviewer appreciates the improvements included in the revised version of the manuscript. I recommend the article to be published in the Fermentation Journal.

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for this new version that is much clear and answer to all remarks.