Comparison of the Responses of Soil Enzymes, Microbial Respiration and Plant Growth Characteristics under the Application of Agricultural and Food Waste-Derived Biochars
, Jiri Holatko 1,4, Tereza Hammerschmiedt 1, Jiri Kucerik 2, Petr Skarpa 1, Antonin Kintl 1,5, Jakub Racek 6, Tivadar Baltazar 1, Ondrej Malicek 1 and Martin Brtnicky 1,2,*Round 1
Reviewer 1 Report
This work focused on the difference between two types of biochars in altering lettuce and soil properties. The results show us some useful information. However, some concerns should be answered at first.
1. The source and production methods have profound impact on the physiochemical characteristics of biochars, which affects the application of biochar in soil amendment and agronomy. Therefore, in this work, final conclusions should be drawn after knowing the properties of biochars. For example, what was the source and composition of food waste? Differences of particle size, microstructure and porosity of two biochars? Composition and metal content of two biochars? Before adding into soil, have the two biochars been grinded to similar particle size? The above information should be supplemented in the manuscript.
2. The method section is too simple to be followed.
3. What software was used for statistical analysis? What method was used to compare results between different groups where homogeneity of variance didn’t meet? Please add these descriptions.
4. Figure 1C shows interesting results. Both FWB and AB increased the root fresh weight while FWB+AB lowered that value significantly. It seems unreasonable. Similar inconsistence can be found in Figure 4C. Please explain them.
5. In my opinion the Pearson correlation matrix showed small contribution to the work, as many indices were practically irrelevant. A clustered heatmap incorporating all indices and specimens may be a better option.
6. This work didn’t give clear conclusion on the reason why biochars yielded different results.
Author Response
This work focused on the difference between two types of biochars in altering lettuce and soil properties. The results show us some useful information. However, some concerns should be answered at first.
Response: We thank you for appreciation and time devoted to review this manuscript. We tried to incorporate your suggestions closely as below.
- The source and production methods have profound impact on the physiochemical characteristics of biochars, which affects the application of biochar in soil amendment and agronomy. Therefore, in this work, final conclusions should be drawn after knowing the properties of biochars. For example, what was the source and composition of food waste? Differences of particle size, microstructure and porosity of two biochars? Composition and metal content of two biochars? Before adding into soil, have the two biochars been grinded to similar particle size? The above information should be supplemented in the manuscript.
Response: We thank you very much for the suggestions. The source of food waste biochar was mixed food waste coming from a University canteen. The information has been supplemented. The properties we measured for the biochars are added in the manuscript (Table 1). We did not specifically measured the properties like, microstructure and porosity. However, the particle size was homogenized before application to <2mm for both biochars. As for the metal contents are concerned, neither we measured, nor the commercial provider of AB provided the metal contents. However, we assume that these contain no toxic elements. The relative information has been added in the manuscript where applicable.
- The method section is too simple to be followed.
Response: We have now tried to improve the methods section. Please see track changed text.
- What software was used for statistical analysis? What method was used to compare results between different groups where homogeneity of variance didn’t meet? Please add these descriptions.
Response: We thank you for the comment. Data normality and homogeneity of variance was performed using the Shapiro–Wilk and the Levene tests. The (Program R, version 3.6.1) was utilized for statistical analyses. The required information has been added in lines 165-170.
- Figure 1C shows interesting results. Both FWB and AB increased the root fresh weight while FWB+AB lowered that value significantly. It seems unreasonable. Similar inconsistence can be found in Figure 4C. Please explain them.
Response: Thanks. The figure 1C does not contain the data on reduced root fresh weight. However, figure 4c contains the data on reduced SIR. The discussion has been made on it. Please see track changed text in discussion.
- In my opinion the Pearson correlation matrix showed small contribution to the work, as many indices were practically irrelevant. A clustered heatmap incorporating all indices and specimens may be a better option.
Response: Thanks. The figure has been removed.
- This work didn’t give clear conclusion on the reason why biochars yielded different results.
Response: We have now revised the conclusion section.
Reviewer 2 Report
It is one of the current research focuses to improve soil and promote plant growth by using biochar prepared from waste. The theme of the manuscript conforms to the theme of the journal, and the research method has credibility. However, I have some questions about the manuscript.
1. The manuscript lacks the detection of soil physical and chemical properties by biochar, so it is difficult to prove the phenomenon reflected by NDVI index. It is necessary for the author to explain the relationship.
2. The discussion in Figure 6 is too few and needs to be added.
3. The coordinate axis in the image needs to be improved
Author Response
It is one of the current research focuses to improve soil and promote plant growth by using biochar prepared from waste. The theme of the manuscript conforms to the theme of the journal, and the research method has credibility. However, I have some questions about the manuscript.
Response: Dear reviewer, we thank you very much for the time devoted to review our manuscript and appreciating this work.
- The manuscript lacks the detection of soil physical and chemical properties by biochar, so it is difficult to prove the phenomenon reflected by NDVI index. It is necessary for the author to explain the relationship.
Response: Dear reviewer, The NDVI evaluated in the greenhouse pot experiment was determined using hand-held, battery-powered devices PlantPen. The device helps to characterize plants by means of reflectance. PlantPens measure and calculate specific reflectance indices to monitor chlorophyll content, photoprotecting carotenoids, and other important aspects of live foliage. It is a reflectance-based device that provides a convenient, low-cost method of measuring the relative chlorophyll content of attached or detached leaves.
In the present study food waste biochar and agricultural waste derived biochar either alone or combination (FWB+AB) were tested for their effects on soil microbiological characteristics, and plant growth and photosynthetic parameters.
We are confident that biochar affected soil physical and chemical properties in addition to the soil biome and plant growth characteristics we observed. However, monitoring the effect of biochar on soil physical and chemical properties was not in our interest.
- The discussion in Figure 6 is too few and needs to be added.
- The coordinate axis in the image needs to be improved
Response: We thank you. However, the figure has been removed as per reviewer 1 suggestions.
Reviewer 3 Report
Please check the attached file.
Comments for author File: 
Comments.pdf
Author Response
This paper addresses an important and interesting problem—Comparison of the responses
of soil enzymes, microbial respiration and plant growth characteristics under the application
of agricultural and food waste derived biochars. In the present study, food waste biochar
(FWB) and agricultural waste derived biochar (AB) either alone or combination (FWB+AB)
were tested for their effects on soil microbiological characteristics (Soil enzymes related to
C, N and P cycling, soil basal as well as substrate-induced respiration) and plant growth and
photosynthetic parameters.
Overall, a lot of work have been done and some innovative conclusions were drawn.
However, some issues still need to be improved before published, as follows:
Response: Dear reviewer thank you very much for the time devoted to review our manuscript and suggesting valuable changes. We closely incorporated your suggestions.
- The detailed experimental methods and conditions should be provided,
Response: We have tried to elaborate the details in M&M section. Please see track changed text.
- The unit of ordinate of Fig.1-4 ?
Response: Thanks. The units are already mentioned in figures. However, the parameters in Fig. 2 bear no units.
- What is the purpose and function of Section 3.4 Results from Pearson correlation and
principal component analysis ? What is the relationship between Section 3.4 and Section 3.1-
3.3 ?
Response:Thanks for your comment. The pearson correlation has been emitted from the results as per other reviewers suggestions. However, PCA retained as such. It shows the mutual dpendence of observed parameters and applied amendments. The relationship between section 3.1-3.3 and 3.4 is clear from the PCA showing the mutually positiove as well as negative relationships among observed parameters as shown by distribution of parameters in 2 principál components. This has been mentioned in discussion at relevant places e.g., lines 290-292.
- The article should be more logically organized.
Response: Thanks. We have tried to present the manuscript in a logical way. We believe that the revision according to reviewers suggestion has improved the quality of the manuscript.
- Abstract should conclude some specific data or values of parameters.
Response: Thanks. The suggestion has been incorporatged.
- Population structure of microorganisms should be added, in order to analyze the results of
soil enzymes and microbial respiration.
Response: Dear reviewer, we appreciate your valid comment. However, at present the population of microbes was not tested for this study.
- Page4, lines 152-156: The methods for determining the activities of these enzymes (GLU,
PHOS, URE, NAG) are not specified.
Response: Thanks. The method for enzymes determination has been specified. Lines 160-161.
- Page4, line159: “D-glucose (hereinafter referred to as Glc-IR)”. The “Glc-IR” is not
correct.
Response: Thanks the whole sentence has been revised and mistake corrected. Please see lines 166-170.
- Page4, lines 169-176:
(1) The expression of “applied biochars (Food and agricultural waste derived biochars)”
should be written as “applied biochars (FWB and AB)”.
(2) “agricultural waste biochar (AB)”. The preceding abbreviation “AB” can be used directly.
Response: Thanks. The suggestions are incorporated.
- Page5, lines 194-207:
(1) “application of foodwaste (FWB) and agricultural (AB) derived biochar (FWB+AB
Figure 3A) which was also statistically significant as compared to control.” The “B” in “FWB”
and “AB” is duplicated with the “biochar”; “(FWB+AB Figure 3A)” should be changed to
“(FWB+AB Figure. 3A)”; There is no significant difference of pH between FWB+AB and
control.
Response: Thanks the correction has been made.
(2) The abbreviations should be used in the whole paragraph.
Response: Thanks. The abbreviated names are already used.
(3) The “combined AB+FWB application” in “however the combined AB+FWB application
resulted in similar values as control (Figures. 3D, E). ” should be changed to “combined
application (AB+FWB)” or “combined application of AB+FWB”.
Response: corrected.
(4) “food-waste biochar” should be written as “FWB”.
Response: corrected.
- Page6, lines 212-218:
(1) The abbreviations should be used in the whole paragraph.
Response: Thanks. Suggestion incorporated.
(2) “the alanine-induced ” should be written as “Ala-IR”.
Response: done
- Page8, lines 267-268: The abbreviations should be used in the whole paragraph.
Response: done
- There is no any reason why wheat is chosen as the experimental plant.
Response: Dear reviewer, its not wheat, but lettuce. Line (133)
- It would be better to include DOI in all references.
Response: Thanks. We retained it as such as the MDPI allows format free submission.
- Before submitting a revision, be sure that your material is properly prepared and
Formatted
Response: Thanks for the comment. Done.
Round 2
Reviewer 1 Report
The authors make revisions according to comments. The recent version of manuscript has better quality than the initial one. Some questions are listed below.
1. Line 239, it should be “principal component 2”. The words “hereinafter referred to as” are unnecessary.
2. Section 3.4, data interpretation and the corresponding discussion are not enough. The significance (P) and correlation (R2) should be provided to show the confidence of PCA assay. Figure 5 shows clear clusters of FWB and AB groups. They contribute differently to the tested aspects. Discussion about the above variation can add scores to this work. Unfortunately, I do not see related content in this article.
3. Same as question 2, the discussion section concerns about the common problems that how biochar affects the soil properties. However, difference between FWB and AB is less described. Relation between biochar source/properties and functionality should be the central point of this work.
Author Response
Comments and Suggestions for Authors
The authors make revisions according to comments. The recent version of manuscript has better quality than the initial one. Some questions are listed below.
Response: We thank the reviewer for the appreciation and recommending some minor comments for improvement. We have closely followed your suggestion and incorporated within the manuscript.
- Line 239, it should be “principal component 2”. The words “hereinafter referred to as” are unnecessary.
Response: Thanks. Corrected.
- Section 3.4, data interpretation and the corresponding discussion are not enough. The significance (P) and correlation (R2) should be provided to show the confidence of PCA assay. Figure 5 shows clear clusters of FWB and AB groups. They contribute differently to the tested aspects. Discussion about the above variation can add scores to this work. Unfortunately, I do not see related content in this article.
Response: We thank for the suggestion. The suggestion has been incorporated. The required information extracted from PCA has been added. Please see lines (237-262). We have discussed now on the distribution of AB and FWB as indicated by PCA in discussion section. Please see yellow highlighted track changed text.
Same as question 2, the discussion section concerns about the common problems that how biochar affects the soil properties. However, difference between FWB and AB is less described. Relation between biochar source/properties and functionality should be the central point of this work.
Response: We appreciate your suggestion. The discussion section has been improved in accordance with the suggestion. The differences b/w AB and FWB are now rigorously described by referring their composition and properties observed where applicable. Please see yellow highlighted track changed text throughout the discussion.
Reviewer 2 Report
The authors have refined the manuscript as requested by the reviewers, and I think it has made great progress.
Although the authors believe that the physical and chemical properties of soil do not conform to their research interests, the improvement of the physical and chemical properties of biochar in soil directly affects the microbial community structure and plant growth. The impact of biochar on plants is mostly indirect. It is recommended to refer to the following literature.
(https://doi.org/10.1016/j.scitotenv.2018.04.127;
Agronomy 2022, 12, 1003. https://doi.org/10.3390/agronomy12051003)
Therefore, I suggest that the author should at least briefly discuss the direct or indirect relationship between biochar and microbial communities and plant growth by improving soil.
Author Response
The authors have refined the manuscript as requested by the reviewers, and I think it has made great progress.
Although the authors believe that the physical and chemical properties of soil do not conform to their research interests, the improvement of the physical and chemical properties of biochar in soil directly affects the microbial community structure and plant growth. The impact of biochar on plants is mostly indirect. It is recommended to refer to the following literature.
(https://doi.org/10.1016/j.scitotenv.2018.04.127;
Agronomy 2022, 12, 1003. https://doi.org/10.3390/agronomy12051003)
Therefore, I suggest that the author should at least briefly discuss the direct or indirect relationship between biochar and microbial communities and plant growth by improving soil.
Response: Dear Reviewer, Thank you very much for appreciation. Yes, we have closely followed your suggestion and found the shared manuscripts quite handy. We have modified the discussion section as per your suggestions. Please see yellow highlighted track changed text in discussion section. Please see lines 292-304 in the revised version.
