Liming Alters the Soil Microbial Community and Extracellular Enzymatic Activities in Temperate Coniferous Forests

Round 1

Reviewer 1 Report

Liming is important treatment which can  minimize negative effect of acid rains but also calcium depletion in pine forest to maintain good health of trees and high production ability. Naturally, liming change properties of soil and quantification of such changes was the aim of this study

authors compared soil and microbial properties in 5 sites where on lime was applied. Design of experiment seems to be properly chosen, number of replication seems to be sufficient and wide spectrum of properties was evaluated, supposed fine quality of results.

Authors used proper statistic methods, but I would see some hypothesis testing about simmilarity of microbial community such as PERMANOVA or ANOSIM

Author Response

Liming is important treatment which can minimize negative effect of acid rains but also calcium depletion in pine forest to maintain good health of trees and high production ability. Naturally, liming change properties of soil and quantification of such changes was the aim of this study.

Authors compared soil and microbial properties in 5 sites where on lime was applied. Design of experiment seems to be properly chosen, number of replication seems to be sufficient and wide spectrum of properties was evaluated, supposed fine quality of results.

Authors used proper statistic methods, but I would see some hypothesis testing about similarity of microbial community such as PERMANOVA or ANOSIM.

Line 225 – 227

- Thank you for your constructive comments, which helped us to improve the manuscript. We have added testing about similarity of microbial community by ANOSIM.

Reviewer 2 Report

Dear Authors,

I have studied your article “Liming Alters the Soil Microbial Community and Extracellular Enzymatic Activities in Temperate Coniferous Forests”. However, it will be necessary to make some changes and edit the article. Your manuscript describes an important topic of the impact of liming on forest ecosystems.

Abstract

Lines 11 – 15: ... Environmental pollution caused by anthropogenic activities is disturbing the ecosystem in various ways. The damage caused by acid precipitation, which is referred to as acid rain, is representative of these effects. Soil pH is an important factor in soil quality and function, so soil acidification by acidic precipitation is detrimental to forest ecosystems. Liming is one suggested way to solve this problem ...

This text is suitable for the introduction rather than the abstract. I recommend adding the achieved results to the abstract. How much was the enzymatic activity affected by liming compared to the variant without liming? Differences in percentage?

Introduction

This part is brief and clear - general aspects of the topic are described. I have no comments.

Methods

2.1. Site description

The description of the localization of the field experiment is sufficient. I only recommend improving the map so that it is more readable. Furthermore, I would recommend to mark the individual states in the first part of the map. A reader who does not come from an area of interest can get a better idea of where the field experiment was carried out.

It is not clear from the description of the experiment how large area was limed. I assume that a similar acreage was limed in each area and the non-calcified area selected as a control was selected.

2.2. Soil sampling

Line 91: … The three samples were combined and mixed to generate a single sample for 91 analysis …

Again, it is not clear for the description how many samples were taken in total at individual localities within individual variants of experiment.  

What was the time interval between soil sampling and liming?

2.3. Soil chemical properties

I have no comments.

2.4 Soil microbial biomass and extracellular enzyme activity

I have no comments.

2.5 Soil microbial community structure and composition.

I have no comments.

2.6. Statistical analysis

The level of significance (? P < 0.01. or 0.05 ?) at which the analyzes were performed is not stated.

Results

3.1. Soil chemical property

The data are well described, I appreciate that. On the other hand, from Tables 1 is not evident whether there is a significant difference between the control and lime variants. I recommend marking demonstrable differences not only in bold, but also, for example, in red. Above all, did you calculate differences between individual sites?

3.2. Microbial biomass and extracellular enzyme activity

Similar situation as in the previous chapter. The results are well described, but Table 2 is not very clear. It is not entirely clear whether these are only differences within a site or between sites.

3.3. Microbial community structure

Line 214: … bacterial richness increased by 17% with lime treatment (p = 0.001) …

How did you calculate the value 17%? Is this the average of all percentage differences between the control variant and the calcareous variant?

Line 220: Table 3

This type of display values is quite confusing, as in the case of the above tables. If you do not compare the differences between sites, but only within individual localities, then I recommend separating the individual sites/localities with a horizontal line in the table.

3.4. Factors affecting biological property

Line 245: … MBC did not show any significant relationships with soil chemical properties …

This is a very interesting finding because, for example, different results have been found in Central European conditions. I recommend elaborating more in connection with the results in Table 4.

Discussion

4.1. Effects of liming on microbial biomass and extracellular enzyme activity

Lines 270 – 271: … MBC, MBN, and MBP did not show any significant differences between the control and lime treatment … but the effects of lime treatment were confirmed based on the relationships between the changes in soil chemical properties and microbial biomass …

I recommend modifying the sentences or better explaining how it is possible that the effect of lime application was found in some microbial parameters and not elsewhere.

Line 288: … BG activity did not show any differences among treatment sites …

The discussion does not explain “why”. In this chapter, the results should be discussed more, especially the effect of liming on the decomposition of organic matter in the soil and thus microbial activity. In my opinion, this is not mentioned enough.

4.2. Effects of liming on microbial community structure 

This part of the discussion chapter is very well processed and comprehensively explains the measured values in terms of the effect of pH. I have no comments.

4.3. Ecological implications of liming

Line 386: … Our observations indicate that extracellular enzyme activity and the microbial com-386 munity are significantly responsive to lime treatment of the soil …

I recommend comparing it with data from other scientific works - to confirm this statement.

Line 401: … Lime treatment can affect soil microbial communities through direct or indirect processes

I recommend extending these direct and indirect processes more.

Line 413 – 423: … In summary, this study revealed ….

I propose to amend this paragraph so that it does not replace the conclusion.

Conclusion

Line 431: … By decreasing toxicity due to leaching of Ale, liming indirectly increased microbial biomass …

In the results chapter you state: “MBC, MBN, and MBP did not show any significant differences between the control and lime treatment”. I recommend specifying this either in the previous chapter “Results” or here at the Conclusion.

Author Response

Response to the Reviewer 2

I have studied your article “Liming Alters the Soil Microbial Community and Extracellular Enzymatic Activities in Temperate Coniferous Forests”. However, it will be necessary to make some changes and edit the article. Your manuscript describes an important topic of the impact of liming on forest ecosystems.

- Thank you for your valuable comment, which helped us to improve the manuscript.

Abstract

Lines 11 – 15: ... Environmental pollution caused by anthropogenic activities is disturbing the ecosystem in various ways. The damage caused by acid precipitation, which is referred to as acid rain, is representative of these effects. Soil pH is an important factor in soil quality and function, so soil acidification by acidic precipitation is detrimental to forest ecosystems. Liming is one suggested way to solve this problem ...

This text is suitable for the introduction rather than the abstract. I recommend adding the achieved results to the abstract. How much was the enzymatic activity affected by liming compared to the variant without liming? Differences in percentage?

Line 16 – 17

- We corrected the first three sentences that 'suitable for the introduction'. And we added a sentence comparing the difference in enzyme activity by liming.

Introduction

This part is brief and clear - general aspects of the topic are described. I have no comments.

Methods

2.1. Site description

The description of the localization of the field experiment is sufficient. I only recommend improving the map so that it is more readable. Furthermore, I would recommend to mark the individual states in the first part of the map. A reader who does not come from an area of interest can get a better idea of where the field experiment was carried out.

Line 88

- The country name was added to the first part of the map to make it easier to identify the location.

It is not clear from the description of the experiment how large area was limed. I assume that a similar acreage was limed in each area and the non-calcified area selected as a control was selected.

Line 97

- We added information about the lime treatment area.

2.2. Soil sampling

Line 91: … The three samples were combined and mixed to generate a single sample for 91 analysis …

Again, it is not clear for the description how many samples were taken in total at individual localities within individual variants of experiment. 

What was the time interval between soil sampling and liming?

Line 100

- We have added information to know the number of samples and the interval between soil sampling and liming.

2.3. Soil chemical properties

I have no comments.

2.4 Soil microbial biomass and extracellular enzyme activity

I have no comments.

2.5 Soil microbial community structure and composition.

I have no comments.

2.6. Statistical analysis

The level of significance (? P < 0.01. or 0.05 ?) at which the analyzes were performed is not stated.

Line 174

- We have added information about the significance of statistics.

Results

3.1. Soil chemical property

The data are well described, I appreciate that. On the other hand, from Tables 1 is not evident whether there is a significant difference between the control and lime variants. I recommend marking demonstrable differences not only in bold, but also, for example, in red. Above all, did you calculate differences between individual sites?

- The differences between sites were not indicated because the main content was about the effect of lime treatment in our study.

Line 205

- We added a horizontal line to the table to make it easier to distinguish between sites.

3.2. Microbial biomass and extracellular enzyme activity

Similar situation as in the previous chapter. The results are well described, but Table 2 is not very clear. It is not entirely clear whether these are only differences within a site or between sites.

Line 176

- We have added a few information to the Materials and Methods to let the reader know that the result is a comparison of differences within the site and added a horizontal line to the table.

3.3. Microbial community structure

Line 214: … bacterial richness increased by 17% with lime treatment (p = 0.001) …

How did you calculate the value 17%? Is this the average of all percentage differences between the control variant and the calcareous variant?

Line 227 – 229

- This sentence has been deleted. This is not in the table due to the difference between the control variant and the calcareous variant of the entire site.

Line 220: Table 3

This type of display values is quite confusing, as in the case of the above tables. If you do not compare the differences between sites, but only within individual localities, then I recommend separating the individual sites/localities with a horizontal line in the table.

Line 219, 233

- We added horizontal lines to the table.

3.4. Factors affecting biological property

Line 245: … MBC did not show any significant relationships with soil chemical properties …

This is a very interesting finding because, for example, different results have been found in Central European conditions. I recommend elaborating more in connection with the results in Table 4.

- Microbial biomass C is influenced by several factors, but it is generally known to be strongly influenced by the soil organic matter. In our results, MBC showed significant relationship not only with OM, but also with TN, silt, and Ca at the plot level. However, in the manuscript, statistical analysis was conducted at the site level to find clear influencing factor. The variation within the site of the factors related to MBC and MBC is large, so it is not shown significant relationship. These are not considered to be related to the effects of lime treatment and has not been discussed in detail in the manuscript.

Discussion

4.1. Effects of liming on microbial biomass and extracellular enzyme activity

Lines 270 – 271: … MBC, MBN, and MBP did not show any significant differences between the control and lime treatment … but the effects of lime treatment were confirmed based on the relationships between the changes in soil chemical properties and microbial biomass …

I recommend modifying the sentences or better explaining how it is possible that the effect of lime application was found in some microbial parameters and not elsewhere.

- In our study, first, the effect of lime treatment was examined by simply comparing the lime treatment and non-treatment at the site level, and second, soil factors that influence microbial parameters at the plot level were selected, and third, the impact of lime treatment was discussed again by analyzing the linear relationship between microbial parameters at the site level. This process is designed to account for the effect of treatment in situations where several parameters have large variations due to complex soil conditions. Of course, if the results of lime treatment were clear in several parameters, it could be explained in a simpler process, but unfortunately, this was not the case in our study. We think that was a rather complicated but necessary process.

Line 288: … BG activity did not show any differences among treatment sites …

The discussion does not explain “why”. In this chapter, the results should be discussed more, especially the effect of liming on the decomposition of organic matter in the soil and thus microbial activity. In my opinion, this is not mentioned enough.

- The reason why BG activity did not show any difference at the site level is that OM showed little difference at the site level. We did not attach this because we thought it would be meaningless information to explain in the text why activity of BG at the site level did not show a difference. As we describe after this sentence, we think that the activity of BG will be sufficiently explained by showing a significant correlation between OM and linear regression analysis.

4.2. Effects of liming on microbial community structure

This part of the discussion chapter is very well processed and comprehensively explains the measured values in terms of the effect of pH. I have no comments.

4.3. Ecological implications of liming

Line 386: … Our observations indicate that extracellular enzyme activity and the microbial com-386 munity are significantly responsive to lime treatment of the soil …

I recommend comparing it with data from other scientific works - to confirm this statement.

- Line 397–400 are examples of changes in the activity of AP and POX according to changes in soil environment in other scientific works. [33] shows the change in enzyme activity by lime treatment in agricultural soil, and [77] shows the change in the activity of POX as the pH changes. Comparisons with the results of other studies on microbial communities are discussed in line 404–406.

Line 401: … Lime treatment can affect soil microbial communities through direct or indirect processes

I recommend extending these direct and indirect processes more.

- We also think that it is necessary to expand the contents of the direct and indirect processes of lime treatment, especially the indirect process. However, we determined that it is difficult to expand the contents with the data obtained from the design of present study, so we plan to explain it through future study.

Line 413 – 423: … In summary, this study revealed ….

I propose to amend this paragraph so that it does not replace the conclusion.

Line 426 – 436

- We have deleted this paragraph so that it does not replace the conclusion.

Conclusion

Line 431: … By decreasing toxicity due to leaching of Ale, liming indirectly increased microbial biomass …

In the results chapter you state: “MBC, MBN, and MBP did not show any significant differences between the control and lime treatment”. I recommend specifying this either in the previous chapter “Results” or here at the Conclusion.

Line 440

- We specified that MBC, MBN and MBP did not differ by lime treatment at the site level in Conclusion.