Effects of Soil Moisture and Temperature on Microbial Regulation of Methane Fluxes in a Poplar Plantation

Round 1

Reviewer 1 Report

forests-1083931-comments_CM

Microbial regulation of the effects of soil moisture and temperature on methane fluxes from soils under a poplar plantation

 

This paper looks at the environmental control of methane fluxes from poplar plantation soils in Huai River Basin of Jiangsu Province, China. Soil samples taken from two depths (0-20, 20-50 cm) were incubated for two weeks, under controlled 2 moisture and 3 temperature regimes, and analyzed for CH4 flux, microbial composition, and soil parameters such as carbon and nitrogen, organic matter and microbial biomass. The paper then reports on CH4 emission (or uptake) rates, the diversity of methanotroph and methanogenic microbial communities, and their relationship to soil moisture and temperature.

 

The data sets seem solid and the methods and results are quite comprehensively documented (with nevertheless a few problems pointed out below). The methodological set up (2 moisture levels, 3 temperature levels) are clear and well-argued and defined. There is intriguing and complex analysis of the microbial communities (methanogens and methanotrophs) at the varying moisture and temperature levels and the two depths. This is the best part of the data.

 

The paper is nevertheless problematic in its current form as it seems to present the data in an overly complicated, misleading and bungled way. I would allow a resubmission only after a really thorough major revision.

 

One major problem I have with the paper is in the data: up to which point the results from two weeks of laboratory incubation of soil samples from one plantation site allow to draw wide-ranging ecological and climate-related conclusions? The soil under a 13-year-old poplar plantation might still reflect much of the previous land use, and such a plantation is hardly representative of forests. The authors draw very generic conclusions about CH4 fluxes in forest soils – these might be allowed as speculative but there should be more attention paid in the discussion to this methodological limitation and what it could mean for the data. E.g. how would the reported relationships in older, more mature, more diverse forests look like? What speaks in favor of generalizations is the fact that they look at environmental controlling variables – these might work similarly in every site, but we do not have proof of this and so the discussion needs to reflect this in better ways.

 

Another major problem is how the paper’s main idea is presented. There is a lack of one clearly presented hypothesis (or a set of coherent hypotheses), and then a missing presentation of results and a discussion of the results in light of these hypotheses. To be sure, there are hypotheses – the formally introduced two hypotheses in Lines 94-98 of the introduction – but the authors create a rather confusing narrative around them. It starts with the title which claims to study “Microbial regulation” of the effects of soil moisture and temperature on methane fluxes (which could be presented as a model of MIC >> M & T >> CH4, with “>>” meaning leads to, representing a causal loop), but in fact the paper studies soil moisture and temperature effects on methane-related microbial communities and CH4 emissions (M & T + soil parameters >> MIC >> CH4), which is a different (and more compelling) logic.

 

The authors confound this story then even more by touching on the subject, in the rather unnecessarily lengthy introduction, repeatedly and in different ways, providing at least four conceptually different ideas of how M, T, MIC and CH4 fluxes are related:

 

• L35-37 “The metabolic activities of soil microbial communities, commonly known as methanogens and methanotrophs, are the main controllers of production and consumption of CH4 in soils, which are regulated by several environmental factors” (environmental factors, which would seem to be moisture and temperature, regulate microbial communities, which control CH4: M&T >> MIC >> CH4)
• L 90-92: “our objectives were: (1) to determine the relationship between the soil CH4 fluxes and the moisture and temperature changes that naturally occur along with the hydrological shifts” (M& T >> CH4)
• L93-94: “investigate the governing mechanisms by observing the abiotic and biotic factors responsible for CH4 fluxes” (rather generic and vague “abiotic and biotic” factors: M&T + MIC >> CH4)
• L96-98: “soil moisture and temperature govern the rates of soil CH4 fluxes through … microbial community composition and activities.” (M&T >> MIC >> CH4)

 

I find this rather confusing. It is clear from the rest of the paper and the discussion that the hypothesis they are really investigating is M&T + soil parameters >> MIC >> CH4, so they must change the title of the paper and thoroughly revise (and shorten) the introduction.

 

Furthermore, the microbiological part of the study is in my view insufficiently introduced, wrongly summarized, and not fully explored. The authors look at community composition in a rather sophisticated way (as Figures 4 and 5 testify), and this is the interesting part, but they get lost over large passages in results that state the more obvious (e.g. that CH4 emissions go up with temperature, moisture and aeration (soil depth)), while the really interesting microbial data are taking second stage. E.g. what sense does it make in the abstract to cite the average CH4 production rates over all T and M regimes when there is not much insight to be gained from this? Instead, they should dedicate more space in the abstract detailing the microbial results, which are the real strength of the study. Currently, less than half of the abstract (the last 7 lines) deals with the microbial community data.

 

This part of the abstract also summarizes the data in ways that are not correctly representing the data. E.g. take these examples:

 

Lines 21-22, “A decline in α-diversity of methanotrophs in subsurface soils was recorded along the moisture gradient”. First, the authors should not work with the concept of a gradient suggesting that a continuous gradient was studied if only two moisture levels are in the study and they were studied as separate, fixed treatments. There was no study of what happens in a gradient and how methanotrophs would change in such a changing environment. You have two separate points, but you don’t know if they are connected by a line or a more complex relationship. So you can say “… with increasing moisture level”. (The three temperature levels are, like humidity, also just points, not a ‘gradient’, so authors should be careful with the use of that word which denotes a wrong concept about their data)

 

Second, the numbers of methanotrophs go down in some cases (at 5 degrees, and in D2 at 15 degrees) but they also go up (at 15 degrees, D1, and at 30 degrees, D2). So there is no overall decline in diversity with moisture as the sentence claims.

 

 

Lines 23-24: “methanogenic community diversity was independent of soil moisture and temperature at both soil depths” – this is also not true, as yes, methanogens’ diversity at 5 degrees seems to stay stable with the moisture change, but goes up at 15 and 30 degrees. It goes up at low and high moisture when moving from 5 to 15 degrees in D1, and 15 to 30 degrees in D2, and it goes down or stays similar in all the other cases. So there is not this overall independence.

 

I am demonstrating these cases for the abstract, but these over-generalizations also appear in the rest of the text and need to be carefully revised by the authors.

 

Methods: the text talks about “randomized block design” and “trials” in Line 116. It is not clear what these trials are, as soil samples have been taken from the poplar plantation and incubated in the lab. There are no visible trials distinguished in the data presentation.

 

The sampling set up is not really clear. We have these mysterious “trials”, and then we have 30 cores at 2 depths in 5 pits – what are those pits? It is not explained. The paper needs a much better explanation of what was collected, and where, and how often.

 

Reporting the two sampling depths as 0-20 cm and then 21-50 is trying to be over-precise. When you end at 20 cm with the first depth, you start at 20 cm with the second depth. You are not taking and discarding 1 cm (20-21) at this point. Authors, please review this throughout, it should be 0-20 and 20-50 cm.

 

Discussion: Much of what is being discussed on page 13 is overly general, and not really being used to promote one argument or the other related to what the data in this paper mean. This discussion must become much more specific.

 

Conclusion: There is speculation here (L 437-438) and elsewhere in the paper (L 355 ff) that soil aggregates protected anaerobic methanogens from oxygen. This is interesting and could have been tested (e.g. by sieving the soils or disrupting the aggregates and incubate the soil and see what happens then, probably with a control for the disturbance effect (by disturbing yet another set of samples but not breaking up the aggregates)), or at least, if the authors do not want or cannot go back to the lab, be suggested for future research.

 

There are a few typos and missing words. It is likely that these text passages may disappear in a thorough revision, but I point these out as the authors need to pay more attention to these things:

 

L277, Legend to table 1: replace “tow” by “two”

L293: “we found that almost of the” – add “all” after almost

L294-295: replace “On the contrary” with “In contrast”

L363: “This could because” – add “be” after could

L365: “showed that a slight” – remove “that a”

 

In summary, I would expect this paper to be more streamlined towards the ‘correct’ hypothesis, cutting the rather lengthy and repetitive writing around the various competing ideas (hypotheses) in the introduction that I have pointed out above, improve the precision with which the data are being reported, write an abstract that emphasizes the microbial data and not the more trivial findings, and provide a discussion more directed at the data in the paper, and discussing the breadth of conclusions that can be drawn from a specific poplar site for forests more general.

Author Response

We would like to thank referees for their constructive reviews. We have addressed reviewer’ comments point by point and have made changes in the revised manuscript, which are detailed below. The line number mentioned in this response letter indicate in the revised manuscript with track changes (simple markup).

 

Response to Reviewer 1 Comments

 

Point 1: One major problem I have with the paper is in the data: up to which point the results from two weeks of laboratory incubation of soil samples from one plantation site allow to draw wide-ranging ecological and climate-related conclusions? The soil under a 13-year-old poplar plantation might still reflect much of the previous land use, and such a plantation is hardly representative of forests. The authors draw very generic conclusions about CH4 fluxes in forest soils – these might be allowed as speculative but there should be more attention paid in the discussion to this methodological limitation and what it could mean for the data. E.g. how would the reported relationships in older, more mature, more diverse forests look like? What speaks in favor of generalizations is the fact that they look at environmental controlling variables – these might work similarly in every site, but we do not have proof of this and so the discussion needs to reflect this in better ways.

 

Response 1: We discussed the methodological limitation and the meaning of our study. Please see lines 444-460.

 

Point 2: Another major problem is how the paper’s main idea is presented. There is a lack of one clearly presented hypothesis (or a set of coherent hypotheses), and then a missing presentation of results and a discussion of the results in light of these hypotheses. To be sure, there are hypotheses – the formally introduced two hypotheses in Lines 94-98 of the introduction – but the authors create a rather confusing narrative around them. It starts with the title which claims to study “Microbial regulation” of the effects of soil moisture and temperature on methane fluxes (which could be presented as a model of MIC >> M & T >> CH4, with “>>” meaning leads to, representing a causal loop), but in fact the paper studies soil moisture and temperature effects on methane-related microbial communities and CH4 emissions (M & T + soil parameters >> MIC >> CH4), which is a different (and more compelling) logic.

 

Response 2: We revised the hypothesis, and discussed the results in light of these points. Please see text in revised version (lines 92-95).

 

Point 3: The authors confound this story then even more by touching on the subject, in the rather unnecessarily lengthy introduction, repeatedly and in different ways, providing at least four conceptually different ideas of how M, T, MIC and CH4 fluxes are related:

• L35-37 “The metabolic activities of soil microbial communities, commonly known as methanogens and methanotrophs, are the main controllers of production and consumption of CH4 in soils, which are regulated by several environmental factors” (environmental factors, which would seem to be moisture and temperature, regulate microbial communities, which control CH4: M&T >> MIC >> CH4)
• L90-92: “our objectives were: (1) to determine the relationship between the soil CH4 fluxes and the moisture and temperature changes that naturally occur along with the hydrological shifts” (M& T >> CH4)
• L93-94: “investigate the governing mechanisms by observing the abiotic and biotic factors responsible for CH4 fluxes” (rather generic and vague “abiotic and biotic” factors: M&T + MIC >> CH4)
• L96-98: “soil moisture and temperature govern the rates of soil CH4 fluxes through … microbial community composition and activities.” (M&T >> MIC >> CH4)

I find this rather confusing. It is clear from the rest of the paper and the discussion that the hypothesis they are really investigating is M&T + soil parameters >> MIC >> CH4, so they must change the title of the paper and thoroughly revise (and shorten) the introduction.

 

Response 3: We revised the title and shorten the introduction. Specifically, we removed the similar or repeated depiction in the introduction. Please see lines 2-3, and 42-95.

 

Point 4: Furthermore, the microbiological part of the study is in my view insufficiently introduced, wrongly summarized, and not fully explored. The authors look at community composition in a rather sophisticated way (as Figures 4 and 5 testify), and this is the interesting part, but they get lost over large passages in results that state the more obvious (e.g. that CH4 emissions go up with temperature, moisture and aeration (soil depth)), while the really interesting microbial data are taking second stage. E.g. what sense does it make in the abstract to cite the average CH4 production rates over all T and M regimes when there is not much insight to be gained from this? Instead, they should dedicate more space in the abstract detailing the microbial results, which are the real strength of the study. Currently, less than half of the abstract (the last 7 lines) deals with the microbial community data.

 

Response 4: (1) We re-analysed the microbial α-diversity (Figure 4 instead of the Figure S1), community composition (Figure 5 instead of the Figure 4), and db-RDA (Figure 6 instead of the Figure 5), where we removed the samples of prior to incubation (PIS treatment, i.e., initial fresh field samples) to make the results more clear. We have revised the text and improved discussion on the microbiological part. Please see lines 307-367. (2) Moreover, we dedicated more space in the abstract detailing the new microbial results. Please see lines 22-34.

 

Point 5: This part of the abstract also summarizes the data in ways that are not correctly representing the data. E.g. take these examples:

• Lines 21-22, “A decline in α-diversity of methanotrophs in subsurface soils was recorded along the moisture gradient”. First, the authors should not work with the concept of a gradient suggesting that a continuous gradient was studied if only two moisture levels are in the study and they were studied as separate, fixed treatments. There was no study of what happens in a gradient and how methanotrophs would change in such a changing environment. You have two separate points, but you don’t know if they are connected by a line or a more complex relationship. So you can say “… with increasing moisture level”. (The three temperature levels are, like humidity, also just points, not a ‘gradient’, so authors should be careful with the use of that word which denotes a wrong concept about their data)
• Second, the numbers of methanotrophs go down in some cases (at 5 degrees, and in D2 at 15 degrees) but they also go up (at 15 degrees, D1, and at 30 degrees, D2). So there is no overall decline in diversity with moisture as the sentence claims.
• Lines 23-24: “methanogenic community diversity was independent of soil moisture and temperature at both soil depths” – this is also not true, as yes, methanogens’ diversity at 5 degrees seems to stay stable with the moisture change, but goes up at 15 and 30 degrees. It goes up at low and high moisture when moving from 5 to 15 degrees in D1, and 15 to 30 degrees in D2, and it goes down or stays similar in all the other cases. So there is not this overall independence.
• I am demonstrating these cases for the abstract, but these over-generalizations also appear in the rest of the text and need to be carefully revised by the authors.

 

Response 5: (1) We replaced the word “gradient” with “level”, and throughout the revised manuscript. (2) We have revisited the and modified the microbial α-diversity part according to the new analysis result. Please see lines 22-27. (3) We revised the problem of over-generalizations in the text.

 

Point 6: Methods: the text talks about “randomized block design” and “trials” in Line 116. It is not clear what these trials are, as soil samples have been taken from the poplar plantation and incubated in the lab. There are no visible trials distinguished in the data presentation.

 

Response 6: We have removed ambiguous sentences from text. Please see lines 113-116.

 

Point 7: The sampling set up is not really clear. We have these mysterious “trials”, and then we have 30 cores at 2 depths in 5 pits – what are those pits? It is not explained. The paper needs a much better explanation of what was collected, and where, and how often.

 

Response 7: We re-explained the sampling methods. Please see lines 113-116.

 

Point 8: Reporting the two sampling depths as 0-20 cm and then 21-50 is trying to be over-precise. When you end at 20 cm with the first depth, you start at 20 cm with the second depth. You are not taking and discarding 1 cm (20-21) at this point. Authors, please review this throughout, it should be 0-20 and 20-50 cm.

 

Response 8: We replaced the “21-50 cm” with “20-50 cm” throughout the manuscript. Please see lines 17, 115, 275, 280, 286, 303, 337, 343, and 362.

 

Point 9: Discussion: Much of what is being discussed on page 13 is overly general, and not really being used to promote one argument or the other related to what the data in this paper mean. This discussion must become much more specific.

 

Response 9: We revised the discussion to make it more specific accordingly. Please see lines 369-433.

 

Point 10: Conclusion: There is speculation here (L 437-438) and elsewhere in the paper (L 355 ff) that soil aggregates protected anaerobic methanogens from oxygen. This is interesting and could have been tested (e.g. by sieving the soils or disrupting the aggregates and incubate the soil and see what happens then, probably with a control for the disturbance effect (by disturbing yet another set of samples but not breaking up the aggregates)), or at least, if the authors do not want or cannot go back to the lab, be suggested for future research.

 

Response 10: We suggested a further research on the effect of soil aggregates on methanogens in the future.  Please see lines 453-455.

 

Point 11: There are a few typos and missing words. It is likely that these text passages may disappear in a thorough revision, but I point these out as the authors need to pay more attention to these things:

• L277, Legend to table 1: replace “tow” by “two”
• L293: “we found that almost of the” – add “all” after almost
• L294-295: replace “On the contrary” with “In contrast”
• L363: “This could because” – add “be” after could
• L365: “showed that a slight” – remove “that a”

 

Response 11: Done as suggested. Please see lines 274, 290, 291-292, 380, and 388.

 

Point 12: In summary, I would expect this paper to be more streamlined towards the ‘correct’ hypothesis, cutting the rather lengthy and repetitive writing around the various competing ideas (hypotheses) in the introduction that I have pointed out above, improve the precision with which the data are being reported, write an abstract that emphasizes the microbial data and not the more trivial findings, and provide a discussion more directed at the data in the paper, and discussing the breadth of conclusions that can be drawn from a specific poplar site for forests more general.

 

Response 12: We revised the hypothesis, introduction, abstract, microbial analysis results, discussion, and conclusion, according to the comments from reviewer.

 

Reviewer 2 Report

It is a very interesting paper, well conducted and with high quality results, which would be interest for the journal´s readers. I am pleased to suggest its publication in its actual form. 

Author Response

We highly appreciate reviewer 2‘s recognition to our work.

Reviewer 3 Report

In the reviewed article, the effects of various temperatures and moistures on methane flow in the soil - atmosphere system were investigated. Indeed, a large number of studies have been carried out on methane exchange in different zones to present time, but this study is potentially important and interesting anyway. Both quantitative data on methane emission / consumption rates and confirmed dependences of methane flux on different biotic and abiotic soil properties as well as on microbial diversity are the main advantages of this highly qualified research. The manuscript contains original data. The title reflects clearly the content of the manuscript, while the abstract and keywords are sufficiently informative. The aim and objectives of the article are adequate and appropriate to the subject of the research. The description of materials and methods is clear enough to realize how the authors obtained the experimental data. All results have sufficient statistical analysis and clearly represented. The manuscript (organization, structure, length, figures and tables, references) does not conflict with the requirements of the journal.

The article gives a good impression, but there are a few remarks that should be still addressed.

1. Although the discussion topic is detailed enough, I would suggest to compare the results of these studies with similar ones obtained in the temperate zone (Kravchenko et al, 2005; Semenov et al, 2004) and to use a recently published review also (Kharitonov et al, 2021).

Kharitonov S. et al. 2021. Microbial Communities in Methane Cycle: Modern Molecular Methods Gain Insights into Their Global Ecology. Environments. 8(2):16. DOI: 10.3390/environments8020016

Kravchenko I.K. et al. 2005. Physicochemical and Biological Factors Affecting Atmospheric Methane Oxidation in Gray Forest Soils. Microbiology. 74 (2). 216–220.

Semenov V.M. et al. 2004. Seasonal Dynamics of Atmospheric Methane Oxidation in Gray Forest Soils. Microbiology. 73 (3). 356–362.

2. The Q10 values of methane flux versus previous literature data have to be discussed.
3. The coordinates of the experimental site indicate the body of water. Please, update ones.

 

Author Response

We would like to thank referees for their constructive reviews. We have addressed reviewer’ comments point by point and have made changes in the revised manuscript, which are detailed below. The line number mentioned in this response letter indicate in the revised manuscript with track changes (simple markup).

 

Response to Reviewer 3 Comments

 

We highly appreciate reviewer 3 for positive feedback and recognition of our work.

 

Point 1: Although the discussion topic is detailed enough, I would suggest to compare the results of these studies with similar ones obtained in the temperate zone (Kravchenko et al, 2005; Semenov et al, 2004) and to use a recently published review also (Kharitonov et al, 2021).

 

Response 1: We cited the recent review (Kharitonov et al, 2021) in support our result pointing the effect of moisture on methanotrophic and methanogenic populations. Please see lines 423-424.

 

Point 2: The Q₁₀ values of methane flux versus previous literature data have to be discussed.

 

Response 2: We have compared the Q₁₀ values of methane flux versus previous literature. Please see lines 382-387.

 

Point 3: The coordinates of the experimental site indicate the body of water. Please, update ones.

Response 3: We have checked and updated the coordinates of the experimental site. Please see line 98.