Differential Temperature Sensitivity of Intracellular and Extracellular Soil Enzyme Activities

Adekanmbi, Adetunji Alex; Dale, Laurence; Shaw, Liz J.; Sizmur, Tom

doi:https://doi.org/10.5194/egusphere-2022-1091

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https://doi.org/10.5194/egusphere-2022-1091

Preprints

20 Dec 2022

| 20 Dec 2022

Differential Temperature Sensitivity of Intracellular and Extracellular Soil Enzyme Activities

Adetunji Alex Adekanmbi, Laurence Dale, Liz J. Shaw, and Tom Sizmur

Abstract. Predictions concerning the feedback of soil heterotrophic respiration to a warming climate often do not differentiate between the extracellular and intracellular processes involved in soil organic matter decomposition. This study examined the temperature sensitivities of intracellular and extracellular soil enzyme activities and how they are influenced by previous temperatures. We pre-incubated soils at 5 °C, 15 °C or 26 °C to acclimatise the microbial communities to different thermal regimes for 60 days before measuring potential activities of β-glucosidase and chitinase (extracellular enzymes), glucose-induced respiration (intracellular enzymes), and basal respiration at a range of assay temperatures (5 °C, 15 °C, 26 °C, 37 °C, and 45 °C). A higher pre-incubation temperature decreased soil pH and C / N ratio which exerted a strong legacy effect by decreasing β-glucosidase potential activity and respiration, but not chitinase potential activity. It is likely that this legacy effect is an indirect effect of substrate depletion rather than physiological acclimatation or genetic adaptation. There was no overall significant effect of pre-incubation temperature on temperature sensitivity of these enzymes, perhaps because of the short (60 day) duration of the pre-incubation. However, we found that the intracellular and extracellular enzyme activities differ in their temperature sensitivity and this observation differs depending on the range of temperature used for Q10 estimates of temperature sensitivity. Between 5 °C and 15 °C intracellular and extracellular enzyme activities show equal temperature sensitivity, but between 15 °C and 26 °C intracellular enzyme activity was more temperature sensitive than extracellular enzyme activity and between 26 °C and 37 °C extracellular enzyme activity was more temperature sensitive than intracellular enzyme activity. This result implies that depolymerisation of higher molecular weight carbon is more sensitive to temperature changes at higher temperatures (e.g. higher temperatures on extremely warm days) but the respiration of the generated monomers is more sensitive to temperature changes at moderate temperatures (e.g. mean daily maximum soil temperature). Therefore, since climate change predictions currently indicate that there will be a greater frequency and severity of hot summers and heatwaves, it is possible that global warming may reduce the importance of extracellular depolymerisation relative to intracellular catalytic activity as the rate limiting step of soil organic matter mineralization. We conclude that extracellular and intracellular steps are not equally sensitive to changes in soil temperature and that the previous temperature a soil is exposed to may influence the potential activity, but not temperature sensitivity, of extracellular and intracellular enzymes.

Received: 28 Oct 2022 – Discussion started: 20 Dec 2022

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Journal article(s) based on this preprint

16 Jun 2023

Differential temperature sensitivity of intracellular metabolic processes and extracellular soil enzyme activities

Adetunji Alex Adekanmbi, Laurence Dale, Liz Shaw, and Tom Sizmur

Biogeosciences, 20, 2207–2219, https://doi.org/10.5194/bg-20-2207-2023,https://doi.org/10.5194/bg-20-2207-2023, 2023

Short summary

Adetunji Alex Adekanmbi et al.

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2022-1091', Anonymous Referee #1, 13 Jan 2023

The article of Adekanmbi and coauthors is aiming to set up an interesting comparison between extracellular and intracellular enzymes and evaluate their temperature sensitivity after being exposed to distinct temperatures for 60 days. However, the authors need to better justify the use of glucose-induced respiration as a proxy for intracellular enzymes. Because the glucose-induced respiration will be the result of various processes and also ultimately depends on the microbial community growth efficiency. While the beta-glucosidase and chitinase activities are capturing only the activity of these enzymes. So, making the comparison between extracellular and “intracellular” enzymes becomes difficult in my understanding. Moreover, it is important to remember that the production of extracellular enzymes will also result in CO2 production. I am concerned that authors’ experimental design might not allow to separate between intracellular and extracellular enzymes. Instead of referring to intracellular enzymes authors could refer to “intracellular activity” or “intracellular processes” related to SOM decomposition. This should help to avoid confusion. If authors think that the design allow to make the comparison between extracellular and “intracellular” enzymes they should add an explanation and references to justify their choice.

Nevertheless, I think that the data collected by the authors is valuable and is a good contribution to the field of soil ecology and to the EGU community. It could be interesting to evaluate if the respiration temperature sensitivity and extracellular enzyme temperature sensitivity are coupled or not (are they correlated?). It is also interesting to observe how distinct the two extracellular enzymes responded to the increase in temperatures. I think the authors did a good job in their discussion section.

It is not very clear why authors used a distinct range of temperatures to evaluate the enzyme activation energy for the respiration and extracellular enzymes. Authors could clarify this choice.

Overall, the paper is very well written and is citing the relevant literature in this topic.

Citation: https://doi.org/10.5194/egusphere-2022-1091-RC1
- AC1: 'Reply on RC1', Tom Sizmur, 15 Feb 2023
  
  Thank you very much for reviewing our manuscript. Please find our responses to your comments and those made by reviewer 2 in the attached PDF.
  
  Citation: https://doi.org/10.5194/egusphere-2022-1091-AC1
RC2:
'Comment on egusphere-2022-1091', Anonymous Referee #2, 29 Jan 2023

I have read the manuscript titled "Differential Temperature Sensitivity of Intracellular and Extracellular Soil Enzyme Activities" by Adekanmbi et al.
The study has two main objectives. The first objective is to evaluate the thermal sensitivity of the extra- and intracellular steps of soil organic matter decomposition. The second objective is to evaluate the potential of microbial communities to acclimatize/adapt to a temperature treatment over 60 days.
The study is well written and the introduction and discussion sections are well-supported with relevant hypotheses and current literature. I find that the topic and questions raised in this article are of great interest, as there is still a lack of understanding about the thermal sensitivity of soil microorganisms, their potential to adapt to climate change, and the implications on soil carbon decomposition. The study is well-written and has a clear introduction and discussion with well-stated hypotheses and up-to-date bibliography. However, the study has three main limitations that I highlight below.

1- I understand the idea of removing substrate limitation by feeding microbes with glucose, but using this as a proxy for intracellular enzyme activity is confusing. Other factors, such as diffusion, active transport, and carbon use efficiency of the microbes, among others, can also impact this step. Additionally, comparing the intracellular decomposition process (which involves multiple enzymes) to an extracellular specific enzyme reaction (such as beta glucosidase or chitinase) seems not appropriate. The authors should rephrase this in their manuscript and consider discussing non-limited respiration or maybe glucose-induced respiration.

2- I do not understand why the authors are calculating Q10 at different temperatures. It is known that one of the main limitations of Q10 is that it can change depending on the temperature range chosen for calculation. Why did the authors not use linear regression between the natural logarithm of enzyme activity (Vmax) and temperature, and convert to Q10 values based on the relationship: Q10 = exp (10 × slope) (as cited in Zuo et al, 2021, German et al, 2016 and many other articles)? Can you please provide a strong rationale for why this method was not used or present a single Q10 value calculated in this manner.
Refs
-The effect of soil depth on temperature sensitivity of extracellular enzyme activity decreased with elevation: Evidence from mountain grassland belts. 2021. Yiping Zuo, Hongjin Zhang, Jianping Li, Xiaodong Yao, Xinyue Chen, Hui Zeng, Wei Wang,
-The Michaelis-Menten kinetics of soil extracellular enzymes in response to temperature: A cross-latitudinal study.2016. German, D.P., Marcelo, K.R.B., Stone, M.M., Allison, S.D.
3- This study only uses one soil and three temperature treatments to explore the relative thermal sensitivity of extra- and intracellular steps of decomposition. I acknowledge that determining thermal sensitivity in the laboratory is a lot of work, but using only one soil and three treatments is still very limited compared to other published studies. The authors should clearly state this limitation in the abstract and main conclusion to avoid extrapolating or overstating the main findings (which are indeed interesting).
Line to line comments:
Line 100: “measurement of enzyme activity at different temperatures in the lab is not an experimental treatment in itself (compared to the 60 day of temperature treatment). This sentence is misleading. The experiment did not have "60 experimental units," but 12 (3 incubation temperatures x 4 replicates).
Line 97: A space is needed between the two commas.
Line 128: What does MUB stand for? Was the buffer pre-incubated at different temperatures?
Line 326/327: "Accumulation of monomers" needs to be reformulated.
Line 343: "It is tempting" is not appropriate scientific language. Please rephrase.
Line 346: Please remove the hyphen in "trimeric."
Line 349: Please remove the comma.
Line 361: Double space?
Line 384: Changes in the thermal sensitivity of enzymes could have indicated an adaptation of the enzymes produced by the microbial community.
Line 386: Please use "Vmax" or "apparent Vmax" instead of "concentration," as you did not measure it. Please make sure to use consistent terminology throughout the manuscript.
In the supplementary material: Could you please specify if the curves on the graph are the mean of all samples or just one sample for illustrating the reaction? (Figure S-3: β-glucosidase).
Line 391: You could calculate enzyme-specific activity (normalized by microbial biomass) to test if this statement is correct or not?
Figure 2 caption: Please specify that it is Vmax.

Citation: https://doi.org/10.5194/egusphere-2022-1091-RC2
- AC2: 'Reply on RC2', Tom Sizmur, 15 Feb 2023
  
  Thank you very much for reviewing our manuscript. Please find our responses to your comments and those made by reviewer 1 in the attached PDF.
  
  Citation: https://doi.org/10.5194/egusphere-2022-1091-AC2

Interactive discussion

Status: closed

RC1:
'Comment on egusphere-2022-1091', Anonymous Referee #1, 13 Jan 2023

The article of Adekanmbi and coauthors is aiming to set up an interesting comparison between extracellular and intracellular enzymes and evaluate their temperature sensitivity after being exposed to distinct temperatures for 60 days. However, the authors need to better justify the use of glucose-induced respiration as a proxy for intracellular enzymes. Because the glucose-induced respiration will be the result of various processes and also ultimately depends on the microbial community growth efficiency. While the beta-glucosidase and chitinase activities are capturing only the activity of these enzymes. So, making the comparison between extracellular and “intracellular” enzymes becomes difficult in my understanding. Moreover, it is important to remember that the production of extracellular enzymes will also result in CO2 production. I am concerned that authors’ experimental design might not allow to separate between intracellular and extracellular enzymes. Instead of referring to intracellular enzymes authors could refer to “intracellular activity” or “intracellular processes” related to SOM decomposition. This should help to avoid confusion. If authors think that the design allow to make the comparison between extracellular and “intracellular” enzymes they should add an explanation and references to justify their choice.

Nevertheless, I think that the data collected by the authors is valuable and is a good contribution to the field of soil ecology and to the EGU community. It could be interesting to evaluate if the respiration temperature sensitivity and extracellular enzyme temperature sensitivity are coupled or not (are they correlated?). It is also interesting to observe how distinct the two extracellular enzymes responded to the increase in temperatures. I think the authors did a good job in their discussion section.

It is not very clear why authors used a distinct range of temperatures to evaluate the enzyme activation energy for the respiration and extracellular enzymes. Authors could clarify this choice.

Overall, the paper is very well written and is citing the relevant literature in this topic.

Citation: https://doi.org/10.5194/egusphere-2022-1091-RC1
- AC1: 'Reply on RC1', Tom Sizmur, 15 Feb 2023
  
  Thank you very much for reviewing our manuscript. Please find our responses to your comments and those made by reviewer 2 in the attached PDF.
  
  Citation: https://doi.org/10.5194/egusphere-2022-1091-AC1
RC2:
'Comment on egusphere-2022-1091', Anonymous Referee #2, 29 Jan 2023

I have read the manuscript titled "Differential Temperature Sensitivity of Intracellular and Extracellular Soil Enzyme Activities" by Adekanmbi et al.
The study has two main objectives. The first objective is to evaluate the thermal sensitivity of the extra- and intracellular steps of soil organic matter decomposition. The second objective is to evaluate the potential of microbial communities to acclimatize/adapt to a temperature treatment over 60 days.
The study is well written and the introduction and discussion sections are well-supported with relevant hypotheses and current literature. I find that the topic and questions raised in this article are of great interest, as there is still a lack of understanding about the thermal sensitivity of soil microorganisms, their potential to adapt to climate change, and the implications on soil carbon decomposition. The study is well-written and has a clear introduction and discussion with well-stated hypotheses and up-to-date bibliography. However, the study has three main limitations that I highlight below.

1- I understand the idea of removing substrate limitation by feeding microbes with glucose, but using this as a proxy for intracellular enzyme activity is confusing. Other factors, such as diffusion, active transport, and carbon use efficiency of the microbes, among others, can also impact this step. Additionally, comparing the intracellular decomposition process (which involves multiple enzymes) to an extracellular specific enzyme reaction (such as beta glucosidase or chitinase) seems not appropriate. The authors should rephrase this in their manuscript and consider discussing non-limited respiration or maybe glucose-induced respiration.

2- I do not understand why the authors are calculating Q10 at different temperatures. It is known that one of the main limitations of Q10 is that it can change depending on the temperature range chosen for calculation. Why did the authors not use linear regression between the natural logarithm of enzyme activity (Vmax) and temperature, and convert to Q10 values based on the relationship: Q10 = exp (10 × slope) (as cited in Zuo et al, 2021, German et al, 2016 and many other articles)? Can you please provide a strong rationale for why this method was not used or present a single Q10 value calculated in this manner.
Refs
-The effect of soil depth on temperature sensitivity of extracellular enzyme activity decreased with elevation: Evidence from mountain grassland belts. 2021. Yiping Zuo, Hongjin Zhang, Jianping Li, Xiaodong Yao, Xinyue Chen, Hui Zeng, Wei Wang,
-The Michaelis-Menten kinetics of soil extracellular enzymes in response to temperature: A cross-latitudinal study.2016. German, D.P., Marcelo, K.R.B., Stone, M.M., Allison, S.D.
3- This study only uses one soil and three temperature treatments to explore the relative thermal sensitivity of extra- and intracellular steps of decomposition. I acknowledge that determining thermal sensitivity in the laboratory is a lot of work, but using only one soil and three treatments is still very limited compared to other published studies. The authors should clearly state this limitation in the abstract and main conclusion to avoid extrapolating or overstating the main findings (which are indeed interesting).
Line to line comments:
Line 100: “measurement of enzyme activity at different temperatures in the lab is not an experimental treatment in itself (compared to the 60 day of temperature treatment). This sentence is misleading. The experiment did not have "60 experimental units," but 12 (3 incubation temperatures x 4 replicates).
Line 97: A space is needed between the two commas.
Line 128: What does MUB stand for? Was the buffer pre-incubated at different temperatures?
Line 326/327: "Accumulation of monomers" needs to be reformulated.
Line 343: "It is tempting" is not appropriate scientific language. Please rephrase.
Line 346: Please remove the hyphen in "trimeric."
Line 349: Please remove the comma.
Line 361: Double space?
Line 384: Changes in the thermal sensitivity of enzymes could have indicated an adaptation of the enzymes produced by the microbial community.
Line 386: Please use "Vmax" or "apparent Vmax" instead of "concentration," as you did not measure it. Please make sure to use consistent terminology throughout the manuscript.
In the supplementary material: Could you please specify if the curves on the graph are the mean of all samples or just one sample for illustrating the reaction? (Figure S-3: β-glucosidase).
Line 391: You could calculate enzyme-specific activity (normalized by microbial biomass) to test if this statement is correct or not?
Figure 2 caption: Please specify that it is Vmax.

Citation: https://doi.org/10.5194/egusphere-2022-1091-RC2
- AC2: 'Reply on RC2', Tom Sizmur, 15 Feb 2023
  
  Thank you very much for reviewing our manuscript. Please find our responses to your comments and those made by reviewer 1 in the attached PDF.
  
  Citation: https://doi.org/10.5194/egusphere-2022-1091-AC2

Peer review completion

AR: Author's response | RR: Referee report | ED: Editor decision | EF: Editorial file upload

ED: Reconsider after major revisions (22 Feb 2023) by Alberto Canarini

ED: Reconsider after major revisions (01 Mar 2023) by Michael Bahn (Co-editor-in-chief)

AR by Tom Sizmur on behalf of the Authors (01 Mar 2023) Author's response Author's tracked changes Manuscript

ED: Referee Nomination & Report Request started (07 Mar 2023) by Alberto Canarini

RR by Anonymous Referee #1 (21 Mar 2023)

RR by Anonymous Referee #2 (12 Apr 2023)

Suggestions for revision or reasons for rejection

I have gone through the revised manuscript titled "Differential Temperature Sensitivity of Intracellular and Extracellular Soil Enzyme Activities" by Adekanmbi et al. The manuscript has shown improvement, and the authors have addressed all of my comments. However, there are still some points that require correction or clarification.

1/
I am surprised that the article titled "The Inflection Point Hypothesis: The Relationship between the Temperature Dependence of Enzyme-Catalyzed Reaction Rates and Microbial Growth Rates" by Erica J. Prentice et al. has not been cited in the present study, as its present theory that explain the results obtain in the present study. Therefore, I suggest that the authors cite and integrate this article into their discussion.
Please also correct the title based on reviewer 1 and my comment about “intracellular enzyme” vs “intracellular metabolic processes’”.

2/
I appreciate the authors' clarification on the reason for calculating Q10 at different temperatures and presenting graphs of the relationship between the natural log of enzyme activity/respiration and temperature. The presented graphs should be included in the article, either in the main text or as supplementary information.
It seems that beta glucosidase follows the Arrhenius function, and respiration thermal sensitivity presents a bell-shaped response similar to the one proposed by the MMRT. However, I am more concerned about the thermal response of Chintinase, which appears to be more chaotic. Therefore, I suggest that the authors add a couple of sentences that precisely describe the observed temperature relationship of Chintinase and provide arguments or hypotheses to explain why the Chintinase temperature does not follow what is generally observed.

3/
Was the buffer pre-incubated at different temperatures at the desired temperature? How long did it take for the assay solution to reach the desired temperature (5C or 40C)? As the activity was only assessed based on a 30 min assay, I am wondering if the measure really reflects the activity at 5 degrees or rather an average temperature between room temperature and 5 degrees during the 30 min of cooling. In the latter case, that would mean that extreme temperatures (temperatures that are most different from room temperature, 5 or 40C) are most likely wrong or closer to room temperature than expected (8 C, 35 C, for example). Moreover, did the authors control the temperature of the buffer assay solution during the 30 min, or at least control the temperature at the end?
Overall, the material and method section needs to be much more precise on this point, as the entire article is about the temperature sensitivity of enzymes, and the observed response of one enzyme (chitinase) does not follow proposed/common theory (Arhenius, MMRT).

4/
In the materials and methods section, the authors indicate that the concentration of the substrate used was "1ml 25mM nitrophenyl-β-D-glucopyranoside or 10 mM p-nitrophenyl-N-acetyl-b-D-glucosaminide solution." I suggest that the authors explain why they chose this concentration and whether it was chosen to be most likely in a non-substrate limiting condition (although not tested for the soil under study). In the latter case, this would mean that the authors measured Apparent Vmax.

Hide

ED: Publish subject to minor revisions (review by editor) (02 May 2023) by Alberto Canarini

Dear Authors,
Thanks for submitting your article. We finally received comments from the two appointed reviewers. As you can see form their comments, they both express positive feedback regarding the first round of review and R2 ask to include some of the graphs you used to assess their comments in the manuscript. I agree and I would ask the authors to add these graphs to the supplementary section (relationship between natural log of enzyme activity/respiration and temperature), as they represent an important step to fully understand the study.
There are still a few things that need to be addressed before I can recommend publication. As you see both R1 and R2 have still some doubts on the calculation of activation energy and Q10. Specifically R1 ask to justify better why different temperature ranges were used, as this may impact the comparison. For example an additional graph presenting results where the same ranges were used could additionally show that this would not be an issue for the comparison (e.g. using the range 5-26 also for the extracellular enzyme if it is not possible to extend to 5-37 for the intracellular and basal respiration for conformity reasons). If results are instead different depending on the range used, this should be used to discuss your results. R2 asks to add a sentence in the discussion to explain why chitinase behaves somehow erratic in response to temperature.
Additionally R2 also point at two other important details to clarify in the materials and methods:
1) the potential temperature issue of the buffer;
2) rationale for the choice of substrate concentration for enzyme assays.
Finally, R2 provides an interesting manuscript that should be included in the current study as highly relevant for the topic.
Please take care of addressing these points and I will review your final manuscript before recommending.

Hide

ED: Publish subject to minor revisions (review by editor) (03 May 2023) by Sara Vicca (Co-editor-in-chief)

AR by Tom Sizmur on behalf of the Authors (16 May 2023) Author's response Author's tracked changes Manuscript

ED: Publish as is (17 May 2023) by Alberto Canarini

ED: Publish as is (18 May 2023) by Sara Vicca (Co-editor-in-chief)

AR by Tom Sizmur on behalf of the Authors (18 May 2023) Manuscript

Journal article(s) based on this preprint

16 Jun 2023

Differential temperature sensitivity of intracellular metabolic processes and extracellular soil enzyme activities

Adetunji Alex Adekanmbi, Laurence Dale, Liz Shaw, and Tom Sizmur

Biogeosciences, 20, 2207–2219, https://doi.org/10.5194/bg-20-2207-2023,https://doi.org/10.5194/bg-20-2207-2023, 2023

Short summary

Adetunji Alex Adekanmbi et al.

Supplement

https://doi.org/10.5194/egusphere-2022-1091-supplement

Data sets

Differential Temperature Sensitivity of Intracellular and Extracellular Soil Enzyme Activities Adetunji Alex Adekanmbi, Laurence Dale, Liz Shaw, and Tom Sizmur https://doi.org/10.17632/xvr3dzvdcw.1

Adetunji Alex Adekanmbi et al.

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The decomposition of soil organic matter and flux of carbon dioxide is expected to increase as temperatures rise. However, soil organic matter decomposition is a two step process whereby large molecules are first broken down outside microbial cells and then respired within microbial cells. We show here that these two steps are not equally sensitive to increases in soil temperature and that global warming may cause a shift in the rate limiting step from outside to inside the microbial cell.


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Differential Temperature Sensitivity of Intracellular and Extracellular Soil Enzyme Activities

Journal article(s) based on this preprint

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Interactive discussion

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