Fungal Methane Production Controlled by Oxygen Levels and Temperature

Round 1

Reviewer 1 Report

General comments:

This is a clear outline of work done to identify environmental variables which affect methane production in two species of fungi. The study is well-written and in general requires only minor revisions to be suitable for publication. However, the lack of some controls (any beech, and grass at 40°C) makes it difficult to confirm the biotic nature of CH₄ formation in some experiments. I would prefer that either a) all reference to experiments on beech are removed or b) control experiments are performed under the same conditions and added to the text.

Most of the figures are of poor resolution in this version, which makes it difficult to identify data points noted in the text.

I note L. Sulphureus was not incubated with grass – was there a reason for this not noted in the text?

For “author prominent” citations, the author’s name should be included (e.g. p1, line 39 “...was presented by [4]”, should be “...was presented by Keppler et al [4]”, and p1, line 10 “...the discovery by [10]” should be “...the discovery by Ernst et al [10]”). For ‘information prominent” citations the reference number alone is sufficient.

Specific comments:

p1, line 34: missing a period at the end.

p2, line 54: “...the global carbon recycling” does not need “the”.

p2, line 66: “...further highlight, that...” should be “further highlighted that”.

p2, line 74 and others: “xylothrophic” should be “xylotrophic”.

p3, line 103, and section 2.3: There seems to be a big jump between 21% and >90% O₂. Was there some reason behind this decision? It also would have been interesting if these results were included in Figure 5.

p3, line 119: What was the specific time? Did it vary between experiments?

p4, end of Table 1: Why was the grass control incubated at 27°C when there was no fungal equivalent? Why not at 40°C as with P. sapidus?

p5, p153: If there was not enough beech for controls, I am unsure of the value of carrying out incubation experiments with fungi on beech.

p5, lines 160-181: This seems to be mostly repetition of information in Table 1, and could be shortened to make this easier to read.

p6, figures 1 and 2, p7, figure 3: With no beech controls, it is difficult to confirm the biotic nature of CH₄ formation.

p7, figure 3: please add “at 27°C” (or equivalent) to the legend.

p8, lines 228-230: “Figure 4A” should be “Figures 4A and 4B”; “Figures 4B and 4C” should be “Figures 4C-4F”.

p8, line 234: The rate of CH₄ production appears to be highest at 40°C in Figure 4A, not 27°C.

p8, lines 240-256: There should also be a reference back to the >90 O₂ experiments here.

p9, figure 4: This figure is almost illegible.  Does the pink arrow refer to all the experiments? I assume the black arrow is only for the 40°C incubation for L. sulphureus in 4A, but both temperatures in 4C and 4E? Why are there no arrows on 4B, 4D and 4F?

p10, figure 5: Is this at the “end” of each experiment? What time points? Why not add the >90% O₂ data here?

p10, line 273: “e.g.,” is not needed here.

p10, lines 276-278: The references to Figures 1 and 4 should be after “CH₄ formation ceased”. From Figure 4, it looks like CH₄ formation pauses rather than ceases, but it is very difficult to tell the difference between the control and the fungal data points.

p11, line 304: This is unclear. Were the 1% and 10% figures from the two different studies? Any reasoning behind this difference?

p11, line 321: This sentence is unclear. I suggest “...the most likely explanation for the observed differences is the different enzyme sets of...”.

p12, line 335: Should this be Figure 4? D and F show O₂ not CH4_norm though. Or Figure 5B and C?

p12, line 373: Are T_max or T_opt known for these species?

p14, line 476: Seems to be missing something - “This GC was equipped with an 8; Shimadzu, Japan).”

p15, line 492: But in e.g. Figure 5, the classification is 0, <5 and 5-21%.

p15, line 518: Please revise the  wording of “and likely all fungi” – is there any evidence that this activity is found in non-xylotrophic fungi? Also, per se doesn’t fit here.

Supplementary Information specific comments:

Figure S1: Figure labels or legend are incorrect: ABC labelled as P. sapidus on pine, DEF labeled as P. sapidus on grass and GHI labelled as L. sulphureus on pine. The scale also makes it very difficult to determine individual data points/O₂ addition and removal.

p3, 3^rd paragraph: “...oak wood as well as grass, corn)...” should be “...grass and corn”.

p3, 3^rd paragraph: There is an extra “this” at the end of the paragraph

p3, 3^rd paragraph: “..strong potential to contribute to e.g., to the carbon...” does not need “to e.g.,”

Figure S2: ABC – Where is the control at 27°C? I can only a single point at the start of the CH₄ graph.

Figure S2: DEF – was the 40°C experiment stopped much earlier than the 17°C one? If so, why?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript presents a study that investigates the impacts of O2 levels and temperature on aerobic methane production by saprotrophic fungi. The authors present evidence of O2-dependent CH4 production. 

Comments:

Please indicate (state in the main text) the observed "control" medium levels of methane production for all levels of O2 and T tested.

Is there any evidence of methanol or halomethane production, which could be expected for the condition tested? 

Can any correlations be made between the growth "material" composition and methane production rates? 

Specific comments:

Table 1:

beech 4: O2 mixing ratios are described as 0 to 26, but data for 5 to 20.9% O2 are provided. Is that correct?

Why were only grass experiments run at 40^oC, not beech and pine? 

Please include control medium CH4 production rates for each experimental condition for comparisons. 

Figure 2: 

L.sulphureus data: Why is O2 consumption observed for 5-20% O2 samples but not observed for 10% O2 samples? Please explain differences in O2 ranges presented/ selected for the demonstration. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

This manuscript described a study exploring the influence of oxygen (O₂) levels and temperature (17, 27, and 40°C) on methane (CH₄) production by two fungi, Laetiporus sulphureus and Pleurotus sapidus. It is interesting to observe that methane formation was linked with the high O₂ levels. The authors tested the methane production under different O₂levels and different temperatures for the two fungi. The findings are presented clearly, and the discussion offers some explanations for the results. However, the absence of metabolic mechanism analysis weakens the persuasiveness of the results. The ambiguous experimental design is also perplexing and requires refinement. To enhance the discussion, it should include more detailed and thorough exploration. Below are my suggestions for improvement.

1.     The first keyword repeats with the second one. 

2.     Serval bullets of highlights that summarized the key results and contributions of this study were recommended. Highlights can help to increase the visibility of the manuscript. 

3.     There are abundant citations in the introduction section. Some of them can be reduced. Some can be explained with more details rather than a simple list of citations (e.g. Line 56, Line 63). Please also give the author information when needed (e.g. Line 59), this should also be checked in the discussion section.

4.     I suggest moving the materials and methods section to the front after the Introduction section. A clear experimental design should be added to the materials and methods section. Table 1 is too complicated and confusing. It should be simplified. In addition, some values in column O₂ range (%) seem to be inconsistent with the column of O₂ mixing ratio. Please check. The table caption is too long. Some information can be added to the table notes.

5.     What does data of O₂ mixing ratio and O₂ range (%) mean, 0 to 26.0 for example?

6.     Figures in the manuscript are very vague. The quality of figures should be improved. It is encouraged to use the original figures.

7.     The negative values in Figure 2 should be explained. 

8.     There are only four data points in Figure 3 (B). It is suggested to have more data points. In addition, there are two O₂ levels indicated in the figure caption (“incubation starting at 98% and 95% O2, respectively). However, there is only one data line of methane production and O₂ mixing ratio. If they are the conditions for the two fungi, please add the information to the figure.

9.     A metabolic mechanism analysis is suggested to give a more supportive discussion or justification to the results.

10.  Lines 277-279. This is interesting. But the authors should give more details about the reference information and more clear explanations.

11.  Could it be possible that bacteria and archaea proliferated during the incubation experiment due to insufficiently stringent sterile conditions? If such concerns exist, analysis experiment like a 16s rRNA sequencing of the media may help to provide stronger support for the experimental results of this study.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Dear Authors,

The manuscript ID: ISSN 2674-0389, entitled "Fungal Methane Production Controlled by Oxygen Levels and Temperature" aims to study the effect of the O₂ and temperature on CH₄ production by two fungi, Laetiporus sulphureus, and Pleurotus sapidus. Overall, this study is highly interesting, valuable, and aligns well with the scope of the Methane journal. However, some minor revisions are required before the manuscript can be published.

Abstract

Please include the question addressed in a broad context and highlight the purpose of the study.

Introduction

Line 47: Please define ROS.

Results

Line 143-145: The authors mention “the amount of O₂ initially increased (from 0.05 ± 0.03 to 16.8 ± 0.7 mmol) while that of CH₄ decreased (from 240.0 ± 144 7.8 to 190.3 ± 7 nmol), a result of the dilution effect from the supplemented gas volume.” 
Since the authors are measuring the gas in moles, the dilution effect doesn't seem to make sense. Can the authors explain these results better?

Line 151-152: Here is the same situation. Please revise this information.  

Materials and Methods

Section 4.2.: What was the working volume and the volume of inoculum used? Please include this information.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I appreciate the time the authors have taken to respond to my feedback and improve the manuscript. I believe the text is now clearer, particularly with reference to why beech controls were not performed outlined in the discussion. The higher resolution (or splitting) of the figures has also improved clarity.

One minor issue - my comments appear to have been changed from standard text to italics and therefore my comments on citations look incorrect. Citations should be First author (regular) et al (italics), not the other way around.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf