Do composted bioamendments enhance the resistance of Mediterranean agricultural soils and their microbial carbon use efficiency to extreme heat-stress events?

Boubehziz, Sana; Cooledge, Emily C.; Chadwick, David Robert; Barrón, Vidal; Sánchez-Rodríguez, Antonio Rafael; Jones, Davey Leonard

doi:https://doi.org/10.5194/egusphere-2025-2592

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https://doi.org/10.5194/egusphere-2025-2592

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18 Jun 2025

| 18 Jun 2025

Status: this preprint is open for discussion and under review for SOIL (SOIL).

Do composted bioamendments enhance the resistance of Mediterranean agricultural soils and their microbial carbon use efficiency to extreme heat-stress events?

Sana Boubehziz, Emily C. Cooledge, David Robert Chadwick, Vidal Barrón, Antonio Rafael Sánchez-Rodríguez, and Davey Leonard Jones

Abstract. Mediterranean agroecosystems are vulnerable to extreme heat-stress, especially because of their low organic matter content. Bioamendments may enhance soil nutrient content and microbial resilience to heatwaves. However, their effectiveness under these conditions is still unclear. We investigated the effect of bioamendments (composted olive mill pomace, biosolids and solid urban residue) and a conventional fertiliser (diammonium phosphate) on microbial carbon use efficiency (CUE), and soil biogeochemistry in two different soils, a calcareous Vertisol and a non-calcareous Inceptisol, with low P availability, subjected to extreme heat-stress. We conducted warming experiments (20, 30, 40, or 50 °C), to monitor ¹⁴C-glucose mineralization and to evaluate modifications in soil biochemical properties. As result of warming, both soils microorganisms exhibited thermotolerance until 40 °C, with a critical shift in microbial respiration observed at 50 °C. Consequently, microbial CUE, which was a function of the bioamendments and soil, significantly declined from 0.47–0.65 at 20 °C to 0.27–0.45 at 50 °C (p < 0.05), with the control decreasing by 0.010 ± 0.001 °C^-1 (Vertisol) and 0.007 ± 0.001 °C^-1 (Inceptisol). Moreover, composted olive mill pomace-treated soils enhanced the resistance of soils to heat stress as they produced the highest microbial CUE at 40 °C in the Inceptisol and 50 °C in both soils (0.43 ± 0.02 Inceptisol vs. 0.45 ± 0.02 Vertisol). Soil biogeochemistry varied with temperature and treatment, while available P in soils treated with diammonium phosphate was reduced with temperature in both soils, and available P added with bioamendments was not affected by temperature but was increased with biosolids for all temperatures in the Inceptisol. In conclusion, organic matter rich bioamendments (composted olive mill pomace) may enhance the resistance of Mediterranean agricultural soils subjected to extreme heat-stress events (50 °C).

Received: 04 Jun 2025 – Discussion started: 18 Jun 2025

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Sana Boubehziz, Emily C. Cooledge, David Robert Chadwick, Vidal Barrón, Antonio Rafael Sánchez-Rodríguez, and Davey Leonard Jones

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RC1:
'Comment on egusphere-2025-2592', Anonymous Referee #1, 30 Jul 2025 reply
General
The manuscript is about the effect of a high soil temperature for a certain period on microbial activity (14CO2 from glucose) and microbial carbon use efficiency (14C growth/ 14C uptake). The hypothesis -I think- is that heat results in a lower CUE, and that balanced nutrient supply or organic amendments decrease the magnitude of the lowering of the CUE. If CUE is still high at 50 C, the authors assume that the soils has a high resistance to heat. Two soils are tested and three amendments. The type of microbes in the soils were not determined.
Specific comments
The introduction is not always logical, and reading the text still gives many small questions which is unnecessary. I wonder if the method is correct. CO2 can be precipitated by Ca and Mg rich materials at certain CO2 concentrations. The vertisols but also the composts probably contain carbonates. Should this be tested with a dead soil? For readers not familiar with CUE, the calculation should be given. Is it relevant that the microbial life has not been determined?

Minor comments
Title: I guess “resistance” is an interpretation of the CUE at various circumstances. So the title should be more like …..resistance derived from microbial CUE measurements…
Writing only about western Mediterranean region is strange, it also seems relevant for other regions in the world.

53-53. This does not seem relevant for this manuscript.
59 “poor” suggests that you have a opinion, which is strange if it is a natural state. I guess you mean low.
61”negative impact”, similar comment. Being calcareous is a state, it is not intervention which has an impact: it is the just the state of these soils which is problematic for certain crops.
59-63 I do not agree with the statement: “…. that calcareous soil … often lead to a negative impact on soil fertility and plant productivity”. Impact is a strange word for a state of a soil, but also the idea that calcareous soils give low plant productivity is not correct: there is potential for negative effects. However, the productivity of calcareous soils is high when sufficient water and nutrient are applied (as is true for most soils). Maybe you mean alkaline soils, or the vertisols, when you want to talk about problematic soils.
63 50 degrees Celsius is not that high for a barren dry arable soil. On a global scale many parts of the land have higher temperatures than air temperature. https://doi.org/10.1029/2010JG001486
70 Many authors have studied organic matter mineralisation, for example Kirby et al. showed an effect of nutrient on CO2 loss in a incubation experiment. https://doi.org/10.1016/j.soilbio.2013.09.032. In this paper the focus is on CUE. Please introduce this specific aspect. Why is it better or different?
77 “most”, do you mean “these”? Or do you mean other studies, then you should mention the other studies.
78-79 “such as calcareous soil … in regions?” Why not directly mention that vertisols and alfisols are specifically challenging?
81 and 84 “such as available P, further potentially deceasing CUE……23-24% reduction in CUE due to DAP and SSP..” Both sentences do not agree with each other. Does additional P increase or decrease CUE or do you mean that it is probably more complex?
97 “little is known about their impact on CUE” Is that true, not at a first glance. https://doi.org/10.1186/s13213-024-01780-9, https://doi.org/10.1007/s42832-022-0137-3, https://doi.org/10.7717/peerj.12131, https://doi.org/10.1016/j.soilbio.2024.109531. So, please be more precise.
103 Earlier you mentioned that the calcareous soils were problematic for phosphorous, and now you include a non-calcareous soil. Please explain you choices: for example you chose soils with a low P availability.
104 “soil biogeochemistry”. Please write more precise: you do not study soil biogeochemistry, you have determined P-Olsen and extractable N.
106 “than conventional fertiliser”. This does not seem a fair comparison. If so, then you should add similar amount of nutrients using various mineral fertilisers, including micronutrient. Conventional fertiliser is not a very good term: in many countries this is animal manure, in other mineral fertiliser. So mineral fertiliser is a better term.
In most studies authors use a simple fertilisation advice for the soils, and choosing different fertilisers.
In the current research you might have deficiencies for N, Mg, S etc. By using soils from farms, you probably use well fertilised soils, without deficiencies, at least not in micronutrients.
108 “a more buffered”. This is in contrast to line 79 were you state that the vertisols are prone to high moisture and reduced oxygen.
145-150 Did you make batches of soil+amendment mixtures, and did you sample these mixtures for experiment 1-3? The text does not explain how you did this. This is relevant as these amendments have a structure (compost contain large particles, and the phosphate minerals are also particles). If you sample 2,5 gram soil (mixture of soil+amendment), then the samples are probably heterogeneous. This might explain the large variance in figure 2. Also in figure 3 there is a large variation for soluble N in the treatment with DAP although in every soil you have added the same amount of N+P. It is rather problematic that the variation is so large, when you expect very similar results.
150 It seems like a small amount of water: 0.18 gram water per gram dry material? Normally soils are wetted until a certain percentage of water filled pore space: ±70% of the maximum, to have a good circumstances for plant roots.
190 Soils seem deficient in zinc according to these measurements.
197 data in table 2 are strange for “volatile solid content” or “oxidable organic carbon”. How is it possible that the volatile solid content (proxi for organic matter, and water to clays) is lower than organic carbon? You would expect a factor 2 between both.
200 Can ¹⁴CO2 might also be precipitated as CaCO3? For example:. https://doi.org/10.1016/S0168-1923(02)00231-9. So how sure are you of the measurements? Has it been tested in dead soil?
200-205 Rather unclear how you derive CUE.
209, 238, 250: Experiment 1, 2 and 3 have been performed in different tubes:
1: 2.5 gram soil, 0.2 ml water, 50 ml tube, 0.25 ml labelled glucose.
2: 2.5 gram soil, 0.2 ml water, in a 1,5 ml (?) Eppendorf tube. How does this fit?
3:2.5 gram soil, 0.2 ml water in 50 ml tube, 0.25 ml labelled glucose.
Unclear are the effects of the differences. Unclear: are the tubes closed from air, or open? Does the soil dry out during the 27 days of having a 1 M NaOH trap on top of it?
I would not use the word “soil” here, when you mean a mixture of soil+amendment. A reader expects that you add the amendment afterwards when he/she reads “soil”.
2.5.2: In experiment 2: pH, mineral N, and P Olsen were determined. Did you do this on this small 2.5 gram sample? How?
263-275 Please give calculation of CUE in the methods.
357-364 So nitrification is rather slow. One would expect that all NH4 would be transformed into NO3 after so many days, at least for DAP.
425 “not many”, if so then you should mention these few studies.
440 Do you add microbial live to a sample by adding compost? Or is that negligible to soil?
445 Strange. You Spanish soils have of course been adapted to 50°C. You have given the temperatures, and temperatures of soils are often much higher than air temperatures. Otherwise you should have chosen soils from Scandinavia or some other region without warm summers/sun.
498 “…. Especially DAP, lead to … NH4… this reflects either increased mineralization of organic N or….”. That seems to make no sense. You add NH4 with DAP (NH4 and HPO4), so you do not need a biological process to find NH4.
528 “vulnerability”. I wonder if you can state this on the basis of your two soils.
570-575. That is a rather unrealistic conclusion: the availability of compost per hectare in the EU is very small compared to potential need. It is probably much easier to keep the soil covered with crops, being cover crops or crops.

Reply
Citation: https://doi.org/10.5194/egusphere-2025-2592-RC1
- AC1: 'Reply on RC1', Sana Boubehziz, 13 Aug 2025 reply
  
  Dear reviewer,
  
  On behalf of all the co-authors, I would like to thank you for revising our paper. Your constructive comments have helped us to improve the clarity of the manuscript and the objectives that we wish to convey. We have attached our responses to your comments.
  We hope that this version of the manuscript meets your expectations. Please let us know if you require anything else.
  Best regards,
  Corresponding author
  
  Reply
  
  Citation: https://doi.org/10.5194/egusphere-2025-2592-AC1
RC2: 'Comment on egusphere-2025-2592', Anonymous Referee #2, 03 Sep 2025 reply

General assessment:

This study investigates the effect of bioamendments and heat stress in two soil types on microbial respiration and carbon use efficiency. This is a very relevant topic, and fits well the scope of SOIL. I have, however, strong reservations about the quality of the study.

Overall, I find the introduction and methods hard to read. The introduction is overall a bit confused, and does not clearly frame the interplay between heat stress and amendments in affecting the carbon cycle. The part on microbial adaptation mixes causes and consequences (see detailed comments below). Most importantly, there is no clear or accurate description of what CUE is, how it is conceptualised across scales and also methods, and finally more precisely how it contributes to ecosystem C cycling. The method section is also overall unclear because some elements are mentioned in passing before being explained. Quite a few methodological details are missing (as listed in the detailed comments below), including the calculation method for CUE.

Most importantly, I have concerns about the validity of the method used. The choice of 14C glucose addition is interesting here as a standardised assay to quantify CUE, since the treatments include amendments containing carbon in different forms (different C:N), whose incorporation into microbial biomass probably differ from that of glucose. This choice could potentially be justified, but it needs careful explanation and a clear description of what can be concluded about the c cycle from it in the context of this study with different amendments, both in introduction and discussion.

If 14C glucose as a general method could be, perhaps, justified, different incubation times for different treatments constitutes a methodological bias. It is clearly stated that different treatments were subject to different incubation times. Incubation time (after which 14C remaining into the soil was measured, which I assume was used to estimate incorporation of 14C into microbial biomass) appears to be based on the time it takes for CO2 emission rates to stabilised, which expectedly differed between temperature treatments. In my sense, this does not allow comparison of CUE in the different temperature treatments, thus providing a biased method to address the key question of understanding the impact of heat stress on CUE. This is because incubation time in substrate incorporation methods to calculate CUE determines largely how CUE can be conceptualised, with increasing incubation time increasing the chances of added inputs being exuded, turned-over or maintenance respiration, rather than contributing to growth. If not accounted for, these processes can lead to overestimations of the fraction of substrate assimilated into from the classical equation:

CUE = 14C biomass / (14C biomass + 14 respired).

I recommend reading Geyer et al. (2016) (DOI:10.1007/s10533-016-0191-y) to shed light on how incubation times impacts not only results, but also conceptualisation of CUE.

Due to this lack of clear framing, and particularly of partly inadequate methodology, I cannot recommend publication.

Detailed comments:

Can only Line 50-53; Line 6: Syntax errors

L66-70: The formulations are a bit inaccurate here, and I have issues with the concepts. 1. “Consequently”, line 67; the death or dormancy, and the change in composition are responses of the community that partly define adaptation, not the cause of adaptation; death does not trigger a shift in metabolism, it IS a pretty dramatic shift in metabolism… high temperature is the cause of all that (death, dormancy, shift in metabolism and adaptation)

2. Also, “shift in metabolism to facilitate thermal adaptation”… I think a shift in metabolism is a form of adaptation (acclimation perhaps) itself, like species turnover and de novo genetic mutations. I recommend reading this Bradford (2013): https://doi.org/10.3389/fmicb.2013.00333

3. “inadvertently” is not the right word.

4. increasing OM mineralisation, deleting soil C stocks and reducing CUE… it sounds like all those would be the direct consequence of an increased respiration in the remaining thermotolerant species. I think this is a large oversimplification. It needs to be laid out how increased mineralisation and decreased CUE may contribute to decrease C stocks, and in which condition would this lead to a decrease in C stocks (with respect to plant C inputs particularly).

L70-74: how does the fact that CUE matters to C cycling and is sensitive to various factors justifies the need to understand resilience? We want to know specifically how resilience relates to soil C cycling, and how understanding CUE’s response to drought and temperature is critical, because of this role in C cycling, to understand resilience…

L78: “challenging CUE conditions”… what are those? It was not mentioned before that calcareous soils have low CUE or why.

L81: we need a ref to justify that low P availability would decrease CUE.

L82: compost application is a fairly common practice that is absolutely not unique to “organic agricultural practices”.

METHODS

L200-201: suddenly 14C is mentioned. I am not sure I understand here. The biosolids are obtained from commercial sources, so I guess they are not labelled with 14C. So how would one determine how much 14C from the biosolid has been incorporated into microbial biomass? Or is this using the natural abundance of 14C? but 14C as natural abundance is only useful to date centennial or millennial C, not the incorporation of new inputs into microbes which takes place over a few days to months…

L202: what “monitoring period”? What “each experiment”? does this refer to each treatment (combination soil type/amendment)? Or each of the experiments numbered later ?

L209: I am confused here: n=5, but further up (line 143): n=4. From the 4 times 100g prepared for each combination of bioamendment/soil type (40 pots in total: 2 soils x 5 bioamendments including no addition x 4 reps), line 143, how do we get to 5 replicates of bioamendment/soil type/temperature combinations?

L214: now I get it! 14C labelled glucose… so a glucose incorporation method is used as a standardised assay to quantify CUE.

L225: so when is incorporation into microbial biomass quantified? I suppose the extraction with NaCl extracts what is not in microbial biomass or respired? So one would need to deduce that from what is added originally and what is recovered as 14CO2 cumulatively to deduce what is in microbial biomass?

L239: In experiment 1, 7 days at the different temperatures, including only 5 in the presence of 14C glucose. Ok; but why 9 days in experiment 2?

L254: and now it’s a week of heating. But how long are soils at 20oC before 14C glucose addition?

L256-257: as I suspected line 224-225, microbial biomass C of microbial biomass 14C were never measured, only the remaining 14C in the total soil is quantified. Explanations about how this is used to calculate CUE are needed! It says here “As described above” but I can’t find the calculations/equations anywhere.

L258-260: Now I am a bit confused. L256, it is implied that all treatments are incubated for 16 days before extraction for remaining 14C. For experiment 1, lines 217-220, it is indeed indicated that incubation time difference between temperature treatments. So this seems to apply only to experiment 1. I think diverging incubation time for calculating CUE based on glucose incorporation are hugely problematic, as described in the general comment, and I question the validity of the approach to conclude anything g about the effect of temperature on CUE.

Reply

Citation: https://doi.org/10.5194/egusphere-2025-2592-RC2

Sana Boubehziz, Emily C. Cooledge, David Robert Chadwick, Vidal Barrón, Antonio Rafael Sánchez-Rodríguez, and Davey Leonard Jones

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https://doi.org/10.5194/egusphere-2025-2592-supplement

Sana Boubehziz, Emily C. Cooledge, David Robert Chadwick, Vidal Barrón, Antonio Rafael Sánchez-Rodríguez, and Davey Leonard Jones

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Short summary

Alterations in microbial carbon use efficiency and respiration were evaluated at different temperatures (20 to 50 °C) after adding ¹⁴C-glucose to two Mediterranean soil types (Inceptisol and Vertisol) when fertilized with bioamendments (as P source). The findings proved that the soils show resistance to high temperature (up to 40 °C), which decreased at 50 °C. The bioamendments rich in organic matter helped in increasing this resistance (maintaining microbial carbon use efficiency).


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