Effect of Charcoal on the Properties, Enzyme Activities and Microbial Diversity of Temperate Pine Forest Soils

Round 1

Reviewer 1 Report

This manuscript by Lasota et al. investigated the impact of RCHs on soil physical properties, C and nutrient content, enzymatic activities and microbial communities in a temperate pine forest. Two field experiments were conducted to compare soil and microbial properties between carbon-rich and carbon-free soils. The experiment design make sense to me, providing interesting and reliable data to the readers of Forests journal. In the context of global climate change, forest fire occurs more and more frequently, the post-fire charcoal plays a great role in forest biogeochemical cycling due to its unique morphological and chemical characteristics. Understanding how charcoal production modifies soil environment and interacts with bacteria and fungi are key to develop forest management/restoration strategy.  

Generally, the manuscript is well written and easy to follow. However, the discussion of the manuscript was largely descriptive and more in-depth analysis is needed. For example, apart from only discussing separately the influence of charcoal on soil properties, enzymatic activities and microbial community diversity index, is it possible to reveal the links among soil properties, enzyme activity and microbial diversity? Or is there any links? Is it likely that the changes in soil organic C or AP contents were triggered by associated soil enzyme? Whether these changes were also driven by some microbial taxa? If the missing links could be revealed and deeply discussed, I believe more value will be added to the manuscript. Please look at the specific comments for more details.

Major points:

• The enzyme you selected to measure in the study were related to soil C, N and P transformation respectively. Please clarify the function of each enzyme, maybe also their response to charcoal addition briefly in the Introduction part.
• Try structural Equation Model (SEM) to analyze the enzyme activity and soil properties (Table 5, Figure 6,7). It may be more visible than multiple regression.

Specific points:

• L22：Do you mean “water holding capacity”? Please revise it throughout the manuscript.
• L32: Please define “NGS” in the first appearance.
• Please add a brief introduction of the soil enzymes you selected to measure in this study.
• L337: Figure 4, please illustrate what “x axis” and “y axis” represent in the figure legend, and how you calculate the delta value.
• Is it likely to measure the average C content and pH in the charcoal sample itself? It looks massive in the picture.
• There are too many tables and figures in the text. Please move the less important ones to the supplementary.
• Figure 5 is not clear. Please edit the bar chart.

Overall, I recommend minor revisions for this manuscript.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

High level well structured contribution.

Some minimal additional explanations would be useful.

Lines 119-120

charcoal from burning improves the properties of forest soils, particularly organic carbon content and the content of macro- and micro-elements

This statement is strongly related to the methods of the C determination. C content can bee only the trace of the burning giving nothing to the soil.

Lines 122-123

charcoal modifies the soil environment, which may alter the composition and diversity of microbial communities

Yes. But can you suppose this change as positive for the soil biome, forest soil as a whole?

Line 123-124

charcoal-enriched soils release a higher amount of nutrients than soils that were not subjected to previous charcoal production

Yes. But the statement is a veiled proposal to burn the forest at the climax stage. There are methods of gasification of the non-commercial timber and thinning byproduct. Using this methodology, one will be capable to overcome the excessive CO₂ release, produce syngas (and methane as a next technological step), produce high quality fly ash, and provide the nutrients return to the soil.

The section 2. Materials and Methods and section 3 Results are of highest scientific level, well detailed, and beautifully illustrated.

Don’t you think that increment of the charcoal origin C content can distort the standard analytical procedures result as these procedures were developed for the charcoal free soil? I understand that you can’t use another procedure except standard, but it would be interesting to have your highly valuable expert opinion whether standard procedures are appropriate for the object you study, or we ought to take in mind some corrections in our conclusions in this case? The same DOM function of soil in the Table 6 forced me to put the question. If result is the same, maybe the substrates are of the close properties?

Line 576

charcoal mixed with humus accelerated the decomposition of humus compounds

Yes. But what kind of equipment of the current technological level can you recommend to produce an acceptable for soil biota mixture of biochar and soil for the silviculture practice? This is important concerning your beautiful Figure 3 where charcoal remained intact for centuries.

Lines 686-687

Charcoal from wood burning improved the properties of forest soils

Yes. But not only. For better effect, we ought to exclude eolian transportation of the charcoal as it is a carcinogenic substance, to provide well mixed intra-soil contact of the dispersed charcoal and the soil, to increase the soil layer depth, and to reduce the soil acidity. You can read about our Biogeosystem Technique methodology in: https://doi.org/10.1016/j.envres.2020.110605

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

I have read the manuscript “Effect of charcoal production on the properties, enzymes activities and microbial diversity of temperate pine forest soils” several times and, in my opinion, the paper is of great interest for the entire scientific community. The article is well-written and documented so it should be published and shared with the community. However, I have some small recommendations that will help improve the article. 

General comments:
1. The manuscript has one major weakness related to the English language and style. In my opinion, a moderate editing of English language and style are required. Some examples below.

2. 2. It will be useful to include in the introduction some data from the specialty literature (and from Poland) regarding the general characteristics of soils that are specifc to pine stands located in Europe's temperate areas. This should include the type of soils, pH and humus content for example. This addition would be of interest as the article already refers to them even though it studies a particular aspect of the soil.
3. My recommendation is to divide the results in separate sub-chapters (for Physico-chemical properties of RCH soils and Enzymes activities of RCH soils). This is needed as the two groups have totally different characteristics. The Discussion section has treated these categories separately (physical properties, chemical properties and enzymes) so this continuation will be of impact in the Result section as well. 
4. Figure number 8 mentions that “chemical properties (carbon, nitrogen and phosphorus content) were used in the design of the diagram”. For me it is unclear how you did this. Can you please detail shortly? 
5. In the Discussion section, Lune 531 you mention that “Differences in the physical properties (e.g. structure and water capacity) of soils were detected in the present study”. The results mention that “The charcoal-enriched horizon was thicker (18 to 25 cm), had a clearly developed (subangular blocky) structure”. My question is: how is the structure of charcoal-free soils? This should be mentioned in order for us to see the difference. 
6. In order to have similar subtitles in the Discussion section, please change “Influence of added charcoal on soil chemical properties” with “Influence of charcoal enrichment on soil chemical properties”

Specific comments:

Line 109  “no study has explored how the chemical properties of RCH soils influence microbial activity in this environment”.

A simple internet search reveals articles that have studied this problem, such as:

Gul, S., Whalen, J. K., Thomas, B. W., Sachdeva, V., & Deng, H. (2015). Physico-chemical properties and microbial responses in biochar-amended soils: mechanisms and future directions. Agriculture, Ecosystems & Environment, 206, 46-59.

Kolb, S. E., Fermanich, K. J., & Dornbush, M. E. (2009). Effect of charcoal quantity on microbial biomass and activity in temperate soils. Soil Science Society of America Journal, 73(4), 1173-1181.

Please change no with "a few studies have analysed...."

Usage of ”the”:

Line 14 Relict charcoal hearths (RCHs) in forest ecosystems affect the soil properties by increasing = affect soil properties

Line 21 Significant differences in the physical properties were found between these two soil types in terms = in physical properties

Line 283 The Mann-Whitney U test was used to compare the means between the different soil properties = between different soil properties

Phrases that is too long. Reconsider dividing them in more phrases that are easier to read:

Line 90-93 The most important biotic factor that shapes soil microbiome community assemblages is tree hosts, which are affected by complex interactions such as changes in the microclimate (e.g. temperature and moisture), litter and root exudate production, or by direct interactions with root-symbiotic and root- associated microorganisms [30, 17, 31].

= Tree hosts are the most important biotic factor…. They are affected by…

Line 264-268

To ensure full cross-compatibility of results during fungal identification using culture-based method and Illumina metabarcoding, all fungal OTUs identified in NGS analysis were manually queried against the NCBI GenBank database using BLASTn and corrected accordingly if necessary (using the same taxonomic threshold values previously described for identification using Sanger sequencing).

Line 30 Members of Rhodoplanes dominated the bacterial community at both RCH and non-RCH sites = Rhodoplanes Members or Members of the Rhodoplanes Genys

Line 49 They occur as small embankments 18–20 m in diameter and = embankments of 18-20 m

Line 152 Each sample consisted of 10 sub-samples of soil (each 100 g) stored in = 10 soil sub-samples

Line 230 The number of colony-forming units (CFUs) in every soil sample was determined. = was also determined?

Line 300 Figure 2. Soil profiles with charcoal (a) and soil profile without charcoal production (b). = singular or plural? Soil profiles for both or soil profile for both?

Line 345 Charcoal-enriched horizons showed significantly higher activity of specific enzymes = showed a significantly higher activity / significantly higher activities

Line 412 Members of the genus Oidiodendron (12 species) = of the Oibiodendron Genus

Line 622 However, results of the abundance of soil microorganisms varied between traditional = results for the abundance….

Author Response

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Author Response File: Author Response.docx