Comparison of Biochar- and Lime-Adjusted pH Changes in N2O Emissions and Associated Microbial Communities in a Tropical Tea Plantation Soil
Round 1
Reviewer 1 Report
This work investigates alleviation of soil acification via addition of biochar or lime. The study focuses on acidic tea plantation soil, which is treated with either nothing, biochar or lime. Both lime and biochar resulted in decreased N2O emission but biochar did so to a higher extent. The treatments also decreased the abundance of certain genes involved in nitrification/denitrification and increased others. The findings show that fungal detrifiers were the most important predictor of N2O emissions.
The topic is important because N2O is a greenhouse gas. Soils of tea plantations are acidic and extensive N fertilization leads to high N2O emissions. The introduction provided an appropriate introduction to the topic. The discussion of the different reactions catalyzed by the enzymes coded by the various genes discussed AOA, AOB, NirK, NirS etc. would benefit from showing the chemical equations for each of these enzymes.
Both biochar and lime resulted in increased soil pH values and the pH changed over the course of the experiment. They should review literature where pH was changed via other mechanisms to see how specific the effects they see are to the specific amendments biochar and lime versus general effects of increased soil pH. This issue should be discussed, and references provided.
The caption for Figure 6 has the following: Based on the maximum likelihood phylogenetic tree of representative and reference sequences in 25 Fungi-nirK OTUs and NCBI databases. This needs to be converted to a complete sentence. Check that all your sentences are clear and complete.
The findings are appropriately interpreted but the English language must be significantly improved. Without giving specific examples, there are extensive errors in grammar, spelling, verb/noun agreement and many others. It is important to have the text revised for language.
Author Response
1、This work investigates alleviation of soil acidification via addition of biochar or lime. The study focuses on acidic tea plantation soil, which is treated with either nothing, biochar or lime. Both lime and biochar resulted in decreased N2O emission but biochar did so to a higher extent. The treatments also decreased the abundance of certain genes involved in nitrification/denitrification and increased others. The findings show that fungal detrifiers were the most important predictor of N2O emissions.
Answer: Thank the reviewers for their recognition of this study
2、The topic is important because N2O is a greenhouse gas. Soils of tea plantations are acidic and extensive N fertilization leads to high N2O emissions. The introduction provided an appropriate introduction to the topic. The discussion of the different reactions catalyzed by the enzymes coded by the various genes discussed AOA, AOB, NirK, NirS etc. would benefit from showing the chemical equations for each of these enzymes.
Answer: Thank the reviewers for their recognition of this study
3、Both biochar and lime resulted in increased soil pH values and the pH changed over the course of the experiment. They should review literature where pH was changed via other mechanisms to see how specific the effects they see are to the specific amendments biochar and lime versus general effects of increased soil pH. This issue should be discussed, and references provided.
Answer: Thank you for your suggestion. Other method that using NaOH solutions to increase the soil pH also influence soil nitrification and associated microorganisms (Wang et al., 2019). However, this study aims to compare the effects of lime and biochar amendments on N2O emissions and potential microbial mechanisms in a tropical tea plantation soil. Specifically, we aim to (1) compare the effects of lime and biochar amendment on N2O emissions in an acidic tropical tea soil; (2) assess the effects of biochar and lime amendment on ammonia oxidizers and bacterial and fungal denitrifiers communities; (3) identify the dominant drivers leading to differences of N2O emissions between biochar and lime treatments. Therefore, in our opinion, it is unnecessary to compare the impacts of biochar and lime amendments versus NaOH solutions on N2O emissions and associated microbial communities.
References: Wang, Z.; Meng, Y.; Zhu-Barker, X.; He, X.; Horwath, W.R.; Luo, H.; Zhao Y.; Jiang, X. Responses of nitrification and ammonia oxidizers to a range of background and adjusted pH in purple soils. Geoderma, 2019, 334, 9-14.
4、The caption for Figure 6 has the following: Based on the maximum likelihood phylogenetic tree of representative and reference sequences in 25 Fungi-nirK OTUs and NCBI databases. This needs to be converted to a complete sentence. Check that all your sentences are clear and complete.
Answer: Thank you for your suggestion. We revised it as “Maximum likelihood phylogenetic tree depicting the top 25 Fungi nirK OTUs, along with relevant reference sequences from NCBI databases.” Line 332-333.
5、The findings are appropriately interpreted but the English language must be significantly improved. Without giving specific examples, there are extensive errors in grammar, spelling, verb/noun agreement and many others. It is important to have the text revised for language.
Answer: Thank you for your suggestion. We have sent our manuscript to a language polishing company for revising English grammar.
Reviewer 2 Report
- There is excessive use of abbreviations in the abstract
- What were the actual fungal species under the Ascomycota?
- The soil sampling strategy and design is not clear. what representative area
under the tea plantation was sampled? How many replicate sampling sites? Were the individual soil samples taken for analysis or mixed samples from replicate sites?
- Some more details on CO2 and N2O gas sampling and analysis are required. How were the samples taken? What was involved in destructive
sampling of the soils? What data was taken in chromatograph analysis of
the gases?
- The data on microbial DNA content of the soils should have been converted to the actual microbial density in the soils rather than reporting the data directly as DNA or bacterial gene content only.
- The figures are too clouded and too small in size and require better presentation
Comments for author File: 
Comments.pdf
Author Response
1、There is excessive use of abbreviations in the abstract
Answer: Thank you for your suggestion. We have modified according to the reviewer's requirements.
2、What were the actual fungal species under the Ascomycota?
Answer: According to high throughput sequence analysis results, Ascomycota were the dominant fungal denitrifiers belong to the families Nectriaceae, Aspergillaceae, and Chaetomiaceae, and Chaetomium, Penicillium, and Fusarium were the dominant genera regulating N2O emissions. Line 26-28.
3、The soil sampling strategy and design is not clear. what representative area under the tea plantation was sampled? How many replicate sampling sites? Were the individual soil samples taken for analysis or mixed samples from replicate sites?
Answer: Ten surface layer soils (0–15 cm) were taken and mixed into a composite sample. Field moisture soil samples were sieved (< 2 mm) and kept under 4 ℃ for incubation experiment within a week. Line 125-127.
4、Some more details on CO2 and N2O gas sampling and analysis are required. How were the samples taken? What was involved in destructive sampling of the soils? What data was taken in chromatograph analysis of the gases?
Answer: Before gas sampling, the bottles were ventilated to ensure gas exchange between air and bottle inside. After ventilation, gas samples were collected from the bottle to considered as the blank control. After that, bottles were sealed with rubber stoppers and incubated for 6 h, and then gas samples were taken using a sterile syringe. We measured the concentrations of CO2 and N2O using a gas chromatograph (Agilent 7890A, USA) which was equipped with a flame ionization detector and an electron capture detector. Line 245-252.
5、The data on microbial DNA content of the soils should have been converted to the actual microbial density in the soils rather than reporting the data directly as DNA or bacterial gene content only.
Answer: We revised it as “Total soil DNA used for qPCR was also used for amplification fungal nirK gene using the the barcoded primers. Thermal cycling consisted of an initial denaturation at 95℃ for 30 seconds, followed by 30 cycles of denaturation at 95℃ for 30 seconds, annealing at 55℃ for 30 seconds, and elongation at 72℃ for 30 seconds, with a final elongation step at 72℃ for 7 minutes. The PCR products were purified using the Qiagen Gel Extraction Kit (Qiagen, Germany) and quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). The purified PCR products were mixed in equimolar concentrations and sent to a biotech company and sequenced using the Illumina Miseq platform at the Shenzhen Micro Ecological Technology Co., Ltd., Shenzhen, China. Raw sequences were demultiplexed and quality filtered with QIIME 1.9.1. Low-quality reads < 200 bp in length or reads with ambiguous bases were discarded. Chimeras were subsequently removed using UCHIME 4.3. Remaining reads were grouped into operational taxonomic units (OTUs) at 3% dissimilarity using UPARSE. Representative OTU sequences of fungal nirK OTUs were identified using BLAST in NCBI database. A maximum likelihood phylogenetic tree of the fungal nirK gene was conducted using MEGA 11.0 software with 1000 bootstrap replicates. All sequences have been submitted to the NCBI database under the accession number PRJNA894784.” Line 169-185
6、The figures are too clouded and too small in size and require better presentation
Answer: Thank you for your suggestion. We have modified according to the reviewer's requirements.
Reviewer 3 Report
The article, entitled 'Comparison of biochar- and lime-adjusted pH changes in N2O emissions and associated microbial communities in a tropical tea plantation soil', concerns an experiment using biochar or lime as a factor to influence soil properties and the activity and diversity of the soil microbiome. The authors present results from an experiment using microcosms in which they investigate both N2O, carbon dioxide emissions, the frequency of genes involved in nitrogen cycling and the diversity of fungi present in the soil after the experiment. This is a well-written paper, very nicely illustrated (consistency in choice of style throughout the article). This makes the paper easy to read. In my opinion, due to the subject matter covered, this is a paper worthy of publication in the journal Agronomy. Nevertheless, due to my role as a reviewer, I have to make a few minor comments on the content of the article:
Abbreviations should not be used in the abstract. Therefore, the authors should explain the abbreviations: CK and AOB. There is a redundant dot preceding the N2O formula in line 14. It seems to me that it would be worth considering enriching the introduction with a diagram illustrating the nitrogen transformations discussed in lines 46-59. This would make it easier to read and understand for nonfamiliar readers. Figure 6 is poorly legible, the font is too small (also for the legend). It might be worthwhile, in the case of figure S3, to limit it only to diagrams showing significant relationships (using the relevant p-value or strength of the relationship as a criterion).
Nevertheless, after these changes the article could be accepted for publication.
Author Response
1、The article, entitled 'Comparison of biochar- and lime-adjusted pH changes in N2O emissions and associated microbial communities in a tropical tea plantation soil', concerns an experiment using biochar or lime as a factor to influence soil properties and the activity and diversity of the soil microbiome. The authors present results from an experiment using microcosms in which they investigate both N2O, carbon dioxide emissions, the frequency of genes involved in nitrogen cycling and the diversity of fungi present in the soil after the experiment. This is a well-written paper, very nicely illustrated (consistency in choice of style throughout the article). This makes the paper easy to read. In my opinion, due to the subject matter covered, this is a paper worthy of publication in the journal Agronomy. Nevertheless, due to my role as a reviewer, I have to make a few minor comments on the content of the article: Abbreviations should not be used in the abstract. Therefore, the authors should explain the abbreviations: CK and AOB.
Answer: Thank you for your suggestion. We have modified according to the reviewer's requirements.
2、There is a redundant dot preceding the N2O formula in line 14.
Answer: Delete “dot”.
3、It seems to me that it would be worth considering enriching the introduction with a diagram illustrating the nitrogen transformations discussed in lines 46-59. This would make it easier to read and understand for nonfamiliar readers.
Answer: This is a good suggestion. In the introduction, we explained the nitrogen transformation and mechanisms of N2O emission by ammonia oxidizers. Thank you for your suggestion.
4、Figure 6 is poorly legible, the font is too small (also for the legend). It might be worthwhile, in the case of figure S3, to limit it only to diagrams showing significant relationships (using the relevant p-value or strength of the relationship as a criterion).
Answer: Thank you for your suggestion. We have modified according to the reviewer's requirements.
5、Nevertheless, after these changes the article could be accepted for publication.
Answer: Thank the reviewers for their recognition of this study. We have modified according to the reviewer's requirements.
