Interactive comment on “Distinct microbial composition and functions in an underground high-temperature hot spring at different depths”

Anonymous Referee #1 Received and published: 18 December 2019 This manuscript mainly reported the microbial diversity and functions of three hot springs water samples collected from different depths. The authors found that both bacterial and archaeal community compositions were different along depth, and distinct microbial functions may occur at different depths of hot springs. This study has some merit, because few study focused on surveying the microbial diversity and functions in the subsurface of geothermal systems, which deserves publication. But the authors may need to revise the manuscript thoroughly on its grammar and structure before the ms is acceptable. Some examples were listed below but they are not exhaustive.

1. The main conclusion of this study is that the microbial communities were different at different depths. Most figures were used to indicate how similar or different the C1 three samples were. This conclusion is too vague to deliver useful information from these valuable datasets. More thinking about the results are needed to obtain more specific findings. There was some discussion on specific microbes found in extreme environments from previous studies in the 'discussion' section. However, searching the 16S data in this study for microbial species which were expected in hot springs or analyzing the most abundant species to see whether they were previously recognized as hot spring specialists could make the discussion more solid. 3. Microbial function potential was inferred from both 16S data via FAPROTAX and GeoChip 5.0. There was not any comparison between results from these two methods in the manuscript. Why two different methods were used? Did they tell the same story?
If not, what is the reason? Is there any information we could get from one but not the other?
Detailed comments: Abstract: went straight into the methods without any background information. Please C2 start with the motivation of the study.
Line 200-201 and Line 360-363: the logic seems to be that the environmental conditions at three depths were similar, but microbial communities were different. You cannot say that the environmental conditions were similar based only on two parameters (pH and temperature). How about other important parameters such as oxygen and nutrient concentrations?
Line 239-240: based on Fig 4B, : is 'uncultured archaeon' in the legend supposed to be 'uncultured Desulfurococcales archaeon' as mentioned in the text? The text kept saying uncultured Desulfurococcales archaeon, but it does not even exist in the legend of Fig 4B. If 'uncultured archaeon' is actually 'uncultured Desulfurococcales archaeon' which doesn't belong to Thaumarchaeota, how could it be possible that more than 90% of archaea were from Thaumarchaeota phylum at 19 m ( Fig 4A) and more than 50% of archaea in the same sample were uncultured Desulfurococcales archaeon which does not belong to Thaumarchaeota (Fig 4B)? Please check the taxonomic classification carefully. Line 313-314: this sounds like that the absolute abundance of nitrogen cycling genes was the lowest. Wasn't it the relative abundance of nitrogen cycling genes among all the genes on GeoChip? Line 414-419: I could not follow the logic here: Some studies suggested archaea were rare, but other studies suggested that archaea and bacteria coexisted. Then I expected to hear about whether archaea in this study is rare or not. The data in this study could only indicate that Thaumarchaeota was the most abundant phylum among archaea but archaea could still be rare (i.e. much fewer than bacteria) in hot springs.
Some methods were in the results: The subtitle of the first part of the results 'sampling' sounds like a method section, and the entire section was only one paragraph describing pH and temperature. It could be easily moved to the beginning of another section in the results as one sentence.
Line 216-217: 'random resampling was conducted for further analyses' should be re-C4 moved.
Line 223: 'with a similarity of 97% for OTU classification', Line 225 'at a 97% similarity level.', and line 237 'at a 97% similarity level'. The criterium for OTU classification does need to be mentioned but once is enough.
Unclear, redundant or awkward expressions (some examples): Line 17-18: 'in response to the depths, . . .shifts over the depth profile'. Line 87: 'community functional potential altered by the depth'. I think you wanted to say that the composition of microbial communities depended on the depth.
Line 112: 'three duplicate samples'. Duplicates mean two replicates. Three duplicates mean six samples. I think here you meant 'triplicates'.
Line 200-201: 'According to the temperatures and pH, there were no significant differences between the samples.' The authors may mean that the temperatures and pH of the samples were similar, but the sentence was saying that the samples were considered similar only based on two environmental variables.
Line 216:' For the microbial diversity, the composition and structure of each sample could be compared;' What do you mean? This sentence needs to be re-written.
Line 225-227: 'The bacterial groups at 0 m with the highest relative abundances at the phylum level were members of Aquificae, Gamma-proteobacteria, and Deinococcus-Thermus.' This sentence needs to be re-written and the Gamma-proteobacteria is not a phylum.
Line 231: awkward expression: 'At the genus level, the OTUs were distributed. . .' Line 283-285: the second half of the sentence said the same thing as the first half.