Dear Dr. Peng,
Thank you for submitting your manuscript to BGD
Your manuscripts have been critically reviewed by three independent referees, and these comments have been addressed. However, after a careful check, there are still some concerns that might have been overlooked, and part of results was over-interpreted. For example, it is not appropriate to say “The cell-specific nitrification rates were estimated to be in the range of 0.41 to 0.79 fmol N per AOA cell per hour, which is consistent with earlier estimates in estuary environments. This study demonstrated that AOA were widely involved in nitrification in this hot spring.” The reason is that AOA in estuary environment might not be the same as those detected in this study from hotsprings.
The comments are listed below for your reference.
(1) P2L4. Replace “unclear” with “poorly understood”, and it may be rephrased as follows “However, it remains poorly understood about the link of in situ nitrification activity to taxonomic identities of ammonia oxidizers in high-temperature environments”.
(2) P2L9. Three significant figures might be preferred. For example, 4.81 instead of 4.8
(3) P2L13. “Candidatus Nitrosocaldus yellowstonii”. Candidatus should be in italic, while Nitrosocaldus yellowstonii is NOT ITALIC.
(4) P2L7. The abstract can be rephrased as follows: “”
The in situ ammonia oxidation rates measured by the 15N–NO􀀀 pool dilution technique in the surface sinter and bottom sediments were 4.8 and 5.3 nmolNg􀀀1 h􀀀1, respectively. Real-time quantitative PCR (qPCR) indicated that the archaeal 16S rRNA genes and amoA genes were present in the range of 0.128 to 1.96_108 and 2.75 to 9.80_105 and gene copies g-1 respectively, while bacterial amoA genes were not detected. Phylogenetic analysis of archaeal 16S rRNA genes showed high sequence similarity to thermophilic “Candidatus Nitrosocaldus yellowstonii”, which represented the most abundant operation taxonomic units (OTU) in both surface and bottom sediments. The archaeal predominance was further supported by fluorescence in situ hybridization (FISH) visualization. The cell-specific rate of ammonia oxidation was estimated to range from 0.41 to 0.79 fmol N archaeal cell-1 h-1, being almost ***-fold higher than that of thermophilic “Candidatus Nitrosocaldus yellowstonii”. It implies that phylogenetic relatedness may not necessitate similarity in physiology of ammonia oxidation. These results suggest he importance of archaea rather than bacteria in driving the nitrogen cycle in terrestrial geothermal environments, and highlight the important of effort to cultivate thermophilic AOA in a wide variety of hotsprings.
(1) P3L9. Please add reference for the first evidence of thermophilic AOA (2008 José R. de la Torre et al, 2008 EM; and Wagner group 2008 PNAS)
(2) P3L13-14. Rephrased as: previous studies suggested that AOA play important roles in nitrification in oligotrophic environments such as oceanic water (reference) and acid soils (reference).
(3) P4L6. delete “extremely”
(4) P4L11. In situ nitrification remains largely unknown and very few studies have explicitly…
(5) P5L3. Please state that this is 15N pool dilution technique for nitrification measurement in situ.
(6) P5L5. Rephrased as follows. Meanwhile, potential nitrification activity was determined in the presence of high ammonium concentration: B1 (SS slurry+15NO3+15NH4+)
(7) P5L20. Pls add the brand of the GC-IRMS here.
(8) P7L19. Replace “16S rDNA” with “16S rRNA gene”, replace “and” with “using universal primers”
(9) P7L20-25. Please specify how many different clone libraries constructed. For example, the authors obtained 16S rRNA genes amplicons of total bacteria (27f-1492), archaea (21f-958r).
(10) P9L12. Rephrased as follow: Archaeal and bacterial populations were determined by quantify their 16S rRNA genes with *** (reference) and *** primer pairs (reference), respectively. In addition, the abundance of AOA and AOB were quantified using *** and *** primers, respectively.
(11) P9L23. Pls specify the probes used for AOA visualization.
(12) P10L23-24. As far as I understand, the authors made clone libraries for archaeal communities (21f-958r), bacterial communities (27f-1492r), and archaeal amoA genes. pls specify it clearly here.
(13) P11L10, rephrased as follows. “…, the near-in situ rates of ammonia oxidation were estimated to be *** and *** using 15N pool dilution technique, respectively.”
(14) P11L13. Rephrased as follows: “In the meantime, the nitrate concentration increased from 2.84_2 to 3.25_2 μM in the surface sediments and from 2.33_3 to 2.62_3 μM in the bottom sediments, lending further support for strong nitrification activity under in situ conditions in the hotsprings.
(15) P11L14. Furthermore, the potential activity of ammonia oxidation was determined with ammonium amendments. The nitrate concentration increased significantly upon the addition of NH+4 , and the ammonia rates recorded in the surface sediments and bottom sediments (with NH+4 ) were 5.7_0.6 and 7.1_0.8 nmolNg􀀀1 h􀀀1, respectively.
(16) P11L19. Please specify the number of cloned 16S rRNA genes here.
(17) P12L16-27. As referee suggested, pls refer to the taxonomic classification proposed by Pester 2012 EM paper. For example, Nitrososphaera lineage, Nitrotalea lineage; N. yellowstonii lineage, and N.maritimus lineage. And the old term usually refers to group 1.1b, group 1.1a and group 1.1a-associated. The readers will be confused by cluster A, B and C. The authors can put the representative AOA sequences (e.g from group 1.1a or 1.1b) together with sequences in cluster A and B. Thus it would be easy for readers to understand
(18) P14L25. It seems plausible that archaeal amoA genes were detected ony in the Gongxiaoshe hot spring
(19) P16L2. Please replace “amoA transcripts” with “amoA genes”
(20) P17L1. Rephrased as follow. “Therefore, it implies that phylogenetic relatedness does not necessarily indicate similarity in the physiology of thermophilic AOA, and ammonia oxidation activity of thermophilic AOA in this study might be stronger than those detected in other hotsprings from US (reference) and Iceland (reference). These results also suggest the importance of cultivation studies for comparative analysis of environmentally representative AOA in a wide variety of hotsprings.
Yours Sincerely
Zhongjun Jia

Dear Dr. Peng,
Thank you for submitting your manuscript to BG
The major concerns have been adequately addressed in the revised ms. However, some minor corrections need to be done before publication as follows.
(1) The title might be rephrased. The current title pays more attention to “composition”, and abundance of ammonia oxidizers was indeed analyzed by both qPCR and FISH. In addition, AOB were rarely mentioned in this study. Therefore, it is hard to emphasize “the contribution”. It might be rephrased as follows. Nitrification of archaeal ammonia oxidizers in a high temperature hot spring
(2) L15. Replace “abundance” with “diversity” because both abundance and composition were analyzed in this study.
(3) L27-28. Replace “archaea rather than bactera” with “archaeal rather than bacterial ammonia oxidation”
(4) L183. Replace “16S rDNA” with “16S rRNA genes”
(5) L183. It should be lowercase, i.e., replace “Gene” with “gene”
(6) L250. Insert “of 16S rRNA genes” after clone sequences.
(7) L573. Pls add the primer information for clone library construction.
(8) L588. The color may not be that helpful since there are only two treatments.
Yours Sincerely
Zhongjun Jia