Nitrogen fixation and the diazotroph community in the temperate coastal region of the northwestern North Pacific

Shiozaki, T.; Nagata, T.; Ijichi, M.; Furuya, K.

doi:https://doi.org/10.5194/bg-12-4751-2015

Articles | Volume 12, issue 15

https://doi.org/10.5194/bg-12-4751-2015

© Author(s) 2015. This work is distributed under
the Creative Commons Attribution 3.0 License.

https://doi.org/10.5194/bg-12-4751-2015

© Author(s) 2015. This work is distributed under
the Creative Commons Attribution 3.0 License.

Articles | Volume 12, issue 15

Research article

|

07 Aug 2015

Research article |

| 07 Aug 2015

Nitrogen fixation and the diazotroph community in the temperate coastal region of the northwestern North Pacific

T. Shiozaki, T. Nagata, M. Ijichi, and K. Furuya

Download

Final revised paper (published on 07 Aug 2015)
Supplement to the final revised paper
Preprint (discussion started on 15 Jan 2015)
Supplement to the preprint

Interactive discussion

Status: closed

AC: Author comment | RC: Referee comment | SC: Short comment | EC: Editor comment

- Printer-friendly version

- Supplement

RC C131: 'Seasonal dynamics of nitrogen fixation and the diazotroph community in the temperate coastal region of the northwestern North Pacific', Anonymous Referee #1, 10 Feb 2015
- AC C1440: 'Response to reviewer #1', Takuhei Shiozaki, 20 Apr 2015
RC C366: 'Comments on “Seasonal dynamics of nitrogen fixation and the diazotroph community in the temperate coastal region of the northwestern North Pacific” by T. Shiozaki et al.', Anonymous Referee #2, 28 Feb 2015
- AC C1450: 'Response to reviewer #2', Takuhei Shiozaki, 20 Apr 2015
RC C543: 'Seasonal dynamics of nitrogen fixation and the diazotroph community in the temperate coastal region of the northwestern North Pacific', Anonymous Referee #3, 07 Mar 2015
- AC C1456: 'Response to reviewer #3', Takuhei Shiozaki, 20 Apr 2015

Peer-review completion

AR: Author's response | RR: Referee report | ED: Editor decision

AR by Takuhei Shiozaki on behalf of the Authors (20 Apr 2015) Author's response Manuscript

ED: Referee Nomination & Report Request started (21 Apr 2015) by Koji Suzuki

RR by Anonymous Referee #1 (05 May 2015)

RR by Anonymous Referee #3 (11 May 2015)

RR by Anonymous Referee #2 (11 May 2015)

Suggestions for revision or reasons for rejection

General comment:
Nitrogen fixation data is rare in the temperate marine ecosystems, and the authors clarified the accuracy of their rate measurement data. Therefore, this paper can provide significant contribution to our understanding of nitrogen fixation in the ocean. After the revision, the authors added quantitative PCR (qPCR) data of the three major phylotypes, which provided more useful molecular information and compensated the limitation of their sequencing results. Hence, they can explain (discuss) the seasonal and spatial variations of nitrogen fixation in a more convincing way. However, there are still some problems and questions needed to be considered, especially the influences of environmental factors to nitrogen fixation.

Specific comments:
P.4, L.72: the methods of Pearson’s correlation matrix were missing. At least, the authors should explain what the “surface water” meant (Table 2). Did they average the surface data of different stations for each cruise or input each data point during calculation? It seems that the error bars of the averaged nitrogen fixation rate and nutrient data were very large. Therefore, it is not appropriated if they used averaged data when they were calculating the correlation matrix. The author can simply display the data used for calculation in a table in supplementary information. Also, why didn’t the author use the data of other water layers during calculation? It seems that nitrogen fixation was also significant in other water layers.

P.13, L.222: the unit of nitrogen fixation rate should be uniform throughout the paper. The author used “L” or “l” in different places of the paper.

P.14, L.252: according to Table 2, correlation between nitrate, phosphate and temperature was strong, and all this factors had significant correlation with nitrogen fixation rate. However, it is not necessary that all these factors influenced nitrogen fixation directly. For examples, the negative correlation of nitrate (phosphate) with nitrogen fixation rate can be due to their negative correlations with temperature. Therefore, this issue is needed to be considered during discussion. Especially, both DIN concentration (ammonium should only contribute very small portion) and N:P ratio did not show significant negative correlation with nitrogen fixation rate.

P.18, L.308: It is suggested not to use one page to discuss the two rate measurement methods in the beginning of discussion, as this study was not focusing on this issue.
Moreover, if gas dissolution method is better in this study, further discussion of these two methods is not helpful to the objective of this study.

P.22, L388: Correlations of the qPCR result and nitrogen fixation rate and environmental factor are suggested to be analyzed. The authors tried to discuss numbers of different phylotypes with nitrogen fixation rate and environmental factors. Conducting environmental correlation analysis will support their points and provide clear picture. In order to estimate gene copies of different nifH phylotypes accurately, the authors can use qPCR of 16S rRNA to normalize the data of nifH.

P.24, L.415: “disappearing during in spring” seems grammatically incorrect to me.

p.26, L.452: as the author did not do qPCR of cluster III, it is inappropriate to imply that cluster III was increased in abundance with “suspension of sediment”. The abundance of cluster III might not be changing a lot throughout different seasons, and their higher relative abundance in cold condition may be simply due to repression of the cyanobacterial diazotrophs. Therefore, without qPCR data of cluster III, the author should not strengthen the importance of cluster III in cold conditions. Also, the nitrogen fixation of marine cluster III is still not well confirmed and this study was based on DNA works, so, it is not helpful and convince to mention too much about cluster III in discussion and conclusion. Besides that, relatively abundant cluster III was also reported in Arctic (Farnelid et al 2001).

S3,Phylogenetic tree: the Trichodesmium should not be clustering with cluster I proteobacteria. The tree was not stable, and the reason may be due to insufficient sequences of cyanobacteria. The author can consider adding more reference sequences of cyanobacteria, which should make the tree more stable.

Referee Report: PDF

Hide

ED: Reconsider after major revisions (25 May 2015) by Koji Suzuki

AR by Takuhei Shiozaki on behalf of the Authors (08 Jun 2015) Author's response Manuscript

ED: Referee Nomination & Report Request started (11 Jun 2015) by Koji Suzuki

RR by Anonymous Referee #2 (01 Jul 2015)

Suggestions for revision or reasons for rejection

General comment:
The manuscript improved significantly after the previous revisions. Although insufficient sequence number of clone library is the major limitation of this study, addition of qPCR of some nifH phylotypes is helpful in compensating the limitation. As limited amount of relevant studies have been done in temperate regions of North Pacific Ocean, it is difficult for the authors to further elaborate and discuss with this set of data in a convincing way. On another hand, the data set could provide useful information for the related studies in the future. Therefore, I think this work can be accepted, after some minor revisions. There are some grammatical mistakes found in the manuscript, and the authors should do proof-reading more carefully.

Specific comments:
L.141, What does “when nitrogen fixation was not detected” mean? Did the authors mean “when nitrogen fixation was undetectable” or “not measured”? If the nitrogen fixation rate was not measured, the authors should not assume the missing data to be zero. The authors should clarify their meaning here.

L.199, “underestimates” should be “underestimated”.

L.401, “one or more the factors” should be “one or more factors”

L. 432, It is suggested to use “undetectable” to replace “disappear”

L.455-457, The logic here is not clear enough. Since the author did not use qPCR to quantify the P. stutzeri-like nifH gene, there is no reason to say “P. stutzeri could not be a major diazotroph in this study region”. Also, “γ-24774A11 was not detected on that occasion by qPCR analysis” should not be the evident suggesting “γ-24774A11 was not quantified as P. stutzeri”. The authors can simply compare the sequences of γ-24774A11 and P. stutzeri nifH gene recovered in this study.

L.496, It is suggested to add “coastal” after “temperate”, as the study was conducted in coastal area.

Referee Report: PDF

Hide

RR by Anonymous Referee #3 (01 Jul 2015)

ED: Publish subject to minor revisions (Editor review) (06 Jul 2015) by Koji Suzuki

AR by Takuhei Shiozaki on behalf of the Authors (14 Jul 2015) Author's response Manuscript

ED: Publish as is (16 Jul 2015) by Koji Suzuki

AR by Takuhei Shiozaki on behalf of the Authors (17 Jul 2015)

Download

Article (2616 KB)
Full-text XML

Short summary

We found that nitrogen fixation can proceed at high rates, depending on the season, in the temperate coastal region of the northwestern North Pacific. nifH sequences were omnipresent and recovered throughout the year. UCYN-A was a major diazotroph during summer, whereas Trichodesmium was abundant during fall, despite low temperatures. The Cluster III diazotrophs, putative anaerobic bacteria, were found at the surface throughout the year.