Dear authors,

I am happy to tell you that both reviewers liked your manuscript, although both suggested some changes and modifications. In general, you followed their suggestions in your response letters. Thus, I would like to encourage you to submit the revised version now. Please make sure that the graphics can be read without using a 250% view.

I wish you all a Happy New Year 2016!

Kirsten Küsel

Dear Dr. Gier,

I was informed by the Editorial Support Office that one reviewer comment was not uploaded properly in the System. Thus, the third review was not visible for you.
The reviewer contacted us, since he/she missed it in the Biogeosciences Discussion Forum.

In general, the reviewer was positive, but some suggestions to improve the quality of your mansucript were made. Thus, I would like to ask you to include these suggestions in your revised Version as well.

Thank you very much in advance for your cooperation and understanding keeping the review process open and transparent in Biogeosciences.

Best

Kirsten Küsel
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Third Review:

Nitrogen fixation in marine sediments is an essential part of the nitrogen cycle. In depth knowledge on diazotrophic key players and the regulation of nitrogen fixation are of importance. The present study adresses benthic nitrogen fixation along with sulfate reduction in the oxygen minimum zone of Peru and is thus not without merit. The authors report depth-dependent nitrogen fixation and sulfate reduction potentials along a transect along with biogeochemical data and molecular analyses of diazotrophs. Sulfate reduction and nitrogen fixation potentials basically declined with sediment depth and varied among sampling sites. Organic carbon content rather than sulfate reduction might have been correlated with nitrogen fixation potentials. The authors detected nifH genes that grouped with nifH from various organisms including uncultured taxa, Gammaproteobacteria and gram-positve Clostridia. None of the sequences clustered with Desulfovibrio vulgaris, a sulfate reducer. Transcript analyses indicating active diazotrophs rather than the genetic potential only are lacking. Thus, the conclusion on the importance of sulfate reducers for nitrogen fixation appears not to be supported by the data.

My major concerns are:

1. Lack of appropriate statistics to evaluate correlations of biogeochemical parameters and nitrogen fixation potentials.

2. Incubation times during the acetylene reduction assay were seven days rather than 24 h (L5, 14408), allowing for changes in microbial community. Was the ethylene production linear over time?

3. Low number of sequences and nifH gene analysis. 120 sequences were obtained from about 60 subsamples (same number of samples as for acetylene reduction assays; L16, 14407; L19 14409), suggesting that 2 sequences were retrieved per sample. This is far too low to judge on the diversity of nifH in any environmental sample. In any case, rarefaction analyses or coverages have to be provided in order to demonstrate sufficient sequencing effort for a meaningful diversity analysis. Conclusions on the absence of cyanobacterial diazotrophs are thus not appropriate.

4. Descritption/ interpretation
of nifH data. The legend to Figure 6 describes "expressed nifH genes".
However, DNA rather than RNA was analyzed (L18-25, 14409). Sequencing of nifH transcripts (mRNA/ cDNA) would indeed provide insights into expressed nifH genes and active diazotrophs, and would thus provide meaningful data. However, this was not done and conclusions are thus not supported by teh data (e.g., L14, 14416).

5.Phylogenetic interpretation of nifH gene data. The authors conclude from the clustering of recovered nifH genes that diazotrophic sulfate reducers were present in their samples and associated with nitrogen fixation. Sequences mainly clustered with nifH from uncultured organisms, Gammaproteobacteria and Firmicutes (Clostridia) rather than Desulfovibrio (which was always more distant than the previously named taxa; Figure 6) (L12-13, 14414). Thus, the concusion that the molecular analysis supports the conclusion on a contribution of diazotrophic sulfate reducers to nitrogen fixation in Peruvian oxygen minimum zones is not supported by the data.

Minor comments: L1, 14406. Should read "these bacteria". L6, 14409. A short description of the method would be helpful.

Thank you again for submitting the above manuscript to Biogeosciences. It has been expertly refereed by three anonymous referees who both found merit in your study. In your very detailed response letter you have commented on their concerns in the Interactive Discussion. In general, you followed the suggestions of the referees.

Thus, the revised version of your manuscript can be published in Biogeosciences without further delay.

Your interest in publishing in Biogeosciences is very much appreciated.

Sincerely,

Kirsten Küsel

(Associate Editor)