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Title: Revisiting N2 fixation in Guerrero Negro intertidal microbial mats with a functional single-cell approach

Photosynthetic microbial mats are complex, stratified ecosystems in which high rates of primary production create a demand for nitrogen, met partially by N2 fixation. Dinitrogenase reductase (nifH) genes and transcripts from Cyanobacteria and heterotrophic bacteria (for example, Deltaproteobacteria) were detected in these mats, yet their contribution to N2 fixation is poorly understood. We used a combined approach of manipulation experiments with inhibitors, nifH sequencing and single-cell isotope analysis to investigate the active diazotrophic community in intertidal microbial mats at Laguna Ojo de Liebre near Guerrero Negro, Mexico. Acetylene reduction assays with specific metabolic inhibitors suggested that both sulfate reducers and members of the Cyanobacteria contributed to N2 fixation, whereas 15N2 tracer experiments at the bulk level only supported a contribution of Cyanobacteria. Cyanobacterial and nifH Cluster III (including deltaproteobacterial sulfate reducers) sequences dominated the nifH gene pool, whereas the nifH transcript pool was dominated by sequences related to Lyngbya spp. Single-cell isotope analysis of 15N2-incubated mat samples via high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that Cyanobacteria were enriched in 15N, with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% 15N), whereas the Deltaproteobacteria (identified by CARD-FISH) were not significantly enriched. We investigated the potentialmore » dilution effect from CARD-FISH on the isotopic composition and concluded that the dilution bias was not substantial enough to influence our conclusions. As a result, our combined data provide evidence that members of the Cyanobacteria, especially Lyngbya spp., actively contributed to N2 fixation in the intertidal mats, whereas support for significant N2 fixation activity of the targeted deltaproteobacterial sulfate reducers could not be found.« less
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [3] ;  [5] ;  [6] ;  [7] ;  [8] ;  [6] ;  [4] ;  [9] ;  [3] ;  [4] ;  [6]
  1. Stanford Univ., Stanford, CA (United States); NASA Ames Research Center, Moffett Field, CA (United States); Univ. of Vienna, Vienna (Austria)
  2. Stanford Univ., Stanford, CA (United States); NASA Ames Research Center, Moffett Field, CA (United States)
  3. Univ. of Vienna, Vienna (Austria)
  4. Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
  5. NASA Ames Research Center, Moffett Field, CA (United States); California State Univ., Channel Islands, Camarillo, CA (United States)
  6. NASA Ames Research Center, Moffett Field, CA (United States)
  7. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  8. Northwestern Center for Biological Research (CIBNOR), La Paz (Mexico)
  9. Stanford Univ., Stanford, CA (United States)
Publication Date:
OSTI Identifier:
1251059
Report Number(s):
LLNL-JRNL--672239
Journal ID: ISSN 1751-7362
Grant/Contract Number:
AC52-07NA27344
Type:
Accepted Manuscript
Journal Name:
The ISME Journal
Additional Journal Information:
Journal Volume: 9; Journal Issue: 2; Journal ID: ISSN 1751-7362
Publisher:
Nature Publishing Group
Research Org:
Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
54 ENVIRONMENTAL SCIENCES; 58 GEOSCIENCES; 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY Cyanobacteria; microbial mat; N2 fixation; nanoSIMS; nifH; sulfate reducing bacteria