title: Revisiting N₂ fixation in Guerrero Negro intertidal microbial mats with a functional single-cell approach.
abstract: 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 (15)N₂ 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 (15)N₂-incubated mat samples via high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that Cyanobacteria were enriched in (15)N, with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% (15)N), whereas the Deltaproteobacteria (identified by CARD-FISH) were not significantly enriched. We investigated the potential dilution effect from CARD-FISH on the isotopic composition and concluded that the dilution bias was not substantial enough to influence our conclusions. 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.
authors.affiliation: 1] Departments of Chemical Engineering, and of Civil and Environmental Engineering, Stanford University, Stanford, CA, USA [2] Exobiology Branch, NASA Ames Research Center, Moffett Field, CA, USA [3] Department of Microbiology and Ecosystem Science, Division of Microbial Ecology, University of Vienna, Vienna, Austria.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Woebken
authors.affiliation: 1] Departments of Chemical Engineering, and of Civil and Environmental Engineering, Stanford University, Stanford, CA, USA [2] Exobiology Branch, NASA Ames Research Center, Moffett Field, CA, USA.
authors.forename: Luke C
authors.initials: LC
authors.lastname: Burow
authors.affiliation: Department of Microbiology and Ecosystem Science, Division of Microbial Ecology, University of Vienna, Vienna, Austria.
authors.forename: Faris
authors.initials: F
authors.lastname: Behnam
authors.affiliation: Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA, USA.
authors.forename: Xavier
authors.initials: X
authors.lastname: Mayali
authors.affiliation: Large-Instrument Facility for Advanced Isotope Research, University of Vienna, Vienna, Austria.
authors.forename: Arno
authors.initials: A
authors.lastname: Schintlmeister
authors.affiliation: Exobiology Branch, NASA Ames Research Center, Moffett Field, CA, USA.
authors.forename: Erich D
authors.initials: ED
authors.lastname: Fleming
authors.affiliation: Exobiology Branch, NASA Ames Research Center, Moffett Field, CA, USA.
authors.forename: Leslie
authors.initials: L
authors.lastname: Prufert-Bebout
authors.affiliation: Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Singer
authors.affiliation: Laboratory of Geomicrobiology and Biotechnology, Northwestern Center for Biological Research (CIBNOR), La Paz, Mexico.
authors.forename: Alejandro López
authors.initials: AL
authors.lastname: Cortés
authors.affiliation: Exobiology Branch, NASA Ames Research Center, Moffett Field, CA, USA.
authors.forename: Tori M
authors.initials: TM
authors.lastname: Hoehler
authors.affiliation: Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Pett-Ridge
authors.affiliation: Departments of Chemical Engineering, and of Civil and Environmental Engineering, Stanford University, Stanford, CA, USA.
authors.forename: Alfred M
authors.initials: AM
authors.lastname: Spormann
authors.affiliation: 1] Department of Microbiology and Ecosystem Science, Division of Microbial Ecology, University of Vienna, Vienna, Austria [2] Large-Instrument Facility for Advanced Isotope Research, University of Vienna, Vienna, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Wagner
authors.affiliation: Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA, USA.
authors.forename: Peter K
authors.initials: PK
authors.lastname: Weber
authors.affiliation: Exobiology Branch, NASA Ames Research Center, Moffett Field, CA, USA.
authors.forename: Brad M
authors.initials: BM
authors.lastname: Bebout
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D044822:Biodiversity; D000458:Cyanobacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017037:Dinitrogenase Reductase / Q000235:genetics; D017753:Ecosystem; D008800:Mexico; D009586:Nitrogen Fixation / Q000235:genetics; D059010:Single-Cell Analysis
chemical_list: D017037:Dinitrogenase Reductase