Award: OCE-1335436

Approximately 10% of the global ocean is experiencing moderate to severe oxygen depletion, which consequently has significant impacts on nitrogen and sulfur cycling by microorganisms. Oxygen-depleted water columns shift microbial communities and their activities toward processes that lead to increased release of climate-active trace gases, and depletion of bioavailable nitrogen, which can have detrimental effects on ocean primary production. The overall goal of this work was to examine microbial diversity (Bacteria, Archaea, and Eukarya, plus viruses) and processes along an oxygen gradient in a well-constrained ecosystem (Cariaco Basin, Venezuela) and to determine if microbial processes are partitioned differently between the particle-associated and free-living microorganisms. Our hypothesis was that close coupling of nitrogen and sulfur cycling which can escape detection by traditional analyses actually fuel the anomalously high chemoautotrophic and heterotrophic productivity observed in the water layer transitioning between oxygenated and anoxic (= redoxcline). We produced data on taxonomic diversity (based on SSU rRNA iTAGs), microbial functional potential (metagenome libraries and qPCR of functional genes), and gene activity (metatranscriptome libraries and RT-qPCR of functional genes), in two seasons, in the particle-associated and free-living fractions, and at six primary sampling depths along oxygen and chemical gradients in the Cariaco water column. We interpreted these data in the context of physical structure, geochemistry, and microbial ecology, including observed rates of key microbial processes along the redoxcline. Particle-associated and free-living assemblages were found to be functionally different. Evidence was found for a cryptic sulfur cycle in suboxic/anoxic waters, as was close metabolic coupling of sulfur and nitrogen cycles. Both denitrification and anammox (anaerobic ammonia oxidation) were active in removing bioavailable nitrogen in the Cariaco Basin, while occurring in separate microhabitats. Denitrifiers are primarily particle-associated and responsive to nitrate pulses potentially provided by horizontal water transport in this system. Anammox organisms were primarily free-living and generally unresponsive to nitratepulses. Bacterial assemblage composition across the redoxcline clearly underwent abrupt changes that were detected in 2009 in the course of the Cariaco Ocean Time-Series program. The dominant putative chemoautotrophs in this layer appeared to have switched from e-proteobacteria to g-proteobacteria after the 2009 transition. During the observation period, vertical distributions of oxygen, nitrate, nitrite, ammonium, and sulfide and water density structure changed significantly through the redoxcline. Variations in vertical fluxes of oxidants (oxygen, nitrate and nitrite) and reductants (sulfide, thiosulfate, sulfite, sulfur, and ammonium) appear to be key in niche partitioning between e- and g-proteobacteria and may explain the ecosystem state change in ways that are yet to be discovered. Forty-eight metagenomes and forty-eight metatranscriptomes have been deposited to public databases, as well as 141 iTAG (taxonomic) libraries. Two publications by our group and two presentations by Edgcomb at international events hosted by the Scientific Committee on Ocean Research highlighted this work, pointed out the biases and artifacts introduced by traditional Niskin sampling for RNA-based studies, and made the case for the need for technological advances to provide cost-effective means for rapid in situ sample collection and preservation. This project framed the PhD thesis for one graduate student, Elizabeth Suter, Stony Brook University (SBU), and the work of one Postdoctoral Investigator, Maria Pachiadaki, WHOI. In addition, three undergraduate students were trained at SBU throughout the funding period and at WHOI three high school during summer projects, and at least two high school students annually (2016-17, 2015-16, 2014-15, 2013-14) for Science Fair Projects around the theme of effects of oxygen depletion on marine microbial ecology. Edgcomb taught groups of ~20 high school students annually in each of summers 2014-2017 during 2-day intensive workshops on marine microbiology and oxygen depletion, through the Weston Schools Advanced Biotechnology Institute. Last Modified: 08/25/2017 Submitted by: Gordon T Taylor Data generated by this project has been deposited in several open access databases that will enable other researchers to perform comparative studies. Our data are deposited as follows: All measured hydrographic, geochemical, and ecological parameters are available at www.bco-dmo.org/project/562425 under "Dataset collection". Scripts for multivariate analyses and relative abundance bubble plots are available at https://github.com/lizsuter/Cariaco-iTags. 141 iTAG libraries (3 failed) have been uploaded to GenBank SRA and linked in both the SRA and BioSample databases under one BioProject with the following link: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA326482 and were released on 09/30/2017. iTAGs from all three Domains of life are under the same BioProject, and have the accession numbers SRX1887279-SRX1887324 (Archaea), SRX1890724-SRX1890775 (Bacteria), and SRX1890776- SRX1890825 (Eukarya). 48 Metagenomic and 48 metatransciptomic data have been uploaded to IMG and will be released on 03/30/2018. The description of the datasets and the accession links are available at www.bco-dmo.org/project/562425 under "Dataset collection". Last Modified: 10/10/2017 Submitted by: Gordon T Taylor