Award: OCE-1341178

The ocean is an active reservoir for much of the world?s carbon dioxide (CO2), a potent greenhouse gas linked to climate change. When phytoplankton, the base of the majority of the Ocean?s food webs, have sufficient nutrients, they can use energy generated from photosynthesis to turn CO2 into more phytoplankton that can be grazed by zooplankton or sink into the deep ocean, sequestering this C. However, much of this production is limited by the availability of nitrogen (N) and different N sources will affect the fate of this newly formed biomass. Certain sources of inorganic N, e.g. ammonium and urea, arise from the breakdown of previously formed organic matter (termed recycled N) and do not allow for net C accumulation and sequestration. CO2 taken up this way will ultimately be released back into these surface waters and into the atmosphere where it came from. Other sources of N, e.g. nitrate and N2 fixation (termed new production), add new nitrogen to these waters allowing net accumulation of C to be stored in biomass that will eventually sink out and be sequester in the deep ocean. This can allow even more CO2 to diffuse into these waters causing them to become a sink for CO2. The breakdown of organic matters is often associated with heterotrophic processes undertaken by various microorganisms including diverse bacteria, archaea and protists. Organic matter itself can be deficient in N content. Hence, many marine heterotrophs assimilate inorganic N and can compete with marine phytoplankton for these limited resources. Understanding the communities involved in the response to fluxes of different N sources into marine ecosystems will allow us to better predict how they will respond in this changing climate. Our project was aimed at optimizing a stable isotope probing (SIP) method for studying nitrogen assimilation with a particular goal of identifying heterotrophic N fixers. In nature, only 0.37% of naturally occurring N atoms are 15N, the remainder, 99.6% are the lighter natural isotope, 14N. SIP attempts to identify the organisms directly involved in the uptake and assimilation of specific N substrates by assessing changes in DNA density based on the incorporation of an enriched (99% 15N) heavy substrate. Traditional SIP methodologies separate the labelled from unlabeled DNA by long (+60h) ultra-centrifugation runs in a CsCl gradient after exposure of the sample to the enriched substrate. We coupled our analysis by analyzing each fraction using high throughput sequencing and term this approach Tag-SIP. To develop our methods we used both pure cultures of diazotrophs and heterotrophic bacteria, and in the summer of 2012 surface samples from Big Fisherman?s Cove off the coast of Catalina Island. Incubations were used to first determine the limit of detection of the method and the minimum incubation length required to obtain a labeled signal. If incubations are too long, cross-feeding, i.e. a primary utilizer excretes a transformed version of the original substrate that is secondarily taken up and assimilated into biomass, can occur causing the misidentification of primary utilizers. We were able to couple amplicon sequencing of the 16S rRNA gene commonly used for taxonomic identification with more traditional SIP methodology. This increased the resolution, from 30% to 10% DNA enrichment required, while allowing the assessment of individual organisms. Our initial effort in natural waters indicated activity in populations of heterotrophic diazotrophs was below the detection limit of the method. Hence, we shifted our focus to investigate nitrate and ammonium assimilation in these waters and found that organisms previous shown to be lacking the genes required to incorporate this N sources were showing evidence of assimilation. Some clades included the dominant heterotrophic clades SAR11 (Figure 1) and MG-II (Table 1). As mentioned above, no meaningful results were obtained for N2 fixers. We believe this is likely due to the relatively low densities and growth rates often associated with N2 fixing organisms in nutrient rich coastal areas, particularly heterotrophs, potentially requiring incubation length that would provide confounding results. We did however establish a collaboration with Xavier Mayali at Lawrence Livermore National Laboratory (LLNL) to examine heterotrophic nitrogen fixers using his sophisticated CHiP-SIP method which takes advantage of the higher turnover rate of mRNA. This combined with CHiP-SIP?s use of the highly sensitive nanoSIMS technology to evaluate enrichment may facilitate the detection of isotopic label in slower growing organisms. Several experiments have been undertaken and the samples are in the queue at LLNL to be run. Last Modified: 08/31/2016 Submitted by: Douglas G Capone