Award: OCE-0960806

The oceanÆs microbial realm is the largest and arguably most complex ecosystem on Earth. It plays a central role in controlling global element cycles and is tightly linked to climate-feedback processes. Nitrogen (N) is an essential element for all life on Earth. Organisms need N to form proteins and deoxyribonucleic acids (DNA) and will compete vigorously for N to survive and grow. The total concentrations of the inorganic and organic N pools in the ocean are fairly well understood. What is not well known are the identities of the microbes that take up the different forms of N and the rates with which they do it. Not capturing the æwhoÆ and the æhow muchÆ dimensions of the N cycle can have large consequences for our understanding of global geochemical fluxes. This grant used a stable isotope of N (15N) to trace the incorporation of N into microbial (phytoplankton, bacteria, and archaea) DNA and to determine how quickly different N compounds (for example, nitrate or urea) were taken up. Our goal was to specifically link N uptake from different sources to individual microbial populations. Isotopes of N can be distinguished between one another by their molecular weight. For example, 15N is heavier than the more common 14N isotope that accounts for 99.6% of all global N. Since 15N is heavier, we can determine whose DNA has incorporated the 15N from the added substrate and whose DNA still contains the naturally abundant 14N and thus did not use the added N. This work is done through stable isotope probing (SIP) techniques. SIP has been used as a powerful tool for the investigation of carbon metabolism in microbial communities but technical challenges limited its use in N studies. We used 15N-SIP in a study to investigate N uptake by phytoplankton and bacterial populations in the ocean. Historically, it has been perceived that inorganic forms of N (for example, nitrate) are utilized by phytoplankton, while organic N (for example, urea) is primarily a source of N for bacteria. Evidence suggests that this distinction may be too simplistic and that there can be considerable flexibility in the N substrates used by a given organism. It is also unclear how phytoplankton and bacteria interact in the environment when forced to compete for limited N resources, and what the N substrate ranges of bacterial and phytoplankton populations are under natural conditions. A series of experiments on subtropical (Chesapeake Bay) and polar (Arctic) coastal communities was conducted using a range of 15N-labeled organic (urea and amino acids) and inorganic (ammonium, nitrate and nitrite) substrates. In subtropical coastal environments we observed that multiple organic and inorganic sources of N were fuelling cyanobacterial and diatom primary production simultaneously. This result is particularly interesting because it challenges the traditional hypothesis that nitrate utilization is a good proxy for primary production. Our work shows that this may not always be the case in coastal environments. Our study is also among only a few in the literature that directly demonstrate the uptake of a specific form of N into individual phytoplankton species in the environment. In a subsequent related study we focused on bacterial nitrate utilization. Molecular evidence had suggested that nitrate-utilizing bacteria are abundant, wide-spread, and potentially active in marine systems, but direct evidence of incorporation of N from nitrate into individual bacterial species had not been reported. We combined uptake rate measurements and15N-SIP with ribosomal nucleic acid (RNA)-based functional microarray analysis which is a technique that provides information of genes expressed by the whole community. Through this combination of techniques, we were able to gain a more comprehensive perspective of N cycling by considering all of the active N cycling genes of the whole community, while targeting nitrate utilizatio...