Marine Nitrogen Cycling
The marine nitrogen (N) cycle is complex and involves many biological transformations among different inorganic and organic N reservoirs (Zehr et Ward 2002). N cycling processes can sometimes limit productivity in marine systems and therefore have a bearing on marine ecology, as well as the sequestration of carbon (C) from marine environments. Historically, we understand the relationship between N and C cycling in the context of the concepts of 'new' and 'recycled' productivity (Dugdale et al, 1967, Eppley et al. 1979). In this context, new production is equated with export production and is seen as the result of new nutrient influxes, in particular nitrate, which lead to diatom productivity. Recycled productivity on the other hand results from ammonium uptake and occurs primarily in sub-tropical and tropical oligotrophic surface oceans.
In recent years, studies have shown that the marine N cycle is much more complex than the new vs. recycled productivity paradigm might suggest. However, a significant limitation in many studies has been the inability to test the uptake of specific forms of N into individual phyto- and bacterioplankton species. Traditional uptake measurements are made on bulk communities, which are composed of a large number of different microbial species that may not behave uniformly. In my lab we have significantly improved upon traditional uptake studies by applying DNA stable isotope probing (SIP) using 15N stable isotope tracers of different N sources. 15N SIP allows us to specifically interrogate individual microbial community members for their N uptake patterns in the environment. An example of Synechococcus nitrate uptake examined in culture is shown in figure 1.
|Figure 1. Stable isotope incorporation experiment showing 15NO3 uptake into a culture of Synechococcus WH7803. (A) Relationship between buoyant density of DNA in a CsCl gradient, G+C content, and % 15N incorporation. The solid line indicates the predicted buoyant density of DNA containing only 14N. The dotted line indicates the predicted buoyant density of DNA at 100% 15N incorporation. The horizontal line represents the G+C content of WH7803, and the vertical lines predict the buoyant densities of WH7803 at 0% and 100% 15N incorporation. (B) Two cultures of Synechococcus WH7803 were grown on 14NO3 and 15NO3 as N sources, respectively. DNA was extracted from both cultures and both were fractionated on CsCl gradients. WH7803 DNA was quantified in each fraction by means of quantitative PCR by determining rbcL gene copy numbers in each 100-μl fraction. Shown are the ratios of quantities, which were calculated by dividing the measured amount of Synechococcus rbcL DNA in each fraction by the highest value measured in any of the fractions collected from a CsCl column. Shown are unlabeled (0% 15N) WH7803 DNA (▲) and labeled (100% 15N) WH7803 DNA (●).|
In a recent publised sudy (Wawrik et al 2009), we employed 15N-based SIP techniques to assess the use of a suite of inorganic and organic nitrogen substrates by Synechococcus and diatoms in a coastal marine system. Our goal was to investigate the traditional characterization of Synechococcus as recycled producers (mainly ammonium uptake) and diatoms as new producers (nitrate uptake). To accomplish this goal, seawater was incubated with a series of 15N-labeled N substrates. DNA was then extracted at the end of the incubation, and quantitative PCR was used to determine the amount of Synechococcus and diatom DNA as a function of density in fractionated gradients. Shifts in densities of Synechococcus (Figure 2) and diatom DNA (data not shown) as the result of incubations with 15N- labeled N substrates were interpreted as evidence for uptake. Synechococcus and diatoms both actively incorporated 15N-ammonium, 15N-nitrate, and 15N-urea. Synechococcus appeared to also incorporate N from 15N-glutamate and 15N-amino acids. These data suggest that N flow in communities containing Synechococcus and diatoms has more plasticity than the new versus regenerated production paradigm might suggest, and that these two phytoplankters should not strictly be viewed as recycled and new producers respectively. Our data are also one of the first pieces of evidence showing the uptake of amino acids by a marine phytoplankton species (Synechococcus).
Figure 2. Synechococcus DNA as a function of density as determined by CsCl centrifugation and quantitative PCR, using the Synechococcus rbcL gene as a proxy in field samples. Vertical lines correspond to the vertical lines in Figure 1, and indicate the density of Synechococcus DNA containing only 14N or only 15N, respectively. (A) T0 = ambient population, NTC = incubation without the addition of any 15N-labeled nitrogen source. Additional graphs are data after incubation in the presence of 2 µmol N L-1 (B) 15N-urea, (C) 15NH4+, (D) 15NO3-, (E) 15N-amino acids, and (F) 15N-glutamaic acid. Shown are ratios of quantities, which are calculated by dividing the measured amount of Synechococcus rbcL DNA in each fraction by the highest value measured in any of the fractions collected from a CsCl column.
Wawrik,B., W.B. Boling, J.D. Van No1strand, J. Xie, J. Zhou, and D.A. Bronk. Assimilatory Nitrate Utilization by Bacteria on the West Florida Shelf as Determined by Stable Isotope Probing and Functional Microarray Analysis. Applied and Environmental Microbiology. In press (FEMS Microbiology Ecology).
Dugdale, R. C., and J. J. Goering. 1967. Uptake of new and regenerated forms of nitrogen in primary productivity. Limnol. Oceanogr. 12:196-206.
Eppley, R. W., and B. J. Peterson. 1979. Particulate organic matter flux and planktonic new production in the deep ocean. Nature 282:677-680.
Wawrik, B., A.V. Callaghan, D.A. Bronk. 2009. Inorganic and Organic Nitrogen Use by Synechococcus and Diatoms on the West Florida Shelf Measured Using Stable Isotope Probing. Applied and Environmental Microbiology 75:6662-6670.
Zehr, J. P., and B. B. Ward. 2002. Nitrogen cycling in the ocean: new perspectives on processes and paradigms. Appl. Environ. Microbiol. 68:1015-24.