PP42A-01 INVITED
The evolution and feedbacks on the global N cycle
The global N cycle essentially is a set of three coupled redox reactions. Although N2 is, by far, the most abundant gas in Earth's atmosphere, and its reduction to NH3 is slightly exergonic, the activation energy is extremely high. The biological fixation of N2 is catalyzed by nitrogenase, an ancient, heterodimeric protein complex which contains 19 iron-sulfur clusters. The iron-sulfur clusters are extremely sensitive to molecular oxygen, and hence, nitrogenase must work under anaerobic conditions. However, the chemoautotrophic oxidation of NH3 by nitrifying bacteria, a process requiring free O2, provides a substrate for another set of anaerobic organisms that can remove the oxidized species to the atmosphere, ultimately as N2 gas. Thus, while, in principle, N should be extremely abundant in aquatic ecosystems, it is often limiting in the oceans. Here I will examine whether N2 fixation is limited by iron, or whether denitrification is elevated by low oxygen concentrations in the contemporary ocean – and explore these two processes in an historical context.
PP42A-02 INVITED
Evolution of the Nitrogen Cycle, an ~Omics Perspective
Nitrogen is an essential nutrient for life on planet Earth and the transformations between dinitrogen and
various fixed states of reduced and oxidized Nitrogen comprise the extant Nitrogen cycle. With the beginning
of cellular life, the environmental pools of fixed Nitrogen were differentially integrated into both biosynthetic
(energy consuming) and catabolic (energy harnessing) cellular pathways leading to significant shifts in these
pools. The evolution of pertinent protein inventory involved in Nitrogen transformations from its humble
beginnings in the Archean to the extant complex Nitrogen cycle appears to reflect bioavailability of active site
metals (catalytically active transition elements) and there are indications that extant N cycle enzymes have
homologues involved in extant sulfur and carbon cycles. An analysis of this inventory will be presented with
emphasis on inventory involved in extant respiratory and assimilatory ammonification, classical and aerobic
denitrification as well as aerobic (nitrification) and anaerobic (anammox) ammonia oxidation.
http://mgkmicro.com
PP42A-03 INVITED
The Nitrogen Cycle During the Transition to Euxinia
Nitrogen and phosphorous are essential to life, and their biological availability is hypothesized to regulate marine productivity on short and geologic timescales. The nature of primary production during recurrent intervals of Phanerozoic anoxia is of particular interest because of the redox control of nutrient and trace metal availability. Dissolved phosphate likely increased during transitions from oxic to euxinic marine conditions, while nitrogen availability may have decreased due to extensive denitrification as low-oxygen waters spread. Because nitrogen fixation is both metabolically and trace-metal intensive, a key question in the transition to euxinia is whether nitrogen fixation can "keep pace" with denitrification. If denitrification exceeds nitrogen fixation, diminished export production and oxygen demand in an N-limited ocean would pose a negative feedback that may prevent euxinia altogether or initiate the shift back to oxic conditions. Here we use the GENIE-1 Earth system model to address the biogeochemistry of the oxic-euxinic transition characteristic of some Phanerozoic oceanic anoxic events. As previously demonstrated with box models, phosphate accumulation stimulates both nitrogen fixation and denitrification. While there is an initial transient loss of total fixed nitrogen from the ocean, nitrogen inputs eventually exceed losses, and the marine nitrogen reservoir grows with that of phosphate to significantly exceed its modern value. Nitrogen buildup also corresponds with a shift in ecology of the surface ocean and the unexpected initiation of non-Redfieldian stoichiometry in the chemistry of the deep ocean.
PP42A-04 INVITED
Holocene History of the Oceanic N Cycle from N Isotope Records - Implications for Modern Balance or Imbalance
According to current best estimates, the modern ocean's N cycle is in severe deficit. N isotope budgeting provides an independent geochemical constraint in this regard as well as the only means for past reconstruction. Overall, it is the relative proportion of N2 fixation consumed by water column denitrification that sets average oceanic δ15N under steady-state conditions. If an imbalance in oceanic N sources and sinks changes this proportion then a transient in average oceanic δ15N would occur. Using a simple model, changing water column denitrification ±30% or N2 fixation by ±15% produces detectable (>1‰) changes in average oceanic δ15N over one residence time period or more with corresponding changes in oceanic N inventory. Sediment δ15N records from sites thought to be sensitive to oceanic average δ15N all show no detectible change over the last 3 kyr or so implying a balanced marine N budget over the latest Holocene. A mismatch in time scales is the most likely meaningful interpretation of the apparent conflict with modern flux estimates. Decadal to centennial scale oscillations between net N deficit and net surplus may occur but on the N residence timescale of several thousand years, net balance is achieved in sum. However, sediment δ15N records from the literature covering the period since the last glacial maximum show excursions of up to several ‰ that are consistent with sustained N deficit during the deglaciation followed by readjustment and establishment of balance in the early Holocene. Since imbalance was sustained for one N residence time period or longer, excursions in ocean N inventory of 10 to 30% likely occurred. The climatic and oceanographic changes that occurred over this period evidently overcame, for a time, the capacity of ocean biogeochemistry to maintain N balance.
PP42A-05
Dinitrogen and Cyanide Fixation by Methane Seep Microorganisms Revealed by FISH- SIMS And Implications for AOM Productivity and Nitrogenase Evolution
The anaerobic oxidation of methane (AOM), mediated by methane oxidizing archaea (ANME) and sulfate reducing bacterial symbionts (SRB), minimizes the flux of methane from marine sediment to the overlying water column. Understanding the factors determining AOM productivity, and particularly the rates of methane catabolism and anabolism, is of interest to both modern and ancient investigations of climate and bulk carbon isotopic change. It has been hypothesized that nitrogen availability in methane seeps is temporally variable, and that the seep biomass may be at least partially nitrogen limited. The recent finding of nif genes, those necessary for the production of nitrogenase, in enrichments of ANME and SRB consortia suggested that the organisms mediating AOM have the potential to fix dinitrogen. In the present study we incubated methane seep sediment with nitrogen-deplete artificial marine media and a headspace of methane (CH4) and either 15N-labeled dinitrogen (15N2), cyanide (C15N-), or ammonia (15NH3) in order to (1) test the ability of these currently unculturable microorganisms to fix nitrogen and other triple bonded substrates, (2) investigate which AOM partner was responsible for the fixation, (3) compare growth rates on different nitrogen sources, and (4) characterize the phylogeny of these methane seep-associated nitrogenases. Fluorescence in situ hybridization coupled to nano-scale Secondary Ion Mass Spectroscopy imaging (FISH-SIMS) revealed incorporation of 15N into ANME and SRB biomass of up to 0.06 15N fractional abundance in the 15N2 incubation, and up to 0.02 in the C15N- incubation, after 6 and 4 months, respectively. This represents a nearly ten-fold enrichment of 15N compared to the measured natural 15N fractional abundance (0.0036). The NanoSIMS ion images of ANME/SRB aggregates from 15N2 incubations show evidence for 15N enrichment in both partners with the highest incorporation of 15N within the methanotrophic ANME cells. Cyanide incubations revealed a more heterogeneous pattern of 15N distribution, with localized zones of enrichment within both the SRB and the ANME biomass. From these findings, two alternative explanations are considered: (1) both partners are capable of nitrogenase production, but express the nif genes under different conditions, and (2) the distribution of fixed nitrogen within the ANME and SRB is driven by intimate metabolic coupling and resource sharing, with only one partner serving as the primary diazotroph. In incubations with 15NH3, the AOM biomass 15N fractional abundance was nearly 1.0 after 6 months, demonstrating a much faster growth rate when NH3 rather than N2 or CN- is the nitrogen source, consistent with what is observed in other diazotrophic organisms. Nitrogenase genes recovered from these incubations primarily were associated with the nifH group III clade, but the majority were diverged from known nifH sequences. This suggests that novel nitrogenases are responsible for the N2 fixation observed, and the poor substrate specificity and the potential use as a CN- detoxification mechanism could imply that they are similar to the first nitrogenases. The finding that nitrogen fixation occurs within these potentially ancient organisms therefore may provide a window for examining the history and functional diversity of nitrogenase, and the variable growth rates depending on nitrogen substrate could have implications for AOM productivity through time.
PP42A-06
Simulation of Marine Nitrogen Cycling as Function of Atmospheric Oxygen: Results of a Coupled C,N,P,O,S Biogeochemical Model Including d15N
Bioavailable nitrogen is a critical limiting nutrient in the modern marine biosphere. We expect that the rate of denitrification may have been higher in the geologic past due to decreased atmospheric O2 and expanded ocean anoxia. To examine the consequences of this idea, we present numerical simulations of coupled carbon, nitrogen, phosphorus, oxygen, and sulfur cycling as a function of atmospheric oxygen in an ocean with circulation similar to modern conditions. The model has been specifically developed to function over a wide range of ocean redox conditions and has been successfully tested in simulations of both the modern global ocean and Black Sea. Global rates of nitrogen fixation and pelagic denitrification, which are strongly coupled in our default model, reach maximum rates between 25% and 50% of the present atmospheric level of O2 (PAL O2). At 40% PAL O2, the simulated steady-state pelagic denitrification rate is 82.1 Tmol/yr, and the N- fixation rate is 85.7 Tmol/yr. These rates are 8-15× greater than modern estimates. The maximum simulated rate of N-fixation is determined by the N flux required to entirely support export production. At mid- levels of atmospheric oxygen, large areas of the oceans are characterized by a suboxic to anoxic "oxygen minimum zone" between 100m and 1000m depth which is over- and underlain by oxic water. Under these conditions, denitrification in the upper water column is nearly complete, suppressing the δ15N isotopic signal for this process. To test the impact of limitation of N-fixation (e.g. by trace metals, light, temperature) we imposed a cap on the global N-fixation rate. In these simulations, limitation of N-fixation below 50% PAL O2 results in severe N limitation of primary production and low mean oceanic N:P. Our results imply that N limitation may have been chronic at intermediate levels of atmospheric O2. At the same time, low N:P conditions would create evolutionary pressure for efficient N-fixation pathways and high N use efficiency in non-fixing marine phytoplankton, testing the limits of plasticity in the Redfield ratio. If N-fixation were unable to keep up with high rates of denitrification at intermediate levels of atmospheric O2, intense N limitation of Proterozoic marine primary production may have strongly inhibited any further rise of atmospheric O2, thus stabilizing atmospheric O2 at <25% PAL.
PP42A-07
Compound-specific isotope analysis of nitrogen in sedimentary porphyrins: A novel and powerful approach for reconstructing the nitrogen cycle of the past ocean
Nitrogen isotopic composition of organic matter produced by primary photosynthetic organisms is crucial information on the state of their nitrogen metabolisms and thus can be a key to understand nitrogen cycle of the past ocean. Unlike the isotopic signal of bulk sedimentary samples, sedimentary porphyrins, the diagenetic products of chlorophylls, directly reflect isotopic compositions of photoautotrophs. We have developed methods for compound-specific nitrogen isotopic analyses of (1) sedimentary porphyrins that were isolated from extractable organic matter and (2) maleimides, chemical degradation products of porphyrins, that were oxidatively extracted from residual organic matter after organic extraction. The former includes (1) isolation and purification of various species of sedimentary porphyrins using high-performance liquid chromatography and (2) determination of nitrogen isotopic compositions of isolated porphyrins on a high- sensitivity elemental analyzer-isotope ratio mass spectrometer (IRMS) system that allows determination of isotopic composition on nanomole-scale nitrogen. The latter method analyzes isotopic composition of maleimides on gas chromatography-combustion-IRMS. Applying these methods, we determine nitrogen isotopic composition of sedimentary porphyrins from various geological samples from the Archean to the Miocene. In the black shales deposited during the Cretaceous Oceanic Anoxic Events, the nitrogen isotopic composition of porphyrins generally ranged from -7 to -4‰, which suggests that the average δ15N value for the entire photoautotrophic community at these times ranged -3 to +1‰ considering the empirical isotopic relationships that the tetrapyrrole nuclei of chlorophylls are depleted in 15N by ~5‰ relative to cell. This finding suggests that nitrogen utilized in the primary production was supplied mainly through N2-fixation by diazotrophic cyanobacteria in these oceans.
PP42A-08
Hydrologic Control on Bacterial Nitrogen Fixation in the Holocene Black Sea
Stratified oceans of the Phanerozoic Oceanic Anoxic Events apparently were dominated by bacterial nitrogen fixation. Decreased marine N:P nutrient ratios resulting from increased denitrification and decreased phosphate burial efficiency under anoxic waters drove this nutrient regime. This model is upheld by the presence of cyanobacterial hopanoid biomarkers in sedimentary records and δ15N values indicative of nitrogen fixation. However, in the largest modern redox-stratified marine basin, the Black Sea, bacterial nitrogen fixation seems to be only a minor contributor to the nitrogen cycle. In this study, we use geochemical proxies to evaluate the role of bacterial nitrogen fixation during the deposition of the Holocene Black Sea sapropel, starting 7.8 ka. We report compound-specific nitrogen and carbon stable isotope values of pyropheophytin a, a chlorophyll degradation product, and bacteriochlorophyll e produced by green sulfur bacteria. We also present the surprising finding of scytonemin, a pigment produced only by filamentous cyanobacteria exposed to ultraviolet radiation, in certain intervals in these sediments. In the Holocene, nitrogen fixation in the Black Sea is most prominent during times of reduced river water influx. This directly decreases the external flux of nitrate into the surface waters. Reduced freshwater influx also decreases the volume of low salinity water dispersed around the sea by the Rim Current, allowing the chemocline to shoal along the margins. Previous geochemical studies have described this changing chemocline geometry. The exposure of shallow water sediments to anoxic waters further stimulates nitrogen fixation by releasing more phosphorus to the system. Nitrogen fixation is recorded in the sediments as bulk and compound-specific pyropheophytin a δ15N values near 0 ‰ and -5 ‰, respectively. We have also detected scytonemin in two intervals characterized by especially low δ15N values. This compound suggests abundant filamentous cyanobacteria were living at the sea surface, a marked ecological shift from modern phytoplankton distributions. These data support the hypothesis that bacterial nitrogen fixation, at times, contributed significantly to the Black Sea nitrogen cycle. Interestingly, nitrogen fixation did not dominate the entire time period of sapropel sedimentation and stable stratification. Normal marine δ15N values coincide with periods of Black Sea level high stand and a deeper marginal chemocline.