This project is a collaborative effort between the Joye Research Group and Drs. Mike Madigan (Southern Illinois University Carbondale) and Karen Casciotti (Stanford University). We are working at sites in the Antarctic dry valleys, including Lake Vanda, on Cape Bird, and in the Labryinth. We are examining the processes that produce nitrous oxide and the isotopic composition of that nitrous oxide. We are measuring environmental fluxes of nitrous oxide to the atmosphere and conducting laboratory experiments to identify the production mechanism(s) of nitrous oxide.
Nitrous oxide is an atmospheric trace gas. On Earth, biological processes, mainly nitrification and denitrification, are thought to dominate nitrous oxide production in the environment. We recently measured large areal fluxes of nitrous oxide to the atmosphere in a cold, hypersaline environment: Don Juan Pond, Wright Valley, Antarctica. Stable isotopic measurements showed that this nitrous oxide had a unique depletion of heavy nitrogen (15N) in both the internal and external positions of the linear nitrous oxide molecule. This pattern of 15N distribution resulted in a site preference that was low and sometimes negative (down to -45‰). In contrast, biological processes generate a positive site preference in nitrous oxide. Laboratory experiments of Don Juan Pond samples revealed an abiotic production mechanism, whereby nitrous oxide was produced during reaction of nitrate-rich brine with a variety of Fe(II)-containing minerals. The process of “chemodenitrification” we documented represents a novel mechanism of nitrous oxide production that could be important in other Earth habitats and on Mars.
In this project, we are studying the distribution of chemodenitrification in nitrate-rich, cold, desert habitats in Antarctica to evaluate whether this process is widespread and environmentally relevant. We will describe, in detail, the kinetics of chemodenitrification by evaluating rates of nitrate reduction/nitrous oxide production fueled by Fe(II) derived from pure mineral phases (in the laboratory) or by Fe(II) contained in geologic field samples from Antarctic sites. We will characterize the isotopic signature of chemodenitrification-derived nitrous oxide. At selected field sites, we will compare the magnitude of nitrous oxide fluxes and the isotopic signatures of nitrous oxide at sites where biological vs. abiotic production mechanisms dominate. We will quantify the importance of biological and abiotic processes explicitly. By determining the isotopic composition of reactants (nitrate, nitrite) and product (nitrous oxide), the production mechanism (biotic or abiotic), and the environmental factors that influence the production rate (e.g. temperature), we will develop an index to describe the isotopic composition of nitrous oxide. This index can be used to evaluate whether nitrous oxide in a given environment is derived from biological or abiotic processes. The brine-rock reactions we propose to study could return nitrogen oxides in Martian soils and brines to the atmosphere, providing a potentially dynamic and unexpected link between the geosphere and atmosphere. The isotopic composition of this nitrous oxide may provide a new and robust way to distinguish nitrous oxide arising from biological processes from that arising from chemodenitrification.
Through this project, we will: 1) identify novel sources of an atmospheric trace gas, nitrous oxide; 2) advance the knowledge of environmental cycling of a key elemental building block of life, nitrogen; 3) identify novel geosphere-brine reactions that facilitate nitrogen recycling between the lithosphere and atmosphere; and, 4) evaluate the utility of nitrogen stable isotopes in nitrous oxide as an unambiguous biosignature for life.
Background and Relevance
Nitrogen is an essential building block for microbial life as we know it and, on Earth, nitrogen cycling is dominated by microbially-mediated processes. “Follow the nitrogen” has been proposed as a robust way to identify potential niches for life on extraterrestrial bodies. Studies of nitrogen cycling in Mars analog environments can provide crucial information for understanding pathways of nitrogen flow on Mars, for distinguishing biological versus chemical mechanisms of nitrogen transformation, and for evaluating potential nitrogen biosignatures. On Mars, only a small fraction of the nitrogen presumably received during planetary accretion is now present in the atmosphere. The remaining nitrogen is presumed to be located in the subsurface regolith or in brines on the Martian surface as nitrate salts or lattice-bound ammonium.
Mancinelli and Banin (2003) proposed that a suite of abiotic reactions cycled nitrogen on early Earth and Mars (Figure 1). Thermal shock and lightning converted dinitrogen to nitric oxide, which was further transformed to nitrous oxide, nitrate and nitrite in aqueous solution.
The ultimate product of atmospheric thermal shock was nitrite and nitrate. Nitrate accumulated in solution because it is soluble and because it reacted more slowly with Fe(II) than did nitrite (Figure 1). Such a series of events could lead to accumulation of nitrate in the Mars subsurface. On Earth today, substantial accumulation of nitrate in soils is rare, because nitrate is consumed rapidly by biological processes. However, substantial accumulation of nitrates occurs in desert soils. Atmospheric deposition of nitrates and accumulation over periods varying between 104 to 107 yr has been invoked to explain nitrate accumulation in desert soils from Death Valley (California) and the Atacama Desert (Chile). Similarly, soils in the McMurdo Dry Valleys in Antarctica are rich in nitrate and the isotopic composition of this nitrate indicates an atmospheric source. In this project, we aim to study nitrate dynamics in the cold desert of the Antarctic Dry Valleys where extensive soil nitrate accumulation is well documented.
On Earth, nitrogen exists in three primary ionic forms: ammonium (NH4+), nitrite (NO2-), and nitrate (NO3-); a large variety of simple and complex organic molecules, i.e. urea, proteins, amino acids, DNA; and two dominant gases: nitrous oxide (N2O) and dinitrogen (N2). Fixed nitrogen refers to nitrogen present as ammonium, nitrate, nitrite, or simple organic molecules; this pool of nitrogen is readily available to microorganisms and is rapidly cycled in the geosphere via microbially-mediated processes (Figure 2). In this project, we focus on distinguishing microbiologically-mediated nitrous oxide from chemodenitrification, whereby nitrous oxide is produced abiotically. These processes share a common product, nitrous oxide, which is an important atmospheric trace gas and holds the potential to serve as an atmospheric biosignature for life.
Nitrous oxide is an atmospheric trace gas with a greenhouse warming potential 125 times that of carbon dioxide; it contributes significantly to stratospheric ozone degradation. Nitrification, the microbial oxidation of ammonia to nitrite and nitrite to nitrate, is restricted to oxic and microaerophilic soils and waters while microbial denitrification is restricted to anoxic soils and waters. Despite their different requirements for and tolerance of oxygen, the microorganisms involved in nitrification and denitrification often live in close physical proximity and their activities may be tightly coupled, resulting in efficient conversion of ammonium to nitrite/nitrate, and conversion of this nitrite/nitrate to gaseous end-products. In addition, nitrifying microbes alone can produce N2O, without the assistance of denitrifying bacteria. Nitrous oxide production during nitrification and denitrification results from decomposition of unstable intermediates, enzymatic short-circuits, or enzymatic inhibition (e.g. sulfide inhibition of the nitrous oxide reductase in denitrifying bacteria). Some denitrifying microorganisms lack the enzymatic machinery for complete denitrification (to N2) and produce nitrous oxide as their primary product.
An alternate mechanism of nitrous oxide production is chemodenitrification, the consumption of oxidized nitrogen species (mainly nitrate and nitrite but in some cases hydroxylamine) by non-enzymatic means. Chemodenitrification has thus far been shown to produce nitric oxide, nitrous oxide, and in some cases, ammonium. In most cases nitrous oxide is a minor product, but recent work suggests that nitrous oxide was the primary product of nitrite reduction with siderite (FeCO3). This process, though rarely studied, has been documented in acidic (pH<4) and neutral soils and is facilitated by the presence of ferrous iron or organic matter.
Some Antarctic brines, e.g. Lake Bonney, are characterized by high dissolved nitrate concentrations (>100 µM) and extreme super-saturation of nitrous oxide. Previous studies in Lakes Bonney, Hoare, and Vanda invoked biological processes, mainly denitrification, to explain high nitrous oxide concentrations. In a recent study of an Antarctic extreme environment, Don Juan Pond, we discovered a novel abiotic nitrate reduction pathway that proceeds via brine-rock interaction and produces predominantly nitrous oxide (Samarkin et al. 2010; Figure 3). Samarkin et al. observed nitrous oxide production from brine-derived nitrate only in the presence of dolerite (or other Fe(II)-containing minerals), whereas no nitrous oxide production was observed in the absence of mineral reactants or with dolerite incubated in sterile brine solutions lacking nitrate or nitrite. These results point to the intriguing possibility that abiotic processes, namely chemodenitrification, may contribute to the anomalous accumulation of nitrous oxide previously documented in Antarctic brines. We are evaluating this possibility in detail through the studies.
The fact that nitrous oxide can be produced rapidly via abiotic processes means that the presence of nitrous oxide in the atmosphere or in a particular habitat cannot be used as an unambiguous indicator of biological activity. Nonetheless, it may be possible to distinguish biologically-derived from chemically-derived nitrous oxide using the N isotopic distribution in the linear nitrous oxide molecule (N=N=O). The site preference (SP) is the difference between the nitrogen isotope ratio in the central nitrogen (Na) and the terminal nitrogen (Nb) atoms: SP = d15Na- d15Nb and hypothesized that the preferential distribution of 15N within the nitrous oxide molecule would be largely determined by the mechanism of its production. In Don Juan Pond field samples, the bulk nitrogen (d15N-N2O: -39.9 ± 5.1‰) and oxygen (d 18O-N2O: 58.1 ± 10‰) isotopic compositions were comparable to some of those observed in the east lobe of Lake Bonney, Antarctica. However, the chemodenitrification-derived nitrous oxide from Don Juan Pond soils had a low to negative SP, with values as low as -45‰ and a median of +1.2‰, whereas the average SP in Lake Bonney was +20‰. We postulate that the low, sometimes negative, site preferences observed in Don Juan Pond samples reflect the abiotic production mechanism.
The McMurdo Dry Valleys in Antarctica contains numerous Mars analog habitats. In fact, the Beacon Valley region, Antarctica, is one of the best analogs for the Mars Phoenix landing site (http://phoenix.lpl.arizona.edu/02_03_09_pr.php). The NaCl/CaCl/Na2SO4 nitrate-rich brines present in diverse habitats across the McMurdo Dry Valleys, Antarctica, hold similarities to predicted eutectic brines on Mars. Evidence for flowing liquids on the Martian surface is most easily explained by the presence of highly concentrated brines with low freezing points, as such fluids would be stable at the temperatures and pressures of the Martian surface. Moreover, dry valley soils are rich in iron(II), similar to the basalts of the Martian regolith. Together, the nitrate-rich brine and local geology make the Antarctic Dry Valleys ideal Mars analog sites. The brine-rock reactions we propose to study are not currently considered in models of the Martian nitrogen cycle (Figure 1 above). In fact, transfer of atmospheric nitrogen to the soils by the processes described above has been considered largely unidirectional and irreversible. Using nitrous oxide isotopic signatures to indicate biological versus chemical origin would provide a novel way to assess the potential signatures of microbial life on Mars.
Hypotheses and Objectives
Our overarching hypothesis is that chemodenitrification is a key mechanism of nitrous oxide formation in Mars-analog Antarctic desert habitats. Our work will addresses three major hypotheses:
Hypothesis 1. Brine-derived nitrate and nitrite react abiotically with mineral-derived Fe(II) to produce nitrous oxide.
Hypothesis 2. The spatial distribution of nitrous oxide production via chemodenitrification versus biological processes is distinct and dependent on local mineralogy.
Hypothesis 3. Nitrous oxide derived from chemodenitrification is chemically distinct from biologically-derived nitrous oxide in terms of its N isotopomer distribution and calculated site preference.
To test these three hypotheses, we are conducting fieldwork and laboratory experiments that address the following objectives. The approach for addressing each objective is given in italics.
Objective 1. Determine the kinetics and production mechanism of nitrous oxide production via chemodenitrification in laboratory experiments using sterile artificial brine and sterile pure Fe(II)-containing minerals.
-We are performing controlled laboratory experiments at 8ºC (comparable to the air temperature in Antarctica in summer) with pure mineral phases, including dolerite, basalt, olivine, wusite, augite, pigeonite, siderite, and Fe0, to describe the kinetics and stoichiometry of abiotic nitrous oxide production.
Objective 2. Quantify the bulk and intramolecular isotopic signatures of nitrous oxide produced during laboratory chemodenitrification experiments.
-We are quantifying the bulk and intramolecular stable N and O isotopic signatures of nitrous oxide and substrate (nitrate) in samples from laboratory experiments using isotope ratio monitoring mass spectrometry.
Objective 3. Determine the spatial variability of sediment/soil nitrous oxide concentrations and fluxes in multiple Antarctic habitats.
-We are assessing the spatial variability in brine and soil nitrous oxide concentrations and soil-atmosphere nitrous oxide fluxes at different Antarctic habitats that have different source terms (biotic or abiotic; site details provided Section 4.B.1.).
Objective 4. Quantify rates of abiotic and biotic nitrous oxide production in field samples.
- We are performing controlled experiments with sterilized field samples (autoclaved soil/rock material and sterile-filtered brine) to describe the kinetics and stoichiometry of abiotic nitrous oxide production. In parallel, we will perform experiments to quantify rates of total nitrous oxide production (biotic and abiotic). The relative importance of biological versus abiotic processes will be evaluated by the difference between the sterilized and unsterilized materials.
Objective 5. Quantify the bulk and intramolecular isotopic signature of inorganic nitrogen precursors and nitrous oxide in field samples.
-We are quantifying the bulk and intramolecular stable N and O isotopic signatures of nitrous oxide and substrate (nitrate, nitrite) in samples from abiotic and biological rate assays of field samples.
Objective 6. Develop an index to describe the isotopic composition of nitrous oxide as a function of its production mechanism, biotic or abiotic, and environment (mineralogy, brine composition, temperature, etc.)
-Using data from abiotic and biotic experiments, we are developing a robust index that permits delineation of nitrous oxide as a function of its production mechanism.
Vladimir Samarkin (Research Scientist)
Charles Schutte (PhD student)