Nathaniel E. Ostrom

Distinguishing Nitrous Oxide Production from Nitrification and Denitrification on the Basis of Isotopomer Abundances

ABSTRACT The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 ± 1.2‰, 32.5 ± 0.6‰, and 35.6 ± 1.4‰ for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 ± 4.2‰) was similar to that produced during hydroxylamine oxidation (33.5 ± 1.2‰) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 ± 1.7‰), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (−0.6 ± 1.9‰ and −0.5 ± 1.9‰, respectively) were similar to those during nitrate reduction (−0.5 ± 1.9‰ and −0.5 ± 0.6‰, respectively), indicating no influence of either substrate on site preference. Site preferences of ∼33‰ and ∼0‰ are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.

THERMODYNAMIC CONSTRAINTS ON NITROGENTRANSFORMATIONS AND OTHER BIOGEOCHEMICALPROCESSES AT SOIL–STREAM INTERFACES

There is much interest in biogeochemical processes that occur at the interface between soils and streams since, at the scale of landscapes, these habitats may function as control points for fluxes of nitrogen (N) and other nutrients from terrestrial to aquatic ecosystems. Here we examine whether a thermodynamic perspective can enhance our mechanistic and predictive understanding of the biogeochemical function of soil–stream interfaces, by considering how microbial communities interact with variations in supplies of electron donors and acceptors. Over a two-year period we analyzed >1400 individual samples of subsurface waters from networks of sample wells in riparian wetlands along Smith Creek, a first-order stream draining a mixed forested–agricultural landscape in southwestern Michigan, USA. We focused on areas where soil water and ground water emerged into the stream, and where we could characterize subsurface flow paths by measures of hydraulic head and/or by in situ additions of hydrologic tracers. We found strong support for the idea that the biogeochemical function of soil–stream interfaces is a predictable outcome of the interaction between microbial communities and supplies of electron donors and acceptors. Variations in key electron donors and acceptors (NO3−, N2O, NH4+, SO42−, CH4, and dissolved organic carbon [DOC]) closely followed predictions from thermodynamic theory. Transformations of N and other elements resulted from the response of microbial communities to two dominant hydrologic flow paths: (1) horizontal flow of shallow subsurface waters with high levels of electron donors (i.e., DOC, CH4, and NH4+), and (2) near-stream vertical upwelling of deep subsurface waters with high levels of energetically favorable electron acceptors (i.e., NO3−, N2O, and SO42−). Our results support the popular notion that soil–stream interfaces can possess strong potential for removing dissolved N by denitrification. Yet in contrast to prevailing ideas, we found that denitrification did not consume all NO3− that reached the soil–stream interface via subsurface flow paths. Analyses of subsurface N chemistry and natural abundances of δ15N in NO3− and NH4+ suggested a narrow near-stream region as functionally the most important location for NO3− consumption by denitrification. This region was characterized by high throughput of terrestrially derived water, by accumulation of dissolved NO3− and N2O, and by low levels of DOC. Field experiments supported our hypothesis that the sustained ability for removal of dissolved NO3− and N2O should be limited by supplies of oxidizable carbon via shallow flowpaths. In situ additions of acetate, succinate, and propionate induced rates of NO3− removal (∼1.8 g N·m−2·d−1) that were orders of magnitude greater than typically reported from riparian habitats. We propose that the immediate near-stream region may be especially important for determining the landscape-level function of many riparian wetlands. Management efforts to optimize the removal of NO3− by denitrification ought to consider promoting natural inputs of oxidizable carbon to this near-stream region.

Biphenyl-utilizing bacteria and their functional genes in a pine root zone contaminated with polychlorinated biphenyls (PCBs)

Bacteria and functional genes associated with biphenyl (BP) degradation in the root zone of an Austrian pine (Pinus nigra L.) growing naturally in polychlorinated-BP (PCB)-contaminated soil were identified using stable isotope probing (SIP) integrated with comprehensive functional gene analyses. SIP revealed 75 different genera that derived carbon from 13C-BP, with Pseudonocardia, Kribella, Nocardiodes and Sphingomonas predominating carbon acquisition. Rhodococcus spp. were not detected with SIP, despite being the most abundant BP utilizers isolated from agar plates. Only one organism, an Arthrobacter spp., was detected as a BP utilizer by both cultivation and SIP methods. Time-course SIP analyses indicated that secondary carbon flow from BP-utilizing bacteria into other soil organisms may have occurred largely between 4 and 14 days incubation. Functional gene contents of the BP-utilizing metagenome (13C-DNA) were explored using the GeoChip, a functional gene array containing 6465 probes targeting aromatic degradative genes. The GeoChip detected 27 genes, including several associated with catabolism of BP, benzoate and a variety of aromatic ring hydroxylating dioygenase (ARHD) subunits. Genes associated with the β-ketoadipate pathway were also detected, suggesting a potential role for this plant aromatic catabolic pathway in PCB degradation. Further ARHD analyses using targeted polymerase chain reaction primers and sequence analyses revealed novel dioxygenase sequences in 13C-DNA, including several sequences that clustered distantly from all known ARHDs and others that resembled known Rhodococcus ARHDs. The findings improve our understanding of BP degradation and carbon flow in soil, reveal the extent of culture bias, and may benefit bioremediation research by facilitating the development of molecular tools to detect, quantify and monitor populations involved in degradative processes.

Isotopologue fractionation during N2O production by fungal denitrification

Identifying the importance of fungi to nitrous oxide (N2O) production requires a non-intrusive method for differentiating between fungal and bacterial N2O production such as natural abundance stable isotopes. We compare the isotopologue composition of N2O produced during nitrite reduction by the fungal denitrifiers Fusarium oxysporum and Cylindrocarpon tonkinense with published data for N2O production during bacterial nitrification and denitrification. The fractionation factors for bulk nitrogen isotope values for fungal denitrification were in the range -74.7 to -6.6 per thousand. There was an inverse relationship between the absolute value of the fractionation factors and the reaction rate constant. We interpret this in terms of variation in the relative importance of the rate constants for diffusion and enzymatic reduction in controlling the net isotope effect for N2O production during fungal denitrification. Over the course of nitrite reduction, the delta(18)O values for N2O remained constant and did not exhibit a relationship with the concentration characteristic of an isotope effect. This probably reflects isotopic exchange with water. Similar to the delta(18)O data, the site preference (SP; the difference in delta(15)N between the central and outer N atoms in N2O) was unrelated to concentration during nitrite reduction and, therefore, has the potential to act as a conservative tracer of production from fungal denitrification. The SP values of N2O produced by F. oxysporum and C. tonkinense were 37.1 +/- 2.5 per thousand and 36.9 +/- 2.8 per thousand, respectively. These SP values are similar to those obtained in pure culture studies of bacterial nitrification but quite distinct from SP values for bacterial denitrification. The large magnitude of the bulk nitrogen isotope fractionation and the delta(18)O values associated with fungal denitrification are distinct from bacterial production pathways; thus multiple isotopologue data holds much promise for resolving bacterial and fungal production. Our work further provides insight into the role that fungal and bacterial nitric oxide reductases have in determining site preference during N2O production.

The origin and cycling of particulate and sedimentary organic matter and nitrate in Lake Superior

Abstract The elemental and isotopic composition of water column particulate and sedimentary organic matter and nitrate in Lake Superior was determined to assess the origin and cycling of these materials. The δ 15 N and δ 13 C of sedimentary organic matter and suspended particles at three stations were consistent with an origin primarily from autochthonous production. The δ 15 N of seston was controlled by a balance between the isotope effects associated with nitrate uptake and microbial degradation. The ratio of chlorophyll fluorescence to light hindrance (100-transmittance) was used in this study as an indication of the relative composition of recently produced photosynthetic vs. refractory and non-photosynthetic materials (such as bacteria or microzooplankton). Chlorophyll fluorescence to light hindrance (CF:LH) ratios were greatest within the region of the thermocline at the shallow station and lowest in the near bottom waters of the unstratified deep station. These changes in CF:LH indicated a predominance of recently produced photosynthetic material in surface stratified waters and an increase in refractory or non-photosynthetic material at the deepest station relative to the shallow station. Seston at the deepest station was characterized by the highest δ 15 N value of the three stations suggesting that degradation, bacterial growth, and/or an enrichment in microzooplankton resulted in an increase in the 15 N content of seston. Seston at the shallowest station was characterized by high CF:LH ratios and low δ 15 N values suggesting a greater relative contribution of labile material and an influence of an isotope effect during nutrient assimilation. Suspended particles in the benthic nepheloid layer were characterized by marked depletions in 13 C and 15 N relative to seston and sedimentary organic matter and indicated a unique origin for this material, possibly from recent primary production. The δ 15 N and δ 13 C of particles within the sediment boundary layer were intermediate those of sediments and nepheloid layer particles and were suggestive of an origin from the mixing of these two materials. Nitrate in Lake Superior was characterized by the lowest δ 15 N reported for an aquatic environment (average of −4.1‰). These low δ 15 N values and large input of water from precipitation directly to the lake surface suggests that much of the nitrate in Lake Superior is derived from atmospheric deposition.

Mechanisms of nitrous oxide production in the subtropical North Pacific based on determinations of the isotopic abundances of nitrous oxide and di-oxygen

Abstract In this study, we compare stable isotopic compositions of di-oxygen (O 2 ) and nitrous oxide (N 2 O) in two depth profiles at the well-characterized deep water station ALOHA (A Long-term Oligotrophic Habitat Assessment) in the subtropical North Pacific gyre to attain an understanding of the mechanisms of N 2 O production. The δ 18 O of O 2 varied from values indicative of an atmospheric origin near the surface ( 24.7‰ ), to minimum values reflective of a predominance of photosynthesis over respiration between the surface and 200 m (as low as 22.2 ‰ ), to maximum values as high as 36.6‰ in association with the O 2 minimum near 800 m. A similar pattern of isotopic variation was evident in the δ 18 O of N 2 O, however, values were enriched by approximately 20‰ . The similar pattern of variation in δ 18 O with depth is consistent with an origin of O in N 2 O from dissolved O 2 via the nitrification of intermediate compounds NH 2 OH or NO. Between the depths of 350 and 500 m, however, the distinction in the isotopic composition of N 2 O and O 2 was reduced to as little as 12‰ . This decrease in the difference between the δ 18 O of N 2 O and that of O 2 with depth indicates either a reduction in the magnitude of isotopic discrimination during nitrification or a contribution of O in N 2 O from water via the reduction of NO 2 − . Two mechanisms of N 2 O production via nitrification may, therefore, occur in the subtropical Pacific; release from the nitrification of NH 2 OH or NO at most depths and reduction of NO 2 − between 350 and 500 m. In that, the carbon flux decreases markedly over a similar depth interval at this locale (Karl, D.M., Knauer, G.A., Martin, J.H., 1988. Downward flux of particulate organic matter in the ocean: A particle decomposition paradox. Nature 332, 438–441), this distinct mechanism of N 2 O production between 350 and 500 m may be associated with the mineralization of organic matter from sinking particles. Low O 2 or anoxic micro-environments within particles within this depth interval may be maintained by lower ambient O 2 than at the surface and high rates of microbial activity supported by the mineralization of organic matter. Such conditions may facilitate an environment conducive to N 2 O production via NO 2 − reduction.

Stable nitrogen isotope dynamics of dissolved nitrate in a transect from the North Pacific Subtropical Gyre to the Eastern Tropical North Pacific

Abstract The stable nitrogen isotopic composition of nitrate, concentrations of inorganic nitrogen and phosphorus, dissolved oxygen and nitrification rates were determined at six stations ranging from the oligotrophic North Pacific Subtropical Gyre (NPSG) to the more productive Eastern Tropical North Pacific (ETNP). Nitrification rates increased along the transect from a maximum rate of 1 nmol L −1 d −1 at station ALOHA to 23.7 nmol L −1 d −1 at station 6. In oxic surface waters, nitrate isotopically enriched in 15 N (maximum δ 15 N-NO 3 − value of 12.5‰) was most likely the result of assimilatory nitrate reduction. In contrast, high δ 15 N-NO 3 − values (maximum of 12.3‰) in association with high nitrate deficits and anoxic conditions supported the interpretation of isotopic fractionation due to denitrification. A one-dimensional vertical advection and diffusion model was used to estimate the fractionation factor for denitrification at two stations in the ETNP. A comparison of modeled to observed δ 15 N-NO 3 − data indicated an isotopic enrichment factor (e) of 30‰ at station 4 and 30 to 35‰ at station 5. Isotopically light nitrate (1.1 and 3.2‰) was observed in the upper 200 m of the water column at stations in the ETNP. Tracer studies of 15 NH 4 and biogeochemical indicators of nitrogen fixation supported the interpretation of nitrification as the most plausible explanation for low δ 15 N-NO 3 − values observed in water column samples. Our results are consistent with the occurrence of nitrification within the euphotic zone and for the first time provide corroborating stable nitrogen isotopic evidence for this process.

Microbial life at −13 °C in the brine of an ice-sealed Antarctic lake

The permanent ice cover of Lake Vida (Antarctica) encapsulates an extreme cryogenic brine ecosystem (−13 °C; salinity, 200). This aphotic ecosystem is anoxic and consists of a slightly acidic (pH 6.2) sodium chloride-dominated brine. Expeditions in 2005 and 2010 were conducted to investigate the biogeochemistry of Lake Vida’s brine system. A phylogenetically diverse and metabolically active Bacteria dominated microbial assemblage was observed in the brine. These bacteria live under very high levels of reduced metals, ammonia, molecular hydrogen (H2), and dissolved organic carbon, as well as high concentrations of oxidized species of nitrogen (i.e., supersaturated nitrous oxide and ∼1 mmol⋅L−1 nitrate) and sulfur (as sulfate). The existence of this system, with active biota, and a suite of reduced as well as oxidized compounds, is unusual given the millennial scale of its isolation from external sources of energy. The geochemistry of the brine suggests that abiotic brine-rock reactions may occur in this system and that the rich sources of dissolved electron acceptors prevent sulfate reduction and methanogenesis from being energetically favorable. The discovery of this ecosystem and the in situ biotic and abiotic processes occurring at low temperature provides a tractable system to study habitability of isolated terrestrial cryoenvironments (e.g., permafrost cryopegs and subglacial ecosystems), and is a potential analog for habitats on other icy worlds where water-rock reactions may cooccur with saline deposits and subsurface oceans.

NITROGEN TRANSFORMATIONS AND NO3− REMOVAL AT A SOIL–STREAM INTERFACE: A STABLE ISOTOPE APPROACH

The natural removal of NO3− by denitrification within riparian zones of streams and rivers is an area of considerable interest owing to its potential to minimize the impacts of excess anthropogenic loadings. In this study we utilize natural variations in stable N isotopic compositions of NO3− and NH4+ within a transect of shallow wells extending 4 m inland from Smith Creek, a southwestern Michigan stream, to provide insight into microbial processes and the extent of NO3− removal within a soil–stream interface. Within this region three water masses with unique biogeochemical characteristics intersect: a shallow flow rich in NH4+ and dissolved organic carbon (DOC), a deep groundwater mass rich in NO3− but depleted in DOC, and stream water low in NO3−, NH4+, and DOC. N isotope values for NO3− within the well transect were highly variable (−7.7–34.1‰) and reflected intense microbial activity within this narrow region. Isotopic variation was primarily controlled by upwelling of deep groundwater near the stream...

Insights into the origin of perylene from isotopic analyses of sediments from Saanich Inlet, British Columbia

Perylene is an abundant and common polycyclic aromatic hydrocarbon in sedimentary settings, yet its origin remains puzzling. We have investigated the relation of perylene to the amount and type of organic matter in the sediments of Saanich Inlet, a coastal marine anoxic basin. Organic matter is predominantly marine in origin, but the proportions of marine and land-derived components have varied. Perylene concentrations generally increase with sediment depth, relative to TOC, which indicates continued formation of this compound by microbially mediated diagenesis. Perylene δ13C values range between −27.7 and −23.6‰, whereas TOC δ13C values vary narrowly from −21.7 to −21.2‰ over the same sediment depth interval. The variation in isotopic difference suggests that perylene originates from more than one precursor material, both aquatic and continental organic matter, different microbial processes, or some combination of these possibilities.