Abundance and diversity of archaeal ammonia oxidizers in a coastal groundwater system

Author Posting. © The Author(s), 2010. This is the author's version of the work. It is posted here by permission of American Society for Microbiology for personal use, not for redistribution. The definitive version was published in Applied and Environmental Microbiology 76 (2010): 7938-7948, doi:10.1128/AEM.02056-09.Nitrification, the microbially-catalyzed oxidation of ammonia to nitrate, is a key process in the nitrogen cycle. Archaea have been implicated in the first part of the nitrification pathway (oxidation of ammonia to nitrite), but the ecology and physiology of these organisms remain largely unknown. This work describes two different populations of sediment-associated ammonia-oxidizing archaea (AOA) in a coastal groundwater system on Cape Cod, Massachusetts. Sequence analysis of the ammonia monooxygenase subunit A gene (amoA) shows that one population of putative AOA inhabits the upper meter of the sediment where they may experience frequent ventilation with tidally-driven overtopping and infiltration of bay water supplying dissolved oxygen, ammonium and perhaps organic carbon. A genetically distinct population occurs deeper in the sediment, in a mixing zone between a nitrate- and oxygen-rich freshwater zone and a reduced, ammonium-bearing salt water wedge. Both of these AOA populations are coincident with increases in the abundance of Group I crenarchaeota 16S rRNA gene copies.Funding was provided by WHOI’s Coastal Ocean Institute to DRR and KLC, as well as funding from NSF/OCE project #05-24994 to KLC

Biogeochemical controls and isotopic signatures of nitrous oxide production by a marine ammonia-oxidizing bacterium

Nitrous oxide (N2O)[N subscript 2 O] is a trace gas that contributes to the greenhouse effect and stratospheric ozone depletion. The N2O [N subscript 2 O] yield from nitrification (moles N2O-N [N subscript 2 O - N] produced per mole ammonium-N consumed) has been used to estimate marine N2O [N subscript 2 O] production rates from measured nitrification rates and global estimates of oceanic export production. However, the N2O [N subscript 2 O] yield from nitrification is not constant. Previous culture-based measurements indicate that N2O [N subscript 2 O] yield increases as oxygen (O2) [O subscript 2] concentration decreases and as nitrite (NO2−) [NO subscript 2 overscore] concentration increases. Here, we have measured yields of N2O [N subscript 2 O] from cultures of the marine β-proteobacterium [beta-proteobacterium] Nitrosomonas marina C-113a as they grew on low-ammonium (50 μM)[50 mu M] media. These yields, which were typically between 4 × 10−4 [10 superscript -4] and 7 × 10−4 [10 superscript -4] for cultures with cell densities between 2 × 102 [10 super script 2] and 2.1 × 104 [10 superscript 4] cells ml−1 [ml superscript -1], were lower than previous reports for ammonia-oxidizing bacteria. The observed impact of O2 [O subscript 2] concentration on yield was also smaller than previously reported under all conditions except at high starting cell densities (1.5 × 106 cells ml−1) [1.5 x 10 superscript 6 cells ml superscript -1], where 160-fold higher yields were observed at 0.5% O2 [O subscript 2](5.1 μM [mu M] dissolved O2 [O subscript 2]) compared with 20% O2 [O subscript 2] (203 μM [mu M] dissolved O2 O subscript 2]). At lower cell densities (2 × 102 [10 superscript 2] and 2.1 × 104 [10 superscript 4] cells ml−1 [ml superscript -1]), cultures grown under 0.5% O2 [O subscript 2] had yields that were only 1.25- to 1.73-fold higher than cultures grown under 20% O2 [O subscript 2]. Thus, previously reported many-fold increases in N2O [N subscript 2 O] yield with dropping O2 [O subscript 2] could be reproduced only at cell densities that far exceeded those of ammonia oxidizers in the ocean. The presence of excess NO2− [NO subscript 2 overscore] (up to 1 mM) in the growth medium also increased N2O [N subscript 2 O] yields by an average of 70% to 87% depending on O2 [O subscript 2] concentration. We made stable isotopic measurements on N2O [N subscript 2 O] from these cultures to identify the biochemical mechanisms behind variations in N2O [N subscript 2 O] yield. Based on measurements of δ15Nbulk [delta superscript 15 N superscript bulk], site preference (SP = δ15Nα−δ15Nβ [delta superscript 15 N superscript alpha - delta superscript 15 N superscript beta]), and δ18O [delta superscript 18 O] of N2O [N subscript 2 O] (δ18O-N2O [delta superscript 18 O - N subscript 2 O]), we estimate that nitrifier-denitrification produced between 11% and 26% of N2O [N subscript 2 O] from cultures grown under 20% O2 [O subscript 2] and 43% to 87% under 0.5% O2 [O subscript 2]. We also demonstrate that a positive correlation between SP and δ18O-N2O [delta superscript 18 O - N subscript 2 O] is expected when nitrifying bacteria produce N2O [N subscript 2 O]. A positive relationship between SP and δ18O-N2O [delta superscript 18 O - N subscript 2 O] has been observed in environmental N2O [N subscript 2 O] datasets, but until now, explanations for the observation invoked only denitrification. Such interpretations may overestimate the role of heterotrophic denitrification and underestimate the role of ammonia oxidation in environmental N2O [N subscript 2 O] production

Nitrite cycling in the primary nitrite maxima of the eastern tropical North Pacific

The primary nitrite maximum (PNM) is a ubiquitous feature of the upper ocean, where nitrite accumulates in a sharp peak at the base of the euphotic zone. This feature is situated where many chemical and hydrographic properties have strong gradients and the activities of several microbial processes overlap. Near the PNM, four major microbial processes are active in nitrite cycling: ammonia oxidation, nitrite oxidation, nitrate reduction and nitrite uptake. The first two processes are mediated by the nitrifying archaeal/bacterial community, while the second two processes are primarily conducted by phytoplankton. The overlapping spatial habitats and substrate requirements for these microbes have made understanding the formation and maintenance of the PNM difficult. In this work, we leverage high-resolution nutrient and hydrographic data and direct rate measurements of the four microbial processes to assess the controls on the PNM in the eastern tropical North Pacific (ETNP). The depths of the nitrite maxima showed strong correlations with several water column features (e.g., top of the nitracline, top of the oxycline, depth of the chlorophyll maximum), whereas the maximum concentration of nitrite correlated weakly with only a few water column features (e.g., nitrate concentration at the nitrite maximum). The balance between microbial production and consumption of nitrite was a poor predictor of the concentration of the nitrite maximum, but rate measurements showed that nitrification was a major source of nitrite in the ETNP, while phytoplankton release occasionally accounted for large nitrite contributions near the coast. The temporal mismatch between rate measurements and nitrite standing stocks suggests that studies of the PNM across multiple timescales are necessary.</p

Oxygen isotopic composition of nitrate and nitrite produced by nitrifying cocultures and natural marine assemblages

Author Posting. © Association for the Sciences of Limnology and Oceanography, 2012. This article is posted here by permission of Association for the Sciences of Limnology and Oceanography for personal use, not for redistribution. The definitive version was published in Limnology and Oceanography 57 (2012): 1361-1375, doi:10.4319/lo.2012.57.5.1361.The δ18O value of nitrate produced during nitrification (δ18ONO3,nit) was measured in experiments designed to mimic oceanic conditions, involving cocultures of ammonia-oxidizing bacteria or ammonia-oxidizing archaea and nitrite-oxidizing bacteria, as well as natural marine assemblages. The estimates of ranged from −1.5‰ ± 0.1‰ to +1.3‰ ± 1.4‰ at δ18O values of water (H2O) and dissolved oxygen (O2) of 0‰ and 24.2‰ vs. Vienna Standard Mean Ocean Water, respectively. Additions of 18O-enriched H2O allowed us to evaluate the effects of oxygen (O) isotope fractionation and exchange on . Kinetic isotope effects for the incorporation of O atoms were the most important factors for setting overall values relative to the substrates (O2 and H2O). These isotope effects ranged from +10‰ to +22‰ for ammonia oxidation (O2 plus H2O incorporation) and from +1‰ to +27‰ for incorporation of H2O during nitrite oxidation. values were also affected by the amount and duration of nitrite accumulation, which permitted abiotic O atom exchange between nitrite and H2O. Coculture incubations where ammonia oxidation and nitrite oxidation were tightly coupled showed low levels of nitrite accumulation and exchange (3% ± 4%). These experiments had values of −1.5‰ to +0.7‰. Field experiments had greater accumulation of nitrite and a higher amount of exchange (22% to 100%), yielding an average value of +1.9‰ ± 3.0‰. Low levels of biologically catalyzed exchange in coculture experiments may be representative of nitrification in much of the ocean where nitrite accumulation is low. Abiotic oxygen isotope exchange may be important where nitrite does accumulate, such as oceanic primary and secondary nitrite maxima.This research was funded by the National Science Foundation Chemical Oceanography grants 05-26277 and 09- 610998 to K.L.C

Nitrogen cycling in the secondary nitrite maximum of the eastern tropical North Pacific off Costa Rica

Author Posting. © American Geophysical Union, 2015. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Global Biogeochemical Cycles 29 (2015): 2061–2081, doi:10.1002/2015GB005187.Nitrite is a central intermediate in the marine nitrogen cycle and represents a critical juncture where nitrogen can be reduced to the less bioavailable N2 gas or oxidized to nitrate and retained in a more bioavailable form. We present an analysis of rates of microbial nitrogen transformations in the oxygen deficient zone (ODZ) within the eastern tropical North Pacific Ocean (ETNP). We determined rates using a novel one-dimensional model using the distribution of nitrite and nitrate concentrations, along with their natural abundance nitrogen (N) and oxygen (O) isotope profiles. We predict rate profiles for nitrate reduction, nitrite reduction, and nitrite oxidation throughout the ODZ, as well as the contributions of anammox to nitrite reduction and nitrite oxidation. Nitrate reduction occurs at a maximum rate of 25 nM d−1 at the top of the ODZ, at the same depth as the maximum rate of nitrite reduction, 15 nM d−1. Nitrite oxidation occurs at maximum rates of 10 nM d−1 above the secondary nitrite maximum, but also in the secondary nitrite maximum, within the ODZ. Anammox contributes to nitrite oxidation within the ODZ but cannot account for all of it. Nitrite oxidation within the ODZ that is not through anammox is also supported by microbial gene abundance profiles. Our results suggest the presence of nitrite oxidation within the ETNP ODZ, with implications for the distribution and physiology of marine nitrite-oxidizing bacteria, and for total nitrogen loss in the largest marine ODZ.National Science Foundation. Grant Numbers OCE 05-26277, OCE 09-610998; WHOI Coastal Ocean Institute2016-06-1

Testing the influence of light on nitrite cycling in the eastern tropical North Pacific

Light is considered a strong controlling factor of nitrification rates in the surface ocean. Previous work has shown that ammonia oxidation and nitrite oxidation may be inhibited by high light levels, yet active nitrification has been measured in the sunlit surface ocean. While it is known that photosynthetically active radiation (PAR) influences microbial nitrite production and consumption, the level of inhibition of nitrification is variable across datasets. Additionally, phytoplankton have light-dependent mechanisms for nitrite production and consumption that co-occur with nitrification around the depths of the primary nitrite maximum (PNM). In this work, we experimentally determined the direct influence of light level on net nitrite production, including all major nitrite cycling processes (ammonia oxidation, nitrite oxidation, nitrate reduction and nitrite uptake) in microbial communities collected from the base of the euphotic zone. We found that although ammonia oxidation was inhibited at the depth of the PNM and was further inhibited by increasing light at all stations, it remained the dominant nitrite production process at most stations and treatments, even up to 25 % surface PAR. Nitrate addition did not enhance ammonia oxidation in our experiments but may have increased nitrate and nitrite uptake at a coastal station. In contrast to ammonia oxidation, nitrite oxidation was not clearly inhibited by light and sometimes even increased at higher light levels. Thus, accumulation of nitrite at the PNM may be modulated by changes in light, but light perturbations did not exclude nitrification from the surface ocean. Nitrite uptake and nitrate reduction were both enhanced in high-light treatments relative to low light and in some cases showed high rates in the dark. Overall, net nitrite production rates of PNM communities were highest in the dark treatments.</p

Modeling oceanic nitrate and nitrite concentrations and isotopes using a 3-D inverse N cycle model

Nitrite (NO2-) is a key intermediate in the marine nitrogen (N) cycle and a substrate in nitrification, which produces nitrate (NO3-), as well as water column N loss processes denitrification and anammox. In models of the marine N cycle, NO2- is often not considered as a separate state variable, since NO3- occurs in much higher concentrations in the ocean. In oxygen deficient zones (ODZs), however, NO2- represents a substantial fraction of the bioavailable N, and modeling its production and consumption is important to understand the N cycle processes occurring there, especially those where bioavailable N is lost from or retained within the water column. Improving N cycle models by including NO2- is important in order to better quantify N cycling rates in ODZs, particularly N loss rates. Here we present the expansion of a global 3-D inverse N cycle model to include NO2- as a reactive intermediate as well as the processes that produce and consume NO2- in marine ODZs. NO2- accumulation in ODZs is accurately represented by the model involving NO3- reduction, NO2- reduction, NO2- oxidation, and anammox. We model both 14N and 15N and use a compilation of oceanographic measurements of NO3- and NO2- concentrations and isotopes to place a better constraint on the N cycle processes occurring. The model is optimized using a range of isotope effects for denitrification and NO2- oxidation, and we find that the larger (more negative) inverse isotope effects for NO2- oxidation, along with relatively high rates of NO2-, oxidation give a better simulation of NO3- and NO2- concentrations and isotopes in marine ODZs.</p

Inverse kinetic isotope fractionation during bacterial nitrite oxidation

Author Posting. © Elsevier B.V., 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Geochimica et Cosmochimica Acta 73 (2009): 2061-2076, doi:10.1016/j.gca.2008.12.022.Natural abundance stable isotopes in nitrate (NO3-), nitrite (NO2-), and nitrous oxide (N2O) have been used to better understand the cycling of nitrogen in marine and terrestrial environments. However, in order to extract the greatest information from the distributions of these isotopic species, the kinetic isotope effects for each of the relevant microbial reactions are needed. To date, kinetic isotope effects for nitrite oxidation and anaerobic ammonium oxidation (anammox) have not been reported. In this study, the nitrogen isotope effect was measured for microbial nitrite oxidation to nitrate. Nitrite oxidation is the second step in the nitrification process, and it plays a key role in the regeneration of nitrate in the ocean. Surprisingly, nitrite oxidation occurred with an inverse kinetic isotope effect, such that the residual nitrite became progressively depleted in 15N as the reaction proceeded. Three potential explanations for this apparent inverse kinetic isotope effect were explored: 1) isotope exchange equilibrium between nitrite and nitrous acid prior to reaction, 2) reaction reversibility at the enzyme level, and 3) true inverse kinetic fractionation. Comparison of experimental data to ab initio calculations and theoretical predictions leads to the conclusion that the fractionation is most likely inverse at the enzyme level. Inverse kinetic isotope effects are rare, but the experimental observations reported here agree with kinetic isotope theory for this simple N-O bond-forming reaction. Nitrite oxidation is therefore fundamentally different from all other microbial processes in which N isotope fractionation has been studied. The unique kinetic isotope effect for nitrite oxidation should help to better identify its role in the cycling of nitrite in ocean suboxic zones, and other environments in which nitrite accumulates.Funding from NSF award OCE 05-26277 to KLC is also gratefully acknowledged

Multiple Metabolisms Constrain the Anaerobic Nitrite Budget in the Eastern Tropical South Pacific

The Eastern Tropical South Pacific is one of the three major oxygen deficient zones (ODZs) in the global ocean and is responsible for approximately one third of marine water column nitrogen loss. It is the best studied of the ODZs and, like the others, features a broad nitrite maximum across the low oxygen layer. How the microbial processes that produce and consume nitrite in anoxic waters interact to sustain this feature is unknown. Here we used 15N-tracer experiments to disentangle five of the biologically mediated processes that control the nitrite pool, including a high-resolution profile of nitrogen loss rates. Nitrate reduction to nitrite likely depended on organic matter fluxes, but the organic matter did not drive detectable rates of denitrification to N2. However, multiple lines of evidence show that denitrification is important in shaping the biogeochemistry of this ODZ. Significant rates of anaerobic nitrite oxidation at the ODZ boundaries were also measured. Lodate was a potential oxidant that could support part of this nitrite consumption pathway. We additionally observed N2 production from labeled cyanate and postulate that anammox bacteria have the ability to harness cyanate as another form of reduced nitrogen rather than relying solely on ammonification of complex organic matter. The balance of the five anaerobic rates measured—anammox, denitrification, nitrate reduction, nitrite oxidation, and dissimilatory nitrite reduction to ammonium—is sufficient to reproduce broadly the observed nitrite and nitrate profiles in a simple one-dimensional model but requires an additional source of reduced nitrogen to the deeper ODZ to avoid ammonium overconsumption. ©2017. American Geophysical Union. All Rights Reserved

Protocols for Assessing Transformation Rates of Nitrous Oxide in the Water Column

Nitrous oxide (N2O) is a potent greenhouse gas and an ozone destroying substance. Yet, clear step-by-step protocols to measure N2O transformation rates in freshwater and marine environments are still lacking, challenging inter-comparability efforts. Here we present detailed protocols currently used by leading experts in the field to measure water-column N2O production and consumption rates in both marine and other aquatic environments. We present example 15N-tracer incubation experiments in marine environments as well as templates to calculate both N2O production and consumption rates. We discuss important considerations and recommendations regarding (1) precautions to prevent oxygen (O2) contamination during low-oxygen and anoxic incubations, (2) preferred bottles and stoppers, (3) procedures for 15N-tracer addition, and (4) the choice of a fixative. We finally discuss data reporting and archiving. We expect these protocols will make 15N-labeled N2O transformation rate measurements more accessible to the wider community and facilitate future inter-comparison between different laboratories