Quantifying potential N turnover rates in hypersaline microbial mats by 15N tracer techniques.

Microbial mats, due to stratification of the redox zones, have a potential to include a complete N cycle, however an attempt to evaluate a complete N cycle in these ecosystems has not been yet made. In this study, occurrence and rates of major N cycle processes were evaluated in intact microbial mats from Elkhorn Slough, Monterey Bay, CA, USA, and Baja California Sur, Mexico under oxic and anoxic conditions using 15N-labeling techniques. All of the major N transformation pathways, with the exception of anammox, were detected in both microbial mats. Nitrification rates were found to be low at both sites for both seasons investigated. The highest rates of ammonium assimilation were measured in Elkhorn Slough mats in April and corresponded to high in situ ammonium concentration in the overlying water. Baja mats featured higher ammonification than ammonium assimilation rates and this, along with their higher affinity for nitrate compared to ammonium and low dissimilatory nitrate reduction to ammonium rates, characterized their differences from Elkhorn Slough mats. Nitrogen fixation rates in Elkhorn Slough microbial mats were found to be low implying that other processes such as recycling and assimilation from water are main sources of N for these mats at the times sampled. Denitrification in all of the mats was incomplete with nitrous oxide as end product and not dinitrogen. Our findings highlight N cycling features not previously quantified in microbial mats and indicate a need of further investigations in these microbial ecosystems.Importance: Nitrogen is essential for life. The nitrogen cycle on Earth is mediated by microbial activity and has had a profound impact on both the atmosphere and the biosphere throughout geologic time. Microbial mats, present in many modern environments, have been regarded as living records of the organisms, genes, and phylogenies of microbes, as they are one of the most ancient ecosystems on Earth. While rates of major nitrogen metabolic pathways have been evaluated in a number of ecosystems, it remains elusive in microbial mats. In particular it is unclear what factors affect nitrogen cycling in these ecosystems and how morphological differences between mats impact nitrogen transformations. In this study we investigate nitrogen cycling in two microbial mats having morphological differences. Our findings provide insight for further understanding of biogeochemistry and microbial ecology of microbial mats.

Dynamics of localised nitrogen supply and relevance for root growth of Vicia faba ('Fuego') and Hordeum vulgare ('Marthe') in soil.

Root growth responds to local differences in N-form and concentration. This is known for artificial systems and assumed to be valid in soil. The purpose of this study is to challenge this assumption for soil mesocosms locally supplied with urea with and without nitrification inhibitor. Soil column experiments with Vicia faba ('Fuego') and Hordeum vulgare ('Marthe') were performed to investigate soil solution chemistry and root growth response of these two species with contrasting root architectures to the different N-supply simultaneously. Root growth was analysed over time and separately for the fertiliser layer and the areas above and below with X-ray CT (via region growing) and WinRHIZO. Additionally, NO3- and NH4+ in soil and soil solution were analysed. In Vicia faba, no pronounced differences were observed, although CT analysis indicated different root soil exploration for high NH4+. In Hordeum vulgare, high NO3- inhibited lateral root growth while high NH4+ stimulated the formation of first order laterals. The growth response to locally distributed N-forms in soil is species specific and less pronounced than in artificial systems. The combination of soil solution studies and non-invasive imaging of root growth can substantially improve the mechanistic understanding of root responses to different N-forms in soil.

Gas entrapment and microbial N2O reduction reduce N2O emissions from a biochar-amended sandy clay loam soil.

Nitrous oxide (N2O) is a potent greenhouse gas that is produced during microbial nitrogen transformation processes such as nitrification and denitrification. Soils represent the largest sources of N2O emissions with nitrogen fertilizer application being the main driver of rising atmospheric N2O concentrations. Soil biochar amendment has been proposed as a promising tool to mitigate N2O emissions from soils. However, the underlying processes that cause N2O emission suppression in biochar-amended soils are still poorly understood. We set up microcosm experiments with fertilized, wet soil in which we used 15N tracing techniques and quantitative polymerase chain reaction (qPCR) to investigate the impact of biochar on mineral and gaseous nitrogen dynamics and denitrification-specific functional marker gene abundance and expression. In accordance with previous studies our results showed that biochar addition can lead to a significant decrease in N2O emissions. Furthermore, we determined significantly higher quantities of soil-entrapped N2O and N2 in biochar microcosms and a biochar-induced increase in typical and atypical nosZ transcript copy numbers. Our findings suggest that biochar-induced N2O emission mitigation is based on the entrapment of N2O in water-saturated pores of the soil matrix and concurrent stimulation of microbial N2O reduction resulting in an overall decrease of the N2O/(N2O + N2) ratio.

Effects of grass species and grass growth on atmospheric nitrogen deposition to a bog ecosystem surrounded by intensive agricultural land use.

We applied a (15)N dilution technique called "Integrated Total Nitrogen Input" (ITNI) to quantify annual atmospheric N input into a peatland surrounded by intensive agricultural practices over a 2-year period. Grass species and grass growth effects on atmospheric N deposition were investigated using Lolium multiflorum and Eriophorum vaginatum and different levels of added N resulting in increased biomass production. Plant biomass production was positively correlated with atmospheric N uptake (up to 102.7 mg N pot(-1)) when using Lolium multiflorum. In contrast, atmospheric N deposition to Eriophorum vaginatum did not show a clear dependency to produced biomass and ranged from 81.9 to 138.2 mg N pot(-1). Both species revealed a relationship between atmospheric N input and total biomass N contents. Airborne N deposition varied from about 24 to 55 kg N ha(-1) yr(-1). Partitioning of airborne N within the monitor system differed such that most of the deposited N was found in roots of Eriophorum vaginatum while the highest share was allocated in aboveground biomass of Lolium multiflorum. Compared to other approaches determining atmospheric N deposition, ITNI showed highest airborne N input and an up to fivefold exceedance of the ecosystem-specific critical load of 5-10 kg N ha(-1) yr(-1).

Nitrogen transforming community in a horizontal subsurface-flow constructed wetland.

Constructed wetlands are important ecosystems with respect to nitrogen cycling. Here we studied the activity and abundance of nitrogen transforming bacteria as well as the spatial distribution of nitrification, anaerobic ammonium oxidation (anammox), and denitrification processes in a horizontal subsurface-flow constructed wetland. The functional genes of the nitrogen cycle were evenly distributed in a linear way along the flow path with prevalence at the superficial points. The same trend was observed for the nitrification and denitrification turnover rates using isotope labeling techniques. It was also shown that only short-term incubations should be used to measure denitrification turnover rates. Significant nitrate consumption under aerobic conditions diminishes nitrification rates and should therefore be taken into account when estimating nitrification turnover rates. This nitrate consumption was due to aerobic denitrification, the rate of which was comparable to that for anaerobic denitrification. Consequently, denitrification should not be considered as an exclusively anaerobic process. Phylogenetic analysis of hydrazine synthase (hzsA) gene clones indicated the presence of Brocadia and Kuenenia anammox species in the constructed wetland. Although anammox bacteria were detected by molecular methods, anammox activity could not be measured and hence this process appears to be of low importance in nitrogen transformations in these freshwater ecosystems.

Release of nitrous oxide and dinitrogen from a transition bog under drained and rewetted conditions due to denitrification: results from a [15N]nitrate-bromide double-tracer study.

Denitrification is well known being the most important nitrate-consuming process in water-logged peat soils, whereby the intermediate compound nitrous oxide (N(2)O) and the end product dinitrogen (N(2)) are ultimately released. The present study was aimed at evaluating the release of these gases (due to denitrification) from a nutrient-poor transition bog ecosystem under drained and three differently rewetted conditions at the field scale using a (15)N-tracer approach ([(15)N]nitrate application, 30 kg N ha(-1)) and a common closed-chamber technique. The drained site is characterized by a constant water table (WT) of -30 cm (here referred to as D30), while rewetted sites represent a constant WT of -15 cm, a constant WT of 0 cm (i.e. waterlogged), and an initial WT of 0 cm (which decreased slightly during the experiment), respectively, (here referred to as R15, R0, and R0(d), respectively). The highest N(2)O emissions were observed at D30 (291 µg N(2)O-N m(-2) h(-1)) as well as at R0d (665 µg N(2)O-N m(-2) h(-1)). At the rewetted peat sites with a constant WT (i.e. R15 and R0), considerably lower N2O emissions were observed (maximal 37 µg N(2)O-N m(-2) h(-1)). Concerning N(2) only at the initially water-logged peat site R0d considerable release rates (up to 3110 µg N(2)-N m(-2) h(-1)) were observed, while under drained conditions (D30) no N(2) emission and under rewetted conditions with a constant WT (R15 and R0) significantly lower N(2) release rates (maximal 668 µg N(2)-N m(-2) h(-1)) could be detected. In addition, it has been found that natural WT fluctuations at rewetted peat sites, in particular a rapid drop down of the WT, can induce high emission rates for both N(2)O and N(2).

Oxygen availability and distance to surface environments determine community composition and abundance of ammonia-oxidizing prokaroytes in two superimposed pristine limestone aquifers in the Hainich region, Germany.

We followed the abundance and compared the diversity of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in the groundwater of two superimposed pristine limestone aquifers located in the Hainich region (Thuringia, Germany) over 22 months. Groundwater obtained from the upper aquifer (12 m depth) was characterized by low oxygen saturation (0-20%) and low nitrate concentrations (0-20 µM), contrasting with 50-80% oxygen saturation and 40-200 µM nitrate in the lower aquifer (48 m and 88 m depth). Quantitative PCR targeting bacterial and archaeal amoA and 16S rRNA genes suggested a much higher ammonia oxidizer fraction in the lower aquifer (0.4-7.8%) compared with the upper aquifer (0.01-0.29%). In both aquifers, AOB communities were dominated by one phylotype related to Nitrosomonas ureae, while AOA communities were more diverse. Multivariate analysis of amoA DGGE profiles revealed a stronger temporal variation of AOA and AOB community composition in the upper aquifer, pointing to a stronger influence of surface environments. Parallel fluctuations of AOA, AOB, and total microbial abundance suggested that hydrological factors (heavy rain falls, snow melt) rather than specific physicochemical parameters were responsible for the observed community dynamics.

Nitrate turnover in a peat soil under drained and rewetted conditions: results from a [(15)N]nitrate-bromide double-tracer study.

Under natural conditions, peatlands are generally nitrate-limited. However, recent concerns about an additional N input into peatlands by atmospheric N deposition have highlighted the risk of an increased denitrification activity and hence the likelihood of a rise of emissions of the greenhouse gas nitrous oxide. Therefore, the aim of the present study was to investigate the turnover of added nitrate in a drained and a rewetted peatland using a [(15)N]nitrate-bromide double-tracer method. The double-tracer method allows a separation between physical effects (dilution, dispersion and dislocation) and microbial and chemical nitrate transformation by comparing with the conservative Br(-) tracer. In the drained peat site, low NO3(-) consumption rates have been observed. In contrast, NO3(-) consumption at the rewetted peat site rises rapidly to about 100% within 4 days after tracer application. Concomitantly, the (15)N abundances of nitrite and ammonium in soil water increased and lead to the conclusion that, besides commonly known NO3(-) reduction to nitrite (i.e. denitrification), a dissimilatory nitrate reduction to ammonium has simultaneously taken place. The present study reveals that increasing NO3(-) inputs into rewetted peatlands via atmospheric deposition results in a rapid NO3(-) consumption, which could lead to an increase in N2O emissions into the atmosphere.

Analysis of the coexisting pathways for NO and N2O formation in Chernozem using the (15)N-tracer SimKIM-Advanced model.

The nitrogen (N) cycle consists of a variety of microbial processes. These processes often occur simultaneously in soils, but respond differently to local environmental conditions due to process-specific biochemical restrictions (e.g. oxygen levels). Hence, soil nitrogen cycling (e.g. soil N gas production through nitrification and denitrification) is individually affected through these processes, resulting in the complex and highly dynamic behaviour of total soil N turnover. The development and application of methods that facilitate the quantification of individual contributions of coexisting processes is a fundamental prerequisite for (i) understanding the dynamics of soil N turnover and (ii) implementing these processes in ecosystem models. To explain the unexpected results of the triplet tracer experiment (TTE) of Russow et al. (Role of nitrite and nitric oxide in the processes of nitrification and denitrification in soil: results from (15)N tracer experiments. Soil Biol Biochem. 2009;41:785-795) the existing SimKIM model was extended to the SimKIM-Advanced model through the addition of three separate nitrite subpools associated with ammonia oxidation, oxidation of organic nitrogen (Norg), and denitrification, respectively. For the TTE, individual treatments with (15)N ammonium, (15)N nitrate, and (15)N nitrite were conducted under oxic, hypoxic, and anoxic conditions, respectively, to clarify the role of nitric oxide as a denitrification intermediate during N2O formation. Using a split nitrite pool, this analysis model explains the observed differences in the (15)N enrichments in nitric oxide (NO) and nitrous oxide (N2O) which occurred in dependence on different oxygen concentrations. The change from oxic over hypoxic to anoxic conditions only marginally increased the NO and N2O release rates (1.3-fold). The analysis using the model revealed that, under oxic and hypoxic conditions, Norg-based N2O production was the dominant pathway, contributing to 90 and 50 % of the total soil N2O release. Under anoxic conditions, denitrification was the dominant process for soil N2O release. The relative contribution of Norg to the total soil NO release was small. Ammonia oxidation served as the major pathway of soil NO release under oxic and hypoxic conditions, while denitrification was dominant under anoxic conditions. The model parameters for soil with moderate soil organic matter (SOM) content were not scalable to an additional data set for soil with higher SOM content, indicating a strong influence of SOM content on microbial N turnover. Thus, parameter estimation had to be re-calculated for these conditions, highlighting the necessity of individual soil-dependent parameter estimations.

Microbial communities along biogeochemical gradients in a hydrocarbon-contaminated aquifer.

Micro-organisms are known to degrade a wide range of toxic substances. How the environment shapes microbial communities in polluted ecosystems and thus influences degradation capabilities is not yet fully understood. In this study, we investigated microbial communities in a highly complex environment: the capillary fringe and subjacent sediments in a hydrocarbon-contaminated aquifer. Sixty sediment sections were analysed using terminal restriction fragment length polymorphism (T-RFLP) fingerprinting, cloning and sequencing of bacterial and archaeal 16S rRNA genes, complemented by chemical analyses of petroleum hydrocarbons, methane, oxygen and alternative terminal electron acceptors. Multivariate statistics revealed concentrations of contaminants and the position of the water table as significant factors shaping the microbial community composition. Micro-organisms with highest T-RFLP abundances were related to sulphate reducers belonging to the genus Desulfosporosinus, fermenting bacteria of the genera Sedimentibacter and Smithella, and aerobic hydrocarbon degraders of the genus Acidovorax. Furthermore, the acetoclastic methanogens Methanosaeta, and hydrogenotrophic methanogens Methanocella and Methanoregula were detected. Whereas sulphate and sulphate reducers prevail at the contamination source, the detection of methane, fermenting bacteria and methanogenic archaea further downstream points towards syntrophic hydrocarbon degradation.

Automated and rapid online determination of 15N abundance and concentration of ammonium, nitrite, or nitrate in aqueous samples by the SPINMAS technique.

On the basis of the principle of reaction continuous-flow quadrupole mass spectrometry, an automated sample preparation unit for inorganic nitrogen (SPIN) species was developed and coupled to a quadrupole Mass Spectrometer (MAS). The SPINMAS technique was designed for an automated, sensitive, and rapid determination of 15N abundance and concentration of a wide variety of N-species involved in nitrogen cycling (e.g. NH4+, NO3-, NH2OH etc.). In this paper, the SPINMAS technique is evaluated with regard to the determination of 15N abundance and concentration of the most fundamental inorganic nitrogen compounds in ecosystems such as NH4+, NO2-, and NO3-. The presented paper describes the newly developed system in detail and demonstrates the general applicability of the system. For a precise determination of 15N abundance and concentration, a minimum total N-amount of 10 microg NH4+ - N, 0.03 microg NO2- - N, or 0.3 microg NO3- - N has to be supplied. Currently, the SPINMAS technique represents the most rapid and only fully automated all-round method for a simultaneous determination of 15N abundance and total N-amount of NH4+, NO2-, or NO3- in aqueous samples.

A new mathematical approach for calculating the contribution of anammox, denitrification and atmosphere to an N2 mixture based on a 15N tracer technique.

Denitrification and anaerobic ammonium oxidation (anammox) have been identified as biotic key processes of N2 formation during global nitrogen cycling. Based on the principle of a 15N tracer technique, new analytical expressions have been derived for a calculation of the fractions of N2 simultaneously released by anammox and denitrification. An omnipresent contamination with atmospheric N2 is also taken into account and is furthermore calculable in terms of a fraction. Two different mathematical approaches are presented which permit a precise calculation of the contribution of anammox, denitrification, and atmosphere to a combined N2 mixture. The calculation is based on a single isotopic analysis of a sampled N2 mixture and the determination of the 15N abundance of nitrite and nitrate (simplified approach) or of ammonium, nitrite, and nitrate (comprehensive approach). Calculations are even processable under conditions where all basal educts of anammox and denitrification (ammonium, nitrite, and nitrate) are differently enriched in 15N. An additional determination of concentrations of dissolved N compounds is unnecessary. Finally, the presented approach is transferable to studies focused on terrestrial environments where N2 is formed by denitrification and simultaneously by codenitrification or chemodenitrification.

A 15N-aided artificial atmosphere gas flow technique for online determination of soil N2 release using the zeolite Köstrolith SX6.

N2 is one of the major gaseous nitrogen compounds released by soils due to N-transformation processes. Since it is also the major constituent of the earth's atmosphere (78.08% vol.), the determination of soil N2 release is still one of the main methodological challenges with respect to a complete evaluation of the gaseous N-loss of soils. Commonly used approaches are based either on a C2H2 inhibition technique, an artificial atmosphere or a 15N-tracer technique, and are designed either as closed systems (non-steady state) or gas flow systems (steady state). The intention of this work has been to upgrade the current gas flow technique using an artificial atmosphere for a 15N-aided determination of the soil N2 release simultaneously with N2O. A 15N-aided artificial atmosphere gas flow approach has been developed, which allows a simultaneous online determination of N2 as well as N2O fluxes from an open soil system (steady state). Fluxes of both gases can be determined continuously over long incubation periods and with high sampling frequency. The N2 selective molecular sieve Köstrolith SX6 was tested successfully for the first time for dinitrogen collection. The presented paper mainly focuses on N2 flux determination. For validation purposes soil aggregates of a Haplic Phaeozem were incubated under aerobic (21 and 6 vol.% O2) and anaerobic conditions. Significant amounts of N2 were released only during anaerobic incubation (0.4 and 640.2 pmol N2 h(-1) g(-1) dry soil). However, some N2 formation also occurred during aerobic incubation. It was also found that, during ongoing denitrification, introduced [NO3]- will be more strongly delivered to microorganisms than the original soil [NO3]-.