Peter Dörsch

Production of NO, N2O and N2 by extracted soil bacteria, regulation by NO2− and O2 concentrations

The oxygen control of denitrification and its emission of NO/N2O/N2 was investigated by incubation of Nycodenz-extracted soil bacteria in an incubation robot which monitors O2, NO, N2O and N2 concentrations (in He+O2 atmosphere). Two consecutive incubations were undertaken to determine (1) the regulation of denitrification by O2 and NO2(-) during respiratory O2 depletion and (2) the effects of re-exposure to O2 of cultures with fully expressed denitrification proteome. Early denitrification was only detected (as NO and N2O) at <or=80 microM O2 in treatments with NO2(-), and the rates were three orders of magnitude lower than the rates observed after oxygen depletion (with N2 as the primary product). When re-exposed to O2, the cultures continued to denitrify (8-55% of the rates during the foregoing anoxic phase), but its main product was N2O. The N2O reductase activity recovered as oxygen was being depleted. The results suggest that expression of the denitrifying proteome may result in significant subsequent aerobic denitrification, and this has profound implications for the understanding and modelling of denitrification and N2O emission. Short anoxic spells caused by transient flooding during rainfall, could lead to subsequent unbalanced aerobic denitrification, in which N2O is a major end product.

Genetic characterization of denitrifier communities with contrasting intrinsic functional traits

Microorganisms capable of denitrification are polyphyletic and exhibit distinct denitrification regulatory phenotypes (DRP), and thus, denitrification in soils could be controlled by community composition. In a companion study (Dörsch et al., 2012) and preceding work, ex situ denitrification assays of three organic soils demonstrated profoundly different functional traits including N(2) O/N(2) ratios. Here, we explored the composition of the underlying denitrifier communities by analyzing the abundance and structure of denitrification genes (nirK, nirS, and nosZ). The relative abundance of nosZ (vs. nirK + nirS) was similar for all communities, and hence, the low N(2) O reductase activity in one of the soils was not because of the lack of organisms with this gene. Similarity in community composition between the soils was generally low for nirK and nirS, but not for nosZ. The community with the most robust denitrification (consistently low N(2) O/N(2) ) had the highest diversity/richness of nosZ and nirK, but not of nirS. Contrary results found for a second soil agreed with impaired denitrification (low overall denitrification activity, high N(2) O/N(2) ). In conclusion, differences in community composition and in the absolute abundance of denitrification genes clearly reflected the functional differences observed in laboratory studies and may shed light on differences in in situ N(2) O emission of the soils.

Sorption of Pure N2O to Biochars and Other Organic and Inorganic Materials under Anhydrous Conditions

Suppression of nitrous oxide (N2O) emissions from soil is commonly observed after amendment with biochar. The mechanisms accounting for this suppression are not yet understood. One possible contributing mechanism is N2O sorption to biochar. The sorption of N2O and carbon dioxide (CO2) to four biochars was measured in an anhydrous system with pure N2O. The biochar data were compared to those for two activated carbons and other components potentially present in soils-uncharred pine wood and peat-and five inorganic metal oxides with variable surface areas. Langmuir maximum sorption capacities (Qmax) for N2O on the pine wood biochars (generated between 250 and 500 °C) and activated carbons were 17-73 cm(3) g(-1) at 20 °C (median 51 cm(3) g(-1)), with Langmuir affinities (b) of 2-5 atm(-1) (median 3.4 atm(-1)). Both Qmax and b of the charred materials were substantially higher than those for peat, uncharred wood, and metal oxides [Qmax 1-34 cm(3) g(-1) (median 7 cm(3) g(-1)); b 0.4-1.7 atm(-1) (median 0.7 atm(-1))]. This indicates that biochar can bind N2O more strongly than both mineral and organic soil materials. Qmax and b for CO2 were comparable to those for N2O. Modeled sorption coefficients obtained with an independent polyparameter-linear free-energy relationship matched measured data within a factor 2 for mineral surfaces but underestimated by a factor of 5-24 for biochar and carbonaceous surfaces. Isosteric enthalpies of sorption of N2O were mostly between -20 and -30 kJ mol(-1), slightly more exothermic than enthalpies of condensation (-16.1 kJ mol(-1)). Qmax of N2O on biochar (50000-130000 μg g(-1) biochar at 20 °C) exceeded the N2O emission suppressions observed in the literature (range 0.5-960 μg g(-1) biochar; median 16 μg g(-1)) by several orders of magnitude. Thus, the hypothesis could not be falsified that sorption of N2O to biochar is a mechanism of N2O emission suppression.

Phenotypic and genotypic heterogeneity among closely related soil-borne N2- and N2O-producing Bacillus isolates harboring the nosZ gene

Little is known about the genetic and phenotypic diversity of Gram-positive denitrifying bacteria. We compared the production of gaseous denitrification products for 14 closely related Bacillus soil isolates at pH 6 and 7 during 48-h batch incubations using a robotic gas-sampling apparatus. Primers targeting the nosZ gene encoding the nitrous oxide reductase were designed to confirm the presence of this gene in the isolates. The variation in the production of gaseous nitrogen products was compared with the genetic variation based on 16S rRNA gene sequences, genomic fingerprinting and nosZ sequences. The nosZ gene was detected in all isolates and all produced N(2) as the dominant end product at pH 7. Production of gaseous nitrogen products was more variable at pH 6, with different levels of NO and N(2) O production among the isolates, although minimal variation was observed among the 16S rRNA and nosZ gene sequences. One isolate was more divergent from the others based on genomic fingerprinting, and had two different nosZ gene copies, which coincided with the highest production of N(2) at pH 7 and the lack of intermediates at pH 6. Overall, our analysis suggests that genetic variation plays a role in the variation in N(2) O and N(2) production, but the variation in activity caused by acidification can be substantially greater than genotypic variation among closely related Bacillus.

Community-specific pH response of denitrification: experiments with cells extracted from organic soils.

Denitrifying prokaryotes are phylogenetically and functionally diverse. Little is known about the relationship between soil denitrifier community composition and functional traits. We extracted bacterial cells from three cultivated peat soils with contrasting native pH by density gradient centrifugation and investigated their kinetics of oxygen depletion and NO2 -, NO, N(2) O and N(2) accumulation during initially hypoxic batch incubations (0.5-1 μM O(2)) in minimal medium buffered at either pH 5.4 or 7.1 (2 mM glutamate, 2 mM NO3 -). The three communities differed strikingly in NO2 - accumulation and transient N(2) O accumulation at the two pH levels, whereas NO peak concentrations (24-53 nM) were similar across all communities and pH treatments. The results confirm that the communities represent different denitrification regulatory phenotypes, as indicated by previous denitrification bioassays with nonbuffered slurries of the same three soils. The composition of the extracted cells resembled that of the parent soils (PCR-TRFLP analyses of 16S rRNA genes, nirK, nirS and nosZ), which were found to differ profoundly in their genetic composition (Braker et al., ). Together, this suggests that direct pH response of denitrification depends on denitrifier community composition, with implications for the propensity of soils to emit N(2) O to the atmosphere.

Effect of soil pH Increase by Biochar on NO, N2O and N2 production during denitrification in acid soils

Biochar (BC) application to soil suppresses emission of nitrous- (N2O) and nitric oxide (NO), but the mechanisms are unclear. One of the most prominent features of BC is its alkalizing effect in soils, which may affect denitrification and its product stoichiometry directly or indirectly. We conducted laboratory experiments with anoxic slurries of acid Acrisols from Indonesia and Zambia and two contrasting BCs produced locally from rice husk and cacao shell. Dose-dependent responses of denitrification and gaseous products (NO, N2O and N2) were assessed by high-resolution gas kinetics and related to the alkalizing effect of the BCs. To delineate the pH effect from other BC effects, we removed part of the alkalinity by leaching the BCs with water and acid prior to incubation. Uncharred cacao shell and sodium hydroxide (NaOH) were also included in the study. The untreated BCs suppressed N2O and NO and increased N2 production during denitrification, irrespective of the effect on denitrification rate. The extent of N2O and NO suppression was dose-dependent and increased with the alkalizing effect of the two BC types, which was strongest for cacao shell BC. Acid leaching of BC, which decreased its alkalizing effect, reduced or eliminated the ability of BC to suppress N2O and NO net production. Just like untreated BCs, NaOH reduced net production of N2O and NO while increasing that of N2. This confirms the importance of altered soil pH for denitrification product stoichiometry. Addition of uncharred cacao shell stimulated denitrification strongly due to availability of labile carbon but only minor effects on the product stoichiometry of denitrification were found, in accordance with its modest effect on soil pH. Our study indicates that stimulation of denitrification was mainly due to increases in labile carbon whereas change in product stoichiometry was mainly due to a change in soil pH.

pH-driven shifts in overall and transcriptionally active denitrifiers control gaseous product stoichiometry in growth experiments with extracted bacteria from soil.

Soil pH is a strong regulator for activity as well as for size and composition of denitrifier communities. Low pH not only lowers overall denitrification rates but also influences denitrification kinetics and gaseous product stoichiometry. N2O reductase is particularly sensitive to low pH which seems to impair its activity post-transcriptionally, leading to higher net N2O production. Little is known about how complex soil denitrifier communities respond to pH change and whether their ability to maintain denitrification over a wider pH range relies on phenotypic redundancy. In the present study, we followed the abundance and composition of an overall and transcriptionally active denitrifier community extracted from a farmed organic soil in Sweden (pHH2O = 7.1) when exposed to pH 5.4 and drifting back to pH 6.6. The soil was previously shown to retain much of its functioning (low N2O/N2 ratios) over a wide pH range, suggesting a high functional versatility of the underlying community. We found that denitrifier community composition, abundance and transcription changed throughout incubation concomitant with pH change in the medium, allowing for complete reduction of nitrate to N2 with little accumulation of intermediates. When exposed to pH 5.4, the denitrifier community was able to grow but reduced N2O to N2 only when near-neutral pH was reestablished by the alkalizing metabolic activity of an acid-tolerant part of the community. The genotypes proliferating under these conditions differed from those dominant in the control experiment run at neutral pH. Denitrifiers of the nirS-type appeared to be severely suppressed by low pH and nirK-type and nosZ-containing denitrifiers showed strongly reduced transcriptional activity and growth, even after restoration of neutral pH. Our study suggests that low pH episodes alter transcriptionally active populations which shape denitrifier communities and determine their gas kinetics.

Multiyear dual nitrate isotope signatures suggest that N-saturated subtropical forested catchments can act as robust N sinks.

In forests of the humid subtropics of China, chronically elevated nitrogen (N) deposition, predominantly as ammonium (NH4+ ), causes significant nitrate (NO3- ) leaching from well-drained acid forest soils on hill slopes (HS), whereas significant retention of NO3- occurs in near-stream environments (groundwater discharge zones, GDZ). To aid our understanding of N transformations on the catchment level, we studied spatial and temporal variabilities of concentration and natural abundance (δ15 N and δ18 O) of nitrate (NO3- ) in soil pore water along a hydrological continuum in the N-saturated Tieshanping (TSP) catchment, southwest China. Our data show that effective removal of atmogenic NH4+ and production of NO3- in soils on HS were associated with a significant decrease in δ15 N-NO3- , suggesting efficient nitrification despite low soil pH. The concentration of NO3- declined sharply along the hydrological flow path in the GDZ. This decline was associated with a significant increase in both δ15 N and δ18 O of residual NO3- , providing evidence that the GDZ acts as an N sink due to denitrification. The observed apparent 15 N enrichment factor (ε) of NO3- of about -5‰ in the GDZ is similar to values previously reported for efficient denitrification in riparian and groundwater systems. Episode studies in the summers of 2009, 2010 and 2013 revealed that the spatial pattern of δ15 N and δ18 O-NO3- in soil water was remarkably similar from year to year. The importance of denitrification as a major N sink was also seen at the catchment scale, as largest δ15 N-NO3- values in stream water were observed at lowest discharge, confirming the importance of the relatively small GDZ for N removal under base flow conditions. This study, explicitly recognizing hydrologically connected landscape elements, reveals an overlooked but robust N sink in N-saturated, subtropical forests with important implications for regional N budgets.

Mechanism leading to N2O production in wastewater treating biofilm systems

Wastewater treatment plants are known to be important point sources for nitrous oxide (N2O) in the anthropogenic N cycle. Biofilm based treatment systems have gained increasing popularity in the treatment of wastewater, but the mechanisms and controls of N2O formation are not fully understood. Here, we review functional groups of microorganism involved in nitrogen (N) transformations during wastewater treatment, with emphasis on potential mechanism of N2O production in biofilms. Biofilms used in wastewater treatment typically harbour aerobic and anaerobic zones, mediating close interactions between different groups of N transforming organisms. Current models of mass transfer and biomass interactions in biofilms are discussed to illustrate the complex regulation of N2O production. Ammonia oxidizing bacteria (AOB) are the prime source for N2O in aerobic zones, while heterotrophic denitrifiers dominate N2O production in anoxic zones. Nitrosative stress ensuing from accumulation of NO2− during partial nitrification or denitrification seems to be one of the most critical factors for enhanced N2O formation. In AOB, N2O production is coupled to nitrifier denitrification triggered by nitrosative stress, low O2 tension or low pH. Chemical N2O production from AOB intermediates (NH2OH, HNO, NO) released during high NH3 turnover seems to be limited to surface-near AOB clusters, since diffusive mass transport resistance for O2 slows down NH3 oxidation rates in deeper biofilm layers. The proportion of N2O among gaseous intermediates (NO, N2O, N2) in heterotrophic denitrification increases when NO or nitrous acid (HNO2) accumulates because of increasing NO2−, or when transient oxygen intrusion impairs complete denitrification. Limited electron donor availability due to mass transport limitation of organic substrates into anoxic biofilm zones is another important factor supporting high N2O/N2 ratios in heterotrophic denitrifiers. Biofilms accommodating Anammox bacteria release less N2O, because Anammox bacteria have no known N2O producing metabolism and reduce NO2− to N2, thereby lowering nitrosative stress to AOB and heterotrophs.

Nitrous Oxide Emission and Global Changes: Modeling Approaches

Publisher Summary A brief survey of the critical aspects of denitrification modeling starts with a discussion of the product stoichiometry as controlled by the biology of denitrifiers. It is followed by a discussion of attempts to model O2 distribution within the soil matrix and then by the examples of various approaches to simulate denitrification and N2O emission as a part of complex soil–plant biogeochemical models. Denitrification results in the emission of three gases, NO, N2O, and N2, and the stoichiometry of the emitted gas mixture depends on the relative activities of the three enzymes, NO2−, NO−, and N2O−reductases, which are encoded by the nir, nor, and nos genes, respectively. This chapter deals with the models of soil anaerobiosis as a regulator for denitrification. A more refined approach uses a power function of soil-moisture content as a dimensionless reduction factor (0–1), which is multiplied by an estimated “maximum” or “potential” denitrification rate. Several of the biogeochemical models along with microbial kinetics that simulate both the C- and N-transformations in soil–plant ecosystems are available and work at different scales. A common feature of most of these models is that they rely on an explicit simulation of heat and water transport in the soil. New models with more elaborate and legitimate representations of the biology of denitrifying bacteria may hypothetically improve predictions, but the epistemological gain is probably a more important reason for such exercises.