Search

Inoculating agricultural soils with nitrous oxide respiring bacteria (NRB) can reduce N2O-emission, but would be impractical as a standalone operation. Here we demonstrate that digestates obtained after biogas production are suitable substrates and vectors for NRB. We show that indigenous NRB in digestates grew to high abundance during anaerobic enrichment under N2O. Gas-kinetics and meta-omic analyses showed that these NRB’s, recovered as metagenome-assembled genomes (MAGs), grew by harvesting fermentation intermediates of the methanogenic consortium. Three NRB’s were isolated, one of which matched the recovered MAG of a Dechloromonas, deemed by proteomics to be the dominant producer of N2O-reductase in the enrichment. While the isolates harbored genes required for a full denitrification pathway and could thus both produce and sequester N2O, their regulatory traits predicted that they act as N2O sinks in soil, which was confirmed experimentally. The isolates were grown by aerobic respiration in digestates, and fertilization with these NRB-enriched digestates reduced N2O emissions from soil. Our use of digestates for low-cost and large-scale inoculation with NRB in soil can be taken as a blueprint for future applications of this powerful instrument to engineer the soil microbiome, be it for enhancing plant growth, bioremediation, or any other desirable function.

Microbial nitrogen (N) transformations in soil, notably denitrification, result in the production of the potent greenhouse and ozone depleting gas nitrous oxide (N2O). Soil chemistry and microbiome composition impact N2O emission potential but the relative importance of these factors as determinants of N2O emission in denitrifying systems is rarely tested. In addition, previous linkages between microbiome composition and N2O emission potential rarely demonstrate causality. Here, we determined the relative impact of microbiome composition (i.e. soil extracted cells) and chemistry (i.e. water extractable chemicals) on N2O emission potential utilizing an anoxic cell based assay system. Cells and chemistry for assays were sourced from soils with contrasting N2O/N2O+N2ratios, combined in various combinations and denitrification gas production was measured in response to nitrate addition. Average directionless effects of cell and chemical extract on N2O/N2O+N2(Cell: Δ0.16, Chemical extract: Δ0.22) and total N2O hypothetically emitted (Cell: Δ2.62 μmol-N, Chemical extract: Δ4.14 μmol-N) indicated chemistry is the most important determinant of N2O emissions. Independent pH differences of just 0.6 points impacted N2O/N2O+N2on par with independent chemical extract differences, supporting the dominance of this variable in previous studies. However, impacts on overall N2O hypothetically emitted were smaller suggesting that soil pH manipulation may not necessarily be a successful approach to mitigate emissions over a fixed time period. In addition, we observed increased N2O accumulation and emission potential at the end of incubations concomitant with predicted decreases in carbon availability suggesting that carbon limitation increases N2O emission transiently with the magnitude of emission dependent on the both chemical and microbiome controls.

Soil denitrification produces the potent greenhouse gas nitrous oxide (N2O) and by further reduction of N2O, the harmless inert gas N2. N2O emission is determined by rate and timing of the N2O producing and reducing steps which are sensitive to a series of proximal and distal regulators such as pH and microbial community composition. Microbial community associations to N2O emission potential (N2O/(N2O+N2)) are commonly entangled with pH leaving the true role of community composition unclear. Here, we leverage a set of soil microbiomes strongly linked to rainfall above pH to test the hypothesis that microbiome vs. N2O emission potential (N2O/(N2O+N2)) correlations will be maintained across alternative distal drivers. N2O emission potential (N2O/(N2O+N2)) and denitrification gas (NO, N2O, N2) kinetics were assessed by automated gas chromatography while community composition was assessed by 16S rRNA gene sequencing and qPCR of nosZI and II genes. Analyses revealed a sustained correlation between microbiome and N2O emission potential (N2O/(N2O+N2)) in the absence of a pH effect. Further, a continuum of gas accumulation phenotypes linked to NO accumulation and sensitive to carbon addition are identified. Separate phenotypes carried out N2O production and reduction steps more concurrently or sequentially and thus determined N2O accumulation and emission potential (N2O/(N2O+N2)). Concurrent N2O producing/reducing soils typically contained NO accumulation to a low steady state, while carbon addition manipulations which increased NO accumulation also increased sequentiality of N2O production/reduction and thus emission potential (N2O/(N2O+N2)). These features may indicate a conserved NO inhibitory mechanism across multiple effectors (rainfall, community composition, carbon availability).HighlightsN2O emission potential is linked to microbiome changes associated with rainfall, but not to pH.Sequential vs. concurrent denitrification phenotypes differing in NO and N2O accumulation are identified.High N2O accumulation is associated with increased NO accumulation.Sequentiality of N2O production/reduction determines soil N2O emission potential.Sequentiality of N2O reduction was susceptible to manipulation via carbon addition.

Soil denitrification produces the potent greenhouse gas nitrous oxide (N2O) and by further reduction of N2O, the harmless inert gas N2. N2O emission is determined by rate and timing of the N2O producing and reducing steps which are sensitive to a series of proximal and distal regulators such as pH and microbial community composition. Microbial community associations to N2O emission potential (N2O/(N2O + N2)) are commonly entangled with pH leaving the true role of community composition unclear. Here, we leverage a set of soil microbiomes strongly linked to rainfall above pH to test the hypothesis that microbiome vs. N2O emission potential (N2O/(N2O + N2)) correlations will be maintained across alternative distal drivers. N2O emission potential (N2O/(N2O + N2)) and denitrification gas (NO, N2O, N2) kinetics were assessed by automated gas chromatography while community composition was assessed by 16 S rRNA gene sequencing and qPCR of nosZI and II genes. Analyses revealed a sustained correlation between microbiome and N2O emission potential (N2O/(N2O + N2)) in the absence of a pH effect. Further, a continuum of gas accumulation phenotypes linked to NO accumulation and sensitive to carbon addition are identified. Separate phenotypes carried out N2O production and reduction steps more concurrently or sequentially and thus determined N2O accumulation and emission potential (N2O/(N2O + N2)). Concurrent N2O producing/reducing soils typically contained NO accumulation to a low steady state, while carbon addition manipulations which increased NO accumulation also increased sequentiality of N2O production/reduction and thus emission potential (N2O/(N2O + N2)). These features may indicate a conserved NO inhibitory mechanism across multiple effectors (rainfall, community composition, carbon availability).

Oxygen is known to repress denitrification at the transcriptional and metabolic levels. It has been a common notion that nitrous oxide reductase (N2 OR) is the most sensitive enzyme among the four N-oxide reductases involved in denitrification, potentially leading to increased N2 O production under suboxic or fluctuating oxygen conditions. We present detailed gas kinetics and transcription patterns from batch culture experiments with Paracoccus denitrificans, allowing in vivo estimation of e(-) -flow to O2 and N2 O under various O2 regimes. Transcription of nosZ took place concomitantly with that of narG under suboxic conditions, whereas transcription of nirS and norB was inhibited until O2 levels approached 0 μM in the liquid. Catalytically functional N2 OR was synthesized and active in aerobically raised cells transferred to vials with 7 vol% O2 in headspace, but N2 O reduction rates were 10 times higher when anaerobic pre-cultures were subjected to the same conditions. Upon oxygen exposure, there was an incomplete and transient inactivation of N2 OR that could be ascribed to its lower ability to compete for electrons compared with terminal oxidases. The demonstrated reduction of N2 O at high O2 partial pressure and low N2 O concentrations by a bacterium not known as a typical aerobic denitrifier may provide one clue to the understanding of why some soils appear to act as sinks rather than sources for atmospheric N2 O.

Homeostatic control of nitric oxide (NO) at nanomolar concentrations appears common among denitrifying bacteria, often ascribed to synchronized expression of nitrite and nitric oxide reductase (Nir and Nor). We questioned whether this is sufficient: using the reported substrate affinities for cytochrome cd1 nitrite reductase (cNor), our model of batch cultures of Paracoccus denitrificans predicted NO concentrations orders of magnitude higher than measured. We rejected a hypothesis that the homeostatic control is due to a negative feedback by NO on the activity of NirS because the inclusion of such feedback resulted in too slow anaerobic growth and N2 production. We proceeded by determining the kinetic parameters for cNor in vivo by a carefully designed experiment, allowing the estimation of NO concentration at the cell surface while anoxic cultures depleted low headspace doses of NO. With the new parameters for cNor kinetics in vivo {v = vmax /[1 + K2 /(NO) + K1 × K2 /(NO)(2) ]; vmax = 3.56 fmol NO cell(-1) h(-1) , K1 1 nM, and K2 = 34 nM}, the model predicted NO concentrations close to that measured. Thus, enzyme kinetics alone can explain the observed NO homeostasis. Determinations of enzyme kinetic parameters in vivo are not trivial but evidently required to understand and model NO kinetics in denitrifying organisms in soils and aquatic environments.

Oxygen is known to repress denitrification at the transcriptional and metabolic levels. It has been a common notion that nitrous oxide reductase (N2 OR) is the most sensitive enzyme among the four N-oxide reductases involved in denitrification, potentially leading to increased N2 O production under suboxic or fluctuating oxygen conditions. We present detailed gas kinetics and transcription patterns from batch culture experiments with Paracoccus denitrificans, allowing in vivo estimation of e(-) -flow to O2 and N2 O under various O2 regimes. Transcription of nosZ took place concomitantly with that of narG under suboxic conditions, whereas transcription of nirS and norB was inhibited until O2 levels approached 0 μM in the liquid. Catalytically functional N2 OR was synthesized and active in aerobically raised cells transferred to vials with 7 vol% O2 in headspace, but N2 O reduction rates were 10 times higher when anaerobic pre-cultures were subjected to the same conditions. Upon oxygen exposure, there was an incomplete and transient inactivation of N2 OR that could be ascribed to its lower ability to compete for electrons compared with terminal oxidases. The demonstrated reduction of N2 O at high O2 partial pressure and low N2 O concentrations by a bacterium not known as a typical aerobic denitrifier may provide one clue to the understanding of why some soils appear to act as sinks rather than sources for atmospheric N2 O.

As genomic data for bacteria are unraveled at an increasing speed, there is a need for more efficient and refined techniques to characterize metabolic traits. The regulatory apparatus for denitrification, for instance, has been explored extensively for type strains, but we lack refined observations of how these and wild type denitrifiers respond metabolically to changing environmental conditions. There is a need for new “phenomic” approaches, and the present paper describes one; an automated incubation system for the study of gas kinetics in 15 parallel bacterial cultures. An autosampler with a peristaltic pump takes samples from the headspace, and replaces the sampled gas with He by reversing the pump. The sample flows through the injector of a micro GC (for determination of N2 ,O 2 ,C H4 ,C O2 ,N 2O) to the inlet of a chemoluminescence NO analyzer. The linear range for NO is 0.5–10 4 ppmv (CV=2%, detection limit 0.2 ppmv). The gas leakage of N2 into the system is low and reproducible, allowing the quantification of N2 production (in flasks with He+O2 atmosphere) with a detection limit of 150–200 nmol N2 for a single time increment. The gas loss by each sampling is taken into account, securing mass balance for all gases, thus allowing accurate estimation of electron flows to the various terminal acceptors (O2 ,N O2 , NO, N2O) throughout the culture's depletion of O2 and NOx. We present some experimental results with Agrobacterium tumefaciens, Paracoccus denitrificans and denitrifying communities, demonstrating the system's potential for unraveling contrasting patterns of denitrification gene expression as a function of concentrations of O2 and NO in the medium. © 2007 Elsevier B.V. All rights reserved.