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Stimulating litho-autotrophic denitrification in aquifers with hydrogen is a promising strategy to remove excess NO

Rhizobia living as microsymbionts inside nodules have stable access to carbon substrates, but also must survive as free-living bacteria in soil where they are starved for carbon and energy most of the time. Many rhizobia can denitrify, thus switch to anaerobic respiration under low O(2) tension using N-oxides as electron acceptors. The cellular machinery regulating this transition is relatively well known from studies under optimal laboratory conditions, while little is known about this regulation in starved organisms. It is, for example, not known if the strong preference for N(2)O− over NO(3)(−) reduction in bradyrhizobia is retained under carbon limitation. Here, we show that starved cultures of a Bradyrhizobium strain with respiration rates 1 to 18% of well-fed cultures reduced all available N(2)O before touching provided NO(3)(−). These organisms, which carry out complete denitrification, have the periplasmic nitrate reductase NapA but lack the membrane-bound nitrate reductase NarG. Proteomics showed similar levels of NapA and NosZ (N(2)O reductase), excluding that the lack of NO(3)(−) reduction was due to low NapA abundance. Instead, this points to a metabolic-level phenomenon where the bc1 complex, which channels electrons to NosZ via cytochromes, is a much stronger competitor for electrons from the quinol pool than the NapC enzyme, which provides electrons to NapA via NapB. The results contrast the general notion that NosZ activity diminishes under carbon limitation and suggest that bradyrhizobia carrying NosZ can act as strong sinks for N(2)O under natural conditions, implying that this criterion should be considered in the development of biofertilizers. IMPORTANCE Legume cropped farmlands account for substantial N(2)O emissions globally. Legumes are commonly inoculated with N(2)-fixing bacteria, rhizobia, to improve crop yields. Rhizobia belonging to Bradyrhizobium, the microsymbionts of several economically important legumes, are generally capable of denitrification but many lack genes encoding N(2)O reductase and will be N(2)O sources. Bradyrhizobia with complete denitrification will instead act as sinks since N(2)O-reduction efficiently competes for electrons over nitrate reduction in these organisms. This phenomenon has only been demonstrated under optimal conditions and it is not known how carbon substrate limitation, which is the common situation in most soils, affects the denitrification phenotype. Here, we demonstrate that bradyrhizobia retain their strong preference for N(2)O under carbon starvation. The findings add basic knowledge about mechanisms controlling denitrification and support the potential for developing novel methods for greenhouse gas mitigation based on legume inoculants with the dual capacity to optimize N(2) fixation and minimize N(2)O emission.

To improve the nitrogen fixation, legume crops are often inoculated with selected effective rhizobia. However, there is large variation in how well the inoculant strains compete with the indigenous microflora in soil. To assess the success of the inoculant, it is necessary to distinguish it from other, closely related strains. Methods used until now have generally been based either on fingerprinting methods or on the use of reporter genes. Nevertheless, these methods have their shortcomings, either because they do not provide sufficiently specific information on the identity of the inoculant strain, or because they use genetically modified organisms that need prior authorization to be applied in the field or other uncontained environments. Another possibility is to target a gene that is naturally present in the bacterial genomes. Here we have developed a method that is based on amplicon sequencing of the bacterial housekeeping gene rpoB, encoding the beta-subunit of the RNA polymerase, which has been proposed as an alternative to the 16S rRNA gene to study the diversity of rhizobial populations in soils. We evaluated the method under laboratory and field conditions. Peanut seeds were inoculated with various Bradyrhizobium strains. After nodule development, DNA was extracted from selected nodules and the nodulating rhizobia were analysed by amplicon sequencing of the rpoB gene. The analyses of the sequence data showed that the method reliably identified bradyrhizobial strains in nodules, at least at the species level, and could be used to assess the competitiveness of the inoculant compared to other bradyrhizobia.

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.

Rhizobia living as microsymbionts inside nodules have stable access to carbon substrates, but also have to survive as free-living bacteria in soil where they are starved for carbon and energy most of the time. Many rhizobia can denitrify, thus switch to anaerobic respiration under low O2tension using N-oxides as electron acceptors. The cellular machinery regulating this transition is relatively well-known from studies under optimal laboratory conditions, while little is known about this regulation in starved organisms. It is, for example, not known if the strong preference for N2O-over NO3--reduction in bradyrhizobia is retained under carbon limitation. Here we show that starved cultures of aBradyrhizobiumstrain with respiration rates 1-18% of well-fed cultures, reduced all available N2O before touching provided NO3-. Proteomics showed similar abundance of Nap (periplasmic NO3-reductase) and NosZ (N2O reductase), suggesting that competition between electron pathways to Nap and NosZ favoured N2O reduction also in starved cells, similar to well-fed cultures. This contrasts the general notion that NosZ activity diminishes under carbon limitation. The results suggest that bradyrhizobia carrying NosZ can act as strong sinks for N2O under natural conditions and that this criterion should be considered in the development of biofertilizers.ImportanceLegume cropped farmlands account for substantial N2O emissions globally. Legumes are commonly inoculated with N2-fixing bacteria, rhizobia, to improve crop yields. Rhizobia belonging toBradyrhizobium, the micro-symbionts of several economically important legumes, are generally capable of denitrification but many lack genes encoding N2O reductase and will be N2O sources. Bradyrhizobia with complete denitrification will instead act as sinks since N2O-reduction efficiently competes for electrons over nitrate reduction in these organisms. This phenomenon has only been demonstrated under optimal conditions and it is not known how carbon substrate limitation, which is the common situation in most soils, affects the denitrification phenotype. Here we demonstrate that bradyrhizobia retain their strong preference for N2O under carbon starvation. The findings add basic knowledge about mechanisms controlling denitrification and support the potential for developing novel methods for greenhouse gas mitigation based on legume inoculants with the dual capacity to optimize N2-fixation and minimize N2O emission.

Soil pH is a key controller of denitrification. We analysed the metagenomics/transcriptomics and phenomics of two soils from a long-term liming experiment, SoilN (pH 6.8) and un-limed SoilA (pH 3.8). SoilA had severely delayed N2O reduction despite early transcription of nosZ (mainly clade I), encoding N2O reductase, by diverse denitrifiers. This shows that post-transcriptionally hampered maturation of the NosZ apo-protein at low pH is a generic phenomenon. Identification of transcript reads of several accessory genes in the nos cluster indicated that enzymes for NosZ maturation were present across a range of organisms, eliminating their absence as an explanation for the failure to produce a functional enzyme. nir transcript abundances (for NO2− reductase) in SoilA suggest that low NO2− concentrations in acidic soils, often ascribed to abiotic degradation, are primarily due to biological activity. The accumulation of NO2− in neutral soil was ascribed to high nar expression (nitrate reductase). The -omics results revealed dominance of nirK over nirS in both soils while qPCR showed the opposite, demonstrating that standard primer pairs only capture a fraction of the nirK pool. qnor encoding NO reductase was strongly expressed in SoilA, implying an important role in controlling NO. Production of HONO, for which some studies claim higher, others lower, emissions from NO2− accumulating soil, was estimated to be ten times higher from SoilA than from SoilN. The study extends our understanding of denitrification-driven gas emissions and the diversity of bacteria involved and demonstrates that gene and transcript quantifications cannot always reliably predict community phenotypes.

Improved understanding of the microbes producing and reducing N2O spurs the invention of direct and manipulative methods to reduce N2O emissions, beyond that achieved by ‘good agronomic practice’. A core issue is the kinetics of N2O-reductase by denitrifying organisms during anoxic/hypoxic spells; the ‘hot moments’ of N2O emission. Soil pH has a pervasive effect on the Expression of the enzyme, and liming soils will reduce the N2O emission. In this review we present some novel options for mitigation of N2O emissions, beside liming. During recent years, an increasing number of strongly N2O reducing organisms have been identified, and ways to enhance their numbers and activity in soil are emerging. One suggestion is to develop inoculants for legumes that are strong N2O reducers. Another is to grow N2O reducing organisms in digestates from biogas production. These promising options need further elaboration and validation.

The greenhouse gas nitrous oxide (N

The strong greenhouse gas nitrous oxide (N2O) can be emitted from wastewater treatment systems as a byproduct of ammonium oxidation and as the last intermediate in the stepwise reduction of nitrate to N2 by denitrifying organisms. A potential strategy to reduce N2O emissions would be to enhance the activity of N2O reductase (NOS) in the denitrifying microbial community. A survey of existing literature on denitrification in wastewater treatment systems showed that the N2O reducing capacity (VmaxN2O→N2) exceeded the capacity to produce N2O (VmaxNO3→N2O) by a factor of 2–10. This suggests that denitrification can be an effective sink for N2O, potentially scavenging a fraction of the N2O produced by ammonium oxidation or abiotic reactions. We conducted a series of incubation experiments with freshly sampled activated sludge from a wastewater treatment system in Oslo and found that the ratio α = VmaxN2O→N2/VmaxNO3→N2O fluctuated between 2 and 5 in samples taken at intervals over a period of 5 weeks. Adding a cocktail of carbon substrates resulted in increasing rates, but had no significant effect on α. Based on these results – complemented with qPCR and metaproteomic data – we discuss whether the overcapacity to reduce N2O can be ascribed to gene/protein abundance ratios (nosZ/nir), or whether in-cell competition between the reductases for electrons could be of greater importance.

Manipulating soil metabolism by heavy inoculation with microbes is deemed realistic if waste from anaerobic digestion (digestate) is utilized as substrate and vector, but requires organisms that can grow both in digestate and soil (=generalist). We designed a strategy to enrich and isolate such generalist N2O-respiring bacteria (NRB) in soil and digestate, to provide inoculum for reducing N2O-emissions from agricultural soil. Sequential anaerobic enrichment cultures were provided with a small dose of O2 and unlimited N2O, alternating between sterilized digestate and soil as substrates. The cultures were monitored for gas kinetics and community composition (16SrDNA), and cluster-analysis identified generalist-OTUs which became dominant, digestate/soil-specialists which did not, and a majority that were diluted out. Several NRBs circumscribed by generalist-OTU’s were isolated, genome sequenced to screen for catabolic capacity, and phenotyped, to assess their capacity as N2O-sinks in soil. The two isolates Cloacibacterium sp., carrying only N2O-reductase (Clade-II) and Pseudomonas sp., with full-fledged denitrification-pathway, were both very effective N2O-sinks in soil, with Pseudomonas sp., showing a long-lasting sink effect, suggesting better survival in soil. This avenue for utilizing waste to bioengineer the soil microbiota holds promise to effectively combat N2O-emissions but could also be utilized for enhancing other metabolic functions in soil.Graphical abstract

Bradyrhizobia are common members of soil microbiomes and known as N2 -fixing symbionts of economically important legumes. Many are also denitrifiers, which can act as sinks or sources for N2 O. Inoculation with compatible rhizobia is often needed for optimal N2 -fixation, but the choice of inoculant may have consequences for N2 O emission. Here, we determined the phylogeny and denitrification capacity of Bradyrhizobium strains, most of them isolated from peanut-nodules. Analyses of genomes and denitrification end-points showed that all were denitrifiers, but only ~1/3 could reduce N2 O. The N2 O-reducing isolates had strong preference for N2 O- over NO3 - -reduction. Such preference was also observed in a study of other bradyrhizobia and tentatively ascribed to competition between the electron pathways to Nap (periplasmic NO3 - reductase) and Nos (N2 O reductase). Another possible explanation is lower abundance of Nap than Nos. Here, proteomics revealed that Nap was instead more abundant than Nos, supporting the hypothesis that the electron pathway to Nos outcompetes that to Nap. In contrast, Paracoccus denitrificans, which has membrane-bond NO3 - reductase (Nar), reduced N2 O and NO3 - simultaneously. We propose that the control at the metabolic level, favoring N2 O reduction over NO3 - reduction, applies also to other denitrifiers carrying Nos and Nap but lacking Nar. This article is protected by copyright. All rights reserved.

Soil pH is a key controller of denitrification. We analysed the metagenomics/transcriptomics and phenomics of two soils from a long-term liming experiment, SoilN (pH 6.8) and un-limed SoilA (pH 3.8). SoilA had severely delayed N

Bradyrhizobia are common members of soil microbiomes and known as N

Liming acidic soils is often found to reduce their N2O emission due to lowered N2O/(N2O + N2) product ratio of denitrification. Some field experiments have shown the opposite effect, however, and the reason for this could be that liming stimulates nitrification-driven N2O production by enhancing nitrification rates, and by favoring ammonia oxidizing bacteria (AOB) over ammonia oxidizing archaea (AOA). AOB produce more N2O than AOA, and high nitrification rates induce transient/local hypoxia, thereby stimulating heterotrophic denitrification. To study these phenomena, we investigated nitrification and denitrification kinetics and the abundance of AOB and AOA in soils sampled from a field experiment 2–3 years after liming. The field trial compared traditional liming (carbonates) with powdered siliceous rocks. As expected, the N2O/(N2O + N2) product ratio of heterotrophic denitrification declined with increasing pH, and the potential nitrification rate and its N2O yield (YN2O: N2O-N/NO3–-N), as measured in fully oxic soil slurries, increased with pH, and both correlated strongly with the AOB/AOA gene abundance ratio. Soil microcosm experiments were monitored for nitrification, its O2-consumption and N2O emissions, as induced by ammonium fertilization. Here we observed a conspicuous dependency on water filled pore space (WFPS): at 60 and 70% WFPS, YN2O was 0.03-0.06% and 0.06–0.15%, respectively, increasing with increasing pH, as in the aerobic soil slurries. At 85% WFPS, however, YN2O was more than two orders of magnitude higher, and decreased with increasing pH. A plausible interpretation is that O2 consumption by fertilizer-induced nitrification cause hypoxia in wet soils, hence induce heterotrophic nitrification, whose YN2O decline with increasing pH. We conclude that while low emissions from nitrification in well-drained soils may be enhanced by liming, the spikes of high N2O emission induced by ammonium fertilization at high soil moisture may be reduced by liming, because the heterotrophic N2O reduction is enhanced by high pH.

Denitrifier community phenotypes often result in transient accumulation of denitrification (NO3−→NO2−→NO→N2O→N2) intermediates. Consequently, anoxic spells drive NO-, N2O- and possibly HONO-emissions to the atmosphere, affecting both climate and tropospheric chemistry. Soil pH is a key controller of intermediate levels, and while there is a clear negative correlation between pH and emission of N2O, NO2− concentrations instead increase with pH. These divergent trends are probably a combination of direct effects of pH on the expression/activity of denitrification enzymes, and an indirect effect via altered community composition. This was studied by analyzing metagenomics/transcriptomics and phenomics of two soil denitrifier communities, one of pH 3.8 (Soil3.8) and the other 6.8 (Soil6.8). Soil3.8 had severely delayed N2O reduction despite early transcription of nosZ, encoding N2O reductase, by diverse denitrifiers, and of several nosZ accessory genes. This lends support to a post-transcriptional, pH-dependent mechanism acting on the NosZ apo-protein or on enzymes involved in its maturation. Metagenome/metatranscriptome reads of nosZ were almost exclusively clade I in Soil3.8 while clade II dominated in Soil6.8. Reads of genes and transcripts for NO2−-reductase were dominated by nirK over nirS in both soils, while qPCR-based determinations showed the opposite, demonstrating that standard primer pairs only capture a fraction of the nirK community. The -omics results suggested that low NO2− concentrations in acidic soils, often ascribed to abiotic degradation, are primarily due to enzymatic activity. The NO reductase gene qnor was strongly expressed in Soil3.8, suggesting an important role in controlling NO. Production of HONO, for which some studies claim higher, others lower, emissions from NO2− accumulating soil, was estimated to be ten times higher from Soil3.8 than from Soil6.8. The study extends our understanding of denitrification-driven gas emissions and the diversity of bacteria involved and demonstrates that gene and transcript quantifications cannot always reliably predict community phenotypes.

Inoculating agricultural soils with N2O-respiring bacteria (NRB) can reduce N2O-emissions, 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.

International audience; The interaction between rhizobia and their legume host plants conduces to the formation of specialized root organs called nodules where rhizobia differentiate into bacteroids which fix atmospheric nitrogen to the benefit of the plant. This beneficial symbiosis is of importance in the context of sustainable agriculture as legumes do not require the addition of nitrogen fertilizer to grow. Interestingly, nitric oxide (NO) has been detected at various steps of the rhizobium–legume symbiosis where it has been shown to play multifaceted roles. Both bacterial and plant partners are involved in NO synthesis in nodules. To better understand the role of NO, and in particular the role of bacterial NO, at all steps of rhizobia–legumes interaction, the enzymatic sources of NO have to be elucidated. In this review, we discuss different enzymatic reactions by which rhizobia may potentially produce NO. We argue that there is most probably no NO synthase activity in rhizobia, and that instead the NO2− reductase nirK, which is part of the denitrification pathway, is the main bacterial source of NO. The nitrate assimilation pathway might contribute to NO production but only when denitrification is active. The different approaches to measure NO in rhizobia are also addressed.

SummaryBradyrhizobia are common members of soil microbiomes and known as N2-fixing symbionts of economically important legumes. Many are also denitrifiers, which can act as sinks or sources for N2O. Inoculation with compatible rhizobia is often needed for optimal N2-fixation, but the choice of inoculant may also have consequences for N2O emission. Here, we analyzed the phylogeny and denitrification capacity of Bradyrhizobium strains, most of them isolated from peanut-nodules. All were dinitrifiers, but only ~1/3 could reduce N2O while most others were net N2O producers. The N2O-reducing isolates showed strong preference for N2O- over NO3−-reduction. Such preference was also observed in a study of other bradyrhizobia and tentatively ascribed to competition between the electron pathways to Nap (periplasmic NO3− reductase) and Nos (N2O reductase). Another possible explanation is lower abundance of Nap than Nos. Here, proteomics revealed that Nap was instead more abundant than Nos, supporting the hypothesis that the electron pathway to Nos outcompetes that to Nap. In contrast, Paracoccus denitrificans, which has membrane-bond NO3− reductase (Nar), reduced N2O and NO3− simultaneously. We propose that the control at the metabolic level, favoring N2O reduction over NO3− reduction, applies also to other denitrifiers carrying Nos and Nap but lacking Nar.Originality-Significance StatementThis study extends the current knowledge on denitrification in bradyrhizobia, which mostly originates from studies of one model strain, by investigating the denitrification phenotypes of a diverse collection of Bradyrhizobium isolates. Only 1/3 of them could reduce N2O while the others were net sources for this potent greenhouse gas. All N2O-reducers showed strong preference for N2O over NO3−. We revealed by proteomics that this was not explained by differences in the abundances of Nap (periplasmic nitrate reductase) and Nos (N2O reductase), which strengthens our hypothesis (Mania et al., 2020) of a metabolic control mechanism by which Nos competes efficiently with Nap for electrons, making these organisms strong sinks for N2O. The findings highlight the potential importance of these organisms as N2O sinks in natural and agricultural ecosystems and pinpoint the need to take N2O reduction into account, along with N2-fixation effectiveness, when searching for strains suitable for production of inoculants.

International audience; Fungal denitrification is claimed to produce non‐negligible amounts of N2O in soils, but few tested species have shown significant activity. We hypothesized that denitrifying fungi would be found among those with assimilatory nitrate reductase, and tested 20 such batch cultures for their respiratory metabolism, including two positive controls, Fusarium oxysporum and Fusarium lichenicola, throughout the transition from oxic to anoxic conditions in media supplemented with urn:x-wiley:14622912:media:emi14980:emi14980-math-0001. Enzymatic reduction of urn:x-wiley:14622912:media:emi14980:emi14980-math-0002 (NIR) and NO (NOR) was assessed by correcting measured NO‐ and N2O‐kinetics for abiotic NO‐ and N2O‐production (sterile controls). Significant anaerobic respiration was only confirmed for the positive controls and for two of three Fusarium solani cultures. The NO kinetics in six cultures showed NIR but not NOR activity, observed through the accumulation of NO. Others had NOR but not NIR activity, thus reducing abiotically produced NO to N2O. The presence of candidate genes (nirK and p450nor) was confirmed in the positive controls, but not in some of the NO or N2O accumulating cultures. Based on our results, we conclude that only the Fusarium cultures were able to sustain anaerobic respiration and produced low amounts of N2O as a response to an abiotic NO production from the medium.

Emissions of the potent greenhouse gas N2O is one of the environmental problems associated with intensive use of synthetic N fertilizers, and novel N2O mitigation strategies are needed to minimize fertilizer applications and N2O release without affecting agricultural efficiencies. Increased incorporation of legume crops in agricultural practices offers a sustainable alternative. Legumes, in their symbiosis with nitrogen fixing bacteria, rhizobia, reduce the need for fertilizers and also respond to the need for increased production of plant-based proteins. Not all combinations of rhizobia and legumes result in efficient nitrogen fixation, and legume crops therefore often need to be inoculated with compatible rhizobial strains. Recent research has demonstrated that some rhizobia are also very efficient N2O reducers. Several nutritionally and economically important legumes form root nodules in symbiosis with bacteria belonging to Bradyrhizobium. Here, the host-ranges of fourteen N2O reducing Bradyrhizobium strains were tested on six legume hosts; cowpea, groundnut, mung bean, haricot bean, soybean, and alfalfa. The plants were grown for 35 days in pots in sterile sand supplemented with N-free nutrient solution. Cowpea was the most promiscuous host nodulated by all test strains, followed by groundnut (11 strains) and mungbean (4 strains). Three test strains were able to nodulate all these three legumes, while none nodulated the other three hosts. For cowpea, five strains increased the shoot dry weight and ten strains the shoot nitrogen content (pairwise comparison; p < 0.05). For groundnut the corresponding results were three and nine strains. The symbiotic effectiveness for the different strains ranged from 45 to 98% in cowpea and 34 to 95% in groundnut, relative to fertilized controls. The N2O reduction capacity of detached nodules from cowpea plants inoculated with one of these strains confirmed active N2O reduction inside the nodules. When released from senescent nodules such strains are expected to also act as sinks for N2O produced by denitrifying organisms in the soil microbial community. Our strategy to search among known N2O-reducing Bradyrhizobium strains for their N2-fixation effectiveness successfully identified several strains which can potentially be used for the production of legume inoculants with the dual capacities of efficacious N2-fixation and N2O reduction.

Fungal denitrification is claimed to produce non-negligible amounts of N

Bradyrhizobia are abundant soil bacteria, which can form nitrogen-fixing symbioses with leguminous plants, including important crops such as soybean, cowpea and peanut. Many bradyrhizobia can denitrify, but studies have hitherto focused on a few model organisms. We screened 39 diverse Bradyrhizobium strains, isolated from legume nodules. Half of them were unable to reduce N

Bradyrhizobia are abundant soil bacteria, which can formnitrogen-fixing symbioses with leguminous plants, including important crops such as soybean, cowpea and peanut. Many bradyrhizobia can denitrify, but studies have hitherto focused on a few model organisms. We screened 39 diverse Bradyrhizobium strains, isolated from legume nodules. Half of them were unable to reduce N2O, making them sources of this greenhouse gas. Most others could denitrify NO3 − to N2. Timeresolved gas kinetics and transcription analyses during transition to anaerobic respiration revealed a common regulation of nirK, norCB and nosZ (encoding NO2 −, NO and N2O reductases), and differing regulation of napAB (encoding periplasmic NO3 − reductase). A prominent feature in all N2-producing strains was a virtually complete hampering of NO3 − reduction in the presence of N2O. In-depth analyses suggest that this was due to a competition between electron transport pathways, strongly favouring N2OoverNO3 − reduction. In a natural context, bacteria with this feature would preferentially reduce available N2O, produced by themselves or other soil bacteria,making them powerful sinks for this greenhouse gas. One way to augment such populations in agricultural soils is to develop inoculants for legume crops with dual capabilities of efficient N2-fixation and efficient N2O reduction. A common mechanism for efficient N2O reduction in diverse isolates of nodule-forming bradyrhizobia

Bacillus vireti is a nitrate-ammonifying bacterium and a partial denitrifier, reducing NO3 (-) , NO2 (-) , NO and N2 O with NarG, NrfA, CbaA and NosZ respectively. Growth is optimized through successive use of the electron acceptors O2 and NO3 (-) , followed by NO2 (-) , NO and N2 O. Fermentation takes place simultaneously with anaerobic respiration. When grown in batch culture with 5 mM initial NO3 (-) , transcription of nrfA was high and most NO3 (-) was reduced to NH4 (+) . With 20 mM initial NO3 (-) , nrfA transcription was lower and more than 50% of the nitrate was recovered as NO, N2 O and N2 . Analysis of gene transcription patterns and corresponding gas kinetics indicated that O2 and NO2 (-) or NO are main controllers of nrfA, nirB, cbaA and nosZ transcription. This was corroborated by analyses of putative binding regions for specific transcriptional regulators. Furthermore, we demonstrate that N2 O reduction in B. vireti supports growth. The high nosZ transcription but low N2 O production seen at 5 mM NO3 (-) implies that this organism can use N2 O reductase to scavenge N2 O from other organisms in the soil, thus possibly acting as a net sink for N2 O.

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.

Significance Denitrifying microorganisms reduce nitrate to N2 via nitrite, NO, and N2O under hypoxic/anoxic conditions. Since these organisms are the main sources and sinks for N2O in the environment, their regulatory biology controls the emission of this potent greenhouse gas. We demonstrate bet hedging in Paracoccus denitrificans when facing hypoxia: a minority of the cells synthesize NIR + NOR, the N2O source, while all synthesize NOS, the N2O sink, hence the population becomes a strong net sink for N2O. Bet hedging is prominent below 20 °C and data indicate that it is widespread in soil organisms. This suggests a prominent role of bet hedging in controlling N2O emissions that can now be tested for in other strains and in natural environments., When oxygen becomes limiting, denitrifying bacteria must prepare for anaerobic respiration by synthesizing the reductases NAR (NO3− → NO2−), NIR (NO2− → NO), NOR (2NO → N2O), and NOS (N2O → N2), either en bloc or sequentially, to avoid entrapment in anoxia without energy. Minimizing the metabolic burden of this precaution is a plausible fitness trait, and we show that the model denitrifier Paracoccus denitrificans achieves this by synthesizing NOS in all cells, while only a minority synthesize NIR. Phenotypic diversification with regards to NIR is ascribed to stochastic initiation of gene transcription, which becomes autocatalytic via NO production. Observed gas kinetics suggest that such bet hedging is widespread among denitrifying bacteria. Moreover, in response to oxygenation, P. denitrificans preserves NIR in the poles of nongrowing persister cells, ready to switch to anaerobic respiration in response to sudden anoxia. Our findings add dimensions to the regulatory biology of denitrification and identify regulatory traits that decrease N2O emissions.

The strong greenhouse gas nitrous oxide (N

Source at https://doi.org/10.1038/s41598-018-24082-y. Here we show that a commercial blocking reagent (G2) based on modified eukaryotic DNA significantly improved DNA extraction efficiency. We subjected G2 to an inter-laboratory testing, where DNA was extracted from the same clay subsoil using the same batch of kits. The inter-laboratory extraction campaign revealed large variation among the participating laboratories, but the reagent increased the number of PCR-amplified16S rRNA genes recovered from biomass naturally present in the soils by one log unit. An extensive sequencing approach demonstrated that the blocking reagent was free of contaminating DNA, and may therefore also be used in metagenomics studies that require direct sequencing.

Anoxic spells in soil induce denitrification, i.e. the sequential reduction NO3-→NO2-→NO→N2O→N2, catalysed by the four enzymes NAR, NIR, NOR and NOS, respectively. Transient accumulation of all intermediates is inevitable, but the concentrations depend on the regulation of gene expression and the physical/chemical properties of the soil. Nitrite is chemically unstable at low pH, decomposing via a conglomerate of abiotic reactions with metals and organic compounds which can result in production of NO, N2O, N2 and nitrosated organic compounds (R-NO). There is evidence that acidic soils accumulate less nitrite than neutral soils, but it is unclear if this is due to high abiotic decomposition rate (VADEC) or fast enzymatic reduction of nitrite (VNIR) at low pH. To investigate this, we monitored the kinetics of NO2-, NO, N2O and N2 during anoxic incubations of three organic soils with pHCaCl2 ranging from 3.4 to 7.2, taken from a long-term liming experiment. In parallel, we determined the rate of abiotic nitrite decay (VADEC) and its product stoichiometry (NO, N2O and R-NO) in gamma-irradiated soils. VADEC was clearly first-order with respect to HNO2 (kHNO2 = 1.4 h-1), N-gas production (NO, N2O and N2) accounted for only ~50% of VADEC, the rest was ascribed to nitrosation (R-NO). During denitrification (live soil incubation), the nitrite concentrations reached 2-3 mM in the soils with pH 4.9 and 7.2, while the soil with pH 3.4 kept nitrite concentrations at 20-50 µM , except for a short spike reaching 160 μM. Estimated rates of nitrite scavenging by the two competing sinks (NIR and ADEC) showed that NIR was the strongst nitrite sink in soil with pH 3.4 (VNIR>VADEC), while VNIR ≈ VADEC in the soil with pH 5.9. In the soil with pH 7.2, VADEC was insignificant. Thus, the regulation of denitrification (high VNIR relative to VNAR) played a crucial role in determining nitrite kinetics, hence the fate of nitrite in acid soils. High nitrite reductase activity effectively minimized abiotic nitrite decomposition and nitrosation of soil organic matter. The results shed light on regulation of denitrification in acid soils, and its implications for the fate of nitrogen during denitrification events. Nitrite kinetics during anoxia: the role of abiotic reactions versus microbial reduction

Quinoline is biodegradable under anaerobic conditions, but information about the degradation kinetics and the involved microorganisms is scarce. Here, the dynamics of a quinoline-degrading bacterial consortium were studied in anoxic batch cultures containing nitrate. The cultures removed 83.5% of the quinoline during the first 80 hours, which were dominated by denitrification, and then switched to methanogenesis when the nitrogen oxyanions were depleted. Time-resolved community analysis using pyrosequencing revealed that denitrifiying bacteria belonging to the genus Thauera were enriched during the denitrification stage from 12.2% to 38.8% and 50.1% relative abundance in DNA and cDNA libraries, respectively. This suggests that they are key organisms responsible for the initial attack on quinoline. Altogether, 13 different co-abundance groups (CAGs) containing 76 different phylotypes were involved, directly or indirectly, in quinoline degradation. The dynamics of these CAGs show that specific phylotypes were associated with different phases of the degradation. Members of Rhodococcus and Desulfobacterium, as well as Rhodocyclaceae- and Syntrophobacteraceae-related phylotypes, utilized initial metabolites of the quinoline, while the resulting smaller molecules were used by secondary fermenters belonging to Anaerolineae. The concerted action by the different members of this consortium resulted in an almost complete anaerobic mineralization of the quinoline.

Ammonia oxidising bacteria (AOB) are thought to emit more nitrous oxide (N

Present-day knowledge on the regulatory biology of denitrification is based on studies of selected model organisms. These show large variations in their potential contribution to NO2-, NO, and N2O accumulation, attributed to lack of genes coding for denitrification reductases, but also to variations in their transcriptional regulation, as well as to post-transcriptional phenomena. To validate the relevance of these observations, there is a need to study a wider range of denitrifiers. We designed an isolation protocol that identifies all possible combinations of truncated denitrification chains (NO3-/NO2-/NO/N2O/N2). Of 176 isolates from two soils (pH 3.7 and 7.4), 30 were denitrifiers sensu stricto, reducing NO2- to gas, and five capable of N2O reduction only. Altogether, 70 isolates performed at least one reduction step, including two DNRA isolates. Gas kinetics and electron flow calculations revealed that several features with potential impact on N2O production, reported from model organisms, also exist in these novel isolates, including denitrification bet-hedging and control of NO2-/NO/N2O accumulation. Whole genome sequencing confirmed most truncations but also showed that phenotypes cannot be predicted solely from genetic potential. Interestingly, and opposed to the commonly observed inability to reduce N2O under acidic conditions, one isolate identified as Rhodanobacter reduced N2O only at low pH.

Several Gram-positive bacteria carry genes for anaerobic reduction of NO3(-) via NO2(-) to NH4(+) or gaseous nitrogen compounds, but the processes are understudied for these organisms. Here, we present results from a whole-genome analysis of the soil bacterium Bacillus vireti and a phenotypic characterization of intermediate and end-products, formed under anoxic conditions in the presence of NO3(-). Bacillus vireti has a versatile metabolism. It produces acetate, formate, succinate and lactate from fermentation and performs dissimilatory nitrate reduction via NO2(-) to ammonium (DNRA) using NrfA, while NirB may detoxify NO2(-) in the cytoplasm. Moreover, it produces NO from an unknown source and reduces it via N2O to N2 using two enzymes connected to denitrification: an unusual NO reductase, qCuA Nor encoded by cbaA, and a z-type N2O reductase, encoded by nosZ. In batch cultures, B. vireti reduced all NO3(-) to NO2(-) before the NO2(-) was reduced further. The quantities of all products varied with the initial NO3(-) concentration. With 5 mM NO3(-) , 90% was reduced to NH4 (+) while with ≥ 20 mM NO3(-), 50% was reduced to NO, N2O and N2. This organism is thus an aggressive NO2(-) accumulator and may act as a net source and sink of NO and N2O.

Bacteria belonging to the genus Bradyrhizobium nodulate various leguminous woody plants and herbs, including economically important crops such as soybean, peanut and cowpea. Here we analysed 39 Bradyrhizobium strains originating from root nodules of the leguminous trees and crops Acacia saligna , Faidherbia albida , Erythrina brucei , Albizia gummifera , Millettia ferruginea , Cajanus cajan , Vigna unguiculata and Phaseolus vulgaris , growing in southern Ethiopia. Multilocus sequence analyses (MLSA) of the 16S rRNA, glnII , recA , gyrB and dnaK genes and the ITS region grouped the test strains into seven well-supported genospecies (I–VII), six of which occupied distinct positions excluding all hitherto defined Bradyrhizobium species. Analyses of the nodA , nodC and nifH genes suggested different evolutionary history of the chromosomal and symbiosis-related genes. Our study corroborates earlier findings that Ethiopia is a hotspot for rhizobial biodiversity, justifying further search for novel strains from this region and calling for intensified research on the ecology and biochemistry of these organisms.

A total of 18 strains, representing members of the genus Mesorhizobium , obtained from root nodules of woody legumes growing in Ethiopia, have been previously shown, by multilocus sequence analysis (MLSA) of five housekeeping genes, to form three novel genospecies. In the present study, the phylogenetic relationship between representative strains of these three genospecies and the type strains of their closest phylogenetic neighbours Mesorhizobium plurifarium , Mesorhizobium amorphae , Mesorhizobium septentrionale and Mesorhizobium huakuii was further evaluated using a polyphasic taxonomic approach. In line with our earlier MLSA of other housekeeping genes, the phylogenetic trees derived from the atpD and glnII genes grouped the test strains into three well-supported, distinct lineages that exclude all defined species of the genus Mesorhizobium . The DNA–DNA relatedness between the representative strains of genospecies I–III and the type strains of their closest phylogenetic neighbours was low (≤59 %). They differed from each other and from their closest phylogenetic neighbours by the presence/absence of several fatty acids, or by large differences in the relative amounts of particular fatty acids. While showing distinctive features, they were generally able to utilize a wide range of substrates as sole carbon and nitrogen sources. The strains belonging to genospecies I, II and III therefore represent novel species for which we propose the names Mesorhizobium shonense sp. nov., Mesorhizobium hawassense sp. nov. and Mesorhizobium abyssinicae sp. nov. The isolates AC39aT ( = LMG 26966T = HAMBI 3295T), AC99bT ( = LMG 26968T = HAMBI 3301T) and AC98cT ( = LMG 26967T = HAMBI 3306T) are proposed as type strains for the respective novel species.

Direct extraction and characterization of microbial community DNA through PCR amplicon surveys and metagenomics has revolutionized the study of environmental microbiology and microbial ecology. In particular, metagenomic analysis of nucleic acids provides direct access to the genomes of the “uncultivated majority.” Accelerated by advances in sequencing technology, microbiologists have discovered more novel phyla, classes, genera, and genes from microorganisms in the first decade and a half of the twenty-first century than since these “many very little living animalcules” were first discovered by van Leeuwenhoek. The unsurpassed diversity of soils promises continued exploration of a range of industrial, agricultural, and environmental functions. The ability to explore soil microbial communities with increasing capacity offers the highest promise for answering many outstanding who, what, where, when, why, and with whom questions such as: Which microorganisms are linked to which soil habitats? How do microbial abundances change with changing edaphic conditions? How do microbial assemblages interact and influence one another synergistically or antagonistically? What is the full extent of soil microbial diversity, both functionally and phylogenetically? What are the dynamics of microbial communities in space and time? How sensitive are microbial communities to a changing climate? What is the role of horizontal gene transfer in the stability of microbial communities? Do highly diverse microbial communities confer resistance and resilience in soils?, La lista completa de autores que integran el documento puede consultarse en el archivo., Facultad de Ciencias Exactas