Project description:Anthropogenic perturbations to the nitrogen cycle, primarily through use of synthetic fertilizers, is driving an unprecedented increase in the emission of nitrous oxide (N2O), a potent greenhouse gas, and an ozone depleting substance, causing urgency in identifying the sources and sinks of N2O. Microbial denitrification is a primary contributor to the biotic production of N2O in anoxic regions of soil, marine systems, and wastewater treatment facilities. Here, through comprehensive genome analysis, we show that pathway partitioning is a ubiquitous mechanism of complete denitrification by microbial communities. We have further investigated the mechanisms and consequences of process partitioning through detailed physiological characterization and kinetic modeling of a synthetic community of Rhodanobacter R12 and Acidovorax 3H11. We have discovered that these two bacterial isolates from a heavily NO3- contaminated superfund site complete denitrification through the exchange of nitrite (NO2-) and nitric oxide (NO). Our findings further demonstrate that cooperativity within this denitrifying community emerges through process partitioning of denitrification and other processes, including amino acid metabolism. We demonstrate that certain contexts, such as high NO3-, cause unbalanced growth of community members, due to differences in their substrate utilization kinetics and inter-enzyme competition. The altered growth characteristics of community members drives accumulation of toxic NO2- , which disrupts denitrification causing N2O off gassing.
Project description:Oxygen deficient zones (ODZs) are major sites of net natural oceanic nitrous oxide (N2O) production and emissions. In order to understand changes in the magnitude of N2O production in response to global change, knowledge on the individual contributions of the major microbial pathways (nitrification and denitrification) to N2O production and their regulation is needed. In the ODZ of the coastal area off Peru, the sensitivity of N2O production to oxygen and organic matter was investigated using 15N-tracer experiments in combination with qPCR and microarray analysis of total and active functional genes targeting archaeal amoA and nirS as marker genes for nitrification and denitrification, respectively. Denitrification was responsible for the highest N2O production with mean 8.7 nmol L-1 d-1 but up to 118 ± 27.8 nmol L-1 d-1 just below the oxic-anoxic interface. Highest N2O production from AO of 0.16 ± 0.003 nmol L-1 d-1 occurred in the upper oxycline at O2 concentrations of 10 - 30 µmol L-1 which coincided with highest archaeal amoA transcripts/genes. Oxygen responses of N2O production varied with substrate, but production and yields were generally highest below 10 µmol L-1 O2. Particulate organic matter additions increased N2O production by denitrification up to 5-fold suggesting increased N2O production during times of high particulate organic matter export. High N2O yields from ammonium oxidation of 2.1% were measured, but the overall contribution to N2O production stays an order of magnitude behind denitrification as an N2O source. Hence, these findings show that denitrification is the most important N2O production process in low oxygen conditions fueled by organic carbon supply which implies a positive feedback of the total oceanic N2O sources in response to increasing oceanic deoxygenation. [SUBMITTER_CITATION]: Frey, C., Bange, H. W., Achterberg, E. P., Jayakumar, A., Löscher, C. R., Arévalo-Martínez, D. L., León-Palmero, E., Sun, M., Sun, X., Xie, R. C., Oleynik, S., and Ward, B. B.: Regulation of nitrous oxide production in low-oxygen waters off the coast of Peru, Biogeosciences, 17, 2263-2287
Project description:Oxygen deficient zones (ODZs) are major sites of net natural oceanic nitrous oxide (N2O) production and emissions. In order to understand changes in the magnitude of N2O production in response to global change, knowledge on the individual contributions of the major microbial pathways (nitrification and denitrification) to N2O production and their regulation is needed. In the ODZ of the coastal area off Peru, the sensitivity of N2O production to oxygen and organic matter was investigated using 15N-tracer experiments in combination with qPCR and microarray analysis of total and active functional genes targeting archaeal amoA and nirS as marker genes for nitrification and denitrification, respectively. Denitrification was responsible for the highest N2O production with mean 8.7 nmol L-1 d-1 but up to 118 ± 27.8 nmol L-1 d-1 just below the oxic-anoxic interface. Highest N2O production from AO of 0.16 ± 0.003 nmol L-1 d-1 occurred in the upper oxycline at O2 concentrations of 10 - 30 µmol L-1 which coincided with highest archaeal amoA transcripts/genes. Oxygen responses of N2O production varied with substrate, but production and yields were generally highest below 10 µmol L-1 O2. Particulate organic matter additions increased N2O production by denitrification up to 5-fold suggesting increased N2O production during times of high particulate organic matter export. High N2O yields from ammonium oxidation of 2.1% were measured, but the overall contribution to N2O production stays an order of magnitude behind denitrification as an N2O source. Hence, these findings show that denitrification is the most important N2O production process in low oxygen conditions fueled by organic carbon supply which implies a positive feedback of the total oceanic N2O sources in response to increasing oceanic deoxygenation. [SUBMITTER_CITATION]: Frey, C., Bange, H. W., Achterberg, E. P., Jayakumar, A., Löscher, C. R., Arévalo-Martínez, D. L., León-Palmero, E., Sun, M., Sun, X., Xie, R. C., Oleynik, S., and Ward, B. B.: Regulation of nitrous oxide production in low-oxygen waters off the coast of Peru, Biogeosciences, 17, 2263-2287
Project description:Anthropogenic perturbations to the nitrogen cycle, primarily through use of synthetic fertilizers, have caused unprecedented increases in the emission of nitrous oxide (N2O) in recent decades. As a potent greenhouse gas, and an ozone depleting substance, understanding the sources and sinks of N2O is of vital importance. Nitrate (NO3-) reducing microbes are a primary contributor to the biotic production of N2O in anoxic regions of soil, marine systems, and wastewater treatment facilities through the process of denitrification. Thus, developing a better understanding of denitrifying microbial communities, and the environmental factors that influence N2O emissions may provide strategies to mitigate emissions in agriculture and wastewater treatment. Here, through comprehensive genome analysis, we show that pathway partitioning is a common strategy utilized by microbial communities to perform complete denitrification. Through detailed physiological characterization and kinetic modeling of a cooperative synthetic community (SynCom) assembled by pairing bacterial isolates from a field site heavily contaminated with NO3-, we also provide insight into the controls of N2O emissions. We demonstrate that members of this SynCom cooperate to perform complete denitrification through exchange of nitrite (NO2-) and nitric oxide (NO), and that community context drives global physiological changes in each member. We identify links between amino acid metabolism and denitrification activity as well as indicators of competition and amino acid exchange. We also show that NO2- toxicity with unbalanced growth of community members drives N2O production, suggesting that this SynCom provides a simplified, environmentally relevant, model of pathway partitioning in denitrifying communities. This SynCom should provide a framework with which to further explore how environmental context can impact cooperation and lead to the production of N2O
Project description:Nitrosomonas europaea is a chemolithoautotrophic bacterium that oxidizes ammonia (NH3) to obtain energy for growth on carbon dioxide (CO2), and can also produce nitrous oxide (N2O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses of N. europaea to replete (> 5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2CO3) supplemented with atmospheric CO2. IC-limited cultures oxidized 25 to 58% of available NH3 to nitrite, depending on dilution rate and Na2CO3 concentration. IC limitation resulted in a 1.5-fold increase in cellular maintenance energy requirements compared to NH3-limited cultures. Rates of N2O production increased 2- and 6.3 fold under the two IC-limited conditions increasing the percentage of oxidized NH3-N being transformed to N2O-N from 0.5% (replete) to 4.4% (0.25 mM Na2CO3). Transcriptome analysis showed differential expression (p ≤ 0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits, and increased expression of genes involved in C1 metabolism including RuBisCO (cbb gene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924-0927) correlated with increased production of N2O. Together, these data suggest that N. europaea adapts physiologically during IC-limited steady state growth, which leads to uncoupling of NH3 oxidation from growth and increased N2O production.
Project description:Nitrosomonas europaeais a chemolithoautotrophic bacterium that oxidizes ammonia (NH3) to obtain energy for growth on carbon dioxide (CO2), and can also produce nitrous oxide (N2O), a greenhouse gas. We interrogated the growth, physiological, and transcriptome responses ofN. europaeato replete (> 5.2 mM) and limited inorganic carbon (IC) provided by either 1.0 mM or 0.2 mM sodium carbonate (Na2CO3) supplemented with atmospheric CO2 IC-limited cultures oxidized 25 to 58% of available NH3to nitrite, depending on dilution rate and Na2CO3concentration. IC limitation resulted in a 2.3-fold increase in cellular maintenance energy requirements compared to NH3-limited cultures. Rates of N2O production increased 2.5- and 6.3-fold under the two IC-limited conditions increasing the percentage of oxidized NH3-N being transformed to N2O-N from 0.5% (replete) up to 4.4% (0.2 mM Na2CO3). Transcriptome analysis showed differential expression (p⤠0.05) of 488 genes (20% of inventory) between replete and IC-limited conditions, but few differences were detected between the two IC-limiting treatments. IC-limited conditions resulted in decreased expression of ammonium/ammonia transporter and ammonia monooxygenase subunits, and increased expression of genes involved in C1 metabolism including RuBisCO (cbbgene cluster), carbonic anhydrase, folate-linked metabolism of C1 moieties, and putative C salvage due to oxygenase activity of RuBisCO. Increased expression of nitrite reductase (gene cluster NE0924-0927) correlated with increased production of N2O. Together, these data suggest thatN. europaeaadapts physiologically during IC-limited steady state growth, which leads to uncoupling of NH3oxidation from growth and increased N2O production. Transcriptome responses of N. europaea in 60 mM NH4+ to suboptimum levels of carbonate (1.0 mM and 0.2 mM) in continuous steady-state culture in a bioreactor.
Project description:Nitrite-oxidizing bacteria are vital players in the global nitrogen cycle that convert nitrite to nitrate during the 2nd step of nitrification. Within this functional guild, the genus Nitrospira is among the most widespread and phylogenetically and physiologically diverse nitrite oxidizers and its members drive nitrite oxidation in many natural and biotechnological ecosystems. Despite their ecological and biotechnological importance, our understanding of Nitrospira’s energy metabolism is still limited. The main bottleneck for a detailed biochemical characterization of Nitrospira is biomass production, since they are slow-growing organisms and fastidious to culture. In this study, we cultured Nitrospira moscoviensis in a continuous stirred tank reactor system (CSTR) allowing constant biomass harvesting. Additionally, this cultivation setup enabled accurate control of physicochemical parameters and thus avoided fluctuating levels of nitrite and accumulation of nitrate. We performed transcriptome analysis and confirmed constant gene expression profiles in the chemostat culture over a period of two weeks. The transcriptomic data supports the predicted core metabolism of N. moscoviensis, including the reductive TCA cycle as a CO2 fixation pathway, the novel bd-like oxidase as terminal oxidase and the octaheme nitrite reductase involved in nitrogen assimilation. Additionally, the expression of multiple copies of respiratory complexes suggests functional differentiation of these copies within the respiratory chain. Transcriptome analysis also suggests a soluble and a membrane-bound gamma subunit as part of the nitrite oxidoreductase (NXR), the enzyme catalyzing nitrite oxidation. Overall, the transcriptome data provided novel insights into the metabolism of Nitrospira supporting the genome-based prediction of key pathways. Moreover, the application of a CSTR to cultivate Nitrospira is an important foundation for future proteomic and biochemical characterizations, which are crucial for a better understanding of canonical and complete nitrifying microorganisms.
Project description: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 N2O. Inoculation with compatible rhizobia is often needed for optimal N2-fixation, but the choice of inoculant may have consequences for N2O 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 N2O. The N2O-reducing isolates had 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.
Project description:Mitigation of N2O-emissions from soils is needed to reduce climate forcing by food production. Inoculating soils with N2O-reducing bacteria would be effective, but costly and impractical as a standalone operation. Here we demonstrate that digestates obtained after biogas production may provide a low-cost and widely applicable solution. Firstly, we show that indigenous N2O-reducing bacteria in digestates grow to high levels during anaerobic enrichment under N2O. Gas kinetics and meta-omic analysis show that the N2O respiring organisms, recovered as metagenome-assembled genomes (MAGs) grow by harvesting fermentation intermediates of the methanogenic consortium. Three digestate-derived denitrifying bacteria were obtained through isolation, one of which matched the recovered MAG of a dominant Dechloromonas-affiliated N2O reducer. While the identified N2O-reducers encoded 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 the current system. Secondly, moving towards practical application, we show that these isolates grow by aerobic respiration in digestates, and that fertilization with these enriched digestates reduces N2O emissions. This shows that the ongoing implementation of biogas production in agriculture opens a new avenue for cheap and effective reduction of N2O emissions from food production.
2022-02-16 | PXD023233 | Pride
Project description:bacteria community for autotrophic sulfide-driven denitrification Genome sequencing and assembly