Various microorganisms responsible for denitrification in soil

Marine nitrogen cycle ultimately depends on the biological responses of oceanic microbial communities. It indirectly regulates primary production and influences the strength of the biological pump, which contributes to the oceanic uptake of atmospheric carbon dioxide (CO2). As the microbial community structure and functional capacities remain underestimated in terms of temporal and geographical coverage in the Baltic Sea, our understanding of the nitrogen cycle with respect to ecosystem functioning and climate change is limited. Therefore, in this study, we investigated the seasonal and spatial structure of microbial community abundance involved in the nitrogen loss (denitrification, anammox), reduction processes (dissimilatory nitrate reduction (DNR), dissimilatory nitrite reduction to ammonium (DNRA), and oxidation process (nitrification) in the Baltic Proper (Bornholm Deep, Gdańsk Deep, and Gotland Deep). Specifically, we focused on waters below the halocline at depths ranging from 75 to 135 m, characterized by changeable oxygen conditions. The potential of selected nitrogen processes was resolved by mapping raw reads against nitrogen cycle genes identified in de novo assembled metagenomes. Taxonomic analysis of bacterial and archaeal communities, based on paired-end raw reads, revealed that nitrification, DNR, and denitrification potential were primarily associated with the Nitrosopumilaceae and Thioglobaceae families within these phyla. Ammonia oxidation products likely fueled the production of nitrous oxide (N2O), with nitric oxide reductase (NOR)—an enzyme encoded by the Thioglobaceae genome—being responsible for further reduction. Anammox-related genes were not present within sites, thus denitrification pathway enzymes, namely, NOR and N2O reductase (NOS) were responsible for nitrogen loss. At all sites, genes encoding nitrogen reduction enzymes were most abundant, while the presence of NOS encoding genes was found in Bacteroidetes and Proteobacteria phyla within all sites. Our findings revealed no significant spatial variation, suggesting that the studied ecosystem exhibits a consistent nitrogen processing capacity across different locations. However, seasonality emerged as a key factor, as changes in nutrient and oxygen conditions throughout the year significantly influence microbial activity and the associated nitrogen-cycling processes.

Nitric oxide reductase (NOR) is a key enzyme in denitrification, reforming the N-N bond (making N2O from two NO molecules) in the nitrogen cycle. It is a cytochrome bc complex which has apparently only two subunits, NorB and NorC. It contains two low-spin cytochromes (c and b), and a high-spin cytochrome b which forms a binuclear center with a non-heme iron. NorC contains the c-type heme and NorB can be predicted to bind the other metal centers. NorB is homologous to the major subunit of the heme/copper cytochrome oxidases, and NOR thus belongs to the superfamily, although it has an Fe/Fe active site rather than an Fe/Cu binuclear center and a different catalytic activity. Current evidence suggests that NOR is not a proton pump, and that the protons consumed in NO reduction are not taken from the cytoplasmic side of the membrane. Therefore, the comparison between structural and functional properties of NOR and cytochrome c- and quinol-oxidizing enzymes which function as proton pumps may help us to understand the mechanism of the latter. This review is a brief summary of the current knowledge on molecular biology, structure, and bioenergetics of NOR as a member of the oxidase superfamily.

Nitrification and denitrification are two critical processes in the global nitrogen cycle. Many microorganisms have been shown to contribute to these biological nitrogen transformation processes. However, our understanding of the nitrogen cycle is still evolving. In the recent years, novel microorganisms and metabolic pathways involved in the nitrogen cycle have been discovered.n Therefore, the overall aim of this thesis is to enrich and characterise novel microorganisms involved in the nitrogen cycle, in order to improve our understanding of the microbial nitrogen conversion processes and their interactions with other important nutrients cycles including carbon and metals.In terms of ammonium oxidation, ammonia-oxidising archaea (AOA) are critical ammonium oxidizers that regulate the nitrogen transformation in ubiquitous environments. AOA may have advantages when competing with ammonia-oxidising bacteria (AOB) in certain extreme environments such as acidic soils. Although 30% of the soils in the world are acidic soils, our understanding of the AOA living under low pH is insufficient. Therefore, the first research objective of this thesis is to enrich and identify a novel AOA under low pH. Using a fresh water reservoir sediments as inoculum, two novel microorganisms (an AOA and a NOB) were enriched in a bioreactor operated at pH 4.5, and proved to play a critical role in ammonium oxidation to nitrate. The AOA strain was assigned to the genus of Nitrosotalea and identified as a novel species Candidatus Nitrosotalea sp. GC1, while the NOB was clustered into a novel lineage within genus Nitrospira. According to the metagenomic analysis on Candidatus Nitrosotalea sp. GC1, the genes encoding enzymes for ammonium oxidation to nitrite were all identified and the Thaumarcheal HP/HB pathway was used for carbon fixation.As a member affiliating in the genus of Nitrosotalea, Candidatus Nitrosotalea sp. GC1 might have similar features to the other members, such as its acidophily, substrate affinity and nitrous oxide (N2O) production. As a critical step to help understand this novel AOA culture, the second research objective of this thesis is to characterise the effect of environmental conditions on Candidatus Nitrosotalea sp. GC1 and its N2O emission potential. With a series batch tests, the acidophily of strain GC was verified, with an optimal pH of 4.5. The Km of ammonium and O2 for Candidatus Nitrosotalea sp. GC1 are 39.7 p 3.2 and 35.2 p 3.0 mM, respectively, which were much higher than other AOA isolates, although still lower than most AOB. In addition, N2O emission from Candidatus Nitrosotalea sp. GC1 has a remarkable yield of 6.7%, which suggested the critical role of acidophilic AOA in greenhouse gas emission.In terms of nitrate reduction, dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are the main processes that convert nitrate to ammonium or inert dinitrogen gas respectively. Although organic carbon compounds are the main energy and electron sources for the most of known DNRA microorganisms, the mechanisms of methane driven DNRA process have not been investigated. Therefore, the third research objective of this thesis is to characterise the microorganisms and pathways involved in a culture performing methane driven DNRA process. In a bioreactor fed with methane and limited nitrate supply, continuous ammonium production from nitrate coupled with anaerobic oxidation of methane was observed, while an anaerobic methanotrophic archaea, Candidatus Methanoperedens nitroreducens (M. nitroreducens), dominated the microbial community. During batch tests, metagenomic and metatranscriptomic analyses showed that the DNRA related genes in Candidatus M. nitroreducens have significantly higher expression levels when the culture was producing ammonium, compared to their levels when the culture was fed with large quantity of nitrate to inhibit DNRA activity. Overall, the results confirmed that Candidatus M. nitroreducens can facilitate the methane-driven DNRA process.It has been shown that denitrification can couple to the oxidation of methane or metals (e.g. Fe or Mn), thus linking the nitrogen cycle to the methane and metal cycles, respectively. However, research works focusing on the interactions between nitrogen, methane and metal cycles in one system are rarely done. Therefore, the last research objective is to investigate interactions between anaerobic oxidation of methane and nitrate and metals reduction processes in one reactor. By setting up and incubating the bioreactor with methane, nitrate and ferrihydrite for more than 900 days, simultaneous methane oxidation and nitrate reduction were observed. Mass and electron balances suggested that in this system, nitrite/iron-dependent anaerobic oxidation of methane processes are coupled with nitrate-dependent iron (Fe) oxidation (NDFO) process. The community analysis suggested that Azospira sp. and Methylomirabiliaceae sp. may be responsible for these processes, while an unknown microorganism facilitated metal-dependent anaerobic oxidation of methane process.Overall, the discovery and characterisation of these novel microorganisms and metabolic pathways mediating ammonium oxidation and nitrate reduction will provide important insights into the understanding of the nitrogen cycle.n

Abstract. The isotopic composition of soil-derived N2O can help differentiate between N2O production pathways and estimate the fraction of N2O reduced to N2. Until now, δ18O of N2O has been rarely used in the interpretation of N2O isotopic signatures because of the rather complex oxygen isotope fractionations during N2O production by denitrification. The latter process involves nitrate reduction mediated through the following three enzymes: nitrate reductase (NAR), nitrite reductase (NIR) and nitric oxide reductase (NOR). Each step removes one oxygen atom as water (H2O), which gives rise to a branching isotope effect. Moreover, denitrification intermediates may partially or fully exchange oxygen isotopes with ambient water, which is associated with an exchange isotope effect. The main objective of this study was to decipher the mechanism of oxygen isotope fractionation during N2O production by soil denitrification and, in particular, to investigate the relationship between the extent of oxygen isotope exchange with soil water and the δ18O values of the produced N2O. In our soil incubation experiments Δ17O isotope tracing was applied for the first time to simultaneously determine the extent of oxygen isotope exchange and any associated oxygen isotope effect. We found that N2O formation in static anoxic incubation experiments was typically associated with oxygen isotope exchange close to 100 % and a stable difference between the 18O ∕ 16O ratio of soil water and the N2O product of δ18O(N2O ∕ H2O) = (17.5 ± 1.2) ‰. However, flow-through experiments gave lower oxygen isotope exchange down to 56 % and a higher δ18O(N2O ∕ H2O) of up to 37 ‰. The extent of isotope exchange and δ18O(N2O ∕ H2O) showed a significant correlation (R2 = 0.70, p < 0.00001). We hypothesize that this observation was due to the contribution of N2O from another production process, most probably fungal denitrification. An oxygen isotope fractionation model was used to test various scenarios with different magnitudes of branching isotope effects at different steps in the reduction process. The results suggest that during denitrification, isotope exchange occurs prior to isotope branching and that this exchange is mostly associated with the enzymatic nitrite reduction mediated by NIR. For bacterial denitrification, the branching isotope effect can be surprisingly low, about (0.0 ± 0.9) ‰, in contrast to fungal denitrification where higher values of up to 30 ‰ have been reported previously. This suggests that δ18O might be used as a tracer for differentiation between bacterial and fungal denitrification, due to their different magnitudes of branching isotope effects.

Denitrification is an important process of global nitrogen cycle as it removes reactive nitrogen from the biosphere, and acts as the primary source of nitrous oxide (N2O). This thesis seeks to gain better understanding of the biogeochemistry of denitrification by investigating the process from four different aspects: genetic basis, enzymatic kinetics, environmental interactions, and environmental consequences. Laboratory and field experiments were combined with modeling efforts to unravel the complexity of denitrification process under microbiological and environmental controls. Dynamics of denitrification products observed in laboratory experiments revealed an important role of constitutive denitrification enzymes, whose presence were further confirmed with quantitative analysis of functional genes encoding nitrite reductase and nitrous oxide reductase. A metabolic model of denitrification developed with explicit denitrification enzyme kinetics and representation of constitutive enzymes successfully reproduced the dynamics of N2O and N2 accumulation observed in the incubation experiments, revealing important regulatory effect of denitrification enzyme kinetics on the accumulation of denitrification products. Field studies demonstrated complex interaction of belowground N2O production, consumption and transport, resulting in two pulse pattern in the surface flux. Coupled soil gas diffusion/denitrification model showed great potential in simulating the dynamics of N2O below ground, with explicit representation of the activity of constitutive denitrification enzymes. A complete survey of environmental variables showed distinct regulation regimes on the denitrification activity from constitutive enzymes and new synthesized enzymes. Uncertainties in N2O estimation with current biogeochemical models may be reduced as accurate simulation of the dynamics of N2O in soil and surface fluxes is possible with a coupled diffusion/denitrification model that includes explicit representation of denitrification enzyme kinetics. In conclusion, denitrification is a complex ecological function regulated at cellular level. To assess the environmental consequences of denitrification and develop useful tools to mitigate N2O emissions require a comprehensive understanding of the regulatory network of denitrification with respect to microbial physiology and environmental interactions.

The influence of revetment types on soil denitrification in the adjacent tidal urban riparian zones

The accumulation of nitrous oxide (N 2 O) in the atmosphere is a significant manifestation of human perturbations of the nitrogen cycle. This thesis reports the development and first applications of a novel isotopic technique for characterizing nitrous oxide sources. Chapter 1 describes the development of methods to use the newly available technology of high- resolution dual-inlet multi-collector mass spectrometry to measure six isotopic parameters in nitrous oxide. It reports the standardization and initial biological application of these methods. Chapter 2 presents a model for the generation of isotope effects in an important N 2 O generating enzyme, the bacterial nitric oxide reductase; this model and published isotopic constraints are used to provide insights into the mechanism of that enzyme. Chapter 3 describes the six-dimensional isotopic characterization of nitrous oxide from bacterial denitrifiers, while Chapter 4 describes nitrous oxide generated by ammonia oxidizing bacteria.

Effects of chicken manure-derived black soldier fly organic fertilizer on soil carbon and nitrogen cycling: insights from metagenomic and microbial network analysis.

Denitrifying and anammox bacteria are involved in the nitrogen cycling in marine sediments but the environmental factors that regulate the relative importance of these processes are not well constrained. Here, we evaluated the abundance, diversity, and potential activity of denitrifying, anammox, and sulfide-dependent denitrifying bacteria in the sediments of the seasonally hypoxic saline Lake Grevelingen, known to harbor an active microbial community involved in sulfur oxidation pathways. Depth distributions of 16S rRNA gene, nirS gene of denitrifying and anammox bacteria, aprA gene of sulfur-oxidizing and sulfate-reducing bacteria, and ladderane lipids of anammox bacteria were studied in sediments impacted by seasonally hypoxic bottom waters. Samples were collected down to 5 cm depth (1 cm resolution) at three different locations before (March) and during summer hypoxia (August). The abundance of denitrifying bacteria did not vary despite of differences in oxygen and sulfide availability in the sediments, whereas anammox bacteria were more abundant in the summer hypoxia but in those sediments with lower sulfide concentrations. The potential activity of denitrifying and anammox bacteria as well as of sulfur-oxidizing, including sulfide-dependent denitrifiers and sulfate-reducing bacteria, was potentially inhibited by the competition for nitrate and nitrite with cable and/or Beggiatoa-like bacteria in March and by the accumulation of sulfide in the summer hypoxia. The simultaneous presence and activity of organoheterotrophic denitrifying bacteria, sulfide-dependent denitrifiers, and anammox bacteria suggests a tight network of bacteria coupling carbon-, nitrogen-, and sulfur cycling in Lake Grevelingen sediments.

BackgroundDenitrification is defined as the dissimilatory reduction of nitrate or nitrite to nitric oxide (NO), nitrous oxide (N2O), or dinitrogen gas (N2). N2O is a powerful atmospheric greenhouse gas and cause of ozone layer depletion. Legume crops might contribute to N2O production by providing nitrogen-rich residues for decomposition or by associating with rhizobia that are able to denitrify under free-living and symbiotic conditions. However, there are limited direct empirical data concerning N2O production by endosymbiotic bacteria associated with legume crops. Analysis of the Ensifer meliloti 1021 genome sequence revealed the presence of the napEFDABC, nirK, norECBQD and nosRZDFYLX denitrification genes. It was recently reported that this bacterium is able to grow using nitrate respiration when cells are incubated with an initial O2 concentration of 2%; however, these cells were unable to use nitrate respiration when initially incubated anoxically. The involvement of the nap, nirK, nor and nos genes in E. meliloti denitrification has not been reported.ResultsE. meliloti nap, nirK and norC mutant strains exhibited defects in their ability to grow using nitrate as a respiratory substrate. However, E meliloti nosZ was not essential for growth under these conditions. The E. meliloti napA, nirK, norC and nosZ genes encode corresponding nitrate, nitrite, nitric oxide and nitrous oxide reductases, respectively. The NorC component of the E. meliloti nitric oxide reductase has been identified as a c-type cytochrome that is 16 kDa in size. Herein, we also show that maximal expression of the E. meliloti napA, nirK, norC and nosZ genes occurred when cells were initially incubated anoxically with nitrate.ConclusionThe E. meliloti napA, nirK, norC and nosZ genes are involved in nitrate respiration and in the expression of denitrification enzymes in this bacterium. Our findings expand the short list of rhizobia for which denitrification gene function has been demonstrated. The inability of E. meliloti to grow when cells are initially subjected to anoxic conditions is not attributable to defects in the expression of the napA, nirK, norC and nosZ denitrification genes.

BackgroundThe lithospheric microbiome plays a vital role in global biogeochemical cycling, yet their mutual modulation mechanisms remain largely uncharted. Petroleum reservoirs are important lithosphere ecosystems that provide desirable resources for understanding microbial roles in element cycling. However, the strategy and mechanism of modulating indigenous microbial communities for the optimization of community structures and functions are underexplored, despite its significance in energy recovery and environmental remediation.ResultsHere we proposed a novel selective stimulation of indigenous functional microbes by driving nitrogen and sulfur cycling in petroleum reservoirs using injections of an exogenous heterocycle-degrading strain of Pseudomonas. We defined such bacteria capable of removing and releasing organically bound sulfur and nitrogen from heterocycles as “bioredox triggers”. High-throughput 16S rRNA amplicon sequencing, metagenomic, and gene transcription-level analyses of extensive production water and sandstone core samples spanning the whole oil production process clarified the microbiome dynamics following the intervention. These efforts demonstrated the feasibility of in situ N/S element release and electron acceptor generation during heterocycle degradation, shifting microbiome structures and functions and increasing phylogenetic diversity and genera engaged in sulfur and nitrogen cycling, such as Desulfovibrio, Shewanella, and Sulfurospirillum. The metabolic potentials of sulfur- and nitrogen-cycling processes, particularly dissimilatory sulfate reduction and dissimilatory nitrate reduction, were elevated in reservoir microbiomes. The relative expression of genes involved in sulfate reduction (dsrA, dsrB) and nitrate reduction (napA) was upregulated by 85, 28, and 22 folds, respectively. Field trials showed significant improvements in oil properties, with a decline in asphaltenes and aromatics, hetero-element contents, and viscosity, hence facilitating the effective exploitation of heavy oil.ConclusionsThe interactions between microbiomes and element cycling elucidated in this study will contribute to a better understanding of microbial metabolic involvement in, and response to, biogeochemical processes in the lithosphere. The presented findings demonstrated the immense potential of our microbial modulation strategy for green and enhanced heavy oil recovery.7ucoPBoC4m39tpZT7x_42NVideo Graphical

Quorum sensing mediates spatiotemporal microbial community dynamics and nitrogen metabolism in biofloc-based Litopenaeus vannamei aquaculture systems.

Chapter 7 - Purification and Functional Analysis of Fungal Nitric Oxide Reductase Cytochrome P450nor

Nitrogen deposition is a crucial factor in global change, which is widespread across various regions globally. It has drawn extensive attention due to its direct modification of soil nitrogen retention and nitrogen species distribution, thereby influencing nitrogen metabolism across entire ecosystems. Previous studies on its influence on nitrogen metabolism have not reached a consensus. In an urban ryegrass lawn mesocosm experiment, we set two levels of nitrogen deposition and soil nutrients respectively, aiming to study the impacts of these factors on the N-cycling process through metagenomic analysis. The results demonstrated nitrogen deposition increased nitrification, nitrogen fixation, denitrification, and dissimilatory nitrate reduction, but decreased assimilatory nitrate reduction in the nitrogen metabolism process by changing soil nitrogen availability and the abundance of N-cycling functional genes in the soil microbial community. The soil nutrient levels exhibited effects opposite to those of nitrogen deposition, negatively impacting nitrification, denitrification, and nitrogen fixation in the nitrogen metabolism process. This work further elucidates the impacts of nitrogen deposition on the ecological functions of the ryegrass lawn with different soil nutrient levels, and predicts the potential impacts of intensified nitrogen deposition on these ecological functions. It provides valuable theoretical support for understanding and evaluating complex ecological interactions.

Haloalkaliphilic denitrifiers-dependent sulfate-reducing bacteria thrive in nitrate-enriched environments