Aerobic Denitrifying Bacteria That Produce Low Levels of Nitrous Oxide

ABSTRACT Most denitrifiers produce nitrous oxide (N2O) instead of dinitrogen (N2) under aerobic conditions. We isolated and characterized novel aerobic denitrifiers that produce low levels of N2O under aerobic conditions. We monitored the denitrification activities of two of the isolates, strains TR2 and K50, in batch and continuous cultures. Both strains reduced nitrate (NO3 −) to N2 at rates of 0.9 and 0.03 μmol min−1 unit of optical density at 540 nm−1 at dissolved oxygen (O2) (DO) concentrations of 39 and 38 μmol liter−1, respectively. At the same DO level, the typical denitrifier Pseudomonas stutzeri and the previously described aerobic denitrifier Paracoccus denitrificans did not produce N2 but evolved more than 10-fold more N2O than strains TR2 and K50 evolved. The isolates denitrified NO3 − with concomitant consumption of O2. These results indicated that strains TR2 and K50 are aerobic denitrifiers. These two isolates were taxonomically placed in the β subclass of the class Proteobacteria and were identified as P. stutzeri TR2 and Pseudomonas sp. strain K50. These strains should be useful for future investigations of the mechanisms of denitrifying bacteria that regulate N2O emission, the single-stage process for nitrogen removal, and microbial N2O emission into the ecosystem.

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Potential of Aerobic Denitrification by Pseudomonas stutzeri TR2 To Reduce Nitrous Oxide Emissions from Wastewater Treatment Plants

Applied and Environmental Microbiology ◽

10.1128/aem.01983-09 ◽

2010 ◽

Vol 76 (14) ◽

pp. 4619-4625 ◽

Cited By ~ 84

Author(s):

Morio Miyahara ◽

Sang-Wan Kim ◽

Shinya Fushinobu ◽

Koki Takaki ◽

Takeshi Yamada ◽

...

Keyword(s):

Nitrous Oxide ◽

Bacterial Species ◽

Pseudomonas Stutzeri ◽

Sharp Contrast ◽

Nitrous Oxide Emissions ◽

Aerobic Denitrification ◽

Piggery Wastewater ◽

Lower Growth ◽

Aerobic Conditions ◽

Denitrification Activity

ABSTRACT In contrast to most denitrifiers studied so far, Pseudomonas stutzeri TR2 produces low levels of nitrous oxide (N2O) even under aerobic conditions. We compared the denitrification activity of strain TR2 with those of various denitrifiers in an artificial medium that was derived from piggery wastewater. Strain TR2 exhibited strong denitrification activity and produced little N2O under all conditions tested. Its growth rate under denitrifying conditions was near comparable to that under aerobic conditions, showing a sharp contrast to the lower growth rates of other denitrifiers under denitrifying conditions. Strain TR2 was tolerant to toxic nitrite, even utilizing it as a good denitrification substrate. When both nitrite and N2O were present, strain TR2 reduced N2O in preference to nitrite as the denitrification substrate. This bacterial strain was readily able to adapt to denitrifying conditions by expressing the denitrification genes for cytochrome cd 1 nitrite reductase (NiR) (nirS) and nitrous oxide reductase (NoS) (nosZ). Interestingly, nosZ was constitutively expressed even under nondenitrifying, aerobic conditions, consistent with our finding that strain TR2 preferred N2O to nitrite. These properties of strain TR2 concerning denitrification are in sharp contrast to those of well-characterized denitrifiers. These results demonstrate that some bacterial species, such as strain TR2, have adopted a strategy for survival by preferring denitrification to oxygen respiration. The bacterium was also shown to contain the potential to reduce N2O emissions when applied to sewage disposal fields.

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nosX is essential for whole-cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase

Microbiology ◽

10.1099/mic.0.000955 ◽

2020 ◽

Vol 166 (10) ◽

pp. 909-917 ◽

Cited By ~ 1

Author(s):

Sophie P. Bennett ◽

Maria J. Torres ◽

Manuel J. Soriano-Laguna ◽

David J. Richardson ◽

Andrew J. Gates ◽

...

Keyword(s):

Nitrous Oxide ◽

Type Species ◽

Paracoccus Denitrificans ◽

Pseudomonas Stutzeri ◽

Reduction Reaction ◽

Nitrous Oxide Reductase ◽

Content Type ◽

Link Type ◽

Reverse Transcription Pcr

Nitrous oxide (N2O) is a potent greenhouse gas that is produced naturally as an intermediate during the process of denitrification carried out by some soil bacteria. It is consumed by nitrous oxide reductase (N2OR), the terminal enzyme of the denitrification pathway, which catalyses a reduction reaction to generate dinitrogen. N2OR contains two important copper cofactors (CuA and CuZ centres) that are essential for activity, and in copper-limited environments, N2OR fails to function, contributing to rising levels of atmospheric N2O and a major environmental challenge. Here we report studies of nosX, one of eight genes in the nos cluster of the soil dwelling α-proteobaterium Paraccocus denitrificans. A P. denitrificans ΔnosX deletion mutant failed to reduce N2O under both copper-sufficient and copper-limited conditions, demonstrating that NosX plays an essential role in N2OR activity. N2OR isolated from nosX-deficient cells was found to be unaffected in terms of the assembly of its copper cofactors, and to be active in in vitro assays, indicating that NosX is not required for the maturation of the enzyme; in particular, it plays no part in the assembly of either of the CuA and CuZ centres. Furthermore, quantitative Reverse Transcription PCR (qRT-PCR) studies showed that NosX does not significantly affect the expression of the N2OR-encoding nosZ gene. NosX is a homologue of the FAD-binding protein ApbE from Pseudomonas stutzeri , which functions in the flavinylation of another N2OR accessory protein, NosR. Thus, it is likely that NosX is a system-specific maturation factor of NosR, and so is indirectly involved in maintaining the reaction cycle of N2OR and cellular N2O reduction.

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In-situ characterization of microbial community in an A/O submerged membrane bioreactor with nitrogen removal

Water Science & Technology ◽

10.2166/wst.2004.0483 ◽

2004 ◽

Vol 50 (8) ◽

pp. 41-48 ◽

Cited By ~ 31

Author(s):

A. Sofia ◽

W.-T. Liu ◽

S.L. Ong ◽

W.J. Ng

Keyword(s):

Bacterial Community ◽

Nitrogen Removal ◽

Membrane Bioreactor ◽

Ammonia Oxidation ◽

Paracoccus Denitrificans ◽

Pseudomonas Stutzeri ◽

Laboratory Scale ◽

Negative Results

The bacterial community involved in removing nitrogen from sewage and their preferred DO environment within an anoxic/oxic membrane bioreactor (A/O MBR) was investigated. A continuously operated laboratory-scale A/O MBR was maintained for 360 d. At a sludge age of 150 d and a C/N ratio of 3.5, the system was capable of removing 88% of the influent nitrogen from raw wastewater through typical nitrogen removal transformations (i.e. aerobic ammonia oxidation and anoxic nitrate reduction). Characterization of the A/O MBR bacterial community was carried out using fluorescence in situ hybridization (FISH) techniques. FISH results further showed that Nitrosospira spp. and Nitrospira spp. were the predominant groups of ammonia and nitrite oxidizing group, respectively. They constituted up to 11% and 6% of eubacteria at DO below 0.05 mg/l (low DO), respectively, and about 14% and 9% of eubacteria at DO between 2–5 mg/l (sufficient DO), respectively, indicating preference of nitrifiers for a higher DO environment. Generally low counts of the genus Paracoccus were detected while negative results were observed for Paracoccus denitrificans, Alcaligenes spp, and Pseudomonas stutzeri under the low and sufficient DO environments. The overall results indicate that Nitrosospira spp., Nitrospira spp. and members of Paracoccus spp. can be metabolically functional in nitrogen removal in the laboratory-scale A/O MBR system.

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Nature’s way of handling a greenhouse gas: the copper-sulfur cluster of purple nitrous oxide reductase

Biological Chemistry ◽

10.1515/hsz-2012-0177 ◽

2012 ◽

Vol 393 (10) ◽

pp. 1067-1077 ◽

Cited By ~ 29

Author(s):

Anja Wüst ◽

Lisa Schneider ◽

Anja Pomowski ◽

Walter G. Zumft ◽

Peter M.H. Kroneck ◽

...

Keyword(s):

Nitrous Oxide ◽

Paracoccus Denitrificans ◽

Copper Ions ◽

Bacterial Species ◽

Three Dimensional ◽

Pseudomonas Stutzeri ◽

Structural Data ◽

Nitrous Oxide Reductase ◽

Bridging Ligand ◽

Marinobacter Hydrocarbonoclasticus

Abstract The tetranuclear CuZ cluster is the unique active site of nitrous oxide reductase, the enzyme that catalyzes the reduction of nitrous oxide to dinitrogen as the final reaction in bacterial denitrification. Three-dimensional structures of orthologs of the enzyme from a variety of different bacterial species were essential steps in the elucidation of the properties of this center. However, while structural data first revealed and later confirmed the presence of four copper ions in spectroscopically distinct forms of CuZ, the exact structure and stoichiometry of the cluster showed significant variations. A ligand bridging ions CuZ1 and CuZ2 was initially assigned as a water or hydroxo species in the structures from Pseudomonas nautica (now Marinobacter hydrocarbonoclasticus) and Paracoccus denitrificans. This ligand was absent in a structure from ‘Achromobacter cycloclastes’, and could be reconstituted by iodide that acted as an inhibitor of catalysis. A recent structure of anoxically isolated nitrous oxide reductase from Pseudomonas stutzeri revealed the bridging ligand to be sulfide, S2-, and showed an unprecedented side-on mode of nitrous oxide binding to this form of CuZ.

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