Denitrifying Paracoccus Denitrificans Research Articles

Response characteristics of nirS-type denitrifier Paracoccus denitrificans under florfenicol stress

Florfenicol (FF) is widely used in aquaculture and can interfere with denitrification when released into natural ecosystems. The aim of this study was to analyze the response characteristics of nirS-type denitrifier Paracoccus denitrificans under FF stress and further mine antibiotic-responsive factors in aquatic environment. Phenotypic analysis revealed that FF delayed the nitrate removal with a maximum inhibition value of 82.4% at exponential growth phase, leading to nitrite accumulation reached to 21.9-fold and biofilm biomass decreased by ~38.6%, which were due to the lower bacterial population count (P < 0.01). RNA-seq transcriptome analyses indicated that FF treatment decreased the expression of nirS, norB, nosD and nosZ genes that encoded enzymes required for NO2− to N2 conversion from 1.02- to 2.21-fold (P < 0.001). Furthermore, gene associated with the flagellar system FlgL was also down-regulated by 1.03-fold (P < 0.001). Moreover, 10 confirmed sRNAs were significantly induced, which regulated a wide range of metabolic pathways and protein expression. Interestingly, different bacteria contained the same sRNAs means that sRNAs can spread between them. Overall, this study suggests that the denitrification of nirS-type denitrifiers can be hampered widely by FF and the key sRNAs have great potential to be antibiotic-responsive factors.

Tetracycline-induced effects on the nitrogen transformations in sediments: Roles of adsorption behavior and bacterial activity

Nitrification and denitrification are the most important nitrogen transformation processes in the environment. Recently, due to widespread use, antibiotics have been reported to lead to environmental risks. Tetracycline (TC) is one of the most extensively used antibiotics in many areas. However, its reported effects on nitrogen transformations were conflicting in previous studies. In this study, the effects of TC on nitrogen transformations in sediment were investigated by analyzing TC transport and bacterial activity. It was found that the adsorption of TC onto the sediment was favorable and spontaneous, with adsorption capacity 54.3 mg/kg. The adsorption kinetics of TC onto the sediment and the isotherm fitted the Elvoich and Freundlich models, respectively, indicating that the adsorption was a chemisorption process, including electrostatic interactions and chemical bonding between TC and the sediment. TC showed no effect on nitrification in the sediment, but significantly inhibited the reduction of nitrate and nitrite during denitrification, consistent with observations made for the model denitrifier Paracoccus denitrificans under TC stress. Mechanistic study indicated that TC at 130 μg/g-cell inhibited 50.7% of P. denitrificans growth and 61.6% of cell viability. Meanwhile, the catalytic activities of the key denitrifying enzymes, nitrate reductase (NAR) and nitrite reductase (NIR), decreased to 29.1% and 68.0% of the control levels when the TC concentration was 130 μg/g-cell, suggesting that NAR was more sensitive to the TC than NIR, which contributed to a delay in nitrite accumulation.

A bet-hedging strategy for denitrifying bacteria curtails their release of N2O

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.

Bioremoval of nutrients from wastewater by a denitrifier Paracoccus denitrificans ISTOD1

The introduction of plant nutrients like nitrogen and phosphorus in water bodies cause a natural ageing process called eutrophication. In the present study, 71.9% NO3−N and 93% ortho-P removal was achieved within 72h by an aerobic denitrifier P.denitrificans ISTOD1, suggesting the strain as a potential denitrifying phosphorus accumulating organism (DPAO). To make wastewater treatment cost-effective, different concentration of molasses were optimized for maximum EPS production. Application of strain ISTOD1 in real wastewater samples with 5% molasses resulted in NH4+-N, NO2−N, NO3−N, TN, COD and TP removal efficiencies of 99.6%, 79.2%, 83.8%, 97.3%, 84.2% and 100%. Moreover, 1.9g/L EPS production was also observed between late exponential- stationary phase. Assessment on wastewater demonstrated the capability of ISTOD1 in overall nutrient removal along with EPS production. Bioflocculation efficiency of the domestic wastewater produced EPS makes the strain an efficient candidate for enhanced wastewater remediation.

Enzyme level N and O isotope effects of assimilatory and dissimilatory nitrate reduction

AbstractTo provide mechanistic constraints to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate ( ), 15N/14N and 18O/16O, in the environment, we measured the enzymatic N and O isotope effects (15ε and 18ε) during its reduction by reductase enzymes, including (1) a prokaryotic respiratory reductase, Nar, from the heterotrophic denitrifier Paracoccus denitrificans, (2) eukaryotic assimilatory reductases, eukNR, from Pichia angusta and from Arabidopsis thaliana, and (3) a prokaryotic periplasmic reductase, Nap, from the photoheterotroph Rhodobacter sphaeroides. Enzymatic Nar and eukNR assays with artificial viologen electron donors yielded identical 18ε and 15ε of ∼28‰, regardless of [ ] or assay temperature, suggesting analogous kinetic mechanisms with viologen reductants. Nar assays fuelled with the physiological reductant hydroquinone (HQ) also yielded 18ε ≈ 15ε, but variable amplitudes from 21‰ to 33.0‰ in association with [ ], suggesting analogous substrate sensitivity in vivo. Nap assays fuelled by viologen revealed 18ε:15ε of 0.50, where 18ε ≈ 19‰ and 15ε ≈ 38‰, indicating a distinct catalytic mechanism than Nar and eukNR. Nap isotope effects measured in vivo showed a similar 18ε:15ε of 0.57, but reduced 18ε ≈ 11‰ and 15ε ≈ 19‰. Together, the results confirm identical enzymatic 18ε and 15ε during assimilation and denitrification, reinforcing the reliability of this benchmark to identify consumption in the environment. However, the amplitude of enzymatic isotope effects is apt to vary in vivo. The distinctive signature of Nap is of interest for deciphering catalytic mechanisms but may be negligible in most environments given its physiological role.

Planktonic and biofilm-grown nitrogen-cycling bacteria exhibit different susceptibilities to copper nanoparticles.

Proper characterization of nanoparticle (NP) interactions with environmentally relevant bacteria under representative conditions is necessary to enable their sustainable manufacture, use, and disposal. Previous nanotoxicology research based on planktonic growth has not adequately explored biofilms, which serve as the predominant mode of bacterial growth in natural and engineered environments. Copper nanoparticle (Cu-NP) impacts on biofilms were compared with respective planktonic cultures of the ammonium-oxidizing Nitrosomonas europaea, nitrogen-fixing Azotobacter vinelandii, and denitrifying Paracoccus denitrificans using a suite of independent toxicity diagnostics. Median inhibitory concentration (IC50) values derived from adenosine triphosphate (ATP) for Cu-NPs were lower in N. europaea biofilms (19.6 ± 15.3 mg/L) than in planktonic cells (49.0 ± 8.0 mg/L). However, in absorbance-based growth assays, compared with unexposed controls, N. europaea growth rates in biofilms were twice as resilient to inhibition than those in planktonic cultures. Similarly, relative to unexposed controls, growth rates and yields of P. denitrificans in biofilms exposed to Cu-NPs were 40-fold to 50-fold less inhibited than those in planktonic cells. Physiological evaluation of ammonium oxidation and nitrate reduction suggested that biofilms were also less inhibited by Cu-NPs than planktonic cells. Furthermore, functional gene expression for ammonium oxidation (amoA) and nitrite reduction (nirK) showed lower inhibition by NPs in biofilms relative to planktonic-grown cells. These results suggest that biofilms mitigate NP impacts, and that nitrogen-cycling bacteria in wastewater, wetlands, and soils might be more resilient to NPs than planktonic-based assessments suggest.

Aerobic denitrifying bacteria that produce low levels of nitrous oxide.

Most denitrifiers produce nitrous oxide (N(2)O) instead of dinitrogen (N(2)) under aerobic conditions. We isolated and characterized novel aerobic denitrifiers that produce low levels of N(2)O 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 (NO(3)(-)) to N(2) at rates of 0.9 and 0.03 micro mol min(-1) unit of optical density at 540 nm(-1) at dissolved oxygen (O(2)) (DO) concentrations of 39 and 38 micro mol liter(-1), respectively. At the same DO level, the typical denitrifier Pseudomonas stutzeri and the previously described aerobic denitrifier Paracoccus denitrificans did not produce N(2) but evolved more than 10-fold more N(2)O than strains TR2 and K50 evolved. The isolates denitrified NO(3)(-) with concomitant consumption of O(2). These results indicated that strains TR2 and K50 are aerobic denitrifiers. These two isolates were taxonomically placed in the beta 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 N(2)O emission, the single-stage process for nitrogen removal, and microbial N(2)O emission into the ecosystem.

Nitrification and denitrification as a source for NO and NO2 production in high-strength wastewater

Laboratory and half-technical scale experiments were performed to evaluate nitric oxide (NO) and nitrogen dioxide (NO2) production during biological N-elimination from wastewater with high ammonium concentration (about 700mgNL–1). In a laboratory scale bioreactor with biomass retention, the ammonia oxidizer Nitrosomonas europaea and the denitrifier Paracoccus denitrificans were grown as reference organisms in co-culture in order to simulate the nitrifying and denitrifying community of wastewater treatment plants. Synthetic wastewater and sludge liquor from the municipal wastewater treatment plant in Lueneburg (Germany) were used. In the laboratory scale reactor, during the treatment of synthetic wastewater, 0.28% of the oxidized ammonium-N was released as NO-N by a pure culture of Nitrosomonas. A simultaneously nitrifying and denitrifying co-culture only released 0.04 to 0.2%. NO2 formation was not observed. NO production was much higher in sludge liquor. A pure culture of Nitrosomonas produced 0.52% NO+NO2-N (=NOx-N), a co-culture of Nitrosomonas and Paracoccus even 1.64% NOx-N. The production rate strongly depended on the media and the organisms used. In a co-culture of N. europaea and P denitrificans, Nitrosomonas was shown to be the most efficient NO producer. NO production increased with ammonium oxidation rate and with nitrite concentration of the medium. In synthetic wastewater, NO production was not influenced by reduced oxygen content. However, in sludge liquor NO production rate increased with decreasing O2 concentration. Here, for the first time, the formation of significant amounts of NO2 during simultaneous nitrification/denitrification could be demonstrated. In half-technical scale experiments, only 0.07% of the oxidized ammonium-N was released as NO-N from the nitrification stage. NO2 was not detectable. Release of nitric oxide from the denitrification stage was mainly diffusion limited and the amount produced did not exceed 0.0001%. A calculation on the basis of the results presented, revealed that biological treatment of nitrogen-rich wastewater is not a significant source for pollution of the atmosphere with NOx in industrial areas.

Nitrification, denitrification and growth in artificial Thiosphaera pantotropha biofilms as measured with a combined microsensor for oxygen and nitrous oxide

Cells of the aerobic denitrifier and heterotrophic nitrifier Thiosphaera pantotropha and of the traditional denitrifier Paracoccus denitrificans were immobilized in a 1.5 mm thick agar layer (biofilm) and submersed in liquid medium. A combined microsensor for O2 and N2O was used to record microprofiles of these two species in biofilms where the reduction of N2O was inhibited by acetylene. Nitrification in T. pantotropha was not affected by the addition of acetylene and by using a diffusion-reaction model to simulate the N2O profiles it was possible to calculate depth profiles of both nitrification and denitrification. The validity of the calculations when both nitrification and denitrification were operating in concert was confirmed by performing identical calculations on data obtained for a P. denitrificans biofilm. At high NO3− concentrations, part of the NO3− reduced by T. pantotropha biofilms was reduced only to NO2− and N2O production thus did not reflect total NO3− reduction. When NO2− and no NO3− was present in the water above the biofilm N2O production was recorded in the anoxic zone directly below the oxic zone. Nitrous oxide production was never detected in the oxic zone of the biofilms, although aerobic denitrification was described for the original isolate of this bacterium. The growth rate of T. pantotropha in the oxic region of the biofilms was estimated to be 0.42 h−1 which is slightly higher than rates previously obtained in liquid culture. In the T. pantotropha biofilms nitrification was calculated to account for more than 50% of the O2 consumption whereas this process only consumed about 10% of the O2 in liquid culture.

Nitrification, denitrification and growth in artificial Thiosphaera pantotropha biofilms as measured with a combined microsensor for oxygen and nitrous oxide

Cells of the aerobic denitrifier and heterotrophic nitrifier Thiosphaera pantotropha and of the traditional denitrifier Paracoccus denitrificans were immobilized in a 1.5 mm thick agar layer (biofilm) and submersed in liquid medium. A combined microsensor for O 2 and N 2O was used to record microprofiles of these two species in biofilms where the reduction of N 2O was inhibited by acetylene. Nitrification in T. pantotropha was not affected by the addition of acetylene and by using a diffusion-reaction model to simulate the N 2O profiles it was possible to calculate depth profiles of both nitrification and denitrification. The validity of the calculations when both nitrification and denitrification were operating in concert was confirmed by performing identical calculations on data obtained for a P. denitrificans biofilm. At high NO 3 − concentrations, part of the NO 3 − reduced by T. pantotropha biofilms was reduced only to NO 2 − and N 2O production thus did not reflect total NO 3 − reduction. When NO 2 − and no NO 3 − was present in the water above the biofilm N 2O production was recorded in the anoxic zone directly below the oxic zone. Nitrous oxide production was never detected in the oxic zone of the biofilms, although aerobic denitrification was described for the original isolate of this bacterium. The growth rate of T. pantotropha in the oxic region of the biofilms was estimated to be 0.42 h −1 which is slightly higher than rates previously obtained in liquid culture. In the T. pantotropha biofilms nitrification was calculated to account for more than 50% of the O 2 consumption whereas this process only consumed about 10% of the O 2 in liquid culture.

Kinetics of H2 oxidation in respiring and denitrifying Paracoccus denitrificans

Kinetics of H2 oxidation in respiring and denitrifying Paracoccus denitrificans

Rationalization of properties of nitrate reductases in Rhodopseudomonas capsulata

1. The properties of nitrate reductase activities have been compared in several strains of Rhodopseudomonas capsulata grown phototrophically in the presence of nitrate as sole nitrogen source. 2. Strains AD2 and BK5 resemble the spontaneous mutant N22DNAR+ (described by McEwan et al. 1982 FEBS Lett. 150, 277\2-280) in that reduction of nitrate was inhibited by either illumination or oxygen but not by NH 4 + , and that electron flow to nitrate under dark anaerobic conditions generated a cytoplasmic membrane potential (as judged by an electrochromic shift in the absorbance spectrum of endogenous carotenoid pigments). In contrast disappearance of nitrate from suspensions of strains N22 and St. Louis was dependent upon illumination and was inhibited by NH 4 + . Membrane potentials were not generated by addition of nitrate in the dark to N22, St. Louis or strain Kbl. 3. Nitrate reductase was shown to be located in the periplasmic space of both strain AD2 and mutant N22DNAR+. The nitrate reductase activity in cells of AD2 and N22DNAR+ was relatively insensitive to azide, with 0.5mM azide required for 50% inhibition. The nitrate reductase of strain BK5 was more strongly associated with the cytoplasmic membrane and no conclusion could be reached about whether it was located on the periplasmic or cytoplasmic surface. In BK5 cells nitrate reductase activity was sensitive to low concentrations of azide (50% inhibition with 2 \gmM azide). It is proposed that functionally the nitrate reductase activity in strains AD2, BK5 and N22DNAR+ has identical roles. These roles are suggested to include: (i) The first step in the assimilation of nitrate. (ii). Provision of an alternative electron acceptor to oxygen for generating a membrane potential. (iii). A mechanism for disposing of excess reducing equivalents in the maintenance of balanced growth. This type of nitrate reductase, especially in AD2 and N22DNAR+, appears to resemble that described in a denitrifying strain of Rps. sphaeroides, but to differ markedly from its membrane-bound counterpart in other bacteria including the denitrifying Paracoccus denitrificans and Escherichia coli. 4. In other strains of Rps. capsulata including St. Louis, N22 and Kbl, only an assimilatory nitrate reductase, whose activity in intact cells is relatively sensitive to azide, is present in anaerobic, phototrophic cultures grown with nitrate as nitrogen source. As this reductase cannot be detected after breakage of cells, no conclusion can be made as to its location in the cell.

Dissimilatory nitrate uptake in Paracoccus denitrificans via a [formula omitted] system and a nitrate-nitrite antiport system

Respiration-driven proton translocation has been studied with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing H 2 during reduction of O 2, NO − 3, NO − 2 or N 2O. A simplified scheme of anaerobic electron transport and associated proton translocation is shown that is consistent with the measured H + oxidant ratios . Furthermore, the kinetics and energetics of NO − 3 uptake in whole cells of P. denitrificans were studied. For this purpose, we measured H 2 consumption or N 2O production after addition of NO − 3 to a cell suspension, which indirectly gave information about uptake (and reduction) of NO − 3. It was found that a lag phase in H 2 consumption or N 2O production appeared whenever the membrane potential was dissipated by addition of thiocyanate, carbonyl cyanide m-chlorophenylhydrazone or triphenyl-methylphosphonium bromide. However, these lag phases were not observed when NO − 2 was present at the moment of introduction of NO − 3. On the basis of these findings we conclude that there are two uptake systems for NO − 3. One system is dependent on the proton-motive force and is probably used for initiation of NO − 3 uptake. The other is an NO − 3 NO − 2 antiport and its function is to take over NO − 3 uptake from the first system.

Proton translocation and proline uptake associated with reduction of nitric oxide by denitrifying Paracoccus denitrificans

Summary Minimum →H + /2e − ratios for both the NO→1/2N 2 O and NO→1/2N 2 reactions were about 3.7 with denitrifying Paracoccus denitrificans . These ratios are only slightly less than those obtained with other N-oxides similarly linked to the oxidation of endogenous substrate. Active transport of [ 14 C]L-proline was supported by the above two reactions of NO in denitrifying cells. NO failed to promote proline uptake with aerobically grown cells. The uncoupler, carbonyl cyanide m -chlorophenylhydrazone, abolished proline uptake and greatly diminished proton translocation with NO and other N-oxides. The results directly demonstrate for the first time that reduction of exogenous NO is energy yielding in a denitrifying bacterium.

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) H +/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O 2, NO − 2 or N 2O. Under optimal H +-translocation conditions, the ratios H + O , H + N 2 O , H + NO − 2 for reduction to N 2 and H + NO − 2 for reduction to N 2O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/ N, N, N′, N′-tetramethyl- p-phenylenediamine as exogenous substrate, addition of NO − 2 or N 2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. H + N 2 O , H + NO − 2 for reduction to N 2 and H + NO − 2 for reduction to N 2O were −0.84, −2.33 and −1.90, respectively. (3) The H + oxidant ratios, mentioned in item 2, were not altered in the presence of valinomycin K + and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O 2, NO − 2 and N 2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO − 2, N 2O or O 2 pass two H +-translocating sites. The H + electron acceptor ratios predicted by this scheme are in good agreement with the experimental values.