Heterotrophic nitrification and aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001

A strain YZN-001 was isolated from swine manure effluent and was identified as Pseudomonas stutzeri. It can utilise not only nitrate and nitrite, but also ammonium. The strain had the capability to fully remove as much as 275.08 mg L⁻¹ NO₃⁻–N and 171.40 mg L⁻¹ NO₂⁻–N under aerobic conditions. Furthermore, At 30 °C, the utilization of ammonium is approximately 95% by 18 h with a similar level removed by 72 h and 2 weeks at 10 and 4 °C, respectively. Triplicate sets of tightly sealed serum bottles were used to test the heterotrophic nitrifying ability of P. stutzeri YZN-001. The results showing that 39% of removed NH₄⁺–N was completely oxidised to nitrogen gas by 18 h. Indicating that the strain has heterotrophic nitrification and aerobic denitrification abilities, with the notable ability to remove ammonium at low temperatures, demonstrating a potential using the strain for future application in waste water treatment.     

Effects of cultural conditions on denitrification by Pseudomonas stutzeri measured by Membrane Inlet Mass Spectrometry

Denitrification is a globally important process leading to loss of fertiliser efficiency and the production of the greenhouse gas nitrous oxide and nitric oxide, an ozone depleter. Membrane inlet mass spectrometry (MIMS) was employed to study the effect of different variables on the process of denitrification by Pseudomonas stutzeri in a defined salts medium. MIMS was used for concomitant measurements of nitrous oxide, nitrogen and oxygen and showed that denitrification occurred in the presence of dissolved oxygen. A nitrate concentration of 15 mmol l⁻¹ and a nitrite concentration of 5 mmol l⁻¹ were found to be optimum for complete denitrification of nitrate or nitrite to nitrogen and varying these concentrations had a marked effect on the ratio of gaseous products released. Denitrification products were also dependant on pH with neutral or alkaline conditions being best for production of gaseous end products. Our results suggest that under nutrient rich conditions the most important factor in the regulation of denitrification by Ps. stutzeri is the amount of nitrite generated at the first enzymatic stage of the process. This appears to cause inhibition of the denitrification pathway above 5 mmol l⁻¹ and at high enough concentrations (15 mmol l⁻¹) restricts growth.     

Fe3O4对施氏假单胞菌反硝化过程的影响

生物反硝化是目前废水深度处理中应用最为广泛的硝酸盐氮处理技术,但该方法一般停留时间较长,在冬季因低温处理效果欠佳,因此有必要开发反硝化强化技术。以施氏假单胞菌Pseudomonas stutzeri为研究对象,考察了不同投加量下Fe3O4对P. stutzeri反硝化过程的影响。结果显示当Fe3O4投加量由0 mg/L增至4 000 mg/L时,硝酸盐氮最大比降解速率由18.0 h–1增加至23.7 h–1,体系中的总蛋白含量以及细菌体内的铁含量显著增加。RT-qPCR和非标记 (Label-free) 定量蛋白组学分析表明,投加4 000 mg/L Fe3O4体系中的P. stutzeri,其反硝化功能基因napA、narJ、nirB、norR、nosZ表达量分别提高了55.7%、24.9%、24.5%、36.5%、120%,对应反硝化还原酶Nap、Nar、Nir、Nor、Nos表达量提高了85.0%、147%、16.5%、47.1%、95.9%。对比体系中“游离细菌”和“Fe3O4粘附细菌”,发现二者的反硝化功能基因以及反硝化相关酶没有显著差别;而Fe3O4粘附细菌电子传递相关蛋白表达量有所提高,说明了Fe3O4通过与细菌直接接触促进其生长代谢,导致体系中细菌总量的增加,从而提高反硝化速率。该结果可为反硝化强化技术的开发提供理论支撑。     

Continuous measurement of NOaq during denitrification by immobilized Pseudomonas stutzeri

Summary An ISO-NO sensor was used for continuous measurement of nitric oxide release and consumption during denitrification. The sensor was selectively responsive to NO in the presence of other denitrification-associated nitrogen oxides. Evolution of NO signal was coupled with the metabolism of NO₂ ^–. The immobilized Pseudomonas stutzeri seems able to to restrict its physiological NO pool to less than 100 nM (about 2 × 10^–5 mole/mole of the NO₃ ^– or NO₂ ^– reduced), a level being one hundredth of the concentration required to inactivate a population in 45 min.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.     

Siderophore production by Pseudomonas stutzeri under aerobic and anaerobic conditions

The siderophore production of the facultative anaerobe Pseudomonas stutzeri, strain CCUG 36651, grown under both aerobic and anaerobic conditions, was investigated by liquid chromatography and mass spectrometry. The bacterial strain has been isolated at a 626-m depth at the Äspö Hard Rock Laboratory, where experiments concerning the geological disposal of nuclear waste are performed. In bacterial culture extracts, the iron in the siderophore complexes was replaced by gallium to facilitate siderophore identification by mass spectrometry. P. stutzeri was shown to produce ferrioxamine E (nocardamine) as the main siderophore together with ferrioxamine G and two cyclic ferrioxamines having molecular masses 14 and 28 atomic mass units lower than that of ferrioxamine E, suggested to be ferrioxamine D₂ and ferrioxamine X₁, respectively. In contrast, no siderophores were observed from anaerobically grown P. stutzeri. None of the siderophores produced by aerobically grown P. stutzeri were found in anaerobic natural water samples from the Äspö Hard Rock Laboratory.In order to facilitate iron(III) acquisition, plants and microorganisms, such as fungi and bacteria, produce and excrete strong iron(III) chelators, i.e., siderophores (18, 22, 23, 33, 34). While fungal siderophores bind to iron(III) by hydroxamate ligands, bacterial siderophores are more structurally diverse, and common ligands are catecholates, hydroxamates, and carboxylates (21). The iron(III) stability constants for bacterial siderophores vary in the range of 10²⁰ to 10⁵² (6). In addition to iron(III), other metals can be complexed by siderophores. For the trihydroxamate siderophore desferrioxamine B, sometimes called proferrioxamine B (10), some actinides have been shown to have stability constants in the same range as the ferric stability constant (10^30.6), e.g., 10^26.6 with thorium(IV) and 10^30.8 with plutonium(VI) (32), while the stability constant for uranium(VI) was lower, i.e., 10¹⁸ (2).Concerning bacteria, there are several reports on siderophore production by Pseudomonas spp. (1, 3, 4, 19). More than 50 structurally related siderophores, i.e., pyoverdins, produced by the fluorescent Pseudomonas spp., especially Pseudomonas fluorescens and Pseudomonas aeruginosa, have been characterized (3). All pyoverdins emit yellow fluorescent light due to the presence of a 5-amino-2,3-dihydro-8,9-dihydroxy-1-H-pyrimido-quinoline-carboxylic chromophore, to which a peptide chain and a carboxyl chain are attached (1, 3). Nonfluorescent Pseudomonas has also been shown to produce siderophores, such as ferrioxamine E, also called nocardamine (Fig. ​(Fig.1),1), which was produced by one strain of Pseudomonas stutzeri (19). In addition to ferrioxamines, the P. stutzeri strain KC produced a smaller siderophore, i.e., pyridine-2,6-bis(thiocarboxylic acid) (35). Conversely, a catecholate-type siderophore was shown to be produced by another strain of P. stutzeri, which did not produce any hydroxamate siderophores (4).Open in a separate windowFIG. 1.Structures, molecular masses (mw), and stability constants (K_s) of ferric complexes of the three ferrioxamines: ferrioxamine B (B), ferrioxamine E (E), and ferrioxamine G (G) (5, 18).Most of the studies on bacterial siderophore production have been conducted on microorganisms growing under aerobic conditions. One field-based report, however, indicates the occurrence of putative siderophores in anaerobic environments also (29). In the present study, siderophore production has been studied with both aerobic and anaerobic cultures of P. stutzeri. This species is a facultative aerobe, able to grow with oxygen or nitrate as the electron acceptor, meaning that it can be active under both anaerobic and aerobic conditions. The P. stutzeri strain CCUG 36651, studied here, has been isolated from a depth of 626 m below ground at the Äspö Hard Rock Laboratory (16), where research concerning the geological disposal of nuclear waste is performed. The possibility of mobilizing radionuclides by complexing compounds from bacteria is an important research area in the context of nuclear waste disposal research. It is unknown if such compounds are produced in aquifers under conditions relevant to a disposal site, which would be approximately 500 m underground in granitic rock (27).A study from 2004 shows that P. stutzeri growing aerobically in the presence of uranium-containing shale leached Fe, Mo, V, and Cr from the shale material (17). More recently it was shown that the supernatant of aerobically and anaerobically cultured P. stutzeri was able to increase the partitioning of added Fe, Pm, Am, and Th into the aqueous phase in samples where quartz sand was used as a solid surface (16). Aerobic supernatants maintained 60% or more of the added metals in solution, while anaerobic supernatants were best at maintaining Am in solution, reaching a value of 40% in solution. The increased partitioning to the aqueous phase in the presence of the supernatants was ascribed to the production of organic ligands. Supernatants of both aerobically and anaerobically grown P. stutzeri strain CCUG 36651 yielded a positive response on the universal siderophore assay, the CAS assay (16). This assay is based on ligand competition for iron bound to the colored chrome azurol complex (25, 30).In this study, siderophore production by P. stutzeri strain CCUG 36651 was investigated using mass spectrometry (MS) and liquid chromatography (LC) followed by mass spectrometric detection. Electrospray ionization mass spectrometry (ESI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) are useful tools in characterizing siderophores such as ferrioxamines (10, 13, 14, 28, 31). In order to detect iron(III)-chelating compounds, the ferric iron can be replaced by gallium(III) through ascorbate-mediated reduction of iron(III) (8, 20). In mass spectra, gallium-bound substances are easily recognized due to the characteristic isotope pattern of gallium, where the intensity of the ⁷¹Ga signal is about 66% of that of the ⁶⁹Ga signal. The use of ESI provides so-called soft ionization; thus, information about the molecular weight is obtained. However, by employing MS/MS, fragmentation is achieved, providing more information about the compound structure.In order to verify the chemical difference between the siderophores found by ESI-MS, chromatographic separation was performed. In this case, one reversed-phase C₁₈ column and one column containing a porous graphitic carbon (PGC) stationary phase were used. The separation mechanism of PGC is a combination of hydrophobic interactions, as in C₁₈, and electrostatic interactions between π-electrons. In order to detect substances at low concentrations, column-switched capillary chromatography with MS detection was used. The detection limits of the combined LC-MS/MS system used in this study are in the range of 1 to 5 nM for hydroxamate siderophores of the ferrichrome and ferrioxamine families (9). In order to facilitate analysis of lower concentrations of ferrioxamines, natural water samples were preconcentrated by solid-phase extraction (SPE), resulting in minimum detectable concentrations in the range of 0.02 to 0.1 nM, depending on the initial sample volume.     

Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans.

A comparison was made of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans. Although all three organisms reduced nitrate to dinitrogen gas, they did so at different rates and accumulated different kinds and amounts of intermediates. Their rates of anaerobic growth on nitrate varied about 1.5-fold; concomitant gas production varied more than 8-fold. Cell yields from nitrate varied threefold. Rates of gas production by resting cells incubated with nitrate, nitrite, or nitrous oxide varied 2-, 6-, and 15-fold, respectively, among the three species. The composition of the gas produced also varied markedly: Pseudomonas stutzeri produced only dinitrogen; Pseudomonas aeruginosa and Paracoccus denitrificans produced nitrous oxide as well; and under certain conditions Pseudomonas aeruginosa produced even more nitrous oxide than dinitrogen. Pseudomonas stutzeri and Paracoccus denitrificans rapidly reduced nitrate, nitrite, and nitrous oxide and were able to grow anaerobically when any of these nitrogen oxides were present in the medium. Pseudomonas aeruginosa reduced these oxides slowly and was unable to grow anaerobically at the expense of nitrous oxide. Furthermore, nitric and nitrous oxide reduction by Pseudomonas aeruginosa were exceptionally sensitive to inhibition by nitrite. Thus, although it has been well studied physiologically and genetically, Pseudomonas aeruginosa may not be the best species for studying the later steps of the denitrification pathway.     

Analysis of denitrification by Pseudomonas stutzeri under nutrient-limited conditions using membrane inlet mass spectrometry

Membrane inlet mass spectrometry (MIMS) was used to investigate denitrification by Pseudomonas stutzeri in a static lake water column. Continuous real-time measurement of gases enabled the dynamics of the process to be investigated. Concentrations of 17 mmol l-1 nitrate and 10 mmol l-1 nitrite were identified as optimal for denitrification under nutrient-limited conditions (i.e., produced the highest concentrations of N2). Available carbon was the major rate-limiting factor in lake water when nitrate or nitrite was present. No stratification of the process with depth was observed, and aerobic denitrification was apparent under all the conditions employed. The rate of denitrification was dependent on cell concentration, and possible limitations of the usefulness of MIMS under environmentally modelled conditions were identified for environments containing low numbers of bacteria.     

Isolation of a Pseudomonas stutzeri strain that degrades o-xylene

A Pseudomonas stutzeri strain capable of growing on o-xylene was isolated from enrichment cultures. The organism grew on 2,3- and 3,4-dimethylphenol but not on 2-methylbenzyl alcohol, o-tolualdehyde, or o-toluate. P. stutzeri was not able to utilize m-xylene, p-xylene, or 1,2,4-trimethylbenzene, but growth was observed in the presence of the corresponding alcohols and acids. From the Pseudomonas cultures supplied with o-xylene, 2,3-dimethylphenol was isolated and identified. When resting P. stutzeri cells were incubated with 2,3-dimethylphenol, the reaction mixture turned greenish yellow and showed spectral properties identical to those of the 3,4-dimethylcatechol meta ring fission product. Catechol 2,3-oxygenase was induced by growth on o-xylene or on 2,3- or 3,4-dimethylphenol. The suggested hypothesis is that the first metabolic steps of growth on o-xylene involve the direct oxygenation of the aromatic nucleus, followed by meta pathway reactions.     

Degradation of phenol through ortho-cleavage pathway by Pseudomonas stutzeri strain SPC2

P.Y. ANEEZ AHAMAD AND A.A.M. KUNHI. 1996. Generally pseudomonads degrade phenol through the meta -pathway, but Pseudomonas stutzeri strain SPC2 isolated by flask enrichment of municipal sewage degraded phenol through the ortho -pathway. The strain utilized up to 1200 ppm of phenol as a sole source of carbon and energy. The strain also degraded benzoate and 4-hydroxy and 3,4-dihydroxybenzoates via the ortho -pathway. Cell-free extracts of the strain grown on these substrates showed fairly good catechol 1,2-dioxygenase (C1,2-D) and protocatechuate 3,4-dioxyenase (PCA 3,4-D) activities, the induction of both activities being increased by benzoate. No meta -cleavage activities were detected.     

Structural and functional analysis of denitrification genes in Pseudomonas stutzeri A1501

Denitrification is the process by which nitrates are converted to nitrogen gas under the action of microor-ganism, and in a bioenergetics viewpoint, a kind of respiration of bacteria in anoxia condition. In such a process, nitrogen in oxidation state replaces oxygen as the terminal electron acceptor in cell membrane, gen-erates potential gradient with the action of a series of oxidoreductase, and finally converts nitrate into nitro-gen[1]. Denitrification is widely present in nature, and resea…     

Survival of the aerobic denitrifier Pseudomonas stutzeri strain TR2 during co-culture with activated sludge under denitrifying conditions

The aerobic denitrifier Pseudomonas stutzeri TR2 (strain TR2) has the potential to reduce nitrous oxide emissions during the wastewater treatment process. In this application, it is important to find the best competitive survival conditions for strain TR2 in complex ecosystems. To that end, we examined co-cultures of strain TR2 with activated sludge via five passage cultures in a medium derived from treated piggery wastewater that contained a high concentration of ammonium. The results are as follows: (i) The medium supported the proliferation of strain TR2 (P. stutzeri strains) under denitrifying conditions. (ii) Nitrite was a better denitrification substrate than nitrate for TR2 survival. (iii) Strain TR2 also demonstrated strong survival even under aerobic conditions. This suggests that strain TR2 is effectively augmented to the wastewater treatment process, aiding in ammonium-nitrogen removal and reducing nitrous oxide production with a partial nitrification technique in which nitrite accumulates.     

Hyperproduction of polyesters consisting of medium-chain-length hydroxyalkanoate monomers by strain Pseudomonas stutzeri 1317

Pseudomonas stutzeri strain 1317 was found to grow on various fatty acids, alcohols, diols, as well as glucose and gluconate for the synthesis of polyhydroxyalkanoates (PHA) with various monomer units. The PHA monomer structures were dependent on the type of fatty acids and alcohols, as well as the diols in the culture media. Only even number monomers, such as 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO) and 3-hydroxydecanoate (HD), were accumulated when even numbered fatty acids, alcohols, glucose and gluconate, as well as diol were used as carbon sources. Odd numbered fatty acids and odd numbered alcohols led to the formation of odd numbered monomers, such as 3-hydroxyvalerate (HV), 3-hydroxyheptanoate (HHp), 3-hydroxynonanoate (HN) and 3-hydroxyundecanoate (HU). The strain tolerated up to 1.5% of ethanol and made 8.3% of PHA when growth was conducted in 1.2% of ethanol. PHA formed up to 77% of cell dry weight when the strain was grown in tridecanoate. PHA synthesis was highly dependent on the nitrogen source. A depletion in nitrogen supply immediately resulted in PHA accumulation in cells grown in the glucose mineral medium.     

Pleuropneumonia caused by Pseudomonas stutzeri

Biological characteristics and antibiotic sensitivity of P. stutzeri strain, isolated from a child with pleuropneumonia, are presented. Formation of rugous colonies, growth at 41 degrees C and in the presence of 6.5% of NaCl, the positive results of the oxidase and nitrate reductase tests, the negative signs of arginine hydrolase and lysine decarboxylase activity permit the identification of this Pseudomonas species. The isolated culture has proved to be sensitive to amino glycoside antibiotics, carbonicillin and polymyxin.     

Efficient production of pyruvate from DL-lactate by the lactate-utilizing strain Pseudomonas stutzeri SDM

Background

The platform chemical lactate is currently produced mainly through the fermentation of sugars presented in biomass. Besides the synthesis of biodegradable polylactate, lactate is also viewed as a feedstock for the green chemistry of the future. Pyruvate, another important platform chemical, can be produced from lactate through biocatalysis.

Methodology/Principal Findings

It was established that whole cells of Pseudomonas stutzeri SDM catalyze lactate oxidation with lactate-induced NAD-independent lactate dehydrogenases (iLDHs) through the inherent electron transfer chain. Unlike the lactate oxidation processes observed in previous reports, the mechanism underlying lactate oxidation described in the present study excluded the costliness of the cofactor regeneration step and production of the byproduct hydrogen peroxide.

Conclusions/Significance

Biocatalysis conditions were optimized by using the cheap dl-lactate as the substrate and whole cells of the lactate-utilizing P. stutzeri SDM as catalyst. Under optimal conditions, the biocatalytic process produced pyruvate at a high concentration (48.4 g l⁻¹) and a high yield (98%). The bioconversion system provides a promising alternative for the green production of pyruvate.