Initial reactions in anaerobic ethylbenzene oxidation by a denitrifying bacterium, strain EB1.

Initial reactions in anaerobic oxidation of ethylbenzene were investigated in a denitrifying bacterium, strain EB1. Cells of strain EB1 mineralized ethylbenzene to CO2 under denitrifying conditions, as demonstrated by conversion of 69% of [14C]ethylbenzene to 14CO2. In anaerobic suspensions of strain EB1 cells metabolizing ethylbenzene, the transient formation and consumption of 1-phenylethanol, acetophenone, and an as yet unidentified compound were observed. On the basis of growth experiments and spectroscopic data, the unknown compound is proposed to be benzoyl acetate. Cell suspension experiments using H2(18)O demonstrated that the hydroxyl group of the first product of anoxic ethylbenzene oxidation, 1-phenylethanol, is derived from water. A tentative pathway for anaerobic ethylbenzene mineralization by strain EB1 is proposed.     

A Novel C1-Using Denitrifier Alcaligenes sp. STC1 and Its Genes for Copper-containing Nitrite Reductase and Azurin

A novel denitrifier Alcaligenes sp. STC1 was identified. The strain efficiently denitrifies under an atmosphere of 10% oxygen (O₂) where Paracoccus denitrificans, one of the most studied aerobic denitrifiers, had less denitrifying activity, indicating that the strain has an O₂-torelant denitrifying system. It denitrified by using C1-carbon sources such as formate and methanol as well as glucose, glycerol, and succinate. The genes for the copper-containing nitrite reductase and azurin of this C1-using denitrifier were cloned. Their predicted products of them were similar to those of their counterparts and the maximum similarities were 90% and 92%, respectively.     

Metabolism of both 4-chlorobenzoate and toluene under denitrifying conditions by a constructed bacterial strain.

T1, a dentrifying bacterium originally isolated for its ability to grow on toluene, can also metabolize 4-hydroxybenzoate and other aromatic compounds under denitrifying conditions. A cosmid clone carrying the three genes that code for the 4-chlorobenzoate dehalogenase enzyme complex isolated from the aerobic bacterium Pseudomonas sp. strain CBS3 was successfully conjugated into strain T1. The cloned enzyme complex catalyzes the hydrolytic dechlorination of 4-chlorobenzoate to 4-hydroxybenzoate. Since molecular oxygen is not required for the dehalogenation reaction, the transconjugate strain of T1 (T1-pUK45-10C) was able to grow on 4-chlorobenzoate in the absence of O2 under denitrifying conditions. 4-Chlorobenzoate was dehalogenated to 4-hydroxybenzoate, which was then further metabolized by strain T1. The dehalogenation and metabolism of 4-chlorobenzoate were nitrate dependent and were coupled to the production of nitrite and nitrogen gas. 4-Bromobenzoate was also degraded by this strain, while 4-iodobenzoate was not. Additionally, when T1-pUK45-10C was presented with a mixture of 4-chlorobenzoate and toluene, simultaneous degradation of the compounds was observed. These results illustrate that dechlorination and degradation of aromatic xenobiotics can be mediated by a pure culture in the absence of oxygen. Furthermore, it is possible to expand the range of xenobiotic substrates degradable by an organism, and it is possible that concurrent metabolism of these substrates can occur.     

Isolation and characterization of novel denitrifying alkalithermophiles, AT-1 and AT-2

Two novel denitrifying alkalithermophilic bacteria, AT-1 and AT-2, were isolated from manure-amended soil. The isolates grew at 35–65°C with an optimum temperature at 50–60°C, and pH 6.5–10.0 with an optimum pH at 9.5. Both isolates were Gram-positive, facultative anaerobic, non-motile rod-shaped bacteria. A phylogenetic analysis based on 16S rRNA sequence data indicated that both AT-1 and AT-2 are members of the genus Anoxybacillus. DNA-DNA hybridization revealed moderate relatedness between AT-1 and AT-2 and one phylogenetically related strain, A. pushchinensis K1 (69.5 and 69.1%, respectively). Comparative analysis of morphology and biochemical characteristics of the two isolates also showed similarity to A. pushchinensis K1. Based on these results, we identified AT-1 and AT-2 as A. pushchinensis. To our knowledge, this is the first report of denitrifying bacterium isolated from alkalithermophilic Anoxybacillus spp.     

Nitrous oxide reduction in nodules: denitrification or n(2) fixation?

Detached cowpea nodules that contained a nitrous oxide reductase-positive (Nor) rhizobium strain (8A55) and a nitrous oxide reductase-negative (Nor) rhizobium strain (32H1) were incubated with 1% N(2)O (95 atom% N) in the following three atmospheres: (i) aerobic with C(2)H(2) (10%), (ii) aerobic without C(2)H(2), and (iii) anaerobic (argon atmosphere) without C(2)H(2). The greatest production of N(2) occurred anaerobically with 8A55, yet very little was formed with 32H1. Although acetylene reduction activity was slightly higher with 32H1, about 10 times more N(2) was produced aerobically by 8A55 than by 32H1 in the absence of acetylene. The major reductive pathway of N(2)O reduction by denitrifying rhizobium strain 8A55 is by nitrous oxide reductase rather than nitrogenase.     

Anaerobic oxidation of 2-chloroethanol under denitrifying conditions by<Emphasis Type="Italic"> Pseudomonas stutzeri</Emphasis> strain JJ

A bacterium that uses 2-chloroethanol as sole energy and carbon source coupled to denitrification was isolated from 1,2-dichloroethane-contaminated soil. Its 16 S rDNA sequence showed 98% similarity with the type strain of Pseudomonas stutzeri (DSM 5190) and the isolate was tentatively identified as Pseudomonas stutzeri strain JJ. Strain JJ oxidized 2-chloroethanol completely to CO(2) with NO(3)(- )or O(2) as electron acceptor, with a preference for O(2) if supplied in combination. Optimum growth on 2-chloroethanol with nitrate occurred at 30 degrees C with a mu(max) of 0.14 h(-1) and a yield of 4.4 g protein per mol 2-chloroethanol metabolized. Under aerobic conditions, the mu(max) was 0.31 h(-1). NO(2)(-) also served as electron acceptor, but reduction of Fe(OH)(3), MnO(2), SO(4)(2-), fumarate or ClO(3)(-) was not observed. Another chlorinated compound used as sole energy and carbon source under aerobic and denitrifying conditions was chloroacetate. Various different bacterial strains, including some closely related Pseudomonas stutzeri strains, were tested for their ability to grow on 2-chloroethanol as sole energy and carbon source under aerobic and denitrifying conditions, respectively. Only three strains, Pseudomonas stutzeri strain LMD 76.42, Pseudomonas putida US2 and Xanthobacter autotrophicus GJ10, grew aerobically on 2-chloroethanol. This is the first report of oxidation of 2-chloroethanol under denitrifying conditions by a pure bacterial culture.     

Growth and phenol activity of Pseudomonas aeruginosa strain 101/1 in batch cultures

Strain 101/1, isolated from petroleum wastewater sediment was classified as Pseudomonas aeruginosa. In wild type condition the strain tolerated phenol in concentration 1,000 mg/L under aerobic conditions and 800 mg/L under denitrifying conditions. As a result of adaptation to phenol the resistance of the strain to the compound increased to 1,600 and 1,400 mg/L, respectively. Maximum phenol activity under aerobic and denitrifying conditions was 350 and 65 mg/L x day-1, respectively. Under denitrifying conditions a reduction in incubation temperature from 30 degrees C to 20 degrees C resulted in two-fold drop in phenol activity of the adapted strain and reduction in tolerance to phenol by 400 mg/L.     

Enumeration of Flexibacter canadensis in environmental samples by using a bacteriophage isolated from soil.

The denitrifier Flexibacter canadensis, in the presence of sulfide, can reduce N2O in the presence of concentrations of C2H2 which normally inhibit N2O reduction. Most-probable-number estimates of naturally occurring F. canadensis populations in various soils and sediments were made with a bacteriophage which is active against and specific for a strain of denitrifying F. canadensis (Is-11). Our survey suggests that F. canadensis is common in the natural environment.     

Enumeration of Flexibacter canadensis in environmental samples by using a bacteriophage isolated from soil.

The denitrifier Flexibacter canadensis, in the presence of sulfide, can reduce N2O in the presence of concentrations of C2H2 which normally inhibit N2O reduction. Most-probable-number estimates of naturally occurring F. canadensis populations in various soils and sediments were made with a bacteriophage which is active against and specific for a strain of denitrifying F. canadensis (Is-11). Our survey suggests that F. canadensis is common in the natural environment.     

反硝化除磷菌筛选及其特性研究

【目的】研究反硝化除磷菌特性。【方法】通过微生物筛选和生物学特性研究方法,从对虾养殖池塘中筛选出多株可在有氧条件下同时具有反硝化除磷功能的菌种。【结果】菌株LY-1可在18 h内将初始量为10 mg/L的亚硝酸盐氮降低至0.04 mg/L,PO43?-P降低至0.05 mg/L。在DO浓度为5.0?5.9 mg/L时,该菌反硝化除磷率近100%。试验选取具有反硝化除磷功能的枯草芽孢杆菌为阳性对照菌,大肠杆菌为阴性对照菌,比较研究了菌株LY-1在不同pH、温度、盐度、PO43?-P浓度、亚硝酸盐浓度时反硝化除磷的强弱,在pH为5?9范围时,该菌亚硝酸盐氮去除率近99%,PO43?-P去除率86%;温度为30°C时,该菌反硝化除磷率近100%;盐度为5‰?15‰、PO43?-P浓度为10 mg/L、亚硝酸盐氮浓度为20 mg/L时,该菌亚硝酸盐氮和PO43?-P去除率均可达99%。【结论】菌株LY-1反硝化除磷性能显著高于对照菌(P<0.05)。通过菌株LY-1形态学观察、生理生化及16S rRNA基因序列分析,初步鉴定为蜡样芽孢杆菌(Bacillus cereus)。     

好氧反硝化菌的筛选及其脱氮除磷性质的研究

利用富集培养基, 从用生活污水驯化后的活性污泥中筛选得到一株具有好氧反硝化兼具除磷功能的细菌。通过形态学及生理生化指标鉴定其为假单胞菌属。利用此好氧反硝化菌处理模拟废水及生活废水, 通过监测总氮、无机磷及CODcr变化确定在C/N摩尔比为3:1、接种量为10%、pH 6.8、30°C条件下处理2 d, 该菌株脱氮、除磷及去除有机物的效果最佳, 活性污泥经此好氧反硝化菌强化后, 对生活废水的处理能力得到明显提升。     

Effect of acetylene on nitrous oxide reduction and sulfide oxidation in batch and gradient cultures of Thiobacillus denitrificans.

Anaerobic enrichment cultures with H2S and N2O as substrates which were inoculated with a biofilm sample showed rapid growth and gas formation after 2 to 3 days at 27 degrees C. By using the deep-agar dilution technique, a pure culture was obtained. The strain was tentatively identified as Thiobacillus denitrificans. The isolate was used for batch and gradient culture studies under denitrifying conditions, oxidizing H2S with concomitant reduction of N2O to N2. In batch culture, oxidation of H2S was stepwise, with transient accumulation of elemental sulfur; the final oxidation product was SO4(2-). In gradient culture, there was no notable accumulation of elemental sulfur and microsensor measurements of H2S and N2O showed that H2S was oxidized directly to SO4(2-). In the presence of C2H2, however, oxidation of H2S stopped at the level of elemental sulfur and no SO4(2-) was produced in either batch or gradient cultures. This is a hitherto unknown inhibitory effect of C2H2. The inhibition is suggested to occur at the level of sulfite reductase, which catalyzes the oxidation of elemental sulfur to SO3(2-) in T. denitrificans. However, reduction of N2O in this strain was, surprisingly, not affected by C2H2. The isolate is the first chemolithoautotrophic organism shown to reduce N2O in the presence of C2H2. Denitrification in natural ecosystems is often quantified as N2O accumulation after C2H2 addition. However, the presence of large numbers of similar organisms with C2H2-insensitive N2O reduction could lead to underestimation of in situ rates.     

Development and application of a novel and effective screening method for aerobic denitrifying bacteria

Herein we describe a novel and effective screening method for aerobic denitrifying bacteria. For this procedure, we utilized KCN to inhibit the electron transference from Cytaa3 to oxygen in the bacteria respiratory chain. We employed a 3-h aeration operation cycle and intermittent rotations. The resultant bacterial suspensions were plated on a KCN-screening medium and incubated aerobically. Single colonies were selected and incubated in an aerobic culture medium. Culture nitrate and nitrite levels were determined over time, and ultimately four bacterial strains that performed denitrifying under aerobic conditions were identified by this method. Of these, strain Y2-1-1 demonstrated the best aerobic denitrifying ability. In a 5-day test, the NO3--N of the aerobic culture medium was reduced from 282.0+/-8.3 mg L(-1) to 149.2+/-17.1 mg L(-1), with little nitrite or N2O production. The morphological, physiological and biochemical characteristics and the 16S rRNA gene sequence homology comparison data for this strain were consistent with the classification of the genus Pseudomonas. We named this strain Pseudomonas sp. Y2-1-1.     

一株海洋好氧反硝化细菌的鉴定及其好氧反硝化特性

【目的】从处理海洋养殖循环水的生物滤器生物膜中分离到1株具有好氧反硝化活性的细菌(菌株2-8),并进一步研究了该菌的分类地位及反硝化特性。【方法】采用16S rRNA基因序列分析对菌株进行初步鉴定,采用好氧培养技术,探讨了碳源种类、起始pH、NaCl浓度、C/N、温度和摇床转速对菌株2-8好氧反硝化活性的影响。【结果】该菌株的16S rRNA基因序列与Pseudomonas segetis FR1439T(AY770691)的相似性最高,达到99.9%,因此初步鉴定菌株2-8属于假单胞菌属(Pseudomonas sp.2-8)。碳源类型和C/N对其好氧反硝化作用的影响最为显著,以柠檬酸钠为唯一碳源,C/N为15时脱氮效率最高,低C/N导致亚硝酸盐的积累;其好氧反硝化的最适温度和pH分别为30℃和7.5;菌株2-8在摇床转速为160r/min下脱氮效果最好;NaCl浓度对其反硝化活性的影响不明显。【结论】在初始硝酸氮浓度为140mg/L,以柠檬酸钠为唯一碳源、C/N为15、pH为7.5、NaCl浓度为30g/L,30℃以及160r/min摇床培养的条件下,菌株2-8在48h内脱氮率可达92%且无亚硝酸盐积累。     

Effect of acetylene on nitrous oxide reduction and sulfide oxidation in batch and gradient cultures of Thiobacillus denitrificans.

Anaerobic enrichment cultures with H2S and N2O as substrates which were inoculated with a biofilm sample showed rapid growth and gas formation after 2 to 3 days at 27 degrees C. By using the deep-agar dilution technique, a pure culture was obtained. The strain was tentatively identified as Thiobacillus denitrificans. The isolate was used for batch and gradient culture studies under denitrifying conditions, oxidizing H2S with concomitant reduction of N2O to N2. In batch culture, oxidation of H2S was stepwise, with transient accumulation of elemental sulfur; the final oxidation product was SO4(2-). In gradient culture, there was no notable accumulation of elemental sulfur and microsensor measurements of H2S and N2O showed that H2S was oxidized directly to SO4(2-). In the presence of C2H2, however, oxidation of H2S stopped at the level of elemental sulfur and no SO4(2-) was produced in either batch or gradient cultures. This is a hitherto unknown inhibitory effect of C2H2. The inhibition is suggested to occur at the level of sulfite reductase, which catalyzes the oxidation of elemental sulfur to SO3(2-) in T. denitrificans. However, reduction of N2O in this strain was, surprisingly, not affected by C2H2. The isolate is the first chemolithoautotrophic organism shown to reduce N2O in the presence of C2H2. Denitrification in natural ecosystems is often quantified as N2O accumulation after C2H2 addition. However, the presence of large numbers of similar organisms with C2H2-insensitive N2O reduction could lead to underestimation of in situ rates.     

Growth and denitrifying activity of Xanthobacter autotrophicus CECT 7064 in the presence of selected pesticides

The effects of the application of nine pesticides used commonly in agriculture (aldrin, lindane, dimetoate, methylparathion, methidation, atrazine, simazine, captan and diflubenzuron) on growth, CO₂ production, denitrifying activity [as nitrous oxide (N₂O) released] and nitrite accumulation in the culture medium by Xanthobacter autotrophicus strain CECT 7064 (Spanish Type Culture Collection) (a micro-organism isolated from a submerged fixed-film) were studied. The herbicide atrazine and the insecticide dimetoate totally inhibited growth and biological activity of X. autotrophicus at 10 mg l⁻¹, while the rest of the tested pesticides delayed the growth of strain CECT 7064 but did not drastically affect the bacterial growth after 96 h of culture. The denitrifying activity of X. autotrophicus was negatively affected by the pesticides application with the exception of fungicide captan. The release of N₂O was strongly inhibited by several pesticides (aldrin, lindane, methylparathion, methidation and diflubenzuron), while dimetoate, atrazine and simazine inhibited totally the denitrifying activity of the strain. The effects of the pesticides on denitrifying submerged fixed-film reactor are discussed.     

土壤中反硝化酶活性变化与N2O排放的关系

研究施肥条件下,土壤反硝化酶活性硝酸还原酶(NR)活性、亚硝酸还原酶(NiR)活性及羟胺还原酶(HyR)活性在玉米生长季节中的变化及其与土壤含水量、硝态氮含量、N₂O排放之间的关系。结果表明,3种还原酶都有明显的季节变化规律并受土壤水分含量及施肥的影响。通过研究3种反硝化酶活性与土壤含水量及N₂O排放量之间的关系后指出,反硝化酶活性变化可作为一个区分旱田N₂O产生途径的指标.     

Loss of N2O reductase activity as an explanation for poor growth of Pseudomonas aeruginosa on N2O.

N2O uptake activity of cells and N2O reductase activity of the soluble fraction from denitrifying bacteria were assayed. Pseudomonas aeruginosa strains PAO1 and P1 lost most of their N2O uptake activity and the ability to grow well on N2O within 2 to 5 h after exposure to N2O. Extensive loss of N2O reductase activity accompanied the nearly complete loss of N2O uptake activity under N2O. Paracoccus denitrificans retained much, but not all, of both activities and the ability to grow vigorously on N2O. The pattern with P. aeruginosa strain P2 resembled that for PAO1 and P1 except that loss of the activities proceeded at a slower rate and growth could continue for up to 12 h after exposure to N2O. The inability of a number of P. aeruginosa strains to grow well on N2O is therefore a direct consequence of the nearly complete loss of N2O reductase activity. Turnover-dependent inactivation of N2O reductase and its reactivation under reducing conditions occurred in vitro for the enzyme from P. aeruginosa and Paracoccus denitrificans. These events may be significant in determining the activity level of N2O reductase in denitrifying bacteria during N2O respiration.     

Diaphorobacter nitroreducens gen nov,sp nov,a poly(3-hydroxybutyrate)-degrading denitrifying bacterium isolated from activated sludge

Three denitrifying strains of bacteria capable of degrading poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were isolated from activated sludge and characterized. All of the isolates had almost identical phenotypic characteristics. They were motile gram-negative rods with single polar flagella and grew well with simple organic compounds, as well as with PHB and PHBV, as carbon and energy sources under both aerobic and anaerobic denitrifying conditions. However, none of the sugars tested supported their growth. The cellular fatty acid profiles showed the presence of C16:1omega7cis and C16:0 as the major components and of 3-OH-C10:0 as the sole component of hydroxy fatty acids. Ubiquinone-8 was detected as the major respiratory quinone. A 16S rDNA sequence-based phylogenetic analysis showed that all the isolates belonged to the family Comamonadaceae, a major group of beta-Proteobacteria, but formed no monophyletic cluster with any previously known species of this family. The closest relative to our strains was an unidentified bacterium strain LW1 (=DSM 13225) (99.9% similarity), reported previously as a 1-chloro-4-nitrobenzene degrading bacterium. DNA-DNA hybridization levels among the new isolates were more than 60%, whereas those between our isolates and strain DSM 13225 were less than 50%. The G+C content of genomic DNA of the new strains was 64 to 65 mol%. Based on these results, we concluded that the PHBV-degrading denitrifying isolates should be classified as a new genus and a new species, for which we propose the name Diaphorobacter nitroreducens. The type strain is strain NA10B (=JCM 11421=CIP 107294). We also propose to classify strain DSM 13225 as a genospecies of Diaphorobacter.     

Nitrate removal by a novel autotrophic denitrifier (Microbacterium sp.) using Fe(II) as electron donor

A novel denitrifying bacterium was isolated using bicarbonate as the sole carbon source in a defined medium. Strain W3 was isolated from deep sediments of East Lake (Wuhan, China). In this study, analysis of 16S rRNA genes showed that strain W3 was affiliated with Microbacterium sp. When using Fe2+ as the only electron donor, this strain could convert 88.6 % of NO3 −-N to N2, corresponding to an Fe2+ oxidation rate of 80 %. Meanwhile, neither NO2 −-N nor NH4 +-N was accumulated after the experiment. In similar experiments with Fe(II)-EDTA, cell encrustations did not occur and supplementary substrates were consumed. The accumulated NO2 −-N was below 2.5 mg L−1. In addition, PCR revealed five kinds of key denitrifying genes: narG, napA, nirS, norB and nosZ. These results indicated that strain W3 could be used as an alternative autotrophic denitrifier for the treatment of groundwater and low C/N ratio wastewater.