JP2009220007A - Compound for controlling growth of bacteria and application thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus and a method for treating wastewater by finding a factor, which directly affects denitrification, and utilizing the factor. <P>SOLUTION: A trivalent iron ion is a factor that promotes the growth of denitrifying bacteria and directly affects denitrification. An apparatus 100 for treating wastewater is characterized by comprising a denitrification tank 10 that reduces nitric acid ions and/or nitrous acid ions with denitrifying bacteria to nitrous oxide and/or nitrogen, and a growth promotor supply means 20 for supplying a denitrifying bacteria growth promotor comprising trivalent iron ions into the denitrification tank. The denitrifying bacteria activated by the trivalent iron ions can efficiently reduce nitric acid ions and/or nitrous acid ions to nitrous oxide and/or nitrogen. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to compounds that control bacterial growth and applications thereof. More specifically, the present invention relates to a growth promoting agent for denitrifying bacteria, a wastewater treatment apparatus and a wastewater treatment method using the same. Furthermore, the present invention relates to an antibacterial agent.

Certain bacteria reduce nitrate ions (NO ₃ ⁻ ) and nitrite ions (NO ₂ ⁻ ) to release nitrogen (N ₂ ) and nitrous oxide (N ₂ O), and nitrate respiration that synthesizes ATP. I do. Such bacteria are called denitrifying bacteria, and this process is called denitrifying. More specifically, nitrate ions are reduced to nitrogen through a route of NO ₃ ⁻ → NO ₂ ⁻ → NO → N ₂ O → N ₂ , and nitrate reductase (NAR) and nitrite reductase (NIR) are respectively used in each stage. ), Nitric oxide reductase (NOR) and nitrous oxide reductase (NOS) are involved. A typical denitrifying bacterium is Pseudomonas aeruginosa.

  Denitrification of denitrifying bacteria has important significance in the field of environmental engineering, and a denitrifying method using denitrifying bacteria has been developed and applied to wastewater treatment (Patent Document 1, etc.). Various devices have been devised for the purpose of improving the treatment efficiency of denitrification, and methods for blowing hydrogen gas into the denitrification tank (Patent Document 1, etc.) and methods for controlling the pH of the denitrification tank (Patent Document 2, etc.) have been studied. ing.

  Pseudomonas aeruginosa, on the other hand, is less likely to infect healthy individuals due to its low pathogenicity, but it is often the cause of opportunistic infections by infecting people with reduced immunity due to aging, disease, drug administration . Since Pseudomonas aeruginosa has a strong resistance to antibacterial agents, multidrug-resistant Pseudomonas aeruginosa appears, making it difficult to treat Pseudomonas aeruginosa infections. Therefore, various antibacterial agents against opportunistic pathogens such as Pseudomonas aeruginosa have been developed (Patent Document 3 etc.).

Japanese Patent Laid-Open No. 05-317881 JP 2000-285883 A JP 2006-273796 A JP 2003-1292 A

  The existing method for improving the denitrification treatment efficiency is merely to optimize the growth environment of the denitrification bacteria and does not directly affect the denitrification of the denitrification bacteria. If denitrification can be directly affected, it is thought that the efficiency of denitrification can be dramatically improved. Therefore, an object of the present invention is to find a factor that directly affects denitrification, and to provide a wastewater treatment apparatus and a wastewater treatment method using the factor.

  In addition, as long as an antibacterial agent is used, resistant bacteria to it always appear, so it does not lead to a fundamental solution. However, it is considered that resistant bacteria are unlikely to occur if the antibacterial agent can directly affect denitrification (ie, nitrate respiration). Therefore, an object of the present invention is to find a factor that directly affects denitrification, and to provide an antibacterial agent using the factor.

  As a result of diligent studies, the present inventors have clarified that 2-heptyl-3-hydroxy-4-quinolone (also referred to as PQS) is also capable of suppressing denitrification. It was also clarified that the denitrification inhibitory action of 2-heptyl-3-hydroxy-4-quinolone was due to chelating activity, and that other chelating agents also suppressed denitrification. Furthermore, it was clarified that the addition of trivalent iron ions promotes denitrification. Based on these findings, the inventors have completed the present invention.

  The growth promoting agent for denitrifying bacteria of the present invention is characterized by comprising trivalent iron ions. The growth promoting agent for denitrifying bacteria of the present invention can improve the denitrifying activity of the denitrifying bacteria and grow the denitrifying bacteria. Therefore, the treatment efficiency of denitrification can be improved, and it can be used for a wastewater treatment method and a wastewater treatment apparatus. Note that Patent Document 4 discloses a denitrification method using divalent iron ions as a growth promoter. However, nothing is mentioned regarding trivalent iron ions, and the growth promoting effect when divalent iron ions are used alone is insignificant (Example 12 of Patent Document 4).

  The first waste water treatment apparatus of the present invention is a waste water treatment apparatus for denitrifying waste water containing nitrate ions and / or nitrite ions, and the nitrate ions and / or nitrite ions in the introduced waste water are removed by denitrifying bacteria. The denitrification tank which carries out reduction treatment to nitrous oxide and / or nitrogen, and the proliferation promoter supply means which supplies the proliferation promoter of the denitrifying bacteria which consists of a trivalent iron ion in the said denitrification tank are provided. In the first wastewater treatment method of the present invention, wastewater containing nitrate ions and / or nitrite ions is treated with nitrous oxide and / or nitrite ions and / or nitrite ions by denitrifying bacteria in a denitrification tank. In the wastewater treatment method of treating by reducing to nitrogen, the denitrifying bacteria are grown in the denitrifying tank in the presence of a growth promoting agent for denitrifying bacteria comprising trivalent iron ions. The waste water treatment apparatus and waste water treatment method of the present invention utilize the growth promoting agent for denitrifying bacteria of the present invention, and can efficiently perform denitrification.

The second wastewater treatment apparatus of the present invention is a wastewater treatment apparatus that denitrifies wastewater containing nitrate ions and / or nitrite ions, and the nitrate ions and / or nitrite ions in the introduced wastewater are removed by denitrifying bacteria. A denitrification tank that performs reduction treatment to nitrous oxide and / or nitrogen, and a trivalent iron ion chelating agent supply unit that supplies a trivalent iron ion chelating agent to the denitrification tank are provided. In the second wastewater treatment method of the present invention, wastewater containing nitrate ions and / or nitrite ions is treated with nitrite and / or nitrite ions and / or nitrite ions by denitrifying bacteria in a denitrification tank. In the wastewater treatment method for treating by reducing to nitrogen, the denitrifying bacteria are grown in the denitrifying tank in the presence of a chelating agent of trivalent iron ions. The trivalent iron ion chelating agent (particularly 2,2′-bipyridyl) has the effect of reducing the N ₂ O production amount without reducing the N ₂ production amount in the denitrification of the denitrifying bacteria. N ₂ O has a greenhouse effect that is approximately 300 times that of CO ₂ and is subject to emission regulations under the Kyoto Protocol. From the viewpoint of preventing global warming, it is very important to reduce N ₂ O emissions in wastewater treatment.

  The antibacterial agent of the present invention is characterized by comprising a chelating agent of trivalent iron ions. The antibacterial agent of the present invention can suppress denitrification of denitrifying bacteria and inhibit the growth of denitrifying bacteria.

The growth promoting agent for denitrifying bacteria of the present invention can improve the denitrifying activity of the denitrifying bacteria and allow the denitrifying bacteria to grow, and can be used in a wastewater treatment apparatus and a wastewater treatment method. The first waste water treatment apparatus and waste water treatment method of the present invention can efficiently perform denitrification by using the growth promoting agent for denitrifying bacteria of the present invention. The second waste water treatment apparatus and waste water treatment method of the present invention can reduce the amount of N ₂ O discharged along with waste water treatment.

  In addition, the antibacterial agent of the present invention can suppress denitrification of denitrifying bacteria and inhibit the growth of denitrifying bacteria.

  DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings.

  The growth promoting agent for denitrifying bacteria of the present invention is characterized by comprising trivalent iron ions. Trivalent iron ions function as denitrification promoters for denitrifying bacteria. When denitrifying bacteria are cultured in the presence of the growth promoting agent for denitrifying bacteria of the present invention, the denitrifying activity of the denitrifying bacteria is improved, and the denitrifying bacteria efficiently denitrify and grow. When denitrifying bacteria are cultured in a complex system, denitrifying bacteria are in a state in which denitrification and growth are suppressed by PQS produced by the denitrifying bacteria themselves. However, when denitrifying bacteria are cultured in the presence of trivalent iron ions, denitrification and growth suppressed by PQS are recovered, and the denitrifying ability and growth ability inherent to denitrifying bacteria are maximized. It becomes possible. In order to exert an appropriate growth promoting activity, the concentration of the denitrifying bacteria growth promoter is preferably 5 μM to 500 μM, and more preferably 10 μM to 200 μM.

  As the trivalent iron ion, a salt of trivalent iron ion can be used, and examples thereof include iron chloride (III) and iron sulfate (III).

  FIG. 10 is a schematic view showing an embodiment of the first waste water treatment apparatus of the present invention. As shown in FIG. 10, the wastewater treatment apparatus 100 includes a denitrification tank 10 in which denitrifying bacteria are accommodated, and a growth promoter supply means 20 that supplies trivalent iron ions to the denitrification tank. The denitrification tank 10 has a drain line L1 containing nitrate ions and / or nitrite ions, a line L2 for supplying trivalent iron ions from the growth promoter supply means 20 into the denitrification tank 10, and a denitrification reaction. A line L3 for discharging the treated water containing nitrous oxide and / or nitrogen to the outside of the denitrification tank 10 is connected. The denitrification tank 10 is kept under anaerobic conditions.

  Next, a wastewater treatment method using the wastewater treatment apparatus 100 having the above-described configuration will be described.

  First, the waste water to be treated is introduced into the denitrification tank 10 through the line L1 and mixed with the denitrifying bacteria in the denitrification tank 10. Next, trivalent iron ions are supplied into the denitrification tank 10 from the growth promoter supply means 20 via the line L2, and mixed with the denitrifying bacteria in the denitrification tank 10. By supplying trivalent iron ions, the denitrifying bacteria in the denitrifying tank 10 are activated, and nitrate ions and / or nitrite ions contained in the waste water to be treated supplied from the line L1 are nitrous oxide and / or Efficiently reduced to nitrogen. The treated water after the denitrification reaction containing nitrous oxide and / or nitrogen is discharged to the outside of the denitrification tank 10 through the line L3.

  FIG. 11 is a schematic view showing an embodiment of the second waste water treatment apparatus of the present invention. As shown in FIG. 11, the wastewater treatment apparatus 200 includes a denitrification tank 30 in which denitrifying bacteria are accommodated, and a trivalent iron ion chelating agent supply unit 40 that supplies a trivalent iron ion chelating agent to the denitrification tank. And. The denitrification tank 30 is supplied with a trivalent iron ion chelating agent into the denitrification tank 30 from a drain line L4 containing nitrate ions and / or nitrite ions and a trivalent iron ion chelating agent supply means 40. A line L5 for discharging the treated water containing nitrous oxide and / or nitrogen after the denitrification reaction to the outside of the denitrification tank 30 is connected. The denitrification tank 30 is kept under anaerobic conditions.

  Next, a wastewater treatment method using the wastewater treatment apparatus 200 having the above-described configuration will be described.

First, the waste water to be treated is introduced into the denitrification tank 30 through the line L4 and mixed with the denitrifying bacteria in the denitrification tank 30. Next, the trivalent iron ion chelating agent is supplied into the denitrification tank 30 from the trivalent iron ion chelating agent supply means 40 via the line L5 and mixed with the denitrifying bacteria in the denitrification tank 30. By supplying the chelating agent in trivalent iron ions, denitrifying bacteria denitrification tank 30 can reduce the production of N _{2 O} with little reduction in the production of N _2. The nitrate ions and / or nitrite ions contained in the waste water to be treated supplied from the line L4 are reduced to nitrous oxide and / or nitrogen, but compared with the case where no chelating agent of trivalent iron ions is supplied. The rate of reduction to nitrogen increases. The treated water after the denitrification reaction containing nitrous oxide and / or nitrogen is discharged to the outside of the denitrification tank 30 through the line L6.

  Next, the antibacterial agent of the present invention will be described. The antibacterial agent of the present invention comprises a chelating agent of trivalent iron ions. If a chelating agent of trivalent iron ions is present, the denitrification reaction of denitrifying bacteria, that is, nitrate respiration is suppressed, leading to suppression of ATP synthesis and suppression of denitrifying bacteria growth. Trivalent iron ion chelating agents also suppress the growth of bacteria other than denitrifying bacteria. The antibacterial agent of the present invention is considered to be particularly effective in suppressing bacterial growth at an early stage of infection. Moreover, it is thought that the proliferation of bacteria can be effectively suppressed by combining the antibacterial agent of the present invention and the existing antibacterial agent.

  Known compounds can be used as chelating agents for trivalent iron ions, such as 2-heptyl-3-hydroxy-4-quinolone, 2,2'-bipyridyl, deferoxamine and its salts, deferasirox, ethylenediamine, ethylenediamine Examples include tetraacetic acid and its salt, 1,10-phenanthroline, porphyrin and the like, and 2-heptyl-3-hydroxy-4-quinolone, 2,2′-bipyridyl, deferoxamine and its salt and deferasirox are particularly preferable.

  2-Heptyl-3-hydroxy-4-quinolone has an excellent property of high specificity. That is, 2-heptyl-3-hydroxy-4-quinolone can specifically inhibit the denitrification reaction of denitrifying bacteria and is difficult to function as a chelating agent for trivalent iron ions in other situations. In addition, the growth of bacteria can be suppressed and side effects are unlikely to occur.

  Deferoxamine (N '-[5- (acetyl-hydroxy-amino) pentyl] -N- [5- [3- (5-aminopentyl-hydroxy-carbamoyl) propanoylamino] pentyl] -N-hydroxy-butanedivalent) Combines with iron ions to form ferrioxamine, a stable water-soluble chelate compound. Clinically, deferoxamine mesylate is used as a treatment for iron overload. The dosage and dosage of deferoxamine mesylate for iron overload is as follows: deferoxamine mesylate is injected intramuscularly in daily doses of 500 mg to 1000 mg, or up to a daily dose of 80 mg / kg. Intravenously infuse at a rate of 15 mg / kg per hour. When deferoxamine and its salt are used as an antibacterial agent, the usage and dosage can be determined with reference to this.

  Deferasirox ([4-[(3Z, 5E) -3,5-bis (6-oxo-1-cyclohexa-2,4-diylenedene) -1,2,4-triazolidin-1-yl] benzoic acid) Is also used clinically as a treatment for iron overload. It has been shown that oral administration of 20-30 mg / kg once a day shows a clinically effective iron chelating effect in pediatric patients. When deferasirox is used as an antibacterial agent, the usage and dose can be determined with reference to this.

  The antibacterial agent of the present invention can be formulated into various preparations by adding pharmaceutically acceptable additives according to the administration form. As the additive, various additives commonly used in the pharmaceutical field can be used, for example, gelatin, lactose, sucrose, titanium oxide, starch, crystalline cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, corn starch, microcrystalline wax, White petrolatum, magnesium aluminate metasilicate, anhydrous calcium phosphate, citric acid, trisodium citrate, hydroxypropylcellulose, sorbitol, sorbitan fatty acid ester, polysorbate, sucrose fatty acid ester, polyoxyethylene, hydrogenated castor oil, polyvinylpyrrolidone, stearic acid Magnesium, light silicic anhydride, talc, vegetable oil, benzyl alcohol, gum arabic, propylene glycol, polyalkylene glycol, B dextrin, hydroxypropyl cyclodextrin, and the like.

  Examples of the dosage form formulated as a mixture with these additives include solid preparations such as tablets, capsules, granules, powders or suppositories; liquid preparations such as syrups, elixirs or injections, etc. It is done. These can be prepared according to a usual method in the pharmaceutical field. The liquid preparation may be dissolved or suspended in water or other appropriate medium at the time of use. In particular, in the case of injections, they may be dissolved or suspended in physiological saline or glucose solution as necessary, and buffering agents and preservatives may be added.

  (Bacterial strain, plasmid and growth conditions) Escherichia coli strains JM109 and DH5α for light conversion were purchased from Takara Bio. E. coli S17-1 has been described by Simon et al., Methods Enzymol. 118: 640-659 (1986). Cloning vector pHSG398 (chloramphenicol resistance) was purchased from Takara Bio. pK19mobsac-derived suicide vector pG19II (gentamicin resistance) is described in Maseda et al., Antimicrob. Agents Chemother. 48: 1320-1328 (2004). The broad host range vector pUCP24 (gentamicin resistance) was obtained by the method described in West et al., Gene, 148: 81-86 (1994). pMEX9, pMEXnarK, pMEXnirS, pMEXnorC and pMEXnosR are described in Toyofuku et al. Bacteriol. 189: 4969-72 (2007).

  The primers used are as shown in the table below.

Bacterial strains were grown at 37 ° C. on Luria-Bertani (LB) medium or LB agar medium. Gentamicin at a concentration of 10 μg / ml was added to E. coli, and gentamicin at a concentration of 80 μg / ml was added to P. aeruginosa as needed. Prior to the test culture, Pseudomonas aeruginosa is anaerobically grown in a 24 ml test tube containing 4 ml of LB medium and inoculated into the anaerobic medium so that the absorbance at 600 nm (OD ₆₀₀ ) at the start is 0.01. did. For anaerobic culture, the air in the Hangate test tube or Erlenmeyer flask capped with butyl rubber was replaced with argon. Growth, denitrification activity and transcriptional activity were measured using 17 ml Hangate tubes containing 5 ml LBN medium (LB medium supplemented with 100 mM KNO ₃ ), incubated at 37 ° C. and shaken at 200 rpm. Cells used to measure denitrifying enzyme activity were cultured at 37 ° C. and 200 rpm in a 500 ml Erlenmeyer flask containing 80 ml of LBN.

  (Construction of Pseudomonas aeruginosa mutants) Plasmids pG19pqsA, pG19pqsR and pG19pqsE carrying deletion cassettes of pqsA, pqsR and pqsE were obtained from Maseda et al., Antimicrob. Agents Chemother. 48: 1320-1328 (2004). The PAO1 chromosome was amplified with pqsAF1 / pqsAR2, pqsRF1 / pqsRR2 and pqsEF1 / pqsER2 primer pairs and ligated to the multiple cloning site of the cloning vector pHSG398 treated with XbaI / HindIII. The adjacent DNA fragments of pqsA, pqsR and pqsE of pHSG398 were amplified by inverse PCR using pqsAF2 / pqsAR1, pqsRF2 / pqsRR1 and pqsEF2 / pqsER1 primer pairs. The amplified adjacent fragment of pqsA was digested with SpeI, and the amplified adjacent fragment of pqsR and prsE was digested with BglII. These restriction enzyme sites are added to the primer. The pqsA, pqsR and pqsE deleted DNA fragments were subcloned into the XbaI-HindIII site of the multicloning site of pG19II to prepare pG19pqsA, pG19pqsR and pG19pqsE. Conjugated to E. coli S17-1 (Simon et al., Methods Enzymol., 118: 640-659 (1986)), Maseda et al., Antimicrob. Agents Chemother. 48: 1203-1328 (2004), homologous recombination was performed, and a plasmid derived from pG19II was introduced into PAO1. Mutants were analyzed by PCR.

  (Preparation of cell fraction) Cells grown in LBN medium were collected in the middle logarithmic growth phase (incubation time of 6 hours), centrifuged at 7000 g at 4 ° C, and 100 mM potassium phosphate buffer containing 10% glycerol. And washed twice at pH 7.5. Cells were suspended in the same buffer and sonicated. The sonicated cells were centrifuged at 2000 g, 4 ° C. for 10 minutes to remove unbroken cells, and centrifuged at 104000 g, 4 ° C. for 60 minutes. The supernatant was collected as a cell soluble fraction, and the pellet was resuspended in the same buffer and collected as a membrane fraction.

(Enzyme activity measurement) NAR activity was measured by quantifying the product NO ₂ ^- by colorimetry (Nicholas et al., Methods Enzymol., 3: 981-984 (1957)). Benzylviologen-dithionate-related NAR activity has been reported using Kobayashi et al., J. Biol., Benzylviologen instead of methylviologen. Biol. Chem. , 271: 16263-16267 (1996). 100 μl Na ₂ S in 400 μl of 100 mM potassium phosphate buffer (pH 7.5) containing 10 mM NaNO ₃ , 0.2 mM benzyl viologen and an appropriate concentration of anaerobically cultured Pseudomonas aeruginosa cell membrane fraction ₂ O ₄ was added to a final concentration of 10 mM. The reaction air was replaced with N ₂ before Na ₂ S ₂ O ₄ was added. The reaction solution was incubated at 30 ° C. for 5 minutes and stirred until the color of the reaction solution became transparent, which is an indicator of methyl viologen oxidation. NAR activity associated with the respiratory chain was measured using NADH as an electron donor (Radcliffe et al., Biochim. Biophys. Acta, 205: 273-287 (1970)). A 500 μl reaction solution for measuring NADH-derived NAR activity contains 10 mM NaNO ₃ and an anaerobically cultured Pseudomonas aeruginosa membrane fraction. The air in the reaction mixture was replaced with N _2. The reaction was started by adding NADH at 30 ° C. so that the final concentration was 5 mM, and the reaction was stopped by boiling at 100 ° C. for 15 minutes.

NIR activity was assayed by measuring NO ₂ ⁻ consumption (Lam et al., Biochim. Biophys. Acta, 180: 459-472 (1969)). By adding 100 μl of NADH to a 400 μl reaction solution containing 200 μM NaNO ₂ , 200 μM phenazine methosulfate (PMS) and anaerobically cultured soluble fraction of Pseudomonas aeruginosa to a final concentration of 1 mM. The reaction was started. Before adding NADH, the air in the reaction solution was replaced with N ₂ and reacted at 30 ° C. for 10 minutes.

NOR activity was assayed by measuring its product N ₂ O by gas chromatography. 100 μl of 20 mM NO donor, nitroprusside sodium, was added to the reaction solution containing 4 mM NADH, 2 mM PMS with air replaced with N ₂ and anaerobically cultured Pseudomonas aeruginosa membrane fraction. The reaction was performed at 30 ° C. for 6 minutes.

Catechol-2,3-dioxygenase specific activity was assayed by measuring absorbance A ₃₇₅ at ₃₇₅ nm (Maseda et al., Antimicrob. Agents Chemother., 48: 1320-1328 (2004)) and Sawada et al., Microbiol. Immunol. 48: 435-439 (2004)).

(Analytical method) Growth of bacteria in liquid culture was monitored by absorbance at 600 nm. The amount of protein was quantified by the Bradford method (Bradford, Anal. Biochem., 72: 248-254 (1976)). The concentration of NO ₃ ⁻ was measured by the brucine method (Nicholas, Methods Enzymol., 3: 981-984 (1957)). The concentrations of N ₂ O and N ₂ were measured by gas chromatography (GC-8AIT; Shimadzu Corporation) equipped with a heat conduction detector. Helium was used as the carrier gas. A Shincarbon ST or Molecular Sieve 5A column was used for detection of N ₂ O or N ₂ , respectively.

(Pyocyanin assay) Pyocyanin was measured according to the method of Essar et al. (Essar et al., J. Bacteriol., 172: 884-900 (1990)). The cells were stoppered with LB or LBN medium (2 ml medium in a 16 ml Hangate tube), with silicone rubber for aerobic culture and with butyl rubber for anaerobic culture and incubated for 12 hours. 1 ml of medium was collected in a 2 ml test tube and centrifuged. The supernatant was transferred to a new tube and 750 μl of chloroform was added. The sample was agitated and centrifuged, and 500 μl of the lower layer was transferred to a new tube. 100 μl of 0.2N HCl was added to the test tube, and the absorbance at 520 nm of the upper layer was measured. The number of A ₅₂₀ was divided by the OD ₆₀₀ of the medium.

(Test Example 1) As described above, denitrification is a step of reducing NO ₃ ^- to N ₂ O or N ₂ and is also an important step for acquiring energy under physiological anaerobic conditions. In order to investigate the effect of PQS on denitrification, ΔpqsA mutant that does not produce PQS and related 2-alkyl-4-quinolones (AHQs) (PAO1 mutant with deletion in pqsA gene) was anaerobically treated in LBS medium. And exogenous PQS was added to a final concentration of 50 μM. When PQS was added, the turbidity at 600 nm decreased to 50% or less of the middle logarithmic growth phase, suggesting that denitrification was suppressed by PQS (see FIG. 1A). In order to investigate in more detail, the decrease amount of NO ₃ ⁻ and the production amounts of N ₂ O and N _{2 after} incubation for 12 hours were measured. Along with the suppression of growth due to the addition of PQS, the amount of NO ₃ ⁻ decreased by 59% (see FIG. 1B), the amount of N ₂ O produced decreased by 51% (see FIG. 1C), and the amount of N ₂ produced was 48 % (See FIG. 1D). These data indicate that PQS suppresses P. aeruginosa denitrification.

  (Test Example 2) In order to investigate which step of denitrification is suppressed by PQS, the influence of PQS on the transcription of denitrification enzyme involved in each step was examined. Promoter fusion plasmids in which the xylE gene was fused to the promoter regions of narK1, irS, norC and noSR denitrification genes were each transferred to a ΔpqsA mutant. As a control, the base catechol-2,3-dioxygenase (C23O) activity in the ΔpqsA mutant lightly transformed with the pMEX9 plasmid was examined with and without the addition of PQS. C23O activity was measured in the mid-log growth phase (6 hour incubation). Promoter-dependent C23O activity was determined by dividing the C23O activity by the base C23O activity. When PQS was added to the medium so that the final concentration was 50 μM, no effect was observed on any promoter activity (see FIG. 2). This result indicates that the denitrification control by PQS is not based on transcription control but based on other post-transfer mechanisms.

  (Test Example 3) In order to examine whether or not denitrification is controlled at the level after transcription, the denitrification enzyme activity in the middle logarithmic growth phase was measured. ΔpqsA mutants were cultured under conditions with or without the addition of PQS (final concentration 50 μM), and NAR, NIR, and NOR denitrase activities were measured. As a result, NAR activity was suppressed when PQS was added and cultured (66% compared to the activity when cultured without PQS; see FIG. 3A). Interestingly, NIR activity increased 1.8-fold with the addition of PQS (see FIG. 3B). NOR activity decreased to 64% with the addition of PQS (see FIG. 3C). Changes in the activity of these denitrification enzymes may be due to post-transcriptional control or direct interaction with PQS. In order to examine whether PQS directly affects denitrifying enzyme activity, PQS was added to the enzyme assay reaction solution of Δpqs mutant, and the enzyme activity was measured. As shown in FIG. 3C, NOR activity is inhibited by adding PQS to the reaction solution, which indicates that PQS directly inhibits NOR activity. On the other hand, NAR and NIR activities were not directly affected even when PQS was added to the reaction solution (FIGS. 3A and B). Combined with the result that transcription of the denitrification gene is not regulated by PQS, this result indicates that the activity of NAR and NIR is post-transcriptionally regulated by PQS, and that NOR activity is directly inhibited by PQS. ing.

(Test Example 4) The denitrification enzyme is related to the respiratory chain from which energy is produced. NO ₃ ^- during respiration, electrons donated from NADH is transmitting respiratory chain, eventually NO ₃ ^- receives, this last emperor are mediated by NAR. In NAR activity assay (Fig. 3A), the electron donor to donate electrons directly to the NAR (benzyl viologen) is used, NO ₃ ^- activity of the respiratory chain is not in the calculation. NO ₃ ^- respiratory chain To investigate whether affected by PQS, was measured NADH derived NAR activity. When NADH was used as an electron donor, NADH-NAR activity was suppressed by the addition of PQS to the medium, and was also directly inhibited when PQS was added to the reaction solution (final concentration 50 μM) (FIG. 3D). This result, NO ₃ ^- indicates that the breath is directly inhibited by PQS. NADH-derived NAR activity is directly inhibited by PQS (FIG. 3D), whereas benzylviologen-derived NAR activity is not inhibited (FIG. 3A), indicating that PQS is responsible for either electron transfer from NADH to NO ₃ ⁻ Indicates direct inhibition.

(Test Example 5) PQS has been reported not only to regulate transcription in Pseudomonas aeruginosa but also to chelate iron. Therefore, it was considered that the chelating effect of PQS on iron has an effect on the denitrification enzyme. To test this hypothesis, the medium supplemented with PQS (final concentration 50 μM) was supplemented with FeCl ₃ (final concentration 100 μM). As a result, the addition of FeCl ₃ completely restored the NO ₃ ⁻ decrease and the N ₂ O production in the cultures added with PQS (FIGS. 4A and B). This indicates that the denitrification control of PQS is due to iron chelation. In order to confirm that iron chelation affects denitrification, 2,2′-bipyridyl, an iron chelator, was added to the medium (final concentration 120 μM), and denitrification activity was measured. NO ₃ ^- reduction and _{N 2} O production amount is suppressed as in the case of PQS, restored by supplementation of FeCl ₃ (FIGS. 4A and B). Interestingly, N ₂ production when PQS was added was only partially recovered by FeCl ₃ and 2,2′-bipyridyl did not reduce N ₂ production (FIG. 4C). These results suggest that NOS-catalyzed N ₂ O to N ₂ reduction is regulated by other mechanisms other than iron chelation of PQS.

(Test Example 6) Many of the genes controlled by PQS require a cognate response control factor PqsR or PqsE whose function is unknown but is thought to promote the response to PQS. To investigate whether the reduction of N ₂ O to N ₂ is regulated by this regulatory pathway, ΔpqsAΔpqsR (PAO1 mutant with deletion in pqsA and pqsR genes) and ΔpqsAΔpqsE (deletion in pqsA and pqsE genes) Some PAO1 mutants) double mutants were constructed. PQS (final concentration 50 μM) was added to the mutants and the amount of NO ₃ ⁻ decreased and the amount of N ₂ O and N ₂ produced after 12 hours of incubation was measured. The decrease in NO ₃ ⁻ and production of N ₂ O in ΔpqsAΔpqsR and ΔpqsAΔpqsE were the same as in ΔpqsA (FIGS. 5A-B). As shown in FIG. 5C, _{N 2} production inhibition by PQS, not observed in ΔpqsAΔpqsR and any double mutant of DerutapqsAderutapqsE, whereas, PQS inhibited _{N 2} production in DerutapqsA (Figure 1D). This result indicates that N ₂ production is regulated by PQS and PqsR transcriptional regulators through PqsE. Since PQS does not regulate transcription of nos (FIG. 2), this regulation may be involved in transcription regulation of other factors involved in N ₂ production other than nos. Reduction of NO ₃ ⁻ to N ₂ O is inhibited by PQS even in the absence of PqsE or PqsR (FIGS. 5A and B), as it was also observed with ΔpqsA (FIGS. 1B and C), NO ₃ ^- reduction to _{N 2} O from supports results (Fig. 4A-C) that is regulated by iron chelation of PQS. In order to confirm whether the results obtained with the ΔpqsAΔpqsR and ΔpqsAΔpqsE strains are due to the polar effect, a complementation experiment in which each mutant is complemented with an appropriate wild type copy (ΔpqsAΔpqsR / pUCP-pqsR, ΔpqsAΔpqsE / pUCP-pqsE) went. Surprisingly, NO ₃ ^- reduction and _{N 2} O and _{N 2} production amount when the pqsR and pqsE were expressed plasmid, even in the absence of PQS, was strongly suppressed. Taken together with the results of the iron chelation experiments (FIGS. 4A-C), these data indicate that PQS is responsible for the production of N ₂ by a pathway that mediates PqsR and PqsE and differs from the iron chelation properties of PQS. It shows that it is adjusting.

(Test Example 7) Under aerobic conditions, PQS is reported to prolong the lag phase at a high concentration (150 μM or more) and to slightly reduce the turbidity in the stationary phase compared to the growth in LB medium alone. Has been. On the other hand, under anaerobic conditions, PQS reduces proliferation to 50%, indicating that the influence of PQS on proliferation is significant under anaerobic conditions. Recent studies (Diggle et al., Chem. Biol., 14: 87-96 (2007)) have shown that the iron chelation properties of PQS affect aerobic growth of Pseudomonas aeruginosa. It has a strong effect in pvdDpchEF double mutants that cannot produce iron-chelating siderophores (pyocyanin and pyokelin). This result indicates that siderophore production is involved in the suppression of PQS growth, and it was speculated that the iron chelation effect of PQS on denitrification may be due to reduced production of siderophore under anaerobic conditions. In order to investigate this, the pyocyanin production of the cultures in LB aerobic medium, LBN aerobic medium and LBN anaerobic medium was measured. As expected, pyocyanin production was reduced in LBN anaerobic culture (FIG. 6). Interestingly, pyocyanin production, NO ₃ ^- has decreased even in aerobic culture with added, pyocyanin production NO ₃ ^- suggests that it is regulated by both and O _2. These results suggest that denitrification is inhibited by PQS under aerobic conditions and anaerobic conditions in the presence of NO ₃ ⁻ due to insufficient production of siderophores. NO ₃ ^- reduced pyocyanin production and iron chelation dependence of de窒調clause of absence, NO ₃ ^- N-oxides and iron or the like, whether important environmental growth by PQS is affected Suggests that it is a factor.

(Test Example 8) It was examined whether PQS had an effect on bacterial growth. Pseudomonas putida, Delftia acidovorans, Pseudomonas stutzeri and Comonas terigena were grown aerobically in LB medium. When PQS and FeCl ₃ were added, they were added so as to have final concentrations of 50 μM and 100 μM, respectively. As is clear from FIG. 8, the growth of any bacterium was suppressed by the addition of PQS, and the growth was recovered by the addition of FeCl ₃ . It was revealed that PQS suppressed bacterial growth, and trivalent iron recovered the growth suppression by PQS. Also, when Pseudomonas putida was grown anaerobically on LBN medium, growth was suppressed by the addition of PQS, and growth was restored by the addition of FeCl ₃ . From this result, it is expected that PQS suppresses the growth of many denitrifying bacteria as well as Pseudomonas aeruginosa, and trivalent iron recovers the growth suppression caused by PQS.

  (Test Example 9) The effect of 2,2-bipyridyl on the growth of Pseudomonas aeruginosa was examined. P. aeruginosa was grown aerobically on LB medium in the presence (200 μM) or absence of 2,2-bipyridyl or anaerobically on LBN medium. As is clear from FIG. 9, when P. aeruginosa was grown aerobically, growth was not suppressed by 2,2-bipyridyl, but when P. aeruginosa was grown anaerobically, 2, 2 -Growth was suppressed by bipyridyl.

It is a graph showing the effect of PQS on anaerobic growth and denitrification. A is a graph showing anaerobic growth with or without addition of exogenous PQS to the ΔpqsA mutant. Three independent experiments are performed and representative data are shown. The NO ₃ B ^- the decrease, C is the _{N 2} O production amount, D is a graph showing the _{N 2} production amount. Data of the mean ± standard deviation of three independent experiments. It is a graph showing the effect of PQS on the transcription | transfer of a denitrification gene. Data of mean ± standard deviation of 3 or more independent experiments. De窒酵containing activity and NO ₃ ^- is a graph showing the effect of PQS on respiratory chain. A is a graph showing NAR activity, B is NIR activity, C is NOR activity, and D is NADH-dependent NAR activity. PQS is added to the medium or reaction solution to a final concentration of 50 μM. 1 represents a culture of the ΔpqsA mutant without PQS, 2 represents a culture of the ΔpqsA mutant without PQS, and 3 represents the reaction of the cell fraction recovered from the culture of the ΔpqsA mutant without PQS. It represents the liquid with PQS added. Data of mean ± standard deviation of 3 or more independent experiments. It is a graph showing the effect of iron chelation on denitrification. A is NO ₃ ^- the decrease, B is a _{N 2} O production amount, C is a graph showing the _{N 2} production amount. Data of the mean ± standard deviation of three independent experiments. The _{N 2} production regulated by PQS is PqsR and PqsE is a graph indicating the involvement. A is NO ₃ ^- the decrease, B is a _{N 2} O production amount, C is a graph showing the _{N 2} production amount. Data of the mean ± standard deviation of three independent experiments. NO ₃ on pyocyanin production ^- is a graph showing the effect of and _{O 2.} LB represents the case where aerobic culture was performed in LB medium, LBN represents the case where aerobic culture was performed in a medium (LBN medium) supplemented with 100 mM NO ₃ ⁻ in LB medium, and LBN / −O ₂ represents The case where it culture | cultivated anaerobically with the LBN culture medium is represented. Data of the mean ± standard deviation of three independent experiments. ND indicates that it could not be detected. NO ₃ by PQS ^- a diagram representing a model of the regulation of respiration and de窒酵element. The solid line represents direct adjustment and the dashed line represents indirect adjustment. DH represents NADH dehydrogenase, OM represents the outer membrane, PP represents the periplasmic space, and IM represents the inner membrane. NO ₃ ^- respiratory chain and NOR enzyme activity is directly inhibited by PQS through iron chelation. NAR and NOS activity are indirectly suppressed by PQS, and NIR activity is indirectly increased by PQS. PqsE and PqsR are involved in NOS activity suppression. It is a graph showing the effect of PQS on the growth of various bacteria. It is a graph showing the effect of 2,2'-bipyridyl on the growth of Pseudomonas aeruginosa. It is the schematic which shows embodiment of the 1st waste water treatment apparatus of this invention. It is the schematic which shows embodiment of the 2nd waste water treatment equipment of this invention.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 10 and 30 ... Denitrification tank, 20 ... Growth promoter supply means, 40 ... Trivalent iron ion chelating agent supply means, 100 and 200 ... Waste water treatment equipment, L1-L6 ... line.

Claims (8)

  Denitrifying bacteria growth promoter consisting of trivalent iron ions. In a wastewater treatment apparatus for denitrifying wastewater containing nitrate ions and / or nitrite ions,
A denitrification tank for reducing nitrate ions and / or nitrite ions in the introduced waste water to nitrous oxide and / or nitrogen by denitrifying bacteria;
A waste water treatment apparatus comprising a growth promoter supply means for supplying a growth promoter for denitrifying bacteria comprising trivalent iron ions into the denitrification tank. In a wastewater treatment method for treating wastewater containing nitrate ions and / or nitrite ions by reducing the nitrate ions and / or nitrite ions to nitrous oxide and / or nitrogen by denitrifying bacteria in a denitrification tank,
A wastewater treatment method, wherein the denitrifying bacteria are grown in the denitrifying tank in the presence of a growth promoting agent for denitrifying bacteria comprising trivalent iron ions. In a wastewater treatment apparatus for denitrifying wastewater containing nitrate ions and / or nitrite ions,
A denitrification tank for reducing nitrate ions and / or nitrite ions in the introduced waste water to nitrous oxide and / or nitrogen by denitrifying bacteria;
A wastewater treatment apparatus comprising: a trivalent iron ion chelating agent supplying means for supplying a trivalent iron ion chelating agent into the denitrification tank. In a wastewater treatment method for treating wastewater containing nitrate ions and / or nitrite ions by reducing the nitrate ions and / or nitrite ions to nitrous oxide and / or nitrogen by denitrifying bacteria in a denitrification tank,
A wastewater treatment method, wherein the denitrifying bacteria are grown in the denitrification tank in the presence of a chelating agent of a trivalent iron ion.   An antibacterial agent consisting of a chelating agent of trivalent iron ions.   The antibacterial agent of Claim 6 whose chelating agent of a trivalent iron ion is 2-heptyl-3-hydroxy-4-quinolone.   The antibacterial agent according to claim 6, wherein the chelating agent of trivalent iron ions is 2,2'-bipyridyl.

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