KR101222602B1 - Castellaniella sp. and mixotrophic denitrification process using castellaniella sp - Google Patents

Abstract

PURPOSE: A novel Castellaniella sp. which performs heterotrophic and autotrophic denitrification is provided to effectively resolve nitrogen pollution. CONSTITUTION: A novel Castellaniella sp.(KFCC 115214P) has heterotrophic and autotrophic denitrification ability. An electron donor for heterotrophic denitrification is glucose, lactose, starch, cellulose, sucrose, maltose, protein, lipid, alcohol, hydrocarbon, butyrate, acetate, propionate, or glucose. An electron donor for autotrophic denitrification is sulfur, sulfur compounds, iron, hydrogen, or ammonia. A composition for denitrification contains the strain, a culture liquid thereof, or a mixture thereof as an active ingredient. A denitrification method comprises a step of supplying the strain, culture liquid, or mixture thereof to a water system or soil.

Description

New Castellaniella strains with mixed nutritional denitrification capacity and denitrification process using the same {Castellaniella sp. and mixotrophic denitrification process using Castellaniella sp.}

The present invention relates to a novel strain having denitrification ability, and more particularly, to a novel strain having both heterotrophic and autotrophic denitrification ability, a composition for denitrification comprising the strain or culture medium and the strain or strain of It relates to a denitrification method comprising the step of supplying a culture solution.

Wastewater containing nitrogen leads to eutrophication of streams and lakes, dissolved oxygen deficiency, direct toxicity of ammonia to aquatic organisms, and adversely affects the aquatic environment.

In addition, organic nitrogen components such as nitrogen fertilizer continuously administered to the soil and livestock wastewater generated by livestock raising for food production has become an important environmental problem. The accumulation of nitrogen above the self-cleaning capacity of the ecosystem directly affects the acidification of the soil and eutrophication of the water quality. In addition, when nitrogen is accumulated in the environment, poor soil conditions are created and the final nitrogen decomposer in the soil is shortened, resulting in a vicious cycle of nitrogen accumulation.

Nitrogen accumulates in the environment in the form of organic nitrogen (Org-N), ammonia nitrogen (NH ₃ ⁺ -N), nitrite nitrogen (NO ₂ ^-- N) and nitrate nitrogen (NO ₃ ^-- N) The main forms of nitrogen remaining in the environment are organic nitrogen and ammonia nitrogen. In order to completely remove nitrogen accumulated in the environment, both nitrification and denitrification of nitrogen must be carried out. Nitrogen contamination is primarily present as ammonia nitrogen, but as the contamination is restored, it is oxidized back to nitrate nitrogen in an aerobic state. This oxidation process is called nitrification. The main products of nitrification by oxidation are in the form of nitrites (NO ₂ −) and nitrates (NO ₃ −). Natural circulation of the ecosystem nitrogen component finally reduces these two nitrification substances to nitrogen gas (N ₂ ) and returns them to the environment. After all, if the above two nitrification products are not removed quickly, the excess accumulation of nitrogen in the environment is not solved.

In the denitrification action of the present inventors, as a result of diligent efforts to invent a microorganism strain having excellent denitrification function, the present inventors have found a new strain having both heterotrophic and autotrophic denitrification ability, and containing this strain and its culture solution. To invent a composition for denitrification and a method for denitrification using the same.

It is an object of the present invention to provide a novel strain having both heterotrophic and autotrophic denitrification.

In addition, another object of the present invention to provide a denitrification treatment composition containing the cells of the monarch, culture, or a mixture thereof as an active ingredient and a denitrification treatment method comprising the step of supplying the same to the aqueous or soil.

In order to achieve the above object, the present invention provides a novel Castellaniera genus strain (KFCC11514P) having heterotrophic and autotrophic denitrifying ability.

Also preferably, the heterotrophic denitrification has glucose as an electron donor, and preferably the autotrophic denitrification has a sulfur compound as an electron donor. Also preferably, the strain performs autotrophic denitrification after performing heterotrophic denitrification.

In another aspect, the present invention provides a composition for denitrification containing the cells of the above strain, the culture medium or a mixture thereof as an active ingredient.

In another aspect, the present invention provides a denitrification method comprising the step of supplying a cell, a culture medium or a mixture thereof to the aqueous or soil.

In case of using the new Castellaniera genus strain (KFCC11514P) provided by the present invention, nitrogen from groundwater and soil as well as sewage and wastewater can be treated with both inorganic and organic substances, thereby improving effective and environmentally friendly nitrogen pollution. To make it possible.

1 is a neighbor-joining phylogenetic tree based on 16S rRNA gene sequence, showing the phylogenetic location of SORA1. Accumulation shows a 2% difference in nucleic acid sequence.
FIG. 2 is a graph showing nitrogen removal characteristics under sulfur use conditions, in which nitrate, nitrite and sulphate in inorganic nutrient growth of SORA1 in MBM medium supplemented with bicarbonate (2mM). This is a graph of the change in ion concentration. The medium contains thiosulfate as an electron donor, and nitrate as an electron acceptor. ●: nitrite, ○: nitrate, □: sulfate, ◇: absorbance measurement result. Cell growth was followed by absorbance measurements at 650 nm wavelength.
Figure 3 is a graph showing the nitrogen removal characteristics in the organic use conditions. It is a graph of the ion concentration change of nitrate, nitrite and sulfate when MBM medium contains 2 mL / L spent caustic instead of thiosulfate. ●: nitrite, ○: nitrate, □: sulfate.
4 is a graph showing heterotrophic denitrification of SORA01 and concentration changes of nitrate, nitrite, COD _Cr and sulfate therefrom in a MBM medium containing glucose at 30 ° C. ●: nitrite, ○: nitrate, □: sulfate, △: COD _Cr , ◇: absorbance measurement results. Cell growth was followed by absorbance measurements at 650 nm wavelength.
Figure 5 shows the concentration changes of nitrate, nitrite, COD _Cr and sulfate in mixed nutrient conditions of 30 ℃.
(a) is the result of providing electron donors with sufficient organics and thiosulfate to remove nitrite. (b) is the result of adding half the amount of organic matter and thiosulfate required to remove nitrite.
●: nitrite, ○: nitrate, △: sulfate, ▼: COD _Cr (organic)

Hereinafter, the present invention will be described in more detail.

The present invention provides a novel Castellaniera genus strain (KFCC11514P) having heterotrophic and autotrophic denitrifying ability.

Castellaniera genus strain (KFCC11514P) was isolated from the sewage treatment process and can be used as an electron donor for denitrification.

As used herein, the term “denitrification” means that the microorganism reduces nitrate nitrogen, which acts as a final electron acceptor for respiration in the absence of oxygen. Depending on the electron donor used by the microorganism, it is divided into autotrophic and heterotrophic denitrification.

In the present invention, "dependent nutrient denitrification" means to perform denitrification by using an organic substance as an electron donor, and if the organic substance is not limited in scope, for example, glucose, lactose, starch, cellulose, sucrose , Maltose, proteins, fats, alcohols, hydrocarbons, butyrate, acetate and propionate glucose, and the like. Also preferably, glucose may be used as the electron donor.

In the present invention, "independent nutrient denitrification" means to perform denitrification by using an inorganic material as an electron donor, and the inorganic material as an electron donor does not limit the range, and examples thereof include sulfur, sulfur compounds, iron, hydrogen, Ammonia and the like. Also preferably, a sulfur compound may be used as the electron donor, and more preferably, thiosulfate or Spent Caustic (SC).

In addition, the strain of the present invention performs heterotrophic denitrification after performing heterotrophic denitrification. As organic and inorganic materials are mixed in sewage, wastewater, and soil, the denitrification characteristics under mixed nutrient conditions where organic and inorganic materials exist at the same time. First, after denitrification using organic materials, the sulfur source is consumed after about 5 days. I could see.

In another aspect, the present invention provides a composition for denitrification containing the cells of the above strain, the culture medium or a mixture thereof as an active ingredient.

In the present invention, the "culture medium" can be used as long as it is obtained by a medium capable of culturing the new Castellaniella sp. Strain (KFCC11514P) of the present invention.

It is easy to fix the strain in the composition, a general carrier such as organic carriers, ceramic carriers, plastic carriers, such as zeolites, ochers, clays and the like, which are known to have a large physical fixing effect, which maintains a stable structure in water for a long time. In addition, a carbon source for heterotrophic denitrification may be included, and the carbon source may preferably be bicarbonate.

In another aspect, the present invention provides a denitrification method comprising the step of supplying a cell, a culture medium or a mixture thereof to the aqueous or soil.

In the present invention, "water system" is preferably sewage, wastewater, groundwater and sewage, and "soil" includes both natural and artificial soils.

Supplying in the present invention means a process of adding a cell or a culture solution by spraying or inflowing into an aqueous system or soil, and is not limited to the means of the addition process.

Hereinafter, the present invention will be described more specifically based on examples. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.

< Example 1  > Experimental Materials and Methods

1-1: Inoculator  Ready

Sludge samples were taken from an oxygen-free tank in a pilot-scale Bardenpho process reactor at a southern sewage treatment plant in Busan, South Korea, to isolate denitrifying bacteria using “Spent caustic (SC)”, a sulfur-containing liquid waste from a petrochemical plant. Got it.

1-2: Medium used for isolation of strains

Thiobacillus to isolate SC- derived denitrifying bacteria The culture medium of denitrificans (medium 832 produced by German Collection of Microorganisms and Cell Cultures) was used. Medium 832: 1.2 g of Na ₂ HPO ₄ , 1.8 g of KH ₂ PO ₄ , 0.1 g of MgSO ₄ · 7H ₂ O, 0.1 g of (NH ₄ ) ₂ · SO ₄ , and 0.03 of CaCl ₂ , with respect to 1 liter of solvent. g, FeCl ₃ .6H ₂ O, 0.02 g, MnSO ₄ .4H ₂ O, 0.02 g, Na ₂ S ₂ O ₃ · 5H ₂ O 10.0 g, NaHCO ₃ 0.5 g, KNO ₃ containing 5.0 g have. MBM medium was used for the physiological test, which was performed by NaNO ₃ per 1 liter of solvent. 0.2 g, 0.2 g of KH ₂ PO ₄ , 0.2 g of NH ₄ Cl, 0.4 g of MgCl ₂ · 6H ₂ O, 0.2 g of KCl, 0.1 g of CaCl ₂ · 2H ₂ O, Na ₂ S ₂ O ₃ 2.5 g of 5H ₂ O, 2.0 mL of SL-4 trace element solution, 1.0 mg of resazurin.

The configuration of SL-4 is as follows. 0.5 g of EDTA, 0.2 g of FeSO ₄ · 7H ₂ O, and 100 mL of a trace element solution per 1 liter of solvent; 0.1 g of ZnSO _{4 ·} 7H ₂ O, 0.03 g, per 1 liter of solvent in a trace element solution MnCl _{2 ·} 4H ₂ O, 0.3 g H ₃ BO ₃ , 0.2 g CoCl _{2 ·} 6H ₂ O, 0.01 g CuCl _{2 ·} 2H ₂ O, 0.02 g NiCl _{2 ·} 6H ₂ O, 0.03 g Na ₂ MoO _{4 ·} 2H ₂ O is included. All culture media were used after sterilization.

1-3: Separation

Enrichment of microorganisms in the sludge obtained from the anoxic tank was carried out using 100 mL of the serum vial containing 832 medium and shaken at 100 rpm in a dark incubator at 30 ℃. The concentrated culture was transferred to 832 medium (1% vol / vol) and incubated for 3 to 5 days. This thickening process was repeated eight times. The concentrated culture and its dilutions were plated on agar plates containing 832 medium. This plate was incubated at 30 ° C. in a GasPack Jar (GasPack 100, BBL, USA). Colonies formed on the plates were separated and plated again on agar plates to purify.

1-4: Systematic  analysis

Colonies of 832 medium plates were isolated to isolate 1 μL template DNA, 0.25 μL forward and reverse primer (10 pmol), 2.5 μL 10 × Taq buffer, 10 μL 10 mM dNTP, 0.125 μL DNA polymerase (Solgent, Daejeon, Korea) was suspended in a PCR solution consisting of. Colonies were amplified using a Mastercycler gradient automated thermal cycler (Bio-Rad Laboratories, Inc., Hercules, USA) and performed in the following order. Initial denaturation was at 95 ° C. for 2 minutes; 29 cycles of denaturation (20 sec at 95 ° C.), annealing (at 55 ° C. for 40 seconds) and extension (at 72 ° C. for 30 seconds) were repeated and the final extension was carried out at 72 ° C. for 5 minutes. To amplify 16S rDNA, forward primer eub 10F (5'AGAGTTTGATCMTGGCTCAG-3 '(SEQ ID NO: 1) and reverse primer eub 1392R (5'ACGGGCGGTGTGTACAAG-3' (SEQ ID NO: 2)) were used. Primers eub 10F and eub PCR of colonies using the 1392R was followed by sequence analysis using an ABI 3730XL capillary DNA sequencer MEGA 4.0.

1-5: Physiological Experiment

Growth experiments were performed on modified MBM media. 2 mM NaHCO ₃ Was supplied as a carbon source. Cell growth was measured using an DU-70 spectrophotometer (SPECTRONIC20D +, Thermo Fisher scientific, USA) to measure the OD ₆₀₀ value. Serum vials headspace is pure N ₂ (99.9% or more) filled with gas. Heterotrophic denitrification experiments of 0.075 gL ^{- 1} The addition of glucose was performed in MBM medium without thiosulfate. All physiological tests were performed at 30 ° C. The following two media were used to test the hybrid nutritional denitrification characteristics. (1) modified MBM medium containing 0.15 g glucose and (2) 0.4 g NaNO ₃ And modified MBM medium containing 0.15 g of glucose. All physiological experiments were charged to the serum vial with 50 mL medium and additional nitrogen gas.

1-6: Spent caustic Testing of electron donor availability

SC (Spent caustic) used in this application was taken from a petrochemical company in Korea, sterilized and injected into Serum vial. SC was used as the electron donor to test the ability of the isolate, and 2 mL of SC was added to the thiosulfate depleted MBM medium. 50 mL of SC containing medium was sealed using Teflon-coated butyl rubber septum and aluminum cap. The serum vial was filled with N ₂ and then incubated with 0.5 mL of culture grown in LB medium. Cultures were shaken at 30 ° C. at 100 rpm.

1-7: Analysis Method

Chemical oxygen demand (sCOD _Cr ) concentration and NH ₄ ⁺ -N concentration were analyzed using an automatic analyzer (AA3, Bran + Luebbe, Germany), and the sample was analyzed by filtration with 0.45 μm membrane filter. The concentrations of NO ₂ ^-- N and NO ₃ ^-- N were measured using ion chromatography (DX-300, DIONEX, Sunnyvale, USA). pH values were measured using an Orion Research pH meter (230A, Thermo Fisher Scientific Inc., Waltham, USA).

1-8: KCCM Deposit to

The bacterium of the present invention was deposited in the Korean Culture Center of Microorganisms (KCCM), the name of the strain Castellaniella sp. Defined as SORA01.

< Example 2 Determination of separation and separationism

To determine the systematic location of strain SORA01, 16S rRNA gene sequences were compared with microorganisms belonging to Castellaniella spp., Strain SORA01 was castellaniella It showed 98% homology with daejeonesis MJ06 and was found to be a sulfur-derived denitrifier (FIG. 1). Based on 16S rRNA gene homology, strain SORA1 is located closest to C. dajeonensis and C. denitrificans . Strain SORA1 belongs to the Betaproteobacteria class, which is known as sulphur-derived denitrifier Corresponds to the river to which Thiobacillus denitrificans belongs.

< Example 3 > Autotrophic denitrification  Confirmation of action MBM  Growth and Autotrophic Denitrification in Medium

The growth pattern of strain SORA1 in MBM medium in anoxic conditions is shown in FIG. 2. The strain SORA1 showed a constant growth pattern when thiosulfate was an electron donor and nitrate was an electron acceptor. On the 8th day, OD ₆₅₀ reached 0.52. Nitrate reduction and thiosulfate oxidation were not significant in aseptic conditions, whereas nitrate reduction and thiosulfate oxidation were simultaneously performed in MBM medium containing strain SORA1. Autotrophic denitrification with thiosulphate is produced, and the sulfate, and the strain is SORA1 ^{_{2.69 g SO 4 2 - -S /}} - phosphorus (g NO ₃ -N) which was produced a quantitative chemical equation NO ₃ ^- + 0.625S ₂ O ^{_{3 2 - + 0.125H 2 O →}} 0.5N 2 + 1.25SO 4 2 - + 0.25H + a matching and (McCarty, 1975). It was found that strain SORA1 performs autotrophic denitrification with thiosulphate upon nitrate removal and the production of sulphate, which is not known before.

C. denitrificans or C. defragrans belonging to the conventional Castellaniella species It has been reported that the back has denitrifying ability, but no evidence is available on whether their capacity is autotrophic denitrification.

< Example 4 > Autotrophic denitrification  Confirmation of action spent caustic Using Autotrophic denitrification

Strain SORA01 was isolated in a reactor injected with conventional SC and tested for autotrophic denitrification in the presence of SC. Thiosulfate was removed from the MBM medium for testing and 2 mL / L of SC was injected.

Figure 3 shows that SORA01 removes nitrates when SC is supplemented with electron donors, indicating that SORA01 is capable of autotrophic denitrification using SC. The denitrification rate was 32.7%, which was not high compared to the condition in which thiosulfate was present. This low denitrification rate is expected to be due to the SC injection in MBM medium at 2 mL / L, which does not match the stoichiometrically required amount of sulfur (the average concentration of sulfur in SC is 13,977 mg / L).

For complete denitrification, 4 mL / L of SC should be injected into the MBM medium, but 4 mL / L of SC increases the pH of the medium to 9.5 because the SC raw material contains excess alkalinity than is needed for autotrophic denitrification. , Which inhibits denitrification.

However, SC can be sufficiently supplied for denitrification in the process of nitrification and denitrification at the same time because nitrification consumes alkalinity. We also found that SORA01 did not grow in the absence of bicarbonate when SC was provided, indicating that SORA1 does not use SC as a carbon source (data not shown).

< Example 5 > Autotrophic denitrification  Confirmation of Action-Using Glucose Heterotrophic denitrification

0.15 g of glucose was supplemented with electron donor to test whether SORA01 can perform heterotrophic denitrification. Strain SORA01 immediately depleted 99.0% nitrate within 2 days using glucose. Since ionized ammonia (NH ₄ ⁺ -N) is provided to the cells as nitrogen nutrients and the nitrogen of nitrate is not contained in the cells, it is believed that the nitrates in MBM medium denitrate while reducing COD. SORA01 requires 3.18 g COD for heterotrophic denitrification, a value consistent with prior literature. OD ₆₅₀ shows the growth of cells using glucose as a carbon source and glucose within two days, as in previous studies. After the denitrification was complete, about 61.8 g of COD was left, because an excess of the required amount of glucose was provided.

This result shows that SORA01 can use not only thiosulfate but also glucose as an electron donor, which has not been reported before.

< Example 6 > Autotrophic denitrification  Confirmation of action-under mixed nutrient conditions Denitrification

Since the wastewater contains mostly organic matter, the characteristics of denitrification under mixed nutrient conditions in which thiosulfate and organic matter exist simultaneously were tested. First, glucose and thiosulfate were supplied to a vial containing MBM medium in a sufficient amount. The pattern of hybrid nutrition was similar to when glucose was present only as an electron donor. When a sufficient amount of organics was fed into the vial, all of the COD was not consumed and no acid was produced. This clearly shows that if sufficient electron donors are present under mixed nutrient conditions, strain SORA01 performs autotrophic denitrification using sulfur after using organics.

Strain SORA1 was the first to use glucose under insufficient organic conditions. Heterotrophic denitrification of SORA01 using glucose was complete within 2 days, followed by autotrophic denitrification with thiosulfate. In addition, it took more than 5 days to convert from heterotrophic to autotrophic denitrification, which is expected to take time to adapt to different electron donors.

As a result of the mixed nutrient denitrification properties, it was concluded that this isolate could use both organics and sulfur under mixed nutrient conditions. This indicates that microorganisms in activated sludge can use SC and can be used after the organics in the wastewater are used. In particular, this is the first report that Castellaniella species can be denitrified using sulfur.

Korea Microbial Conservation Center (Domestic) KFCC11514P 20110407

Claims (6)

A new strain of Castellaniella spp. (KFCC11514P) with heterotrophic and autotrophic denitrifying ability.
The method of claim 1, wherein the heterotrophic denitrification is glucose, lactose, starch, cellulose, sucrose, maltose, protein, fat, alcohol, hydrocarbons, butyrate, acetate and propionate ( propionate) and a strain characterized in that it has an electron donor selected from the group consisting of glucose.
[Claim 2] The strain according to claim 1, wherein the autotrophic denitrification has an electron donor selected from the group consisting of sulfur, sulfur compounds, iron, hydrogen and ammonia.
According to claim 1, wherein the strain is characterized in that after performing heterotrophic denitrification autotrophic denitrification.
A composition for denitrification containing the cells of the strain according to any one of claims 1 to 4, a culture solution or a mixture thereof as an active ingredient.
A denitrification method comprising the step of supplying a cell, a culture medium or a mixture thereof according to any one of claims 1 to 4 to an aqueous system or soil.

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