KR101036806B1 - Alcaligenes sp. kc-1, method for purifying oil- and heavy metal-contaminated soilusing the same - Google Patents

Abstract

PURPOSE: Alcaligenes microorganism sp., microorganism formulation including the same, a method for purifying oil and heavy metal contaminated soil using the same are provided to obtain the high resistance with respect to heavy metals and accelerate the growth of plant. CONSTITUTION: Alcaligenes microorganism sp. is the alcaligenes KC-1 strain which is separated from wetland. Microorganism formulation includes the alcaligenes KC-1 strain as an effective component. The alcaligenes KC-1 strain generates 1-aminocyclopropane-1-carboxylate deaminase(ACC deaminase) and indole acetic acid(IAA). The ACC deaminase is the precursor of ethylene which is the stress material of plant. The IAA is plant hormone.

Description

Microorganisms of the genus Alcaliginius and the method for the purification of soils contaminated with oil and heavy metals using it {Alcaligenes sp. KC-1, method for purifying oil- and heavy metal-contaminated soilusing the same}

The present invention relates to a microorganism of the genus Alcaligenes sp., Heavy metals and oil contaminated soil using the same, and more specifically, to the production capacity of the plant hormone indoleacetic acid (IAA) and the stress of the plant ethylene A novel microbial Alcaligenes sp. KC-1 strain, Alcaligenes sp., Which produces the precursor aminocyclopropane-1-carboxylate (ACC) deaminase. To clean soil contaminated with oil and heavy metals using the genus KC-1 strain, and to clean soil contaminated with oil and heavy metals using the KC-1 strain from Alcaligenes sp. It is about.

Soil pollution by oil and heavy metals emitted from various industrial activities such as ore smelting and refining process has become a major environmental problem. The biopurification method using microorganisms and plants to purify soil contaminated with heavy metals and oils has recently been commercialized as an economical and environmentally friendly method. Oil-separating microorganisms that can be used to clean oil-contaminated soils include Pseudomonas, Arthrobacter, Rhodococcus and Bacillus. In addition, as plants that can be used to purify oil-contaminated soil, Lolium multiflorum, Leguminosae, Poplar, Pinus densiflora, Festuca arundinacea, Zea mays, etc. have been reported.

Heavy metals are removed from the soil by plant accumulation rather than decomposing themselves, so heavy metal removal is very important for the development of the root zone. Heavy metal overaccumulation There are many studies on microorganisms that help plants and plants accumulate heavy metals well. Metal-hyperaccumulators studied so far include Asteraceae, Brassicaceae, Caryophyllaceae, Cyperaceae, Beanaceae, Lacourtiaceae, Honeysuckle ( Lamiaceae, Poaceae, and Violaceae. Heavy metal-resistant microorganisms include Bacillus sp., Pityrogramma calomelanos, and Serratia sp.

Most of the studies to date have dealt with the use of microorganisms or plants to remove each pollutant when contaminated with oil or heavy metals alone. Soils contaminated with oil and heavy metals are often more difficult to remove than soils contaminated with one type of pollutant. Therefore, it is necessary to establish relevant information through research on soil purification contaminated with two or more pollutants.

In addition, studies to date have been limited to simply planting contaminant-resistant plants or artificially injecting microorganisms that degrade the contaminants. However, this purification method has a problem that the efficiency is not high in removing contaminants. In order to compensate for this problem, recently, microorganisms that are resistant to contaminants and which can accumulate heavy metals can be combined with microorganisms that can tolerate or decompose contaminants and promote plant growth. rhizoremediation technique is used. However, because these purification techniques are new technologies that have just begun, there are very few microorganisms available for this purification technique. Therefore, there is an urgent need for the development of microorganisms that are resistant to contaminants and degrade while at the same time promoting plant growth.

Therefore, the present inventors have studied to solve this problem, aminocyclopropane carboxylate which is a precursor of plant hormone indoleacetic acid (IAA) and ethylene, a stress substance of plants (1-aminocyclopropane-1-carboxylate (ACC)) A novel microorganism that produces AAC deaminase Alcaligenes sp. It separates KC-1, and this microorganism accelerates plant growth while simultaneously breaking down oil and being resistant to heavy metals. The invention was completed by confirming that the microorganisms were able to purify soil contaminated with oil and heavy metals through mesocosm experiments using corn and gangnan beans.

An object of the present invention is to provide a microorganism belonging to the genus Alcaliginus isolated from the root region soil of reeds.

It is still another object of the present invention to provide a method for decomposing oil and heavy metals from the root zone soils of plants, or for separating microorganisms that are resistant and promote plant growth.

In addition, another object of the present invention is to provide a method for purifying soil contaminated with oil and heavy metals using the above microorganisms.

The present invention provides the production capacity of the plant hormone indole acetic acid (IAA) from the reed root soil of wetland and aminocyclopropane-1-carboxylate (ACC), which is a precursor of ethylene, a stress agent of plants. ) A novel microorganism Alcaligenes sp. That produces ACC deaminase. This can be achieved by separating KC-1 (KCTC11801BP).

The novel microbial alkaline genus KC-1 according to the present invention (KCTC11801BP) has a strong resistance to heavy metals and at the same time can decompose oil. In addition, KC-1 of the genus Alkalineinus promotes the growth of plants, decomposes oils, is resistant to heavy metals, and can clean soils contaminated with heavy metals and oils using plants.

1 is a graph showing IAA production capacity and ACC diamine AI synthesis capacity in the evaluation of plant growth promoting ability of the genus Alkalinein according to the present invention.
Figure 2 is a graph showing the oil resolution of the genus KC-1 of the algaline in accordance with the present invention.
3A to 3E are graphs showing the heavy metal resistance characteristics of the genus KC-1 according to the present invention.
Figure 4 is a graph of the results of the investigation of the effect of the KC-1 strains and plants of the genus Alkalinian according to the present invention to remove diesel in the soil.

The present invention provides the production capacity of the plant hormone indole acetic acid (IAA) from the reed root soil of wetland and aminocyclopropane-1-carboxylate (ACC), which is a precursor of ethylene, a stress agent of plants. ) A novel microorganism Alcaligenes sp. That produces ACC deaminase. This can be achieved by separating KC-1 (KCTC11801BP).

Alcaligenes sp. Of the present invention. KC-1 promotes plant growth and degrades oil and is resistant to heavy metals. KC-1 is a strain isolated from reeds in the wetlands and has strong resistance to heavy metals. It has the characteristic of breaking down oil.

The identification method of KC-1 in Alkaliginus is sampled from the root area of wetland soil and reed from 12 wetlands and placed in Burk's, Congo Red and NFB medium and cultured for one week. Thickening cultures are all subcultured with four new medium and smeared on solid medium. Depending on the type of medium, the strains spread on Burk's Agar medium were cultured for 7 days, and the strains spread on Congo Agar medium and NFB ager medium for 3 days. The cultured strains were separated according to color and shape, and spread on new media, and nitrogen fixing ability, indoleacetic acid production capacity, and carboxylate (1-aminocyclopropane-1-carboxylate (ACC)) were investigated to investigate the ability of each colony to promote plant growth. ) AAC deaminase is generated and the sideropore synthesis ability is evaluated.

Investigate the degradability of diesel in strains that have two or more of the above-mentioned plant growth promoting abilities, and examine whether it is resistant to heavy metals, and then select microorganisms capable of promoting plant growth and decomposing pollutants at the same time. 16S rDNA partial sequencing is analyzed by rDNA analysis, and the isolated strains are identified by comparing databases of strains in GenBank.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples.

< Example >

Example 1 Isolation and Identification of Alkaliginus KC-1

(1) Isolation of Strains from Reed Roots and Root Soil

Sampling to isolate the strain was based on the reed root soil and the lake water inhabited by plants. Samples collected at 12 locations were put into a 250mL Erlenmeyer flask and 90mL of sterile water and 10g of the sample were mixed and incubated for 15 minutes in a shaker incubator. The shaker was operated at 30 degrees and 180rpm. 5 mL of the turbid solution in the shake cultured sample for 15 minutes was placed in a 250 mL Erlenmeyer flask, and then placed in 50 mL of liquid medium (Burk's, Congo Red and NFB) and concentrated for one week. The enrichment cultures were all subcultured with four new media. The culture medium was plated in a solid medium and strains were cultured. The cultured strains were separated according to color and shape, and plated in a fresh medium, and each colony was evaluated for its ability to promote plant growth.

(2) Evaluation of plant growth promoting ability of isolated strain

① Evaluation of IAA (indole-3-acetic acid) production capacity of isolated strain

Among the isolates, the best nitrogen-fixing strains were evaluated in IAA (indole-3-acetic acid) production capacity as the following method, and all experiments were repeated three times. The strains were inoculated in 5 mL of DF medium to which 0.5 mg / mL of tryptophane was added, respectively, and incubated at 30 ° C. for 180 days at 180 rpm. The composition of the DF medium is as follows: (NH ₄ ) ₂ SO ₄ 2g; ₄ g KH ₂ PO ₄ ; Na ₂ HPO ₄ .12H ₂ 0 15 g; 0.2 g MgSO ₄ 7H ₂ O; 1.0 mg FeSO ₄ 7H ₂ O; 10 μg of B (as H ₃ BO ₃ ); 11 μg Mn (as MnSO ₄ H ₂ O); 125 μg Zn (as ZnSO ₄ H ₇ O); 78 μg Cu (as CuSO ₄ H ₂ O); 17 μg of Mo (as Na ₂ MoO ₄ .2H ₂ O); 1 L of distilled water. After incubating the strain, the culture solution and Salkowski's reagent (150 mL of concentrated H ₂ SO ₄ ; 250 mL of distilled water; 0.5 mL of 0.5M FeCl ₃ · 6H ₂ O) were mixed at a ratio of 1: 2 (v / v), and then pink color was developed. The mixture was allowed to stand at room temperature for 20 minutes. The degree of color development was measured by absorbance at 530 nm using an absorbance photometer (DR5000 UV-Visible Spectrophotometer, HACH, USA).

② ACC (1-aminocyclopropane-1-carboxylic acid) deaminase activity

ACC (1-aminocyclopropane-1-carboxylic acid) deaminase activity was evaluated by the following method (the medium used in the experiment was DF medium containing 3 mM ACC instead of (NH ₄ ) ₂ SO ₄ ). Each colony was inoculated in medium and incubated at 30 ° C. for 180 hours at 180 rpm. During the incubation period, the absorbance was measured at 600 nm every 4 hours using an absorbance spectrometer. SPSS (12.0K) was used to assess the ACC utilization of the strain by performing a significant sample analysis of two sample t-test with the control group not inoculated with the strain.

③ Siderophores Synthesis

When iron is deficient as an essential component of plant growth, it produces ethylene, creating a stressful environment for plants. Iron is present in Fe (OH) ₃ , which is poorly soluble in soils with neutral and anaerobic conditions. In order to metabolize low iron concentrations, organisms secrete a substance called "siderophores" out of cells. Siderophores are low-molecular iron traps and transporters with high affinity with iron. They bind to iron in soil and are then absorbed back into the body and play an important role in supplying iron to organisms. Siderophores synthesis was performed using chrom azurol S (CAS) blue agar plate assay. The procedure for making 1 L of CAS agar is as follows: (1) Dark-blue dye solution as a siderophores indicator (60.5 mg CAS is dissolved in 50 mL distilled water and then 10 mL of iron (III) solution (1 mM FeCl ₃ · 6H ₂ O, 10 mM). HCl) and a solution of 72.9mg HDTMA dissolved in 40mL distilled water are added together and sterilized. (2) Medium solution (100mL 10 × MM9 salts in 750mL distilled water (60g / L Na ₂ HPO ₄ ; 0.9g / L KH) ₂ PO ₄ ; 5g / L NaCl; 10g / L NH ₄ Cl) 15g agar, 30.24g PIPES and 12.0g of 50% (w / w) NaOH, sterilized and cooled to 50 ℃, 30mL casamino acids (10% , w / v), 10 mL glucose (20%, w / v), 1 mL thiamine HCl (0.2%, w / v), and 3 mL L-tryptophane (1%, w / v)) In the petri dish, 30 mL was hardened to make a blue agar plate. Each microorganism was inoculated on a CAS agar plate and incubated at 30 ° C. for 24 hours. The formation of orange halo around colony was evaluated as siderophores positive.

(2) Identification of KC-1

Among the rhizosphere bacteria isolated from above, the plant growth promoting ability was excellent, and microorganisms capable of decomposing oil and heavy metals were selected and identified. Identification method was used 16S rDNA analysis method. Chlory extracted genomic DNA using the BIO101 kit (FastDAN SPIN Kit for Soil, Q-BIO gene, USA), and then carried out a polymerase chain reaction (RCR) using this genomic DNA sample as a template. Primers used for PCR were 27f (5'-AGA GTT TGA TCM TGG CTC AC-3 ') and 1492r (5'-TAC GGY TAC CTT GTT ACG ACT-3'). PCR conditions were pre-denaturation at 95 ° C for 5 minutes, denaturation at 95 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension cycling at 72 ° C for 30 seconds, and final extension at 72 ° C for 5 minutes.

Alcaligenes sp. The results of 16S rDNA sequencing analysis of the KC-1 strain are shown in SEQ ID NO: 1, and as a result of comparing the databases of strains in GenBank, it was identified as genus Alkaline. Therefore, the inventors of 'Alcaligenes sp. It was named KC-1 'strain and was deposited with Korea Research Institute of Bioscience and Biotechnology and was given accession number KCTC11801BP.

In addition, as shown in Figure 1, Alcaligenes sp. KC-1 (Accession No .: KCTC11801BP) strains have IAA production capacity and ACC diamine AI synthesis ability in plant growth promotion activity evaluation. Therefore, this strain is capable of producing IAA, a plant hormone that can improve the growth of plant roots, and when contaminants such as heavy metals are present in the root zone of the plant, it releases ethylene as a stressor of plants. By synthesizing a substance capable of diamineating ACC to protect the plant from stresses such as heavy metals, the present invention will promote the growth of plants when purifying contaminated soil with plants, thereby increasing the purification efficiency of contaminated soil. It is expected.

Example 2 Oil Resolution Test of Alkaligius KC-1

The isolated strain was inoculated in MSM medium to which 10,000 mg / L diesel was added and shaken for 3 days in an incubator at 30 ° C. and 250 rpm. Residual diesel concentration of the culture was analyzed by gas chromatography, and diesel resolution was confirmed. The composition of the MSM medium was 0.15 g of MgSO ₄ .7H ₂ O, 0.01 g of CaCl ₂ , 1.5 g of KH ₂ PO ₄ , 9.0 g of Na ₂ HPO ₄ .12H ₂ O 9.0 g, 3.0 g of NH ₄ NO, and 0.01 g of FeCl ₃ (pH 7.0 7.2) and 1 liter of distilled water. In addition, the residual TPH concentration was analyzed by gas chromatography (5890 series, Hewlett Packard, USA) equipped with a flame ionization detector and is shown in FIG. 2. GC analysis conditions after the oven temperature was maintained for 3 minutes at the initial 40 ℃, the temperature was raised to 70 ℃ at 4 ℃ / min, the temperature was raised to 200 ℃ at 10 ℃ / min, and after heating up to 300 ℃ at 8 ℃ / min 15 Hold for a minute. The sample inlet and detector temperatures were 300 ° C and 320 ° C, respectively, and an HP-5 capillary column (0.25mm × 30m, 0.25μm) was used, and N ₂ was used as the carrier gas. After the calibration curve was prepared with the diesel concentrations of 0, 312.5, 625, 1,250, 10,000, 20,000, and 40,000 mg / L, the TPH concentration was converted from the calibration equation.

As shown in Figure 2, the result of the experiment by the above method, as a result of culturing KC-1 by adding 10,000 mg / L of diesel as the only carbon source to the inorganic salt medium, 100% of diesel at the 4th day of culture Digested. These results confirm that the strain isolated in this study can be useful for plant restoration of oil contaminated soil as a strain capable of degrading oil while having plant growth ability.

Example 3 Heavy Metal Resistance Test of Alkaligius KC-1

In order to evaluate the heavy metal resistance of the isolated strain 100mL LB-broth medium in a 250mL Erlenmeyer flask was inoculated with the heavy metal strain, and evaluated with strain growth in the medium containing heavy metal. Growth of the strain was measured for absorbance at 600nm at intervals of 3 to 12 hours using an absorbance spectrometer during the culture period. The heavy metals evaluated were lead, copper, cadmium, hexavalent chromium, and zinc, respectively, 1000, 500, 30, 30, and 2,000 mg / L. 3a to 3e.

As shown in Figures 3a to 3e, when lead concentration of 0 ~ 1,000mg / L absorbance value of 0.86 ~ 0.81 even if the concentration of lead increased KC-1 growth was not significantly inhibited. Hexavalent chromium had an absorbance value of 0.80-0.90 at 0-30 mg / L, and the presence of hexavalent chromium did not inhibit the growth of KC-1. Copper was incubated at concentrations of 0, 100, 200, 300 and 500 mg / L, and the growth of KC-1 strain was not inhibited even if the copper concentrations were increased to 1.07, 1.05, 1.12, 1.11 and 1.08, respectively. . These results suggest that KC-1 isolated from this study can be used as a very useful strain for the purification of soil contaminated with oil and heavy metals. It means.

Example 4 Confirmation of Pollutant Purification Efficiency of Oil and Heavy Metal Contaminated Soil Using Strains and Plants

The purification efficiency of oil and heavy metal contaminated soil using the isolated strains and plants was evaluated. To carry out the experiment, 60kg of soil from the oil-free area was taken and transported to the laboratory. The 60 kg of soil transported was divided into 20 kg and 40 kg. The 40 kg soil was kept in a cool place for 4 days in a cool place for 4 days so as to have a concentration of 5,000 mg / L of diesel and 500 mg / L of copper, which is a measure of pollutants specified by the Ministry of Environment. Mix up and down.

KC-1 strain was incubated for 3 days at 30 ℃ in a medium prepared by putting LB (LB broth 200 g, Difco, USA) in 6L DW. The culture solution was centrifuged at 10,000 rpm for 10 minutes and then suspended by adding sterile water to the recovered strain and centrifuged again under the same conditions. This process was repeated twice to wash the cells. The washed cells were suspended in 50 mL of sterile water, and the strain suspension was injected into 20 kg of soil contaminated with diesel, and then sufficiently mixed.

The experimental design prepared a total of six pots by dividing 10 kg each of the soil inoculated with 20 kg of non-contaminated soil, 20 kg of contaminated soil and 20 kg of soil contaminated with contaminants. In this case, the soil injected with the strain to 20kg of soil contaminated with contaminants promoted plant growth, resistant to copper, and injected KC-1 to 3.59 x 108 cfu / kg to decompose diesel. Three of the six pots planted 10 kidney beans, and the other three pots each planted 10 corn seeds. The experiment was carried out for 17 days, and all pots were grown in an environment of 20-25 ° C. Until germination, enough water was given once a day, and after that, it was watered every other day.

After 17 days of cultivation, plants were carefully collected from pollen, dried for 2 days at 70 ° C. dry-oven, and the biomass of stem and root was measured.

Soil samples were collected from each pollen, and the residual TPH concentration remaining in the soil was measured by placing 5 g of soil in a test tube and adding 5 mL of hexane-acetone solution [1: 1 (v / v)]. It stirred for 30 minutes in the stirrer of 200 degreeC and 200 rpm. After the test tube was allowed to stand at room temperature for 30 minutes, the supernatant was collected, and the concentration of residual TPH dissolved in the solvent was analyzed using gas chromatography (5890 series II, Hewlett Packard, USA). (In Fig. 4, I is the diesel concentration of the initial soil, NC is the diesel concentration of the soil without plants and KC-1, B is the diesel concentration of soil planted only with kidney beans, B + R is a combination of kidney beans and KC-1 Soil diesel concentration, C is the diesel concentration of the corn-only soil, C + R is the diesel concentration of the soil with corn and KC-1).

As shown in the graph of FIG. 4 and Table 1, as a result of investigating the effect of KC-1 strains and plants on the removal of diesel from the soil, the TPH concentration in the control without plants and KC-1 strains was 5200.89 mg-TPH. / kg, no natural degradation occurred during the short growing period of 17 days. Under the condition of plants but without the KC-1 strain, kidney beans had a TPH concentration of 23.47 mg-TPH / kg, and corn had a TPH concentration of 969.35 mg-TPH / kg with 90% or more of both conditions. In the presence of -1 strain, kidney beans were 0mg-TPH / kg, corn was 245.02mg-TPH / kg and the inoculation of the strain improved the diesel removal. These results suggest that the KC-1 strain has two plant growth-promoting abilities, and because of its excellent diesel degradability in soils, it can increase the efficiency of rhizosphere restoration of soils contaminated with heavy metals and oils at the same time. There will be.

Concentration (mg / L) Reduction effect (%) Early soil 4816.93 ± 825.74 Soil free from plants and strains 5200.89 ± 156.80 Kidney Bean Plants, Unstrained 23.47 ± 40.66 99.54 Kidney beans plant, strain inoculation 0.00 ± 0.00 100 Corn Plant, Unstrained 969.35 ± 286.43 81.36 Corn plant, strain inoculation 245.02 ± 46.47 95.28

Korea Research Institute of Bioscience and Biotechnology KCTC11801BP 20101101

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Claims (4)

Alcaligenes sp. KC-1 (KCTC11801BP) strain isolated from wetlands.
Microbial agent containing Alcaligenes sp. KC-1 (KCTC11801BP) strain according to claim 1 as an active ingredient.
Method of purifying soil contaminated with oil and heavy metals using Alcaligenes sp. KC-1 (KCTC11801BP) strain according to claim 1.
A method for purifying soil contaminated with oil and heavy metals using Alcaligenes sp. KC-1 (KCTC11801BP) strain and plant according to claim 1.

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