KR102012630B1 - A method for treating oil polluted soil with using a microorganism - Google Patents

Abstract

The present invention provides a method for treating oil-contaminated soils containing a high concentration of TPH, and thus can administer biodegradation by administering specific microbial cells and culturing them under specific conditions, thereby effectively treating oil-contaminated soils containing a high concentration of TPH. Can be.

Description

A method for treating oil polluted soil with using a microorganism}

The present invention relates to a method for purifying TPH contaminated soil using specific microorganisms to purify a high concentration of TPH contaminated soil contaminated with petroleum compounds.

Today's industry is based on the mechanical industry, and almost every machine needs oil directly, such as lubricants and fuels. Especially in modern life, where the demand for vehicles is high, the amount of oil consumed by various vehicles is so large that the quantity cannot be measured. You have no choice but to contaminate your back.

Oil removal methods can be roughly classified into physical, chemical and biological methods.

Physical methods include soil washing and incineration. However, in the case of soil washing, the excavated soil is washed after excavating contaminated land, and thus the inconvenience of transferring the excavated soil to a washing facility is inconvenient. It can cause secondary environmental pollution such as sludge and sewage. Chemical methods include solidification, stabilization, solvent extraction, oxidation treatment, and the like, and have a relatively short treatment period but a disadvantage in that the treatment cost is high. Oxidation treatment of the above chemical method produces OH- which has much stronger oxidizing power than general oxidizing agent to finally oxidatively decompose various pollutants into CO2 and H2O or to convert biodegradable organic components into biodegradable organic materials. Examples of the oxidizing agent to be mentioned include Cl2, Ca (OCl), NaOCl, KMnO4, O3, H2O2 and the like. Currently, the most widely used oxidation treatment method is a method using a Fenton reaction. The Fenton reaction is a method of a high oxidation reaction in which H 2 O 2 reacts with Fe + 2 to generate OH- in an acidic solution. Although accompanied by side reactions, the main reaction of organics removal is to produce the OH-, which reacts with organics to produce organic radicals, which are oxidized as Fe +3 is reduced to Fe +2. Decompose Therefore, in recent years, hydrogen peroxide or hydrogen peroxide and iron salts in contaminated soil using the principle of the fenton reaction, which is caused by the oxidation treatment by ozone injection or by the catalytic action of iron ions during the oxidation reaction of hydrogen peroxide. The method of removing contaminants through oxidation reaction with organic contaminants in contaminated soil is injected into the soil, and Fenton-like reaction using iron ore contained in the soil as an activation catalyst for hydrogen peroxide is used to remove the diesel contaminated soil. Chemical oxidation treatment methods and the like have also been proposed. However, most of the studies on the oxidation treatment described above are in-situ techniques, which make it difficult to establish uniform reaction reagent injection conditions for contaminated soil and increase reagent costs due to excessive injection. Soil contaminated with oil has many challenges to be solved in terms of its treatment efficiency and economics. In addition to the physical or chemical methods described above, biological methods include soil cultivation, composting, bioventing, and plant restoration. Compared to the method, it is inexpensive and does not generate secondary environmental pollution. In particular, based on the fact that the oil which fatally pollutes the soil is decomposed completely by microorganisms although it takes time, in recent years, microbiological treatments have been developed as a biological method to make oil polluted soil more safe and economical. I was able to recover

However, studies related to the above treatments have many problems to be solved in terms of treatment efficiency and economic efficiency in case of soil contaminated by high concentration of oil.

In particular, existing biological treatments have treated contaminated soil with microorganisms in the range of about 2,000 TPH. Therefore, there was a limit to the purification of oil contaminated soil contaminated with high concentration of TPH by biological treatment.

In addition, in the case of contaminated soil exceeding 2,000 TPH of high concentration, it has been relied on physicochemical engineering such as thermal desorption, chemical neutralization, and contaminated soil movement. It is difficult and has many disadvantages in terms of uniform soil purification and cost.

Therefore, there is a need for a new soil recovery method that satisfies the conditions of uniform soil purification, cost savings, environmentally friendly decomposition and complete soil recovery as a method for purifying oil contaminated soils in the range of 10,000 ~ 20,000 TPH.

The inventors of the present invention have led to the present invention by studying to solve the above problems.

Republic of Korea Patent Publication No. 10-2013-0069281 Republic of Korea Patent Publication No. 10-2017-0045951 Republic of Korea Patent Registration 10-1671756 Republic of Korea Patent Registration 10-1551456 Republic of Korea Patent Registration 10-1112742 Republic of Korea Patent Registration 10-1196987

One aspect of the present invention is to provide a method for treating oil polluted soil containing a new high concentration of TPH that solves the above problems.

One aspect of the present invention is to provide a method for administering a specific bacterial community in the TPH treatment method of soil oil containing a high concentration of TPH.

In addition, one aspect of the present invention in the TPH treatment method of oil-contaminated soil containing a high concentration of TPH, before or after administration of the bacterial community, comprising a biodegradation step of a specific condition, to provide a method for treating the oil-contaminated soil TPH do.

In order to achieve the above object,

One aspect of the present invention is a method for treating soil containing high concentration of petroleum-based total hydrocarbon (TPH),

Administering a TPH treated bacterial community,

The TPH-treated bacterial community is Bacillus stearothermophilus Bs100 strain, Pseudomonas aeruginosa Sp300 strain selected from NBC2000 bacterial community with an accession number of KCTC 10623 BP, Pseudoosa aeruginosa (Pseudoosas aeruginosa) ) Tnh strain, Aeromonas hydrophila Aeng 17 strain, Serratia sp. Aeng18 strain, tar degradation gram positive bacterium Bs101 strain, Stennotrophomonas maltophialla Ntar 1 One or more of the strain, the W24 strain which is an oil degradation gram negative bacterium, and the Nz2001 strain which is a sulfur strain; Provided is a Bacillus cereus EBC106 strain selected from EBC1000 bacterial community with an accession number of KCTC 0652 BP. Provides a method for treating soil containing high concentrations of TPH.

In one aspect of the invention, the TPH treated bacterial community is Bacillus stearothermophilus Bs100 strain, Pseudomonas aeruginosa Sp300 strain, Pseudomonas aeruginosa Tnh strain, Aeromonas hydrophila Aeng 17 strain, Serratia sp.Aeng18 strain, tar degradation gram positive bacterium Bs101 strain, Stennotrophomonas maltophialla Ntar 1 strain, oil degradation Provided are methods for treating soil containing high concentrations of TPH, including Gram negative bacteria W24 strain, sulfur strain Nz2001 strain and Bacillus cereus EBC106 strain.

In one aspect of the invention, the soil containing a high concentration of TPH provides a method for treating soil containing a high concentration of TPH, the concentration of TPH is 2,000 mg / kg or more.

In one aspect of the invention, the method comprises the steps of importing TPH contaminated soil; Crushing the soil; Inverting the crushed soil; And administering the TPH treated bacterial community to the inverted contaminated soil. Provides a method for treating soil comprising a high concentration of TPH.

In one aspect of the invention, the method after the step of administering the bacterial community, supplying water; And feeding nutrients; provides a method of treating soil comprising high concentration of TPH.

In one aspect of the invention, in the step of inverting the soil contaminated soil is characterized in that over 50 times, provides a method for treating soil containing a high concentration of TPH.

In one aspect of the present invention, the TPH treated bacterial community is administered at a concentration of 140L or more for about 110 tons of soil to be purified by diluting 10 ⁸ to 10 ¹⁰ cfu / ml culture stock 100 times or more. Provide a method for treating soil containing TPH.

In one aspect of the invention, the soil containing a high concentration of TPH is to provide a method for treating soil containing a high concentration of TPH, the concentration of TPH is 2,000mg / kg to 20,000 mg / kg.

In one aspect of the invention, the nutrients are nutrients containing nitrogen (N) and phosphorus (P) at a concentration of 2 to 5: 1, to provide a method for treating soil containing a high concentration of TPH. .

In one aspect of the invention, the step of crushing the contaminated soil is to provide a method for treating soil containing a high concentration of TPH, crushing the particles of contaminated soil to a uniform size.

In one aspect of the invention, the method is to provide a method for treating soil containing high concentration of TPH, further comprising the step of biodegradation for 4 to 30 days.

The method according to an aspect of the present invention can significantly reduce the concentration of TPH in soil contaminated with a high concentration of TPH while at the same time making the concentration of TPH in the soil much lower than the legal standard.

The method according to an aspect of the present invention can significantly reduce the concentration of PCBs in insulating oil without contamination problems such as solid waste and soil.

The method according to an aspect of the present invention can significantly reduce the concentration of TPH in the soil at economical facility costs.

The method according to an aspect of the present invention can purify the contaminated oil soils in the concentration of TPH range 10,000 ~ 20,000 ppm.

The method according to an aspect of the present invention can purify the contaminated oil contaminated soil with a TPH concentration of 10,000 to 20,000 ppm to a TPH concentration of 500 ppm or less.

The method according to an aspect of the present invention can purify the contaminated oil contaminated soil with a TPH concentration of 10,000 ~ 20,000 ppm to a TPH concentration of 300 ppm or less.

The method according to an aspect of the present invention can purify the contaminated oil contaminated soil with a TPH concentration of 10,000 ~ 20,000 ppm to a TPH concentration of 200 ppm or less.

The method according to an aspect of the present invention can be purified to contaminated oil contaminated soil with a TPH concentration of 10,000 ~ 20,000 ppm to a TPH concentration of 150ppm or less.

Method according to an aspect of the present invention can be a uniform soil purification.

The method according to an aspect of the present invention can be purified soil at low cost.

The method according to one aspect of the present invention is capable of complete soil recovery compared to the physicochemical method.

The method according to an aspect of the present invention can treat the soil contaminated with high concentration of TPH at low cost and high efficiency.

1 is a photograph of a process method for treating soil containing a high concentration of TPH.
2 is a photograph of the soil cultivation method during the treatment method of the soil containing a high concentration of TPH.
Figure 3 is a TPH analysis table according to the present invention empirical test results.
Figure 4 is a graph of TPH according to the number of days passed, the number of turnover, microbial supply.

In the present specification, when a range is described for a variable, it will be understood that the variable includes all values within the described range including the listed endpoints of the range. For example, the range "5 to 10" includes any subrange such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, as well as values of 5, 6, 7, 8, 9, and 10. And any value between integers that are within the scope of the described range, such as 5.5, 6.5, 7.5, 5.5-8.5, 6.5-9, and the like. Also for example, the range of “10% to 30%” ranges from 10% to 15%, 12% to 10%, 11%, 12%, 13%, etc. as well as all integers including up to 30%. It will be understood to include any subranges such as 18%, 20% to 30%, etc., and to include any value between valid integers within the range of the stated range, such as 10.5%, 15.5%, 25.5% and the like.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention is a method for treating soil containing high concentration of petroleum-based total hydrocarbon (TPH),

Administering a TPH treated bacterial community,

The TPH-treated bacterial community is Bacillus stearothermophilus Bs100 strain, Pseudomonas aeruginosa Sp300 strain selected from NBC2000 bacterial community with an accession number of KCTC 10623 BP, Pseudoosa aeruginosa (Pseudoosas aeruginosa) ) Tnh strain, Aeromonas hydrophila Aeng 17 strain, Serratia sp. Aeng18 strain, tar degradation gram positive bacterium Bs101 strain, Stennotrophomonas maltophialla Ntar 1 One or more of the strain, the W24 strain which is an oil degradation gram negative bacterium, and the Nz2001 strain which is a sulfur strain; Provided is a Bacillus cereus EBC106 strain selected from EBC1000 bacterial community with an accession number of KCTC 0652 BP. Provides a method for treating soil containing a high concentration of TPH.

In one aspect of the invention, the TPH treated bacterial community is Bacillus stearothermophilus Bs100 strain, Pseudomonas aeruginosa Sp300 strain, Pseudomonas aeruginosa Tnh strain, Aeromonas hydrophila Aeng 17 strain, Serratia sp.Aeng18 strain, tar degradation gram positive bacterium Bs101 strain, Stennotrophomonas maltophialla Ntar 1 strain, oil degradation Provided are methods for treating soil containing high concentrations of TPH, including Gram negative bacteria W24 strain, sulfur strain Nz2001 strain and Bacillus cereus EBC106 strain.

In one aspect of the invention, the soil containing a high concentration of TPH provides a method for treating soil containing a high concentration of TPH, the concentration of TPH is 2,000 mg / kg or more.

In one aspect of the invention, the method comprises the steps of importing TPH contaminated soil; Crushing the soil; Inverting the crushed soil; And administering the TPH treated bacterial community to the inverted contaminated soil. Provides a method for treating soil comprising a high concentration of TPH.

In one aspect of the invention, the method after the step of administering the bacterial community, supplying water; And feeding nutrients; provides a method of treating soil comprising high concentration of TPH.

In one aspect of the invention, in the step of inverting the soil contaminated soil is characterized in that over 50 times, provides a method for treating soil containing a high concentration of TPH.

In one aspect of the present invention, the TPH treated bacterial community is administered at a concentration of 140L or more for about 110 tons of soil to be purified by diluting 10 ⁸ to 10 ¹⁰ cfu / ml culture stock 100 times or more. Provide a method for treating soil containing TPH.

In one aspect of the invention, the soil containing a high concentration of TPH is to provide a method for treating soil containing a high concentration of TPH, the concentration of TPH is 2,000mg / kg to 20,000 mg / kg.

In one aspect of the invention, the nutrients are nutrients containing nitrogen (N) and phosphorus (P) at a concentration of 2 to 5: 1, to provide a method for treating soil containing a high concentration of TPH. .

In one aspect of the invention, the step of crushing the contaminated soil is to provide a method for treating soil containing a high concentration of TPH, crushing the particles of contaminated soil to a uniform size.

In one aspect of the invention, the method is to provide a method for treating soil containing high concentration of TPH, further comprising the step of biodegradation for 4 to 30 days.

Hereinafter, the isolation, identification and activity of the NBC2000 bacterial community and the EBC1000 bacterial community included in the TPH treated bacterial community used in the present invention will be described in detail.

NBC2000  Isolation, Identification and Activity of Bacterial Communities

One. Environmental hormones  Isolation of Degradable New Bacterial Communities

(1) Isolates bacteria that degrade PCBs, PCP, dioxin, PCE, toluene, taric acid, etc. from soil collected in Korea, Southern Europe, Oceania, etc.

1 gram of collected soil was shaken for 2 to 3 days in Luria-Bertani liquid medium (10 g of Bacterium-Tryptone, 5 g of Bacterial yeast extract, 10 g of NaCl + 1 liter of demineralized water), and then 1 ml was taken to take 1 ml Each colony was isolated from 10 g of Bakto-Tryptone, 5 g of Bakto yeast extract, 10 g of NaCl, 1.5% of agar, and 950 ml of demineralized water. Select each colony that appeared at this time and select the minimum liquid medium containing PCBs, PCP, PCE, toluene and taric acid (K ₂ HPO ₄ 0.065g, KH ₂ PO ₄ 0.017g, MgSO ₄ 0.1g, NaNO ₃ 0.5g + Demineralized water 1 Liters) were sequentially inoculated and shaken at 25 to 30 ^o C for at least 3 days. In addition to confirming the reduction of each harmful substance, 1 ml of shaking culture solution was taken and sequentially inoculated into Luria-Bertani agar medium and incubated at 25 to 30 ^o C for 3 to 5 days.

 Each colony separated from pure water was reinoculated into the above-described minimal liquid medium, followed by shaking culture, followed by different cultures of colonies of different shapes formed in the Luria-Bertani solid medium, and the same colonies appearing in the passage of the colonies. About 50 species were separated.

(2) The bacteria isolated from the above inoculated sequentially in a minimal medium containing PCBs, PCP, PCE, toluene, tartaric acid at a high concentration step by step and repeat in the same manner as in (1) to survive at a higher concentration And 26 species were separated according to morphological differences.

The 26 bacteria obtained here were composed of a community named NBC2000, and each of the bacteria constituting the bacterial community was Cy100, Cy101, Cy102, Cy103, Cy104, Cy105, Cy106, Cy107, Ntar1, Ntar2, Ntar3, Gc300, Gc500, Gc501, Bs100, Aeng17, Aeng18, Sp300, Tnh, Djhc, Pcpts, EBC106, EBC107, Bs101, W24 and Nz2001.

The bacterial community NBC2000 according to the present invention was deposited on April 16, 2004 at the Genetic Resource Center of the Korea Research Institute of Bioscience and Biotechnology under accession number KCTC 10623 BP.

 2. Bacterial Community NBC2000  Establishment of Optimum Conditions for Growth

Luria-Bertani nutritional medium [10 g of bacto-tryptone, 5 g of bacto-yeast extract, 10 g of sodium chloride (NaCl) / 1 liter of demineralized water] 8, temperature 25 ~ 30 ^o C, shaking (shaking) rotation at 80 ~ 120rpm 48 ~ 96 hours incubation for optimum growth, even in subcultures grow well under the same conditions.

3. Bacterial Community NBC2000  Biochemical Identification

23 bacteria were identified by API Kits API20E, API20NE, API50CH and API50CHB purchased from bioMerieux (bioMerieux sa 69280 Marcy I'Etoile / France). To Table 5).

4. Bacterial Community NBC2000  Isolation of each bacterium

1) Strain isolation and method

Each strain consists of NBC2000 purely separated from soils such as Korea, New Zealand, Sweden, and Cyprus, all of which have mobility characteristics and can be applied to a wide variety of soils. The method of morphological characterization of individual strains in bacterial community NBC2000 is as follows.

(A) Cultivation in Luria-Bertani agar medium (10 g of Bacterium-Tryptone, 5 g of Bacterial yeast extract, 10 g of NaCl, 1.5% agar, 950 ml of demineralized water)

Cy100: After 48 hours of incubation, the surface appears as an uneven irregular yellow colony and grows to a size of 4 mm.

Cy101: Appears as translucent ivory convex colonies after 48 hours of incubation and grows to a size of 2.5 mm.

Cy102: Appears as yellow round colonies after 48 hours of incubation and grows to 2 mm in size.

Cy103: After 48 hours of incubation, it appears as an ivory round and flat colony, growing to a size of 4 mm.

Cy104: Appears as translucent ivory round colonies after 48 hours of incubation and grows to 4 mm in size.

Cy105: Appears as ivory round colonies after 48 hours of incubation and grows to a size of 2.5 mm.

Cy106: After 48 hours of incubation, it appears as a round colony with a matt white color and grows to a size of 1.5 mm.

Cy107: After 48 hours of incubation, it appears as a matt white round colony and grows to a size of 1 mm.

Ntar1: After 24 hours of incubation, it appears as a translucent yellowish, round, glossy colony, growing to a size of 1 mm.

Ntar2: After 24 hours of incubation, it appears as a round, glossy colony of beige and ivory, growing to a size of 2 mm.

Ntar3: After 48 hours of incubation, it appears as an ivory-colored round and glossy colony, growing to a size of 1.5 mm.

Gc300: After 24 hours of cultivation, it appears as an ivory round, glossy convex colony and grows to a size of 3 mm.

Gc500: After 24 hours of incubation, it appears as an ivory round, glossy convex colony, growing to a size of 1.5 mm.

Gc501: After 24 hours of cultivation, it appears as an ivory round, glossy convex colony and grows to a size of 2.5 mm.

Bs100: Appears as bright ivory round colonies after incubation for 24 hours and grows to 2 mm in size.

Aeng17: After 24 hours of incubation, it appears as round or irregular colonies of red or ivory color and grows to 3mm in size.

Aeng18: After 24 hours of incubation, it appears as round or irregular colonies of red or ivory color and grows to 3mm in size.

Sp300: After 24 hours of incubation, it appears as round or irregular colonies of beige and brown color, growing to a size of 3 mm.

Tnh: After 40 hours of incubation, the translucent ivory surface appears as an uneven metallic colony and grows to 4 mm in size. Over time, the medium changes color to indigo blue.

Djhc: After 24 hours of cultivation, the ivory is a round, smooth surface with viscous, glossy colonies that grow to a size of 3 mm.

Pcpts: After 24 hours of incubation, they appear as beige and brown round or irregular colonies and grow to a size of 3 mm.

EBC106: Appears as a flat matte colony that grows to an uneven surface after culturing for 24 hours and grows to a size of 7 mm.

EBC107: After 40 hours of incubation, the semi-transparent ivory-colored surface appears as uneven irregular colonies and grows to a size of 4 mm.

Bs101: After culturing for 24 hours, it is yellowish ivory, rounded and surrounded by a rim, growing to a size of 5 mm. It is a gram-positive bacterium that has motility.

W24: Pale yellow, round, glossy and convex colonies that grow to a size of 1.5 mm after 48 hours of incubation. Gram-negative bacteria are motility.

Nz2001: Appears as white matte colonies after 48 h of incubation and grows to 2.5 mm. During prolonged incubation, hyphae appear at the edges of the colony, and there is mobility.

(B) Maconkey solid medium [MacConkey agar: 17g peptone, Proteose peptone 3g, 10g lactose, 1.5g Bil Salts No.3, 5g sodium chloride, 13.5g agar, 0.03g neutral red, 0.001 g of crystal violet, 1 liter of demineralized water, pH7.3 ~ 7.5]

Cy100 appears in light brown small colonies,

Ntar1 is transparent light brown with uneven surface,

Ntar2 is a small beige colony,

Ntar3 is a light brown transparent brown colony,

Gc300 is a dark pink colony with beige edges,

Gc500 is most of the beige and pink colonies,

Gc501 is a dark pink colony with beige edges.

Aeng17 and Aeng18 are dark red colonies,

Sp300 is a beige colony

Tnh is a dark khaki colony,

Djhc is pink in the center of the colony and beige at the edges,

Pcpts appear in light khaki

EBC107 appears as a very pale pink colony,

Nz2001 appears as a pink beige colony,

Cy101, Cy102, Cy103, Cy104, Cy105, Cy106, Cy107, Bs100, EBC106, Bs101, W24 strains do not show colonies even after 48 hours of incubation.

(C) Desoxycholate agar [Desoxycholate agar: Protez peptone 10g, lactose 10g, sodium desoxycholate 0.5g, sodium chloride 5g, sodium citrate 2g, agar 15g, neutral red 0.03g, deionized water 1 liter, pH7.3 ... 7.5] colony color after 48 h incubation

Cy101, Cy102, Cy103, Cy104, Cy105, Cy106, Cy107, Bs100, EBC106, Bs101, 24 strains do not show colonies even after 48 hours of culture.

Cy100 is a pale pink colony that looks like a flower

Ntar1, Ntar2, and Ntar3 are transparent orange colonies,

Gc300 and Gc501 are dark pink colonies with beige edges,

Gc500 is a light reddish beige colony,

Aeng17 and Aeng18 are dark red colonies,

Sp300 is a transparent light brown colony,

Tnh is a light brown colony metallic,

Djhc is a mix of pink and beige

Pcpts are transparent brown colonies,

EBC107 appeared as a yellow colony.

Nz2001 appeared as a red beige colony.

Bs101, sulfur strain Nz2001, and oil degradation strain W24, which decompose tar, were identified as unidentified strains because it was difficult to identify by the present method.

5. NBC2000  Each of the individual strains Environmental hormone  Resolution

1) Range of environmental hormone degradation of each isolate

PCBs, dioxins, PCE, toluene and taric acid are measured by the bacterial community constituent bacteria under optimal conditions in the laboratory, and sulfur and PCP are average values measured at individual strain levels.

Pseudomonas sp. Cy100: 700 ppm PCBs;

Serratia sp. Aeng18: PCP 500 ppm, taric acid, dioxin 100 ng / kg;

Serratia sp. Ntar2: taric acid;

Pseudomonas sp. Djhc: 1000 ppm PCP, 300 ng / kg dioxin;

Pseudomonas sp. Ntar 3: taric acid;

Pseudomonas sp. EBC107: 700 ppm PCBs, 100 ppm PCP, dioxin 100 ng / kg, PCE 50,000 mg / kg;

Pseudomonas sp. Tnh: 700 ppm PCBs, 500 ppm PCP, dioxin 300 ng / kg, PCE 50,000 mg / kg, taric acid;

Aeromonas sp. Aeng17: taric acid, PCP 100 ppm, dioxin 50 ng / kg;

Pseudomonas sp. Pcpts: PCP 1,000 ppm, dioxin 500 ng / kg;

Stenotrophomonas sp. Ntar1: Taric acid;

Pseudomonas sp. Sp300: taric acid, PCP 700 ppm, dioxin 100 ng / kg;

Chryseomonas sp. Gc501: PCP 500 ppm, dioxin 100 ng / kg;

Chryseomonas sp. Gc500: PCP 500 ppm, dioxin 100 ng / kg;

Chryseomonas sp. Gc300: PCP 300 ppm, dioxin 100 ng / kg;

Brevundimonas sp. Cy101: PCBs 150 ppm;

Brebundimonas sp. Cy102: PCBs 150 ppm;

Brevundimonas sp. Cy103: 700 ppm PCBs;

Bacillus sp. Bs100: Taric acid;

Bacillus sp. Cy104: PCBs 800 ppm;

Bacillus sp. Cy105: 700 ppm PCBs;

Bacillus sp. Cy106: 1,000 ppm PCBs;

Bacillus sp. Cy107: 150 ppm PCBs;

Bacillus sp. EBC106: 700 ppm PCBs, 1,000 ppm PCP, dioxin 300 ng / kg, taric acid, PCE 50,000 mg / kg, toluene 50,000 mg / kg;

Gram positive bacterium Bs101: taric acid;

Gram negative bacteria W24: 100 ppm TPH, 100 ppm toluene;

Sulfur Strain Nz2001: Sulphur, Taric Acid, Dioxin 50 ng / kg

EBC1000  Isolation, Identification and Activity of Bacterial Communities

Hereinafter will be described in detail the isolation, identification and activity of the EBC1000 bacterial community according to the present invention.

1.Hydrolysis  toxicity Waste  Which can disassemble waste New  Isolation of Bacterial Communities

(1) separating 40 kinds of microorganisms by shaking culture of samples collected from the soil and wastewater, such as Ulsan Industrial Complex, in a liquid medium mixed with difficult-to-dissolve wastewater

1 g of soil collected from Ulsan Industrial Complex and 10 ml of waste water were collected from waste acid and waste alkaline wastewater liquid media (K ₂ HPO ₄ 0.65 g, KH ₂ PO ₄ 0.17 g, MgSO ₄ A medium formed by diluting 10% of waste acid alkaline wastes containing 0.1g, 0.5g NaNO ₃ and the non-degradable chemicals of pharmaceutical wastes and petrochemical wastes in 1l of deionized water, pH 0 ~ 14) and chlorine Liquid medium (K ₂ HPO ₄ 0.65 g, KH ₂ PO ₄ 0.17 g, MgSO ₄ 0.1 g, 0.5 g NaNO ₃ and 50 ppm contaminated phenolic compound (pentachlorophenol: PCP) were sequentially inoculated in a medium formed by dissolving 1 l of deionized water (pH 7.2) and shaken for 5 days at 20-30 ° C.

Take 1 ml of the shake culture solution, waste acid, waste alkali solid medium (waste acid, waste alkali, medium formed by adding only 1.5% of agar (agar) to medium, pH neutralization) and chlorine compound solid medium (BTB 20 in chlorine compound liquid medium) ㎎, medium formed by adding 1.5% agar) was sequentially inoculated and incubated at 20-30 ° C for 3-10 days.

Each colony was purified by re-inoculation on the same medium as above, and then shaken and cultured, and 40 kinds of useful bacteria having separate colonies of different shapes and the same colonies when subcultured were isolated.

(2) Separating 9 strong bacteria by culturing the bacteria isolated in step (1) in a medium gradually increasing the concentration of the hardly degradable waste liquid

Bacteria isolated in step (1) were used for pharmaceutical wastes and petrochemical wastes. Inoculated sequentially in each of the minimum medium was added and repeated in the same manner as in step (1), nine species were isolated according to colony type mainly viable strain at a higher concentration.

The nine bacteria obtained here were composed of one community and named EBC1000, and each bacteria constituting the bacterial community was named EBC100, 101, 103, 104, 105, 106, 107, 108 and 109, respectively.

The bacterial community EBC1000 according to the present invention was deposited internationally with the accession number KCTC 0652 BP at the Genetic Resource Center in Korea Biotechnology Research Institute on August 12, 1999.

2. Bacterial Community Of EBC1000  Establish optimal conditions for growth

PH 5-8 at Luria-Bertani nutrient medium (10 g of bacto-tryptone, 5 g of bacto-yeast extract, 10 g of NaCl / l of demineralized water), temperature 25-35 ℃, shaking (shaking) 50 ~ 100rpm per minute incubation for 24 to 48 hours shows the optimum growth, even in subcultures grow well under the same conditions.

3. Bacterial Community Of EBC1000  Sympathy

EBC 100, 101, 103, 104, 105, 106, 107, 108, and 109 strains of the bacterial community EBC1000 were mixed with Gram-negative bacteria and Gram-positive bacteria. After 24 hours of incubation in LB solid medium, EBC100 is a circular colony with a diameter of 1 mm, EBC101 is a large round about 2 mm, EBC103 is a thick round about 2 mm, EBC104 and EBC105 are a small round about 0.5 mm, and EBC106 is 3 mm EBC107 is about 1.2mm brown colony, EBC108 is about 1mm yellow colony, EBC109 is about 2mm double circle. Nine strains are aerobic and breathable strains, and EBC100, 101, 103, 106, 107, and 108 have strong viability even under strong acid (pH3 ~ 4) and strong alkali (pH9 ~ 11) conditions, and EBC104, 105, and 109 grow. This was slow. Motility is shown in EBC100, 104, 105, and 109.

Looking at the genus of the strains that make up the bacterial community EBC1000, EBC100, 101 and 103 belong to the genus Klebsiella, EBC105 belong to the genus Providencia, EBC104 and EBC109 belong to the genus Escherichia, EBC106 belong to the genus Bacillus, EBC107 is Gram-negative bacteria and EBC108 is Gram-positive bacteria.

To summarize the characteristics of each strain as described above are shown in Table 6 to Table 8.

sample EBC 100 EBC 101 EBC 103 Gram strain
Catalase
Oxidase
Urease
Citrase utilizat.
Glucose utilizat.
VP test
Lysine decarboxylase
Ornithine decarboxylase -
+
-
+
+
+
+
+
- -







-

sample EBC 100 EBC 101 EBC 103 Inositol
Arabinos
Mannitol
Rhamnos
Glucose
Sorbitol
α-cyclodextrin
dextrin
Glycogen
Adonitol
D-Arabitol
Cellobiose
D-fructose
L-fucose
D-galactose
α-lactose
Maltose
D-rapinose
D-trehalose
Methyl-pyruvate
Citric acid
Formic acid
Malonic acid
Succinic acid
D-alanine
L-alanine
L-glutamic acid
L-serine
D, L-lactic acid
motility
D-Mannose +
+
+
+
+
+
-
+

+


+
+
+



+
-
+

-


+

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sample EBC 104 EBC 105 EBC 109 Gram strain
ONPG
Arginine
Lysine
Ornithine
Sodium citrate
Sodium thiosulfate
Urea
Tryptophan
Indole
Pyruvate creatine sodium
Kohn Charcoal Gelatin
Glucose, potassium nitrate
Manantiol
Inositol
Rhamnos
Sucrose
Melibiose
Amigdalin
Arabinos
Oxidase
Reduction of Nitrate to Nitrite
Reduction of Nitrate to N ₂ Gas
Mobility
Oxidation of glucose
Fermentation of glucose
Antibiotic resistance

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Km ^R
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Km ^S
Ap ^R
Tc ^R

Meanwhile, fatty acid methyl esters (FAMEs) of each strain were analyzed by gas chromatography.

Hewlett Packard series II Gas Chromatograph model 5890A; Microbial ID. Inc., Delaware, USA was used for the analysis of FAMEs.The separation column was 25m × 0.22mm × 0.33㎛ methylphenylsilicone. This fused capillary column (HP 19091B-102) was used.

In the gas chromatography, the carrier gas is hydrogen, the column head pressure is 10 psi, the split ratio is 100: 1, and the split vent is 50 ml / min, Septum Purge is 5 ml / min, FID hydrogen is 30 ml / min, FID nitrogen is 30 ml / min, FID air is 400 ml / min, initial temperature is 170 ° C., The program rate is 5 ° C./min, the final temperature is 270 ° C., the FID temperature is 300 ° C., the injection port is 250 ° C. and the injection volume is 2 μl.

The FAMEs graph used Microbial Identification System Software (Microbial ID, Inc., Delaware, USA) and peak identification and stagnation compared to a standard calibration mixture (Microbial ID, Inc., Delaware, USA). The time, peak area, and peak percentage were obtained.

As a result of analysis of FAMEs for each strain of EBC100, 101 and 103, the cellular fatty acids composition of EBC100 was C12: 0, C14: 0, C16: 0, C16: 1, C17: 0 cyclo, C14: 0 3OH, EBC101 is C12: 0, C14: 0, C15: 0, C16: 0, C17: 0 cyclo, C14: 0 3OH. EBC103 was found to be C14: 0, C15: 0, C16: 0, C17: 0 cyclo, C14: 0 3OH.

4. Bacterial Community EBC1000  Isolation of each bacterium

The method of separating individual strains from bacterial community EBC1000 is as follows.

EBC106 (genus Bacillus), EBC107 (Gram-negative bacteria) and EBC108 (Gram-positive bacteria) were identified as Luria-Bertan agar (10 g of Bacterium-Tryptone, 5 g of Bacterium-Yeast Extract, 10 g of NaCl, 1.5% of agar, 950 ml of demineralized water) When cultured in nutrient solid medium of EBC106, the typical Bacillus-shaped thick wrinkles, EBC107 can be distinguished by the morphological features of brown colony, EBC108 is yellow colony.

Other strains include Desoxycholate agar; 10 g of Bactobacillus peptone, 10 g of Bactobacillus lactose, 1 g of sodium desoxycholate, 5 g of sodium chloride, 2 g of dipotassium, 1 g of Fe citrate, 1 g of sodium citrate, 15 g of bacto agar, neutral The colony morphology of each strain can be confirmed by diluting the bacterial community EBC1000 in 0.03 g of red (neutral red) / deionized water 1-Difco manual, 1984]. After incubation in desoxycholate agar for 24 hours, EBC100, 101, and 103 showed reddish white viscosities and slight differences in size. EBC104 is red and white and viscous, EBC105 is brown, EBC106, 107 is light brown, EBC108 is colorless, and EBC109 is red (Dictionary of Microbiology and Molecular Biology, 2nd, Paul Singleton Diana Sainsbury 1987).

5. Bacterial Community Of EBC1000  characteristic

(1) Degradation rate of sodium alkylarylnaphthalene sulfonate (Tamol-SN) by bacterial community EBC1000

K ₂ HPO ₄ 0.065 g, KH ₂ PO ₄ 0.017 g, MgSO ₄ In a minimum medium (pH 7.2) made by dissolving 0.1 g and 0.5 g of NaNO _{3 in} 1 L of demineralized water, Tamol-SN was gradually increased to 500, 1000, 2000, and 4000 ppm, and the bacterial community EBC1000 was inoculated. The concentration of tamol was measured.

Tamol-SN (ppm) Degradation rate (mg / l / h) 500
1000
2000
4000 1.3
3.8
5.2
4.0

As shown in Table 9, the decomposition rate of tamol per hour was 1.3 ppm (l / h) at 500 ppm, and 4.0 (mg / l / h) at 4000 ppm.

(2) Degradation Rate of Digested Phenol Compounds (PCP) by Bacterial Community EBC1000

After dissolving BTB 20mg in 1L of demineralized water under the minimum liquid medium conditions as shown in (1), the contaminated digestive phenolic compound (PCP) was administered as shown in Table 4, and the inoculation of bacterial community EBC1000 was followed by absorbance and contaminated digestive phenolic compound over time. The concentration of (PCP) was measured. The results are shown in Table 10.

Digestive Phenolic Compounds (PCP) (ppm) Degradation rate (mg / l / h) 200
500
1000
2000 0.9
6.5
5.0
4.5

Bacterial community NBC2000  And EBC1000  According to the combination of each strain Environmental hormone  Resolution

Although the resolution of individual strains is limited, treatment of the bacterial community of the whole NBC2000 or EBC1000 or each combination of its constituent strains in a sample or contaminated environment results in a more extensive and efficient degradation effect. This is a synergistic effect of emergence due to mutual cooperation as a bacterial community rather than the function of individual strains constituting NBC2000 and EBC1000. None of the strains can degrade complex TPH with a single strain, but according to the combination of each strain of NBC2000 and each strain of EBC1000 (KCTC0652 BP), the strains can be effectively complemented by the complementary strains and the gene transfer exchange in the community. Will be displayed. In order to prove this, the inventor performed the following experiment.

Hereinafter, the present invention will be described in detail based on the following experimental examples, but the present invention is not limited thereto.

NBC2000 bacterial community  And EBC1000 bacteria community  Determination of TPH Degradation Effect of Selected Mixed Bacteria

3 kg of tartaric acid from Sweden beach + Luria Verdani liquid medium + strain + small liquid medium [TPH (total petroleum hydrocarbons) 42,600mg / kg, PAH (polycyclic aromatic hydrocarbon) 190mg / kg, BTEX (benzene, toluene, ethylbenzene, Xylene) 3.76 mg / kg, benzene 0.33 mg / kg, ethylbenzene <0.05 mg / kg, toluene <0.05 mg / kg, xylene 0.59 mg / kg, EOX (extractable organic halogens) 76.5 mg / kg, POX (purgeable organic halogens) ) 61.8mg / kg, Halogenated hydrocarbons <0.01mg / kg, Chloro-benzene <0.1mg / kg, Chloro-phenol <0.1mg / kg, PCBs <0.01mg / kg, Cyanide <0.01mg / kg , Ria-bertani liquid medium sterilized with 100% 1 gram of tartaric acid containing arsenic 0.76mg / kg, lead 933mg / kg, cadmium 0.14mg / kg, mercury 0.66mg / kg, 1.8% sulfur, pH 1.2 (10 g of Baktotrypton, 5 g of Bakto yeast extract, 10 g of NaCl, 1 liter of demineralized water) After mixing to 50 ml, the pH was adjusted to 8 using Na4OH.

Then, EBC106, strain was inoculated at 10 ⁵ cfu / ml among BS100, Ntar1, W-24, SP300, Tnh, Aeng17, Aeng18, Bs101, Nz2001 and ebc1000 in strains of NBC2000. Nitrogen and phosphorus were added by using a minimum liquid medium of 20ml at 50 days and 70 days, and 20ml of demineralized water was replenished at 90 days, 100 days, 120 days and 130 days. As a result, inoculation of individual strains showed little degradation, whereas inoculation with a combination of two or more strains showed good degradation efficiency.

Inoculation strain  analysis:

NBC2000: Bacillus stearothermophilus BS100, Pseuomonas aeruginosa SP300, Pseudomonas aeruginosa Tnh, Aeromonas hydrophila Aeng17, Serratia sp. Aeng18, tar degradation gram positive bacteria Bs101, Stenotrophomonas maltophialla Ntar1, oil degradation gram negative bacteria W24, sulfur strain Nz2001

EBC1000 : Bacillus cereus EBC106

In the experiment, Aeng 17 and Aeng18 strains increased by about 1,000 times, and Tnh also increased by about 10 times. According to the linkage metabolism of NBC2000 and EBC1000-related strains, which withstands 20 kinds of poisons including petroleum compounds, the metabolites are completely decomposed with carbon dioxide.

It has been shown to endure and decompose crude oil and more than 20 complex poisons, and has been found to efficiently decompose and purify petroleum compounds contaminated with high concentrations in a wide variety of areas. .

The strains selected as described above were tested through an empirical investigation that the concentration of TPH in oil-contaminated soil can be significantly reduced through the following process.

Of high TPH soil pollution In thermal desorption  Restoration technology by microorganism

1. Overview of test

1-1. summary

-Site Name: TKP Waste Reservoir Environmental Pollution Purification Project

-Location: Jangseong-dong, Buk-gu, Pohang, Gyeongsangbuk-do

-TPH concentration: Reduced initial average concentration of 18,596ppm to completion average of 166ppm

-Scope of inspection: Demonstrating the contaminated soil using mixed microorganisms by Landfarming of oil-contaminated soils during ex-situ

1-2. purpose

-Development of national cleanup standard model, national environmental management capacity

-Proven optimized purification method

-Development of technology to purify oil polluted soil in compliance with legal standards

1-3. term

-Construction period: The total construction period is 175 days, and the cultivation period is 148 days

1-4. Destination location

-Address: Within the site of 858-1, Jangseong-dong, Buk-gu, Pohang, Gyeongsangbuk-do

2. Test objectives and plans

2-1. Pollution Status Survey

-Soil pollution in the project area required cooperation with the supervisory authorities.

-The inspection of empirical test samples confirmed the current state of oil pollution.

2-2. Purification Standards and Purification Goals

-The inspection item criteria of the Enforcement Decree of the Soil Environment Conservation Act are as follows. The unit is mg / kg.

matter Standard of Concern An approximate criterion Autumn area My area Autumn area My area Benzene, Toluene, Ethylbenzene, Xylene (BTEX) - 80 - 200 Petroleum Hydrocarbons
(TPH) 500 2,000 1,200 5,000

2-3. Validation plan

After submitting an empirical inspection plan (3 days), a plan for the operation of soil dressing, purification verification and completion report (about 5 months) was established.

3. Details of the test

3-1. Process photo

Process pictures were as shown in Figures 1a to 1b.

3-2. Soil tillage

It was constructed by the soil cultivation method as shown in Figure 2 below.

4. Test result

4-1.TPH Analysis Table

As a result of the empirical test, the TPH analysis table was as shown in FIG. 3.

4-2. TPH graph

As a result of the empirical test, the TPH graph is as shown in FIGS. 4A to 4C.

TPH contaminated soil purification method using the bacterial community of the present invention as described above was confirmed that can effectively purify the high concentration of TPH.

Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Korea Research Institute of Bioscience and Biotechnology KCTC10623BP 20040416 Korea Research Institute of Bioscience and Biotechnology KCTC0652BP 19990811

Claims (10)

In the processing method of the soil containing the high concentration of petroleum-based total hydrocarbon (TPH) whose concentration of petroleum-based total hydrocarbon is 10,000 mg / kg or more,
Importing TPH contaminated soil;
Crushing the soil;
Inverting the crushed soil;
Administering the TPH treated bacterial community to the inverted contaminated soil;
Supplying moisture after administration of the bacterial community;
Supplying nutrients; And
Biodegrading for 4 to 30 days; includes,
The TPH-treated bacterial community is Bacillus stearothermophilus Bs100 strain, Pseudomonas aeruginosa Sp300 strain selected from NBC2000 bacterial community with an accession number of KCTC 10623 BP, Pseudoosa aeruginosa (Pseudoosas aeruginosa) ) Tnh strain, Aeromonas hydrophila Aeng 17 strain, Serratia sp. Aeng18 strain, tar degradation gram positive bacterium Bs101 strain, Stennotrophomonas maltophialla Ntar 1 Strain, W24 strain which is an oil degradation gram negative bacterium, and Nz2001 strain which is a sulfur strain; Bacillus cereus EBC106 strain selected from the EBC1000 bacterial community with an accession number of KCTC 0652 BP;
In the step of inverting the crushed contaminated soil is contaminated soil more than 50 times,
The TPH-treated bacterial community is administered at 140 L or more with respect to 110 tons of soil to be purified by diluting 10 ⁸ to 10 ¹⁰ cfu / ml culture stock 100 times or more,
The nutrients are characterized in that the nutrients containing nitrogen (N) and phosphorus (P) in a concentration of 2 to 5: 1,
Method of treating soil containing high concentration of TPH.
delete delete delete delete delete delete The method of claim 1,
The soil containing the high concentration of TPH is a method of treating a soil containing a high concentration of TPH, the concentration of TPH is 10,000mg / kg to 20,000 mg / kg.


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