JP7161599B2 - Microbacterium paraoxidans, methods for preparing its broad-spectrum polychlorinated biphenyl enzyme preparations, and applications - Google Patents

Description

CGMCC CGMCC15836

The present invention relates to Microbacterium paraoxidans, its broad-spectrum polychlorinated biphenyl enzyme formulation preparation method and application, and belongs to the technical field of microorganisms.

Polychlorinated biphenyls (PCBs), persistent organic pollutants, are highly toxic, bioaccumulative and long-distance mobile, have long half-lives, severely contaminate soils, aquatic ecosystems and drinking water sources, and cause malformations. and cause cancer. Both the Yusho Incident in Taiwan and the Kanemi Yusho Incident in Japan caused by PCBs contamination caused serious loss of life and property. Due to its thermal and chemical stability, flame resistance, insulation and oxidation resistance, PCBs are widely applied in the chemical, power, electronic and mechanical industries. Biomass is high, and large amounts of PCBs-contaminated sites are being generated by e-waste and industrial land clearing. In 2015, the Ministry of Environmental Protection issued the National Environmental Protection Standard (HJ743-2015) "Gas Chromatography for Determination of Polychlorinated Biphenyls in Soil and Sediment-Mass Spectrometry Method" to measure PCBs in soil and sediment. standardized the method. Timely and effective treatment of PCBs pollution, protection of the environment and guarantee of human health are already urgent needs. PCBs isomers are diverse, coplanar structure PCBs have toxicity similar to that of dioxin, and the indicator PCBs are monomers specified by the United Nations GEMS/FOOD as indicators for monitoring the status of PCBs contamination. The main PCB pollutants in the environment, trichlorobiphenyl PCB28 and pentachlorobiphenyl PCB101, are representative compounds.

Polychlorinated biphenyls (PCBs) have thermal and chemical stability, flame retardancy, insulation and oxidation resistance, so they are widely applied in the chemical industry, power, electronic and mechanical industries. Mainly insulating oil, flame retardant, heat transfer agent, hydraulic oil, plasticizer, railway transformer, mining equipment, electromagnetic equipment, carbonless carbonized paper, pigment, wax additive, dust remover, insecticide additive, lubricant agents, cutting oils, sealants, and occlusive agents. In China, the cumulative production of polychlorinated biphenyls over the years is nearly 10,000 tons.In the beginning of the 1980s, China basically stopped its production, but some developed countries started to produce power capacitors, including PCBs, Due to the import of power transformers, and after the 1990s, foreign countries imported polychlorinated biphenyls into China, mainly through e-waste. Therefore, there is a large amount of PCBs contaminated sites. PCBs enter the soil and water environment through waste discharge, leaking oil storage tanks, volatilization and dry and wet sedimentation, resulting in serious pollution of soil, aquatic ecosystems and drinking water sources. Combined with poor management, improper handling and storage, the problem of secondary and persistent contamination of PCBs is quite serious.

Patent Document 1: Chinese Patent 107287134A (Application No. 201710508652.X) discloses Pseudomonas sp. Deposited at the center, the deposit number is CGMCC No. 13960. The present invention isolates the Pseudomonas sp. ECO-1 strain from POPs-contaminated soil for the first time, and uses this strain to produce a bifunctional enzyme preparation that can efficiently degrade polychlorinated biphenyls and atrazine for the first time. However, this enzyme preparation has a remarkable activity to decompose highly chlorinated polychlorinated biphenyls, which are difficult to decompose especially under aerobic conditions, and is completely functional with conventionally known Pseudomonas (Pseudomonas sp.) and its enzyme preparations. are expected to be applied to mass production.

However, the above strains still cannot meet the needs of practical treatment in terms of treatment speed and the types of polychlorinated biphenyls that can be treated, and rapid remediation of PCBs complex contamination under aerobic conditions is the focus of current research. It has become.

Chinese Patent 107287134A (Application No. 201710508652.X)

The present invention addresses the deficiencies of the prior art by providing Microbacterium paraoxidans, its broad-spectrum polychlorinated biphenyl enzyme formulation preparation methods, and applications.

The technical solution of the present invention is as follows.

On June 1, 2018, it was deposited at the General Microbiology Center of the Chinese Microbial Species Storage and Management Committee (Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing). CGMCC No. Microbacterium paraoxydans ECO-2, which is 15836;

As a pure cultured monoclonal form of this strain, the colonies are yellow, round with smooth edges and surfaces, as shown in FIG.

The method of culturing the above strain is
a step (1) of streaking Microbacterium paraoxydans strain ECO-2 on a solid activation medium and culturing for activation to obtain an activated strain;
A step (2) in which the activated strain obtained in step (1) is inoculated into a liquid medium and cultured with shaking to obtain a seed liquid;
a step (3) of transferring the seed solution obtained in step (2) to an expansion medium at a volume percentage of 2% to 10% and performing expansion culture to obtain a bacterial solution;

According to the present invention, preferably in said step (1), the solid activation medium is LB solid medium, and the composition is:
10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, adjusted to 1 L with water, and pH in the natural state.

According to the present invention, preferably, in step (1), the activation condition is inversion culture at 28-37° C. for 1-2 days.

According to the present invention, preferably both the liquid medium in step (2) and the expansion medium in step (3) are LB liquid medium, and the composition is:
10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, volume to 1 L with water, pH in natural state.

According to the present invention, preferably, the shaking culture conditions in step (2) are 28 to 37° C. and a rotation speed of 100 to 200 rpm for 1 to 2 days. .

According to the present invention, the expansion culture conditions in step (3) are preferably 28-37° C. and 20-70% dissolved oxygen for 1-2 days.

Application of said Microbacterium paraoxydans ECO-2 in the preparation of an enzymatic preparation to remediate polychlorinated biphenyl contaminated soil.

The above application is
Mineral salts induction medium, which is a mineral salts medium containing a bacterial suspension of Microbacterium paraoxydans ECO-2 at a volume percent of 1-10% and a concentration of biphenyl at a concentration of 0.3-0.8 g/L and induced culture for 3 to 5 days at a temperature of 28 to 37 ° C. and a rotation speed of 100 to 200 rpm to obtain an induced Microbacterium paraoxydans ECO-2 bacterial solution. step (i);
The induced Microbacterium paraoxydans ECO-2 bacterial solution obtained in step (i) is subjected to solid-liquid separation, the cells are suspended in a phosphate buffer, the cells are disrupted, and further 4- a step (ii) of performing solid-liquid separation at 25° C. and collecting the supernatant to obtain an enzyme preparation.

According to the present invention, preferably in said step (i), the composition of the mineral salts medium per liter is:
0.5 g potassium dihydrogen phosphate, 0.5 g disodium hydrogen phosphate, 0.2 g magnesium sulfate, 0.1 g calcium chloride, 0.2 g sodium chloride, 1.0 g ammonium sulfate, 2.0 g peptone, pH 7.0 is.

According to the present invention, preferably, in step (ii), the solid-liquid separation is performed by centrifuging at 3000-10000 rpm for 2-10 minutes.

According to the present invention, preferably, in step (ii), the cell disruption employs a high-pressure homogenization cell disruption method.

According to the present invention, preferably, in step (ii), the phosphate buffer is pH 5.0-8.0 phosphate buffer, preferably the amount of phosphate buffer used is 5-50 Double capacity.

Application of the above enzyme preparation in remediation of polychlorinated biphenyl contaminated soil.

According to the invention, preferably said polychlorinated biphenyl is 2,4,4′-trichlorobiphenyl (PCB28), 2,2′,4,5,5′-pentachlorobiphenyl (PCB101) and/or 2 , 3,4,4′,5-pentachlorobiphenyl (PCB114).

1. In the present invention, Microbacterium paraoxydans strain ECO-2 is disclosed for the first time. , PCB28, PCB101 and/or PCB114. Compared to physical and chemical treatment methods, it is more environmentally friendly and convenient to handle on a large scale. The decomposition efficiency is increased, and future applications can be expected.
2. A broad-spectrum polychlorinated biphenyl enzyme preparation prepared with Microbacterium paraoxydans ECO-2 has a coplanar structure of PCB114 and environmental pollution indicators trichlorobiphenyl PCB28 and pentachlorobiphenyl PCB101. It has high decomposing activity against all of them, and the decomposition rate for pentachlorobiphenyl PCB101 reaches 100%. It has a wide range of application, and there is no need to use highly toxic perchlorinated PCBs for induction during preparation. Therefore, the preparation process is simple, and application to mass production is expected.

Photograph of purely cultured monoclonal forms of Microbacterium paraoxydans strain ECO-2.

The technical solution of the present invention will be further described in the following examples, but the patent scope of the present invention is not limited thereto.

Biological Origin Microbacterium paraoxydans ECO-2 was approved on June 1, 2018 by the General Microbiology Center of the China Microbial Species Storage and Management Committee (Address: No. 1, Beichen West Road, Chaoyang District, Beijing). No. 3 Institute of Microbiology, Chinese Academy of Sciences), and the accession number of the bacterial species is CGMCC No. 3. 15836.

Medium LB solid medium has the following composition per liter.
10g peptone, 5g yeast extract, 10g sodium chloride, 20g agar, volume to 1L with water, natural pH
In the LB liquid medium, the composition per liter is as follows.
10g peptone, 5g yeast extract, 10g sodium chloride, volume to 1L with water, natural pH
In the mineral salt medium, the composition per liter is as follows.
0.5 g potassium dihydrogen phosphate, 0.5 g disodium hydrogen phosphate, 0.2 g magnesium sulfate, 0.1 g calcium chloride, 0.2 g sodium chloride, 1.0 g ammonium sulfate, 2.0 g peptone, water to 1 L Constant volume, pH 7.0.

Example 1
A POPs-contaminated soil exudate was prepared and diluted to five concentration gradients of concentrations 10 ⁻¹ , 10 ⁻² , 10 ⁻³ , 10 ⁻⁴ and 10 ⁻⁵ , respectively. The diluted bacterial suspension was spread on a biphenyl-containing solid medium and cultured at 30° C. for 1-3 days. Bacterial colonies with a high growth rate and typical morphology were selected, separated and purified by 3 times of flat plate streaking, and then a single colony was placed in an inorganic salt liquid medium and placed at 30°C at 150 rpm. 0.5 mL of glycerol was added to 1.5 mL of the culture, mixed uniformly, and stored in a refrigerator at -80°C for a long period of time.

The solid medium to which the bacterial suspension is applied is LB solid medium, and the composition is as follows.
10g peptone, 5g yeast extract, 10g sodium chloride, 20g agar, volume to 1L with water, natural pH.

The composition of the mineral salt liquid medium for culturing a single colony is as follows.
0.5 g potassium dihydrogen phosphate, 0.5 g disodium hydrogen phosphate, 0.2 g magnesium sulfate, 0.1 g calcium chloride, 0.2 g sodium chloride, 1.0 g ammonium sulfate, 2.0 g peptone, water to 1 L Constant volume, pH 7.0.

The strains obtained above were added to polychlorinated biphenyl PCB28 (2,4,4′-trichlorobiphenyl), PCB101 (2,2′,4,5,5′-pentachlorobiphenyl), PCB114 at a concentration of 25 mg/L, respectively. (2,3,4,4′,5-Pentachlorobiphenyl) was inoculated into an inorganic salt liquid medium containing 150 rpm and 30° C. for 72 hours, and the state of turbidity of the bacterial solution was observed. The absorbance value at 600 nm was detected for the suspension. Enzyme-producing strains were selected based on the above indicators. The strain with the highest absorbance value was cultured on LB solid medium and stored as ECO-2.

The monoclonal was sent to Qingdao Qing Kezixi Biotechnology Co., Ltd. for sequencing and detection, containing 1357 bp of 16S rDNA sequence, and the nucleotide sequence is SEQ ID NO. 1. When the strain was identified, it was Microbacterium paraoxydans, named Microbacterium paraoxydans ECO-2. Deposited at the General Microbiology Center of the Committee (Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing), and assigned a CGMCC No. 15836.

Identification of the species is as follows.
Sample: Bacterial fungus solution Bacterial genome DNA extraction kit: DP302, Tianjin Technology (Beijing) Co., Ltd.
TAE buffer (50×, 1 L): 242 g Tris, 57.1 ml glacial acetic acid, Na2EDTA. 2H2O 37.2 g, up to 1 L Hydrogenated agarose: BIOWET, AGAROSE G-10
2x Pfu PCR MasterMix, D2000 DNA Marker, nucleic acid dye, loading buffer, etc.: Tianjin Chemical Technology (Beijing) Co., Ltd. DNA purification and recovery kit: Tianjin Chemical Technology (Beijing) Co., Ltd., DP214
Consumables such as centrifuge tubes and pipette tips: Gene Era Biotech (GEB), USA

Primer: Synthesized by Qingdao Qin ▼ Kezixi Biotechnology Co., Ltd. ddH ₂ O was added according to the synthesis manual to give a 10 μM solution.
1, Genomic DNA extraction was performed according to the DP302 kit.
2. PCR amplification

2.3 PCR cycle parameters Pre-denaturation: 94°C, 3 min, denaturation 94°C, 30 s, annealing 55, 30 s, extension, 72°C, 1.5 min (total 35 cycles), extension 72°C, 10 min; store at 4°C .

3. Agarose gel electrophoresis detection A 1.0% agarose gel was prepared, and electrophoresis was performed at a voltage of 18 V/cm and an electrophoresis time of 20 minutes.

4. Purification and recovery Using a general agarose gel DNA recovery kit, agarose gel was recovered for the target fragment, and the recovered product was sequenced at Qingdao Qing Kezixi Biotechnology Co., Ltd.

Example 2
The steps of the method for preparing a broad-spectrum polychlorinated biphenyl degrading enzyme preparation using said Microbacterium paraoxydans ECO-2 of Example 1 are as follows.
(1) Microbacterium paraoxydans ECO-2 was streaked on LB solid medium and subjected to inversion activation culture at 35° C. for 2 days to obtain an activated strain.
(2) The activated strain obtained in step (1) was inoculated into an LB liquid medium and cultured with shaking at 35° C. and a rotation speed of 200 rpm for 2 days to obtain a seed solution.
(3) The seed solution obtained in step (2) was transferred to a mineral salt medium containing 0.5 g/L of biphenyl at a volume percentage of 10%, and maintained at 35°C and 70% dissolved oxygen for 2 days. It was expanded and cultured to obtain a bacterial solution.
(4) The bacterial solution obtained in step (3) is centrifuged at 3000 rpm for 10 minutes to collect the bacterial cells and suspended in 30 times the volume of pH 7.0 phosphate buffer. After turbidity and high-pressure homogenization, the cells were centrifuged at 4° C. and 3000 rpm for 2 minutes, and the supernatant was collected to obtain a broad-spectrum polychlorinated biphenyl degrading enzyme preparation.

Comparative example 1
An enzyme preparation was prepared by the method of Example 2 using Microbacterium paraoxydans strain MA25, which has the most similar genetic relationship, and was cultured in the mineral salt medium of step (3). was not observed, and when the absorbance value at 600 nm was detected for the bacterial suspension, the value of OD _{600 nm} was zero. Paraoxydans Microbacterium paraoxydans strain MA25 was found to be non-viable.

Comparative example 2
An enzyme preparation was prepared by the method of Example 2 using the (Pseudomonas sp.) ECO-1 strain. When cultured in the inorganic salt medium of step (3), turbidity of the bacterial solution was observed, and when the absorbance value at 600 nm was detected for the bacterial suspension, the value of OD _{600 nm} was 1.05. Therefore, it was clarified that Pseudomonas sp. ECO-1 can survive in the mineral salt medium of step (3).

Experimental Examples Pentachlorobiphenyl PCB114, PCB101, and trichlorobiphenyl PCB28 at a concentration of 25 mg/L, respectively, were mixed with broad-spectrum polychlorinated biphenyl enzyme formulations and PBS buffer in a ratio of 1:5:19 (by volume), The mixture was reacted at 30° C. and pH 7.0 for 10 hours, 13 hours, and 10 hours, respectively, and 10 mL of n-hexane was added for extraction three times. The extract was subjected to the GC-MS method to detect the decomposition rate of polychlorinated biphenyl.
As a result of the detection, the broad-spectrum polychlorinated biphenyl degrading enzyme preparation obtained in Example 2 reached a decomposition rate of 90% within 10 hours for trichlorobiphenyl PCB28 under aerobic conditions, and decomposed dioxin-like pentachlorobiphenyl PCB114. The rate reached 57.1% within 10 hours, and the degradation rate for pentachlorobiphenyl PCB101, which serves as an indicator, reached 100% within 13 hours.

In Comparative Example 2, the decomposition rate reached 60.3% within 10 hours only for dioxin-like pentachlorobiphenyl PCB114, and the decomposition rate for the others was zero.

As can be seen from the above data, the Microbacterium paraoxydans ECO-2 of the present invention produces broad-spectrum polychlorinated biphenyl enzyme preparations against PCB114, PCB101, PCB28 when induced with a single substrate. It has a high application value compared to conventional known enzyme preparations that can only decompose a single substance when induced with a single substrate.

Claims (10)

Bacterial strains deposited at the General Microbiology Center of the Chinese Microbial Species Storage and Management Committee (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China) on June 1, 2018 (Acceptance number: CGMCC) No. 15836) of Microbacterium paraoxydans ECO-2. The method for culturing Microbacterium paraoxydans ECO-2 according to claim 1,
a step (1) of streaking Microbacterium paraoxydans strain ECO-2 on a solid activation medium and culturing for activation to obtain an activated strain;
A step (2) in which the activated strain obtained in step (1) is inoculated into a liquid medium and cultured with shaking to obtain a seed liquid;
A step (3) of transferring the seed solution obtained in step (2) to an expansion medium at a volume percentage of 2% to 10%, and expanding and culturing to obtain a bacterial solution. . In step (1) above, the solid activation medium is LB solid medium, and the composition is:
10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar, adjusted to 1 L with water, pH in natural state,
3. The culture method according to claim 2, wherein in step (1), the activating condition is inversion culture at 28 to 37° C. for 1 to 2 days. Both the liquid medium in step (2) and the expansion medium in step (3) are LB liquid medium, and the composition is as follows:
10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, adjusted to 1 L with water, pH in natural state,
The culture method according to claim 2, wherein the shaking culture conditions in step (2) are 28 to 37°C and a rotation speed of 100 to 200 rpm for 1 to 2 days. . The culture method according to claim 2, wherein the expansion culture conditions in step (3) are conditions of 28 to 37°C and 20 to 70% dissolved oxygen for 1 to 2 days. An application of Microbacterium paraoxydans ECO-2 according to claim 1, comprising:
An application characterized in that it is in the preparation of an enzyme preparation for remediation of polychlorinated biphenyl contaminated soil. Mineral salts induction medium, which is a mineral salts medium containing a bacterial suspension of Microbacterium paraoxydans ECO-2 at a volume percent of 1-10% and a concentration of biphenyl at a concentration of 0.3-0.8 g/L and induced culture for 3 to 5 days at a temperature of 28 to 37 ° C. and a rotation speed of 100 to 200 rpm to obtain an induced Microbacterium paraoxydans ECO-2 bacterial solution. step (i);
The induced Microbacterium paraoxydans ECO-2 bacterial solution obtained in step (i) is subjected to solid-liquid separation, the cells are suspended in a phosphate buffer, the cells are disrupted, and further 4- The application according to claim 6, comprising a step (ii) of performing solid-liquid separation at 25°C and collecting the supernatant to obtain the enzyme preparation. In step (i), the composition of the mineral salts medium per liter is
0.5 g potassium dihydrogen phosphate, 0.5 g disodium hydrogen phosphate, 0.2 g magnesium sulfate, 0.1 g calcium chloride, 0.2 g sodium chloride, 1.0 g ammonium sulfate, 2.0 g peptone, pH 7.0 and
In the step (ii), solid-liquid separation is centrifugation for 2 to 10 minutes at 3000 to 10000 rpm,
In the step (ii), the cell disruption is to adopt a high-pressure homogenization cell disruption method;
The phosphate buffer according to claim 7, wherein in step (ii), the phosphate buffer is a phosphate buffer with a pH of 5.0 to 8.0, and the amount of the phosphate buffer used is 5 to 50 times its volume. application. An application of the enzyme preparation obtained in claim 8,
An application characterized in that it is in the remediation of polychlorinated biphenyl contaminated soil. Said polychlorinated biphenyl is 2,4,4'-trichlorobiphenyl, 2,2',4,5,5'-pentachlorobiphenyl and/or 2,3,4,4',5-pentachlorobiphenyl Application according to claim 9.

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