JP6782036B1 - PAHs-Heavy Metals Composite Pollution Degradation / Adsorbed Bacteria and Its Applications in Environmental Pollution Restoration - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide PAHs-heavy metal composite pollution decomposition / adsorbed bacteria and its application in environmental pollution repair. The strain is Sphingobium sp. PM1B, which was deposited at the China Typical Culture Depositary Center on March 28, 2019, and the deposit number is CCTCC NO: M 2019212. The strain has a good decomposing effect on polycyclic aromatic hydrocarbons, while it has an adsorptive effect on heavy metals. It can be applied to the coverage and restoration of PAHs-heavy metal complex contaminated environment including water and soil environment such as electronic waste dismantling area, sewage irrigation area, industrial and mining area, contaminated area. There is a wide range of application prospects. [Selection diagram] None

Description

The present invention relates to the field of treatment of environmental pollutants by living organisms, and more particularly to its application in PAHs-heavy metal composite pollutant decomposition / adsorbed bacteria and environmental pollution repair.

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants, primarily resulting from oil spills, incomplete combustion of coal, petroleum, wood, organic macromolecular compounds or chemical fuels. As the economic development and the process of industrialization accelerate, more PAHs enter the environment and cause serious pollution. Due to mining, exhaust gas, accumulation of solid waste, sewage irrigation, improper use of pesticides, etc., a large amount of heavy metals enter the environment and continue to accumulate. Pollutants in the environment do not exist in a single form, and research and research have shown that air, water and soil in many parts of China are affected by varying degrees of PAHs heavy metal pollution. There is. For example, it has been detected simultaneously in the soil and air of the water, sediments, aquatic organisms and e-waste dismantling areas of the Neri River, the Oyan River and the Pearl River Haikou.

Heavy metals are toxic pollutants that are difficult to decompose, accumulate in vivo, and have highly toxic properties. PAHs have effects such as carcinogenicity, teratogenicity and mutagenicity. Heavy metals and PAHs not only damage the ecological environment, but also enter the human body through direct contact or the food chain and pose a serious threat to human health. The two types of pollutants are long-lasting in the environment, and the resulting complex pollution is more harmful to the environment and humans. Therefore, there is an urgent need to carry out coverage and restoration of PAHs-heavy metal complex contaminated environments. Heavy metals and PAHs in the environment coexist for a long period of time, and the difference in physicochemical properties between them is large, and complex interactions occur, which makes it difficult to repair complex contamination.

Currently, there are mainly physicochemical and biological methods for repairing PAHs-heavy metal complex contamination. Physicochemical restoration techniques include adsorption methods, redox methods, guest soil methods, soil cleaning methods and improvement methods. Easy to operate, fast to repair, but expensive, treatment effect on wastewater or soil containing both PAHs and heavy metals is unclear, damage to ecological environment, prone to problems such as secondary pollution .. Microbial repair is one of the effective ways to remove pollutants from the environment and has the advantages of being economical, convenient and less prone to new pollution. Microorganisms can break down highly toxic and structurally complex pollutants into low-toxic or non-toxic compounds through their own metabolic activity and are the main method of removing PAHs from the environment. In recent years, domestic and foreign scholars have conducted intensive research on microorganisms that decompose PAHs, and isolated more than 70 genera and 200 types of microorganisms that can decompose PAHs including bacteria, fungi, actinomycetes and algae. Was done. On the other hand, since a large number of functional groups (for example, carboxyl group, hydroxyl group, amino group and amide group) and active sites are present on the cell surface of the microorganism, it has a good adsorption action on heavy metals. The use of microorganisms as an effective adsorbent for removing heavy metals in environmental media has received widespread attention.

Chinese Patent Application Publication No. 10513815

To the above-mentioned problems, the present invention provides PAHs-heavy metal composite polluting / adsorbing bacteria and their applications.

The above object of the present invention is achieved by the following technical proposals disclosed.

(. Sphingobium sp) sphingolipid bi Umm bacteria belonging to the genus according to the present invention PM1B is gunna international deposit based on the Budapest Treaty on China typical culture Depositary Center on March 28, 2019, deposit number CCTCC NO: in M 2019212 Yes, the place is China. Wuhan. It is Wuhan University and its address is Wuchang Coffee Mountain, Wuhan City, Hubei Province.

The Sphingobium sp. PM1B was obtained after domestication, screening, and isolation at an incineration site in the electronic waste dismantling area of Longtang Town, Qingyuan Province, Guangdong Province. The morphology of the Sphingobium bacterium PM1B on a flat plate is bright yellow, spherical, regular edges, raised surface, smooth, moist, opaque surface, and the shape of the bacterium observed with an electron microscope. Is rod-shaped.

16SrDNA sequence analysis is used to classify and appraise the strain, and the strain is outsourced to Biotechnology (Shanghai) Co., Ltd. to complete the sequence to obtain a sequence with a length of 1428 bp. By comparing the sequence homology with respect to the strain, the homology between the 16S rDNA sequence of PM1B and the Sphingobium bacterium (Sphingobium sp.) Reached 99%. It belongs to bacteria.

The nucleotide sequence of 16S rDNA of the Sphingobium sp. PM1B is shown in Seq ID No: 1.

As a second object of the present invention, the Sphingobium sp. Provides an application in the decomposition of PAHs and / or the adsorption conversion of heavy metals.

It is preferred that the PAHs are pyrene and / or phenanthrene.

It is preferable that the heavy metal is copper and / or cadmium.

A third object of the present invention is to provide a degrading bacterium capable of degrading PAHs and / or adsorbing and converting heavy metals, including a strain of the Sphingobium sp. PM1B.

Current research focuses on the removal of single heavy metals or PAHs by microorganisms, as most strains only degrade PAHs, have no resistance and adsorption to heavy metals, or only adsorb heavy metals and cannot degrade PAHs. ing. The present invention can acquire Sphingobium bacteria by screening, decompose various PAHs, adsorb heavy metals, and can be applied to the coverage and restoration of the aquatic environment of PAHs-heavy metal complex contamination. It can effectively reduce the damage of heavy metals and PAHs to the ecological environment and human health, including electronic waste dismantling areas, sewage irrigation areas, industrial and mining areas, contaminated areas, etc. and their surrounding environments. ..

Treating PAHs and heavy metal contamination using the Sphingobium bacteria provided in the present invention is more effective, less costly, environmentally friendly, etc. than physical adsorption, chemical improvement and redox methods. Has an advantage.

The present invention will be further described with reference to the drawings. However, the embodiments of the drawings do not limit the present invention, and it is possible for those skilled in the art to obtain other drawings from these drawings without any creative effort.
Flat plate morphology of Sphingobium bacterium PM1B. Electron microscopic morphology of the Sphingobium bacterium PM1B. It is a figure which shows the relationship between the decomposition effect of bacterium PM1B on phenanthrene and pyrene, and time in the complex system of pyrene, phenanthrene, copper and cadmium. It is a figure which shows the relationship between the adsorption effect of bacteria PM1B on copper and cadmium, and time in the complex system of pyrene, phenanthrene, copper and cadmium. The bacterial solution is the effect of removing pyrene, phenanthrene, copper and cadmium in soil samples.

The present invention will be further described with reference to specific examples.

Example 1:
Isolation and analysis of strains of Sphingobium bacterium PM1B.

1. Soil sample The test soil sample is taken from the incineration site in the electronic waste dismantling area of Longtang Town, Guangdong Province, and after the topsoil is removed, the soil in the depth range of 5-20 cm is taken and put into a sealed bag. Put it in, take it back to the laboratory, and store it in a refrigerator at -20 ° C.

2, composition of the medium and the solution prepared mineral salts medium (MSM): 5 ml phosphate buffered saline _{(KH 2 PO 4 8.5 g /} L, K 2 HPO 4 · 3H 2 O
21.75 g / L, Na ₂ HPO ₄ /12H ₂ O 33.4 g / L, NH ₄ Cl 5.0 g / L); 3 mL DDL ₄ solution (22.5 g / L); 1 mL FeCl ₃ aqueous solution (0.25 g / L); 1 mL CaCl ₂ solution (36.4 g / L); 1 mL trace element solution (MnSO ₄ · H ₂ O 39.9 mg / L, ZnSO ₄ · H ₂ O 42.8 mg / L, (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O 34.7 mg / L), the volume is up to 1 L, and the pH value is 7.2 to 7.4.
PAHs stock solution: Prepare the mother liquor of pyrene (1 g / L) and phenanthrene (1 g / L) using acetone as a solvent, and store at 4 ° C in the refrigerator for backup.
Heavy metal stock solution: Weigh a certain amount of Cd (NO ₃ ) ₂ and Cu (NO ₃ ) ₂ and dissolve them in deionized water to prepare a Cd ²⁺ and Cu ²⁺ stock solution with a concentration of 1 g /. To do.

3. Domestication, screening and isolation of bacteria that decompose and adsorb PAHs-heavy metals
Weigh 10g soil placed in 250 mL Erlenmeyer flask, 100 mL sterile sodium acid pyrophosphate _{(Na 2 P 2 O 7 ·} 7H 2 O, 2. 8 g / L) were charged, uniformly ultrasonic Shake overnight (3 min) and shake overnight in a shaker to obtain a standby bacterial solution and stock.

First, 10 mL of the standby bacterial solution was inoculated into MSM medium containing pyrene and phenanthrene, cultured at 150 rpm and 30 ° C. at 7d, and then 10 mL of the supernatant was taken from the medium, and phenanthrene (50 mg / L) and pyrene (10 mg) were taken. Transfer to MSM medium containing / L), Cd ²⁺ (5 mg / L), Cu ²⁺ (20 mg / L). The total volume of the culture system is 100 mL, and the above steps are repeated to domesticate 5 times.

Take 5 mL of the domesticated cultured bacterial solution of the last cycle, dilute the bacterial solution into a suspension with a gradient of 10 ^-1 to 10 ^-7 by a 10-fold dilution method, and then take 0.1 mL and take 0.1 mL of meat juice solid medium. Invert the plate and place it in a biochemical incubator at 30 ° C. After 3-4 days, after the colonies have grown well, observe the morphology of the colonies.

Select colonies with different morphological characteristics on a flat plate, transfer to MSM medium containing 100 mL of 50 mg / L phenanthrene, 10 mg / L pyrene and 5 mg / L Cd ²⁺ , 20 mg / L Cu ^2+, and culture by vibration. To verify the ability to decompose PAHs and adsorb heavy metals. Select a flask whose medium color is turbid, repeat the above steps, and perform purification three times to ensure the purity of the strain and the stability of the decomposition / adsorption performance. Finally, the strain with the highest decomposition and adsorption capacity of pyrene / phenanthrene-copper / cadmium complex contamination and moderate growth performance is numbered PM1B as the target strain.

Example 2
Degradation and adsorption performance of Sphingobium sp. PM1B strains.

Strains were prepared in bacterial suspension with MSM solution, OD ₆₀₀ was adjusted to 0.6, 50 mg / L phenanthrene, 10 mg / L pyrene, 5 mg / L Cd ²⁺ and 20 mg / L in 10% proportions. subscribing to 18 mL MSM culture system containing Cu ² +, established three overlapping any treatment, 30 ° C., subjected to vibration of culture under the condition of 0.99 rpm, regularly pyrene remain in growth volume and solution of PM1B , Phenanthrene, copper, and cadmium concentrations are measured. Set up a fungus-free blank control to analyze the non-biological loss of contaminants during the experiment.

The concentrations of phenanthrene and pyrene are measured by high performance liquid chromatography (HPLC). Place an appropriate amount of chromatography-grade methanol chromatography in the flask, sonicate for a period of time to completely dissolve phenanthrene and pyrene, transfer to a 50 mL volumetric flask and volume with methanol. Enter HPLC using a 0.22 μm organic phase filter to measure the concentration of phenanthrene. The measurement wavelengths of phenanthrene and pyrene are 234 and 250 nm, respectively, and the mobile phase is methanol / ultrapure water (v / v, 90/10), and each reaction system solution is centrifuged to quantify. The supernatant is taken, filtered through a 0.22 μm filter, and the concentrations of cadmium and copper are measured with an atomic absorption spectrometer.

As shown in Fig. 3, the experimental results showed that the decomposition effect of the fungus PM1B on pyrene and phenanthrene gradually increased over time in the complex system of pyrene, phenanthrene, copper and cadmium. At 120h, the removal rates of phenanthrene and pyrene reached 89.58% and 38.81%. In addition, PM1B has a certain adsorption effect on copper and cadmium (see Fig. 4), and the adsorption rate also increases with the passage of time, and at 36h, the adsorption rate on copper and cadmium is the highest. They were 63.03% and 48.04%, respectively. After that, the fungus PM1B had a slight desorption effect on copper, but it was maintained at about 40%.

The bacterium belonging to the genus Sphingobium according to the present invention can effectively decompose various PAHs and can also adsorb heavy metals. PAHs-Can be applied to the coverage and restoration of aquatic environment of heavy metal complex contamination.

Example 3
Sphingobium bacterium PM1B repairs soil in the electronic waste demolition area.

Soil samples were taken from farmland around the e-waste dismantling area in Longtang Town, Qingyuan Province, Guangdong Province. It contains various heavy metals and PAHs contaminants. A certain amount of contaminated soil is weighed and placed in a glass incubator, the soil is inoculated with bacterial solution, the soil humidity is maintained at 60% of the maximum water retention capacity of the field, and a bacteria-free control group is set up. After culturing to 60d in natural light under the condition of 30 ° C., the residual concentrations of pyrene, phenanthrene, copper and cadmium in the soil are measured. As shown in Fig. 5, the results of the experiment showed that the removal rates of pyrene, phenanthrene, copper and cadmium in the soil were 79.47%, 34.92%, 59.12% and 37.98%, respectively, through the decomposition / adsorption of microorganisms for 2 months. Sphingobium bacterium PM1B actually has a good repair effect on PAHs-heavy metal complex contaminated soil.

Lastly, the above-described embodiment is a technical proposal for easily understanding the technical concept of the present invention, and does not limit the scope of protection of the present invention. Although the present invention has been described in detail by examples, all other examples obtained under the premise that creative labor is not imparted are included within the scope of the present invention without departing from the spirit of the present invention. Should be understood by those skilled in the art.

Claims (3)

Sphingobium sp. PM1B,
The deposit number is CCTCC NO: M 2019212. The Sphingobium sp. PM1B according to claim 1, wherein the nucleotide sequence of 16S rDNA of the Sphingobium sp. PM1B is shown in Seq ID No: 1. It is a degrading bacterial agent
It is characterized by containing the Sphingobium sp. PM1B strain according to claim 1 or 2.