A strain of Bacillus cereus MD1W capable of simultaneously degrading microplastics in mulch and adsorbing heavy metals and its application

Abstract

The present invention discloses a strain of Bacillus cereus MD1W and an application thereof. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center of China on August 25, 2022, with a deposit number of GDMCC No: 62744. The strain can simultaneously degrade mulch microplastics and adsorb heavy metals, has strong environmental adaptability, and can be used for the treatment and restoration of mulch microplastic-heavy metal composite pollution environments.

Description

Technical Field

[0001] The invention relates to the field of biological treatment of environmental pollutants, and specifically to a strain of Bacillus cereus MD1W capable of synchronously degrading mulch microplastics and adsorbing heavy metals, and application thereof. Background Art

[0002] The use of agricultural mulch has greatly promoted the development of agriculture. Mulch mulch can maintain soil moisture and temperature, improve the utilization rate of fertilizers, slow down soil erosion, reduce pests and diseases, and achieve increased production and income while ensuring the safety of agricultural products. Due to the lack of mechanical equipment for recycling mulch, a large amount of mulch remains in the soil and gradually accumulates. The residual mulch is constantly worn and broken, forming mulch microplastics with a particle size of less than 5mm. The mulch microplastics can also enter rivers, lakes and oceans with surface runoff and rainwater erosion. Mulch microplastics will block soil pores and affect the migration of water and nutrients, hinder the growth of plant roots, and lead to reduced crop yields. Mulch microplastics entering the water environment will reduce the visibility of the water body and inhibit the photosynthesis of phytoplankton. After being ingested by aquatic organisms, mulch microplastics will affect their metabolism, growth and reproduction. In addition, with the acceleration of industrialization, mining activities, sewage discharge, excessive application of pesticides and fertilizers, etc., have caused a large amount of heavy metals to enter the environment. Heavy metals are difficult to degrade and have the characteristics of concealment and high toxicity. Since microplastics in mulch film have a large specific surface area and strong hydrophobicity, they are easy to interact with heavy metals, forming a complex pollution, which not only has an adverse impact on the ecological environment, but can also be transmitted along the food chain and pose a serious threat to human health. Therefore, choosing an efficient and reasonable restoration technology has become an urgent problem to be solved.

[0003] Physical and chemical remediation technologies are costly and have poor remediation effects on polluted environments. They can damage water / soil structures and cause dysfunction in water and soil ecosystems. Microbial remediation technology has good effects, low costs, no secondary pollution, and can maintain the stability of the ecosystem. It is one of the effective methods to remove pollutants from the environment. Microorganisms can use their own metabolic activities to decompose complex pollutants into low-toxic or non-toxic substances. Plastics have been proven to be degraded by microorganisms (bacteria and fungi): microorganisms first attach to the surface of plastics to form biofilms, and then decompose plastics into oligomers, dimers and monomers by secreting extracellular enzymes, and finally mineralize them into CO 2 and H 2O. The groups and active sites on the surface of microbial cell walls can form chemical bonds with heavy metals through complexation / coordination, thereby achieving extracellular adsorption of heavy metal ions; heavy metals adsorbed on the cell surface will enter the cell through the ion transfer system and accumulate by combining with certain molecules. However, current research mainly focuses on the removal of single film microplastics or heavy metals. Existing strains can only degrade film microplastics but cannot adsorb heavy metals, or can only adsorb heavy metals but cannot degrade film microplastics. There is no efficient strain that can simultaneously degrade film microplastics and adsorb heavy metals. Summary of the invention

[0004] In view of the above problems, the present invention provides a strain of Bacillus cereus MD1W which can simultaneously degrade mulch microplastics and adsorb heavy metals, and application thereof.

[0005] The purpose of the present invention is achieved by the following technical solutions:

[0006] The present invention provides a strain of Bacillus cereus MD1W, with a preservation number of GDMCC No: 62744, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center of China on August 25, 2022.

[0007] The Bacillus cereus MD1W was domesticated, screened and purified from the soil of agricultural land covered with mulch for a long time in Zhaoqing City, Guangdong Province. The morphology of the Bacillus cereus MD1W on the plate can be found in Figure 1 The colonies are large, like molten wax or frosted glass, with a rough, opaque, slightly shiny white surface.

[0008] The strain was classified and identified by 16S rDNA sequence analysis. The DNA of MD1W was extracted by entrusting Shanghai Biotech Co., Ltd. 27F / 1492R primers for 16S rDNA amplification and sequencing, and a sequence of 1455 bp was obtained. The obtained MD1W sequence was compared with the ribosomal database http: / / rdp.cme.msu.edu / index.jsp for homology analysis, and the similarity with Bacillus cereus was 99.93%, and the strain was classified as Bacillus cereus.

[0009] The nucleotide sequence of 16S rDNA of Bacillus cereus MD1W is shown in Seq ID No: 1.

[0010] Another object of the present application is to provide the use of the Bacillus cereus MD1W in degrading mulch microplastics and / or adsorbing and converting heavy metals.

[0011] Preferably, the ground film microplastic is polyethylene.

[0012] Preferably, the heavy metal is cadmium and / or lead.

[0013] The beneficial effects of the present invention are:

[0014] The present invention separates and screens a strain of Bacillus cereus MD1W, which can adsorb multiple heavy metals and degrade mulch microplastics at the same time. It has strong environmental adaptability and can grow in the range of pH 4-10, temperature 20-40°C, and salinity 0.01%-8%. It can be applied to the treatment and restoration of mulch-heavy metal composite polluted environments, and its application objects include water / soil in mining areas, farmland soil, industrial wastewater, coastal zones and offshore environments. It can effectively reduce the harm of mulch microplastics and heavy metals to the ecological environment and human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention is further described using the accompanying drawings, but the embodiments in the accompanying drawings do not constitute any limitation to the present invention. A person skilled in the art can obtain other drawings based on the following drawings without creative work.

[0016] Figure 1 This is the morphology of Bacillus cereus MD1W on NB plate;

[0017] Figure 2 It is the result of measuring the growth status of Bacillus cereus MD1W under different pH environmental conditions;

[0018] Figure 3 It is the result of measuring the growth status of Bacillus cereus MD1W under different temperature environmental conditions;

[0019] Figure 4 It is the result of the growth test of Bacillus cereus MD1W under different salinity conditions;

[0020] Figure 5 The degradation effect of Bacillus cereus MD1W on mulch microplastics in the mulch microplastic-heavy metal composite system;

[0021] Figure 6 It is the surface microscopic morphology of mulch microplastics before and after degradation in the mulch microplastic-heavy metal composite system;

[0022] Figure 7 It is the adsorption effect of Bacillus cereus MD1W on heavy metals in the mulch microplastic-heavy metal composite system. DETAILED DESCRIPTION

[0023] The present invention is further described in conjunction with the following examples.

[0024] Example 1

[0025] Isolation and Identification of Bacillus cereus MD1W Strain

[0026] 1. Preparation of culture medium and solution

[0027] Mineral salt medium (MSM): 5 mL phosphate buffer solution (Na 2 HPO 4 12H 2 O 33.4g / L, K 2 HPO 4 ·3H 2 O21.75g / L, KH 2 PO 4 8.5g / L, NH 4 Cl 5.0 g / L); 3 mL MgSO 4 Solution (22.5 g / L); 1 mL FeCl 3 Solution (0.25 g / L); 1 mL CaCl 2 solution (36.4 g / L); 1 mL trace element solution (ZnSO 4 ·H 2 O 42.8mg / L, MnSO 4 ·H 2 O39.9mg / L, (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 34.7mg / L), adjust the volume to 1L.

[0028] Heavy metal stock solution: Weigh a certain amount of Cd(NO 3 ) 2 、Pb(NO 3 ) 2 Dissolve in deionized water to prepare a cadmium solution with a concentration of 500 mg / L (Cd 2+ ), lead (Pb 2+ ) stock solution.

[0029] Nutrient broth agar solid medium (NB): 18g nutrient broth, 18g technical agar powder, 1L distilled water.

[0030] 2. Domestication, screening, isolation and purification of strains

[0031] The polyethylene mulch was cut into small pieces (2×1.5 cm), crushed with a pulverizer, passed through a 10-mesh sieve, and washed with 2% SDS, 75% ethanol, and 95% ethanol for more than 4 hours, respectively. The mulch was rinsed with sterile water for 3 times on a clean bench, dried naturally, and sterilized with ultraviolet light for 15 minutes to obtain mulch microplastics.

[0032] Weigh 10g of agricultural soil covered with mulch for a long time, put it into a triangular flask containing MSM culture medium, add the mulch microplastics at a concentration of 1g / L, and culture at 30℃, 140rpm shaking for 60 days. Then take 10mL of culture solution and transfer it to the mulch microplastics (1g / L), Cd 2+ (8mg / L), Pb 2+ (5 mg / L) MSM medium, shake culture for 60 days to complete the first acclimation, repeat this step and acclimate for another 4 times;

[0033] 100 μL of the culture solution from the last acclimation cycle was taken and spread on NB medium. The plate was placed upside down in a 30°C biochemical incubator. After 2 to 3 days, colonies of different morphologies were picked from the plate for streak purification to obtain the purified strain, which was numbered MD1W.

[0034] The strain was identified by 16S rDNA sequence analysis and was classified as Bacillus cereus.

[0035] Example 2

[0036] Environmental adaptability of Bacillus cereus MD1W

[0037] The strain MD1W was adapted to different pH values, temperatures and salinities. The bacterial solution was inoculated into MSM medium (1 g / L mulch microplastics, 8 mg / L Cd 2+ and 5mg / L Pb 2+ ), initial OD 600 The pH value was set at 4, 5, 6, 7, 8, 9 and 10, the temperature was set at 20°C, 25°C, 30°C, 35°C and 40°C, the salinity was set at 0.01%, 0.1%, 0.5%, 1%, 2%, 4%, 6%, 8% and 10% with NaCl (w / v), three parallels were set for each treatment, 140r / min, 30°C shaking culture, and samples were taken after 60 days to measure the OD of the bacteria 600 .

[0038] The results of the measurement are shown in the attached Figure 2-4, Bacillus cereus MD1W can grow in acidic, neutral and alkaline environments (pH 4-10), with the optimum pH being 7, indicating that the strain MD1W has a wide range of acid-base adaptability; the strain MD1W can grow in the range of 20℃ to 40℃, with the optimum temperature being 30℃, and it grows well at 40℃, indicating that the strain has a certain tolerance to high temperature; the strain MD1W also has a strong salt tolerance, showing a good growth state when the NaCl range is 0.01% to 8%. These experimental results show that Bacillus cereus MD1W has a strong adaptability to changes in environmental conditions, can adapt to acidic, neutral and alkaline environments, and has a wide range of growth temperature and strong salt tolerance.

[0039] Example 3

[0040] Degradation and adsorption performance of Bacillus cereus MD1W

[0041] The MD1W bacterial suspension was transferred into the soil containing mulch microplastics (1 g / L), Cd 2+ (8mg / L) and Pb 2+ The culture medium was incubated with 5 mg / L of MSM as the treatment group; a control group without MD1W was set up, and three parallels were set up for each treatment. The culture was shaken at 30°C and 140 r / min for 60 days, and the supernatant was collected for quantification and filtered through a 0.22 μm filter head. Cadmium (Cd 2+ ) and lead (Pb 2+ ) concentration, and calculate the removal rates of cadmium and lead;

[0042] The mulch microplastics and bacteria obtained by centrifugation were separated, and the mulch microplastics were soaked in 75% ethanol, 95% ethanol and 100% ethanol for more than 4 hours respectively. They were rinsed with sterile water on a clean bench for 3 times and dried naturally. They were weighed to determine the degradation rate of the mulch microplastics, and the microscopic characteristics of the mulch microplastics were observed using a scanning electron microscope.

[0043] The weight reduction of microplastics in ground film is one of the indicators to determine whether they have been degraded. In the composite pollution system of ground film microplastics, cadmium and lead, the degradation effect of MD1W on ground film microplastics is as follows: Figure 5 As shown in Figure 2, after adding strain MD1W, the degradation rate of MD1W on mulch microplastics gradually increased with the extension of culture time, and the removal rate of mulch microplastics reached 8.87% at 60 days. The surface morphology of mulch microplastics observed by scanning electron microscopy was shown in Figure 2. Figure 6As shown in the figure, the surface of the original film microplastics (a) is smooth and flat. After 60 days of cultivation in the composite pollution system of film microplastics, cadmium and lead, the surface of the film microplastics in the control group (b) is very complete, with only a very slight roughness. The film microplastics in the treatment group (c) have obvious cracks, wrinkles and holes, showing a certain degree of damage and shedding, which shows that Bacillus cereus MD1W has a good ability to degrade film microplastics. At the same time, MD1W also has a good adsorption effect on cadmium and lead (see Figure 7 ), the adsorption rates of MD1W for cadmium and lead were 51.37% and 98.28% respectively. It can be seen that MD1W has a good degradation effect on film microplastics while adsorbing heavy metals.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A strain of Bacillus cereus MD1W, characterized in that: The deposit number is GDMCC No: 62744, and it was deposited in the Guangdong Provincial Microbiological Culture Collection Center, China on August 25, 2022.

2. The use of Bacillus cereus MD1W according to claim 1 in degrading mulch microplastics and adsorbing and converting heavy metals, wherein the mulch microplastics are polyethylene, and the heavy metals are cadmium and lead.

3. A degradation bacterial agent comprising the Bacillus cereus MD1W according to claim 1.

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