Bacillus cereus resistant to severe combined pollution of heavy metals As and Cd and preparation of solid microbial inoculum of bacillus cereus resistant to severe combined pollution of heavy metals As and Cd

Abstract

The invention discloses a bacterium capable of resisting serious combined pollution of arsenic and cadmium. The bacillus cereus strain is identified as bacillus cereus, is named as bacillus cereus L1. GLUT, and is preserved in the China General Microbiological Culture Collection Center on April 10, 2024, the address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.30292. The bacillus cereus strain is named as bacillus cereus L1. GLUT. The bacillus cereus L1. GLUT can resist 200 mg / L of As and 100 mg / L of Cd at the same time, can be prepared into a microbial agent to be combined with a hyperaccumulator, and has considerable application value in the aspect of remediation of arsenic and cadmium combined contaminated soil.

Description

Technical Field

[0001] This invention relates to the field of soil heavy metal pollution remediation technology, specifically to a study on the tolerance of bacteria to severe combined pollution of arsenic and cadmium and the preparation of bacterial agents. Technical Background

[0002] Arsenic (As) is a naturally occurring element found in rocks, soil, and water. As can be obtained from minerals such as pyrite, stansite, and pyrite, and can also enter the soil through industrial emissions, such as from the metallurgical and chemical industries. Pesticide residues are also a source of arsenic in soil. As in the soil can be absorbed by plants, adversely affecting their metabolism, and accumulate in edible parts, posing a risk to humans and livestock. As compounds have been identified as carcinogenic, teratogenic, and mutagenic. As poisoning is a systemic disease primarily affecting the skin; it can harm the skin, respiratory, digestive, urinary, cardiovascular, nervous, and hematopoietic systems, and in severe cases, can cause neurological abnormalities, respiratory distress, heart failure, and even death. Soil cadmium (Cd) pollution mainly originates from atmospheric cadmium deposition, the use of pesticides, fertilizers, and plastic films, sewage irrigation, sludge fertilization, the accumulation of heavy metal-containing waste, and acidic wastewater pollution from metal mines. Cd damages crop cells, inhibits chloroplast photosynthesis, weakens the crop's antioxidant capacity, and thus significantly reduces crop yield. Furthermore, excessive Cd absorption by crops can "steal" the space that belongs to nutrients such as iron (Fe), manganese (Mn), and zinc (Zn), leading to nutrient deficiencies and reduced quality in crops, seriously harming normal crop growth and agricultural production. Excessive Cd absorption in the human body is stored in organs and tissues such as fat, bones, liver, and muscles, causing adverse symptoms such as joint pain, skin diseases, intestinal flora imbalance, and nerve damage, leading to a series of diseases including Itai-itai disease, glaucoma, and Alzheimer's disease.

[0003] Microorganisms play a crucial role in the remediation of heavy metal-contaminated soils. Firstly, microorganisms can reduce the toxicity of heavy metals in soil by utilizing them as energy and nutrient sources for metabolism, thereby reducing their bioavailability and mobility. Secondly, microorganisms can decompose organic matter in the soil, such as organic acids and polyphenols, promoting an increase in soil organic matter content and improving soil fertility. Furthermore, microorganisms can utilize their metabolic characteristics and enzyme activity to absorb, transport, and deposit heavy metals through transformation. Some sulfate-reducing bacteria can reduce heavy metal ions to alcohols, sulfates, or metal sulfides, thus mitigating the toxicity of heavy metals in the soil. Some heavy metal-tolerant microorganisms have the ability to degrade organic pollutants, degrading the intracellular organic complexes of heavy metals through biotransformation, thereby reducing the toxicity of heavy metals in the soil and the subsequent environmental risks. Therefore, the research and application of microbial remediation technologies are of great significance for solving the problem of heavy metal pollution in soil. Summary of the Invention

[0004] The purpose of this invention is to isolate microorganisms from soil contaminated with arsenic and cadmium, conduct heavy metal tolerance studies, and prepare microbial agents for the remediation of heavy metal pollution in soil.

[0005] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0006] The strain capable of simultaneously tolerating arsenic and cadmium co-contamination was deposited on April 10, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It belongs to the Bacillus cereus family, with accession number CGMCC NO.:30292, and is named Bacillus cereus L1.GLUT strain. The optimal growth pH for the arsenic-resistant strain L1.GLUT is 7, the optimal temperature is 30℃, and the optimal rotation speed is 180 r / min.

[0007] The method for preparing solid microbial agents based on the above-mentioned resistant strains includes the following steps:

[0008] (1) Activation of strain: Place the strain at the appropriate temperature and incubate for a period of time.

[0009] (2) Seed fermentation broth: The strain was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of liquid culture medium and cultured with shaking at the optimal growth temperature and rotation speed for 48 h.

[0010] (3) Expanded culture: The strain was inoculated into a 500 mL Erlenmeyer flask containing 250 mL of liquid culture medium and cultured with shaking at the optimal growth temperature and rotation speed for 48 h.

[0011] (4) After centrifugation, resuspend in physiological saline and repeat the operation three times.

[0012] (5) Collect the bacterial cells and a certain amount of glycerol and skim milk powder, and freeze-dry them to obtain bacterial powder.

[0013] (6) Select wood shavings, fish bone meal, wheat bran, and crushed peanut shells as carriers. Mix the bacterial powder obtained in (5) with the carriers and incubate in an incubator for a certain period of time (72 h) to obtain the desired microbial inoculum. The proportions are: bacterial powder 15%, wood shavings 15%, fish bone meal 15.0%, wheat bran 15.0%, and crushed peanut shells 40%.

[0014] The strain was sent to a sequencing company (Shanghai Ling'en Biotechnology Co., Ltd.) for sequencing. The obtained sequence was then compared with the NCBI (National Center for Biotechnology Information) using Nucleotide Blast. Figure 7 ), and used MEGA software based on the Neighbor Joining Method to perform phylogenetic analysis and construct a phylogenetic tree ( Figure 8 ).from Figure 7 It can be seen that L1.GLUT has 99% sequence homology with Bacillus cereus (MZ746191.1), and L1.GLUT was identified as Bacillus cereus.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] 1. The strain Bacillus cereus L1.GLUT provided by this invention is resistant to both arsenic and cadmium contamination, and the concentrations of arsenic and cadmium reach 200 and 100 mg / L, respectively, which are considered to be at the level of severe contamination.

[0017] 2. The microbial agent preparation method provided by this invention is simple. After being mixed with a certain amount of carrier, it can be used for the bioremediation of soil contaminated with arsenic and cadmium, or combined with heavy metal hyperaccumulating plants. It has extremely important significance and application value for improving soil environmental quality. Attached Figure Description

[0018] Figure 1 This is a colony appearance image of strain L1.GLUT.

[0019] Figure 2 This shows the growth of strain L1.GLUT under a contamination concentration of As 200 mg / L + Cd 100 mg / L.

[0020] Figure 3 This shows the growth of strain L1.GLUT under different pH conditions.

[0021] Figure 4 This shows the growth of strain L1.GLUT under different temperature conditions.

[0022] Figure 5 This shows the growth of strain L1.GLUT under different rotation speeds.

[0023] Figure 6 This is the sequence listing of strain L1.GLUT.

[0024] Figure 7 This is proof of the identification of strain L1.GLUT.

[0025] Figure 8 This is the phylogenetic tree of strain L1.GLUT.

[0026] Figure 9 It is a solid microbial agent prepared using L1.GLUT strain as the active ingredient. Detailed Implementation

[0027] The present invention will be further explained and described below with reference to specific embodiments, but the scope of the present invention is not limited thereto.

[0028] Example:

[0029] The isolation, purification, and screening of the strains specifically include the following steps:

[0030] (1) Pouring plates: Melt the LB solid medium by heating, and pour the plates when the temperature drops to 55~60℃. Pour 3 plates for each type of medium.

[0031] (2) Preparation of soil dilution: Weigh 10 g of arsenic-cadmium contaminated soil sample and place it in a 90 mL sterile Erlenmeyer flask containing glass beads. Shake for about 20 min to fully mix the soil sample with the water and disperse the cells. Use a 1 mL sterile pipette to draw 1 mL of the soil suspension and add it to a large test tube containing 9 mL of sterile water. Mix thoroughly. This is 10 mL of the soil suspension. -1 Diluent, and so on, to prepare 10 -2 10 -3 10 -4 10 -5 and 10 -6 Soil solutions at several dilutions.

[0032] (3) Spreading: Write 10 on the bottom of the culture medium plate or around the edge of the petri dish lid with a marker. -4 10 -5 and 10 -6 Three dilution labels were used, with three dishes labeled for each dilution. Then, using sterile pipettes, 10 dishes were prepared. -4 10 -5 and 10 -6Take an appropriate amount of the diluted soil solution from 3 tubes and place it in the center of the plate with the dilution marked. Accurately place 0.2 mL in each plate. Use a sterile glass spreader to gently spread the solution evenly on the surface of the culture medium. The method is to first gently push the bacterial solution back and forth along a straight line to distribute it evenly, and then change the direction by 90° and push it back and forth along another vertical line. You can change the direction and spread it several more times at the inner edge of the plate. Let it stand at room temperature for 5-10 minutes.

[0033] (4) Incubation: Invert the container and incubate for 1-2 days.

[0034] (5) Purification: Select suitable colonies, streak them to purify until a single colony is obtained, and then store them for later use.

[0035] (6) Metal tolerance study: Certain concentrations of NaAsO2 and CdCl2 were added to the culture medium, with As concentration gradients of 100, 200, 300, 400, and 500 mg / L, and Cd concentration gradients of 20, 40, 60, 80, and 100 mg / L. The highest concentration combination of As and Cd contamination tolerating the strains was investigated by pairwise combinations. The strains were then transferred to slant culture medium and cultured for 24 h before being stored at 4 ℃ for later use. Each experiment was repeated three times, with one blank control group.

[0036] The growth conditions of the strain were investigated, including pH (4, 5, 6, 7, 8), temperature (25, 28, 30, 33, 35℃), and rotation speed (90, 120, 150, 180, 210 r / min). The specific steps are as follows:

[0037] Each experiment was repeated three times, with uninoculated culture medium as a reference.

[0038] (1) Effect of pH on bacterial growth

[0039] After activating the bacterial strain in liquid medium for 24 h, 0.6 ml (2% inoculum) of bacterial suspension was inoculated into sterile Erlenmeyer flasks containing 30 ml of medium. The pH gradient of the medium was (4, 5, 6, 7, 8). The OD was measured after incubation at 30℃ and 180 r / min for 24 h. 600 .

[0040] (2) Effect of temperature on bacterial growth

[0041] After activating the bacterial strain in liquid culture medium for 24 h, 0.6 ml (2% inoculum) of bacterial solution was inoculated into sterile Erlenmeyer flasks containing 30 ml of culture medium. The pH of the culture medium was the optimal value obtained in (1). The flasks were placed in shakers at different temperatures, with a gradient of (25℃, 28℃, 30℃, 33℃, 35℃) and a shaker speed of 180 r / min. After culturing for 24 h, the OD was measured.600 value.

[0042] (3) Effect of shaking speed on cell growth

[0043] After activating the corresponding bacterial strains in liquid culture medium for 24 h, 0.6 ml (2% inoculum) of bacterial suspension was inoculated into sterilized Erlenmeyer flasks containing 30 ml of culture medium. The pH of the culture medium and the temperature of the shaker were the optimal values ​​obtained in (1) and (2). The flasks were placed in shakers with different rotation speeds (90, 120, 150, 180, 210 r / min) and cultured for 24 h. The OD was measured after each incubation. 600 value.

[0044] The strain was cultured as follows:

[0045] LB solid medium: 3 g yeast extract, 10 g peptone, 5 g NaCl, 1000 mL distilled water, 15-20 g agar.

[0046] The preparation of the solid inoculum of the strain is as follows:

[0047] (1) Activation of strain: Inoculate the strain preserved on slant onto a plate and incubate for 24 h.

[0048] (2) Seed fermentation broth: The strain was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of liquid culture medium and cultured with shaking at the optimal growth temperature and rotation speed for 48 h.

[0049] (3) Expanded culture: The fermented bacterial solution was inoculated into a 500 mL Erlenmeyer flask containing 250 mL of liquid culture medium (2% inoculation amount), and cultured with shaking at the optimal growth temperature and rotation speed for 48 h.

[0050] (4) After centrifugation, resuspend in physiological saline. Repeat the centrifugation at least three times until the supernatant is clear and transparent.

[0051] (5) Collect the centrifuged bacterial cells, mix them with a certain amount of glycerol and skim milk powder, refrigerate them in a -20℃ refrigerator, and freeze-dry them for 24 hours to obtain bacterial powder.

[0052] (6) Wood shavings, fish bone meal, wheat bran, and crushed peanut shells were selected as carriers. The mycelium powder obtained in (5) was mixed with a certain amount of carriers and placed in a constant temperature incubator for 72 h. The ratio was: 15% mycelium powder, 15% wood shavings, 15.0% fish bone meal, 15.0% wheat bran, and 40% crushed peanut shells.

[0053] (7) Store in a refrigerator at 4°C for later use.

Claims

1. A strain of Bacillus cereus resistant to severe combined contamination by heavy metals As and Cd, characterized in that... The strain was named Bacillus cereus L1.GLUT and was deposited on April 10, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.:30292.

2. Based on the strain Bacillus cereus L1.GLUT as described in claim 1, characterized in that, This strain is tolerant to both 200 mg / L As and 100 mg / L Cd.

3. Based on the strain Bacillus cereus L1.GLUT as described in claim 1, characterized in that, The optimal growth pH for this strain is 7, the optimal temperature is 30℃, and the optimal rotation speed is 180 r / min.

4. A method for preparing solid microbial inoculants, characterized in that, The active ingredient of the bacterial agent includes Bacillus cereus L1.GLUT as described in claim 1.

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