Application of clostridium amazinii in promoting degradation of methyl mercury

Abstract

The invention relates to the technical field of microorganisms, in particular to application of clostridium amazinii in promoting degradation of methyl mercury. The preservation number of the clostridium amazonense is DSM 23598, the preservation number of the clostridium amazonense is CCUG 59712, or the preservation number of the clostridium amazonense is CCAM 1539. The clostridium amazonense disclosed by the invention has the capability of degrading the toxic pollutant methyl mercury for the first time, and is of great significance in deeply knowing the morphological transformation effect of microorganisms on mercury, understanding the biogeochemical cycle of mercury and repairing and treating microorganisms polluted by methyl mercury in the environment.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to application of Clostridium amazonense in promoting the degradation of methylmercury. Background Art

[0002] Mercury is a global pollutant of international concern. Methylmercury (MeHg) is one of the most toxic forms of mercury. It is easily absorbed and accumulated by organisms in the food chain, and can cause serious damage to the human nervous system. For a long time, eating fish has been considered the main source of human exposure to methylmercury. However, new research has found that eating rice is one of the main ways for people to be exposed to methylmercury. The reason was found to be that rice is a crop that hyperaccumulates methylmercury. Paddy field soil is the main source of methylmercury absorption and enrichment in rice. The average methylmercury concentration in sediments of five typical ecosystems in the world (rice fields, wetlands, lakes, oceans, and forests) ranges from 1.70 μg·Kg -1 The average methylmercury concentration in paddy soil is 2.5-3 μg·Kg -1 , which is significantly higher than the other four typical ecosystems.

[0003] At present, the research on prevention and control measures of methylmercury pollution in paddy soil is mainly based on physical and chemical methods. However, physical and chemical remediation methods have disadvantages such as secondary pollution and high cost. The discovery of strains with high efficiency in methylmercury degradation can provide strain resources for microbial remediation of methylmercury pollution, which is of great significance to reducing environmental methylmercury pollution. Summary of the invention

[0004] The object of the present invention is to provide application of Clostridium amazonense in promoting degradation of methylmercury.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] Provided is the use of Clostridium amazonense in promoting the degradation of methylmercury. The deposit number of Clostridium amazonense is DSM 23598, CCUG 59712 or CCAM 1539.

[0007] Furthermore, Clostridium amazonense can greatly reduce the concentration of methylmercury in the liquid phase of the culture medium in a short period of time.

[0008] Furthermore, Clostridium amazonense can reduce methylmercury concentrations in soil.

[0009] The beneficial effects of the present invention are:

[0010] The present invention discloses for the first time that Clostridium amazonense has the ability to degrade the toxic pollutant methylmercury, which is of great significance for in-depth understanding of the morphological transformation of mercury by microorganisms, understanding the biogeochemical cycle of mercury, and microbial remediation and control of methylmercury pollution in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a schematic diagram of the change in methylmercury concentration in the LB medium 24 hours after Clostridium amazonense was inoculated into the LB medium in Experimental Example 1;

[0012] Figure 2 This is a schematic diagram of the change in methylmercury concentration in the soil after Clostridium amazonense was inoculated into the paddy soil and cultured for 20 days in Experimental Example 2;

[0013] Figure 3 This is the phylogenetic tree of Clostridium amazonense in Experimental Example 1. DETAILED DESCRIPTION

[0014] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0015] Experimental Example 1: Degradation of Methylmercury by Clostridium amazonense in LB Medium

[0016] 1. The strain used in this test example is Clostridium amazonense, which was isolated from the topsoil of paddy fields in Wanshan mercury mining area in Guizhou Province.

[0017] The method for isolating Clostridium amazonense comprises the following steps:

[0018] In an anaerobic glove box under anaerobic conditions (85% N 2 +10%H 2 +5% CO 2), mix 10mL of sterile liquid culture medium with 1.5g of paddy field topsoil in a 50mL sterile centrifuge tube, and put in 10 sterile glass beads with a diameter of 2mm, vortex and shake for 15 minutes, and then let it stand. Then take the upper layer of liquid as the bacterial source for subculture experiments. During subculture, add 19.5mL of liquid culture medium to a 30mL glass bottle, inoculate 0.5mL of bacterial source, and seal it with a butyl rubber stopper and an aluminum-plastic cap. The reaction bottle, rubber stopper and culture medium are sterilized with high-pressure steam (121°C, 20min). Cultivate in the dark at 30°C for about 10 days, and continue to subculture and enrichment culture for 6 times to obtain the subculture bacterial solution. The subculture bacterial solution is heated at 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Gradient dilution. Take 200 μL of the diluted bacterial solution and add it to the agar plate (2% agar) containing LB medium, and use a spreading rod to spread it evenly. Place the plate in an anaerobic environment of an anaerobic glove box for constant temperature culture at 30°C. After colonies grow on the plate, select different single colonies for three-zone streaking on a new plate containing agar medium, and then continue to culture. After streaking the plate multiple times, select colonies for strain identification to obtain pure strains.

[0019] LB medium: add 10 g of trypsin, 5 g of yeast extract powder, and 10 g of sodium chloride to every 1000 mL of oxygen-free water.

[0020]

[0021] The sequence obtained by sequencing was compared in NCBI, and the sequence with the greatest similarity was selected as the species identification result. After comparison, the sequence had a similarity of up to 99.86% with the 16SrDNA gene sequence of Clostridium amazonense.

[0022] After searching, the Clostridium amazonense isolated in this example can be purchased from the German Collection of Microorganisms and Cell Cultures (accession number: DSM 23598), the Culture Collection of the University of Gothenburg, Sweden (accession number: CCUG 59712) or the China Anaerobic Microbial Resources Collection Center (CCAM 1539).

[0023] 2. Preparation of LB anaerobic culture medium. LB culture medium ingredients: tryptone 10g / L, yeast extract powder 5g / L, sodium chloride 10g / L. In a 30mL glass bottle, add 19.2mL LB liquid culture medium, and seal it with a rubber stopper and aluminum plastic cover under anaerobic conditions in an anaerobic glove box. Then sterilize it in an autoclave at 121℃ for 20min. After cooling, use a syringe to add 0.3mL glutathione solution (20g / L, filtered with a 0.22μm filter membrane) to the culture medium.

[0024] 3. Add methylmercury to anaerobic LB medium. Use a syringe to puncture the rubber stopper and inject the methylmercury standard solution into the anaerobic LB medium. The final concentration of methylmercury added is 0.5 μg·L -1 .

[0025] 4. Inoculate Clostridium amazonense into LB medium. Use a syringe to puncture and inoculate 0.5mL of Clostridium amazonense bacterial solution into LB anaerobic medium. At the same time, set up a group without inoculation of Clostridium amazonense bacterial solution as a blank control. Carry out dark culture in a 30℃ constant temperature incubator. Set up 3 parallel samples for each group. After 24 hours of culture, terminate the culture, add 2% concentrated sulfuric acid to the reaction system to terminate the reaction, and test the residual methylmercury content in the culture medium.

[0026] like Figure 1 The results showed that after Clostridium amazonense was inoculated into LB medium and cultured for 24 hours, the methylmercury concentration was reduced by 98% compared with the non-inoculated group (sterile medium).

[0027] Test Example 2: Degradation of Methylmercury in Paddy Field Soil by Clostridium amazonense

[0028] 1. Collection of paddy field soil and preparation of mud. Paddy field soil was collected from Wanshan mercury mining area in Guizhou, and the paddy field soil was prepared into mud at a mass ratio of 5:1 (paddy field soil: water). In an anaerobic glove box, 8 mL of mud was added to a 30 mL glass bottle and sealed with a butyl rubber stopper and an aluminum cap.

[0029] 2. Inoculate Clostridium amazonense into methylmercury contaminated soil. Use a syringe to puncture 1mL of Clostridium amazonense bacterial solution and add it to 8mL of paddy soil mud, and culture it at 30℃ in the dark. At the same time, use mud without inoculation of Clostridium amazonense bacterial solution as a control. Set up 3 parallel samples for each group.

[0030] 3. Test on the change of soil methylmercury concentration. After 20 days of soil slurry cultivation, the slurry was freeze-dried and the methylmercury concentration in the soil was measured.

[0031] like Figure 2 The results showed that after Clostridium amazonense was inoculated into methylmercury-contaminated paddy soil and cultured for 20 days, the methylmercury concentration in the paddy soil was reduced by 33.65% compared with the non-inoculated group (paddy soil).

[0032] In summary, the present invention discloses for the first time that Clostridium amazonense has the ability to degrade the toxic pollutant methylmercury, which is of great significance for in-depth understanding of the morphological transformation of mercury by microorganisms, understanding the biogeochemical cycle of mercury, and microbial remediation of methylmercury pollution in the environment.

[0033] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. The use of Clostridium amazonense in promoting the degradation of methylmercury, characterized in that: The deposit number of the Clostridium amazonense is DSM 23598, CCUG 59712 or CCAM1539.

2. The use according to claim 1, characterized in that: The Clostridium amazonense can reduce the concentration of methylmercury in the liquid phase of the culture medium.

3. The use according to claim 1, characterized in that: The Clostridium amazonense can reduce the concentration of methylmercury in the soil.

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