Application of paraclostridium benzene to promotion of degradation of methyl mercury

Abstract

The invention relates to the technical field of microorganisms, in particular to application of paraclostridium benzene to promotion of degradation of methyl mercury. The preservation number of the paraclostridium behenolyticum is KCTC (Korean Center of Type Culture Collection) 15476 or LMG (Leukocyte Molecular Growth) 28745, and the preservation number of the paraclostridium behenolyticum is KCTC 15476. The invention discloses the capability of degrading the toxic pollutant methyl mercury of the paraclostridium behenoelyticum for the first time, and the paraclostridium behenoelyticum has important significance for deeply knowing the form 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 Paraclostridium benzoelyticum in promoting the degradation of methylmercury. Background Art

[0002] Mercury is a global pollutant of international concern. Mercury in the environment is mainly present in the form of elemental mercury (Hg 0 ), inorganic mercury (Hg + , Hg 2+ ), organic mercury (methylmercury, ethylmercury). Methylmercury (MeHg) is one of the most toxic forms of mercury and is easily bioavailable and accumulated in the food chain. Methylmercury can cause serious damage to the human nervous system, cardiovascular disease and other health problems.

[0003] Many studies have shown that the net accumulation of methylmercury in the environment is the result of competition between the methylation process and the demethylation process. In an environment suitable for the survival of microorganisms, microorganisms play a very important role in driving the methylation of mercury and the demethylation of methylmercury. The methylation and demethylation processes of microorganisms are commonly found in aquatic sediments, wetlands, paddy soils, oceans, glaciers, peat, intestines and other environments. Methylmercury-degrading microorganisms are distributed in different microbial fields such as bacteria, fungi, and archaea. However, the strains that can degrade methylmercury confirmed by current studies are still a minority. At present, the research on methylmercury pollution prevention and control measures is mainly based on physical and chemical methods. However, physical and chemical remediation methods have the disadvantages of secondary pollution and high cost. The discovery of strains with high efficiency in methylmercury degradation provides strain resources for microbial remediation of methylmercury pollution, which is of great significance to reducing environmental methylmercury pollution. Summary of the invention

[0004] The present invention aims to provide the use of Paraclostridium benzoelyticum in promoting the 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 Paraclostridium benzoelyticum in promoting the degradation of toxic pollutant methylmercury. The preservation number of Paraclostridium benzoelyticum is KCTC 15476 or LMG28745.

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

[0008] Furthermore, Paraclostridium benzoelyticum can reduce the concentration of methylmercury in soil.

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

[0010] The present invention discloses for the first time that Paraclostridium benzoelyticum 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 Paraclostridium benzoelyticum 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 Paraclostridium benzoelyticum was inoculated into the paddy soil and cultured for 5 days in Experimental Example 2;

[0013] Figure 3 This is the phylogenetic tree of Paraclostridium benzoelyticum 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 Paraclostridium benzoelyticum in LB medium

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

[0017] A method for isolating Paraclostridium benzoelyticum comprises the following steps:

[0018] Under the anaerobic conditions of an anaerobic glove box (85% N2 + 10% H2 + 5% CO2), 10 mL of sterile liquid culture medium was mixed with 1.5 g of paddy field topsoil in a 50 mL sterile centrifuge tube, and 10 sterile glass beads with a diameter of 2 mm were placed at the same time. After vortexing for 15 minutes, the mixture was allowed to stand. The upper layer of liquid was then taken as the bacterial source for subculture experiments. During subculture, 19.5 mL of liquid culture medium was added to a 30 mL glass bottle, inoculated with 0.5 mL of bacterial source, and sealed with a butyl rubber stopper and an aluminum-plastic cap. The reaction bottle, rubber stopper, and culture medium were sterilized with high-pressure steam (121°C, 20 min). After dark culture at 30°C for about 10 days, the subculture enrichment culture was continued for 6 times to obtain the subculture bacterial solution. The subculture bacterial solution was stirred 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] The purified cultured strain was selected and sequenced by 16SrDNA, and the sequence was:

[0021]

[0022] 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 was found to have a similarity of 100% with the 16SrDNA gene sequence of Paraclostridium benzoelyticum.

[0023] After searching, it was found that the Paraclostridium benzoelyticum isolated in this example can be purchased from the Korean Collection of Type Cultures (accession number: KCTC 15476) and the Belgian Collection of Microorganisms (accession number: LMG28745).

[0024] 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 as a reducing agent (20g / L, filtered with a 0.22μm filter membrane) to the culture medium.

[0025] 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 .

[0026] 4. Inoculate Paraclostridium benzoelyticum into LB medium. Use a syringe to puncture and inoculate 0.5mL of Paraclostridium benzoelyticum bacterial solution into LB anaerobic medium. At the same time, set up a group without inoculation of Paraclostridium benzoelyticum bacterial solution as a blank control. Carry out dark culture in a 30℃ constant temperature box. 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.

[0027] like Figure 1 The results showed that after Paraclostridium benzoelyticum was inoculated into LB medium and cultured for 24 hours, the methylmercury concentration was reduced by 97% compared with the non-inoculated group (sterile medium).

[0028] Experimental Example 2: Degradation of Methylmercury by Paraclostridium benzoelyticum in Paddy Field Soil

[0029] 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.

[0030] 2. Paraclostridium benzoelyticum was inoculated into methylmercury contaminated soil. Using a syringe, 1 mL of Paraclostridium benzoelyticum bacterial solution was punctured and added to 8 mL of paddy soil mud, and cultured at 30°C in the dark. At the same time, mud without bacterial solution was cultured synchronously as a control. Three parallel samples were set up for each group.

[0031] 3. Test on the change of soil methylmercury concentration. After the soil slurry was incubated for 5 days, the slurry was freeze-dried and the methylmercury concentration in the soil was measured.

[0032] like Figure 2 The results showed that after Paraclostridium benzoelyticum was inoculated into methylmercury-contaminated paddy soil and cultured for 5 days, the methylmercury concentration in the paddy soil was reduced by 53.55% compared with the non-inoculated group (paddy soil).

[0033] In summary, the present invention discloses for the first time that Paraclostridium benzoelyticum 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.

[0034] 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.

[0035] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. Use of Paraclostridium benzoelyticum in promoting the degradation of methylmercury, characterized in that: The accession number of the Paraclostridium benzoelyticum is KCTC 15476 or LMG28745.

2. The use according to claim 1, characterized in that: The Paraclostridium benzoelyticum 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 Paraclostridium benzoelyticum can reduce the concentration of methylmercury in the soil.

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