Anaerobic acidobacterium and its application

Abstract

The present invention discloses an anaerobic acid soil Clostridium and its application, and relates to the field of microbial technology. Clostridium acidisoli CK74X was deposited with the Guangdong Provincial Center for Microbial Culture Collection on October 17, 2022, at Building 59, 5th Floor, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province, with the deposit number GDMCC No. 62805. The acidoterpene Clostridium CK74X of the present invention has strong viability and tolerance, and is capable of remediating arsenic contamination in oligotrophic, highly arsenic-toxic tailings areas. The acidoterpene Clostridium CK74X of the present invention has a highly efficient volatilization effect on inorganic arsenic, with a volatilization rate of up to 92% under low concentration conditions and up to 55% under high concentration conditions.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to an anaerobic acidobacterium and an application thereof. Background Art

[0002] my country leads the world in nonferrous metal mining production. While mining brings enormous economic benefits, it also inevitably poses serious environmental threats. As ore is mined, large quantities of heavy metals (metalloids) are activated and released. These metals enter plants and animals through the solid-liquid-gas cycle, and then enter the human body through the food chain, posing a significant threat to human production and life. Arsenic, a metalloid, is widely distributed, presents high concentrations, and is highly toxic in ores, posing a significant threat to human production and life. Arsenic primarily exists in inorganic forms with valences of -3, 0, +3, and +5. -3 arsenic primarily exists in the atmosphere as arsine, a colorless gas. While non-toxic, 0 valence arsenic is highly metabolized and rarely found in the natural environment. +3 valence arsenic is the most toxic and is often found in anaerobic environments. +5 valence arsenic primarily exists in aerobic environments and is less mobile and toxic than +3 valence arsenic.

[0003] Currently, arsenic contamination remediation primarily involves physical, chemical, and biological methods. Physical remediation methods include arsenic adsorption using materials such as biochar, iron-based materials, silica gel, and resins, and filtration using materials such as grids and filters. Physical remediation methods are often used for large-scale field remediation. They can rapidly improve soil and water quality, but their effectiveness is limited in duration, and some materials are difficult to recycle, leading to high remediation costs and the possibility of secondary pollution. Chemical remediation primarily utilizes chemical reagents to detoxify and / or immobilize arsenic through oxidation, precipitation, ion exchange, and passivation, often in combination with physical methods. Chemical methods offer advantages such as high efficiency and commercial viability, but drawbacks include the difficulty in controlling the dosage of chemical reagents. Lower doses can result in incomplete remediation, while higher doses can cause secondary environmental contamination. Bioremediation, on the other hand, is a promising remediation method with low investment, minimal environmental disturbance, and sustained effectiveness. It primarily leverages the inherent arsenic defense mechanisms of animals, plants, and microorganisms to alter the form of arsenic in the environment, thereby achieving its immobilization or detoxification goals. Plants and animals need oxygen, and pollution remediation is mainly targeted at the surface, while the anaerobic environment of deep water and soil requires the use of microorganisms for remediation.

[0004] The use of microorganisms to remediate arsenic contamination in soil and water through volatilization has been a research hotspot in recent years. However, few strains have been discovered and successfully cultivated, particularly those capable of volatilizing arsenic under anaerobic conditions. Furthermore, the majority of these microorganisms are found in nutrient-rich soil and water, and have never been found in nutrient-poor, non-ferrous metal mining areas, where arsenic concentrations are higher. Consequently, the use of previously studied microbial agents to remediate oligotrophic, highly arsenic-toxic groundwater in mining areas presents technical challenges. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide an anaerobic acid soil Clostridium and its application. The anaerobic acid soil Clostridium of the present invention has strong survival ability and tolerance, can volatilize As(III) in contaminated water and soil through arsenic methylation behavior, thereby reducing the arsenic content in the contaminated water and soil, and has great potential in the remediation of arsenic contamination in oligotrophic deep water and soil.

[0006] To achieve the above object, the technical solution adopted by the present invention is: an anaerobic acid soil Clostridium, the strain is named acid soil Clostridium ( Clostridium acidisoli ) CK74X, has been deposited in the Guangdong Provincial Microbial Culture Collection Center on October 17, 2022, address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, with a deposit number of GDMCC No: 62805. The inventors of the present application finally screened out an anaerobic acid soil clostridium from a mining area with oligotrophic and high arsenic toxicity through a large number of experimental screenings. Experiments have found that the acid soil clostridium CK74X has the function of efficiently volatilizing inorganic arsenic in deeply contaminated water and soil. The acid soil clostridium CK74X of the present invention can volatilize arsenic through methylation, and can be combined with other physical and chemical means to collect the volatilized gaseous arsenic, which can achieve the reduction of arsenic in contaminated water and soil and the recovery and reuse of arsenic. Compared with microorganisms that repair arsenic contamination through other means, the method of the present invention does not have the risk of arsenic being released again after being converted, and can realize the recovery and reuse of arsenic. The repair of arsenic is more efficient and thorough, with less environmental disturbance, and can also generate certain economic benefits.

[0007] As a preferred embodiment of the anaerobic acidobacterium of the present invention, the 16SrRNA gene sequence of the acidobacterium CK74X is shown as SEQ ID NO.1.

[0008] As a preferred embodiment of the anaerobic acidulospora strain of the present invention, the acidulospora strain CK74X was isolated from groundwater sediments 60 m below the mining site. Most strains discovered so far are found in nutrient-rich soil and water. The acidulospora strain CK74X of the present invention was isolated from groundwater sediments 60 m below the oligotrophic and highly arsenic-toxic Shuilongwei lead-zinc mining area in Qingyuan City, Guangdong Province, and exhibits strong survival and tolerance.

[0009] The present invention also provides the use of the anaerobic acidulosibirum in repairing arsenic pollution.

[0010] As a preferred embodiment of the application described in the present invention, the remediation is that anaerobic acid soil Clostridium has the ability to volatilize arsenic. Existing microorganisms with the ability to repair arsenic pollution mainly reduce the activity or toxicity of arsenic in contaminated sites through oxidation or methylation, and there may be a risk of reduction or demethylation. The anaerobic acid soil Clostridium of the present invention can quickly and completely methylate and then volatilize inorganic arsenic, and can directly extract arsenic from deep water and soil with a high volatilization rate.

[0011] As a preferred embodiment of the application of the present invention, the remediation is carried out under anaerobic conditions.

[0012] As a preferred embodiment of the application of the present invention, the arsenic pollution includes arsenic pollution of groundwater bodies under oligotrophic conditions and / or arsenic pollution of deep soil.

[0013] The present invention also provides a bacterial agent for repairing arsenic pollution, wherein the bacterial agent comprises the anaerobic acidobacterium.

[0014] The present invention also provides a method for repairing arsenic pollution in deep water and soil in a mining area, wherein the anaerobic acid soil Clostridium is added to the arsenic-contaminated site to methylate the arsenic through its arsenic methylation function, thereby volatilizing the arsenic.

[0015] As a preferred embodiment of the method of the present invention, the arsenic pollutants include arsenic pollution in oligotrophic groundwater and / or arsenic pollution in deep soil.

[0016] The beneficial effects of the present invention are as follows: the present invention provides an anaerobic acid soil Clostridium. Since the acid soil Clostridium CK74X of the present invention is derived from a mining area with low nutrient content and high arsenic pollution concentration, the strain has strong survival ability and tolerance to arsenic, and can still exert the ability to repair arsenic pollution in mining areas with harsh environments; the acid soil Clostridium CK74X of the present invention has a highly efficient volatilization effect on inorganic arsenic, with a volatilization rate of up to 92% under low concentration conditions and a volatilization rate of up to 55% under high concentration conditions; the acid soil Clostridium CK74X of the present invention can volatilize arsenic through a methylation process, thereby significantly reducing the content of As(III) in deep contaminated water and soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The growth curves of the strain under 0.1 mM, 1 mM and 10 mM As(III) stress;

[0018] Figure 2 is the colony morphology of Clostridium acidulosum CK74X;

[0019] Figure 3 This is a transmission electron micrograph (TEM) of Clostridium acidulosum CK74X;

[0020] Figure 4is the arsenic volatilization rate of the strain under 0.1 mM, 1 mM and 10 mM As(III) stress;

[0021] Figure 5 The graph shows the changes in arsenic forms of the strain under 0.1 mM, 1 mM and 10 mM As(III) stress. DETAILED DESCRIPTION

[0022] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0023] Example 1 Deposit Information of Anaerobic Acidobacterium

[0024] The present invention provides an anaerobic acid soil Clostridium, which is named acid soil Clostridium ( Clostridium acidisoli )CK74X was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 17, 2022, at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, with the deposit number GDMCC No: 62805.

[0025] Example 2 Acquisition and Identification of Clostridium acidophilum CK74X

[0026] 1. Acquisition of Clostridium acidulosum CK74X: Groundwater and sediment samples were collected from a 60 m underground well in the Shuilongwei lead-zinc mine area in Qingyuan City, Guangdong Province. 1 mL of the sample was added to the anaerobic chamber with ST10 -1 Cultured in liquid medium for 7 days. Then 50 μL of bacterial solution was spread on ST10 -1 Place on a solid plate and incubate at 37°C. After 4 days, pick out the grown single bacteria and place them in 3 mL ST10 -1 The strain was cultured in liquid medium for 4 days, and the arsenic species in the culture medium were determined. The strain was purified by plate plating and liquid culture three times to obtain a single strain.

[0027] 2. Strain identification

[0028] (1) Observation of colony and bacterial morphology: Clostridium acidophilum CK74X was grown in ST10 -1 The strain was streaked onto a solid plate medium and cultured at 37°C for 48 h. The growth of the colonies on the plate was observed and recorded. Figure 2 、 Figure 3 shown.

[0029] Depend on Figure 2 It can be seen that the colonies formed by the strain of this example are white and convex.

[0030] Depend on Figure 3 It can be seen that the strain of this example has a volume of 3~7×1 μm and has flagella.

[0031] (2) Molecular biological identification: The bacterial genomic DNA of strain CK74X was extracted and amplified by PCR using universal primers for 16S rRNA gene. The nucleotide sequence of 16S rRNA of the strain was obtained by sequencing as shown in SEQ ID NO.1. According to the sequence homology comparison of the standard strain, the sequence of 16S rRNA of strain CK74X was similar to that of the nitrogen-fixing bacteria that have been discovered. Clostridium acidisoli DSM 12555 has a similarity of over 99%, and genetic analysis indicates that the bacterium is Clostridium acidulosum ( Clostridium acidisoli ).

[0032] Based on the morphological characteristics, physiological and biochemical identification, 16S rRNA sequencing and homology analysis of the strain in this example, the strain CK74X was identified as Clostridium acidulosum ( Clostridium acidisoli ), named Clostridium acidoterreus ( Clostridium acidisoli )CK74X.

[0033] Example 3 Cultivation of Clostridium acidophilum CK74X

[0034] This example provides a method for culturing the above-mentioned Clostridium acidulosum CK74X.

[0035] Culture medium: 0.5 g tryptone, 0.05 g yeast extract, 5 g glucose, 1 L deoxygenated ultrapure water;

[0036] Culture temperature: 28-37 ℃;

[0037] Culture method: Take out the frozen Clostridium acidulosum CK74X from the -80 ℃ refrigerator, take 1 mL of Clostridium acidulosum CK74X bacterial solution and inoculate it into 20 mL of ST10 -1 Incubate in anaerobic liquid culture tube at 30℃ in the dark for 4 days to activate the strain. -1 2.5 mL of activated bacterial solution was inoculated into the liquid culture medium and cultured statically at 30°C in the dark.

[0038] Example 4 Methylation and volatilization of arsenic by Clostridium acidulosum CK74X

[0039] 1. Preparation of As(III) stock solution: Dissolve 129.9 mg of AsNaO2 in 100 mL of oxygen-free water in an anaerobic workstation. Prepare a 10 mM As(III) stock solution and store it in an anaerobic bottle. Prepare a 0.1 mM As(III) working stock solution by diluting 1 mL of the 10 mM As(III) stock solution with anaerobic water in a 100 mL anaerobic bottle.

[0040] 2. Volatilization of arsenic by Clostridium acidulosum CK74X: 1 mL of Clostridium acidulosum CK74X bacterial solution was inoculated into a 20 mL ST10 -1 To the anaerobic liquid culture tube, add 10 μL, 100 μL, and 1000 μL of 0.1 mM As(III) working stock solution to the final concentrations of 0.1 μM, 1 μM, and 10 μM, respectively. Mix thoroughly by inversion. These solutions are recorded as the experimental group solutions. Take 1 mL of the Clostridium acidulosum CK74X bacterial solution and inoculate it into a 20 mL ST10 -1 Anaerobic liquid culture tubes were incubated in a 30°C incubator without the addition of As(III) working solution as a control group (CK).

[0041] 3. Growth of Clostridium acitrinum CK74X at 30°C: All treatment groups were placed in a 30°C incubator, and the OD values were measured every 4 hours for the first four days. 600 , and then tested once every two days. Figure 1 shown.

[0042] 4. Analysis of the methylation ability of As(III) at 30°C: Samples from all treatment groups were taken every two days. The samples were directly aspirated with a sterile syringe with a needle, passed through a 0.22 μm filter membrane, and placed in a 4°C refrigerator for testing. The arsenic form was detected by plasma mass spectrometry coupled with liquid chromatography (ICP-MS-LC). The chromatographic column selected was , with an injection volume of 10 μL and a flow rate of 1.0 mL / min. The mobile phase was 20 mM diammonium hydrogen phosphate, and the pH was adjusted to 6.0 with high-grade pure nitric acid. The elution method was isocratic elution. The results are shown in Figure 2. Figure 4 shown.

[0043] Depend on Figure 4 It can be seen that under 30 ℃ conditions, the concentration of As(III) in the culture medium was significantly reduced by Clostridium acidulosum CK74X, accompanied by the detection of monomethylarsenic (MMA) and dimethylarsenic (DMA), the sum of which accounted for up to 21% of the total arsenic concentration.

[0044] 5. Analysis of As(III) Volatility at 30°C: After 20 days of incubation, all samples were incubated using a gas collection device to collect headspace gas. The gas collection device was configured as follows: a long needle was inserted into the top of each sample bottle below the liquid level to provide nitrogen flow, and a short needle was connected to an arsenic gas capture tube to collect gaseous arsenic in the headspace. Nitrogen flow was maintained at a steady rate of 1 mL / min for 4 hours. The arsenic gas capture tube was capped at both ends with 0.3 g of glass wool and filled with 0.1 g of treated silica gel beads. The silica gel beads were treated by soaking them in a 10% AgNO₃ solution overnight, freeze-drying them, and sealing them for later use. The silica gel beads, which had adsorbed gaseous arsenic, were ultrasonically extracted using a 1 M phosphoric acid solution with a solid-to-liquid ratio of 1:50 (v:v) at 80°C. The adsorbed gaseous arsenic was dissolved in the phosphoric acid solution, and the arsenic-containing phosphoric acid solution was analyzed using inductively coupled plasma mass spectrometry coupled to liquid chromatography (ICP-MS-LC). The chromatographic column used in the liquid chromatograph was an anion exchange column, with an injection volume of 10 μL and a flow rate of 1.0 mL / min. The mobile phase was a 20 mM diammonium phosphate solution with a pH of 6.0. The pH of the mobile phase was adjusted with chromatographic-grade nitric acid and ammonia. The elution method was isocratic. Figure 5 shown.

[0045] Depend on Figure 5 The results indicate that Clostridium acitrinum CK74X exhibited excellent As(III) volatilization at 30°C, with five to six arsenic compounds detected in the dissolved gas. Calculated based on the difference between the initial addition amount and the arsenic content in the culture medium, the arsenic volatilization rate for this functional bacterium ranged from 55% to 92%.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An anaerobic acidobacterium, characterized in that The clostridium was named Clostridium acidoterreus ( Clostridium acidisoli )CK74X was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 17, 2022, at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, with the deposit number GDMCC No: 62805.

2. The use of the anaerobic acidobacterium Clostridium in remediation of arsenic pollution according to claim 1, characterized in that: The arsenic pollution remediation method is to convert the inorganic trivalent arsenic compound into methylated arsenic and then volatilize it.

3. The use according to claim 2, characterized in that The remediation is carried out under anaerobic conditions.

4. The use according to claim 2, characterized in that The arsenic pollution includes arsenic pollution of groundwater bodies under oligotrophic conditions and / or arsenic pollution of deep soil under oligotrophic conditions.

5. A bacterial agent for repairing arsenic pollution, characterized in that: The bacterial agent includes the anaerobic acidobacterium Clostridium according to claim 1.

6. A method for remediating arsenic contamination of groundwater and / or deep soil in mining areas, characterized in that: The anaerobic acidobacterium according to claim 1 is added to an arsenic-contaminated environment to methylate inorganic trivalent arsenic and volatilize it.

7. The method according to claim 6, characterized in that The arsenic pollution includes arsenic pollution of groundwater bodies under oligotrophic conditions and / or arsenic pollution of deep soil under oligotrophic conditions.

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