Rhizobium bacteria and application thereof in degrading 1, 4-dioxane

Abstract

The invention discloses a rhizobium bacterium and application of the rhizobium bacterium in degradation of 1, 4-dioxane, and belongs to the technical field of environmental microorganisms. The bacterial classification of the rhizobium is named as rhizobium sp., and the rhizobium is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.25779. The invention further discloses a preparation method of the rhizobium. The strain can rapidly degrade 1, 4-dioxane, and has the advantages of strong tolerance, wide applicable pH range, wide applicable concentration range for degradation, wide applicable environmental condition range and strong pollutant tolerance. And by using the strain as a strain resource, economic, green and efficient microbial remediation of 1, 4-dioxane pollution can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental microorganisms, and in particular to the application of a strain of Rhizobium sp. in the degradation of an emerging pollutant 1,4-dioxane. Background Art

[0002] With the rapid development of science and technology and the continuous advancement of detection technology, emerging pollutants have gradually attracted attention in the fields of environmental science and health. 1,4-Dioxane is also known as 1,4-dioxane, with an English name of 1,4-Dioxane and a molecular formula of C4H8O2. It has a stable structural composition, is easily soluble in water and is not easy to volatilize in water, thus having strong water mobility. 1,4-Dioxane was first used in industrial production as a stabilizer for chlorinated hydrocarbon solvents, and was later used in the production of paints, fuels, deodorants, detergents, cosmetics, electronic products, etc. In addition, it is often produced as an industrial by-product in the production process of ethylene oxide, ethanol, and terephthalic acid. Due to problems such as incomplete treatment of industrial wastewater containing 1,4-dioxane, illegal dumping, and garbage leakage, 1,4-dioxane enters the surface water environment and penetrates the soil and gravel into the groundwater with water, thus forming a widespread pollution plume and accumulating persistently in the environment such as surface water, groundwater, and sediments. At present, it has been found in surface water around the world (260 μg·L in the United States -1 , Japan 46μg·L -1 , Netherlands 10μg·L -1 , Germany 2.2μg·L -1 ), groundwater (USA 220mg·L -1 ), industrial wastewater (Japan 4020μg·L -1 ) and landfill leachate (Japan 2000 μg·L -1 ) has been widely detected in China. There are also reports in my country that 1,4-dioxane has been detected in high concentrations in leachate extracted from landfills, and its accumulation has also been detected in the viscera of fish along the Yellow River.

[0003] Toxicological studies have shown that 1,4-dioxane has chronic toxicity, mutagenicity, teratogenicity, carcinogenicity and other hazards to organisms, and ingestion will cause anesthesia and irritation. The World Health Organization has classified it as a Class 2B substance and stipulates that the permissible limit of 1,4-dioxane is 30 mg·L -1 Some countries have also set standards for its detection in water environments. Japan and South Korea set the national discharge standard for 1,4-dioxane at 500 μg·L -1 The maximum detection limit of 1,4-dioxane in the drinking water quality standard is 50 μg·L -1 ; The U.S. Environmental Protection Agency recommends that the detection limit for tap water be 0.67 μg·L -1The German Federal Environment Agency classifies it as a non-regulated toxic substance and recommends that the detection limit for 1,4-dioxane in drinking water be 5 μg·L -1 my country requires in the "Limits of Hazardous Substances in Cosmetics" that the residual concentration of 1,4-dioxane should be less than 30 mg kg -1 .

[0004] There are physical, chemical and biological methods for the degradation of 1,4-dioxane. Conventional physical methods have poor removal effects on 1,4-dioxane, and it is usually necessary to prepare modified new adsorbents to enhance their adsorption capacity for 1,4-dioxane. The chemical method uses advanced oxidation technology to utilize the strong oxidizing properties of free radicals to degrade 1,4-dioxane. However, this type of method is costly when used to remediate 1,4-dioxane in contaminated sites and is difficult to promote in practical applications. In comparison, biological remediation is more feasible. The use of plants or microorganisms for remediation can not only reduce costs but also generate no harmful by-products. It is an economical and green environmentally friendly remediation technology without secondary pollution. The time period for plant remediation of 1,4-dioxane is longer, while microbial remediation of 1,4-dioxane is more efficient. Although it was previously believed that the epoxy heterocyclic molecular structure containing COC bonds of 1,4-dioxane was difficult to be degraded by microorganisms, with the continuous deepening of research, some 1,4-dioxane-degrading bacteria have been isolated to prove that 1,4-dioxane can be biodegraded, mainly through two pathways: metabolism and cometabolism. Most of the strains isolated so far have shortcomings such as low degradation efficiency, the need for cometabolism cultivation, and poor tolerance to environmental stress. Therefore, it is of great practical significance to isolate and screen efficient 1,4-dioxane-degrading strains in the environment and to explore environmental factors such as pH and temperature that affect biodegradation efficiency. Summary of the invention

[0005] 1. Problem to be solved

[0006] The purpose of the present invention is to provide an efficient 1,4-dioxane-degrading strain, and further to provide a strain that can use 1,4-dioxane as the sole carbon source and can completely degrade it, and has a high tolerance to high concentrations of 1,4-dioxane and a strong adaptability to temperature and pH changes, so that the strain can be applied to the bioremediation of 1,4-dioxane-contaminated environments and solve the problem of residual 1,4-dioxane pollution in the environment.

[0007] 2. Technical solution

[0008] The technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides a rhizobium bacterium, which is classified and named as Rhizobium sp., and is deposited in the General Microbiological Culture Collection Center of the China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No. 25779. The deposit date is September 23, 2022, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The second aspect of the present invention provides a Rhizobium bacterium comprising the nucleotide sequence of SEQ ID NO:1.

[0011] The third aspect of the present invention provides a bacterial suspension, which contains a rhizobium bacterium. The rhizobium bacterium is classified and named as Rhizobium sp., and is deposited in the General Microbiological Culture Collection Center of China Microorganism Culture Collection Committee with a deposit number of CGMCC NO.25779.

[0012] As a preferred embodiment of the technical solution of the third aspect of the present invention, the carbon source for the culture of the rhizobium sp. contains 1,4-dioxane. The carbon source for the culture can be a commonly used carbon source for microbial culture, such as Luria-Bertani medium (LB medium for short) or R2A medium for culture, or an inorganic salt medium (MSM medium) with 1,4-dioxane as the carbon source.

[0013] As a preferred embodiment of any solution of the third aspect of the present invention, the bacterial suspension is obtained by expanding the culture under the conditions of 30°C and 150rpm constant temperature shaker. The bacterial cell density of the rhizobium after the bacterial suspension is collected is measured by OD 600 It is generally believed that its OD 600 Should be greater than or equal to 0.05.

[0014] The fourth aspect of the present invention provides a composition, which comprises rhizobium bacteria. The rhizobium bacteria are classified and named as Rhizobium sp., and are deposited in the General Microbiological Culture Collection Center of China Microorganism Culture Collection Committee with a collection number of CGMCC NO.25779.

[0015] As a preferred embodiment of the fourth technical solution of the present invention, the mass percentage of Rhizobium sp. in the composition is 0.1-99.9%.

[0016] The fifth aspect of the present invention provides use of the rhizobium bacteria of the first or second aspect of the present invention, or the bacterial suspension of the third aspect, or the composition of the fourth aspect in degrading 1,4-dioxane.

[0017] As a preferred embodiment of any solution of the fifth aspect of the present invention, the rhizobium sp. is first cultured under a specific concentration of 1,4-dioxane to a degradation end point, wherein the preferred 1,4-dioxane concentration is 100 to 800 mg·L -1 The more preferred 1,4-dioxane concentration is 200 mg·L -1 ; The culture solution is collected, and the rhizobium sp. is inoculated into the medium containing 1,4-dioxane to be degraded in a certain proportion. From the perspective of the activity of the rhizobium sp., when cultured to the degradation end point under a specific concentration of 1,4-dioxane, the activity of the rhizobium sp. is higher, so it is preferably first treated and then inoculated into the medium containing 1,4-dioxane to be degraded.

[0018] As a preferred embodiment of any of the fifth aspects of the present invention, the OD after inoculation of the rhizobium Rhizobium sp. 600 The value is not less than 0.02. From the perspective of ensuring degradation rate and efficiency, it is particularly preferred that the OD after inoculation of Rhizobium sp. 600 The value should not be less than 0.05.

[0019] As a preferred embodiment of any solution of the fifth aspect of the present invention, the concentration of 1,4-dioxane used by the rhizobium sp. is in the range of 0 to 1000 mg·L -1 , excluding 0. Further preferably, the concentration of 1,4-dioxane used by the rhizobium sp. is in the range of 0 to 800 mg·L -1 , excluding 0. Further preferably, the concentration of 1,4-dioxane used by the rhizobium sp. is in the range of 50 to 800 mg·L -1 .

[0020] As a preferred embodiment of any scheme of the fifth aspect of the present invention, the pH range of the application of the rhizobium sp. is 5.0 to 8.0. In this pH range, the degradation rate of 1,4-dioxane by the rhizobium sp. is higher than that of the case where the pH is less than or equal to 4.0 or the pH is greater than or equal to 9.0.

[0021] As a preferred embodiment of any solution of the fifth aspect of the present invention, the temperature at which the rhizobium sp. is applied is 20-37° C. In this temperature range, the degradation rate of 1,4-dioxane by the rhizobium sp. is relatively high.

[0022] As a preferred embodiment of any solution of the fifth aspect of the present invention, the rotation speed of the shaking table is controlled at 100-200 rpm during degradation.

[0023] As the use of the rhizobium Rhizobium sp. in the degradation of 1,4-dioxane provided in any scheme of the fifth aspect of the present invention, the 1,4-dioxane is in any water body (groundwater or sewage treatment plant), leachate or sediment.

[0024] The sixth aspect of the present invention provides a method for isolating 1,4-dioxane-degrading strains from contaminated environmental samples. The isolation and culture of the strains first collects activated sludge from a municipal sewage treatment plant that has been contaminated by 1,4-dioxane for a long time for enrichment culture, and then performs subculture by transfer. After determining that the enrichment can degrade 1,4-dioxane stably for a long time, the bacteria with 1,4-dioxane-degrading ability are isolated and purified by a dilution coating method.

[0025] 3. Beneficial effects

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention provides a highly efficient 1,4-dioxane-degrading strain, Rhizobium sp. (5113), which can grow with 1,4-dioxane as the sole carbon source and completely degrade 1,4-dioxane; the strain is adaptable to a wide range of environmental conditions and has strong tolerance to pollutants; using this strain as a strain resource can achieve economical, green and efficient microbial remediation of 1,4-dioxane pollution.

[0028] (2) The use of the rhizobium Rhizobium sp. (5113) of the present invention in the degradation of 1,4-dioxane is firstly cultured under a condition of a specific concentration of 1,4-dioxane to a degradation end point, the culture fluid is collected, and inoculated into a medium containing 1,4-dioxane to be degraded in a certain proportion; when the bacterial suspension at the degradation end point is used to further degrade 1,4-dioxane in the medium, the degradation rate is faster. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The colony morphology of the rhizobium strain Rhizobium sp.5113 of the present invention is shown;

[0030] Figure 2 is a phylogenetic tree of the rhizobium strain Rhizobium sp.5113 of the present invention;

[0031] Figure 3 The rhizobium strain Rhizobium sp.5113 of the present invention was subjected to 100 mg·L -1 The degradation effect diagram and growth curve of 1,4-dioxane;

[0032] Figure 4 The degradation curves of the rhizobium strain Rhizobium sp.5113 of the present invention to 1,4-dioxane at different concentrations;

[0033] Figure 5 The rhizobium strain Rhizobium sp.5113 of the present invention was subjected to 100 mg·L -1 The degradation curve of 1,4-dioxane;

[0034] Figure 6 The rhizobium strain Rhizobium sp.5113 of the present invention was subjected to 100 mg·L -1 Degradation curve of 1,4-dioxane. DETAILED DESCRIPTION

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0036] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0037] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement or value. The degree of flexibility for a particular variable can be easily determined by one skilled in the art.

[0038] Concentration, amount and other numerical data can be presented in range format herein.It should be understood that such range format is only used for convenience and simplicity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all single numerical values ​​or sub-ranges contained in the range, just as each numerical value and sub-range are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including single numerals (such as 2,3,4) and sub-ranges (such as 1 to 3,2 to 4, etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all the above-mentioned values ​​and ranges.In addition, no matter how the breadth of the described range or feature is, this explanation should be applicable.

[0039] The present invention is further described below in conjunction with specific embodiments.

[0040] The invention provides a 1,4-dioxane efficient degradation strain, namely Rhizobium sp.5113, which is classified and named as Rhizobium sp.strain and preserved in the General Microbiological Center of China Microbiological Culture Collection Administration with a preservation number of CGMCC No.25779.

[0041] The inorganic salt (MSM) medium includes (per liter of sterile deionized water): Na2HPO4·12H2O 4.5 g, K2HPO4 1.0 g, NH4SO4 1.0 g, MgSO4·7H2O 0.2 g, CaCl2·2H2O 0.02 g, 1 mL of trace element stock solution, and 1 mL of vitamin stock solution.

[0042] The trace element stock solution included (per liter of sterile deionized water): MnSO4·H2O 0.1 g, Na2MoO4·2H2O 0.02 g, H3BO3 0.014 g, FeSO4·7H2O 1.0 g, ZnSO4·7H2O 0.1 g, CuSO4·5H2O 0.02 g, and CoCl2·6H2O 0.02 g.

[0043] The vitamin stock solution includes (per liter of sterile deionized water): folic acid 2.0 g, pyridoxine hydrochloride 10.0 g, riboflavin 5.0 g, biotin 2.0 g, thiamine 5.0 g, niacin 5.0 g, calcium pantothenate 5.0 g, vitamin B12 0.1 g, p-aminobenzoic acid 5.0 g, and lipoic acid 5.0 g.

[0044] LB medium includes (per liter of sterile deionized water): tryptone 10 g, yeast extract 5 g, NaCl 10 g.

[0045] R2A medium includes (per liter of sterile deionized water): tryptone 0.25 g, acid hydrolyzed casein 0.5 g, yeast extract 0.5 g, soluble starch 0.5 g, K2HPO4 0.3 g, MgSO4 0.1 g, sodium pyruvate 0.3 g, peptone 0.25 g, and glucose 0.5 g.

[0046] The solid agar medium of the above culture medium is obtained by adding 1.5% agar powder to the above liquid culture medium.

[0047] The nutrient agar medium includes: 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride, and 15-20 g of agar.

[0048] The concentration of 1,4-dioxane was detected by gas chromatography-hydrogen flame ionization detector (GC-FID), and the detection instrument model was Agilent Technologies' Agilent 8860 gas chromatography system. When detecting the concentration of 1,4-dioxane, an HPUltra2 chromatographic analysis column was used, and high-purity nitrogen was used as the carrier gas, and the carrier gas flow rate was set to 2 mL min -1 , the column box temperature was programmed to 40°C, the injection port temperature was 120°C, and the detector temperature was 300°C. Pretreatment was required to detect the concentration of 1,4-dioxane. The sample was first filtered through a 0.22μm filter membrane, then extracted with 200μL of dichloromethane at a volume ratio of 1:1, and placed in a -20°C refrigerator for 0.5h. The organic phase was then taken out of the injection bottle for testing.

[0049] Example 1: Enrichment, separation, purification, preservation and identification of rhizobium strain Rhizobium sp.5113

[0050] Sample collection: The original samples used for enrichment of degradation bacteria were collected from activated sludge of a municipal sewage treatment plant in Nanjing, whose receiving water body was affected by 1,4-dioxane for a long time.

[0051] Laboratory enrichment culture: The collected activated sludge samples were enriched and transferred in the laboratory with 1,4-dioxane as the sole carbon source. Take 5g of activated sludge sample and put it into 100mL of inorganic salt MSM liquid culture medium, add 1,4-dioxane to make the concentration 5% of the activated sludge culture solution was transferred to fresh sterile MSM medium three times, and 1,4-dioxane was added to make the concentrations of 100, 250, and 1000 mg·L -1 , put it into a shaker at 150rpm and 30℃ for enrichment sample culture. Finally, after 4 months of enrichment culture, the final enrichment culture was verified and found to be able to quickly degrade 1,4-dioxane in a short time.

[0053] Isolation and purification of degradation strains: The degradation strains were isolated and purified by plate gradient dilution method. The enriched culture was gradiently diluted with physiological saline or phosphate buffered saline (PBS). The enriched culture solution was diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 6 dilutions were applied on the surface with 200 mg·L -1The bacteria were cultured on an inorganic salt agar solid culture dish containing 1,4-dioxane and incubated in a 30°C constant temperature incubator for about 1 week until a single colony grew out. Then, strains with different morphology and size were selected for streaking on the plate and cultured in a 30°C constant temperature incubator until pure bacterial colonies that could grow in 1,4-dioxane were obtained.

[0054] Preservation of degradation strains: Pick a single colony on the solid culture medium and place it in a carbon source medium (LB, R2A, inorganic salt MSM medium containing 1,4-dioxane) and expand the culture at 150rpm and 30℃. When the bacteria are cultured to the logarithmic phase, collect the bacteria after centrifugation at 4℃, 5000rpm and store them in 25% glycerol and store them in a -80℃ refrigerator for long-term storage, or store them in a 4℃ refrigerator in the form of bacterial suspension and solid culture medium colonies for short-term storage.

[0055] Identification of degradation strains: 16S rDNA universal primers (27F5'-AGTTTGATCMTGGCTCAG-3' and 1492R5'-GGTTACCTTGTTACGACTT-3') were used for amplification and sequencing, and the sequence is shown in SEQ ID NO: 1. The strain was identified as a Gram-negative, aerobic bacterium, and oxidase-positive. The sequencing results were spliced ​​using ContigExpress, and the inaccurate parts at both ends were removed. The spliced ​​sequences were compared in the NCBI database (blast.ncbi.nlm.nih.gov), and a phylogenetic tree was constructed using MEGA software, as shown in Figure 1. Figure 2 shown.

[0056] Combined with the physiological and biochemical strain characteristics and phylogenetic tree analysis, this strain was identified as Rhizobium sp. Strain and was deposited in the General Microbiology Center of China Microorganism Culture Collection on September 23, 2022, with the deposit number CGMCC No.25779.

[0057] The morphology of the bacterial colonies on the plate is shown in Figure 1 The colonies are all round in shape, about 3 to 5 mm in size, with smooth surfaces, neat edges, a protrusion in the center, and light yellow in color.

[0058] Example 2: Growth ability and degradation characteristics of Rhizobium sp. 5113 in 1,4-dioxane

[0059] The culture conditions of the strain were combined with the commonly used conditions for expressing 1,4-dioxane-degrading strains reported previously. The culture medium was incubated at 30°C and 150 rpm in a 100 mg·L -1 The bacterial biomass OD was measured by spectrophotometer at regular intervals. 600 The measurement results are as follows Figure 3 .

[0060] First, the strain was cultured at 200 mg·L -1 The strain was cultured in 1,4-dioxane MSM medium until the degradation end point. At this time, the culture fluid was collected and inoculated into fresh MSM medium containing 1,4-dioxane at a volume ratio of 5%. The strain OD was measured. 600 The degradation experiment is to use gas chromatography GC-FID to measure the residual concentration of 1,4-dioxane at regular intervals, and to calibrate the average degradation rate at the end point of degradation. This method is used to measure the residual concentration of 1,4-dioxane at 50, 100, 200, 400, 600, and 800 mg·L -1 The degradation experiment of 1,4-dioxane was carried out, and the results were as follows Figure 4 As shown. The degradation kinetics were fitted by the Monod equation, and the maximum specific degradation rate of the strain for 1,4-dioxane was 0.273 mg-1,4-Dioxane·(mg-protein) -1 ·h -1 Rhizobium sp. 5113 showed excellent degradation effect on different concentrations of 1,4-dioxane and had good tolerance to high concentrations of 1,4-dioxane.

[0061] Example 3: Effect of Rhizobium sp. 5113 on 1,4-dioxane degradation characteristics under different environmental conditions

[0062] This example studies the degradation ability of Rhizobium sp. 5113 on 1,4-dioxane at different pH values ​​and temperatures.

[0063] The initial pH range was set to 4.0-9.0, including: 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, 9.0. The culture was carried out at 30°C and 150 rpm, and the initial 1,4-dioxane concentration was 100 mg·L -1 .like Figure 5 As shown, the degradation rate of 1,4-dioxane by strain Rhizobium sp.5113 at pH 4.0, 5.0, 6.0, 7.0, 7.5, 8.0, and 9.0 was 0.14 mg·(L·h) respectively. -1 , 1.42mg·(L·h) -1 , 1.82mg·(L·h) -1 , 2.04mg·(L·h) -1 , 2.86mg·(L·h) -1 , 1.78mg·(L·h) -1 , 0.05mg·(L·h)-1.

[0065] Set the initial temperature range to 20-37°C, including 20, 30, and 37°C. Culture at pH 7.5, 150 rpm, and an initial 1,4-dioxane concentration of 100 mg·L -1 .like Figure 6 As shown in the figure, the degradation rate of 1,4-dioxane by strain Rhizobium sp.5113 at 20, 30, and 37°C was 1.40 mg·(L·h) respectively. -1 , 2.81mg·(L·h) -1 , 2.79mg·(L·h)-1.

[0067] The above content is a schematic description of the present invention and its implementation methods, which is not restrictive. What is shown in the embodiment is only one of the implementation methods of the present invention, and the actual implementation method is not limited thereto. Therefore, if a person of ordinary skill in the art is inspired by it and designs implementation methods and embodiments similar to the technical solution without creative design without departing from the purpose of the invention, they should all fall within the scope of protection of the present invention.

Claims

1. Rhizobium bacteria, the rhizobium bacteria are classified and named as Rhizobium sp., and are deposited in the General Microbiological Culture Collection Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.25779.

2. Rhizobium bacteria, characterized in that Comprising the nucleotide sequence of SEQ ID NO:

1.

3. A bacterial suspension, characterized in that: The bacterial suspension contains rhizobium bacteria, which are classified and named as Rhizobium sp. and are preserved in the General Microbiological Culture Collection Center of China Microbiological Culture Collection Administration, with a preservation number of CGMCC NO.25779.

4. The bacterial suspension according to claim 3, characterized in that The carbon source for the culture of the rhizobium sp. contains 1,4-dioxane.

5. A composition, characterized in that The composition comprises a rhizobium bacterium, which is classified and named as Rhizobium sp. and is preserved in the General Microbiological Culture Collection Center of China Microbiological Culture Collection Committee with a preservation number of CGMCC NO.25779.

6. Use of the rhizobium bacteria according to claim 1 or 2, or the bacterial suspension according to claim 3 or 4, or the composition according to claim 5 in degrading 1,4-dioxane.

7. The use according to claim 6, characterized in that: First, the rhizobium Rhizobium sp. is cultured under a certain concentration of 1,4-dioxane to a degradation end point, and the culture solution is collected and inoculated into a medium containing 1,4-dioxane to be degraded in a certain proportion.

8. The use according to claim 7, characterized in that: The OD after inoculation of Rhizobium sp. 600 The value is not less than 0.

02.

9. The use according to claim 7 or 8, characterized in that: The concentration range of 1,4-dioxane used by the rhizobium sp. is 0 to 1000 mg·L -1 , not including 0; and / or The pH range of the Rhizobium sp. application is 5.0 to 8.0; and / or The temperature for applying the rhizobium sp. is 20-37°C.

10. The use according to any one of claims 6 to 9, characterized in that: The 1,4-dioxane is in water, leachate or sediment.

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