An Acetobacter strain capable of anaerobic degradation of 1,3-butadiene and its application

Abstract

The present invention discloses the field of environmental pollution remediation, specifically an Acetobacterium strain capable of anaerobically degrading 1,3-butadiene and its application. The Acetobacterium wieringae strain N capable of anaerobically degrading 1,3-butadiene was deposited at the China General Microbiological Culture Collection Center on July 20, 2022, with the deposit number CGMCC No. 40099. The strain N provided by the present invention can convert 1,3-butadiene pollutants into 1-butene at a rate of 13.6 μmol D⁻¹ under anaerobic conditions; and convert isoprene pollutants into 2-methyl-1-butene and 3-methyl-1-butene at rates of 4.66 μmol D⁻¹ and 0.16 μmol D⁻¹, respectively. Therefore, the Acetobacterium strain obtained in this invention can efficiently detoxify aquifers contaminated with diene compounds.

Description

Technical Field

[0001] The invention discloses the field of environmental pollution restoration, in particular to an acetobacillus strain capable of anaerobically degrading 1,3-butadiene and application thereof. Background Art

[0002] 1,3-Butadiene (CAS 106-99-0, C4H6) is a colorless gas at room temperature with a gasoline-like odor. It is an organic compound containing two C=C unsaturated double bonds. 1,3-Butadiene is often used to produce synthetic rubber products such as tires, resins, plastics and other chemicals. The U.S. Environmental Protection Agency (EPA) stated in its 2016 Chemical Information Report that the annual production of 1,3-butadiene can reach 1-5 billion pounds. Automobile exhaust, tobacco smoke, and polluted air and water near plastic or rubber facilities are the main sources of human exposure to 1,3-butadiene. The median lethal concentration (LC50) of 1,3-butadiene in mice is 259-270g / m 3 LC50 in rats is 285 g / m 3 , accompanied by symptoms such as an increase in the total number of white blood cells and a decrease in neutrophils. Therefore, relevant toxicological studies have received great attention. Studies have consistently shown that occupational exposure to 1,3-butadiene is associated with an increased incidence of leukemia; and long-term exposure to this environment can cause symptoms such as eye pain, blurred vision, coughing, and drowsiness. The International Agency for Research on Cancer (IARC) has also included 1,3-butadiene in the list of Class I suspected human carcinogens. 1,3-Butadiene can undergo photochemical reactions with gases such as O3 and NO2 to produce a variety of mixed products of unknown hazard; in the soil environment, 1,3-butadiene can produce butene, butane and other substances under the catalytic action of granular iron. However, physical and chemical remediation methods are not thorough and are prone to secondary pollution. Therefore, the use of biological, especially microbial catalytic degradation of organic pollutants can minimize the concentration of pollutants, with low cost and little impact on the environment. It is a green remediation method. Anaerobic environments contain a large number of microbial resources with unknown functions, and research on the biological transformation of 1,3-butadiene is extremely scarce. Obtaining an anaerobic microbial resource that can efficiently degrade 1,3-butadiene is a milestone in the field of environmental bioremediation, and exploring the physiological and biochemical properties and functions of related strains is also extremely important. Summary of the invention

[0003] The present invention aims to provide an acetobacillus strain capable of anaerobically degrading 1,3-butadiene and application thereof.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0005] A strain of Acetobacterium wieringaeN that anaerobically degrades 1,3-butadiene, the strain is Acetobacterium wieringaeN, which was deposited in the General Microbiology Center of China Culture Collection Administration on July 20, 2022, with the deposit number CGMCC No.40099, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The strain is derived from river sediment, is enriched in an inorganic salt liquid anaerobic culture medium by adding 1,3-butadiene, and is finally obtained by extinction dilution in a semi-solid agarose culture medium.

[0007] The semi-solid agarose medium is prepared by adding 1 g of agarose having low temperature (<30° C.) gel properties and a gel solid strength greater than 200 g / cm2 to 10 mL of an inorganic salt medium. 2 Made from low hardness agarose.

[0008] The invention discloses an application of Acetobacterium wissenii for degrading 1,3-butadiene, and an application of the strain in degrading 1,3-butadiene pollutants.

[0009] Furthermore, the strain degrades 1,3-butadiene pollutants under anaerobic conditions.

[0010] The invention discloses an application of Acetobacterium wissenii for degrading 1,3-butadiene, and an application of the strain in degrading isoprene pollutants.

[0011] Furthermore, the strain is also capable of degrading isoprene contaminants under anaerobic conditions.

[0012] The invention discloses an application of Acetobacterium wissenii for degrading 1,3-butadiene, and an application of the strain in producing acetate by fermentation.

[0013] A preparation for degrading 1,3-butadiene pollutants, the preparation containing Acetobacterium wieringae N.

[0014] The preparation contains Acetobacterium wieringae N bacteria, glycerol culture, culture liquid concentrate or culture suspension.

[0015] A preparation for degrading isoprene pollutants, the preparation containing Acetobacteriumwieringae N.

[0016] The preparation contains Acetobacterium wieringae N bacteria, glycerol culture, culture liquid concentrate or culture suspension.

[0017] A preparation for preparing acetate bioenergy, the preparation containing Acetobacterium wieringae N.

[0018] A method for preparing acetate comprises fermenting Acetobacterium wieringae N using carbon dioxide and hydrogen under anaerobic conditions to produce acetate.

[0019] The advantages of the present invention are:

[0020] The present invention enriches and separates the strain from a fine river sediment sample, and belongs to Acetobacterium wieringae at the bacterial classification level, and is a new type of anaerobic acetic acid bacillus (N), which supplements the new microbial resources of this genus; in addition, the present invention obtains strain N for the first time with high efficiency in anaerobic degradation of 1,3-butadiene pollutants; the strain N can also degrade isoprene pollutants and produce a large amount of acetate using carbon dioxide and hydrogen. Ultimately, the present invention not only provides a promising bacterial agent remediation solution for solving the problem of 1,3-butadiene and isoprene pollution in soil and water environments, but also provides a new direction for the green production of acetate in industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the consumption rate of 1,3-butadiene by the mixed culture solution of Acetobacterium wieringae N of the present invention at different transfer times.

[0022] Figure 2 The invention discloses an identification method for the degradation products of 1,3-butadiene by the acetic acid bacterium Acetobacterium wieringae N of the present invention.

[0023] Figure 3 The figure is a graph showing the degradation performance of 1,3-butadiene pollutants by Acetobacterium wieringae N of the present invention at 30°C, pH=7.2, and NaCl concentration of 0.1%.

[0024] Figure 4 The figure is a rate diagram of 1,3-butadiene degradation by Acetobacterium wieringae N of the present invention at different temperatures, pH values ​​and salinities.

[0025] Figure 5 This is a graph showing the degradation performance of the acetic acid bacterium Acetobacterium wieringae N of the present invention on isoprene pollutants.

[0026] Figure 6 The phylogenetic classification evolution tree of the 16S rRNA gene of Acetobacterium wieringae N of the present invention. DETAILED DESCRIPTION

[0027] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0028] The experimental methods, materials and reagents used in the following examples are conventional methods unless otherwise specified.

[0029] Example 1. Establishment of enrichment culture of Acetobacterium strain N

[0030] The anaerobic microcosm system was established using the bottom mud of Xihe River in Shenyang City, Liaoning Province (N 41°44′41″, E 123°17′35″) as inoculum and 1,3-butadiene as substrate. The specific operation is as follows: First, the sediment at a depth of 70-90 cm and 10 m from the river bank was collected in a clean collection bottle using a high temperature and high pressure sterilized collector. River water was injected into the bottle to remove air, and the bottle was quickly transported to a 4°C refrigerator in the laboratory for storage.

[0031] Use a 3 L three-necked round-bottom flask to prepare the inorganic salt anaerobic culture medium. The components of each liter of mixed culture medium are as follows: inorganic salt components are 30mM sodium bicarbonate, 1.0g / L sodium chloride, 0.5g / L magnesium chloride hexahydrate, 0.2g / L potassium dihydrogen phosphate, 0.3g / L ammonium chloride, 0.3g / L potassium chloride, 1.5g / L calcium chloride dihydrate; trace elements are 1.5mg / L ferrous chloride tetrahydrate, 0.19mg / L cobalt chloride hexahydrate, 0.1mg / L manganese chloride tetrahydrate, 0.07mg / L zinc chloride tetrahydrate, 0.006mg / L boric acid, 0.036mg / L sodium molybdate dihydrate, 0.024mg / L nickel chloride hexahydrate, 0.002mg / L cupric chloride dihydrate, 0.006mg / L sodium selenate pentahydrate, 0.008mg / L sodium tungstate; reducing agents are 0.2mM sodium sulfide nonahydrate and 0.5mM dithiothreitol. After adjusting the pH of the culture medium to 7.2-7.3 with CO2, a mixture of N2 and CO2 (v / v, 80:20%) was introduced into the headspace of the culture medium. 80 mL of anaerobic culture medium was dispensed into clean, anaerobic serum bottles using a syringe, and the bottle mouth was sealed with a butyl rubber stopper and an aluminum cap. The dispensed culture medium was sterilized at 121 ° C for 30 min at high temperature and high pressure. The sterilized and cooled culture medium and the collected sediment were transferred to the anaerobic glove box, and about 3 g of sediment was added to each bottle of culture medium with a spoon, and the seal was continued with a rubber stopper. Finally, 1 mL of 1,3-butadiene as a substrate, 10 mL of hydrogen as an electron donor, 5 mM sodium lactate as a carbon source, and 0.1 mL of a mixed vitamin solution (each vitamin concentration 10-50 mg / L) were added to each bottle of inorganic salt culture medium with a syringe. The negative control group was treated in the same way as the experimental treatment group, but the culture bottle was sterilized again after inoculation of sediment to kill the active microorganisms in the sediment. The experimental treatment group and the negative control group were placed in a constant temperature incubator at 30°C for static culture. The enrichment culture was performed eight times under the same culture conditions and passage method.

[0032] Finally, an enrichment culture that grew stably in an inorganic salt medium containing 1,3-butadiene was obtained. The average degradation rate of the microcosm containing strain N was 2.22 ± 0.03 μmol D -1 After the transfer, the degradation rate became faster with the deepening of enrichment, and finally reached 8.34±0.39μmol D at the eighth transfer. -1 (See Figure 1 ).

[0033] Example 2. Isolation of strain N that degrades 1,3-butadiene from enrichment culture

[0034] In order to isolate and purify strain N capable of anaerobically degrading 1,3-butadiene from the enriched culture fluid, the twelfth generation enriched culture fluid was used for liquid gradient dilution and semi-solid separation culture. The liquid gradient dilution medium was prepared by adding 9 mL of inorganic salt medium, 1 mL of hydrogen, 0.2 mL of 1,3-butadiene, and 5 mM sodium acetate to 10 20 mL anaerobic separation bottles. 1 mL of culture fluid was extracted using a 1 mL sterile syringe and transferred to the anaerobic separation bottle as 10 -1 After mixing by inversion, start with 10 mL of 10 -1 Take 1 mL of mixed cell solution from the dilution system and transfer it to the next anaerobic bottle to form 10 -2 Repeat this process until the dilution gradient reaches 10 -10 .

[0035] In addition to the above liquid gradient dilution medium components, the semi-solid separation medium also needs to add 0.1g of agar with a low gel temperature (<30°C). -1 -10 -10 1 mL of culture medium was drawn from each of the liquid gradient dilution bottles into the semi-solid separation bottle, and mixed by inverting to make 10 -1 -10 -10 All separated and purified samples were placed in a constant temperature incubator at 30°C.

[0036] The semi-solid agarose medium is prepared by adding 1 g of agarose having low temperature (<30° C.) gel properties and a gel solid strength greater than 200 g / cm2 to 10 mL of an inorganic salt anaerobic medium. 2 Made from low hardness agarose.

[0037] After half a month of cultivation, gas chromatography detected the conversion of 1,3-butadiene to 1-butene in all liquid gradient dilution culture bottles and semi-solid separation culture bottles. At the same time, the formation of formed colonies was observed with the naked eye in the semi-solid culture medium. A single clone was selected and cultured in a liquid inorganic salt culture medium to obtain strain N that can degrade 1,3-butadiene.

[0038] Example 3. Classification and Identification of Strain N

[0039] The cells of 5 mL of strain N culture medium were collected with a vacuum filter. The cell filter was transferred to a centrifuge tube containing lysis solution. The total genomic DNA of the strain was extracted using the bacterial genome kit of Takara Bio Co., Ltd., Japan. The 16S rRNA gene of strain N was amplified by PCR using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3') of the bacterial 16S rRNA gene. The PCR reaction parameters were 94℃5min pre-denaturation; 94℃30s denaturation; 54℃30s annealing; extension 72℃, 1min 30s, 35 cycles; extension 72℃, 10min, 1 cycle. The amplified product was transported to Suzhou Jinweizhi Biotechnology Co., Ltd. for DNA purification and Sanger sequencing. Geneious Prime was used to splice the forward and reverse sequences after sequencing. Finally, the nearly full-length strain N16S rRNA gene was compared with the NCBI database.

[0040] The results showed that the 16S rRNA gene fragment of strain N was 1433bp and the %GC was 53%. The comparison results of NCBI showed that the homology of strain N with species under the genus Acetobacterium was higher than 96%, and the highest homology with Acetobacterium wieringae CH1 was 99.9%. However, it is still unknown whether CH1 can degrade 1,3-butadiene or isoprene. Therefore, it can be determined that strain N belongs to a new strain of Acetobacterium wieringae (see Figure 6 ).

[0041] The 16S rRNA gene sequence of strain N is as follows:

[0042]

[0043]

[0044] The new anaerobic strain N was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 20, 2022, with the deposit number CGMCC No.40099.

[0045] Example 4. Identification of the performance of strain N in converting 1,3-butadiene

[0046] First, the TRACE 1300 gas-mass spectrometer of Thermo Fisher Scientific was used to identify 1,3-butadiene and the conversion products in the culture medium. The gas-mass spectrometer parameters were as follows: the chromatographic column was a DB-624 capillary column (60mx 320μm x 1.8μm); the injection volume was 20μL; the split ratio was 50:1; the injection port temperature was 200℃; the initial column temperature was 30℃ for 6 minutes, then increased to 75℃ at a rate of 5℃ / min, and then increased to 120℃ at a rate of 10℃ / min, and finally increased to 200℃ at a rate of 60℃ / min and maintained for 3.5min; the full scan range of the mass spectrometer was 15-300m / z; the transfer line and electron impact ion source temperatures were 280℃ and 250℃, respectively.

[0047] The daily monitoring of 1,3-butadiene conversion was performed using an Agilent 7890B gas chromatograph-flame ionization detector (GC-FID). The determination method was as follows: 1 mL of culture solution was periodically taken out from the culture bottle with a gas-tight syringe and quickly injected into a 20 mL headspace gas vial. The headspace bottle containing the culture solution was sealed with a PTFE cap. Finally, the headspace bottle was placed on the instrument sample tray for detection. A standard curve was prepared using known concentrations of 1,3-butadiene (purity of 99.9%) and 1-butene (97%) as standards. The detection parameters of GC-FID were as follows: the chromatographic column was DB-624; the fuel gas was 30 mL / min hydrogen; the combustion aid was 350 mL / min air; the carrier gas was 3 mL / min helium; the tail gas was 30 mL / min; the injection port temperature was 200 °C; the column temperature was 40 °C; the detector temperature was 300 °C; and the split ratio was 50:1. The autosampler model was Agilent 7697A, and the parameters were as follows: sample run cycle time was 17 min; headspace bottle equilibration time was 15 min; injection duration was 0.5 min.

[0048] The results showed that after 11 days of cultivation, part of 1,3-butadiene was converted into 1-butene in strain N. After one week of cultivation, 1,3-butadiene completely disappeared and was completely converted into 1-butene (see Figure 2 ). Therefore, strain N was cultured in the enriched medium at 13.6 μmol D -1 The rate at which 1,3-butadiene contaminants are converted to 1-butene (see Figure 3 ).

[0049] Example 5. Determination of the optimal conditions for strain N to degrade 1,3-butadiene

[0050] (1) Determination of the optimum temperature

[0051] Take 3mL of the activated strain N culture solution and inoculate it into 100mL of the inorganic salt medium described in Example 1 at a ratio of 3% (v / v). Add 1mL of 1,3-butadiene, 10mL of hydrogen, and 0.1mL of mixed vitamins to the inorganic salt medium with a pH of 7.2 and a salinity of 0.1% (sodium chloride concentration). The culture bottles were cultured in a constant temperature and dark environment in an incubator at 4°C, 10°C, 20°C, 30°C, 37°C, and 45°C. The degradation of 1,3-butadiene was monitored regularly by GC-FID, and its conversion rate was calculated.

[0052] The mixed vitamins include 20μg / L biotin, 20μg / L folic acid, 100μg / L pyridoxine hydrochloride, 50μg / L riboflavin, 50μg / L thiamine, 50μg / L pantothenic acid, 50μg / L niacin, and 50μg / L vitamin B 12 , a mixed solution of 50μg / L p-aminobenzoic acid and 50μg / L lipoic acid.

[0053] (2) Determination of the optimal pH

[0054] The embodiment was carried out at a culture temperature of 30°C and a sodium chloride concentration of 0.1%. The tested pH was set to 5.0, 5.5, 6.0, 6.5, 7.0, 7.2, 7.5, 7.8, and 8.0, respectively. This embodiment was carried out using an inorganic salt medium buffered in different pH ranges, as follows: pH 5.0, 5.5, 6.0, 6.5, and 7.0 treatment groups used 20mM 2-morpholineethanesulfonic acid (MES) as a buffer; pH 7.2, 7.5, 7.8, and 8.0 treatment groups used 20mM 4-hydroxyethylpiperazineethanesulfonic acid zwitterionic buffer. Sodium hydroxide or hydrochloric acid was used to adjust the culture medium to the corresponding pH. 20mL CO2 and 10mL H2 (20mL each) and 1mL 1,3-butadiene were added to the prepared culture medium of each group, and the activated strain N was inoculated at a ratio of 1% (v / v). The experiment was carried out at 30 °C and 0.1% NaCl concentration, and the degradation was monitored regularly by GC-FID.

[0055] (3) Determination of optimal salinity

[0056] The salinity test was performed in an inorganic salt medium with different NaCl concentrations, pH 7.2, and a culture temperature of 30°C. Specifically, the NaCl concentration (w / v) in the inorganic salt medium was set to 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, and 2.5 g / L. 1 mL of 1,3-butadiene and 10 mL of H2 were added to each inorganic salt medium with different salinities, and the strain N was inoculated to start the degradation reaction. The conversion of 1,3-butadiene was regularly monitored by GC-FID.

[0057] The results showed that the degradation of 1,3-butadiene could be observed in strain N at 10-40℃, pH 5.5-8.0 and 0.1-1.5g / L NaCl. The optimal culture temperature of strain N was 20-30℃, which could reach 7.53μmol D -1 The optimal culture pH of strain N was 7.0, and the degradation rate was 8.38 μmol D -1 The optimal culture salinity of strain N was 0.1-0.6 g / L, and the degradation rate was 7.53 μmol D -1 Therefore, when strain N is used to repair 1,3-butadiene pollution, the repair site should try to meet the optimal growth conditions of strain N (see Figure 4 ).

[0058] Example 6. Degradation performance of strain N on isoprene pollutants

[0059] Take 1 mL of the activated strain N culture solution and inoculate it into 100 mL of the inorganic salt culture solution described in Example 1 containing 7 μL of isoprene, which also contains 10 mL of hydrogen and 0.1 mL of mixed vitamins. The inoculated culture bottle was placed in a constant temperature culture at 30°C for 5 days. During the culture period, the conversion of isoprene by strain N was regularly monitored. The daily monitoring of isoprene and its conversion products also uses the GC-FID method. The instrument heating program is: initially 60°C for 2 minutes, then rise to 200°C at a heating rate of 25°C / min, and maintain operation for 1 minute.

[0060] The results showed that strain N was able to convert 72.8 μmol of isoprene into 55.9 μmol of 2-methyl-1-butene and 1.95 μmol of 3-methyl-1-butene. The average consumption rate of isoprene was 6.07 μmol D -1 (See Figure 5 ).

[0061] Example 7. Determination of acetic acid production by strain N of the present invention

[0062] After the strain N culture was completed with the addition of 1 mL of 1,3-butadiene and 10 mL of hydrogen, 5 mL of culture solution was taken and centrifuged at 16,000 x g for 10 min to collect the culture supernatant. The supernatant sample was placed in an oven to dry the water. Finally, 200 μL of distilled water was added to the residual components to make up the volume, and 0.5 μL of concentrated sulfuric acid (18N) was added for acidification. The formation of acetic acid was monitored by liquid chromatography 1260LC. The specific parameters are as follows: the chromatographic column is Aminex HPX-87H from Bio-Rad; the single mobile phase is an aqueous solvent containing 4 mM sulfuric acid; the sample injection volume is 20 μL; the column flow rate is 0.6 mL / min, and the total running time is 20 min.

[0063] The results showed that after strain N completely converted 1,3-butadiene into 1-butene, the production of acetic acid was also monitored by liquid chromatography, with a yield of 239.2 μM D -1 Compared with some other acetogenic bacteria, the acetate produced by strain N can not only be used as a bioenergy source for pollution remediation, but also has the potential to solve the problem of low acetic acid production in industrialization.

[0064] The strain is used to prepare a bacterial agent for degrading 1,3-butadiene and isoprene pollutants and producing acetate according to the above embodiment, such as adding sufficient hydrogen as a growth substrate to an inorganic salt culture medium, inoculating 3% of the Acetobacterium wieringae N, and culturing for 5 days in the dark at pH 7.2, 20-30°C and 0.1-0.6% salinity; centrifuging at 16,000xg for 20 minutes to collect cells of strain N, adding sodium alginate, glycerol or other solid and liquid carriers to the cells, thereby obtaining a culture concentrate or culture suspension of strain N, and finally obtaining a repair and industrialized bacterial agent. The bacterial agent is applied to a water body or soil environment containing 1,3-butadiene or isoprene, and has broad application prospects for bioremediation.

Claims

1. A strain of Acetobacterium wissenii that anaerobically degrades 1,3-butadiene, characterized in that: The strain is Acetobacterium Wieringae N, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 20, 2022, with the deposit number CGMCC No. 40099.

2. An application of the Acetobacterium wissenii for degrading 1,3-butadiene according to claim 1, characterized in that: The strain is used in degrading 1,3-butadiene pollutants.

3. An application of the Acetobacterium wissenii for degrading 1,3-butadiene according to claim 1, characterized in that: The strain is used in degrading isoprene pollutants.

4. An application of the Acetobacterium wissenii for degrading 1,3-butadiene according to claim 1, characterized in that: The strain is used in producing acetate by fermentation.

5. A preparation for degrading 1,3-butadiene pollutants, characterized in that: The preparation contains the Acetobacterium wissenii described in claim 1 Acetobacterium wieringae N.

6. The preparation for degrading 1,3-butadiene pollutants according to claim 5, characterized in that: The preparation contains Acetobacterium wismerii Acetobacterium wieringae The culture suspension of N.

7. A preparation for degrading isoprene pollutants, characterized in that: The preparation contains the Acetobacterium wissenii described in claim 1 Acetobacterium wieringae N.

8. The preparation for degrading isoprene pollutants according to claim 7, characterized in that: The preparation contains Acetobacterium wismerii Acetobacterium wieringae The culture suspension of N.

9. A preparation for preparing acetate bioenergy, characterized in that: The preparation contains the Acetobacterium wissenii described in claim 1 Acetobacterium wieringae N.

10. A method for preparing acetate, characterized in that: The Acetobacterium wissenii described in claim 1 Acetobacterium wieringae N utilizes carbon dioxide and hydrogen to produce acetate under anaerobic conditions.

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