CN116179411A - Acetobacter for anaerobic degradation of 1, 3-butadiene and application thereof - Google Patents

Abstract

The invention discloses the field of environmental pollution restoration, in particular to acetobacter for anaerobically degrading 1, 3-butadiene and application thereof. The bacillus cereus for anaerobic degradation of 1, 3-butadiene is Acetobacterium wieringae N, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 40099 in the year 7 and the month 20 of 2022. The strain N provided by the invention can be used for preparing 13.6 mu mol D under anaerobic condition ^‑1 Will be 1, 3-butadieneThe contaminant is converted to 1-butene; at 4.66. Mu. Mol D ^‑1 And 0.16. Mu. Mol D ^‑1 At a rate that converts isoprene contaminants to 2-methyl-1-butene and 3-methyl-1-butene. Therefore, the bacillus aceticus obtained in the invention can be used for effectively detoxify the aquifer polluted by diene compounds.

Description

Acetobacter for anaerobic degradation of 1, 3-butadiene and application thereof

Technical Field

The invention discloses the field of environmental pollution restoration, in particular to acetobacter for anaerobically degrading 1, 3-butadiene and application thereof.

Background

1, 3-butadiene (CAS 106-99-0, C) ₄ H ₆ ) Is a colorless gas at room temperature, has a gasoline-like smell, and contains two organic compounds of 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) has shown in 2016 that annual yields of 1, 3-butadiene can reach 10-50 hundred million pounds. Polluted air and water in the vicinity of automobile exhaust, tobacco smoke, plastic or rubber facilities are major sources of human exposure to 1, 3-butadiene. Mice have a semi-lethal concentration (LC 50) of 259-270g/m for 1, 3-butadiene ³ The method comprises the steps of carrying out a first treatment on the surface of the LC50 of the rat was 285g/m ³ And accompanied by the symptoms of increased total leukocyte count, neutropenia, etc. Thus, related toxicology studies are of great interest. Research agreement suggests that occupational exposure to 1, 3-butadiene is associated with an increase in leukemia incidence; and prolonged exposure to such conditions may manifest as eye pain, blurred vision, coughing, and somnolence. The international agency for research on cancer (IARC) also lists 1, 3-butadiene in the list of human suspected carcinogens class I. 1, 3-butadiene may be combined with O ₃ 、NO ₂ The gases undergo photochemical reaction to generate various mixed products with undesireable harmfulness; in the soil environment, the 1, 3-butadiene can generate substances such as butylene, butane and the like under the catalysis of the granular iron. However, the physical and chemical restoration means is not thorough, and secondary pollution is easily brought. Therefore, the organic pollutant can be degraded by utilizing the catalysis of organisms, particularly microorganisms, so that the concentration of the pollutant can be reduced to the maximum extent, the cost is low, and the environmental impact is small, thereby being a green repairing method. The anaerobic environment contains a large number of microorganism resources with unknown functions, and the biological conversion of 1, 3-butadiene is researchedExtremely deficient. The acquisition of anaerobic microbial resources capable of efficiently degrading 1, 3-butadiene is a milestone in the field of environmental bioremediation, and the exploration of the physiological and biochemical properties and effects of related strains is also extremely important.

Disclosure of Invention

The invention aims to provide acetobacter for anaerobically degrading 1, 3-butadiene and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the strain of the bacillus cereus for anaerobically degrading 1, 3-butadiene is Acetobacterium wieringae N, which is preserved in China general microbiological culture Collection center (CGMCC) No.40099 in the 7 th month and 20 th year of 2022, and the preservation address is North Chen West Lu No. 1 of the Chaoyang area of Beijing city.

The strain is obtained from river sediment, is enriched in an inorganic salt liquid anaerobic culture medium by adding 1, 3-butadiene, and is finally obtained by performing trace-free dilution in a semisolid agarose culture medium.

The semi-solid agarose culture medium is prepared by adding 1g of low-temperature medium into 10mL of inorganic salt culture medium<Gel property at 30 ℃ and gel solid strength of more than 200g/cm ² Is prepared from low-hardness agarose.

Use of a strain of baculous wilsonii for degrading 1, 3-butadiene, said strain being useful in the degradation of 1, 3-butadiene contaminants.

Further, the strain degrades 1, 3-butadiene contaminants under anaerobic conditions.

Use of a baculous weii acetate for degrading 1, 3-butadiene, said strain being useful for degrading isoprene contaminants.

Further, the strain is also capable of degrading isoprene contaminants under anaerobic conditions.

Use of a baculous weii acetate for degrading 1, 3-butadiene, said strain being used in the production of acetate by fermentation.

A preparation for degrading 1, 3-butadiene pollutant contains the Bacillus weii acetate Acetobacterium wieringae N.

The preparation contains thalli of the bacillus valicarpus Acetobacterium wieringae N, a glycerol culture, a culture solution concentrate or a culture bacterial suspension.

A preparation for degrading isoprene pollutant contains the Bacillus weii acetate Acetobacterium wieringae N.

The preparation contains thalli of the bacillus valicarpus Acetobacterium wieringae N, a glycerol culture, a culture solution concentrate or a culture bacterial suspension.

A preparation for preparing acetate bioenergy, which comprises the bacillus weii acetate Acetobacterium wieringae N.

A method for preparing acetate comprises fermenting the Bacillus vallismortis Acetobacterium wieringae N with carbon dioxide and hydrogen under anaerobic condition to obtain acetate.

The invention has the advantages that:

the strain is obtained by enrichment and separation from a river sediment sample, belongs to the bacillus weishi acetate Acetobacterium wieringae on the bacteria classification level, is a novel anaerobic bacillus acetate (N) strain, and supplements new microorganism resources of the genus; in addition, the invention obtains the strain N with high-efficiency anaerobic degradation of 1, 3-butadiene pollutants for the first time; the strain N can also degrade isoprene pollutants and produce a large amount of acetate by utilizing carbon dioxide and hydrogen. Finally, the invention not only provides a microbial inoculum restoration scheme with development prospect for solving the pollution problem of 1, 3-butadiene and isoprene in soil and water environments, but also provides a new direction for the green production of acetate in industry.

Description of the drawings:

FIG. 1 shows the consumption rate of 1, 3-butadiene by the mixed culture solution of Bacillus acetate Acetobacterium wieringae N of the present invention at different times of transfer.

FIG. 2 is a graph showing the identification of degradation products of 1, 3-butadiene by Bacillus acetate Acetobacterium wieringae N of the present invention.

Fig. 3 is a graph showing degradation performance of the bacillus aceticus Acetobacterium wieringae N of the present invention against 1, 3-butadiene contaminants at 30 ℃, ph=7.2, and nacl concentration of 0.1%.

FIG. 4 is a graph showing the degradation rate of 1, 3-butadiene by Bacillus acetate Acetobacterium wieringae N of the invention at various temperatures, pH and salinity.

FIG. 5 is a graph showing degradation performance of the Bacillus acetate Acetobacterium wieringae N of the present invention on isoprene contaminants.

FIG. 6 is a phylogenetic classification tree of 16S rRNA genes of Bacillus acetate Acetobacterium wieringae N of the present invention.

The specific embodiment is as follows:

the following description of the embodiments of the present invention is further provided in connection with the accompanying examples, and it should be noted that the embodiments described herein are for the purpose of illustration and explanation only, and are not limiting of the invention.

The experimental methods, materials and reagents used in the following examples were conventional methods unless otherwise specified.

EXAMPLE 1 establishment of enrichment culture of baculous acetate Strain N

An anaerobic microcosm system was established using the river sediment of Shenyang, liaoning, inc. (N41 DEG 44 '41', E123 DEG 17 '35') as an inoculum and 1, 3-butadiene as a substrate. The specific operation is as follows: first, the sediment at a depth of 70-90cm from the river bank is collected in a clean collection bottle by a collector sterilized under high temperature and high pressure. And (3) injecting river water into the upper part of the bottle to remove air, and rapidly transporting to a 4 ℃ refrigerator in a laboratory for storage.

A 3L three-necked round bottom flask was used to prepare inorganic salt anaerobic medium. The ingredients of the mixed culture medium per liter are as follows: the inorganic salt comprises 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, and 1.5g/L calcium chloride dihydrate; the microelements 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 copper chloride dihydrate, 0.006mg/L sodium selenate pentahydrate and 0.008mg/L sodium tungstate; the reducing agent is 0.2mM sodium sulfide nonahydrate, 0.5mM dithiothreoSugar alcohols. By CO ₂ After the pH value of the culture medium is regulated to 7.2-7.3, N is introduced into the head space of the culture medium ₂ And CO ₂ Mixture (v/v, 80:20%). And (3) subpackaging 80mL of anaerobic culture medium into clean anaerobic serum bottles by using a syringe, and sealing the bottle mouth by using a butyl rubber plug and an aluminum cover. The sub-packaged culture was sterilized at 121℃under high temperature and pressure for 30min. The sterilized and cooled medium and the collected sediment were transferred into an anaerobic glove box, about 3g of sediment was added to each bottle of medium with a spoon, and sealing with a rubber stopper was continued. Finally, 1mL of 1, 3-butadiene as a substrate, 10mL of hydrogen gas as an electron donor, 5mM sodium lactate as a carbon source, and 0.1mL of a mixed vitamin solution (each vitamin concentration 10-50 mg/L) were added to each bottle of the inorganic salt medium using a syringe. The negative control group and the experimental treatment group adopt the same treatment, but the culture flask after inoculating the bottom mud is sterilized again to kill the active microorganisms in the bottom mud. The experimental treatment group and the negative control group are placed in a constant temperature incubator at 30 ℃ for static culture. Enrichment culture was performed eight times under the same culture conditions and passaging method.

Finally, an enriched culture grown stably in an inorganic salt medium containing 1, 3-butadiene is obtained. The initial microcosm average degradation rate of strain N is 2.22+/-0.03 mu mol D ^-1 The method comprises the steps of carrying out a first treatment on the surface of the After transfer, the degradation rate becomes faster with the deepening of the enrichment degree, and finally, 8.34+ -0.39 mu mol D is reached at the eighth transfer ^-1 (see FIG. 1).

Example 2 isolation of 1, 3-butadiene-degrading Strain N in enriched culture

In order to separate and purify the strain N capable of anaerobically degrading the 1, 3-butadiene from the enrichment culture solution, the twelfth-generation enrichment culture solution is utilized for liquid gradient dilution and semisolid separation culture. Liquid gradient dilution medium was prepared by adding 9mL of inorganic salt medium, 1mL of hydrogen, 0.2mL of 1, 3-butadiene and 5mM sodium acetate to 10 20mL anaerobic separation flasks. 1mL of the culture broth was withdrawn using a 1mL sterile syringe and transferred to an anaerobic separation flask as 10 ^-1 Is a dilution gradient of (c). After mixing the materials upside down, 10mL of the mixture is obtained from 10mL of ^-1 1mL of mixed cell liquid is extracted from the dilution system and transferred to the next anaerobic bottle to form 10 ^-2 Dilution gradient, repeating this operation untilUntil the dilution gradient reaches 10 ^-10 。

The semi-solid separation culture medium is added with 0.1g of low gel temperature besides the liquid gradient dilution culture medium components<30 ℃) agar. From 10 before the culture medium is not solidified ^-1 -10 ^-10 1mL of culture solution is respectively pumped into semi-solid separation culture bottles from culture bottles with liquid gradient dilution, and the culture solutions are evenly mixed up and down to prepare 10 ^-1 -10 ^-10 . All the separated and purified samples were placed in a constant temperature incubator at 30 ℃.

The semi-solid agarose culture medium is prepared by adding 1g of low-temperature anaerobic culture medium into 10mL of inorganic salt anaerobic culture medium<Gel property at 30 ℃ and gel solid strength of more than 200g/cm ² Is prepared from low-hardness agarose.

After half a month of incubation, gas chromatography monitored the conversion of 1, 3-butadiene to 1-butene in all liquid gradient dilution flasks and semi-solid isolation flasks. At the same time, the formation of formed colonies in the semi-solid medium was observed visually. And (3) picking the monoclonal antibody into a liquid inorganic salt culture medium for culture, thereby obtaining the strain N capable of degrading the 1, 3-butadiene.

Example 3 Classification and identification of Strain N

5mL of cells of strain N broth were collected with a vacuum filter. The cell-containing filters were transferred to centrifuge tubes containing lysate. Total genomic DNA of the strain was extracted with a bacterial genome kit of Takara Bio Inc. of Japan. The 16S rRNA gene of strain N was PCR amplified using the general primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3') of the bacterial 16S rRNA gene. The PCR reaction parameters were 94℃for 5min pre-denaturation; denaturation at 94℃for 30 s; annealing at 54 ℃ for 30 s; extending for 30s at 72 ℃ and 1min for 35 cycles; the temperature is prolonged to 72 ℃,10min, and 1 cycle. The amplified product was transported to san ger sequencing for DNA purification by scow biotechnology limited. The sequenced forward and reverse sequences were spliced using a Geneious Prime. Finally, near full length strain N16S rRNA genes were aligned to NCBI database.

The result showed that the 16S rRNA gene fragment of strain N was 1433bp and the% GC was 53%. The comparison result of NCBI shows that the homology of the strain N with the species under the Acetobacter genus is higher than 96%, and the homology with the Acetobacter weissei (Acetobacterium wieringae) CH1 is the highest and reaches 99.9%. However, it was not known whether CH1 could degrade 1, 3-butadiene or isoprene, and it was confirmed that this strain N belongs to a novel strain among Acetobacterium wieringae species (see FIG. 6).

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

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the novel anaerobic strain N is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.40099 in the year 7 and the month 20 of 2022.

Example 4 identification of 1, 3-butadiene conversion Property of Strain N

The identification of 1, 3-butadiene and conversion products in the broth was first carried out using a TRACE 1300 type gas mass spectrometer from Thermo Fisher Scientific. The gas mass spectrum parameters were as follows: the chromatographic column is DB-624 capillary column (60 m x 320 mu m x 1.8 mu m); the sample injection amount is 20 mu L; the split ratio is 50:1; the temperature of the sample inlet is 200 ℃; the initial column temperature is maintained at 30 ℃ for 6 minutes, and rises to 75 ℃ at a speed of 5 ℃/min, and rises to 120 ℃ at a speed of 10 ℃/min, and finally rises to 200 ℃ at a speed of 60 ℃/min, and is maintained for 3.5min; the mass spectrum full scanning range is 15-300m/z; the transmission line and electron impact ion source temperatures were 280 ℃ and 250 ℃, respectively.

Daily monitoring of 1, 3-butadiene conversion employed Agilent 7890B gas chromatography-flame ion detector (GC-FID). The measuring method comprises the following steps: 1mL of culture broth was periodically removed from the flask using an airtight syringe and rapidly injected into a 20mL headspace vial. The headspace bottle containing the broth was sealed with PTEE cap gasket. Finally, the headspace vial is placed in an instrument sample tray for detection. A standard curve was prepared using 1, 3-butadiene (99.9% purity) and 1-butene (97%) at known concentrations as standards. The detection parameters for GC-FID are as follows: the chromatographic column is DB-624; the fuel gas is 30mL/min hydrogen; the combustion improver is 350mL/min of air; the carrier gas is 3mL/min helium; tail blowing is 30mL/min; the temperature of the sample inlet is 200 ℃; column temperature is 40 ℃; the detector temperature was 300 ℃; the split ratio was 50:1. The model of the autosampler is Agilent 7697A, and the parameters are as follows: sample running cycle time is 17min; the balance time of the headspace bottle is 15min; the sample injection duration was 0.5min.

The results showed that after 11 days of cultivation, part of 1, 3-butadiene was converted to 1-butene containing strain N. After one additional week of incubation, 1, 3-butadiene was completely eliminated and completely converted to 1-butene (see FIG. 2). Thus, strain N was grown in enriched medium at 13.6. Mu. Mol D ^-1 The rate of (a) converts 1, 3-butadiene contaminants to 1-butene (see figure 3).

EXAMPLE 5 determination of optimal conditions for degradation of 1, 3-butadiene by Strain N

(1) Determination of optimum temperature

3mL of the activated strain N culture solution was inoculated into 100mL of the inorganic salt medium described in example 1 at a ratio of 3% (v/v), and 1mL of 1, 3-butadiene, 10mL of hydrogen gas and 0.1mL of mixed vitamins were added to the inorganic salt medium having a pH of 7.2 and a salinity of 0.1% (sodium chloride concentration). The flasks were incubated at constant temperature and in the dark at 4, 10, 20, 30, 37, and 45℃in an incubator, respectively. The GC-FID periodically monitors the degradation of the 1, 3-butadiene and calculates the conversion rate.

Wherein the vitamin mixture contains 20 μg/L biotin, 20 μg/L folic acid, 100 μg/L pyridoxine hydrochloride, 50 μg/L riboflavin, 50 μg/L thiamine, 50 μg/L pantothenic acid, 50 μg/L niacin, 50 μg/L vitamin B ₁₂ 50. Mu.g/L of p-aminobenzoic acid, 50. Mu.g/L of lipoic acid.

(2) Determination of optimum pH

The examples were carried out at a culture temperature of 30℃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, 8.0, respectively. This example was performed with inorganic salt medium buffered at different pH ranges as follows: the pH 5.0, 5.5, 6.0, 6.5, 7.0 treatment groups were buffered with 20mM 2-morpholinoethanesulfonic acid (MES)Flushing liquid; the pH 7.2, 7.5, 7.8, 8.0 treatment groups used 20mM 4-hydroxyethylpiperazine ethanesulfonic acid zwitterionic buffer. The medium is adjusted to the corresponding pH with sodium hydroxide or hydrochloric acid. Adding 20mL of CO in equal proportion into each group of prepared culture mediums ₂ And 10mL H ₂ (20 mL each) and 1mL of 1, 3-butadiene were inoculated with the activated strain N at a ratio of 1% (v/v). This experiment was carried out at 30℃and NaCl concentration of 0.1%, and the GC-FID was periodically monitored for degradation.

(3) Determination of optimal salinity

The salinity test was performed in medium supplemented with inorganic salts of different NaCl concentrations, pH 7.2, incubation temperature 30 ℃. The method comprises the following steps: 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 2.5g/L, respectively. 1mL of 1, 3-butadiene and 10mL of H were added to each of the inorganic salt media of different salinity ₂ And the strain N is inoculated to start degradation reaction, and the conversion of 1, 3-butadiene is monitored by GC-FID periodically.

The results show that: the degradation of 1, 3-butadiene can be observed in the culture of the strain N at 10-40 ℃ and pH 5.5-8.0 and 0.1-1.5g/L NaCl. The optimal culture temperature of the strain N is 20-30 ℃, and the strain N can reach 7.53 mu mol D ^-1 Is a degradation rate of (a); the optimal pH for strain N was 7.0 and the degradation rate was 8.38. Mu. Mol D ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The optimal culture salinity of the strain N is 0.1-0.6g/L, and the degradation rate is 7.53 mu mol D ^-1 . Therefore, when strain N is used to repair 1, 3-butadiene contamination, the repair site should be as good as possible to meet the optimal growth conditions for strain N (see FIG. 4).

Example 6 measurement of degradation Performance of Strain N on isoprene contaminant

1mL of the activated strain N broth was inoculated into 100mL of the inorganic salt broth described in example 1 containing 7. Mu.L of isoprene, and the broth further contained 10mL of hydrogen gas and 0.1mL of mixed vitamins. The inoculated flask was incubated at 30℃for 5 days. During the culture period, the conversion of isoprene by strain N was monitored periodically. Daily monitoring of isoprene and its conversion products was also performed by the GC-FID method. The instrument temperature-raising program is as follows: the initial 60 ℃ is maintained for 2min, then the temperature is raised to 200 ℃ at a heating rate of 25 ℃/min, and the operation is maintained for 1min.

The results showed that strain N was able to convert 72.8. Mu. Mol of isoprene into 55.9. Mu. Mol of 2-methyl-1-butene and 1.95. Mu. Mol of 3-methyl-1-butene. Average consumption rate of isoprene was 6.07. Mu. Mol D ^-1 (see FIG. 5).

Example 7 determination of acetic acid production by Strain N of the present invention

After the completion of the culture of the strain N with 1mL of 1, 3-butadiene and 10mL of hydrogen gas, 5mL of the culture broth was taken and centrifuged at 16,000Xg for 10min to collect the culture broth supernatant. The supernatant samples were placed in an oven to dry the moisture. Finally, 200. Mu.L of distilled water was added to the residual fraction to fix the volume, and 0.5. Mu.L of concentrated sulfuric acid (18N) was added to acidify the mixture. Acetic acid production was monitored using liquid chromatography 1260 LC. The specific parameters are as follows: the chromatographic column is Aminex HPX-87H of Berle company; the single mobile phase is an aqueous solvent containing 4mM sulfuric acid; the sample injection amount of the sample is 20 mu L; the column flow rate was 0.6mL/min and the total run time was 20min.

The results showed that strain N also monitored acetic acid production by liquid chromatography after complete conversion of 1, 3-butadiene to 1-butene with a yield of 239.2. Mu. M D ^-1 . Compared with other certain acetogenic bacteria, the acetate produced by the strain N can be used as a biological energy source for pollution restoration, and is hopeful to solve the problem of low yield of industrial acetic acid.

Preparing bacterial agents for degrading 1, 3-butadiene and isoprene pollutants and producing acetate by the strain according to the above embodiment, for example, adding enough hydrogen as a growth substrate into an inorganic salt culture medium, inoculating 3% of the bacillus aceticus Acetobacterium wieringae N, and performing light-proof static culture for 5 days at the pH of 7.2, 20-30 ℃ and 0.1-0.6% of salinity; and (3) centrifuging at 16,000Xg for 20min to collect cells of the strain N, adding sodium alginate, glycerol or other solid and liquid carriers into the cells to obtain culture concentrate or culture bacterial suspension of the strain N, and finally obtaining the repairing and industrialization bacterial agent. The microbial inoculum is applied to a water body or soil environment containing 1, 3-butadiene or isoprene, and has wide bioremediation application prospect.

Claims (10)

1. A strain of bacillus cereus for anaerobically degrading 1, 3-butadiene is characterized in that: the strain is Acetobacterium wieringae N which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 40099 in the year 7 and 20 of 2022.

2. Use of a bacillus weii acetate for the degradation of 1, 3-butadiene according to claim 1, characterized in that: the use of said strain for degrading 1, 3-butadiene contaminants.

3. Use of a bacillus weii acetate for the degradation of 1, 3-butadiene according to claim 1, characterized in that: the application of the strain in degrading isoprene pollutants.

4. Use of a bacillus weii acetate for the degradation of 1, 3-butadiene according to claim 1, characterized in that: the application of the strain in acetate production by fermentation.

5. A formulation for degrading a 1, 3-butadiene contaminant, characterized by: the preparation comprises the Bacillus weii acetate Acetobacterium wieringae N as claimed in claim 1.

6. A preparation for degrading a 1, 3-butadiene contaminant according to claim 5, wherein: the preparation contains thalli of the bacillus valicarpus Acetobacterium wieringae N, a glycerol culture, a culture solution concentrate or a culture bacterial suspension.

7. A formulation for degrading isoprene contaminants, characterized by: the preparation comprises the Bacillus weii acetate Acetobacterium wieringae N as claimed in claim 1.

8. The isoprene contaminant degradation formulation of claim 7, wherein: the preparation contains thalli of the bacillus valicarpus Acetobacterium wieringae N, a glycerol culture, a culture solution concentrate or a culture bacterial suspension.

9. A formulation for preparing acetate bioenergy, characterized in that: the preparation contains the Bacillus weii acetate Acetobacterium wieringae N as claimed in claim 1.

10. A process for preparing an acetate salt characterized by: the bacillus weii acetate Acetobacterium wieringae N is fermented to produce acetate by utilizing carbon dioxide and hydrogen under anaerobic condition.

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