CN114657092A

CN114657092A - Isoprene anaerobic degradation bacterium and application thereof in environmental bioremediation

Info

Publication number: CN114657092A
Application number: CN202210208300.3A
Authority: CN
Inventors: 杨毅; 金慧娟; 严俊; 李秀颖
Original assignee: Institute of Applied Ecology of CAS
Current assignee: Institute of Applied Ecology of CAS
Priority date: 2022-03-04
Filing date: 2022-03-04
Publication date: 2022-06-24
Anticipated expiration: 2042-03-04
Also published as: CN114657092B

Abstract

The invention discloses the field of environmental pollution remediation, and particularly relates to a novel isoprene anaerobic degradation bacterium and application thereof in environmental bioremediation. The strain is bacillus aceticus aegypti Z which is preserved in China general microbiological culture Collection center at 11 months and 30 days in 2021, and the preservation number is CGMCC No. 24006. The strain Z provided by the invention can degrade isoprene pollutants by co-metabolism in an anaerobic inorganic salt culture medium by taking carbon dioxide and hydrogen as substrates, and shows that the strain can efficiently degrade isoprene and has a wide application prospect in repairing isoprene pollution in the environment.

Description

Isoprene anaerobic degradation bacterium and application thereof in environmental bioremediation

Technical Field

The invention discloses the field of environmental pollution remediation, and particularly relates to a novel isoprene anaerobic degradation bacterium and application thereof in environmental bioremediation.

Background

Isoprene (also known as 2-methyl-1, 3-butadiene, C)₅H₈) Is the most abundant non-methane Biologically Volatile Organic Compound (BVOC) in the atmosphere. Land plants are the main producers of isoprene, releasing up to 5-7.5 million tons per year; it is not only a key cornerstone for the synthesis of isoprenoids (including chlorophyll, cholesterol, archaeolipids, etc.) in organisms, but also a major component in the industrial production of high quality rubber and aviation fuels. However, photo-oxidation of isoprene released into the atmosphere with hydroxyl radicals (OH ·) leads to increased ozone and carbon monoxide concentrations in the troposphere, global transport of nitrogen-containing compounds, aggregation of secondary organic aerosols, and thus to the greenhouse effect. In addition, animal experimental results show that long-term or repeated exposure to isoprene can cause cancer in a series of organs such as liver, lung and breast, and therefore, international agency for research on cancer (IARC) classifies isoprene as a suspected carcinogen in humans.

It is estimated that the abundance of isoprene degrading microorganisms in the soil is about 10 per gram of dry soil⁵A cell. More and more bacteria such as Rhodococcus sp.AD45, Gordonia sp.i37 and Mycobacterium sp.AT1 have been isolated from phylum Familicendes and Proteobacteria, identified to degrade isoprene under aerobic conditions, and their related functional enzymes and metabolic pathways have been elucidated. In contrast, the research on the anaerobic biodegradation of isoprene is rather deficient. Recently, research groups from australia have found that enriched cultures containing bacillus aceticus are capable of efficiently converting isoprene under anaerobic conditions, but the anaerobic strain has not been isolated so far. Therefore, obtaining a biological material capable of anaerobically degrading isoprene is important in the field of environmental bioremediationAnd breaking through.

Disclosure of Invention

The invention aims to provide a novel isoprene anaerobic degradation bacterium and application thereof in environmental bioremediation.

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

an isoprene anaerobic degradation bacterium is characterized in that: the strain is bacillus aceticus aegypti Z which is preserved in China general microbiological culture Collection center at 11 months and 30 days in 2021, and the preservation number is CGMCC No. 24006.

A method for separating isoprene anaerobic degradation bacteria comprises the steps of conducting sterilization dilution on a sample in a semi-solid agarose culture medium to obtain an approximately pure culture, selecting a single colony to grow in a liquid anaerobic inorganic salt culture medium, and obtaining the single colony of bacillus welshimeri acetic acid bacteria wieringae Z.

The semi-solid agarose medium is low-hardness agarose with low temperature (<30 ℃) gel performance added in 0.6-1.0g per 100ml of anaerobic medium containing 30-50mM sodium bicarbonate buffer.

Wherein the anaerobic culture medium containing 30-50mM sodium bicarbonate buffer solution contains microelements, mixed vitamins and inorganic salts.

The application of an isoprene anaerobic degradation bacterium, wherein the strain can degrade isoprene in an environment under anaerobic conditions.

The strain is treated with CO under anaerobic conditions₂And H₂The application of degrading isoprene in the environment in a co-metabolism mode for fermenting substrates.

Use of the isoprene anaerobic degrading bacterium of claim 1 for producing acetic acid under anaerobic conditions as a biological energy source.

A preparation for degrading isoprene pollutants comprises the bacillus welshimeri strain Z.

The preparation contains culture, culture concentrate, concentrate separation supernatant or culture bacterial suspension of the bacillus welshimeri strain Acetobacter wieringie Z.

A preparation for preparing bioenergy (acetic acid), which comprises the bacillus welshimeri wieringae Z.

The preparation contains culture, culture concentrate, concentrate separation supernatant or culture suspension of bacillus Weishi Acetobacter wieringye Z strain

The invention has the advantages that:

the strain is obtained by separating a sediment sample of a river basin, belongs to Acetobacter winengiae on the classification level, is a novel anaerobic bacillus aceti (Z), and supplements microbial resources of the strain; in addition, the invention obtains the strain Z with the function of efficiently degrading isoprene pollutants in an anaerobic manner for the first time; the strain Z can generate a large amount of acetic acid while degrading pollutants, can provide nutritional support for other microorganisms in an in-situ ecosystem, and reduces the cost of adding exogenous nutrient substances in the environment restoration process. Finally, the invention provides a microbial inoculum repairing scheme with development prospect for solving the problem of isoprene pollution in soil and water environment.

Description of the drawings:

FIG. 1 is a scanning electron microscope image of Bacillus aceticus Brevibacterium wieringie strain Z for anaerobic degradation of isoprene;

FIG. 2 is a phylogenetic tree constructed by homology alignment of 16S rRNA gene sequences of an isoprene anaerobic degradation bacillus aceticus wieringae strain Z and a similar strain;

FIG. 3 is a graph showing the degradation performance of Bacillus aceticus Brevibacterium wieringie strain Z for anaerobic degradation of isoprene at 30 ℃, pH 7.2 and NaCl concentration of 0.1% on isoprene;

FIG. 4 is a graph showing the rate of degrading isoprene by Bacillus aceticus Brevibacterium wieringie strain Z of the present invention at different temperatures, pH and salinity;

FIG. 5 shows the strain Z of Bacillus aceticus Brevibacterium wierine for anaerobic degradation of isoprene in the presence of CO₂And H₂Depend onFigure for sexual results.

The specific implementation mode is as follows:

the following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.

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

Example 1 enrichment culture of Strain Z

The sediment sampling time is 2017 and 7 months. The collection place is a sewage drain outlet (latitude: 41 degrees, 39 '46 degrees, longitude: 123 degrees, 6' 20 degrees) of a fine river in Shenyang city of Liaoning province, and the devices used for sampling are 2L blue thread bottles sterilized at high temperature, sealing butyl rubber plugs, iron shovels and the like. Digging into the deep part of river bottom mud with a shovel, quickly feeding the taken bottom mud into a collecting bottle, adding river water to remove air remained in the headspace of the bottle, covering with a sealing blue rubber plug, reinforcing with a plastic cover with a central hole, and immediately placing into a heat preservation box with an ice bag. The collected samples are stored in a refrigerator at 4 ℃ in time, and the enrichment culture work of the microorganisms is carried out within one month.

In an anaerobic glove box, 2g of the collected bottom mud was inoculated to 10mL of H containing 7. mu.L of isoprene₂、10mL CO₂In 100mL of sterilized anaerobic inorganic salt medium (DCB-1), the formulation of DCB-1 was as follows: NaCl 1.0g/L, MgCl₂·6H₂O 0.5g/L，KH₂PO₄ 0.2g/L，NH₄Cl 0.3g/L，KCl 0.3g/L，CaCl₂·2H₂O 0.015g/L，FeCl₂·4H₂O 1.5mg/L，CoCl₂·6H₂O 190μg/L，MnCl₂·4H₂O 100μg/L，ZnCl₂ 70μg/L，H₃BO₃ 6μg/L，Na₂MoO₄·2H₂O 36μg/L，NiCl₂·6H₂O 24μg/L，CuCl₂·2H₂O 2μg/L，Na₂SeO₃·5H₂O 6μg/L，Na₂WO₄·2H₂O8 mug/L is inorganic salt component of the culture medium, and the sodium celastrium is 0.025% (w/v). AddingAfter thermal deoxidization, 24mg/L of L-cysteine and Na are added₂S·9H₂O48 mg/L and dithiothreitol (DTT, 77mg/L) as reducing agents to further remove oxygen from the medium. 30mM NaHCO was added₃Buffer reagent of CO₂The medium pH was adjusted to 7.2. Sterilizing, cooling, adding vitamin mixed solution which is filtered and sterilized by a 0.22 mu m filter membrane, and comprising the following components: vitamin B250 mug/L, lipoic acid 50 mug/L, vitamin B20 mug/L, vitamin B6100 mug/L, vitamin H20 mug/L, vitamin B550 mug/L, nicotinic acid 50 mug/L, vitamin B150 mug/L and p-aminobenzoic acid 50 mug/L. The culture flask was placed in an incubator at 30 ℃ for ten days in the dark. 3mL of the culture medium was taken and transferred to a medium containing the same amount of isoprene, H₂And CO₂In a fresh medium (i.e., isoprene-containing, H, of the culture substrate sludge described above)₂And CO₂The anaerobic inorganic salt medium (DCB-1)) was cultured in the same culture conditions and subculture method for eight times. Finally, an enriched culture stably grown in an isoprene-containing medium was obtained.

Example 2 isolation of Strain Z having isoprene degrading function

1mL of the culture solution enriched in the eighth passage strain Z was taken by a sterile syringe and inoculated into 9mL of anaerobic inorganic salt medium (DCB-1) containing isoprene and hydrogen as 10^-1And (4) diluting the solution. Sequentially preparing gradient 10 with DCB-1 culture medium by adopting an absolute dilution method^-2、10^-3、10^-4、10^-5、10^-6、10^-7、10^-8、10^-9、10^-10、10^-11、10^-12And (4) diluting the solution. From different dilution bottles, 1mL of each strain Z culture solution was aspirated and injected into the unsolidified semi-solid agar medium, shaken up and down uniformly, and then allowed to stand. When the agar solidified, 1mL of H was added₂And 1mL CO₂And 2. mu.L of isoprene, and cultured in an inverted state in an incubator at 30 ℃. The preparation method of the semi-solid agar separation medium comprises the following steps: the preparation and composition of the medium were as described above for anaerobic inorganic salt medium (DCB-1). When 9mL of DCB-1 medium was dispensed into 20mL serum bottles, the low gel temperature (<30 ℃ agar (from Sigma USA)Company, cat # A9414) to a mass concentration of 1% (w/v).

After one week of culture, colonies with different shapes grow out of the middle part of the semisolid agar gel. And (3) picking white and smooth single bacterial colonies by using a syringe needle to an anaerobic inorganic salt culture medium (DCB-1) for continuous culture to obtain a relatively purified strain Z culture solution. For further purification of the strain, the above semi-solid agar dilution culture and DCB-1 liquid inorganic salt culture dilution separation procedure was repeated 6 times, and finally, at 10^-12Strain Z was obtained in the dilution flask.

Example 3 taxonomic identification of Strain Z

(1) Morphological identification: the pure culture solution of the strain Z obtained in example 2 was centrifuged at 13000 Xg for 10min to collect cells in logarithmic growth phase; soaking and fixing the cells cultured to the logarithmic phase for 4h by using 2.5% glutaraldehyde buffer solution (10mL glutaraldehyde, 1.4g sodium dihydrogen phosphate and 50mL distilled water), and then centrifuging for 10min at 13000 Xg; the treated cells were dehydrated for ten minutes with 30%, 50%, 70%, 85%, 95% and 100% ethanol, respectively, in that order; it was then suspended in t-butanol and the suspended cells were lyophilized overnight in a lyophilizer. Finally, scanning observation was performed using an FEI aspect F50 field emission electron microscope (see FIG. 1).

The results showed that the cells of strain Z were uniformly dispersed in the scanning field, the cells were straight rod-shaped cells, which showed smoothness, a cell length of about 1-2 μm, a diameter of 0.4-0.6 μm, no sporulation at both ends, and no cells of other morphologies were observed, further indicating that strain Z was a pure culture (FIG. 1).

(2)16S rRNA gene sequence homology analysis: extracting total genome DNA from the strain Z obtained by the separation and purification of the steps, and taking the total genome DNA as a gene amplification template of Polymerase Chain Reaction (PCR). PCR reactions were performed with the universal primers 27F and 1492R for the bacterial 16S rRNA gene. The reaction system adopts PCR amplification kit of Baobioengineering (Dalian) Co., Ltd. Sanger sequencing of the amplification products was performed by Jinzhi Biotech, Suzhou. Splicing of upstream and downstream sequences was performed using the geneous Prime 2020.2.4 software; the strain similarity results were obtained by comparison with the National Center for Biotechnology Information (NCBI) database (see fig. 2).

The result shows that the 16S rRNA gene fragment of the strain Z is 1405bp, the mol% of G + C is 53%, and homology comparison analysis shows that the homology of the strain Z and microorganisms in Acetobacter genus is higher than 96.0%, and the homology of the strain Z and Acetobacter winograe model strain DSM 1911 is the highest and reaches 99.3%. However, previous studies have demonstrated that members of the Acetobacter genus, including strain DSM 1911, are not able to degrade isoprene, and thus it was determined that this strain Z belongs to a novel strain of the Acetobacter genus (FIG. 2).

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

tgcttaccat gcagtcgacg agacgagatg gaatgaccct gtcgggggaa tgaaatctta gaaagtggcg aacgggtgag taacgcgtgg gtaacctgcc ctatggaaag gaatagcctc gggaaactgg gagtaatgcc ttataataca gtgaagtcgc atggctttgc tgttaaacgc tccggtgcca taggatggac ccgcgtccca ttagctagtt ggtgagataa cagcccacca aggcgacgat gggtaaccgg tctgagaggg cgaacggtca cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga atattgcgca atgggggaaa ccctgacgca gcaataccgc gtgagtgaag aaggttttcg gatcgtaaag ctctgttatt ggggaagaaa aaagacggta cccaagaaga aagtcccggc taactacgtg ccagcagccg cggtaatacg taggggacaa gcgttgtccg gatttactgg gcgtaaaggg cacgcaggcg gttttttaag tcagatgtga aaggtaccgg ctcaaccggt gacatgcatt tgaaactgaa agacttgagt attggagagg caagtggaat tcctagtgta gcggtgaaat gcgtagatat taggaggaac accggtggcg aaggcggctt gctggacaaa tactgacgct gaggtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aggtgttggg gagactcagt gccgcagcta acgcaataag cactccgcct ggggagtacg accgcaaggt tgaaactcaa aggaattgac ggggacccgc acaagcagcg gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga caatctgaga gatcagactt tcccttcggg gacagagaga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ctgtggttag ttgccatcat tcagttgggc actctaagca gactgccgtg gataacacgg aggaaggtgg ggacgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggtct gaacagaggg ctgcgaaacc gcgaggtgaa gctaatccct taaaacagat ctcagttcgg attgcaggct gcaactcgcc tgcatgaagt tggagttgct agtaatcgca gatcagaatg ctgcggtgaa tgcgttcccg ggtcttgtac acaccgcccg tcacaccacg agagttggca acacccgaag tcagtgaggc aaccgcaagg agccagctgc cgaag

the novel anaerobic strain Z is preserved in the China general microbiological culture Collection center at 11 months and 30 days in 2021, with the preservation number of CGMCC No. 24006.

Example 4 determination of the isoprene degrading Properties of Strain Z

1mL of activated strain Z culture medium was inoculated into 100mL of a culture medium containing 10. mu.L of isoprene and 20mL of H₂And 20mL CO₂The DCB-1 culture medium is cultured for 5 days at the constant temperature of 30 ℃, and 0.1mL of headspace sample is taken every 12 hours during the culture period and injected into a gas chromatograph for quantitatively analyzing isoprene and degradation products thereof. The specific analysis method is as follows: agilent 7890B gas chromatography and Flame Ionization Detector (FID) as detection instrument, and DB-624 capillary chromatographic column (60m × 0.32mm × 1.8 μm) as volatile compound separation column. The chromatographic method parameters are that the split mode of the sample inlet is 50:1, and the temperature is 200 ℃; setting the initial column temperature to 60 ℃ and keeping the initial column temperature for 2min, then raising the temperature to 200 ℃ at the temperature rise speed of 25 ℃/min, and keeping the operation for 1 min; helium is used as analysis carrier gas, and the flow rate of the column is 3 mL/min; the fuel gas and the combustion improver are respectively hydrogen and synthetic air, the flow rates are respectively 30mL/min and 350mL/min, and the temperature of the detector is set to be 300 ℃; the tail gas is blown by nitrogen gas, and the flow rate is 25 mL/min; the running time of the method is 8.6 min. The peak detection time of isoprene and its degradation products, 2-methyl-1-butene and 3-methyl-1-butene, was 4.1min, 3.9min and 3.6min, respectively.

The results show that strain Z is treated with CO₂And H₂For the growth substrate, an initial 110.0. mu. mol of isoprene was completely reduced to 100.1. mu. mol (2-methyl-1-butene and 3-methyl-1-butene) within 4 days, and the average degradation rate reached 27.5. mu. mol/d (FIG. 3).

Example 5 determination of optimal conditions for isoprene degradation by Strain Z

(1) Determination of optimal temperature for degrading isoprene by strain Z

1mL of activated strain Z culture solution was inoculated into a culture flask containing DCB-1 medium at a ratio of 1% (v/v), and 10. mu.L of iso-culture solution was addedPentadiene and 20mL H₂And 20mL CO₂Culturing at constant temperature and in dark at 4 deg.C, 10 deg.C, 16 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 34 deg.C, 37 deg.C and 40 deg.C, respectively, and periodically monitoring the degradation of isoprene by gas chromatography. The experiment was carried out under culture conditions of pH 7.2 and NaCl concentration 0.1%.

The results show that: the degradation phenomenon of isoprene can be observed in the culture temperature range of 10-40 ℃ for the strain Z; the rate of isoprene degradation was highest in the incubation temperature range of 30-37 deg.C (FIG. 4).

(2) Determination of optimum pH for degrading isoprene by strain Z

Test pH was set at 5.0, 5.5, 6.0, 6.5, 7.0, 7.2, 7.5, 7.8, 8.0, respectively. The experiment was performed with DCB-1 medium containing buffers of different pH as follows: modified DCB-1 medium using 30mM 2-morpholinoethanesulfonic acid (MES) as a buffer for each of the treatment groups pH 5.0, pH 5.5, pH 6.0, pH 6.5 and pH 7.0, and modified DCB-1 medium using 20mM HEPES as a buffer for each of the treatment groups pH 7.2, pH 7.5, pH 7.8 and pH 8.0; adjusting pH of each treatment group with NaOH and/or hydrochloric acid, and adding CO at equal ratio into prepared culture medium of each group₂And H₂(20 mL each) and 10. mu.L of isoprene were inoculated with 1% (v/v) of activated strain Z and periodically monitored for isoprene degradation. The experiment was carried out at 30 ℃ and NaCl concentration 0.1%.

The results show that: strain Z was unable to undergo isoprene degradation and cell growth in the medium at pH 5.0, but could degrade isoprene to varying degrees in the pH range of 5.5-8.0, especially at weakly acidic pH 6.5 (FIG. 4).

(3) Determination of optimum salinity for degrading isoprene by strain Z

The salinity test is performed in the modified liquid DCB-1 culture medium, and specifically comprises the following steps: the NaCl concentration (w/v) in DCB-1 medium was set to 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15% and 20%, respectively, and the other medium components were unchanged. Then 10 mul of isoprene and 20mL of H are respectively added into the liquid DCB-1 culture medium with different salinity₂And 20mL CO₂Inoculating strain Z to start degradation reaction, and determiningIsoprene consumption was monitored. This experiment was incubated at 30 ℃ incubation temperature and pH 7.2.

The experimental results showed that the rate of isoprene degradation by strain Z decreased with increasing NaCl concentration, and that 15% and 20% salinity could not support the growth of strain Z, again confirming that strain Z is from a freshwater environment microorganism and has an optimum salinity of 0.1% (fig. 4).

Example 6 Strain Z of the invention as CO₂And H₂Determination of acetogenesis for a substrate

The following tests were performed with modified DCB-1 medium (20mM HEPES instead of 30mM NaHCO in the original DCB-1 medium)₃Buffer, replacing CO by NaOH₂Adjust pH to 7.2). Treatment group 1 Medium was supplemented with 1:1 by volume of CO₂/H₂(20 mL each) treatment group 2 Medium to which only 20mL of CO was added₂ Treatment group 3 Medium to which only 20mL of H was added₂. Adding 10 μ L of isoprene into the above three treatment groups, and adding CO into the culture medium of treatment group 4 at a volume ratio of 1:1₂/H₂(20 mL each) but no isoprene was added. All treatment groups were inoculated with activated 1% strain Z and then incubated at 30 ℃ for static culture and observed for isoprene degradation and acetic acid production.

Acetic acid was monitored by High Performance Liquid Chromatography (HPLC) using the following method: agilent 1260LC series liquid chromatograph equipped with Diode Array Detector (DAD) set at 210nm, elution column of Aminex HPX-87H (300X 7.8mM) from Bio-Rad, column flow rate of 0.6mL/min, mobile phase of 4mM H₂SO₄As a mobile phase; the total running time was 20min, and the acetic acid peak time was 14.3 min.

As can be seen from FIG. 5, in CO₂And H₂In the treatment group 1 existing at the same time, the strain Z can completely degrade isoprene; containing only CO₂In treatment group 2, strain Z only degraded 12% of isoprene; in the presence of only H₂In treatment group 3, strain Z was completely unable to degrade isoprene; without isoprene addition, but with CO₂And H₂In treatment group 4, strain Z still grew and produced 169.9. mu. mol of acetic acid. This indicates that of the strain ZGrowth and isoprene degradation are dependent on CO₂And H₂In the process, a large amount of bioenergy, acetic acid, is produced (fig. 5).

The strain is prepared into microbial inoculum according to the above embodiment, such as 20mL of CO is added into DCB-1 culture medium₂And H₂Inoculating 3% of Acetobacter wieringie strain Z as a growth substrate, and performing light-proof static culture for 5 days at pH 7.2, 30 ℃ and 0.1% salinity; centrifugally collecting and re-suspending in a solid or liquid carrier containing a surfactant to obtain a culture concentrate of the strain Z, a filtrate of a cell culture or a culture bacterial suspension, and obtaining the microbial inoculum. The microbial inoculum is applied to isoprene or a composite polluted water body or soil environment thereof, and has wide application prospect of bioremediation.

Sequence listing

<110> Shenyang application ecological research institute of Chinese academy of sciences

<120> isoprene anaerobic degradation bacterium and application thereof in environmental bioremediation

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1405

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

tgcttaccat gcagtcgacg agacgagatg gaatgaccct gtcgggggaa tgaaatctta 60

gaaagtggcg aacgggtgag taacgcgtgg gtaacctgcc ctatggaaag gaatagcctc 120

gggaaactgg gagtaatgcc ttataataca gtgaagtcgc atggctttgc tgttaaacgc 180

tccggtgcca taggatggac ccgcgtccca ttagctagtt ggtgagataa cagcccacca 240

aggcgacgat gggtaaccgg tctgagaggg cgaacggtca cactggaact gagacacggt 300

ccagactcct acgggaggca gcagtgggga atattgcgca atgggggaaa ccctgacgca 360

gcaataccgc gtgagtgaag aaggttttcg gatcgtaaag ctctgttatt ggggaagaaa 420

aaagacggta cccaagaaga aagtcccggc taactacgtg ccagcagccg cggtaatacg 480

taggggacaa gcgttgtccg gatttactgg gcgtaaaggg cacgcaggcg gttttttaag 540

tcagatgtga aaggtaccgg ctcaaccggt gacatgcatt tgaaactgaa agacttgagt 600

attggagagg caagtggaat tcctagtgta gcggtgaaat gcgtagatat taggaggaac 660

accggtggcg aaggcggctt gctggacaaa tactgacgct gaggtgcgaa agcgtgggga 720

gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aggtgttggg 780

gagactcagt gccgcagcta acgcaataag cactccgcct ggggagtacg accgcaaggt 840

tgaaactcaa aggaattgac ggggacccgc acaagcagcg gagcatgtgg tttaattcga 900

agcaacgcga agaaccttac caggtcttga catcctctga caatctgaga gatcagactt 960

tcccttcggg gacagagaga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg 1020

ttgggttaag tcccgcaacg agcgcaaccc ctgtggttag ttgccatcat tcagttgggc 1080

actctaagca gactgccgtg gataacacgg aggaaggtgg ggacgacgtc aaatcatcat 1140

gccccttatg acctgggcta cacacgtgct acaatggtct gaacagaggg ctgcgaaacc 1200

gcgaggtgaa gctaatccct taaaacagat ctcagttcgg attgcaggct gcaactcgcc 1260

tgcatgaagt tggagttgct agtaatcgca gatcagaatg ctgcggtgaa tgcgttcccg 1320

ggtcttgtac acaccgcccg tcacaccacg agagttggca acacccgaag tcagtgaggc 1380

aaccgcaagg agccagctgc cgaag 1405

Claims

1. An isoprene anaerobic degradation bacterium is characterized in that: the strain is bacillus aceticus aegypti Z which is preserved in China general microbiological culture Collection center at 11 months and 30 days in 2021, and the preservation number is CGMCC No. 24006.

2. A method of isolating isoprene anaerobic degrading bacteria according to claim 1, characterized in that: the sample is sterilized and diluted in a semi-solid agarose culture medium to obtain an approximately pure culture, and a single colony is picked to grow in a liquid anaerobic inorganic salt culture medium to obtain the single colony bacillus welshii acetate bacterium wieringae Z of claim 1.

3. The method of isolating isoprene anaerobic degrading bacteria according to claim 2, wherein: the semi-solid agarose medium is low-hardness agarose with low temperature (<30 ℃) gel performance added in 0.6-1.0g per 100ml of anaerobic medium containing 30-50mM sodium bicarbonate buffer.

4. Use of the anaerobic isoprene degrading bacterium of claim 1, wherein: the strain is applied to degrading isoprene in the environment under the anaerobic condition.

5. Use of the anaerobic isoprene degrading bacteria according to claim 4, characterized in that: the strain uses CO under anaerobic condition₂And H₂The application of degrading isoprene in the environment in a co-metabolism mode for fermenting substrates.

6. Use of the anaerobic isoprene degrading bacterium of claim 1, wherein: the application of the strain in producing acetic acid as a biological energy source under anaerobic conditions.

7. A formulation for degrading isoprene contaminants, comprising: the preparation comprises the Bacillus welshimeri strain Z as defined in claim 1.

8. The formulation for degrading isoprene contaminants of claim 7, wherein: the preparation contains culture, culture concentrate, concentrate separation supernatant or culture bacterial suspension of the bacillus welshimeri strain Acetobacter wieringie Z.

9. A formulation for the production of bioenergy (acetic acid) characterized in that: the preparation comprises the Bacillus welshimeri strain Z as defined in claim 1.

10. The formulation for producing bioenergy (acetic acid) according to claim 9, wherein: the preparation contains culture, culture concentrate, concentrate separation supernatant or culture bacterial suspension of the bacillus welshimeri strain Acetobacter wieringie Z.