CN111378601B - Halogenated phenol degradation strain and microbial inoculum produced by same - Google Patents
Halogenated phenol degradation strain and microbial inoculum produced by same Download PDFInfo
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- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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Abstract
The invention discloses a halogenated phenol degrading strain and a microbial inoculum produced by the same, wherein the strain is a gram-stain reaction negative strain DCP-2 which is identified as Bosea sp. Deposited in China center for type culture Collection in 12 months and 23 days in 2019, and the preservation number of the strain is CCTCC M20191083. The halophenol degrading strain DCP-2 can be applied to degrading 2,4-dichlorophenol (2,4-DCP), o-chlorophenol, p-chlorophenol or m-chlorophenol, and is preferentially applied to degrading 2,4-DCP in soil. The direct application of the degradation microbial inoculum product can reduce the residual amount of 2,4-DCP in the soil by more than 95 percent, and solve the pollution problem of the 2,4-DCP in the soil.
Description
Technical Field
The invention belongs to the field of biological high technology, and relates to a halogenated phenol degrading strain and a microbial inoculum produced by the same.
Technical Field
The halogenated aromatic compounds mostly have the advantages of stable chemical properties and excellent chemical performance, can be used as pesticides, medicaments and various intermediates, are widely applied to industrial and agricultural production, generate great economic and social benefits, and greatly improve the life of human beings. But the halogenated aromatic hydrocarbon has the characteristics of high toxicity and high stability, can stably exist in the environment for a long time, and has certain irritation, carcinogenicity, teratogenicity, nerve and reproductive toxicity and the like. Halogenated aromatic hydrocarbon pollution poses serious threats to ecological environment and human health, so that the treatment of halogenated aromatic hydrocarbon pollution environment becomes a hot spot of current scientific research.
The halogenated phenol is an important chemical intermediate and is also an important intermediate product for metabolizing a plurality of complex halogenated aromatic hydrocarbons in the environment. Because the halogen element has extremely strong electronegativity and is easy to be combined with an enzyme system in a living cell, the compound has high stability and strong toxicity and is easy to migrate in the environment, and serious non-point source pollution such as soil, water and the like is caused. 2,4-dichlorophenol (2,4-dichlorophenol,2,4-DCP) is an important pesticide and medical intermediate. For example, the organophosphorus insecticide prothioconazole, the herbicide 2,4-D, 2,4-D butyl ester, diclofop-methyl, bifenox, metoxyfen, azafrin, oxadiargyl and the new bactericide cyazofamid can be synthesized on the pesticide. Can be used as an important raw material of the medicine thiobis-dichlorophenol in the aspect of medicine. 2,4-DCP is widely detected in the environment due to the large-scale use of 2,4-D herbicide and the like, and forms a great threat to the ecological environment and human health.
The microorganism is the main force for degrading pollutants in the environment, and the method for eliminating the halogenated aromatic hydrocarbon in the soil by utilizing the degradation effect of the microorganism is an economic, safe, effective and secondary pollution-free method, and has wide application prospect.
Disclosure of Invention
Technical problem the present invention develops a novel halophenol pollution remediation microbial inoculum for the practical problem and the important requirement of environmental remediation, and the microbial inoculum can reduce the residual amount of 2,4-DCP in soil and water by more than 95%, and has low production cost.
The technical scheme is as follows:
the invention provides a microbial strain for efficiently degrading halogenated phenol. The strain is a gram staining reaction negative strain DCP-2, is preserved in China center for type culture Collection in 2019, 12 months and 23 days, and has the preservation number of CCTCC NO: m20191083, identified as Bosea sp (fig. 2). The morphological characteristics of the strain DCP-2 are as follows: when growing on an LB flat plate, bacterial colonies are beige, round, small and convex, the surfaces are dry, the edges are neat and opaque; strain DCP-2 was rod-shaped (0.9. mu. m.times.2.0. mu.m) under a transmission electron microscope and had flagella formed at its ends (FIG. 1). The physiological and biochemical characteristics of the strain DCP-2 are as follows: aerobic, motile, gram negative; indole reaction is negative, starch cannot be hydrolyzed, glucose cannot be oxidized to produce acid, and catalase and oxidase are positive, so that litmus milk is solidified; it is resistant to streptomycin, spectinomycin. The strain DCP-2 can grow by taking 2,4-DCP as the only carbon source and energy source, and the degradation rate reaches more than 99 percent under the condition of shake flask culture in a laboratory. The strain can be produced by fermentation equipment commonly used in the fermentation industry.
The application of the degrading strain DCP-2 in degrading halogenated phenol; the halogenated phenol is selected from any one or more of 2,4-DCP, o-chlorophenol, p-chlorophenol and m-chlorophenol; 2,4-DCP is preferred.
The application of the degradation strain DCP-2 in preparing a halogenated phenol degradation microbial inoculum; the halogenated phenol is selected from any one or more of 2,4-DCP, o-chlorophenol, p-chlorophenol and m-chlorophenol; 2,4-DCP is preferred.
A halogenated phenol degrading bacterial agent produced by the halogenated phenol degrading bacterial strain.
The process for producing the microbial inoculum by using the halogenated phenol degrading bacteria comprises the following steps: slant seeding-shake seeding-seeding tank-production tank-product (packaging dosage form is liquid microbial inoculum or solid adsorption microbial inoculum).
The detailed implementation steps of the invention are as follows:
1) inoculating the test tube species of the halogenated phenol degrading bacteria DCP-2 into an LB culture medium shake flask, and carrying out shake culture to a logarithmic phase;
2) inoculating the cultured bacterial liquid into a seeding tank according to the inoculation amount of 10%, culturing to logarithmic phase, wherein the formula of a culture medium used by the seeding tank is as follows: glucose 8g L-1Yeast extract 5g L-1，K2HPO4 1g L-1，NaCl 5g L-1， CaCO32g L-1，MgSO4 0.2g L-10.1% (v/v) of soybean oil and 7.2-7.5 of pH value;
3) inoculating the seed liquid into a production tank according to the inoculation amount of 10% for culture, wherein the culture medium used by the production tank is the same as that of the seed tank;
4) the ventilation quantity of sterile air is 1:0.8 in the culture process of a seeding tank and a production tank, the stirring speed is 180-increased at 240 rpm, the culture temperature is 30 ℃, the whole-process culture time is 96 hours, the number of thalli reaches more than 10 hundred million/mL after the fermentation is finished, and fermentation liquor is directly subpackaged into liquid formulations by a plastic packaging barrel or a packaging bottle or solid microbial inoculum formulations by adopting packaging bags for peat adsorption after being taken out of the tank.
The application of the microbial inoculum in degrading the halogenated phenol is that the halogenated phenol is selected from any one or more of 2,4-DCPP, o-chlorophenol, p-chlorophenol and m-chlorophenol; 2,4-DCP is preferred.
The microbial inoculum disclosed by the invention is preferably applied to degrading 2,4-DCP in soil.
Advantageous effects
The invention provides a strain capable of efficiently and quickly degrading halogenPhenol-substituted bacterial DCP-2. The degrading bacteria DCP-2 has a wide degrading spectrum, and can degrade various halogenated aromatic hydrocarbons such as 2,4-DCP, o-chlorophenol, p-chlorophenol, m-chlorophenol and the like. The degrading bacteria DCP-2 has higher degrading efficiency and can completely degrade 50mg L in 54 hours -12, 4-DCP. Has wide application potential and value. The degrading bacteria agent produced by the bacteria has the advantages of low production and use cost, convenient use and good repairing effect, and is suitable for large-area popularization and use in chemical industry parks, agricultural production areas, grain, oil and vegetable production export bases or places with green food brand marks, which are polluted by halogenated aromatic hydrocarbons in China. The invention has important significance for treating the halogenated phenol pollution, protecting the ecological environment, preventing and treating the underground water pollution, protecting the health of people and the like.
The invention successfully solves the problem of pollution of halogenated phenol caused in industrial and agricultural production activities, thereby protecting the ecological environment and maintaining the human health.
Drawings
FIG. 1 colony morphology and electron micrograph of strain DCP-2 on LB plate
FIG. 2 phylogenetic analysis of 16S rRNA genes of Strain DCP-2
FIG. 3 growth and degradation curves of Strain DCP-2 vs. 2,4-DCP
FIG. 4 Effect of temperature on degradation of 2,4-DCP by Strain DCP-2
FIG. 5 Effect of pH on degradation of 2,4-DCP by Strain DCP-2
FIG. 6 Effect of inoculum size on degradation of 2,4-DCP by Strain DCP-2
FIG. 7 substrate spectra of strain DCP-2
Biological material preservation information
DCP-2, classified and named as Bosea sp.DCP-2, is preserved in China center for type culture Collection, and the preservation number of strains is CCTCC NO: m20191083, the preservation date is 2019, 12 months and 23 days, and the preservation address is Wuhan university in Wuhan, China.
Detailed Description
Example 1 isolation and identification of strains
The invention provides a bacterial strain capable of efficiently degrading halogenated phenol and a microbial inoculum produced by the bacterial strain, wherein the bacterial strain is a gram-negative bacterial strain DCP-2 which is separated from soil of a factory area of certain agricultural chemical factory in Nanjing, Jiangsu. The specific separation and screening method of the strain comprises the following steps:
a sample of contaminated soil (10.0 g) was taken and added to 100mL of a solution containing 50mg of L -12,4-DCP was cultured in medium of inorganic salts (MM hereinafter) at 30 ℃ for 7 days with shaking at 150rpm, transferred to fresh medium treated in the same manner at an inoculum size of 15% (v/v), and subjected to enrichment culture four times in succession. And detecting the degradation condition of the enrichment solution of the fifth generation by using an ultraviolet spectrophotometer. Adding effective fifth generation enrichment solution containing 50mg L -12,4-DCP was applied by dilution to MM solid medium and cultured at 30 ℃ for 5 days. Single colonies on the plates were picked in 3mL liquid LB tube medium, then stored and transferred to 20mL liquid LB tube medium containing 50mg L -12,4-DCP in MM medium at 30 ℃ for 5 days. Extracting with dichloromethane with the same volume, and detecting the effect by an ultraviolet spectrophotometer to obtain the 2,4-DCP degrading strain.
Deposited in China center for type culture Collection in 12 months and 23 days in 2019, and the preservation number of strains is CCTCC NO: m20191083, identified as belonging to Bosea sp. The morphological characteristics of the strain DCP-2 are as follows: when growing on an LB flat plate, bacterial colonies are beige, round, small and convex, the surfaces are dry, the edges are neat and opaque; strain DCP-2 was rod-shaped (0.9. mu. m.times.2.0. mu.m) under a transmission electron microscope and had flagella formed at its ends (FIG. 1). The physiological and biochemical characteristics of the strain DCP-2 are as follows: aerobic, motile, gram negative; indole reaction is negative, starch cannot be hydrolyzed, glucose cannot be oxidized to produce acid, and catalase and oxidase are positive, so that litmus milk is solidified; it is resistant to streptomycin, spectinomycin. The 16S rRNA gene sequence (SEQ ID NO.1) of the strain DCP-2 is compared and analyzed in a database EzBioCloud, and the result shows that the strain DCP-2 has the closest relationship with Bosea genus, wherein the strain DCP-2 has Bosea robiniae DSM 26672TThe similarity reaches 99.65 percent, and is similar to Bosea thiooxidans DSM 9653TThe similarity reaches 99.15 percent. The strain DCP-2 was preliminarily identified as Bosea by combining colony morphology, physiological and biochemical characteristics and phylogenetic analysis of 16S rRNA gene (FIG. 2).
Example 2 laboratory degradation experiment
2.1 growth utilization and degradation of 2,4-DCP by Strain DCP-2
Detecting DCP-2 by high performance liquid chromatography: a3 mL sample was centrifuged at 12,000rpm for 5min, the supernatant carefully aspirated, 25% hydrochloric acid was added, and the pH was adjusted to about 3.0. Then, the mixture is extracted by dichloromethane with the same volume, 2mL dichloromethane phase is dried after anhydrous sodium sulfate is used for removing water, then 0.5mL methanol is used for redissolving, and the mixture is filtered by an organic phase filter membrane with the aperture of 0.22 mu m and then is detected by HPLC. Detection conditions are as follows: the high performance liquid chromatograph is Shimadzu RID-10A; the chromatographic column is a C18 reversed phase column with specification of 250mm multiplied by 4.6 mm; the column temperature is 40 ℃; the mobile phase is methanol, water, acetic acid (80:20:0.5, V: V: V), the flow rate is 1.0mL min L-1(ii) a The detection wavelengths were 220nm and 235 nm.
Bacterial strain DCP-2 was added at a final concentration of 0.2 (OD)600Value) was inoculated to a solution containing 50mg L -12,4-DCP was shake-cultured at 30 ℃ and 150rpm in 100mL MM medium, and 4mL samples were taken every 6 hours to 54 hours. Wherein 1mL of the total amount of the solution was used for determining the concentration (OD) of the cells600Value), drawing a strain growth curve; another 3mL of the sample was used for HPLC to detect the concentration of 2,4-DCP, and the degradation curve was plotted. The results are shown in FIG. 3, strain DCP-2 can completely degrade 50mg L within 54h -12,4-DCP, and can grow by using the same as a unique carbon source, and the thallus concentration (OD)600Value) increased from 0.2 initially to 0.23.
2.2 seed liquid preparation
Picking single colony of strain DCP-2 to 100mL with 50mg L -12,4-DCP, culturing in LB liquid culture medium at 30 deg.C and 150rpm shaking table until thallus growth logarithmic phase, centrifuging at 6,000rpm for 5min to collect thallus, washing thallus with sterilized MM culture medium for 2 times, and re-suspending with 10mL sterilized MM culture medium to obtain thallus seed solution.
2.3 Effect of environmental factors on degradation of 2,4-DCP by Strain DCP-2
2.3.1 Effect of temperature on degradation of 2,4-DCP by Strain DCP-2
50mg L into a 50mL Erlenmeyer flask containing 20mL MM liquid medium -12,4-DCP as the sole carbon source, inoculating the seed liquid until the initial thallus concentration is OD600When the mixture was mixed uniformly, 3mL of the resulting suspension was taken, centrifuged at 12,000rpm for 5min, and the supernatant was collected as a control. Then respectively arranged inCulturing at 150rpm in shaking tables at different temperatures (4 deg.C, 16 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 37 deg.C and 45 deg.C) for 30 hr, centrifuging 3mL, collecting supernatant, adding 25% hydrochloric acid to the supernatant and the control supernatant respectively, and adjusting pH to about 3.0. Then, extracting with dichloromethane with the same volume, removing water with anhydrous sodium sulfate, drying 2mL of dichloromethane phase, redissolving 0.5mL of methanol, filtering with an organic phase filter membrane with the aperture of 0.22 μm, detecting with HPLC, and calculating the degradation rate of the 2, 4-DCP. Three replicates of each treatment were set. As shown in FIG. 4, the optimum degradation temperature of 2,4-DCP by strain DCP-2 was 30 ℃.
2.3.2 Effect of pH on degradation of 2,4-DCP by Strain DCP-2
To a 50mL Erlenmeyer flask containing 20mL MM liquid medium at different pH's (4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) was added 50mg L -12,4-DCP is used as a unique carbon source, and seed liquid is inoculated until the initial thallus concentration is OD600When the mixture was mixed uniformly, 3mL of the resulting suspension was taken, centrifuged at 12,000rpm for 5min, and the supernatant was collected as a control. Shaking-culturing at 30 deg.C and 150rpm for 30 hr, sampling 3mL, centrifuging, collecting supernatant, adding 25% hydrochloric acid to the supernatant and the control supernatant respectively, and adjusting pH to about 3.0. Then, extracting with dichloromethane with the same volume, removing water with anhydrous sodium sulfate, drying 2mL of dichloromethane phase, redissolving 0.5mL of methanol, filtering with an organic phase filter membrane with the aperture of 0.22 μm, detecting with HPLC, and calculating the degradation rate of the 2, 4-DCP. Three replicates of each treatment were set. As shown in FIG. 5, the strain DCP-2 showed the best degradation effect on 2,4-DCP at pH 7.0.
2.3.3 Effect of inoculum size on degradation of 2,4-DCP by Strain DCP-2
Inoculating the seed liquid into the seed liquid containing 50mg L according to the inoculation amount of 1%, 3%, 5%, 8%, 10% and 12% respectively-12,4-DCP in 20mL MM medium, after mixing well, immediately taking 3mL bacterial liquid, 12,000rpm, centrifuging for 5min, and taking supernatant as control. Shaking-culturing at 30 deg.C and 150rpm for 30 hr, sampling 3mL, centrifuging, collecting supernatant, adding 25% hydrochloric acid to the supernatant and the control supernatant respectively, and adjusting pH to about 3.0. Then extracting with dichloromethane of equal volume, removing water with anhydrous sodium sulfate, drying 2mL dichloromethane phase, redissolving with 0.5mL methanol, filtering with organic phase filter membrane with pore diameter of 0.22 μm, and detecting with HPLCAnd calculating the degradation rate of the 2, 4-DCP. As shown in FIG. 6, the amount of the inoculated portion has a direct relationship with the degradation efficiency of 2,4-DCP, and the larger the amount of the inoculated portion, the higher the degradation efficiency of 2, 4-DCP.
2.4 degradation substrate spectra of Strain DCP-2
Respectively inoculating seed liquid of strain DCP-2 to 50mg L-1Different halogenated phenol compounds (2,4-DCP, o-chlorophenol, p-chlorophenol, m-chlorophenol and 2,4, 6-trichlorophenol) are put in 20mL MM liquid culture medium, shake cultivation is carried out for 60h at 30 ℃ and 150rpm, then culture solution is taken, 25% hydrochloric acid is added, and pH is adjusted to about 3.0. Then, the mixture is extracted by dichloromethane with the same volume, and after water is removed by anhydrous sodium sulfate, the degradation conditions of different compounds are detected by an ultraviolet spectrophotometer. The results show that the strain DCP-2 can degrade 2,4-DCP, o-chlorophenol, p-chlorophenol and m-chlorophenol (FIG. 7), but cannot degrade 2,4, 6-trichlorophenol.
Example 3 preparation of microbial inoculum
The stock of the halophenol degrading strain DCP-2 of the present invention was activated on a petri dish and inoculated on a test tube slant for future use. The test tube strain was inoculated into a medium containing 200mL of LB medium (LB medium formulation: peptone 10g L)-1Yeast powder 5g L-1Sodium chloride 5g L-1pH 7.4) was incubated with constant temperature shaking in a 1,000mL shake flask to logarithmic phase, ready to be inoculated into a first seed tank. 50L of first-level seed tank, 40L of batch size and the formula of culture medium as follows: glucose 8g L-1Yeast extract 5g L-1，K2HPO4 1g L-1，NaCl 5g L-1，CaCO3 2g L-1，MgSO4 0.2g L-10.1% (v/v) of soybean oil and 7.2-7.5 of pH value; after the feeding is finished, high-pressure steam sterilization is carried out, after the temperature is cooled to 30 ℃, the cultured shake flask strain is inoculated into a 50L first-class seed tank according to the inoculation amount of 10 percent, the shake flask strain is cultured to the logarithmic phase, the stirring speed is 220rpm, and the introduction amount of sterile air is 1: 0.8. Inoculating the seed liquid reaching logarithmic phase into a secondary seed tank according to the inoculation amount of 10%. 500L of secondary seed tank, 400L of material feeding amount, and the formula and culture conditions of the culture medium are consistent with those of the primary seed tank. Inoculating the seed liquid reaching logarithmic phase into a production tank for culture according to the inoculation amount of 10 percent, and producingThe medium composition used in the tank was the same as the seed tank medium. The capacity of the production tank is 5 tons, and the feeding amount is 4.5 tons. And (3) sterilizing the fed production tank by high-pressure steam, cooling to 30 ℃ after sterilization, and introducing sterile air to keep a sterile state for later use. The temperature of the production tank after inoculation is controlled at 30 ℃, the ventilation quantity of sterile air in the culture process of the production tank is 1:0.8, the stirring speed is 220rpm, and the culture time of the whole process flow is 96 hours. The number of the thalli after fermentation is over 10 hundred million/mL.
After fermentation, the culture solution is directly taken out of the tank and is subpackaged into liquid dosage forms by using a plastic packaging barrel or a packaging bottle or into solid microbial inoculum dosage forms by adopting a packaging bag for peat adsorption.
Example 4 soil degradation experiment
Vegetable garden soil was taken as the soil sample to be tested. The soil sample is sieved by a 2mm sieve, a certain amount of 2,4-DCP powder is taken and dissolved in 100mL of methanol, and then diatomite is soaked to ensure that the compound is completely absorbed. The soaked diatomite is dried in a fume hood, and is mixed into the soil to ensure that the concentration of the 2,4-DCP in the soil is about 50mg kg-1. Taking 500g of soil sample, inoculating the DCP-2 seed solution according to the inoculation amount of 10%, culturing in a constant temperature incubator at 30 ℃, and taking the soil sample inoculated with the same amount of sterile MM liquid as a control, wherein the water holding capacity of the soil is kept at 60%. After 5 days, samples were taken and tested, 3 samples each time for each treatment, each sample weighing 20 g. The sample is extracted by shaking with methanol with the same volume for 2 times, the methanol in the two times is combined, after being dried by nitrogen, the sample is re-dissolved by using 1mL of methanol, and then the residual quantity of the 2,4-DCP is detected by HPLC. The measurement results are shown in Table 1, and after 5 days, the degradation rate of 2,4-DCP by strain DCP-2 reaches 95.6%. The results show that after the strain DCP-2 is applied to soil, the phenomenon of no degradation or rapid degradation efficiency reduction does not occur, the degradation performance is stable, and thus scientific basis is provided for the strain DCP-2 to repair the 2,4-DCP polluted soil.
TABLE 1 degradation of three herbicides by Strain DCP-2 in soil
Sequence listing
<110> Nanjing university of agriculture
<120> halophenol degradation strain and microbial inoculum produced by same
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1448
<212> DNA
<213> genus Bosea (Bosea sp.)
<400> 1
agagtttgat cctggctcag agcgaacgct ggcggcaggc ttaacacatg caagtcgaac 60
gggcacttcg gtgctagtgg cagacgggtg agtaacacgt gggaacgtac ctttcggttc 120
ggaataatcc agggaaactt ggactaatac cggatacgcc cttcggggga aagatttatc 180
gccgatagat cggcccgcgt ctgattagct agttggtgag gtaaaggctc accaaggcga 240
cgatcagtag ctggtctgag aggatgatca gccacattgg gactgagaca cggcccaaac 300
tcctacggga ggcagcagtg gggaatattg gacaatgggc gcaagcctga tccagccatg 360
ccgcgtgagt gatgaaggcc ttagggttgt aaagctcttt tgtccgggaa gataatgact 420
gtaccggaag aataagcccc ggctaacttc gtgccagcag ccgcggtaat acgaaggggg 480
ctagcgttgc tcggaatcac tgggcgtaaa gggcgcgtag gcggactttt aagtcggagg 540
tgaaagccca gggctcaacc ctggaattgc cttcgatact gggagtcttg agttcggaag 600
aggttggtgg aactgcgagt gtagaggtga aattcgtaga tattcgcaag aacaccggtg 660
gcgaaggcgg ccaactggtc cgaaactgac gctgaggcgc gaaagcgtgg ggagcaaaca 720
ggattagata ccctggtagt ccacgccgta aacgatgaat gccagccgtt ggggagcttg 780
ctcttcagtg gcgcagctaa cgctttaagc attccgcctg gggagtacgg tcgcaagatt 840
aaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa 900
gcaacgcgca gaaccttacc agcttttgac atgtccggtt tgatcggcag agatgccttt 960
cttcagttcg gctggccgga acacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag 1020
atgttgggtt aagtcccgca acgagcgcaa ccctcgcccc tagttgccat cattcagttg 1080
ggaactctag ggggactgcc ggtgataagc cgcgaggaag gtggggatga cgtcaagtcc 1140
tcatggccct tacaggctgg gctacacacg tgctacaatg gcggtgacaa tgggcagcga 1200
aagggtgacc tcgagctaat cccaaaaagc cgtctcagtt cagattgcac tctgcaactc 1260
gagtgcatga aggtggaatc gctagtaatc gtggatcagc atgccacggt gaatacgttc 1320
ccgggccttg tacacaccgc ccgtcacacc atgggagttg ggtttacccg aaggcgtcgc 1380
gctaaccgca aggaggcagg cgaccacggt aggctcagcg actggggtga agtcgtaaca 1440
aggtaacc 1448
Claims (10)
1. A halophenol degrading strain DCP-2 is characterized by being classified and named as a Hirschhorn (Bosea sp.), being preserved in the China center for type culture collection 12 months and 23 days in 2019, and having the strain preservation number of CCTCC NO: m20191083.
2. Use of the degrading strain DCP-2 of claim 1 for degrading halophenol; the halogenated phenol is selected from any one or more of 2,4-DCP, o-chlorophenol, p-chlorophenol and m-chlorophenol.
3. The use according to claim 2, characterized in that the degrading strain DCP-2 of claim 1 is used for degrading 2, 4-DCP.
4. The application of the degrading strain DCP-2 of claim 1 in preparing a halogenated phenol degrading bacterial agent; the halogenated phenol is selected from any one or more of 2,4-DCP, o-chlorophenol, p-chlorophenol and m-chlorophenol.
5. The use according to claim 4, characterized in that the use of the degrading strain DCP-2 of claim 1 in the preparation of a microbial inoculum for degrading 2, 4-DCP.
6. A halophenol degrading microbial inoculum produced by using the halophenol degrading strain of claim 1.
7. The method for preparing the microbial inoculum according to claim 6, which is characterized by being produced by the following method:
1) inoculating the test tube species into an LB culture medium shake flask, and carrying out shake culture to a logarithmic phase;
2) inoculating the cultured strain into a seeding tank according to the inoculation amount of 10 percent, culturing to a logarithmic phase, wherein the formula of a culture medium used by the seeding tank is as follows: glucose 8g L-1Yeast extract 5g L-1，K2HPO4 1 g L-1，NaCl 5 g L-1，CaCO3 2 g L-1，MgSO4 0.2 g L-10.1 percent of soybean oil and 7.2 to 7.5 of pH value;
3) inoculating the seed liquid into a production tank according to the inoculation amount of 10% for culture, wherein the culture medium used by the production tank is the same as that of the seed tank;
4) the aeration of sterile air during the cultivation in the seed and production tanks was 1:0.8, the stirring speed is 180-240 rpm, the culture temperature is 30 ℃, the whole-process culture time is 96 hours, the number of the thalli reaches more than 10 hundred million/mL after the fermentation is finished, and the culture solution is taken out of a tank and directly subpackaged into liquid formulations by a plastic packaging barrel or a packaging bottle or subpackaged into solid microbial inoculum formulations by a packaging bag for peat adsorption after the fermentation is finished.
8. The use of the microbial inoculum of claim 6 in degrading halogenated phenol, wherein the halogenated phenol is selected from any one or more of 2,4-DCPP, o-chlorophenol, p-chlorophenol and m-chlorophenol.
9. The use according to claim 8, wherein the halophenol is 2, 4-DCP.
10. The use according to claim 8, characterized in that the use of the bacterial agent according to claim 6 for degrading 2,4-DCP in soil.
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