CN107090411B - Beauveria bassiana HAr19 strain capable of degrading alkylphenol and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biological treatment of environmental pollutants, and particularly discloses a Beauveria bassiana (Beauveria bassiana) HAR19 strain capable of degrading alkylphenol and application thereof. The Beauveria bassiana (Beauveria bassiana) HAR19 strain is preserved in Guangdong province microorganism strain preservation center in 2017 at 25.4.month, and the preservation number is GDMCC No. 60166. The Beauveria bassiana (Beauveria bassiana) HAR19 strain provided by the invention can obviously and rapidly degrade alkylphenol pollutant 4-nonylphenol, and the degradation rate is as high as 97% after 7 days. The Beauveria bassiana (Beauveria bassiana) HAr19 strain and the application method thereof provided by the invention provide a technical basis for the practical application of the strain.
Description
Technical Field
The invention belongs to the technical field of biological treatment of environmental pollutants, and particularly relates to a Beauveria bassiana (Beauveria bassiana) HAR19 strain capable of degrading alkylphenol and application thereof.
Background
Alkylphenol (AP)S) Is degradation product of alkylphenol polyoxyethylene (APEs or APnEO) and has estrogen effect. APnEO is the second largest nonionic surfactant in the world, is widely used as an emulsifier and a dispersant, is a main raw material of a synthetic detergent, has wide application in industrial fields of industrial cleaning, textile printing and dyeing, papermaking, leather chemical industry, chemical fiber oiling agents, oil field auxiliaries, pesticides, emulsion polymerization and the like, and is finally degraded into APs by microorganisms in the environment. 4-nonylphenol is a major pollutant of alkylphenols. It is statistically significant that a large amount of 4-nonylphenol enters the soil or water worldwide every year (73500 tons in europe, 154200 tons in usa, 16000 tons in china, 16500 tons in japan). 4-nonyl phenol is stable in chemical property in the environment and is not easy to degrade; the fat solubility is strong, the fat is easy to be absorbed by the organism and is difficult to be discharged by the organism; can be accumulated by biological enrichment in an ecosystem through a food chain, and causes harm to the ecology; is lipophilic, is easy to accumulate in adipose tissues, and is extremely harmful even if the discharged concentration is low.
4-nonyl phenol as an environmental hormone has not gradually attracted human attention until the 90 s of the 20 th century, and prevention and control means mainly focus on the application and development of easily degradable alternative materials, and the potential of microbial degradation of 4-nonyl phenol has not been gradually recognized by human beings until recently. The benzene ring and the long-chain alkyl of the 4-nonylphenol structure cause the difficult degradation of the 4-nonylphenol, but the microorganism can open the ring of the benzene ring in a phenol path and break the chain of the nonyl in an alkylbenzene path, and no new pollutant is generated, and high cost is not caused. Therefore, microorganisms having the ability to degrade 4-nonylphenol are very valuable for research and development.
Research has shown that filamentous fungi have the potential to degrade alkylphenols. Beauveria bassiana is a filamentous fungus belonging to Deteromycetina, Hyphomycetes, Hyphomycetes, Moniliaceae and Beauveria, has a wide host range, records more than 700 insects of 15 meshes and 149 meshes and more than 6 and 10 mites and ticks of acaridae, and is mainly applied to biological control of pests and diseases in the agriculture and forestry field at present. The beauveria bassiana has no ability of degrading alkylphenol compounds and has application in the technical field of biological treatment of environmental pollutants.
Disclosure of Invention
The invention aims to overcome the technical defects of the existing alkylphenol biological treatment and provides a beauveria bassiana HAr19 strain capable of degrading alkylphenol and application thereof.
The invention is realized by the following technical scheme:
the invention provides a Beauveria bassiana (Beauveria bassiana) HAR19 strain capable of degrading alkylphenol. The beauveria bassiana HAR19 strain is preserved in Guangdong province microorganism culture collection (GDMCC) in 2017, 4 and 25 months, the preservation number is GDMCC No.60166, and the preservation address is as follows: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.
The strain of Beauveria bassiana HAR19 belongs to the genus Beauveria of the family Moniliaceae of the order Aphyllophorales of the class Hyphomycetes of the order Deuteromycotina.
The HAr19 strain is obtained by separating, screening and purifying soil 15cm below the ground surface of a farmland in the freshwater mussel lake town of the white cloud area in Guangzhou, Guangdong province, and is identified as Beauveria bassiana (Beauveria bassiana) through morphology and molecules.
Further, the beauveria bassiana HAr19 strain can degrade alkylphenol.
Still further, the alkylphenol is 4-nonylphenol.
The beauveria bassiana HAR19 strain can rapidly degrade alkylphenol compound 4-nonylphenol in a carbon-source-free culture medium, the complete degradation rate can reach 97% in 7 days, and a strong degradation effect is shown.
One method for degrading alkylphenol by adopting the beauveria bassiana HAR19 strain of the invention comprises the following steps:
s1: culturing the beauveria bassiana HAR19 strain to obtain seed liquid;
s2: adding a sample to be treated containing alkylphenol into an MS culture medium, adding a beauveria bassiana HAR19 strain seed solution into the culture medium containing alkylphenol, adjusting the pH value, and culturing and degrading at 150 rmp.
Preferably the medium described in S1 is MS medium: k2HPO44.36g/L,KH2PO41.7g/L,NH4Cl 2.1g/L,MgSO4·7H2O 0.2g/L,MnSO40.05g/L,FeSO4·7H2O 0.01g/L,CaCl2·2H2O0.03g/L, glucose 20g/L, dH2Make up to 1000mL of O.
Preferably, the culture temperature of S1 is 26 +/-2 ℃, and the culture time is 24 h.
Preferably, the volume ratio of the beauveria bassiana HAR19 strain seed liquid to the culture medium in S2 is 0.5-1.8: 9.
More preferably, the volume ratio of the beauveria bassiana HAR19 strain seed solution to the culture medium in S2 is 1: 9.
Preferably, the concentration of the alkylphenol in the culture medium of the step S2 is 25-100 mg/L.
More preferably, the concentration of alkylphenol in the culture medium of step S2 is 50 mg/L.
Preferably the alkylphenol is 4-nonylphenol.
Preferably the pH value described in step S2 is 3-9.
More preferably the pH value described in step S2 is 7.
Preferably, the degradation temperature in step S2 is 20-45 ℃ and the degradation days are 4-7 days.
More preferably the degradation temperature in S2 is 25 ℃ and the degradation days is 7 days.
The invention also provides a composition containing the beauveria bassiana HAR19 strain, which is prepared by taking effective amounts of fermentation culture of the beauveria bassiana HAR19 strain and the beauveria bassiana HAR19 strain, culture solution obtained by centrifuging the fermentation culture of the strain or dried product thereof as effective components and adding auxiliary materials, and is applied to degrading alkylphenol pollutants remained in the environment.
Has the advantages that:
the invention provides a beauveria bassiana HAr19 strain capable of degrading alkylphenol, which is the first application of beauveria bassiana in the field of environmental pollutant biodegradation. The strain can obviously degrade alkylphenol compounds 4-nonylphenol, the degradation rate is up to 97% after 7 days, and the strain has a good application prospect in the field of biodegradation of soil and water pollutants. The invention provides an application method of beauveria bassiana HAR19 strain for degrading alkylphenol 4-nonylphenol, and provides a technical basis for the practical application of beauveria bassiana HAR19 strain.
Drawings
FIG. 1 is a graph showing the degradation of 4-nonylphenol by Beauveria bassiana HAR19 strain under an external carbon source;
FIG. 2 is a graph showing the degradation of 4-nonylphenol by Beauveria bassiana HAR19 strain at various pH values;
FIG. 3 is a graph showing the degradation of 4-nonylphenol by Beauveria bassiana HAR19 strain at different temperatures;
FIG. 4 is a graph showing the degradation of 4-nonylphenol by Beauveria bassiana HAR19 strain under optimum conditions;
FIG. 5 is a chromatogram of 4-nonylphenol degradation product of 48h experimental group and control group, wherein a is experimental group and b is control group.
Detailed Description
The invention will be further described with reference to specific embodiments and figures, but the invention is not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
Example 1: separation, culture and identification of beauveria bassiana HAr19 strain
1. Strain isolation and culture
(1) Collecting strains: sieving a fresh soil sample to remove larger stone grains and impurities, taking 10g of clean soil, suspending the clean soil in 90mL of 0.05% Tween-80 solution, shaking uniformly, standing for 15min, taking 2mL of supernatant, and diluting the supernatant in 8mL of 0.05% Tween-80.
(2) Domestication: the bacterial solution was aspirated into MS medium (K) containing 50 mg/L4-nonylphenol2HPO44.36g/L,KH2PO41.7g/L,NH4Cl 2.1g/L,MgSO4·7H2O 0.2g/L,MnSO40.05g/L,FeSO4·7H2O 0.01g/L,CaCl2·2H2O 0.03g/L,dH2Supplementing O to 1000mL), culturing for 7 days at 150rmp at a volume ratio of bacterial liquid to culture medium of 1:9 and 26 +/-2 ℃, sucking 0.5mL of bacterial liquid after 7 days, inoculating into a new 4-nonylphenol-MS culture medium, and repeating for 10 times to obtain a flora HR 50. The 4-nonylphenol stock solution was prepared by dissolving 4-nonylphenol in ethanol to prepare 1000mg/L stock solution.
(3) And (3) purification: coating the flora HR50 on a plate, performing three repeated treatments, culturing at the constant temperature of 26 +/-2 ℃ for 5 days to obtain single colonies, selecting the single colonies with the best growth condition, further coating the plate and purifying to obtain a pure strain, naming HAr19, then using an inoculating loop to pick hyphae on the single colonies, inoculating the hyphae on a PDA plate for continuous culture, and storing the hyphae in a refrigerator at 4 ℃ for later use after purification. The culture was stored in PDA medium containing 40% glycerol at-80 ℃ for a long period.
2. Identification of strains
(1) Morphological identification
And (3) inoculating the purified and preserved strains on a PDA (personal digital assistant) plate, culturing at 25 ℃ for 3-5 days, picking a proper amount of hyphae when the bacterial colonies start to produce spores, suspending the hyphae in 0.05% Tween-80 solution, placing a small amount of hyphae on a blood counting plate, observing the spore production structure and the conidium morphological characteristics of the strains under a microscope, and taking pictures for recording.
Hypha of beauveria bassiana HAR19 strain has a diaphragm, and is transparent and colorless; the peduncles or small branches of conidiophores can be branched at right angles for many times and are gathered into clusters, and the implantation parts of main branches or lateral branches of the conidiophores are at right angles for many times; the conidia are spherical, have a diameter of 2.00-2.15 μm, and grow on a zigzag structure formed by extending spore-forming cells, and the spore-forming cells are in a bottle shape and are variable, and gradually taper from the ventral end to the upper part.
Morphology of the strain on PDA medium: the colony is flat, powdery or like a broken chalk, and the surface is white to light milk.
(2) Molecular identification
DNA-ITS sequencing: after extracting genomic DNA of the HAR19 strain using a DNA extraction kit, the DNA-ITS gene fragment was amplified and sent to a Bio-Inc for sequencing. Calling the DAN-ITS gene sequence of a related strain with higher similarity from a GenBank database by using a Blast search program, finding that the sequence similarity of the strain HAr19 and the beauveria bassiana on NCBI is up to 99 percent through sequence multiple comparison and phylogenetic evolution analysis, and showing that the strain and the beauveria bassiana naturally gather into one by the phylogenetic analysis result, thereby indicating that the strain is a member of the beauveria bassiana.
Example 2: preparation of the composition
The beauveria bassiana HAr19 strain is inoculated on a PDA slant culture medium sterilized at high temperature and cultured for 24h at the temperature of 26 plus or minus 2 ℃.
The purified strain was added to 20mL MS shake flask medium (K) sterilized at high temperature2HPO44.36g/L,KH2PO41.7g/L,NH4Cl 2.1g/L,MgSO4·7H2O 0.2g/L,MnSO40.05g/L,FeSO4·7H2O 0.01g/L,CaCl2·2H2O0.03g/L, glucose 20g/L, dH2O to 1000mL), and culturing at 26 + -2 deg.C under 150rmp for 24 hr to obtain seed solution.
Inoculating the seed solution into MS culture medium (K) sterilized at high temperature according to the inoculation amount of 1:92HPO44.36g/L,KH2PO41.7g/L,NH4Cl 2.1g/L,MgSO4·7H2O 0.2g/L,MnSO40.05g/L,FeSO4·7H2O 0.01g/L,CaCl2·2H2O0.03g/L, glucose 20g/L, dH2O is added to 1000mL), and the mixture is cultured for 24 hours at 26 +/-2 ℃ under 150rmp to obtain bacterial suspension.
Centrifuging the bacterial suspension, collecting conidium, mixing with adjuvants, drying, and pulverizing to obtain spore powder with spore content of 50-100 hundred million/g.
Example 3: parameter optimization for degrading 4-nonylphenol by beauveria bassiana HAr19 strain
1. External carbon source
The purified strain was added to 20mL MS shake flask medium (K)2HPO44.36g/L,KH2PO41.7g/L,NH4Cl2.1g/L,MgSO4·7H2O 0.2g/L,MnSO40.05g/L,FeSO4·7H2O 0.01g/L,CaCl2·2H2O0.03g/L, glucose 20g/L, dH2O to 1000mL), 26. + -. 2 ℃ for 24 hours at 150 rmp.
The 4-nonylphenol stock solution was added to MS (A) and MS (B) media to give a 4-nonylphenol concentration of 50 mg/L. Adding the bacterial liquid after 24 hours into two MS (A) culture media and two MS (B) culture media (volume ratio is 1:9), wherein the MS (A) culture media contain 20g/L glucose, and the MS (B) culture media are deficient in glucose. The cells were cultured at 26. + -. 2 ℃ under 150rmp for 7 days, and the content of 4-nonylphenol was measured every 24 hours.
The MS medium containing 4-nonylphenol was removed for the planned sampling time, extracted under reflux with dichloromethane-ethyl acetate (1:1) for 6h, cooled and the extract concentrated in a rotary evaporator. And (4) carrying out gas chromatography-mass spectrometry analysis after the concentrated solution is subjected to constant volume and carrying out quantitative calculation on the result.
Chromatographic conditions are as follows: a chromatographic column: 30 m.times.0.25 mm.times.0.25 μm; carrier gas: (He); flow rate: 1.2 mL/min; an ionization mode: EI; column temperature: 70 ℃ (2min) (30 ℃/min) → 160 ℃ (1min) (8 ℃/min) → 300 ℃ (11 min); ionization voltage: 70 eV; sample inlet temperature: 280 ℃; mass spectrum interface temperature: 280 ℃; mass scan range: 35-550/amu; and (3) sample introduction mode: no flow diversion; sample introduction amount: 1 μ L.
As shown in FIG. 1, the addition of exogenous carbon atoms to the culture medium affected the degradation of 4-nonylphenol by Beauveria bassiana HAR19, and the amount of 4-nonylphenol decomposed in the two culture media differed little with time.
pH value
With 1mol/L NaOH or H2SO4The pH was separately adjusted to 3.05.07.09.011.0. The measurement methods of the inoculum size, MS medium composition and 4-nonylphenol content were the same as those in example 3.
As shown in FIG. 2, the neutral environment (pH7.0) is most beneficial to the degradation of 4-nonylphenol by Beauveria bassiana HAR19, the degradation rate is reduced with the increase or decrease of pH, and the alkaline environment is particularly not beneficial to the degradation of 4-nonylphenol by Beauveria bassiana HAR 19.
3. Temperature of
The culture temperatures were set to 15253545 ℃. The measurement methods of the inoculum size, MS medium composition and 4-nonylphenol content were the same as those in example 3.
As shown in FIG. 3, the degradation rate of 4-nonylphenol increased with an increase in temperature in the set temperature range, and the degradation rate of 4-nonylphenol did not differ much 7 days after the experiment at 253545 ℃.
Example 4: verification of beauveria bassiana HAR19 degradation capability
The beauveria bassiana HAR19 seed liquid in example 2 is mixed with the following components in proportion of 1:9 to MS medium containing 4-nonylphenol (50mg/L), the pH was adjusted to 7.0, and the mixture was put at 25. + -. 2 ℃ in a 150rmp shaker to carry out degradation experiments, and the content of 4-nonylphenol was measured every 24 hours. The 4-nonylphenol decomposition was measured after 48 hours and 7 days.
The control group was set as a blank test without adding beauveria bassiana HAr19 strain. Other treatment steps were the same as experimental groups.
The results are shown in fig. 4, compared with the blank test group, the beauveria bassiana HAr19 provided by the invention under the culture conditions can rapidly degrade 4-nonylphenol, the degradation rate reaches over 90% after 4 days, and the degradation rate of 4-nonylphenol reaches 97% after 7 days.
By analyzing the 4-nonylphenol degradants (Table 1, FIG. 5), it was further clarified that Beauveria bassiana HAR19 rapidly degraded 4-nonylphenol, and 9 major degradants were present at 48h, but no degradants were detected after 7 days under the above-mentioned detection conditions.
TABLE 148 h 4-nonylphenol degradants
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (6)
1. A Beauveria bassiana (Beauveria bassiana) HAR19 strain is characterized in that the accession number of the Beauveria bassiana HAR19 strain is GDMCC No. 60166.
2. A composition comprising the Beauveria bassiana (Beauveria bassiana) HAr19 strain of claim 1, wherein the composition is prepared by adding an auxiliary material to an effective amount of a fermentation culture of Beauveria bassiana (Beauveria bassiana) HAr19 strain and Beauveria bassiana (Beauveria bassiana) HAr19 strain as an effective ingredient.
3. Beauveria bassiana (Beauveria bassiana) HAR19 strain as set forth in claim 1, and use of the composition as set forth in claim 2 for degrading residual 4-nonylphenol contaminants in an environment.
4. The use according to claim 3, wherein the use is performed as follows:
(1) adding the beauveria bassiana HAR19 strain into an MS culture medium, and culturing for 24 hours in a shaker at 26 +/-2 ℃ at 150rmp to obtain a seed solution;
(2) adding a sample to be treated containing alkylphenol into an MS culture medium to obtain a culture medium containing alkylphenol, adding the seed liquid obtained in the step (1) into the culture medium containing alkylphenol, adjusting the pH value to 3-9, and carrying out culture degradation under 150 rmp.
5. Use according to claim 4, characterized in that the composition of the MS medium is: k2HPO44.36g/L,KH2PO41.7g/L,NH4Cl 2.1g/L,MgSO4·7H2O 0.2g/L,MnSO40.05g/L,FeSO4·7H2O 0.01g/L,CaCl2·2H2O0.03g/L, glucose 20g/L, dH2Make up to 1000mL of O.
6. The use according to claim 4, wherein the culturing conditions in step (2) are at a temperature of 20-45 ℃ for 4-7 days.
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| CN104498363A (en) * | 2014-08-30 | 2015-04-08 | 安徽农业大学 | Growth promoting endophytic beauveria bassiana strain and application thereof |
| CN105176828A (en) * | 2014-07-16 | 2015-12-23 | 新疆农业大学 | Beauveria bassiana XNBb-04 strain and culture method thereof |
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| CN105176828A (en) * | 2014-07-16 | 2015-12-23 | 新疆农业大学 | Beauveria bassiana XNBb-04 strain and culture method thereof |
| CN104498363A (en) * | 2014-08-30 | 2015-04-08 | 安徽农业大学 | Growth promoting endophytic beauveria bassiana strain and application thereof |
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Effective date of registration: 20200717 Address after: No. 303-305, Guansheng International Seed Trade Exhibition Center, No. 663, Shinan Road, Dongyong Town, Nansha District, Guangzhou City, Guangdong Province Applicant after: GUANGZHOU HELITIAN BIOTECHNOLOGY Co.,Ltd. Address before: 510507 Guangdong city of Guangzhou province Tianhe District Yanling Road No. 133 Applicant before: Pi Yuting |
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