CN107090411A - The beauveria bassiana HAr19 bacterial strains of one plant of degradable alkyl phenol and its application - Google Patents
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Abstract
The invention belongs to environment pollutant biological treatment technical field, beauveria bassiana (Beauveria bassiana) HAr19 bacterial strains and its application of one plant of degradable alkyl phenol are specifically disclosed.Described beauveria bassiana (Beauveria bassiana) HAr19 bacterial strains are preserved in Guangdong Province's Culture Collection on April 25th, 2017, and deposit number is GDMCC No:60166.Beauveria bassiana (Beauveria bassiana) HAr19 bacterial strains that the present invention is provided can the notable nonyl phenol of fast degradation alkyl phenol pollutant 4, degradation rate is up to 97% after 7 days.Beauveria bassiana (Beauveria bassiana) the HAr19 bacterial strains and its application process that the present invention is provided provide technical foundation for the practical application of the bacterial strain.
Description
Technical field
The invention belongs to environment pollutant biological treatment technical field, and in particular to the ball spore of one plant of degradable alkyl phenol is white
Stiff bacterium (Beauveria bassiana) HAr19 bacterial strains and its application.
Background technology
Alkyl phenol (APS) be APES (APEs or APnEO) catabolite, with estrogen effect.
APnEO is world's second largest nonionic surfactant, is widely used as emulsifying agent, dispersant, is synthetic detergent primary raw material,
Have in industrial circles such as industry cleaning link, textile printing and dyeing, papermaking, leather chemical industry, preparation medium, oilfield additive, agricultural chemicals, emulsion polymerizations
And be widely applied, it is finally degraded to APs by microorganism in the environment.4- nonyl phenols are induced by alkyl hydroxybenzene major pollutants.
According to statistics, the whole world have every year substantial amounts of 4- nonyl phenols enter in soil or water body (73500 tons of Europe, 154200 tons of the U.S., in
16000 tons of state, 16500 tons of Japan).Chemical property is stable in the environment for 4- nonyl phenols, is difficult to be degraded；Fat-soluble strong easily quilt
Body absorbs, and is difficult to be discharged by body；Biological concentration can be carried out in the ecosystem by food chain and accumulated, ecology is caused
Harm；With lipophilicity, easily accumulate in adipose tissue, even if the concentration of discharge is very low, also great harmfulness.
4- nonyl phenols are as environmental hormone until 1990s just progressively triggers the mankind to pay attention to, and preventions are main
Concentrate on the application and exploitation of degradable alternative materials, until microbial degradation 4- nonyl phenols in recent years potentiality just progressively by
The mankind are recognized.Phenyl ring and its chain alkyl in 4- nonyl phenol structures cause the refractory organicses of 4- nonyl phenols, but
Microorganism can make the phenyl ring open loop of phenol approach and the nonyl chain rupture of alkylbenzene approach, and will not produce new pollutant, no
High cost can be caused.Therefore, the microorganism with degraded 4- nonyl phenols is that have very much researching value and potentiality to be exploited.
There are some researches show filamentous fungi has the potentiality of degraded alkyl phenol.Beauveria bassiana is that a kind of filaria life is true
Bacterium, is under the jurisdiction of Deuteromycotina (Deteromycotina), Hyphomycetes (Hyphomycetes), hyphomycetales
(Hyphomycetales), Moniliaceae (Moniliaceac), Beauveria (Beauveria), host range is extremely wide, has remembered
What is carried includes 15 mesh, 6 sections of more than 700 kind insects of 149 sections and Acarina, more than 10 kind mites and tick, and agricultural are mainly used at present
The biological control of field pest and disease damage.The ability that beauveria bassiana there are degraded alkylphenol compoundses is never found before this, and in ring
There is application in border pollutant biologic treating technique field.
The content of the invention
The purpose of the present invention is to overcome the technical deficiency of existing alkyl phenol biological treatment there is provided one plant of degradable alkyl phenol
Beauveria bassiana HAr19 bacterial strains and its application.
The present invention is achieved through the following technical solutions：
The invention provides the beauveria bassiana of one plant of degradable alkylphenol (Beauveria bassiana) HAr19 bacterium
Strain.The beauveria bassiana HAr19 bacterial strains were preserved in Guangdong Province's Culture Collection on April 25th, 2017
(GDMCC), deposit number is GDMCC No.60166, preservation address：5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100.
Beauveria bassiana HAr19 bacterial strains belong to Deuteromycotina, Hyphomycetes, hyphomycetales, Moniliaceae, Beauveria.
The HAr19 bacterial strains of the present invention are divided from 15cm soil under the farmland surface of Guangzhou, Guangdong Baiyun District Bang Hu towns
Obtained from screening purifying, be beauveria bassiana (Beauveria bassiana) through form and Molecular Identification.
Further, the beauveria bassiana HAr19 bacterial strains can degrade alkyl phenol.
Further, the alkyl phenol is 4- nonyl phenols.
The beauveria bassiana HAr19 bacterial strains of the present invention can be with fast degradation alkylphenol compoundses in without carbon source culture medium
4- nonyl phenols, 7 days can degradable rate can reach 97%, it is shown that stronger degradation effect.
Use the present invention beauveria bassiana HAr19 strains for degrading alkyl phenols one of which method for：
S1：The beauveria bassiana HAr19 bacterial strains are cultivated, seed liquor is obtained；
S2：Pending sample containing alkyl phenol is added in MS culture mediums, by beauveria bassiana HAr19 bacterial strain seeds
Liquid is added in culture medium containing alkyl phenol, adjusts pH value, 150rmp culture degradeds.
Culture medium described in preferably S1 is MS culture mediums：K2HPO44.36g/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, glucose
20g/L,dH2O complements to 1000mL.
Cultivation temperature described in preferably S1 is 26 ± 2 DEG C, and described incubation time is 24h.
Beauveria bassiana HAr19 bacterial strains seed liquor and the volume ratio of culture medium are 0.5-1.8 in preferably S2:9.
More preferably beauveria bassiana HAr19 bacterial strains seed liquor and the volume ratio of culture medium are 1 in S2:9.
The concentration of alkyl phenol is 25-100mg/L preferably in step S2 culture mediums.
More preferably the concentration of alkyl phenol is 50mg/L in step S2 culture mediums.
Preferably described alkyl phenol is 4- nonyl phenols.
PH value preferably described in step S2 is 3-9.
More preferably the pH value described in step S2 is 7.
Degradation temperature in preferably step S2 is 20-45 DEG C, and degraded number of days is 4-7 days.
More preferably the degradation temperature in S2 is 25 DEG C, and degraded number of days is 7 days.
The present invention also provides the composition containing beauveria bassiana HAr19 bacterial strains, with effective dose beauveria bassiana HAr19 bacterium
Nutrient solution or its drying obtained by strain, the centrifugation of the fermentation culture medium of beauveria bassiana HAr19 bacterial strains, the strain fermentation culture
Thing, nutrient solution or its dried object obtained by strain fermentation culture centrifugation are active ingredient, add what auxiliary material was made, are applied to
Remaining alkyl phenol pollutant in degraded environment.
Beneficial effect：
The invention provides the beauveria bassiana HAr19 bacterial strains of one plant of degradable alkyl phenol, this is muscardine first Application
In the biodegradable field of environmental contaminants.The bacterial strain can significantly degrade alkylphenol compoundses 4- nonyl phenols, be dropped after 7 days
Solution rate is up to 97%, has preferable application prospect in biodegradable soil and water pollutant field.The invention provides ball spore
The application process of muscardine HAr19 strains for degrading alkyl phenol 4- nonyl phenols, is the practical application of beauveria bassiana HAr19 bacterial strains
Technical foundation is provided.
Brief description of the drawings
Fig. 1 is degradation curve figure of the beauveria bassiana HAr19 bacterial strains under additional carbon to 4- nonyl phenols；
Fig. 2 is degradation curve figure of the beauveria bassiana HAr19 bacterial strains under different pH value to 4- nonyl phenols；
Fig. 3 is degradation curve figure of the beauveria bassiana HAr19 bacterial strains under different temperatures to 4- nonyl phenols；
Fig. 4 is degradation curve figure of the beauveria bassiana HAr19 bacterial strains under optimum condition to 4- nonyl phenols；
Fig. 5 is the chromatogram of 48h experimental groups and control group 4- nonyl phenol degradation products, and wherein a is experimental group, and b is control
Group.
Embodiment
The present invention is further detailed with reference to specific embodiments and the drawings, but the invention is not limited in
This.Unless stated otherwise, the reagent of the invention used, method and apparatus is the art conventional reagent, methods and apparatus.
Unless stated otherwise, the reagent and material used in the present invention are purchased in market.
Embodiment 1：Separation, culture and the identification of beauveria bassiana HAr19 bacterial strains
1st, strain isolation and culture
(1) bacterial strain is collected：Larger stone grain and debris are removed into fresh pedotheque sieving, takes pure land 10g to be suspended in
In 90mL0.05% Tween-80 solution, shake up, stand 15min, take its supernatant 2mL to be diluted in the Tween-80s of 8mL 0.05%
In.
(2) tame：Draw (K in above-mentioned bacterium solution to the MS culture mediums containing 50mg/L 4- nonyl phenols2HPO44.36g/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,dH2O complements to 1000mL) to be tamed, the volume ratio of bacterium solution and culture medium is 1:9,26 ± 2
DEG C, draw 0.5mL bacterium solutions after being cultivated 7 days, 7 days under 150rmp and access in new 4- nonyl phenol-MS culture mediums, be repeated 10 times,
Obtain flora HR50.4- nonyl phenol stock solutions are, by 4- nonyl phenol alcohol mixed dissolutions, 1000mg/L stock solutions to be made.
(3) purify：By flora HR50 coated plates, do at three reprocessings, 26 ± 2 DEG C, incubated 5d obtains single bacterium
Fall, choose the wherein best single bacterium colony of upgrowth situation, obtain one plant of pure bacterial strain by further coated plate after purification, name
HAr19, then with oese in picking mycelia in single bacterium colony, is inoculated on PDA plate and continues to cultivate, 4 DEG C are stored in after purification
It is standby in refrigerator.It is stored in -80 DEG C of PDA culture mediums containing 40% glycerine for a long time.
2nd, bacterial strain is identified
(1) Morphological Identification
The strain preserved after purification is connected on PDA plate, at 25 DEG C, 3~5d is cultivated, when just having started production spore to bacterium colony,
The appropriate mycelia of picking, is resuspended in 0.05% Tween-80 solution, takes on a small quantity on blood counting chamber, is placed under microscope
The conidial fructification and conidium form feature of bacterial strain are observed, and is photographed to record.
The mycelia tool barrier film of beauveria bassiana HAr19 bacterial strains, clear, colorless；Point stalk of its conidiophore or sprig can be with
Multiple right angle bifurcated, and assemble agglomerating, how at a right angle the major branch of conidiophore or the inserted part of side shoot be；Conidium is in ball
Shape, diameter life in the zigzag structure that conidiogenous cell extends, conidiogenous cell is ampuliform between 2.00-2.15 μm,
And many changes, it is tapered upwards by abdomen end.
Form of the bacterial strain in PDA culture medium：Bacterium colony is flat, in powdery or such as flour lip pencil, superficial white to light lacteous.
(2) Molecular Identification
DNA-ITS is sequenced：After the genomic DNA that HAr19 bacterial strains are extracted using DNA extraction kit, to DNA-ITS genes
Fragment is expanded, and is sent to biotech firm and is sequenced.With Blast search utilities similitude is recalled from GenBank databases
The DAN-ITS gene orders of higher related strain, are compared by multiple sequence and phylogenetic evolution is analyzed, and find bacterial strain
Beauveria bassiana sequence similarity is up to 99% on HAr19 and NCBI, and Phylogenetic Analysis result shows the bacterium and ball spore
Muscardine gathers for one naturally, and it is the member of beauveria bassiana to illustrate the bacterial strain.
Embodiment 2：The preparation of composition
Beauveria bassiana HAr19 bacterial strains are taken, are inoculated on the PDA slant mediums of high-temperature sterilization, 26 ± 2 DEG C of culture 24h.
Bacterial strain after purification is added to (K in the 20mL MS Shake flask mediums of high-temperature sterilization2HPO44.36g/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 complements to 1000mL), 26 ± 2 DEG C, cultivated 24 hours under 150rmp
To seed liquor.
By 1:Seed liquor is inoculated in (K in the MS culture mediums of high-temperature sterilization by 9 inoculum concentration2HPO44.36g/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 complements to 1000mL), 26 ± 2 DEG C, cultivate 24 hours, obtain under 150rmp
Obtain bacteria suspension.
Bacteria suspension is centrifuged, conidium is collected, mixes, dry, pulverize with auxiliary material, obtain conidia powder, content is 50-
10000000000 spores/gram.
Embodiment 3：The parameter optimization of beauveria bassiana HAr19 strains for degrading 4- nonyl phenols
1. external carbon source
Bacterial strain after purification is added into (K in 20mL MS Shake flask mediums2HPO44.36g/L,KH2PO41.7g/L,
NH4Cl2.1g/L,MgSO4·7H2O 0.2g/L,MnSO40.05g/L,FeSO4·7H2O 0.01g/L,CaCl2·2H2O
0.03g/L, glucose 20g/L, dH2O complements to 1000mL), 26 ± 2 DEG C, cultivated 24 hours under 150rmp.
4- nonyl phenols stock solution is added in MS (A), MS (B) culture medium, the concentration of 4- nonyl phenols is in culture medium
50mg/L.By the bacterium solution after 24 hours is separately added into two kinds of MS (A), (volume ratio is 1 to MS (B):9), MS (A) culture medium is included
20g/L glucose, MS (B) glucose lacks.26 ± 2 DEG C, cultivated under 150rmp 7 days, determine 4- nonyl phenols within every 24 hours
Content.
Within the sampling time of plan, the MS culture mediums containing 4- nonyl phenols are taken out, dichloromethane-ethyl acetate is used
Extraction (1:1) refluxing extraction 6h, extract solution is moved into rotary evaporator concentrate after cooling.By the laggard promoting the circulation of qi phase of concentrate constant volume
Spectrometry is simultaneously quantitatively calculated result.
Chromatographic condition：Chromatographic column：30m×0.25mm×0.25μm；Carrier gas：(He)；Flow：1.2mL/min；Ionization side
Formula：EI；Column temperature：70℃(2min)(30℃/min)→160℃(1min)(8℃/min)→300℃(11min)；Ionization electricity
Pressure：70eV；Injector temperature：280℃；Mass spectrometer interface temperature：280℃；Mass scan range：35~550/amu；Enter sample prescription
Formula：Do not shunt；Sample size：1μL.
As a result as shown in figure 1, supplemented with exogenous carbon original can influence beauveria bassiana HAr19 to 4- nonyl phenols in the medium
Degraded, over time, the decomposition amount difference of 4- nonyl phenols is little in two kinds of culture mediums.
2.pH values
With 1mol/LNaOH or H2SO4PH value is adjusted to 3.05.07.09.011.0 respectively.Inoculum concentration, MS culture mediums into
Point, the assay method be the same as Example 3 of 4- nonyl phenol contents.
As a result as shown in Fig. 2 neutral environment (pH7.0) most useful for beauveria bassiana HAr19 degrade 4- nonyl phenols, with
PH increases or reduce, degradation rate can be reduced, alkaline environment is particularly disadvantageous in beauveria bassiana HAr19 degraded 4- nonyl benzenes
Phenol.
3. temperature
Cultivation temperature is respectively set as 15253545 DEG C.Inoculum concentration, MS medium components, the measure of 4- nonyl phenol contents
Method be the same as Example 3.
As a result as shown in figure 3, within the temperature range of setting, as temperature is raised, the degradation rate of 4- nonyl phenols increases,
7 days after experiment, in the case where 253545 DEG C are handled, the difference of the degradation rate of 4- nonyl phenols is little.
Embodiment 4：The checking of beauveria bassiana HAr19 degradation capabilities
Beauveria bassiana HAr19 seed liquors in Example 2 are with 1：9 volume ratio, which is added, contains 4- nonyl phenols
(50mg/L), MS culture mediums in, regulation pH value be 7.0, be put into the carry out degradation experiment in 25 ± 2 DEG C, 150rmp shaking tables, often
The content for determining 4- nonyl phenols in 24 hours.The analyte of 4- nonyl phenols is determined after 48 hours and 7d.
Control group is set, to be added without the blank test of beauveria bassiana HAr19 bacterial strains.Other process steps are with experiment
Group.
As a result as shown in figure 4, comparing beauveria bassiana provided by the present invention under blank test group, above-mentioned condition of culture
HAr19 4- nonyl phenols capable of being fast degraded, degradation rate reaches that the degradation rate of 4- nonyl phenols behind more than 90%, 7 days reaches after 4 days
97%.
By the analysis (Fig. 5 of table 1) to 4- nonyl phenol degradation products, further elucidating beauveria bassiana HAr19 can be quick
Degraded 4- nonyl phenols, and have 9 kinds in 48h major degradants, but after 7 days, detected not under above-mentioned testing conditions
To degradation product.
The degradation product of the 48h 4- nonyl phenols of table 1
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention
And scope.
Claims (9)
1. one plant of beauveria bassiana (Beauveria bassiana) HAr19 bacterial strain, it is characterised in that the beauveria bassiana
HAr19 bacterial strains deposit number is GDMCC No:60166.
2. a kind of composition of beauveria bassiana (Beauveria bassiana) HAr19 bacterial strains containing described in claim 1,
Characterized in that, with effective dose beauveria bassiana (Beauveria bassiana) HAr19 bacterial strains, beauveria bassiana
Nutrient solution obtained by the fermentation culture medium of (Beauveria bassiana) HAr19 bacterial strains, strain fermentation culture centrifugation or
Its dried object, nutrient solution or its dried object obtained by strain fermentation culture centrifugation are active ingredient, add what auxiliary material was made.
3. the composition according to right 2, it is characterised in that the composition is liquid or solid.
4. beauveria bassiana (Beauveria bassiana) HAr19 bacterial strains described in claim 1, described in claim 2
Application in composition alkyl phenol pollutant remaining in degraded environment.
5. the application according to right 4, it is characterised in that the application is carried out as follows：
(1) the beauveria bassiana HAr19 bacterial strains are added in MS culture mediums, the 150rmp cultures 24 in 26 ± 2 DEG C of shaking tables
Hour, obtain seed liquor；
(2) the pending sample containing alkyl phenol is added in MS culture mediums, obtains the culture medium containing alkyl phenol, then in institute
The seed liquor that the step (1) is added in the culture medium containing alkyl phenol is stated, adjusts and degraded is cultivated under pH value, 150rmp.
6. the application according to right 5, it is characterised in that the component of the MS culture mediums is：K2HPO4 4.36g/L,KH2PO4
1.7g/L,NH4Cl 2.1g/L,MgSO4·7H2O 0.2g/L,MnSO40.05g/L,FeSO4·7H2O 0.01g/L,CaCl2·
2H2O 0.03g/L, glucose 20g/L, dH2O complements to 1000mL.
7. the application according to right 5, it is characterised in that alkyl phenol is dense in the culture medium of the step (2) containing alkyl phenol
Spend for 25-100mg/L.
8. the application according to right 5, it is characterised in that condition of culture is pH value 3-9, temperature 20-45 in the step (2)
DEG C, cultivated days are 4-7 days.
9. the application according to right 4,5,6,7 or 8, it is characterised in that described alkyl phenol is 4- nonyl phenols.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110819353A (en) * | 2019-05-24 | 2020-02-21 | 华南农业大学 | Application of beauveria bassiana SB010 in biodegradation of nonyl phenol |
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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 |
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