CN104480024B - One plant of aspergillus fumigatus and its application with butyl acetate degradation capability - Google Patents

One plant of aspergillus fumigatus and its application with butyl acetate degradation capability Download PDF

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CN104480024B
CN104480024B CN201410814363.9A CN201410814363A CN104480024B CN 104480024 B CN104480024 B CN 104480024B CN 201410814363 A CN201410814363 A CN 201410814363A CN 104480024 B CN104480024 B CN 104480024B
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aspergillus fumigatus
butyl acetate
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CN104480024A (en
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成卓韦
陈建孟
於建明
陆李超
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Zhejiang University of Technology ZJUT
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Abstract

The present invention relates to a kind of butyl acetate degradation bacteria --- aspergillus fumigatus（Aspergillus fumigatus）HD 2, is preserved in China typical culture collection center, address：China, Wuhan, Wuhan University, 430072, deposit number：CCTCC M:2014175, preservation date：On July 11st, 2014.The invention further relates to application of the above-mentioned aspergillus fumigatus in degraded butyl acetate, chlorobenzene, α firpenes, isopropanol or ethyl acetate.Present invention degraded is efficient, tolerance is strong.

Description

One plant of aspergillus fumigatus and its application with butyl acetate degradation capability
Technical field
The present invention relates to a kind of aspergillus fumigatus (Aspergillus fumigatus) HD-2, and its in microbial degradation second Application in the pollutants such as acid butyl ester.
Background technology
Ester is that sour (carboxylic acid or inorganic oxacid) and alcohol react a class organic compound of generation.Rudimentary ester is that have perfume (or spice) The volatile liquid of gas, senior ester is waxy solid or very thick liquid.Several senior esters are the main components of fat.Ester Water is insoluble in, the organic solvents such as ethanol are soluble in, the esters of wherein low molecule amount are colourless, volatile gas.Low molecule amount Ester can be used as solvent, the larger ester of molecular weight is good plasticizer.If methyl methacrylate is that manufacture lucite is (poly- Methyl methacrylate) monomer；Polyester resin is mainly used in fiber and paint industry, may be made as moudling powder；Many is carried The ester of the alcohol formation of side chain is excellent lubricating oil.Ester can be additionally used in the industry such as spices, essence, cosmetics, soap and medicine.It is small After molecule esters are volatized into atmospheric environment through discharge, stimulation is produced to eye and respiratory tract, high concentration can then produce acute Poisoning.
Purification biotechnology has the features such as removal efficiency is high, processing cost is low, secondary pollution is small, is gradually widely used In the degraded and purification of toxic pollutant.It is to obtain to have efficiently that one of key of benzene and benzene homologues is handled using biotechnology The bacterial strain for its ability of degrading.At present, there is degraded energy to all kinds of volatile contaminants by studying to obtain and be separated to both at home and abroad The fungi of power has including belonging to the flat lead fungi category (Phanerochaete) of raw wool, Paecilomyces varioti Pseudomonas (Paecilomyces), reaping hook Mould category (Fusarium), Eurotium (Aspergillus), trichoderma (Trichoderma), spore Saksenaea fungi (Cladosporium) some strains of the bacterial strain of 10 category such as.Degradable target contaminant is mainly benzene, toluene, ethylbenzene etc. The ketones such as the chlorinated hydrocarbons such as benzene compounds, chlorobenzene, methyl ethyl ketone and some other organic acid.Also studies have found that raw wool Flat lead fungi belongs to, and mucor etc. has some strains to have using the ability that ester type compound is carbon source, shows that fungi has ester of degrading The ability of pollutant.At present, scholar proposes to utilize aspergillus fumigatus pollution administration environment, primarily with regard to dye decolored, The biodegradation of polyphenol compound and PAHs (anthracene), lignin degradation etc..
The 2nd phase of volume 22 that in March, 2006 publishes《Polymer material science and engineering》In, entitled " poly-succinic fourth two Mentioned in alcohol ester and poly-succinic/degradation behavior of the adipic acid-butanediol ester under a microbial action " text：It is " right in compost PBSA and PBS degradation capabilities most strong microorganism is aspergillus versicolor.”
It is above-mentioned in the prior art first, although aspergillus versicolor and aspergillus fumigatus belong to aspergillus, but they are two Entirely different kind.In addition just degrade for object, PBSA and PBS belong to high molecular polymer, and butyl acetate belongs to and had Machine thing, two kinds of structures of matter are entirely different, and do not refer to which kind of material PBSA and PBS are degraded in document.Therefore existing skill Art cannot be used for the biodegradation of butyl acetate.
The content of the invention
The technical problems to be solved by the invention are to overcome of the prior art not enough and provide a kind of efficient, tolerance Strong aspergillus fumigatus and its application with butyl acetate degradation capability.
The present invention solve above-mentioned technical problem use technical scheme be：The butyl acetate degradation bacteria --- aspergillus fumigatus (Aspergillus fumigatus) HD-2, is preserved in China typical culture collection center, address：China, Wuhan, Wuhan University, 430072, deposit number：CCTCC M:2014175, preservation date：On May 3rd, 2014.
Aspergillus fumigatus (Aspergillus fumigatus) the HD-2 colony characteristicses are as follows：In 30 DEG C of constant incubators Just start rudiment after middle inoculation 2d, first spread with white fluff sample, after be changed into faint yellow, the culture medium back side is colourless, 3d, bacterium colony Centre becomes cigarette green, and the culture medium back side is faint yellow, and bacterium colony is significantly increased afterwards, and diameter is up to 1.2cm or so, middle green Deepen, and collapse in powdered, bacterium colony has wide paving property, and radial growth, it is impossible to form single bacterium colony, later stage bacterium colony is complete Bottle green is presented in portion, powdered, and mycelia is fine and close.
Aspergillus fumigatus (Aspergillus fumigatus) HD-2 of the present invention with butyl acetate degradation capability Application in degraded butyl acetate, chlorobenzene, australene, isopropanol or ethyl acetate.Described degraded is 5.4,30 in pH value Carried out under conditions of DEG C.
The strain HD -2 is under conditions of 5.4,30 DEG C in pH value, HD-2 can in 48h by initial concentration for 176mg/L Butyl acetate is substantially completely degraded.The discovery of the degradation bacteria is significant to the high-efficient purification of butyl acetate in waste gas.Should Aspergillus fumigatus HD-2 also degradable other industrial common organic pollutions, especially chlorobenzene, australene, isopropanol, benzene and toluene Deng there is good degradation effect.
The present invention has advantages below compared with prior art：The aspergillus fumigatus HD-2 of the present invention is derived from sewage plant sludge, There is preferably degradation effect for esters, especially butyl acetate, pollutant fully more can be converted into CO2、H2O Deng innocuous substance；Meanwhile, the bacterial strain can also degrade the common pollutant of the industry such as benzene, chlorobenzene, australene to some extent, thus Had broad application prospects in industrial waste gas biological purification of waste water.
Aspergillus fumigatus of the present invention can by butyl acetate it is degradable be inorganic matter (H2O and CO2) and cellular biomass, Permineralization is realized, and 350mg/L is up to for the tolerable concentration of butyl acetate.Therefore, the aspergillus fumigatus is for industrial common dirt Contaminating thing (such as butyl acetate) has efficient degradation capability, and can bear the pollutant of higher concentration.
Brief description of the drawings
Fig. 1 is Aspergillus fumigatus HD-2 spore stereoscan photographs.
Fig. 2 is Aspergillus fumigatus HD-2 systematic growth tree graph.
Fig. 3 is influence of the pH value to Aspergillus fumigatus HD-2 degraded butyl acetates.
Fig. 4 is degradation rates of the Aspergillus fumigatus HD-2 in 24h to different butyl acetates.
Embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This.The method do not stated especially in embodiment is prior art.
Embodiment 1：Aspergillus fumigatus HD-2 separation, purifying and its identification.
1.Aspergillus fumigatus HD-2 separation and purifying.
After the sludge air aeration three days of seven lattice sewage plants, 50ml supernatants are taken to be centrifuged, by the dirt precipitated Mud is added in the saline bottle of the minimal medium equipped with 50ml (through 110 DEG C, sterilizing for 40 minutes), and adds streptomysin 0.003g, gentamicin 0.001g.Cover and 5 μ l butyl acetates are added after bottle stopper (concentration is about 88mg/L).30 DEG C are placed on, Cultivated in 160rpm shaking table.Every thalli growth situation in a period of time observation saline bottle, afterwards in complete salt solution of having degraded 5ml bacteria suspensions are taken into the bottle of the minimal medium equipped with fresh 50ml in bottle, streptomysin 0.003g is still added, and celebrating is big mould Plain 0.001g.Cover and 10 μ l butyl acetates are added after bottle stopper (concentration is about 176mg/L).Blake bottle is placed in 30 DEG C, 160rpm Shaking table in cultivate.Cultivate after certain time, then take 5mL bacteria suspensions to the bottle of the minimal medium equipped with fresh 50ml In, add streptomysin 0.003g, gentamicin 0.001g.Cover and 15 μ l butyl acetates are added after bottle stopper (concentration is about 264mg/ L).It is placed in shaking table and cultivates.2ml mixed bacteria liquids are taken, is coated on the Czapek's medium for removing carbon source, is put in culture dish lid center The filter paper of 1cm diameters and the butyl acetate that 5 μ l are added thereon, continuous line separation are final to obtain purifying bacterial strain, are inoculated into In PDA slant mediums, preserved in 4 DEG C of refrigerator.
2.Aspergillus fumigatus HD-2 identification.
Enter the target DNA fragment that performing PCR amplification is obtained to HD-2 genomic DNAs using fungi ITS universal primer.Should Sequence carries out Blast contrasts with the gene order in ncbi database.As a result the ITS sequence and bacterial strain of HD-2 bacterial strains are shown Aspergillus fumigatus isolate NRRL164(EF669932.1)、Aspergillus fumigatus isolate 11a-2a(KF020294.1)、Aspergillus fumigatus strain ATCC 90906 (KC689327.1) ITS sequence area has 100% homology.10 plants of Aspergillus are chosen from result has representative Property bacterial strain, based on ITS gene homologies, with reference to using Clustal X2.0 and MEGA4.0 (1000 times sampling point Analysis) software building phylogenetic tree.Contrasted by genetic distance and ITS sequence, be accredited as aspergillus fumigatus (Aspergillus Fumigatus), it is named as HD-2.
It is 95 DEG C of pre-degeneration 5min that pcr amplification reaction system, which is shown in Table the reaction condition of 1, PCR amplifications, and 95 DEG C are denatured 30s, 55 DEG C annealing 30s, 72 DEG C extension 40s, circulate 32 times；72 DEG C of extension 10min, 10 DEG C of preservations.
Table 1ITS sequence pcr amplification reaction systems
Reaction system composition Volume (μ l)
10×Ex Taq buffer 2.0
2.5mM dNTP Mix 1.6
5p Primer 1 0.8
5p Primer 2 0.8
Template 0.5
5u Ex Taq 0.2
Ultra-pure water 14.1
Cumulative volume 20
Metabolic condition of the bacterial strain to 95 kinds of different carbon sources is investigated using Biolog automatic microbes identification systems：Bacterial strain is connect Plant in fungi plating medium, 26 DEG C incubated 5 days, is washed down the thalline on flat board with aseptic cotton carrier, with inoculation liquid (FF- IF) mix, bacteria suspension is made, is adjusted with nephelometer to 75%T/FF.Bacteria suspension is added in respectively with the electronic charger in 8 holes In each hole of Biolog FF micropore identification plates, per the μ l of hole 100.Micropore identification plate is placed in 26 DEG C of incubators, respectively in culture It is placed on after 24h, 48h, 72h, 96h, 168h and 240h on Biolog readout instruments and reads result.Analyzed through Biolog readout instruments Metabolic Fingerprinting, strain HD -2 can not be utilized more by force using 50 kinds of carbon sources or Utilization ability is weaker to other 45 kinds of carbon sources. Biolog systems provide 240h qualification results, as shown in table 2.
Fig. 1 and Fig. 2 are respectively Aspergillus fumigatus HD-2 spores stereoscan photographs and Aspergillus Fumigatus HD-2 systematic growth tree graph.
The Utilization ability of 95 kinds of carbon sources on -2 pairs of Biolog FF plates of 2 strain HD of table
Table is noted:+, positive reaction；-, negative reaction；B, boundary response
Embodiment 2：Influence of the pH value to Aspergillus fumigatus HD-2 degraded butyl acetates.
The influence that pH value in environment grows to microorganism is larger, and pH can directly affect the change of cell membrane charge, indirectly Influence the degree of ionization of nutriment.It turns out that, compound of the compound than ionic condition under most non-ionic states Enter cell more easily by cell membrane.In addition, the competence exertion activity under suitable pH value condition of the enzyme in microorganism.With it is thin Bacterium, actinomyces are compared, and fungi is liked growing in the environment of slant acidity.
As shown in figure 3, at 30 DEG C, initial concentration is 88.26mg/L, and bacterium inoculum concentration is about at 3mg (albumen).PH threshold values are set For：4.00th, 4.40,4.90,5.40,5.90,6.40 totally 6 gradients, as a result showing can be in 24 hour under various concentrations Reach 90% or so degradation efficiency.Wherein, 5.40 or so pH has faster degradation efficiency.Butyl acetate is after 48 hrs Basic degraded is complete, shows that fungi HD-2 has higher degradation efficiency to butyl acetate.
Embodiment 3：Aspergillus fumigatus HD-2 are detected to the degradation property of various concentrations butyl acetate.
Strain HD -2 is inoculated in pH for 5.4, using butyl acetate as the shaking flask of sole carbon source in, inoculum concentration is about for 3mg (albumen), as shown in figure 4, inoculation bacterium 24h after, HD-2 to initial concentration in the 48h of 4 concentration for 176.52mg/L can base This degraded is complete, and 264.78mg/L, 353.04mg/L, and more than 60% degradation rate can be also reached after 48h.
Embodiment 4：The broad spectrum activity of fungi Aspergillus fumigatus HD-2 degraded substrates.
In actual applications, not only exist generally comprised in a kind of butyl acetate this organic pollution, industrial waste gas it is many VOCs is planted, therefore, research fungi HD-2 is necessary to the degradation effect of other substrates.It is 5.9 in pH value, temperature is 30 DEG C Under the conditions of, fungi HD-2 to initial concentration be 44mg/L chlorobenzene, 53.2mg/L dichloromethane, 34.4mg/L australene, 79mg/L isopropanol, the degradation effect of 90mg/L ethyl acetate is as shown in table 3.
Degradation effects of the fungi HD-2 of table 3 to different carbon source
Material title Initial concentration (mg/L) Clearance (%)
Benzene 88 28.55
Toluene 88 38.22
Chlorobenzene 44 66.30
Dichloromethane 53.2 23.84
Australene 34.4 53.92
Isopropanol 79 41.91
Ethyl acetate 90 37.62
Strain HD -2 can degrade part benzene and toluene, degrade well chlorobenzene and australene, due to isopropanol, acetic acid second Ester is soluble in water, and degradation effect is also good, and the structure of dichloromethane has larger difference with benzene, therefore degradation effect is bad.
Although the present invention is disclosed as above with embodiment, it is not limited to protection scope of the present invention, any ripe The technical staff of this technology is known, in the change and retouching made without departing from the spirit and scope of the invention, this all should be belonged to The protection domain of invention.

Claims (3)

1. a kind of aspergillus fumigatus with butyl acetate degradation capability（Aspergillus fumigatus）HD-2, in being preserved in State's Type Tissue Collection, deposit number：CCTCC M: 2014175.
2. the aspergillus fumigatus with butyl acetate degradation capability according to claim 1 Bacterium（Aspergillus fumigatus）Applications of the HD-2 in degraded butyl acetate, chlorobenzene, isopropanol or ethyl acetate.
3. the application according to claim 2, it is characterised in that：Described degraded is in the bar that pH values are 5.4,30 DEG C Carried out under part.
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