CN105886407B - Radiation hardness filamentous fungi F35 and its application in Adsorption of Lead biological treatment - Google Patents
Radiation hardness filamentous fungi F35 and its application in Adsorption of Lead biological treatment Download PDFInfo
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- CN105886407B CN105886407B CN201610061361.6A CN201610061361A CN105886407B CN 105886407 B CN105886407 B CN 105886407B CN 201610061361 A CN201610061361 A CN 201610061361A CN 105886407 B CN105886407 B CN 105886407B
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- ulocladium
- fungi
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- radiation hardness
- lead
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/347—Use of yeasts or fungi
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/20—Heavy metals or heavy metal compounds
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Abstract
The present invention discloses a kind of radiation hardness filamentous fungi F35 and its application in Adsorption of Lead biological treatment, pass through separation, screening, breeding, domestication in the pedotheque that Xinjiang Heshuo County periphery arid-desert areas acquires, obtain one plant of radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006 to lead ion with high characterization of adsorption, there is the otherness of apparent physio-biochemical characteristics difference and molecular level with Common fungi strain, the bacterial strain F35 filtered out can be in Pb2+In the case that concentration is 100mg/L, thallus additive amount is 0.01g/ml, pH=5, temperature is 60 DEG C, adsorption time is 10min, to Pb2+It both can use the growth adsorpting lead ion of thallus as the biological bacteria of processing low concentration lead pollutant effluents with optimal adsorption effect, can also directly have been adsorbed using thallus, and had many advantages, such as that removal rate was high, reaction speed is fast, without secondary pollution, easy to operate.
Description
Technical field
The present invention relates to environmental organism Treatment process fields, and in particular to a kind of radiation hardness filamentous fungi is in heavy metal biological
The technical field applied in absorption.
Background technique
With the aggravation of mankind's activity, industrialization, urbanization, intensive agriculture fast development, a more and more huge sum of moneys
Belong to pollutant and is shifted by Industrial " three Waste ", atmospheric sedimentation, untreated solid waste, sewage irrigation to farmland, accumulation
Harm will be generated to the ecosystem to a certain extent.Currently, lead contamination distribution is most wide in China's heavy metal pollution of soil,
With strong accumulative and non mobility environmental contaminants, it has also become the one of the chief elements of water body and soil pollution at present.Agriculture
Field soil can be generated secondary pollution to crops and underground water, directly be endangered diversity of soil microorganism, and lead to by after lead contamination
Crossing food chain influences human health.Therefore to the research of heavy metal lead polluted-water and soil remediation recycling increasingly by people
Attention.
Improvement currently for heavy metal water pollution mainly includes the recovery technique of physics, chemistry and biology, respectively there is excellent lack
The disadvantages of point, but that there are effects is slow for generally current recovery technique, reparation needs the time long, at high cost.How to improve existing
The repair ability of technology is one of people's concern.With the fast development of technology, bioremediation technology is due to its spy
Different superiority is taken seriously, and wherein microorganism remediation is absorbed using antimicrobial surfaces large biological molecules such as bacterium, fungies
The approach such as transhipment, cell metabolism, biological adsorption and redox reaction absorb heavy metal ion, are precipitated, being aoxidized also
The effects of former, is thus the method for reducing heavy metal ion toxicity.Repair relative to physics and chemical method to lead, micro- life
Object reparation have repair time is short, bacterial strain breeding is fast, opposite processing cost is low, effect on environment is small, it is high-efficient, can be carried out in situ
The advantages that processing, but that there is also microorganisms is easily affected by environment, it is unstable the disadvantages of.
Repair of the microorganism to lead waste water mainly passes through absorption method, carrier combined techniques, film bag method, covalently knot
It is legal etc..Microorganism depends primarily on the special construction of cell wall to the suction-operated of lead, and it is fixed to occur with heavy metal ion
The combination of amount.At present absorption heavy metal microbial strains be concentrated mainly on containing a large amount of negative electrical charge functional groups (such as carboxyl,
Phosphoryl, hydroxyl etc.), there is the filamentous fungi etc. of very high complexating properties；Some such as polysaccharide, protein and nucleic acid materials are secreted,
The bacterium of non-toxic or low-toxic metal organic compound can be formed in conjunction with heavy metal by ion exchange or complexing；And
Endo-mycorrhiza and the exotrophic mycorrhiza of heavy metal present in environment are reduced by mycelium suction-operated.And Anti-radiation Microbes
It is a kind of extreme microorganism resource that can be survived under high dose of radiation, there is stronger tolerance and absorption to heavy metal
Property, there is great application potential in terms of Heavy Metal Pollution Control.
Although at present there are many report for administering bad border using microorganism remediation, the related performance that can filter out is stablized
And there are the excellent species of higher tolerance and stronger absorption property to be rarely reported lead contamination water body, it is therefore desirable to further
Microorganism is probed into the adsorption capacity of heavy metal lead, providing for microorganism remediation heavy metal lead pollution has opposite high absorption capacity
And the resistant strain that performance is stable, there is realistic meaning for administering Lead pollution environment.
Summary of the invention
It is reported for having no in the prior art in relation to radiation hardness filamentous fungi and its correlation applied in absorption heavy metal lead
Road, and the strain of separation screening has the state of the art of stronger tolerance and adsorptivity to heavy metal, the present invention is directed to be micro-
Biological prosthetic lead contamination, which provides, has opposite high absorption capacity and the stable resistant strain of performance.The present invention passes through in pedotheque
In separate one plant of radiation hardness Ulocladium fungi (Ulocladium sp.) F35 for having higher characterization of adsorption to lead ion
CGMCC No.11006, using the strain isolated to lead contamination water body carry out biological treatment, and provide it is a kind of using the bacterium into
Application technology scheme in quadrat polluted-water biological treatment.
The main technical schemes that the present invention uses:
Pedotheque of the present invention picks up from Xinjiang Heshuo County periphery arid by Microorgan Application Inst., Xinjiang Agricultural Academy
The pedotheque of acquisition is placed in Desert Area60Co is after 5000 KGy irradiation, to add the PDA culture medium of streptomysin for separation
Culture medium, a large amount of screenings preferably go out the well-grown radiation-resistant fungus bacterial strain of a batch, and being screened out from it one plant has lead ion
The fungal bacterial strain F35 of high characterization of adsorption.The fungal bacterial strain for being F35 using the number that the present invention separates is to lead contamination water body
Biological treatment is carried out, the bacterial strain is in Pb2+Concentration is 100 mg/L, thallus additive amount is 0.01 g/ml, pH=5, temperature is 60 DEG C,
In the case that adsorption time is 10 min, to Pb2+With optimal adsorption effect.
The present invention specifically provides a kind of radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC
No.11006, by providing determining screening technique, it is true to obtain a batch for separation screening and culture in the pedotheque of acquisition
Mushroom microbial strains are screened out from it one plant of fungal bacterial strain F35 to lead ion with high characterization of adsorption, through microorganism credit
Class and identification, belong to Ulocladium fungi (Ulocladium sp.) bacterial strain, are temporarily named as Ulocladium sp. 35.
Specifically, the present invention is by placing acquisition pedotheque60Co5000 KGy irradiation after, separated, screen and
Culture is screened out from it the bacterial strain that one plant of number is F35, and through microbiological classification and identification, it is true which belongs to Ulocladium
Bacterium (Ulocladium sp.) bacterial strain.The bacterial strain was preserved in budapest treaty microorganism international accession list before the applying date
Position: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC).Address: Chaoyang District, Beijing City North Star west
The institute 3 of road 1, Institute of Microorganism, Academia Sinica, postcode: 100101.Preservation date on July 9th, 2015, culture presevation number
For CGMCC No. 11006.Ulocladium fungi (Ulocladium sp.) F35 is accredited as through microbiology.The bacterial strain is most suitable
Growth conditions are as follows: 30 DEG C of temperature, culture medium (potato 200g/L, glucose 20g/L, agar 15g/L, is steamed using PDA culture medium
Distilled water 1L, pH are natural), incubation time 48h；After 48h is cultivated, on PDA culture medium surface, bacterium colony in crineous to black, in
Portion's protuberance is velvet-like, and neat in edge is clear.By strain F35 by the observation of the microscopy of inserted sheet and hydraulic pressure piece, the bacterial strain primary hyphae is white
Color, conidiophore is short and small, mycelioid, and tool branch separates.Conidium is at fasciating in production falx top, ellipse, muriform
Separate, dark brown.According to the above morphological feature, morphology, Physiology and biochemistry mirror are carried out to F35 bacterial strain referring to " Fungal identification handbook "
It is fixed, and binding molecule biology is sequenced, the Preliminary Identification bacterial strain is that sac fungus gate seat capsule Gammaproteobacteria lattice spore chamber mesh lattice spore chamber Cordycepps is single
One kind of lattice spore category, is temporarily named as radiation hardness Ulocladium fungi Ulocladium sp. F35.
Extract bacterial strain F35 total DNA, using the area fungi ITS universal primer, carry out PCR amplification, PCR product through cutting glue purification,
Carry out raw work sequencing.Known array in the gained ITS region sequence of experimental strain and GenBank database is subjected to BLAST ratio
Compared with determining the nearest kind of experimental strain affiliation.From GenBank database, and in binding to fungal biodiversity research
Heart database obtains the sequence of related species, uses adjacent method (Neighbor- Joining using 5.0 software package of MEGA
Method clustering and systematic evolution tree building) are carried out.
It is found by sequence alignment, bacterial strain F35 is belonged to Ulocladium (Ulocladium), systematic evolution tree building display
The independent branch of bacterial strain F35, and is currently known bacterial strain Ulocladium atrum UAMH 7840, Ulocladium
cucurbitae HSAUP_XF030282、Ulocladium chartarum UAMH 7842、Ulocladium botrytis
UB32, Ulocladium consortiale BMP 3151001, Ulocladium botrytis ATCC 18043 have
Maximum homology, similitude 99.17%, with its more than similitude all 98.33% hereinafter, not can determine that still its it is definite classify, temporarily
It is named as Ulocladium sp.F35.
Molecule sequencing result shows that the ITS1 gene order of radiation-resistant fungus F35 CGMCC No.11006 is 547bp.This
Inventing radiation hardness Ulocladium fungi (Ulocladium sp.) F35 provided and common fungi strain has apparent physiology raw
The otherness for changing property difference and molecular level, according to strain Analysis of The Physiological And Biochemical Properties, molecular level analysis and genealogical classification
Comprehensive identification, the strain that number is F35 have some categories of general character although compared with common Ulocladium fungi strain
Property, but according to the difference for having apparent physio-biochemical characteristics difference and molecular level with common Ulocladium fungi strain
Property, show that F35 bacterial strain is a kind of typical novel bacterial, it is stronger to the tolerance of heavy metal lead ion, adsorption capacity, from point
F35 bacterial strain is accredited as radiation hardness Ulocladium fungi (Ulocladium sp.) by class.
By above-mentioned each test to the strain for being F35 is numbered compared with common Ulocladium fungi strain, there is general character
Some attributes, but according to common Ulocladium fungi strain having apparent physio-biochemical characteristics difference and molecular level
Otherness, show that F35 bacterial strain is a kind of typical novel bacterial, the Preliminary Identification bacterial strain is sac fungus gate seat capsule Gammaproteobacteria lattice spore chamber
The bacterial strain comprehensive identification that bacterium numbering is F35 is resistance to from bacterium classification angle by one kind of mesh lattice spore chamber Cordycepps Ulocladium
Radiate Ulocladium fungi (Ulocladium sp.) F35.
The present invention provides radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006's in turn
Separation and cultural method.
1. isolation medium uses: PDA culture medium, every 100ml culture medium add 1% Streptomycin Solution 0.3ml, are added simultaneously
Lead ion solution makes its concentration up to 2000mg/L.
2. separation and screening conditions: take gradient dilution method, weigh 10g irradiation after acquisition pedotheque in 90ml without
In bacterium physiological saline, gradient dilution is carried out after 30 DEG C of activation 30min, chooses stoste, 10-1、10-2、10-3Dilution is respectively coated
In on isolation medium plate, 30 DEG C of cultures are set in 3 repetitions of each processing, after growing fungus colony picking shape, size,
The different bacterium colony difference streak inoculation such as color is in corresponding plate, up to no miscellaneous bacteria falls.
Through determining radiation hardness Ulocladium fungi (Ulocladium sp.) the F35 CGMCC No.11006 of culture screening
Bacterial strain bacterium colony is in crineous to black in PDA culture medium, and middle part protuberance is velvet-like, and neat in edge is clear；PDA culture medium selects horse
Bell potato 200g/L, glucose 20g/L, agar 15g/L, distilled water 1L, pH are natural.
Further, the present invention provides a kind of radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC
No.11006 is applied in the processing of lead contamination aqueous bio.By studying in different plumbum ion concentrations, temperature, pH value, inoculum concentration
Influence under the conditions of adsorption time to thallus absorption, obtains F35 to Pb2+Optimal adsorption condition be Pb2+Concentration 100mg/L,
Thallus additive amount 0.01g/ml, pH=5, temperature 60 C, adsorption time 10min.
By implementing particular technique index of the present invention, realization the content of present invention, can achieve it is following the utility model has the advantages that
(1) radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006 provided by the invention,
It is simple with condition of culture, breed fast advantage.
(2) the radiation hardness Ulocladium fungal bacterial strain that the number of separation screening of the present invention is F35 can be can be in Pb2+Concentration
In the case that for 100mg/L, thallus additive amount be 0.01g/ml, pH=5, temperature is 60 DEG C, adsorption time is 10min, to Pb2+
It is a kind of typical radiation-resistant fungus with optimal adsorption effect.
(3) present invention both can use the growth adsorpting lead ion of thallus during handling low concentration lead pollutant effluents,
It can also directly be adsorbed using thallus, have many advantages, such as that removal rate is high, reaction speed is fast, without secondary pollution, easy to operate.
Detailed description of the invention
Fig. 1 show radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006 bacterium colony and
Thallus photo, wherein A- bacterium colony photo, B- thallus photo.
Fig. 2 show radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006 system
System development dendrogram.
It is dense that Fig. 3 show radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006 lead ion
Spend the influence diagram to adsorption effect.
At the beginning of Fig. 4 show radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006 lead ion
Influence diagram of the beginning pH to adsorption effect.
Fig. 5 show radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006 thallus dosage
To the influence diagram of adsorption effect.
Fig. 6 show radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006 absorption temperature
Spend the influence diagram to adsorption effect.
When Fig. 7 show the absorption of radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006
Between to the influence diagram of adsorption effect.
Fig. 8 show radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006 Adsorption of Lead from
Infrared spectrogram before son.
Fig. 9 show radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006 Adsorption of Lead from
Infrared spectrogram after son.
Specific embodiment
In the following, illustrating the present invention for embodiment, still, the present invention is not limited to following embodiments.It is selected in the present invention
All raw and auxiliary materials, and the Spawn incubation method selected all is well known in the art selection, and the % being related in the present invention is
Be weight percentage, unless otherwise indicated except.
Embodiment one: the separation of radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006,
Screening and identification
1, the separation and screening of strain
Ulocladium fungi (Ulocladium sp. F35) used in the present invention is by Xinjiang Agricultural Sciences institute microorganism
Applied Research Laboratory is largely acquired in soil from Xinjiang Heshuo County periphery arid-desert areas and is sampled, and carries out spoke through 5000 KGy cobalt sources
According to rear separation, the fungi in soil layer is isolated using traditional plating method, plate streak purifies bacterial strain, with different trainings
Supporting temperature, pH value, culture medium is enrichment condition, goes out the well-grown fungal bacterial strain of a batch by optimal screening, therefrom preferably goes out
The bacterial strain that one plant of number is F35.
Separating step:
(1) isolation medium uses: PDA culture medium adds 1% Streptomycin Solution according to the PDA culture medium of every 100ml
0.3ml, while lead ion solution is added, make its concentration up to 2000mg/L.
(2) separation and screening conditions: taking gradient dilution method, and the pedotheque after weighing 10g irradiation is in the sterile life of 90ml
It manages in salt water, gradient dilution is carried out after 30 DEG C of activation 30min, choose stoste, 10-1、10-2、10-3Dilution is respectively coated on point
From on culture medium flat plate, 30 DEG C of cultures are set in 3 repetitions of each processing.Picking shape, size, color after growing fungus colony
Etc. different bacterium colony difference streak inoculations in corresponding plate, up to no miscellaneous bacteria falls.
2, the condition of culture of bacterial strain
(1) growth medium for the bacterial strain that number is F35 is PDA culture medium: potato 200g/L, glucose 20g/L, fine jade
Rouge 15g/L, distilled water 1L, pH through 30 DEG C, 48 h naturally, cultivate.
(2) bacterial strain that number is F35 can be grown under the conditions of 28-30 DEG C, and optimum growth temperature is 30 DEG C, incubation time
For 48h.
(3) the growth pH for the bacterial strain that number is F35 is natural.
(4) number is the heavy metal tolerance experiment of F35 bacterial strain.
The strain is inoculated in respectively containing 8 heavy metal species ions, Pb2+Concentration range is in 1500-3500mg/L, Cd2+It is dense
Range is spent in 50-250mg/L, Hg2+Concentration range in 400-1200mg/L, Cu2+Concentration range 700-1500mg/L,
Cr2+Concentration range in 700-1500mg/L, Co2+Concentration range in 400-1200mg/L, Zn2+Concentration range in 800-
2000mg/L、Ni2+Concentration range on the plate of 700-1500mg/L, cultivated in 30 DEG C, observed after 7d its grow shape
Condition.Tolerance testing result of the radiation hardness Ulocladium fungi Ulocladium sp.F35 to different heavy metal ion are as follows: this hair
Bright strain used is to 8 heavy metal species ion Pb2+、Cd2+、Hg2+、Cu2+、Cr2+、Co2+、Zn2+、Ni2+All have higher tolerance
Characteristic, be resistant to respectively 3500mg/L, 250mg/L, 400mg/L, 700mg/L, 1500mg/L, 1200mg/L, 2000mg/L,
The heavy metal ion solution of 1300mg/L concentration, the results showed that, the strain is for heavy metal ion Pb2+Tolerance highest,
It has a good application prospect in heavy metal biological processing.
Specifically, the present invention by being irradiated to acquisition pedotheque with 5000 KGy cobalt sources, separated, screen and
Culture is screened out from it the bacterial strain that one plant of number is F35, and through microbiological classification and identification, it is true which belongs to Ulocladium
Bacterium (Ulocladium sp.) bacterial strain.The bacterial strain was preserved in budapest treaty microorganism international accession list before the applying date
Position: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC).Address: Chaoyang District, Beijing City North Star west
The institute 3 of road 1, Institute of Microorganism, Academia Sinica, postcode: 100101.Preservation date on July 9th, 2015, culture presevation number
For CGMCC No.11006.Radiation hardness Ulocladium fungi Ulocladium sp.F35 is accredited as through microbiology.The bacterial strain is most
Adaptability elongate member are as follows: 30 DEG C of temperature, culture medium use PDA culture medium, potato 200g/L, glucose 20g/L, agar 15g/L,
Distilled water 1L, pH are naturally, incubation time 48h；After 48h is cultivated, on PDA culture medium surface, bacterium colony in crineous to black, in
Portion's protuberance is velvet-like, and neat in edge is clear.By strain F35 by the observation of the microscopy of inserted sheet and hydraulic pressure piece, the bacterial strain primary hyphae is white
Color, conidiophore is short and small, mycelioid, and tool branch separates.Conidium is at fasciating in production falx top, ellipse, muriform
Separate, dark brown.According to the above morphological feature, morphology, Physiology and biochemistry mirror are carried out to F35 bacterial strain referring to " Fungal identification handbook "
It is fixed, and binding molecule biology is sequenced, the Preliminary Identification bacterial strain is that sac fungus gate seat capsule Gammaproteobacteria lattice spore chamber mesh lattice spore chamber Cordycepps is single
One kind of lattice spore category, is temporarily named as radiation hardness Ulocladium fungi (Ulocladium sp.) F35.
Molecule sequencing result shows radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006
ITS1 gene order be 547bp, radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC provided by the invention
No.11006 and Common fungi strain have the otherness of apparent physio-biochemical characteristics difference and molecular level, according to strain physiology
The comprehensive identification of Analysis of Biochemical Characteristics, molecular level analysis and systematics, the strain that number is F35 is although in Physiology and biochemistry
There is apparent difference with common Ulocladium fungi strain in terms of characteristic and molecular level, with common fungi strain phase
Than, it is stronger to the tolerance of heavy metal lead ion, adsorption capacity, but Ulocladium fungi is accredited as according to taxology
(Ulocladium sp.).
3, the Physiology and biochemistry identification of bacterial strain F35
Morphological feature: strain F35 is separately cultured through PDA culture medium, and bacterium colony is in crineous to black in PDA culture medium
Color, middle part protuberance is velvet-like, and neat in edge is clear, and the doubtful Ulocladium fungus colony of picking carries out inserted sheet and the microscopy of hydraulic pressure piece is seen
It examines, the bacterial strain primary hyphae white, conidiophore is short and small, mycelioid, and tool branch separates.Conidium is at fasciating in production spore
Obstruct top, ellipse, muriform separate, dark brown, radiation hardness Ulocladium fungi (Ulocladium sp.) F35 bacterium colony and
Thalli morphology is referring to attached drawing 1.
Physiological and biochemical property: sugared fermentation, carbon assimilation, nitrogen source assimilation identification have been carried out to the bacterium, bacterial strain F35 is in PDA
Well-grown on culture medium is tested, the results showed that bacterial strain F35 can use N- acetyl group-Portugal-D- through Biolog FF identification plate
Grapes glucosamine, apricot glycosides, D-arabinose, L-arabinose, arbutin, D- cellobiose, dextrin, erythritol, D-Fructose, D- half
Lactose, gentiobiose, a-D- glucose, a-D- Cori ester salt, D- glucuronic acid, glycerine, glycogen, the Portugal 2- ketone-D-
Grape saccharic acid, a-D- lactose, lactulose, maltitol, maltose, maltotriose, D-MANNOSE, D- melezitose, D- melibiose, a-
Methyl D-galactoside ,-methyl-D-glucoside, 6-O-D- glucopyranose acyl-D- fructofuranose, D- melitriose, D- core
Sugar, salicin, D-glucitol, stachyose, sucrose, D- trehalose, turanose, xylitol, D- xylose, y- aminobutyric acid, anti-fourth
Enedioic acid, a-ketoglutaric acid, L MALIC ACID, chinic acid, D-Glucose diacid, succinamic acid, succinic acid, amber acid methyl
Ester, L- alanine amide, L- alanine, L- alanyl amion acetic acid, altheine, L-Aspartic acid, Pidolidone, bird ammonia
Acid, L- phenylalanine, proline, Serine, 2- ethylaminoethanol.
Pass through the thallus of above-mentioned strain radiation hardness Ulocladium fungi Ulocladium sp.F35 CGMCC No.11006
Form, cultural characteristic observation and Determination of Physiological And Biochemical Indices, that is, pass through thalli morphology observation, strain culturing observation of characteristics, growth
The test such as temperature measuring, resistance test, compared with common strain, to 8 heavy metal species ion Pb2+、Cd2+、Hg2+、Cu2+、Cr2 +、Co2+、Zn2+、Ni2+Higher resistance characteristics are all had, are carried out referring to the method for " Fungal identification handbook ", the bacterium that number is F35
Although kind compared with common Ulocladium fungi strain, has some attributes of general character, foundation and common single lattice spore
Belong to the otherness that fungi strain has apparent physio-biochemical characteristics difference and molecular level, shows that F35 bacterial strain is a kind of typical
Novel bacterial, the Preliminary Identification bacterial strain are one kind of sac fungus gate seat capsule Gammaproteobacteria lattice spore chamber mesh lattice spore chamber Cordycepps Ulocladium, from
The bacterial strain comprehensive identification that bacterium numbering is F35 is radiation hardness Ulocladium fungi (Ulocladium sp.) by bacterium classification angle
F35 。
Embodiment two: radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006 molecular level
Identification
1. DNA is extracted: fungi extracting method is as follows:
(1) 200mg thallus, liquid nitrogen grinding are added 3% CTAB Extraction buffer of 3ml, 65 DEG C of water-bath 45min, and 4 DEG C
4000r/min is centrifuged 20min.
(2) supernatant is taken to go in centrifuge tube, 4 μ l 10mg/ml protease of addition, 37 DEG C, water-bath 1h.
(3) 800 μ l Tris saturated phenols are added, shake up 13000r/min centrifugation 10min；Take supernatant.
(4) isometric chloroform/isoamyl alcohol is added, shakes up, 13000r/min is centrifuged 10min, takes supernatant.
(5) plus the RNA enzyme of 10mg/ml, 37 DEG C of water-baths are handled overnight.
(6) 800 μ l chloroforms/isoamyl alcohol is added, shakes up, 13000r/min is centrifuged 10min, takes supernatant.
(7) plus 600 μ l isoamyl alcohol, -20 DEG C of precipitating 30min are collected and are precipitated, and 75% alcohol rinses, and super-clean bench vacuum is dry
It is dry.
(8) 100 μ l TE dissolving DNAs, -20 DEG C save backup.The RNA enzyme of 1 μ l is added, 37 DEG C of water-bath 1h are added 400
μ l chloroform/isoamyl alcohol (24: 1), 12000r/min are centrifuged 10min, are repeated 2 times.
2. the amplification of ITS gene and sequencing, are expanded with fungi ITS gene universal primer:
Primer I TS11:5'-TCCGTAGGTGAACCTGCGG-3'
ITS14:5'-TCCTCCGCTTATTGATATGC-3'
PCR amplification reaction system is 50 μ L, reaction condition are as follows: 95 °C, 5min；95 °C, 45s, 57 °C, 30s, 72 °C,
90s, 30cycles, 72 °C, 7min.Amplified production (about 547bp), pcr amplification product are detected with 1% agarose gel electrophoresis,
Amplified production is sequenced, the ITS1 gene order of bacterial strain F35 is measured, after measured, radiation hardness Ulocladium fungi
(Ulocladium sp.) F35 CGMCC No.11006 gene order is 547bp, referring to the gene order table of attached offer
SEQUENCE LISTING。
3. ITS1 sequence alignment and Phylogenetic Analysis
The present invention is sequenced by the extraction of total DNA, the pcr amplification product of ITS1 gene, obtains 547bp sequence
Column, through GenBank Blast homologous sequence compare analysis, the Ulocladium (Ulocladium reported with the prior art
Sp.) homology is higher.Reference culture ITS1 gene order is obtained from GenBank, carries out homologous evolutionary analysis, CLUSTAL
X carries out Multiple Sequence Alignment, and using the adjacent method (Neighbor of Saitou and Nei in 5.0 software of MEGA
Joining the building for) carrying out systematic evolution tree, as a result referring to attached drawing 2.It is from dendrogram as can be seen that provided by the invention resistance to
Radiation Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006 and Common fungi strain have apparent physiology
The otherness of biochemical characteristic difference and molecular level, according to strain Analysis of The Physiological And Biochemical Properties, molecular level analysis and system point
The comprehensive identification of class, strain that number is F35 although in terms of physio-biochemical characteristics and molecular level with common single lattice spore
Belonging to fungi strain has apparent difference, the tolerance, absorption compared with common fungi strain, to heavy metal lead ion
Ability is stronger, but is accredited as Ulocladium fungi (Ulocladium sp.) according to taxology.
Embodiment three: the preparation of radiation hardness Ulocladium fungi Ulocladium sp.F35 CGMCC No.11006 thallus
By activated bacterial strain radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC of the present invention
No.11006 is inoculated in the 500ml triangular flask equipped with 100ml PDA liquid medium, is cultivated in 30 DEG C, 200rpm oscillation training
Support 72h.Thallus is collected in the way of vacuum pump suction filtration, it is rear stand-by three times with sterile water washing.
Example IV: a kind of radiation hardness Ulocladium fungi Ulocladium sp.F35 CGMCC No.11006 is being adsorbed
Application in lead biological treatment
By the strain inoculated in the liquid PDA culture medium without lead ion, after culture 3-5 days, mycelium is collected, is carried out
Thallus adsorption test, research adsorb thallus under the conditions of different plumbum ion concentrations, temperature, pH value, inoculum concentration and adsorption time
Influence.
(1) different plumbum ion concentrations are to radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC
The influence of No.11006 absorption
Each 0.1g of thallus is weighed, being respectively placed in 10ml plumbum ion concentration is 50mg/L, 100mg/L, 250mg/L, 500mg/
L, the Pb of 750mg/L, 1000mg/L2+In solution, after 3 h of standing adsorption, the shadow that different lead solution concentration adsorb thallus is measured
It rings.The results are shown in attached figure 3, with Pb2+The reduction adsorption rate of concentration gradually rises, in Pb2+Adsorption rate highest when reaching 100mg/L,
Up to 66.19%, then decline, therefore when lead solution concentration is 100 mg/L, embodies the thallus to the adsorption capacity of lead ion
It is most strong.
(2) different lead ion initial pH values are to radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC
The influence of No.11006 absorption
Each 0.1g of thallus is weighed, being respectively placed in 10ml plumbum ion concentration is 100mg/L, pH value 3,4,5,6,7,8,9
Pb2+In solution, after standing adsorption 3h, the influence that different pH value adsorb thallus is measured.The results are shown in attached figure 4, pH value bacterial strain at 5
To pb2+Solution adsorption rate highest, up to 70.66%.When pH value raises and reduces, adsorption rate is reduced, therefore, solution ph 5
When, it is most strong to the adsorption capacity of lead ion to embody the thallus.
(3) different thallus dosages are to radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006
The influence of absorption
Weighing the thallus (0.1g, 0.3g, 0.5g, 0.7g, 1.0g) of different quality respectively, to be respectively placed in 10ml lead ion dense
Degree is after 3 h of standing adsorption, to measure influence of the different quality thallus to absorption in the solution of 100mg/L.The results are shown in attached figure 5, bacterium
Body dosage adsorption rate difference in 0.5 g-1.0 g is little, adsorption rate highest when 1.0g, up to 92.19%.But consider thallus dosage
Excessive, higher cost, practical operation is difficult, therefore also higher 0.1g thallus does follow-up test to selection adsorption rate.
(4) different adsorption temps are to radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006
The influence of absorption
Each 0.1g of thallus is weighed, concussion is suspended in the Pb that 10ml plumbum ion concentration is 100mg/L2+In solution, it is respectively placed in
In 20-80 DEG C of water-bath after standing adsorption 3h, the influence that different temperatures adsorbs thallus is measured.The results are shown in attached figure 6, and temperature is at 60 DEG C
When bacterial strain to pb2+Solution adsorption rate highest illustrates that the bacterial strain also has very high absorption under higher temperature conditions up to 81.91%
Ability.
(5) different adsorption times are to radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC No.11006
The influence of absorption
Each 0.1g of thallus is weighed, concussion is suspended in the Pb that 10ml plumbum ion concentration is 100 mg/L respectively2+In solution, every
It is sampled every 5min, the influence that measurement different time adsorbs thallus.The results are shown in attached figure 7, which completes absorption in 10min,
Adsorption rate variation less, tends to be saturated, illustrates the bacterial strain in 10min to the adsorption rate highest of lead ion after 10min.
In summary step, it is known that F35 is to Pb2+Optimal adsorption condition be Pb2+Concentration is 100mg/L, thallus additive amount
It is 60 DEG C for 0.01g/ml, pH=5, temperature, adsorption time 10min.
Embodiment five: infrared spectrum analysis
By F35 in the Pb containing 0mg/L and 100mg/L2+PDA liquid medium culture after, 3 are washed with deionized respectively
Secondary, rear collected by suction thallus, drying is ground, in 10t/cm with KBr2It pushes flakiness and maintains lmin, use infrared spectroscopy
Instrument measures and records its spectrum.Referring to attached drawing 8 it is found that radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC
No.11006 adsorbs Pb2+The infrared spectroscopy variation of front and back is obvious.
By attached drawing 8, attached drawing 9 is shown, radiation hardness Ulocladium fungi (Ulocladium sp.) F35 CGMCC
No.11006 absorption front and back infared spectrum shows apparent difference, illustrates that the structure and function group of absorption front and back thallus occurs
Change.3229cm-1N-H amide or O-H stretching vibration be offset to 3402cm-1Place, and absorption peak broadens, but absorption intensity
Variation is little；2924cm-1And 2855cm-1C-H saturation vibration there is no significantly deviating, but absorption intensity amplification becomes larger；
1742cm-1C=O ester group vibrate also there is no significantly deviating, but absorption intensity significantly increases；1646cm-1C=O amide
Vibration is slightly displaced from, and absorption intensity amplification becomes larger；1554cm-1N-H vibration be offset to 1545cm-1Place, and absorption intensity increases
Amplitude variation is big；1456cm-1And 1385cm-1Primary amide vibration offset less, but absorption intensity amplification becomes larger.1160cm before absorbing-1
To 1031cm-1For primary alconol C-O and OH vibration；1315cm after absorption-1And 1239cm-1For primary alconol O-H vibration, have occurred biggish
Offset, 1077cm-1And 1038cm-1For primary alconol C-O vibration, deviate smaller；The absorption intensity amplification of these absorption peaks becomes simultaneously
Greatly.It is indicated above that these functional groups of cell surface play the role of ligand complex during adsorpting lead ion.
The above embodiment is merely an example for clearly illustrating the present invention, and does not limit the embodiments.
For those of ordinary skill in the art, other various forms of variations can also be made on the basis of the above description
Or it changes.There is no necessity and possibility to exhaust all the enbodiments.And the obvious variation thus extended
Or it changes and is still in the protection scope of this invention.
SEQUENCE LISTING
<110>Microorgan Application Inst., Xinjiang Agricultural Academy
<120>radiation hardness filamentous fungi F35 and its application in Adsorption of Lead biological treatment
<130> 2015
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 547
<212> DNA
<213>single lattice spore fungi (Ulocladium sp.) F35 CGMCC No.11006
<221> ITS1
<400> 1
tacctgcgga gggatcatta cacaatatga aagcgggctg gcatccttcg gggttacagc 60
ctcgctgaat tattcacccg tgtcttttgc gtacttcttg tttccttggt gggttcgccc 120
accataggac aaaccataaa ccttttgtaa ttgcaatcag cgtcagtaaa aaaaattaat 180
aattacaact tttaacaacg gatctcttgg ttctggcatc gatgaagaac gcagcgaaat 240
gcgataagta gtgtgaattg cagaattcgg tgaatcatcg aatctttgaa cgcacattgc 300
gccctttggt attccaaagg gcatgcctgt tcgagcgtca tttgttccct caagctttgc 360
ttggtgttgg gcgtcttgtc tccagttcgc tggagactcg ccttaaagta attggcagcc 420
ggcctactgg tttcggagcg cagcacaagt cgcgctctct tccagccaag gtcagcatcc 480
acaaagcctt ctttcaactt ttgacctcgg atcaggtagg gatacccgct gaacttaagc 540
atatcaa 547
Claims (2)
1. a kind of single lattice spore fungi (Ulocladium sp.) F35 with radiation hardness characteristic, which is characterized in that described has
The deposit number of single lattice spore fungi (Ulocladium sp.) F35 of radiation hardness characteristic is CGMCC No.11006.
2. single lattice spore fungi (Ulocladium sp.) F35 as described in claim 1 with radiation hardness characteristic is raw in Adsorption of Lead
Application in object processing.
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| Utilizaiton of fungi for biotreatment of raw wastewaters;L Coulibaly et al;《African Journal of Biotechnology》;20031231;第2卷(第12期);620-630 * |
| 核辐射污染区真菌多样性及分布特征初步研究;张志东 等;《中国菌物学会2015年学术年会论文摘要集》;20150920;24 * |
| 耐辐射黑色酵母状真菌的筛选和特性研究;张志东 等;《微生物学通报》;20120520;第39卷(第5期);724-731 * |
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