CN103614300A - Acrogenospora sphaerocephala and application thereof - Google Patents
Acrogenospora sphaerocephala and application thereof Download PDFInfo
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Abstract
The invention discloses acrogenospora sphaerocephala. The acrogenospora sphaerocephala HZ-4 is preserved in the China Center for Type Culture Collection, which is located in Wuhan University, on May 26, 2013, and the preservation number is CCTCC NO:M2013230. The acrogenospora sphaerocephala can be used for decomposing synthetic dyes, such as acid blue 40, vital red 11, acid blue 193, acid blue 62, acid blue 113, and acid red 73.
Description
Technical field
The present invention relates to microorganism field, specifically raw spore bacteria strain in a kind of head shoot shape top and uses thereof---decomposing synthetic dye.
Background technology
Many industrial production, as industries such as weaving, leather, makeup, papermaking, printing and dyeing, plastics, all adopt chemical synthetic dye to dye to product.But the raw material of producing synthetic dyestuff is generally poisonous, carcinogenic even explosive chemical; The waste water discharging in the enterprise of above-mentioned use dyestuff contains serious pollutent, need to process.But it is very difficult that these pollutant effluents are carried out to fade treatment, and many dyestuffs are difficult to effective degraded.General physical chemistry treating processes cost is very high, and easily forms secondary pollution, thereby has limited the popularization of these class methods.And by microorganism, the coloured sewage that contains organic dye is carried out to decolored degradation, be that efficiency is high, cost is low and be eco-friendly treatment process.
About utilizing the method research that microorganism or biological enzyme are carried out a biological disposal upon to coloured sewage mainly to concentrate in the research of bacterium and degraded environmental parameter.But, because the kind of bacteriogenic degrading enzyme is limited, therefore high to the specificity requirement of substrate, and after dyestuff degraded, go back the easily new toxic substance of generation, greatly limited the application of some method.In recent years, research finds that some fungi also has the ability of degradating organic dye, as some kinds of white-rot fungi, aspergillus, head mold, very high to the absorption of some dyestuff, degradation and decolorization efficiency.Due to the generation complicated condition of coloured sewage, the Sewage Environment factor complexity that contains dyestuff is various, need to research and develop new fungi and carry out coloured sewage disposal.
Aquatic fungi is organic main initial decomposer in water body environment, plays very important effect in the material cycle of the water body environment ecosystem.The important feature of this class fungi can produce in various kinds of cell and/or extracellular nonspecific degrading enzyme exactly, to complicated and diversified in water body environment, has pollution machine thing to carry out effectively decomposing and utilizing.Therefore, aquatic fungi is the important resource microorganism of disposing of sewage, but at present research also seldom, need further screening to dyestuff, have the fungi of Degradation, define the scope that it processes dyestuff.
The fungi that the shape raw spore bacterium in top (Acrogenospora) belongs to is saprophytic microorganism, at present it is not also being studied to report aspect biological degradation dyestuff.
Summary of the invention
The technical problem to be solved in the present invention is to provide the raw spore bacterium in a kind of head shoot shape top, this bacterial strain synthetic dyestuff of degrading.
In order to solve the problems of the technologies described above, the invention provides the raw spore bacteria strain in a kind of head shoot shape top: preservation name is called: Acrogenospora sphaerocephala HZ-4, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University; Preservation date: May 26 in 2013, preserving number: CCTCC NO:M2013230.
Head shoot shape of the present invention top raw spore bacteria strain HZ-4(is Acrogenospora sphaerocephala HZ-4CCTCC NO:M2013230) there is following characteristic: on PDA, bacterium colony grey black, poor growth, aerial hyphae is flourishing.Can be observed under the microscope mycelia beige to light brown, smooth, have barrier film.Hyphal diameter 2 – 4 μ m.Conidiophore Vandyke brown, thick, Dan Sheng, branch not, upright or geniculation, smooth surface, centre often has and expands, and has barrier film, cylindric, base portion chocolate, middle Vandyke brown, top look shallow.100-750 * 4.2-7.5 μ m; Conidiogenous cell simple bud is raw, and limited growth is cylindrical, light brown, Xiang Sheng.Conidium top is raw, full wall blastogenesis, and subsphaeroidal, without barrier film, light brown to Vandyke brown, heavy wall, produces spore continuously, the separated 17-30 * 15.5-30 μ of spore breaking type m, spore base portion 3.6-5.4 μ m.
The present invention also provides the purposes of the raw spore bacteria strain in above-mentioned head shoot shape top simultaneously, degraded synthetic dyestuff, and synthetic dyestuff comprise Acid Blue 40, reactive red 11, acid blue 193, Acid blue 62, Acid blue 113 and Xylene Red 73.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is the form of HZ-4 bacterial strain: wherein 1 for conidium on conidiophore raw state; 2 is conidiophore and immature conidium; 3 is the configuration of conidiophore; 4-6 is conidium form.
Fig. 2 is that HZ-4 bacterial strain is cultivated the form of 10 days on PDA substratum: the back side feature that wherein A is culture dish; B is the positive feature of culture dish.
Fig. 3 is that HZ-4 bacterial strain is cultivated 5 days degraded situations to 6 kinds of dyestuffs on MEA solid medium.Can obviously observe color at periphery of bacterial colonies dyestuff obviously shoal (arrow indication position).Wherein dyestuff respectively: A is Acid blue 113; B is reactive red 11; C is Acid Blue 40; D is Acid blue 62; E is acid blue 193; F is Xylene Red 73.
Fig. 4 is that HZ-4 bacterial strain is to the percent of decolourization of 6 kinds of dyestuffs and degradation rate comparison diagram.
Embodiment
The separation of embodiment 1, bacterial strain HZ-4:
Potato dextrose agar (potato dextrose agar, PDA): adding distil water is settled to 1000mL in potato 200g, glucose 20g, agar 20g; Then carry out conventional high-temperature sterilization (1.1 normal atmosphere, 121 ℃ at sterilizing 20min).
2% water agar (WA) substratum: agar powder 20g, water 1000mL, carry out conventional high-temperature sterilization (1.1 normal atmosphere, 121 ℃ at sterilizing 20min).
Paraxin storing solution: 340mg paraxin is dissolved in the dehydrated alcohol of 10mL standby.
Streptomycin sulphate storing solution: 100mg Streptomycin sulphate is dissolved in the aqua sterilisa of 10mL standby.
In laboratory, by following condition separation and Culture, can obtain the raw spore bacterium in head shoot shape of the present invention top.
On the corruption wood in HZ-4 bacterial strain Shi Cong China's Zhejiang Hangzhou streams (80 ° of 12'13.68 ' ' of east longitude, 120 ° of 7'11.28 ' ' of north latitude), separation obtains.The corruption wood collection being immersed in for a long time in streams is taken back to laboratory, clean with tap water rinsing, be placed in the plastics bag of using the absorbent cotton moisturizing of soaking water, 3 months (culture temperature is 20～30 ℃) cultivated in sealing.Every 10 days, check 1 time, find that there is thalline and produce after spore, with the tip pincet spore that takes a morsel, be placed in 1.5 milliliters of centrifuge tubes that contain 0.5 ml sterile water, with pipettor, blow and beat up and down several times, make it to be uniformly dispersed.Get 100 μ L spore liquid and drop on blood counting chamber, under microscope, count.According to count results, in original spore liquid, add appropriate sterilized water, be made into the spore liquid that concentration is 100 spore/mL.After 2% water agar (WA) substratum of 100mL is melted, room temperature is cooled to 50 ℃, then adds paraxin storing solution and each 100mL of Streptomycin sulphate storing solution, after mixing, is down flat plate.Get spore liquid that 10 microlitres mix up concentration and drop in that above-mentioned every ware drips 20 containing on paraxin and Streptomycin sulphate water agar (WA) plate, drip 5 culture dish.25 ℃ of dark cultivations after 2 days, the culture dish back side is upwards placed under 40 power microscopes and observes and drip the position that has spore liquid, by confirming as monospore, sprout the position marking pen mark that forms mycelia, then on aseptic operating platform, move to receive on PDA substratum and cultivate 7 days, obtain pure growth.
This bacterium, for the raw spore bacteria strain in head shoot shape top, has carried out following preservation: preservation name is called: Acrogenospora sphaerocephala HZ-4, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University; Preservation date: May 26 in 2013, preserving number: CCTCC NO:M2013230.
The evaluation of embodiment 2, bacterial strain HZ-4:
2.1 identification of morphology: the HZ-4 bacterial strain of purifying is transferred on PDA substratum, cultivates 10 days for 25 ℃.With choosing a small amount of thalline of pin picking, make slide, examine under a microscope, measure, take pictures.Its morphological specificity is:
On PDA, bacterium colony grey black, poor growth, aerial hyphae is flourishing.Beige, to light brown, smooth, has barrier film.Hyphal diameter 2 – 4 μ m.Conidiophore Vandyke brown, thick, Dan Sheng, branch not, upright or geniculation, smooth surface, centre often has and expands, and has barrier film, cylindric, base portion chocolate, middle Vandyke brown, top look shallow.100-750 * 4.2-7.5 μ m; Conidiogenous cell simple bud is raw, and limited growth is cylindrical, light brown, Xiang Sheng.Conidium top is raw, full wall blastogenesis, and subsphaeroidal, without barrier film, light brown to Vandyke brown, heavy wall, produces spore continuously, the separated 17-30 * 15.5-30 μ of spore breaking type m, spore base portion 3.6-5.4 μ m.As shown in Figure 1, colony growth state as shown in Figure 2 for conidium and conidiophore form.
2.2 Molecular Identification:
2.2.1 a small amount of of fungal genomic DNA is extracted
2.2.1.1 Extraction buffer: contain 1M KCl, 100mM Tris-HCl, 10mM EDTA, pH=8.0.
2.2.1.2 extracting method:
(1) HZ-4 bacterial strain was grown after 14 days on PDA flat board, with the mycelia on toothpick scraping flat board, put into the sterilized 500 μ L Extraction buffer 1.5mL centrifuge tubes that contain;
(2) with electrical grinding machine, the mycelia of picking is smashed to pieces to the centrifugal 10min of 9000rpm;
(3) draw supernatant to another centrifuge tube, abandon precipitation;
(4) add isopyknic Virahol (analytical pure), precipitate nucleic acids, puts upside down gently and mixes after several, the centrifugal 10min of 12000rpm;
(5) remove gently supernatant liquor, on thieving paper, drain moisture;
(6) add 800 μ L70% ethanol, put upside down gently and mix after several, the centrifugal 2min of 12000rpm;
(7) remove gently supernatant liquor, on thieving paper, drain moisture, be placed in 15min at 37 ℃, ethanol is fully volatilized;
(8) by the resuspended precipitation of 50 μ L ddH2O, obtain HZ-4 genomic dna, concentration reaches 30ng/ μ L.
2.2.2. the pcr amplification of fungi 18s rDNA gene
Adopt 50 μ L reaction systems to carry out pcr amplification, in system, contain: each 2 μ M of upstream and downstream primer, dNTPs200 μ M, Mg
2+1.5mM, 10 * PCR buffer5 μ L, masterplate DNA2 μ L, Taq enzyme 2U.
Upstream primer NS1 sequence is 5 '-GTAGTCATATGCTTGTCTC-3 ',
Downstream primer NS4 sequence is 5 '-CTTCCGTCAATTCCTTTAAG-3 '.
Pcr amplification reaction carries out on bright base MG96G type PCR instrument.Reaction conditions: 94 ℃ of denaturation 2min, then 35 circulations comprise: 94 ℃ of sex change 30sec, 57 ℃ of annealing 40sec, 72 ℃ are extended 1min.After last 72 ℃, extend 10min.
2.2.2.3PCR the recovery purifying of product:
After PCR reaction finishes, PCR product is after 1% agarose gel electrophoresis detection, and the DNA gel purification kit that employing love is pursued progress biotech company, carries out according to the step of test kit specification sheets.
Step is as follows:
(1) after electrophoresis finishes, under ultraviolet lamp, with blade, cut the gel containing target DNA fragment, be placed in 2mL centrifuge tube, weigh;
(2) add the DE-A damping fluid of 3 times of volumes, 75 ℃ of insulation 10min, during vibration for several times, to melting completely.
(3) add the DE-B damping fluid of 0.5 times of volume, mix.
(4) prepared by DNA to pipe and put into 2mL centrifuge tube, mixed solution is transferred to DNA and prepare in pipe, the centrifugal 1min of 12000rpm, abandons supernatant.
(5) prepared by DNA to pipe and put back in 2mL centrifuge tube, add 500 μ L damping fluid W1, the centrifugal 30s of 12000rpm.
(6) prepared by DNA to pipe and put back in 2mL centrifuge tube, add 700 μ L damping fluid W2, the centrifugal 30s of 12000rpm.
(7) repeating step (6) once.
(8) prepared by DNA to pipe and put back in 2mL centrifuge tube, the centrifugal 2min of 12000rpm.To drain washings on film;
(9) prepared by DNA to pipe and put back in 2mL centrifuge tube, add the ddH of 50 μ L
2o, the centrifugal 1min of 10000rpm, eluted dna is put at-20 ℃ and is preserved.
2.2.2.4 the order-checking of gene and sequential analysis
The target DNA fragment that purifying is reclaimed is delivered to the raw work in Shanghai and is checked order with ABIPRISMA377 type automatic sequencer after electrophoresis detection.Sequencing result, after strictly checking, obtains the sequence dna fragment of 981bp in sequence table.
The nucleotide sequence recording is searched in GenBank with BLAST and compared homology or similar nucleotide sequence.According to the database annotation of homologous sequence, then in conjunction with the morphological structure of bacterial strain, judge the kind of studied bacterial strain.Through BLAST comparison, this sequence and accession number are the sequential covering rate 100% of GU296129 and AY541482, and similarity reaches 99%.These two sequences are all that it has form is Farlowiella carmichaeliana from the 18s rDNA of the head shoot shape raw spore bacterium in top (Acrogenospora sphaerocephala).These results show that Molecular Identification conforms to identification of morphology result.
3.1 required substratum and dyestuffs:
2% wort agar substratum (malt extract agar, MEA): leach adding distil water in powder 20g, agar 20g at Fructus Hordei Germinatus and be settled to 1000mL; Then carry out conventional high-temperature sterilization (1.1 normal atmosphere, 121 ℃ at sterilizing 20min).
11 kinds of dyestuffs to be measured are dissolved in the water separately, are made into the storing solution of 100mg/mL, filter (aperture of filtering membrane is 0.22 μ m) degerming, obtain filtration sterilization dyestuff (11 kinds).
Contain dye solids substratum: the filtration sterilization dyestuff that adds 50 μ L in the 2% wort agar substratum (MEA substratum) of sterilized 100mL.
Contain dye liquid substratum: Fructus Hordei Germinatus leaches powder 20g adding distil water and is settled to 1000mL, is divided in triangular flask every bottle of 100mL.Then carry out conventional high-temperature sterilization (1.1 normal atmosphere, 121 ℃ at sterilizing 20min).In sterilized 100mL malt extract medium, add the filtration sterilization dyestuff of 50 μ L.
For trial dyeing material, there are 11 kinds: Acid Blue 40, reactive red 11, acid blue 193, Acid blue 62, Acid blue 113, Reactive Blue 19 100 4, Xylene Red 73, Reactive Red 24, reactive yellow 2, REACTIVE YELLOW 18, Reactive blue 74.
3.2, actication of culture: the HZ-4 inoculation of purification storage, to solid PDA culture medium flat plate, is cultivated 10 days for 28 ℃.
3.3, bacterial classification decolored degradation ability, the detection of the synthetic dyestuff ability of degrading
3.3.1 on solid plate, screen degradable kind of dyes:
With the punch tool of diameter 1cm, from the punching of activation strain cultures, be inoculated into above-mentioned containing on the culture medium flat plate of dyestuff, in 28 ℃ of incubators, cultivate 5d.. found that in the bottom of bacterium colony and/or edge occur significantly the fading dyestuff of ring has 6 kinds, is respectively Acid Blue 40, reactive red 11, acid blue 193, Acid blue 62, Acid blue 113 and Xylene Red 73.As shown in Figure 3.
Above-mentioned 6 kinds of dyestuffs are proceeded to following detection.
3.3.2 in liquid nutrient medium, detect the Degradation of HZ-4 bacterial strain to dyestuff:
In order to determine the maximum absorption wavelength of each dyestuff, first we scan within the scope of 300nm-900nm adding the liquid nutrient medium of dyestuff, determines respectively the maximum absorption wavelength of every kind of dyestuff.Then by HZ-4 inoculation in the liquid nutrient medium that contains dyestuff, measure the degraded situation of bacterial strain to every kind of dyestuff.
By HZ-4 inoculation, in the 500mL triangular flask of in-built 300mL malt extract medium, under 28 ℃ of conditions, 150rpm shaking table is cultivated 5d.Then draw 1mL culture (being about 100 bacterium colony/ml) and be inoculated in respectively in the triangular flask containing dye liquid substratum 50mL, 28 ℃ of shaking culture 3d.The centrifugal 10min of 4800rpm gets supernatant subsequently, for detect HZ-4 bacterial strain to various dyestuffs the removal situation in liquid, we measure bacterium liquid OD value in the maximum absorption wave strong point of dyestuff, calculate dye decolored rate.
Percent of decolourization=(A-B)/A * 100% (1)
In formula, A-represents to inoculate the OD value of front substratum;
The OD value of nutrient solution after B-represents to decolour.
In order to detect HZ-4 bacterial strain, to various dyestuffs, the removal effect in liquid is due to by dyestuff degraded or due to the adsorption of thalline to dyestuff, we have measured the degradation rate of HZ-4 bacterial strain to every kind of dyestuff: with ethanol extraction process, the dyestuff being adsorbed is eluted to (substratum: ethanol extract=1:1) by thalline, same centrifuging and taking supernatant, take and do not add the substratum that dyestuff only adds ethanol and be blank, take dyestuff substratum as contrast, in the maximum absorption wave strong point of each dyestuff with Lambda45 type spectrophotometric determination by the OD value of the dyestuff nutrient solution under wash-out, calculate dyestuff degradation rate, the degradation capability that represents bacterial strain with degraded:
Degradation rate=(C-D)/C * 100% (2)
In formula, C-represents to add the OD value of the not inoculation medium of equal-volume ethanol;
The OD value of the nutrient solution after ethanol elution for D-.
Table 1 can be by maximum absorption wavelength, percent of decolourization and the degradation rate of 6 kinds of dyestuffs of HZ-4 strains for degrading
Result table 1, as shown in Figure 4, processes three days 6 kinds of dyestuffs by HZ-4 bacterial strain, and in substratum, the removal effect of dyestuff is obvious.HZ-4 bacterial strain is the strongest to the decolorization of reactive red 11, has reached more than 94%.Decolorization to acid blue 193 reaches 33.64%.Decolorization to other 4 kinds of dyestuffs has all reached more than 50%.And the mensuration of degradation rate by the dyestuff to after processing, HZ-4 bacterial strain has all reached 100% to the degradation rate of 5 kinds of dyestuffs, the degradation rate of Xylene Red 73 has also been reached to 62.56%, illustrate that HZ-4 bacterial strain is that biological degradation is removed 6 kinds of dyestuffs, has good effect with HZ-4 bacterial strain to the sewage that contains above 6 kinds of dyestuffs.
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
<110> Zhejiang University
Raw spore bacterium in <120> head shoot shape top and uses thereof
<160> 1
<210> 1
<211> 981
<212> DNA
The raw spore bacterium (Acrogenospora sphaerocephala) in <213> head shoot shape top
<400> 1
gcgaatggct cattaaatca gttatcgttt atttgatagt accttactac ttggataacc 60
gtggtaattc tagagctaat acatgctaaa aaccccgact tcgggagggg tgtatttatt 120
agataaaaaa ccaacgccct tcggggctcc ttggtgattc ataataacta aacgaatcgc 180
atggccttgc gccggcgatg gttcattcaa atttctgccc tatcaacttt cgatggtagg 240
atagtggcct accatggtat caacgggtaa cggggaatta gggttctatt ccggagaggg 300
agcctgagaa acggctacca catccaagga aggcagcagg cgcgcaaatt acccaatccc 360
gacacgggga ggtagtgaca ataaatactg atacagggct cttacgggtc ttgtaattgg 420
aatgagtaca atttaaatcc cttaacgagg aacaattgga gggcaagtct ggtgccagca 480
gccgcggtaa ttccagctcc aatagcgtat attaaagttg ttgcagttaa aaagctcgta 540
gttgaacctt gggcctggct ggccggtccg cctcaccgcg tgcactggtc cggccgggcc 600
tttccttctg gggatccgca tgccctttac tgggtgtgtc ggggaaccag gacttttact 660
ttgaaaaaat tagagtgttc aaagcaggcc ttcgctcgaa tacattagca tggaataata 720
gaataggacg tgcggttcta ttttgttggt ttctaggacc gccgtaatga ttaataggga 780
tagtcggggg catcagtatt caattgtcag aggtgaaatt cttggattta ttgaagacta 840
actactgcga aagcatttgc caaggatgtt ttcattaatc agtgaacgaa agttagggga 900
tcgaagacga tcagataccg tcgtagtctt aaccataaac tatgccgact agggatcggg 960
cgatgttact attctgactc g 981
Claims (3)
1. the raw spore bacterium in head shoot shape top, is characterized in that: preservation name is called: Acrogenospora sphaerocephala HZ-4, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University; Preservation date: May 26 in 2013, preserving number: CCTCC NO:M2013230.
2. the purposes of the raw spore bacterium in head shoot shape as claimed in claim 1 top, is characterized in that: degraded synthetic dyestuff.
3. the purposes of the raw spore bacterium in head shoot shape according to claim 2 top, is characterized in that: described synthetic dyestuff are at least one in Acid Blue 40, reactive red 11, acid blue 193, Acid blue 62, Acid blue 113 and Xylene Red 73.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1621513A (en) * | 2003-11-28 | 2005-06-01 | 广东省微生物研究所 | Shewanella decolorationis |
| CN1800056A (en) * | 2005-12-29 | 2006-07-12 | 苏州大学 | Dye decolored degradation treatment method for dye-containing waste water |
| CN101851586A (en) * | 2010-05-11 | 2010-10-06 | 林永慧 | Fungus for efficiently decomposing synthetic dye and application thereof |
| CN101948758A (en) * | 2010-08-10 | 2011-01-19 | 南京大学 | Fungus capable of degrading multiple types of dyes under non-sterilization condition and application thereof |
| CN102618462A (en) * | 2012-03-20 | 2012-08-01 | 江南大学 | Rhodococcus and method for degrading and decoloring triphenylmethane dyes by utilizing rhodococcus |
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2013
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1621513A (en) * | 2003-11-28 | 2005-06-01 | 广东省微生物研究所 | Shewanella decolorationis |
| CN1800056A (en) * | 2005-12-29 | 2006-07-12 | 苏州大学 | Dye decolored degradation treatment method for dye-containing waste water |
| CN101851586A (en) * | 2010-05-11 | 2010-10-06 | 林永慧 | Fungus for efficiently decomposing synthetic dye and application thereof |
| CN101948758A (en) * | 2010-08-10 | 2011-01-19 | 南京大学 | Fungus capable of degrading multiple types of dyes under non-sterilization condition and application thereof |
| CN102618462A (en) * | 2012-03-20 | 2012-08-01 | 江南大学 | Rhodococcus and method for degrading and decoloring triphenylmethane dyes by utilizing rhodococcus |
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