CN102286410B - Streptomyces yanii - Google Patents
Streptomyces yanii Download PDFInfo
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- CN102286410B CN102286410B CN 201110231780 CN201110231780A CN102286410B CN 102286410 B CN102286410 B CN 102286410B CN 201110231780 CN201110231780 CN 201110231780 CN 201110231780 A CN201110231780 A CN 201110231780A CN 102286410 B CN102286410 B CN 102286410B
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- phosphorus
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- yima116
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Abstract
The invention relates to the technical field of microorganisms and particularly relates to a microbial strain having high phosphate-accumulating capability. The Streptomyces yanii provided by the invention belongs to Streptomyces, is named Streptomyces yanii YIMA116 and is preserved in a preservation unit approved by the state intellectual property office; and the preservation number is CCTCC NO:M2011188. The analysis based on the PCR (Polymerase Chain Reaction) technology and a DNA sequencer shows that the 16SrDNA of the YIMA116 strain comprises 1411 basic groups. After the YIMA116 strain is cultured in a modified ISP2 (International Streptomyces Project Medium2) liquid culture medium under optimized aerobic conditions for 7 days, the soluble phosphate concentration of the supernate in the culture medium is reduced from 208mg/L to 15.8mg/L, and the phosphate removal rate is up to 92.4%.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to a kind of stronger microorganism strains that gathers the phosphorus ability that has.
Background technology
PolyP bacteria is a kind of of mikrobe to be defined saprobiont dephosphorization research from project angle, and the one type of heterotroph bacterium that causes anaerobic phosphorus release, aerobic excess to inhale phosphorus the aerobic/anaerobic alternate run is called polyP bacteria.Early stage investigator thinks that polyP bacteria belongs to acinetobacter, but the later stage investigator thinks and belongs to moraxella, Rhodopseudomonas, Aeromonas etc.These Biochemical processes of phosphorus define because polyP bacteria just gathers according to excess, also should start with from the microbiological property of bacterium and study so will really define polyP bacteria.
PolyP bacteria also is called takes the photograph the phosphorus bacterium, is meant in the body to store the general name of gathering phosphorus and gathering a bacterioid of beta-hydroxy-butanoic acid.It is generally acknowledged bacterial classifications such as mainly containing acinetobacter calcoaceticus, Rhodopseudomonas.PolyP bacteria is one type of special facultative bacteria in the conventional activated sludge process; Ability excess ground is with in the phosphorus suction body in the sewage under aerobic or anoxic condition; Make intravital phosphorus content surpass the several times of the intravital phosphorus content of general bacterium, biological phosphate-eliminating mainly is the phosphorus effect that gathers that utilizes polyP bacteria.
Summary of the invention
The object of the present invention is to provide a kind ofly from grass cogongrass rhizosphere soil, separate having of obtaining and efficiently gather the active soil actinomycete of phosphorus---gather phosphorus actinomycetes Yan Shi streptomycete.
The phosphorus actinomycetes Yan Shi strepto-fungus strain that gathers of the present invention separates from grass cogongrass rhizosphere soil and obtains.Depositary institution's preservation of State Intellectual Property Office's approval now, depositary institution's title: Chinese typical culture collection center, be called for short: CCTCC, depositary institution address: China, Wuhan University, postcode: 430072.Preservation date is on June 1st, 2011, and deposit number is CCTCC NO:M2011188.
Of the present inventionly gather the mould Pseudomonas of phosphorus actinomycetes Yan Shi streptomycete tethers, called after Yan Shi streptomycete YIM A116 (
Streptomyces yaniiYIM A116), be deposited in the depositary institution of State Intellectual Property Office's approval, deposit number is CCTCC NO:M2011188 at present.
YIM A116 bacterial strain of the present invention is through round pcr, and the dna sequencing appearance is analyzed, and the 16S rDNA sequence of this bacterial strain is by 1411 based compositions.With blast program listed 16S rDNA sequence among the 16S rDNA sequence of YIM A116 and the GenBank is carried out nucleotide homology relatively, result and the Yan Shi streptomycete of having reported
Streptomyces yaniiThe 16S rDNA sequence homology of NBRC 14669T (accession number AB006159) reaches 100%, identifies that it is the Yan Shi streptomycete
Streptomyces yaniiMade up the phylogenetic evolution tree of YIM A116 bacterial strain.
YIM A116 bacterial classification is described:
Aerobic growth, Gram-positive produces a large amount of branched substrate myceliums and aerial hyphae.The most advanced and sophisticated long single spore that has of the partly short branch of substrate mycelium.The long fibrillae of spores that short straight shape spore chain, bending spore chain and multi-branched are arranged of aerial hyphae, the spore ovalize.On the ISP2 substratum, cultivate the substrate mycelium that can grow living spore of a large amount of gray gas and grey to black, non-pigment produces.
YIM A116 solid culture characteristic:
The ISP2 substratum was cultivated 24 hours for 28 ℃, grew a small amount of white to milk yellow small colonies, and smooth surface is protruding slightly, neat in edge, colony edge substratum slight depression; Colony diameter increased in 2 days, all became milk yellow, and a small amount of bacterium colony begins to produce white aerial hyphae; 3 days bacterium colony raised growths, large stretch of white aerial hyphae becomes grey, and newborn bacterium colony produces thin white aerial hyphae, and colony diameter is not from waiting less than 1mm to 2mm, and bacterium colony is protruding, and edge agar caves in obviously, and substrate mycelium is a milk yellow; Most of bacterium colony aerial hyphae became grey in 5 days, and substrate mycelium is a grey black, and a small amount of single bacterium colony aerial hyphae is a white, and colony edge is incised, and substrate mycelium is a milk yellow; 9 days whole bacterium colonies all generate the aerial hyphae and the substrate mycelium of grey black, and non-pigment produces.
The microscopic morphology characteristic of YIM A116 bacterial strain:
Produce a large amount of branched substrate myceliums and aerial hyphae.The most advanced and sophisticated long single spore that has of the partly short branch of substrate mycelium.The long fibrillae of spores that short straight shape spore chain, bending spore chain and multi-branched are arranged of aerial hyphae, spore ovalize, 0.8～1 * 1～1.5 μ m.
YIM A116 strain liquid fermentation culture characteristic:
1. ISP2 substratum, shake-flask culture 5～7 days.28 ℃ of culture temperature.
2. fermentation culture characteristic: cultivated the 1st day, it is muddy that substratum becomes slightly; Cultivated the 2nd day, it is muddy that substratum further becomes; Cultivated the 3rd day, the fermented liquid mycelium is intensive, and fermented liquid is pale brown look, has a small amount of little mycelium pellet to produce; Cultivated the 4th day, and covered with pale brown look mycelium, fermented liquid is thickness slightly, cultivates the 5th～7 day, and mycelial growth is stable, and fermented liquid is fine and close pale brown look, and visible a small amount of pale brown look mycelium pellet produces.
Cogongrass soil actinomycete YIM A116 bacterial strain adopts liquid state fermentation, its flow process of fermenting:
Bacterial classification → test tube slant → seed culture → fermentation culture → medium centrifugal → supernatant → titanium pigment is measured.
The fermentation condition parameter is following:
(1) fermentation mode: liquid state fermentation.
(2) strain inclined plane: the test tube slant spawn culture adopts the ISP2 substratum.
(3) seed culture: seed culture is the ISP2 liquid nutrient medium.
The preparation of ISP2 liquid nutrient medium: weigh malt extract (Malt extract) 10g, yeast extract (Yeast extract) 4g, dextrose anhydrous (Glucose) 4g is dissolved in 1L water, is adjusted to pH 7.2 ± 0.2,121 ℃ of autoclaving 30min.
Add agar 15g in the above-mentioned ISP2 substratum, be solid ISP2 substratum.
The ISP2 medium preparation of improvement: weigh malt extract (Malt extract) 10g, yeast extract (Yeast extract) 4g, dextrose anhydrous (Glucose) 7g, KH
2PO
40.5712g be dissolved in 1L water, be adjusted to pH 7.2 ± 0.2,121 ℃ of autoclaving 30min.
(4) fermentation culture: the ISP2 substratum of improvement.
(5) incubation time: test tube slant strain activation and culture 5 days, seed shaking table incubation time 3～4 days, fermented incubation time 5～7 days.
(6) culture temperature: 28 ℃ ± 1 ℃ of test tube slant spawn culture temperature, 28 ℃ ± 1 ℃ of seed shaking table culture temperature, fermentation culture temperature are 28 ℃ ± 1 ℃.
(7) mensuration of titanium pigment: fermentation finishes, and collects fermented liquid, through centrifugal, obtains supernatant.According to the measuring method of titanium pigment, adopt the molybdenum antimony resistance colorimetric method of improveing (GB 7852-87) to measure the content of titanium pigment in the culture supernatant liquid.
Under the aerobic condition of the present invention; Cultivate after 7 days in ISP2 (the International Streptomyces Project Medium 2) liquid nutrient medium of improvement; Medium supernatant titanium pigment concentration drops to 15.8mg/L by 208mg/ L, and tp removal rate reaches 92.4%.YIM A116 bacterial strain has the stronger phosphorus ability of gathering.The present invention is raw material with ISP2, adopts the pure culture mode, through cogongrass rhizosphere soil actinomycete YIM A116 liquid state fermentation, according to the measuring method of titanium pigment, adopts molybdenum antimony resistance colorimetric method to measure the content of titanium pigment in the culture supernatant liquid.YIM A116 has and efficiently gathers the phosphorus ability, is one type of important microbe of administering the phosphorus contaminate environment.
Mostly the polyP bacteria strain of in the past being reported is to separate in the active sludge of Sewage treatment systems and obtains; At the substratum phosphorus concentration is under 10mg/L or the lower concentration; Gathering the phosphorus rate does not wait at 50%-93% mostly; Mostly bacterial strain is bacterium, gathers the sequence batch (reaction (SBR technology) mostly phosphorus technology is that aerobic/anaerobism hockets.
YIM A116 strains separation involved in the present invention is herbage cogongrass rhizosphere soil from rich phosphorus mountain region, is that a strain has and efficiently gathers the active actinomycetes of phosphorus, and it gathers the phosphorus characteristics and is:
1, gathers the bacterial strain that the phosphorus amount is more than before and reports, can optimize gather in the phosphorus substratum (208mg/L phosphorus concentration) at 100mL and gather phosphorus in a large number, make phosphorus concentration reduce to 15.8mg/L, gather the phosphorus rate and reach 92.4%, gather the phosphorus amount only and reach 19.22mg;
2, under simple aerobic condition, can gather phosphorus in a large number, need not complicated loaded down with trivial details aerobic/anaerobic sequencing batch technology;
3, have that good to gather phosphorus long-lasting, cultured continuously is 28 days in the high phosphorus substratum, can in substratum, not emit phosphorus again, can gather the phosphorus rate by long-acting maintenance height.
Description of drawings
Fig. 1 is YIM A116 cogongrass rhizosphere soil actinomycete of the present invention cultural characteristic (front) on the ISP2 substratum.
Fig. 2 is YIM A116 cogongrass rhizosphere soil actinomycete of the present invention cultural characteristic (reverse side) on the ISP2 substratum.
Fig. 3 is a YIM A116 cogongrass rhizosphere soil actinomycete mycelium of the present invention (opticmicroscope 1000X).
Fig. 4 is a YIM A116 cogongrass rhizosphere soil actinomycete fibrillae of spores of the present invention (opticmicroscope 1000X).
Fig. 5 be YIM A116 cogongrass rhizosphere soil actinomycete mycelium of the present invention (ESEM 9113X, 6800X).
Fig. 6 be YIM A116 cogongrass rhizosphere soil actinomycete fibrillae of spores of the present invention (ESEM 9113X, 6800X).
Fig. 7 is a YIM A116 titanium pigment canonical plotting of the present invention.
Fig. 8 is the phylogenetic evolution tree of YIM A116 bacterial strain of the present invention.
Embodiment
Embodiment:
Get numbering YIM A116 bacterial classification, under aseptic condition (sterilisable chamber or Bechtop),, transfer and (in 15 * 150mm), put the interior 28 ℃ of activation culture of incubator 5 days in sterilized solid ISP2 substratum test tube with a little bacterial classification of sterilization bamboo let picking.
Take out 5 days bacterial classification of activation, under aseptic condition, insert the good bacterial classification of activation in sterilized seed ISP2 liquid nutrient medium, cultivated 3～4 days, be YIM A116 strain fermentation seed at 28 ℃ of following shaking tables with the same manner.
250ml glass triangle bottle is adopted in fermentation, the ISP2 liquid nutrient medium 100ml of the improvement of packing into, and behind 121 ℃ of sterilization 30min, taking-up cooling back is placed 28 ℃ of incubators and is spent the night, and no living contaminants can confirm that sterilization is thorough, can be used for follow-up fermentation culture.Under aseptic condition, get YIM A116 seed, be inoculated in the 250ml glass triangle bottle that 100ml improvement ISP2 liquid nutrient medium is housed, inoculum size is 5～10% (v/w), puts 28 ℃ of aerobic culture 5～7 days.During fermentation, note the proving room temperature variation, inspection has or not the microbiological contamination phenomenon.
Fermentation finishes, and collects fermented supernatant fluid.Join fermented liquid in the 5ml centrifuge tube, 12000rpm, centrifugal 3min transfers to supernatant in the clean centrifuge tube, measures titanium pigment content in the supernatant.
The mensuration of titanium pigment:, adopt molybdenum antimony resistance colorimetric method to measure the content of titanium pigment in the culture supernatant liquid according to the measuring method of titanium pigment.
The preparation of reagent:
Molybdenum antimony stock solution: the 153ml vitriol oil (analytical pure, density 1.84 g/ml), pour into lentamente in about 400ml ultrapure water, stir, cooling, other gets 10g ammonium molybdate ((NH
4)
6Mo
70
24.4H
20, analytical pure), be dissolved in about 60 ℃ 300m1 water cooling.Then sulphuric acid soln slowly in the ammonium molybdate solution of falling people, is added 100ml 0.5% antimonypotassium tartrate (KSbOC again
4H
40
6H
20, analytical pure) solution, be diluted to 1L with ultrapure water at last, lucifuge is stored.This stock solution contains [1% ammonium molybdate, 2.75mol/L sulfuric acid].
The anti-developer of molybdenum antimony: 1.50g xitix (C
6H
80
6, left-handed, specific rotation+21 °～+ 22 °, analytical pure) be dissolved in the 100ml molybdenum antimony stock solution.This liquid must be with joining with usefulness.
5mg/L phosphorus (P) standardized solution: 0.4394g is at the potassium primary phosphate (KH of 50 ℃ of oven dry
2PO
4, analytical pure), add 100ml water, add the 5m1 vitriol oil (anticorrosion), to 1L, concentration is 100mg/L phosphorus (P) with the ultrapure water constant volume, this solution can prolonged preservation.Draw above-mentioned solution 10ml in the 200m1 volumetric flask, add water to scale, concentration is 5mg/L phosphorus (P) standardized solution, and this solution is unsuitable stored for a long time.
Measuring method:
1. join fermented liquid in the 5ml centrifuge tube, 12000rpm, centrifugal 3min transfers to supernatant in the clean centrifuge tube.
2. earlier be diluted to 25～50mg/L to the phosphorus concentration in the fermented liquid.
3. draw 0.5～1ml liquid to be measured in the 50ml volumetric flask, add water to 15～20ml, add the anti-developer of 5m1 molybdenum antimony with suction pipe, the water constant volume shakes up to scale.Behind the 30min, on spectrophotometer,, be reference liquid, transfer absorption value, measure the absorption value (OD value) of colour developing liquid to be measured then to zero with blank test solution with 2cm optical path cuvette (, the cuvette of application 1 cm higher), 700nm wavelength colorimetric like phosphorus content.On standard lines, find the phosphorus mg/L number of colour developing liquid, it is stable that color can keep in 8h.
4. the drafting of typical curve: draw 5mg/L phosphorus (P) standardized solution respectively, 0,1,2,3,4,5,6ml in the 50ml volumetric flask, add water to 15～20ml, add the anti-developer of 5m1 molybdenum antimony with suction pipe, the water constant volume shakes up to scale.Obtain 0,0.1,0.2,0.3,0.4,0.5,0.6mg/L phosphorus (P) standard series colour developing liquid.Behind the 30min, on spectrophotometer,, obtain 0,0.1,0.2,0.3,0.4,0.5,0.6mg/L phosphorus (P) standard series colour developing liquid with 2cm optical path cuvette, 700nm wavelength colorimetric.Make reference with 0mg/L phosphorus standard series colour developing liquid, transfer absorption value, by the absorption value of lower concentration to the accurate series colour developing of high density mark liquid to zero.On squared paper, make ordinate zou with the OD value, phosphorus mg/L number is an X-coordinate drawing standard curve (see figure 7).
Calculation formula:
P mg/L=colour developing liquid phosphorus mg/L number * obtain multiple
Colour developing liquid phosphorus mg/L content: the phosphorus mg/L content that checks in colour developing liquid from typical curve
Colour developing liquid is long-pending: 50ml
YIM A116 bacterial strain gathers the phosphorus rate and measures the result:
| Fermented liquid | OD value/700nm | Dilution back phosphorus concentration (mg/L) | Extension rate | Phosphorus concentration (mg/L) |
| ISP2+P is nutrient solution not | 0.227 | 0.5198 | 400 | 207.9 |
| A116 cultivated after 7 days | 0.072 | 0.1580 | 100 | 15.8 |
Can calculate phosphorus and the concentration of cultivating back phosphorus in the nutrient solution not according to above-mentioned formula, that calculates YIM A116 bacterial strain gathers the phosphorus rate:
The present invention adopts cogongrass rhizosphere soil actinomycete as fermentation strain, and that measures bacterial strain gathers the phosphorus effect.It is that ISP2 liquid nutrient medium with improvement is a raw material, through the liquid state fermentation mode, adopts the combinatorial chemistry means, measures titanium pigment.This bacterial strain is the important solid phosphorus using microbe of soil.
The 16S rDNA of bacterial strain YIM A116 of the present invention is by 1411 based compositions:
ACGATGAAGCCCTTCGGGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTTCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATAACACTCTGTCCCGCATGGGACGGGGTTAAAAGCTCCGGCGGTGAAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTGATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAGAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTTGCGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTGTGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATATACCGGAAAGCATCAGAGATGGTGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATGCCGCGAGGCGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATTGGACGAT。
Claims (1)
1. one kind is gathered phosphorus actinomycetes Yan Shi streptomycete, it is characterized in that called after Yan Shi streptomycete (
Streptomyces yanii) YIM A116, deposit number is CCTCC NO:M2011188.
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| CN 201110231780 CN102286410B (en) | 2011-08-15 | 2011-08-15 | Streptomyces yanii |
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| CN101029298A (en) * | 2006-12-11 | 2007-09-05 | 河南亚神环保科技有限公司 | Production of efficient microbe bacteria combing agent |
| CN101037655A (en) * | 2006-11-29 | 2007-09-19 | 金明记 | High-effective microorganism complex agent and method of using the same for treating city sewage |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101037655A (en) * | 2006-11-29 | 2007-09-19 | 金明记 | High-effective microorganism complex agent and method of using the same for treating city sewage |
| CN101029298A (en) * | 2006-12-11 | 2007-09-05 | 河南亚神环保科技有限公司 | Production of efficient microbe bacteria combing agent |
Non-Patent Citations (4)
| Title |
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| Johwan Ahn et al..Characterization of denitrifying phosphate-accumulating organisms cultivated under different electron acceptor conditions using polymerase chain reaction-denaturing gradient gelel ectrophoresis assay.《Water Rearch》.2002,第36卷(第2期),403-412. * |
| 刘洪波.低浓度城市污水强化反硝化除磷.《同济大学博士学位论文》.2008, * |
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| 肖炜等.几种选择性分离稀有放线菌的方法.《微生物学通报》.2006,第33卷(第1期),133-137. * |
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