CN104498387A - Pseudomonas aeruginosa and its application - Google Patents
Pseudomonas aeruginosa and its application Download PDFInfo
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- CN104498387A CN104498387A CN201410691653.9A CN201410691653A CN104498387A CN 104498387 A CN104498387 A CN 104498387A CN 201410691653 A CN201410691653 A CN 201410691653A CN 104498387 A CN104498387 A CN 104498387A
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- 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
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Abstract
The invention relates to a Pseudomonas aeruginosa and its application. The Pseudomonas aeruginosa is named as Pseudomonas aeruginosa A1, and has a preservation number of CCTCC NO:M2014585. The above strain A1 can dissolve Microcystis aeruginosa through secreting an extracellular substance, and the substance has very strong thermal stability, and is a non-protein substance. A strain A1 supernatant is inoculated to an alga liquid according to a volume ratio of 6:100, treatment group algae are thoroughly etiolated on the fifth day, and the removal rate of chlorophyll a in alga cells is 80.29%.
Description
Technical field
The present invention relates to a strain by the bacterial classification with algae-lysing sieved in eutrophication water, this bacterial strain is to the Main Algae causing body eutrophication---and microcystic aeruginosa (Microcystis aeruginosa) has solvency action.
Pseudomonas aeruginosa alleged by the present invention, called after: Pseudomonas aeruginosa (Pseudomonas aeruginosa) A1, on November 23rd, 2014 in China typical culture collection center preservation, be numbered CCTCC NO:M 2014585.
Background technology
Along with quickening and the economic fast development of industrial process, global water body eutrophication degree aggravation, has become the great environmental problem that countries in the world face.For China, within 2009, Chinese environmental quality condition publication is pointed out: national surface water pollution is still heavier, and wherein lake (reservoir) eutrophication problem is given prominence to.In 26 states's control emphasis lake (reservoir), meet 1 of II class water quality, account for 3.9%; 5 of III class, account for 19.2%; 6 of IV class, account for 23.1%; 5 of V class, account for 19.2%; 9 of bad V class, account for 34.6%.Main contamination index is total nitrogen and total phosphorus.Nutritional status is eutrophic 1 of severe, accounts for 3.8%; Eutrophic 2 of moderate, accounts for 7.7%; Slightly eutrophic 8, account for 30.8%; Other are Middle nutrition, account for 57.7%.The blue-green alga bloom pollution problem that current body eutrophication causes has become the focus of global concern, blue-green alga bloom (water bloom) is the main of poisons in freshwater eutrophication, the poisonous algae kind determined at present comprises microcystic aeruginosa (Microcystis aeruginosa), Anabaena Flos-aquae (Aabaenaflos aguae), aphanizomenon flos aquae (Aphanizomenonflosaquae), A Shi quivers algae (Oscillatoria agardhil), quiver algae (Oscillatoria rubescens), foam joint ball algae (Nodular spumigena) etc., wherein, microcystic aeruginosa is main Bloom-causing Algal kind.Harmful algal blooms breaks out in eutrophication water regularly, and has in China and worldwide outburst frequency the trend increased year by year, explores the approach that effective suppression algal bloom breaks out very urgent.
There is such-and-such shortcoming in the method for administering body eutrophication due to tradition, therefore people have found a kind of better method-biological process, this method is set about from control algae reproduction and adjustment Algal Community Structure aspect, biological method whole aquatic ecosystem tended to balance and to good future development, so should conduct a research as solving the Main way that algal bloom pollutes.The discovery of algae-lysing microorganism, particularly algae-lysing bacterium, for Biological control provides new approaches.
Summary of the invention
In order to overcome the above problems, the invention provides a kind of Pseudomonas aeruginosa (Pseudomonas aeruginosa) A1 microcystic aeruginosa to solvency action.
Pseudomonas aeruginosa alleged by the present invention, called after: Pseudomonas aeruginosa (Pseudomonas aeruginosa) A1, on November 23rd, 2014 in China typical culture collection center preservation, be numbered CCTCC NO:M 2014585.Hereinafter referred to as strains A 1.
Another object of the present invention utilizes the effective constituent of strains A 1 to dissolve microcystic aeruginosa.
The screening method of Pseudomonas aeruginosa provided by the invention (Pseudomonas aeruginosa) A1 is as follows:
From the eutrophication water of Shenyang, gather water sample, be placed in 9mL sterilized water, as 10 with pipette, extract 1mL liquid
-1diluent.Being diluted successively by this solution is 10 again
-2, 10
-3, 10
-4, 10
-5solution, the bacterial classification that line picking colony form is different carries out purifying cultivation, and the final single culture that obtains also is numbered A1 ~ D1 respectively.
Various bacterium is placed in 200mL liquid nutrient medium respectively, and with 160r/min, 24h cultivated by 30 DEG C of shaking tables, and by the bacterium liquid of all same concentrations, equal-volume is inoculated in test algae, and establishes blank, is placed in illumination box and cultivates.Observe by the content and every day that measure chlorophyll a (Chla) growing state understanding microcystic aeruginosa, select the bacterium that algicidal effect is best.Final 5 kinds of bacterial strains can make algae liquid present yellow in various degree, and wherein A1 bacterium makes the speed of microcystic aeruginosa yellow faster, better effects if.
The qualification result of strains A 1 is as follows:
A. the form of strains A 1 and feature:
Bacterium colony is rounded, neat in edge, and smooth surface is glossy, and oyster white is translucent.In shaft-like, be Gram-negative bacteria, without gemma.
B. the physio-biochemical characteristics of strains A 1
Strains A 1 is aerobic bacteria, can not carry out nitrate reduction reaction; Can acetic oxide and liquefy gelatin, but can not hydrolyzed starch; Citrate trianion, grease, V-P test is positive, and indole test is negative.
C. the order-checking of the 16SrDNA of strains A 1 and the making of evolutionary tree
Strains A 1 genome DNA is extracted: according to a conventional method, through strain culturing, microorganism collection, cellular lysate, DNA extraction, precipitation, washing, takes out (drying in the air) dry 7 steps, finally obtains genome DNA, get 10 μ L 1% agarose gel electrophoresis and detect.
The pcr amplification of strains A 1 genomic dna: take STb gene as template, 50 μ L reaction systems are in table 1.
Table 1
First adding distil water, other components add successively from low dose.
Pcr amplification condition: 94 DEG C of denaturation 5min, 94 DEG C of sex change 40s, 55 DEG C of annealing 50s, totally 35 circulations, 72 DEG C extend 1.5min, and 72 DEG C stop 7min.
Pcr amplification product is got 10 μ L 1% agarose gel electrophoresis and is detected.Pcr amplification product electrophoretogram as shown in Figure 1.As seen from Figure 1, the length of pcr amplification product is about 1.6kb.
DNA sequencing is completed by the precious biotech firm in Dalian, and the sequence recorded is as shown in SCQ ID No.1.
D. the Phylogenetic analysis of strains A 1
As shown in Figure 2, wherein Pseudomonas i is Pseudomonas A1 to systematic evolution tree, and strains A 1 is 99.93% with the homology of the 16SrDNA sequence of Pseudomonas aeruginosa (Pseudomonas aeruginosa) as seen from Figure 2.
E. conclusion: the 16SrDNA gene order of acquisition is in GenBank registration, and acquisition accession number is KM263617.Carry out nucleotide sequence homology with blast program to 16SrDNA sequence listed in the 16SrDNA sequence of A1 and GenBank to compare, found that with the similarity of the 16SrDNA gene order of Pseudomonas aeruginosa LMG1242T up to 99.93%.Therefore can draw in conjunction with physiological and biochemical index, strains A 1 belongs to Pseudomonas aeruginosa.
The invention has the beneficial effects as follows: strains A 1 original bacteria liquid provided by the invention, through all having obvious solvency action between the filtered liquid, ultracentrifugal supernatant liquor of 0.45 μm of filter membrane and algicidal effect is substantially identical, this illustrates that strains A 1 is to carry out molten algae by the outer material of secretion born of the same parents, and this material has very strong thermostability, is non-protein substance.Be inoculated in test algae by strains A 1 supernatant liquor with the volume ratio of 6:100, the 5th day, treatment group algae is thoroughly yellow and the clearance of frustule chlorophyll a is 80.29%.
Accompanying drawing explanation
Pseudomonas aeruginosa, called after: Pseudomonas aeruginosa (Pseudomonas aeruginosa) A1, depositary institution's title: China typical culture collection center, be called for short: CCTCC, depositary institution address: China, Wuhan University, postcode: 430072.Preservation date is on November 23rd, 2014, and deposit number is CCTCC NO:M2014585.
Fig. 1 is strains A 1PCR amplified production electrophoretogram;
In figure, M is marker, and 1 is strains A 1.
Fig. 2 is that strains A 1 is evolved tree graph, and wherein Pseudomonas i is Pseudomonas A1.
Fig. 3 is strains A 1 growth curve chart.
Fig. 4 is the algicidal effect figure of different bacterium liquid processing mode;
Wherein, T1-bacterium liquid; T2-is through 0.45 μm of membrane filtration liquid; T3-bacterium liquid supernatant; The supernatant liquor of T4-pyroprocessing.
Fig. 5 is copper capsule green microalgae cell metamorphosis figure;
Wherein, abcd represents that Microcystis aeruginosa Strains is by the process of dissolving.A is normal frustule; B is impaired frustule; C is impaired frustule; D is by dissolving frustule.
Fig. 6 is that strains A 1 supernatant liquor is on the impact of Microcystis aeruginosa Strains Chlorophyll-a Content.
Embodiment
Embodiment 1 dissolves the method for microcystic aeruginosa
(1) cultivation of strains A 1
The cultivation of strains A 1: be that Pseudomonas aeruginosa (Pseudomonas aeruginosa) A1 of CCTCC NO:M 2014585 cultivates in substratum by deposit number; Culture medium prescription: extractum carnis 3g, peptone 10g, NaCl5g, pH value 7.0 ~ 7.2, distilled water 1000mL.
Method: by the bacterium liquid (10 cultivated
8individual/mL) with inoculum size 5% in beef extract-peptone liquid nutrient medium, in 37 DEG C of shaking culture (100rmin
-1), the absorbancy under 2h sampling and measuring 500nm wavelength take time as X-coordinate, and OD500 is that ordinate zou draws growth curve.The results are shown in Figure 3.
As can be seen from Figure 3, the lag phase of A1 bacterial strain is very short, and enter logarithmic phase after 2h, about 18h enters stationary phase, and 18th ~ 28h is in stationary phase substantially, enters senescence phase after 28h.
(2) characteristic research of microcystic aeruginosa is dissolved
The cultivation of microcystic aeruginosa adopts BG11 improvement to cultivate: NaCl 1.024g, K
2hPO
40.04g, MgSO
47H
2o0.075g, Ca (NO
3)
20.077g, citric acid 0.006g, FeCl
30.006g, Triammonium citrate 0.005g, EDTANa
20.001g, Na
2cO
30.02g, A5 solution 1mL, distilled water 999mL.
A5 solution composition is: H3BO3 286mg, MnSO4 159mg, ZnSO47H2O 22mg, NaMoO42H2O 39mg, CuSO45H2O 8mg, distilled water 100mL.
By Pseudomonas aeruginosa (Pseudomonas aeruginosa) A1 bacterial strain, cultivate in nutrient solution; Consisting of of nutrient solution: extractum carnis 3g, peptone 10g, NaCl5g, pH7.0 ~ 7.2, distilled water 1000mL.
Method: get 6mL bacterium liquid respectively, through 0.45 μm of membrane filtration liquid, (inoculum is through 8000r/min for bacterium liquid supernatant, 4 DEG C of centrifugal 15min), (bacterium liquid is through 8000r/min for the supernatant liquor of pyroprocessing, 4 DEG C of centrifugal 15min, get supernatant liquor and process 20min at 121 DEG C) add 100mL be in growth logarithmic phase algae liquid in, cultivate in illumination box, intensity of illumination is 1000lx ~ 1500lx, warm light, Light To Dark Ratio is 14h:10h, light room culture temperature is 30 DEG C, and darkroom is 25 DEG C.Respectively establish 3 parallel laboratory tests and blank, after 5d, measure the slippage of chlorophyll a (Chla).Result is as Fig. 4.
As seen from Figure 4, the A1 bacterium liquid through different treatment all has obvious solvency action to microcystic aeruginosa.Original bacteria liquid, algicidal effect between the filtered liquid, ultracentrifugal supernatant liquor of 0.45 μm of filter membrane are substantially identical, illustrate that strains A 1 is that the indirect mode secreting the outer material of born of the same parents carrys out molten algae.And the supernatant liquor after pyroprocessing still has good algicidal effect, and algicidal effect and undressed original bacteria liquid difference very little, the outer material of these born of the same parents illustrating that strains A 1 is secreted has very strong thermostability, nonprotein class material.In whole test, strains A 1 supernatant liquor is for the removal effect remarkable (P<0.05) of microcystic aeruginosa.
(3) algicidal effect of strains A 1
Method: get 6mL strains A 1 nutrient solution 8000rmin
-1centrifugal 15min, collection supernatant liquor joins 100mL and is in the algae liquid of growth logarithmic phase, if 3 parallel laboratory tests and blank, observes frustule form and measure chlorophyll a (Chla) content every 24h.
1, frustule form: every day aseptically, gets algae liquid, directly observes frustule form under the microscope, and take the algae-lysing figure of the 5th day strains A 1 supernatant liquor, result as shown in Figure 5, as seen from Figure 5, microcystic aeruginosa is counted as 0 under the microscope, illustrates and is all removed by dissolving.
2, frustule Chlorophyll-a Content: every day, aseptically, gets the algae liquid of certain volume, with 4000rmin
-1centrifugal 15min, carefully takes out supernatant liquor with liquid-transfering gun, then adds a certain amount of 90% acetone extract, puts 4 DEG C of refrigerator 24h, and then get the absorbancy that 630nm, 645nm, 663nm, 750nm place surveyed respectively by supernatant liquor 722N type spectrophotometer, result as shown in Figure 6.
V-volume of water sample (L); OD-absorbancy; The volume of V1-supernatant liquor constant volume; δ-cuvette light path.
As seen from Figure 6, by the 5th day, chlorophyll a mass concentration dropped to 95.8ug/L and tends towards stability, and clearance is 80.29%, with control group difference and remarkable (P<0.01) thereof.
Claims (4)
1. a Pseudomonas aeruginosa, is characterized in that: called after Pseudomonas aeruginosa (Pseudomonas aeruginosa) A1, and in China typical culture collection center preservation, deposit number is CCTCC NO:M 2014585.
2. Pseudomonas aeruginosa according to claim 1 is dissolving the application in microcystic aeruginosa.
3. apply as claimed in claim 2, it is characterized in that method is as follows:
1) cultivation of bacterial strain: be that Pseudomonas aeruginosa (Pseudomonas aeruginosa) A1 of CCTCC NO:M 2014585 cultivates in substratum by deposit number;
2) by Pseudomonas aeruginosa (Pseudomonas aeruginosa) the A1 bacterium liquid after cultivation, through 8000r/min, 4 DEG C centrifugal, gets supernatant liquor;
3) be 6% by inoculum size, supernatant liquor added in microcystic aeruginosa liquid.
4. apply as claimed in claim 3, it is characterized in that: described substratum is: extractum carnis 3g, peptone 10g, NaCl 5g, pH value 7.0 ~ 7.2, distilled water 1000mL.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087428A (en) * | 2015-07-09 | 2015-11-25 | 标优美生态工程股份有限公司 | Pseudomonas aeruginosa and application thereof |
| CN105502688A (en) * | 2016-01-21 | 2016-04-20 | 华南理工大学 | Method for synchronously dissolving algae/degrading algal toxins by using microbial combined preparation |
| CN113583906A (en) * | 2021-07-21 | 2021-11-02 | 首都师范大学 | Application of pseudomonas B5 in algae removal |
| CN114806942A (en) * | 2022-04-21 | 2022-07-29 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Pseudomonas, fermentation product thereof and application thereof in controlling growth of algae |
-
2014
- 2014-11-25 CN CN201410691653.9A patent/CN104498387B/en active Active
Non-Patent Citations (3)
| Title |
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| KATHLEEN K.GALLUCCI等: "Pseudomonas aeruginosa Chemotaxis Associated with Blooms of N2-fixing Blue-Green Algae(Cyanobacteria)", 《APPL ENVIRON MICROBIOL》 * |
| 陈庆丽等: "1 株铜绿假单胞菌JM1 的溶藻特征", 《吉林农业大学学报》 * |
| 龚良玉等: "铜绿假单胞菌产吩嗪类色素的分离纯化及其对赤潮生物生长的影响", 《复旦学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087428A (en) * | 2015-07-09 | 2015-11-25 | 标优美生态工程股份有限公司 | Pseudomonas aeruginosa and application thereof |
| CN105502688A (en) * | 2016-01-21 | 2016-04-20 | 华南理工大学 | Method for synchronously dissolving algae/degrading algal toxins by using microbial combined preparation |
| CN105502688B (en) * | 2016-01-21 | 2018-06-22 | 华南理工大学 | A kind of method that molten algae/degradation algae toxin is synchronized using microbial association preparation |
| CN113583906A (en) * | 2021-07-21 | 2021-11-02 | 首都师范大学 | Application of pseudomonas B5 in algae removal |
| CN114806942A (en) * | 2022-04-21 | 2022-07-29 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Pseudomonas, fermentation product thereof and application thereof in controlling growth of algae |
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| CN104498387B (en) | 2017-06-16 |
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