CN103667111A

CN103667111A - Bacillus megaterium capable of dissolving microcystis aeruginosa and application thereof

Info

Publication number: CN103667111A
Application number: CN201310554926.0A
Authority: CN
Inventors: 付保荣; 郭鑫; 杨玉东; 张润洁; 付豪; 苗斌
Original assignee: Liaoning University
Current assignee: Liaoning University
Priority date: 2013-11-07
Filing date: 2013-11-07
Publication date: 2014-03-26
Anticipated expiration: 2033-11-07
Also published as: CN103667111B

Abstract

The invention relates to bacillus megaterium capable of dissolving microcystis aeruginosa and an application thereof. The bacterial strain A0 can be used for dissolving the microcystis aeruginosa. When the adding amount is 7.5%, the dissolving function is very obvious in the 6th day of cocultivation, the amount of frustule is 66.7% lower than the matched group and the content of frustule chlorophyll alpha is 35.2% lower than the matched group in the 6th day.

Description

A kind of bacillus megaterium and application thereof microcystic aeruginosa to solvency action

Technical field

The present invention relates to a strain in eutrophication water, sieve the bacterial classification with algae-lysing, this bacterial strain is to causing the Main Algae of body eutrophication---microcystic aeruginosa ( microcystisaeruginosa) there is solvency action.

A kind of bacillus megaterium called after microcystic aeruginosa to solvency action that the present invention is alleged: bacillus megaterium ( bacillus megaterium) A ₀, on September 27th, 2013 in the center preservation of Chinese Typical Representative culture collection, address is: Wuhan, China Wuhan University, is numbered CCTCC NO:M 2013452.

background technology

In recent years, along with the continuous increase of the raising of industrialization degree, the continuous quickening of urbanization process and population, mankind's activity is more and more frequent and having a deep effect on water surrounding.Water, phosphorus accumulation reach the eutrophication that some amount will cause water body, even cause harmful algae wawter bloom.Because nutritive substances such as entering nitrogen in bay and lake, phosphorus constantly increases, body eutrophication process is accelerated.At present there are more than 66% lake and reservoir in China in eutrophication pollution in various degree, has not only destroyed the balance of aquatic ecosystem, and prevailing Microcystis aeruginosa in the water body that occurs of wawter bloom ( microcystis) can produce the meta-bolitess such as a large amount of algae toxin public health, livestock and water source supply are brought to negative impact.Harmful algae wawter bloom breaks out regularly in eutrophication water, and Bing China and worldwide outburst frequency have year by year the trend increasing, and explores the approach that effective inhibition algal bloom breaks out very urgent.

In the situation that utilizing physics, chemistry and other biological method control algal bloom not satisfactory, utilize molten algae bacterium to remove the study hotspot that algae becomes current Biological control.Molten algae bacterium refers to by direct or indirect mode, the general designation that suppresses algal grown or kill the bacterium of algae, dissolving frustule.Molten algae bacterium, as the important component part of aquatic ecosystem biotic population structure and function, has very important effect to maintaining the biomass balance of algae, therefore, utilizes molten algae bacterial control algal bloom to have broad application prospects.

Summary of the invention

In order to overcome the above problems, the invention provides a kind of to microcystic aeruginosa have solvency action bacillus megaterium ( bacillus megaterium) A ₀, on September 27th, 2013 in the center preservation of Chinese Typical Representative culture collection, be numbered CCTCC NO:M 2013452.Hereinafter to be referred as strains A ₀.

Another object of the present invention is to provide a kind of method of dissolving microcystic aeruginosa.

Bacillus megaterium A provided by the invention ₀screening method as follows:

From certain eutrophication water, gather water sample, by gradient dilution method, be diluted to different concns, dull and stereotyped coating, cultivates 48h for 30 ℃, obtains single bacterium colony, gets single bacterium colony and carries out plate streaking, cultivates 48h for 30 ℃, obtains the bacterial strain of 5 strain pure cultures, is numbered A ₀~ A ₄.

Get each pure inoculation in beef extract-peptone liquid nutrient medium, 30 ℃, in 160r/min constant temperature oscillator, cultivate 24 hours, by the inoculum size of 10% (v/v), be inoculated in the preculture microcystic aeruginosa algae liquid of 10 days, blank is set, finally has 2 strain bacterium can make the yellow of algae liquid, wherein A ₀bacterium makes the speed of microcystic aeruginosa yellow faster, better effects if.

Strains A ₀qualification result as follows:

A. strains A ₀form and feature:

Bacterium colony is rounded, neat in edge, and smooth surface, glossy, oyster white is translucent.Micro-Microscopic observation thalline is shaft-like, is gram-positive microorganism, and central spore, without pod membrane;

B. strains A ₀physio-biochemical characteristics

Bacterial classification metabolism can produce tryptophanase, catalase; Do not decompose or less decomposition glucose, do not go back orthonitric acid, milk reindeer moss peptonizes, liquefy gelatin, hydrolyzed starch.

C. strains A ₀the order-checking of 16S rDNA and the making of evolutionary tree

Strains A ₀genome 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, gets 10 μ L and detects with 1% agarose gel electrophoresis.

Strains A ₀the pcr amplification of genomic dna: take total DNA as template, 50 μ L reaction systems are in Table 1.

Table 1

First adding distil water, other components start to add successively from low dose.Pcr amplification needs 94 ℃ of denaturation 5min, 94 ℃ of sex change 40s, and 55 ℃ of annealing 50s, totally 35 circulations, 72 ℃ are extended 1.5min, and 72 ℃ stop 7min.Pcr amplification product is got 10 μ L and is detected with 1% agarose gel electrophoresis.Pcr amplification product electrophoretogram as shown in Figure 1.

DNA sequencing: order-checking is completed by the precious biotech firm in Dalian.Sequence is as follows:

TATACATGCAGTCGAGCGAACTGATTAGAAGCTTGCTTCTATGACGTTAG

CGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATA

ACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGA

GATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGC

ATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCG

ACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCC

TACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGG

AGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGT

TAGGGAAGAACAAGTACGAGAGTAACTGCTCGTACCTTGACGGTACCTAA

CCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT

GGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTC

TTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAA

CTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGT

GAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGG

TCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGA

TACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTT

TCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGT

ACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCG

GTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCT

TGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAG

AGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGG

TTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGT

TGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATG

ACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAAT

GGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAA

CCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAA

TCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCT

TGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGT

GGAGTAACCGTAAGGAGCTAGCCGCCTAAGGTGGGACAGATGATTGGGGT

GAAGTCGTAACA

D. strains A ₀systematic evolution tree analysis

Systematic evolution tree as shown in Figure 2, strains A as seen from Figure 2 ₀with bacillus megaterium ( bacillus megaterium) the homology of 16S rDNA sequence be 99.5%.

E. conclusion: with reference to < < common bacteria system identification handbook > > and the outstanding Bacteria Identification handbook > > of < < uncle, in conjunction with physiological and biochemical property and 16S rDNA order-checking, identify this strains A ₀for bacillus megaterium ( bacillus megaterium), the homology of 16S rDNA sequence is 99.5%.We name strains A ₀for: bacillus megaterium ( bacillus megaterium) A ₀, on September 27th, 2013 in the center preservation of Chinese Typical Representative culture collection, be numbered CCTCC NO:M 2013452.

The invention has the beneficial effects as follows: strains A provided by the invention ₀microcystic aeruginosa is had to solvency action, and when dosage is 7.5%, its solvency action is very obvious common cultivation the 6th day, and the frustule number of the 6th day is lower by 66.7% than control group, and frustule Chlorophyll-a Content is lower by 35.2% than control group.Visible strains A ₀microcystic aeruginosa is had to solute effect.

Accompanying drawing explanation

Fig. 1 is strains A ₀pcr amplification product electrophoretogram;

In figure, M is marker, and 1,2 is strains A ₀.

Fig. 2 is strains A ₀evolution tree graph.

Fig. 3 is strains A ₀growth curve chart.

Fig. 4 is strains A ₀the impact of different dosages on Microcystis aeruginosa Strains number.

Fig. 5 is Microcystis aeruginosa Strains metamorphosis figure;

Wherein, abcd represents the process that Microcystis aeruginosa Strains is dissolved.A is normal frustule; B is impaired frustule; C is impaired frustule; D is dissolved frustule.

Fig. 6 is strains A ₀add the 6th day Microcystis aeruginosa Strains number.

Fig. 7 is strains A ₀the impact of different dosages on Microcystis aeruginosa Strains Chlorophyll-a Content.

Embodiment

the method that embodiment 1 dissolves microcystic aeruginosa

(1) strains A ₀cultivation

Culture medium prescription: distilled water 1000mL, extractum carnis 3g, peptone 10g, NaCl 5g, pH value 7.0 ~ 7.2.

Method: the substratum preparing is packed into by every bottle of 50mL in the triangular flask of 9 250mL, 121 ℃ of sterilizing 20min, after normal temperature is cooling, by 5%(v/v) inoculum size is by strains A ₀seed liquor accesses in each triangular flask, then putting into constant temperature oscillator vibrates, revolution is 160r/min, temperature is 30 ℃, within every three hours, takes out one bottle, with blood counting chamber, examines under a microscope counting, calculate nectar degree, measure the absorbancy under 660nm wavelength simultaneously, take the time as X-coordinate, nectar degree and OD660 are that ordinate zou is drawn growth curve.The results are shown in Figure 3.

As can be seen from Figure 3, the lag phase that 0 ~ 3h is thalli growth, 3 ~ 9h is logarithmic phase, and 9h enters stationary phase later, and now thalline quantity is maximum, and nectar degree is maximum.

(2) dissolve the method for microcystic aeruginosa

To being cultured in the microcystic aeruginosa algae liquid of logarithmic phase, by volume per-cent, adds respectively 2.5%, 5%, 7.5%, 10% the strains A that is cultured to stationary phase ₀seed liquor is cultivated in illumination box, and intensity of illumination is 1000lx ~ 1500lx, warm light, and Light To Dark Ratio is 14h:10h, and light chamber culture temperature is 30 ℃, and darkroom is 25 ℃.Cultivate altogether 8 days, blank is set simultaneously.

(3) result

1, frustule form and quantity: every 2 days, under aseptic condition, get algae liquid, directly observe under the microscope frustule form, and adopt blood counting chamber method to count frustule, calculate algae cell density, result is as Figure 4-Figure 6.

2, frustule Chlorophyll-a Content: every 2 days, under aseptic condition, get 5mL algae liquid, the centrifugal 10min of 3500r/min, removes supernatant liquor, adds 5mL ethanol acetone 1:1 mixed solution, place after 24h dark place, the centrifugal 10min of 3500r/min, surveys supernatant liquor 663nm, 645nm, 630nm and 750nm place absorbancy, and result as shown in Figure 7.

V---volume of water sample (L); OD---absorbancy; V1---the volume of supernatant liquor constant volume; δ---cuvette light path.

3, brief summary: strains A ₀different dosages are more obvious on the impact of microcystic aeruginosa frustule number, as can be seen from Figure 4, and at A ₀add 0 ~ 2 day, each dosage frustule growth is all subject to inhibition to a certain extent, and 10% treatment group shows obvious solvency action; After 2 ~ 8 days, work as A ₀when dosage is 2.5%, microcystic aeruginosa, without dissolution inhibition effect, is even shown to certain promoter action; Work as A ₀when dosage is 5%, add 2 ~ 6 days initial stages, the growth of microcystic aeruginosa is had to certain promoter action, but frustule number, lower than control group, and there is continuation downward trend during by the 8th day; A ₀dosage is 7.5% treatment group, and microcystic aeruginosa is had to certain solvency action, cultivates altogether solvency action after 4 days and starts to manifest gradually, obvious to solvency action after the 6th day; A ₀dosage is 10% treatment group, since the 2nd day, just microcystic aeruginosa is shown to obvious solvency action.As can be seen from Figure 6,7.5% group of frustule number of the 6th day is lower 66.7%, 10% group lower by 72.3% than control group than control group.Strains A ₀different dosages are shown in Fig. 7 to the impact of Microcystis aeruginosa Strains Chlorophyll-a Content, and it affects situation and strains A ₀frustule number is affected to situation basic identical, 7.5% group of the 6th day Chlorophyll-a Content is lower by 51.9% than control group than low 35.2%, the 10% group of Chlorophyll-a Content of control group.

Therefore, analytical results and the Cost Problems of synthesizing map 4-Fig. 7, determine the strains A that microcystic aeruginosa is produced to solvency action ₀optimum dosage is 7.5%, and algae-lysing is very obvious effect the 6th day, reduces more than 60%.

<110> Liaoning University

<120> bacillus megaterium and application thereof microcystic aeruginosa to solvency action

<160> 1

<210> 1

<211> 1462

<212> DNA

<213> bacillus megaterium ( bacillus megaterium) A ₀

<400> 1

TATACATGCA GTCGAGCGAA CTGATTAGAA GCTTGCTTCT ATGACGTTAG

CGGCGGACGG GTGAGTAACA CGTGGGCAAC CTGCCTGTAA GACTGGGATA

ACTTCGGGAA ACCGAAGCTA ATACCGGATA GGATCTTCTC CTTCATGGGA

GATGATTGAA AGATGGTTTC GGCTATCACT TACAGATGGG CCCGCGGTGC

ATTAGCTAGT TGGTGAGGTA ACGGCTCACC AAGGCAACGA TGCATAGCCG

ACCTGAGAGG GTGATCGGCC ACACTGGGAC TGAGACACGG CCCAGACTCC

TACGGGAGGC AGCAGTAGGG AATCTTCCGC AATGGACGAA AGTCTGACGG

AGCAACGCCG CGTGAGTGAT GAAGGCTTTC GGGTCGTAAA ACTCTGTTGT

TAGGGAAGAA CAAGTACGAG AGTAACTGCT CGTACCTTGA CGGTACCTAA

CCAGAAAGCC ACGGCTAACT ACGTGCCAGC AGCCGCGGTA ATACGTAGGT

GGCAAGCGTT ATCCGGAATT ATTGGGCGTA AAGCGCGCGC AGGCGGTTTC

TTAAGTCTGA TGTGAAAGCC CACGGCTCAA CCGTGGAGGG TCATTGGAAA

CTGGGGAACT TGAGTGCAGA AGAGAAAAGC GGAATTCCAC GTGTAGCGGT

GAAATGCGTA GAGATGTGGA GGAACACCAG TGGCGAAGGC GGCTTTTTGG

TCTGTAACTG ACGCTGAGGC GCGAAAGCGT GGGGAGCAAA CAGGATTAGA

TACCCTGGTA GTCCACGCCG TAAACGATGA GTGCTAAGTG TTAGAGGGTT

TCCGCCCTTT AGTGCTGCAG CTAACGCATT AAGCACTCCG CCTGGGGAGT

ACGGTCGCAA GACTGAAACT CAAAGGAATT GACGGGGGCC CGCACAAGCG

GTGGAGCATG TGGTTTAATT CGAAGCAACG CGAAGAACCT TACCAGGTCT

TGACATCCTC TGACAACTCT AGAGATAGAG CGTTCCCCTT CGGGGGACAG

AGTGACAGGT GGTGCATGGT TGTCGTCAGC TCGTGTCGTG AGATGTTGGG

TTAAGTCCCG CAACGAGCGC AACCCTTGAT CTTAGTTGCC AGCATTTAGT

TGGGCACTCT AAGGTGACTG CCGGTGACAA ACCGGAGGAA GGTGGGGATG

ACGTCAAATC ATCATGCCCC TTATGACCTG GGCTACACAC GTGCTACAAT

GGATGGTACA AAGGGCTGCA AGACCGCGAG GTCAAGCCAA TCCCATAAAA

CCATTCTCAG TTCGGATTGT AGGCTGCAAC TCGCCTACAT GAAGCTGGAA

TCGCTAGTAA TCGCGGATCA GCATGCCGCG GTGAATACGT TCCCGGGCCT

TGTACACACC GCCCGTCACA CCACGAGAGT TTGTAACACC CGAAGTCGGT

GGAGTAACCG TAAGGAGCTA GCCGCCTAAG GTGGGACAGA TGATTGGGGT

GAAGTCGTAA CA

Claims

1. microcystic aeruginosa is there is to a bacillus megaterium for solvency action, it is characterized in that: bacillus megaterium ( bacillus megaterium) A ₀, in the center preservation of Chinese Typical Representative culture collection, be numbered CCTCC NO:M 2013452.

2. the application of bacillus megaterium microcystic aeruginosa to solvency action claimed in claim 1 in dissolving microcystic aeruginosa.

3. application as claimed in claim 2, is characterized in that: in microcystic aeruginosa algae liquid, per-cent by volume, the deposit number claimed in claim 1 that drops into 5%-10% be the bacillus megaterium of CCTCC NO:M 2013452 ( bacillus megaterium) A ₀.