CN105779361A

CN105779361A - Method for degrading cyanophycean toxin from bacterial strain by in-situ separation and purification

Info

Publication number: CN105779361A
Application number: CN201610320144.4A
Authority: CN
Inventors: 李国�; 张六六
Original assignee: ANHUI JINNONG HUIMIN BIOLOGY TECHNOLOGY CO LTD
Current assignee: ANHUI JINNONG HUIMIN BIOLOGY TECHNOLOGY CO LTD
Priority date: 2016-05-13
Filing date: 2016-05-13
Publication date: 2016-07-20

Abstract

The invention discloses a method for degrading cyanophycean toxin from a bacterial strain by in-situ separation and purification. According to the method disclosed by the invention, by taking bottom mud in Dianchi of Yunnan as a cyanophycean toxin-degrading bacterium separating source, an MC-LR degrading bacterium is obtained by dilution of the bottom mud, exposure of a flat plate to toxins and coating, picking of single colonies, lineation and purification, and MC-LR degradation rate testing. The maximum degradation efficiency of the separated Aeromonas sp. DC-132 reaches 64.98 percent. The method is practical and convenient and has a remarkable effect.

Description

A kind of method by the most isolated and purified strains for degrading cyanophycean toxin

Technical field

The present invention relates to field of environmental technology, particularly belong to a kind of antibacterial using original position the most isolated and purified and cyanophycean toxin is entered The method of row degraded.

Background technology

Eutrophication is the universality environmental problem in global range, along with Urbanization in China is constantly accelerated, and industry Polluting agrochemical in addition to use in a large number, China's body eutrophication problem is outstanding day by day.During water body generation eutrophication, swim Biological amount reproduction, the water surface can present different colours because dominant plankton species is different, algal layer Cheng Hong in coastal waters Color, is referred to as " red tide ", and in rivers and lakes, cyanophyceae chlorella etc. is preponderated, and is referred to as " wawter bloom ", when plankton is dead Time, organic corruption can cause Dissolved Oxygen in Water to decline, water quality deterioration, causes some aquatile quantity to reduce the most a large amount of Dead.During breakout of cyanobacteria blooms, the Microcystins in 2/3rds water samples exceedes threshold limit values；Edible polluted-water In aquatic products, the Microcystin of absorption can easily reach allows and takes the photograph even every day considerably beyond World Health Organization's suggestion Enter amount.In recent years, the report of Algae toxins intoxicating event gets more and more, in China, the highest onset of liver cancer rate mainly by The drinking water source polluted by cyanophyceae causes.Current certified toxin kind oneself through more than 70 kinds, most common of which, toxicity are That strong is microcapsule algae toxin (MC-LR).MC-LR is a kind of ring-type heptapeptide, molecular weight, and stable chemical nature is acidproof Alkaline-resisting, it all can not effectively be removed by general heated and boiled, belongs to the biological organic toxin of difficult degradation, in natural water environment Can retain for a long time.

The minimizing technology of generally MCs can be largely classified into 3 classes: physical method, chemical method and biological method.Biodegradation Effect is the main path that Algae toxins converts in natural water, for its cost and effect on environment degree, and biodegradation Technology has the advantage of uniqueness compared with other Algae toxins minimizing technology.Microbial degradation is biodegradable main path, the most micro- Biodegradation has stronger specificity, and miscellaneous microorganism is not particularly suited for Algae toxins of degrading, and therefore separates pure in situ Changing bacterial strain and Algae toxins carries out degraded is effective method.

Summary of the invention

The present invention seeks to be to provide a kind of method by the most isolated and purified strains for degrading cyanophycean toxin, method Simply, high specificity, algal toxin degradation effect is notable.

For reaching above-mentioned purpose, the present invention by the following technical solutions:

A kind of method by the most isolated and purified strains for degrading cyanophycean toxin, using Kunming, Yunnan Sediments of Dian Chi Lake as Algae toxins Degradation bacteria separation source, separates and obtains the bacterial strain to Algae toxins with efficient degradation effect, the mud sample obtained connect in Dianchi Lake, Yunnan Province Kind in beef-protein medium in, natural lighting, 37 DEG C, carry out shake-flask culture twice, then under the conditions of 120rpm Plate streaking, observes colony growth situation；Picking list bacterium colony, carries out repeatedly plate isolation purification, progressively carries in purge process In high isolation medium, the concentration of MCs carries out gradient domestication；Through obtaining pure bacterial strain the most after purification, preserve in 4 DEG C of refrigerators； By the pure inoculation of isolated in basal medium, in 37 DEG C, carry out shake-flask culture under the conditions of 120rpm；Timing sampling Measure the concentration of MCs in water, the preliminary power confirming separating obtained microbial degradation MCs ability, and select advantage therein Bacterial strain is further purified, it is thus achieved that the bacterial strain of efficient degradation MC-LR, belongs to Aeromonas, named Aeromonas sp. DC-132。

Described flat board is coated with flat board for contamination, and contamination is coated with flat board and algal toxin degradation rate test cyanophycean toxin used is MC-LR, purchased from Sigma company, CAS 101043-37-2, molecular weight 995.17.

Described beef-protein medium uses LB fluid medium and LB solid medium, wherein LB liquid culture Base: antibacterial culturing tryptone 10g, the 5mol/L NaOH of yeast extract 5g, NaCl 10g, 0.2ml regulate pH extremely 7.0, it is settled to 1000ml with deionized water, under 0.1MPa, steam sterilization 30min, standby；LB solid medium: often 100ml LB fluid medium adds agar 1.3g-1.5g, steam sterilization 30min under 0.1MPa, is cooled to about 45 DEG C-50 DEG C, it is down flat plate, standby.

Accompanying drawing explanation

Fig. 1 is the Aeromonas sp. DC-132 degradation curve figure to MC-LR.

Detailed description of the invention

The present invention is a kind of isolation and purification method being applicable to cyanophycean toxin bacterium for degrading.

Beef-protein medium uses LB fluid medium and LB solid medium, wherein LB fluid medium: thin Bacterium cultivation tryptone 10g, the 5mol/L NaOH of yeast extract 5g, NaCl 10g, 0.2ml regulate pH to 7.0, use Deionized water is settled to 1000ml, and under 0.1MPa, steam sterilization 30min, standby.

LB solid medium: adding agar 1.3g-1.5g at every 100ml LB fluid medium, steam goes out under 0.1MPa Bacterium 30min, is cooled to about 45 DEG C-50 DEG C, is down flat plate, standby.

1. using Dianchi Lake, Yunnan Province bed mud as algal toxin degradation bacterium separation source, in absorption 1ml bed mud to 9ml sterilized water, as 10^-1Diluent；Mixed hook after, then draw during in this test tube, 1ml liquid is placed in 9ml sterilized water, as 10^-2Diluent, successively class Push away, be respectively prepared 10 by gradient dilution method^-3、10^-4、10^-5、10^-6Diluent；

Drawing 0.2ml different dilution sample instillation LB solid the most respectively and poison in culture medium, LB solid poisons culture medium and is By adding purification MC-LR in LB solid medium, concentration is about 3mg/L, smears 18 flat boards, reference numeral, is inverted in 37 DEG C Biochemical cultivation case cultivates 24h-48h；

3. picking list flora on flat board, takes streaking inoculation, makes strain be further purified；

4. the strain of isolated is inoculated in LB fluid medium, 120rpm, shaken cultivation 24h-48h at 37 DEG C.Then Each bacteria suspension of 50 ml is joined in 200 ml microcystic aeruginosa supernatant, regulate initial Algae toxins concentration to about 1 mg/ L, arranges 3 groups of repetitions, and sets blank, and blank is the Aerugo microcapsule that 200 mL initial Algae toxins concentration is about 1 mg/L Algae supernatant adds 50 mL sterilized water.120 rpm, shaken cultivation 48h-72h at 25 DEG C；

5. before and after being processed by HPLC detection, the MC-LR concentration in each group, determines the strain having algal toxin degradation ability；

6. selecting after having the strain of algal toxin degradation ability, repeat step 4-5, every 8h detects each process group with HPLC method MC-LR concentration, draws degradation curve.Obtain the bacterial strain that degradation capability is the highest；

7. the further purification of bacterial strain that will obtain, and with morphology, molecular biology method, it is identified；

8. obtain the bacterial strain of efficient degradation MC-LR with Dianchi Lake, Yunnan Province bed mud for algal toxin degradation bacterium separation source, belong to aeromonas Belong to, named Aeromonas sp. DC-132.

By embodiments of the invention degradation data effect:

The degradation curve of MC-LR is shown in accompanying drawing 1 by Aeromonas sp. DC-132, and when 48h, degradation rate is close to 60%, afterwards Changing less, most degradation efficiency is 64.98%, and degradation effect is notable.

The foregoing is only the better embodiment of the present invention, protection scope of the present invention with above-mentioned embodiment is not Limit, as long as those of ordinary skill in the art modify or change according to the equivalence that disclosed content is made, all should include power in In profit protection domain described in claim.

Claims

1. the method by the most isolated and purified strains for degrading cyanophycean toxin, it is characterised in that: with Kunming, Yunnan Dian Chi Bed mud, as algal toxin degradation bacterium separation source, separates and obtains the bacterial strain to Algae toxins with efficient degradation effect, by Dianchi Lake, Yunnan Province In the beef-protein medium that the mud sample of middle acquirement is inoculated in, natural lighting, 37 DEG C, carry out shaking flask under the conditions of 120rpm Cultivate twice, then plate streaking, observe colony growth situation；Picking list bacterium colony, carries out repeatedly plate isolation purification, at purification During step up the concentration of MCs in isolation medium and carry out gradient domestication；Through obtaining pure bacterial strain the most after purification, in 4 DEG C refrigerator preserves；By the pure inoculation of isolated in basal medium, in 37 DEG C, carry out shaking flask under the conditions of 120rpm Cultivate；Timing sampling measures the concentration of MCs in water, the preliminary power confirming separating obtained microbial degradation MCs ability, and selects Select dominant strain therein to be further purified, it is thus achieved that the bacterial strain of efficient degradation MC-LR, belong to Aeromonas, named Aeromonas sp. DC-132。

A kind of method by the most isolated and purified strains for degrading cyanophycean toxin the most according to claim 1, its feature Being: described flat board is coated with flat board for contamination, contamination is coated with flat board and algal toxin degradation rate test cyanophycean toxin used is MC- LR, purchased from Sigma company, CAS 101043-37-2, molecular weight 995.17.

A kind of method by the most isolated and purified strains for degrading cyanophycean toxin the most according to claim 1, its feature It is: described beef-protein medium uses LB fluid medium and LB solid medium, wherein LB fluid medium: thin Bacterium cultivation tryptone 10g, the 5mol/L NaOH of yeast extract 5g, NaCl 10g, 0.2ml regulate pH to 7.0, use Deionized water is settled to 1000ml, and under 0.1MPa, steam sterilization 30min, standby；LB solid medium: at every 100ml LB liquid Body culture medium adds agar 1.3g-1.5g, steam sterilization 30min under 0.1MPa, is cooled to about 45 DEG C-50 DEG C, is down flat plate, standby With.