CN1206174C

CN1206174C - Microbe capable of degradation removing microcystin from water bloom

Info

Publication number: CN1206174C
Application number: CN 02156462
Authority: CN
Inventors: 闫海; 潘纲; 张明明
Original assignee: Research Center for Eco Environmental Sciences of CAS
Current assignee: Research Center for Eco Environmental Sciences of CAS
Priority date: 2002-12-18
Filing date: 2002-12-18
Publication date: 2005-06-15
Anticipated expiration: 2022-12-18
Also published as: CN1435383A

Abstract

The present invention relates to a method for degrading and removing microcystin with high efficiency by utilizing one strain of microbial strain (ralstonia solanacearum, an RS bacterium for short), which is characterized in that the RS bacterium is cultivated, and prepared enzyme is used as a biological catalyst; after the RS bacterium is proportionally thrown into a water body polluted by blue algae water bloom, the microcystin is fast degraded and removed with high efficiency. In addition, ralstonia solanacearum cells or the prepared enzyme can be used for rescuing and detoxifying animals poisoned by themicrocystin. The present invention has important significance on the aspects of environment pollution treatment and the research and the application of biological medicine.

Description

A kind of method of utilizing microbiological deterioration to remove Microcystin in the wawter bloom

Technical field

Patent application of the present invention relates to a kind of microbial strains (Ralstonia solanacearum bacterium CGMCC0856 of our screening, be called for short: the RS bacterium, preservation date on December 18th, 2002, Institute of Microorganism, Academia Sinica of depositary institution, deposit number: 0856) and enzyme preparation degrades remove Microcystin (Microcystins, be called for short: method MC), it is characterized in that with the zymin of RS bacterium of cultivating and preparation as a kind of fast, safety and biological catalyst efficiently, after adding the water body that is subjected to the MC pollution by a certain percentage, make MC obtain the method that efficient degradation is removed.Belong to biological technical field.

Background technology

Body eutrophication is meant nutritive substances such as nitrogen that admittance in lake, reservoir and the river is too much and phosphorus, and the ecologic structure of water body and function are changed, and causes the algae particularly unusual breeding growth of blue-green algae and the blue-green alga bloom phenomenon that occurs.When water bloom pollution is serious, the water surface forms thick blue-greenish colour water bloom, give off an unpleasant smell, not only destroyed aquatic ecosystem in a healthy and balanced way, and the drinking water safety of humans and animals has been constituted serious threat because of frustule has discharged multiple algae toxin after breaking.At present, the frequency and the severity that take place of freshwater lake blue-green alga bloom all presents swift and violent rising tendency in the world, the various places that spread all over the world, the place of generation.Europe, Africa, North America and South America have 53,28,48 and 41% lake existence eutrophication phenomenon in various degree respectively, the lake of the Asian-Pacific area 54% is in the eutrophication state, and there is eutrophication phenomenon in various degree in present about 60% the lake of China.From reported first in 1878 animal since the death, the hydrocoles that causes because of the algae toxin, birds, domestic animals even human dead incident frequently take place both at home and abroad owing to drink the water that contains blue-green algae.How controlling the blue-green alga bloom contamination phenomenon and effectively removing the algae toxin is the environmental science difficult problem of pendulum in face of the China and even the world.

It is the algae toxin that discharges number of different types behind poisonous blue-green algae cell rupture in water body that blue-green alga bloom pollutes the main harm of being brought, and wherein MC is a kind of algae toxin kind that the frequency of occurrences is the highest in blue-green alga bloom pollutes, generation is maximum and work the mischief the most serious.Result of study shows that the main target device of MC is a liver, mainly shows as to make the congested enlargement of liver, can cause hepatorrhagia and necrosis when serious.The MC mechanism of toxication is by suppressing the activity of phosphoprotein phosphatase in the liver cell, bring out the cytokeratin hyperphosphorylation, causes the hepatocyte of mammal microfilament to decompose, break and hemorrhage.Find according to another investigation, in the tap water among the existence of MC and the crowd sickness rate of primary hepatocarcinoma and large bowel cancer very big dependency is arranged.

Though microbiological deterioration is a very promising method removing MC, but the ring texture of MC and at interval two key have suitable stability, general polypeptide lytic enzyme can not decompose MC, have only some special microbial strainss just to possess degradation capability to MC, this also is that MC can exist a major reason for a long time in natural water body.Chinese scholar adopts sequencing batch biofilm reactor that the experiment that 3 types of MC degrade is shown that mixed bacterium has certain degradation capability to MC, the degraded aerobic condition of MC is better than anaerobic condition, but the removal amount of MC all is lower than 400 μ g/l in 3d.The Japan scholar finds that from natural water body isolated Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Sphingol single-cell (Sphingomonas) have degradation capability to the MC of 2 types of MC-LR and RR, wherein Sphingol single-cell (Sphingomonas) reaches every day 13.0 and 5.4mg/l respectively to the degradation rate of MC-RR and LR, and this is the maximum MC biodegradation rate of reporting in the world at present.Australia scholar's research the degradation pathway of Sphingol single-cell (Sphingomonas) to MC-LR, discovery has at least 3 kinds of Microcystin enzymes (microcystinase) to participate in the catalytic degradation reaction of MC-LR, and the gene of correspondence has been carried out clone and the identification of molecule characteristics.Although also have other foreign scholar the degradability of MC to be studied at pure strain and mixed bacterium, China utilize pure strain efficient degradation MC aspect yet there are no bibliographical information.Therefore filter out and have China's independent intellectual property right, but efficient degradation being removed the microorganism novel bacterial of MC, still is all to have very important significance aspect the application and development in fundamental research.

Summary of the invention:

In the research work of biological degradation MC, we have isolated the microorganism pure strain of the MC that can degrade, are accredited as Ralstonia solanacearum (RS bacterium) through institute of microbiology of the Chinese Academy of Sciences.Discover this bacterial classification 3d starting point concentration can be respectively in the time 50.2 and MC-RR and the LR of 30.1mg/l all degrade, the speed of per day degraded MC-RR and LR is respectively up to 16.7 and 9.4mg/, far above the day degraded MC-RR of foreign latest report and the level of LR speed 13.0 and 5.4mg/l.Simultaneously, find no the research report of RS bacterium degraded MC both at home and abroad.Content of the present invention be will screening the RS bacterium and contained enzyme as a kind of biological catalyst, by adding the pollution of MC in the efficient removal water body.

Description of drawings

Experiment microorganism used therefor bacterial classification is Ralstonia solanacearum bacterium CGMCC0856, is called for short: RS bacterium preservation date on December 18th, 2002, Institute of Microorganism, Academia Sinica of depositary institution, deposit number 0856.

The dynamic process of Fig. 1 Ralstonia solanacearum bacterium degraded MC-RR and LR

1, centrifugal results are cultivated 3 days RS mycetocyte in batches, after joining the 100mM phosphate buffered saline buffer (pH7.0) that contains MC-RR and LR, the process that MC is removed in the degraded of RS bacterium shows, increase along with Initial R S mycetocyte dry weight concentrations, speeding up of MC removed in degraded, in the initial dry weight concentrations of cell is 0.05g/l and when above, can will initially be respectively 51.5 and the MC-RR of 29.5mg/l and the LR removal (Fig. 2 and 3) of all degrading in 24 hours.

The different dry cell weight concentration of Fig. 2 Ralstonia solanacearum bacterium are removed the degraded of MC-RR

The different dry cell weight concentration of Fig. 3 Ralstonia solanacearum bacterium are removed the degraded of MC-LR

2, with behind the broken RS mycetocyte of cultivating of ultra-sonic oscillation, through 18000 rev/mins of high speed centrifugations after 20 minutes, get supernatant liquor both the cell-free extract of RS bacterium (TOC=47.3mg/l) join respectively in the 100mM phosphate buffered saline buffer (pH7.0) that contains MC-RR and LR as zymin.The HPLC collection of illustrative plates shows that the peak height that reaction only just can be observed MC-RR and LR in 20 minutes reduces (Figure 4 and 5) significantly, has respectively found the mesostate A and the C (Figure 4 and 5) of an enzymatic degradation simultaneously.

The HPLC spectrogram of Fig. 4 Ralstonia solanacearum bacterium enzymatic degradation MC-RR

The HPLC spectrogram of Fig. 5 Ralstonia solanacearum bacterium enzymatic degradation MC-LR

The result of study that we carry out shows, is that RS mycetocyte or RS bacterium cell-free extract (enzyme) all have very strong degraded to remove ability to MC-RR and 2 kinds of MC of LR.

Embodiment

1, at first prepare the growth medium of RS bacterium, it consists of (every liter): MgSO ₄7H ₂O 1.0g, KH ₂PO ₄0.5g, K ₂HPO ₄4.0g, NaCl 1.0g, CaCl ₂20.0mg, FeSO ₄5.0mg, ZnCl ₂5.0mg, MnCl ₂4H ₂O 5.0mg, CuCl ₂0.5mg, add MC-RR and LR starting point concentration be respectively 500 and the blue-green algae cell-free extract 100ml of 300mg/l as the carbon source and the nitrogenous source of RS bacteria growing.The initial pH of the substratum of this preparation is about 7.5.Add 100 milliliters of the liquid nutrient mediums that prepare with 500 milliliters of triangular flasks, bottleneck sterilized 20 minutes under High Temperature High Pressure (124 ℃) with absorbent cotton sealing back, then in clean bench under the uviolizing sterilization 20 minutes.

2, in clean bench under the aseptic condition, inoculate 1 milliliter of RS bacterium liquid in the triangular flask substratum, 30 ℃ of temperature, carry out under 150 rev/mins of conditions of shaking speed after batch cultivates 3 days, adopt (12000 rev/mins of centrifugal backs, 10 minutes) remove the method results RS mycetocyte of supernatant liquor, at last the RS mycetocyte of results is kept in 0.5% the NaCl solution as the RS microbial inoculum ,-80 ℃ of cryogenic refrigerators are medium-term and long-term to be preserved if temporarily need not be placed on.

3, getting RS mycetocyte dry weight is that the suspension liquid 20ml of 1.3g/l joins in the 50ml Glass tubing, after inserting this Glass tubing in the frozen water, with ultrasonic cell disruption instrument the RS mycetocyte is carried out fragmentation, condition is: 2 seconds at interval, sonic oscillation 10 seconds, the broken 15 minutes time (each 5 minutes).After cytoclasis finishes, cytoclasis liquid carried out 18000 rev/mins of 20 minutes centrifugal after, slowly pour out the cell-free extract (zymin) of supernatant liquor as the RS bacterium ,-80 ℃ of cryogenic refrigerators are medium-term and long-term to be preserved if temporarily need not also can be placed on.

4, according to the MC concentration that is subjected in blue-green alga bloom polluted drinking water or other water body, the RS bacterium and the zymin of cultivating preparation added by a certain percentage as a kind of biological catalyst fast, safely and efficiently, reach the purpose that rapid efficient degradation is removed MC.

5, have in the biological wastewater treatment process of MC existence for finding, also can add RS bacterium and zymin by a certain percentage, the efficient that makes the sewage-farm remove MC is greatly improved.

6, for the animal that pollutes acute poisoning because of MC, can explore by modes such as oral RS bacterial enzyme preparations and rescue and the toxicide application approach.

7, in sum, the present invention is in screening and cultivates on the basis of Ralstonia solanacearum bacterium, utilizes RS bacterium and zymin efficiently to remove the utilisation technology of MC in the blue algae polluted water body as a kind of biological catalyst.Having very important significance aspect the degraded removal MC pollution that blue-green alga bloom produced.

Claims

1, a kind of employing Ralstonia solanacearum bacterium CGMCC0856 degrades and removes the method for Microcystin (Microcystins), the method of after it is characterized in that the Ralstonia solanacearum bacterium CGMCC0856 cell that will cultivate or the enzyme that extracts preparation from Ralstonia solanacearum bacterium CGMCC0856 cell add the blue-green alga bloom polluted-water by a certain percentage, reach fast, safety and efficient degradation being removed Microcystin.

2, in the method for 1 record of claim the, the cultivation of Ralstoniasolanacearum bacterium CGMCC0856 comprises in batches and the fermentation culture mode.

3, in the method for 1 record of claim the, the enzyme of Ralstoniasolanacearum bacterium CGMCC0856 cell or preparation is removed the method that Microcystin is used.

4, in the method for 1 record of claim the, the enzyme of Ralstoniasolanacearum bacterium CG MCC0856 cell or preparation is applied to Microcystin poisoning animal and human's rescue and detoxifcation.