CN1206174C - Microbe capable of degradation removing microcystin from water bloom - Google Patents

Abstract

A method for efficiently degrading and removing Microcystin (Microcystin) by using a microbial strain (Ralstonia solanacearum, referred to as: RS bacteria), which is characterized in that the cultured RS bacteria and the prepared enzyme are used as biocatalysts, and they are added to the subject in a certain proportion. A method for quickly and efficiently degrading and removing microcystins from water bodies polluted by cyanobacteria blooms. In addition, the Ralstonia solanacearum bacteria cells or the prepared enzyme can also be applied to the rescue and detoxification of animals poisoned by microcystin. It is of great significance in the research and application of environmental pollution control and biomedicine.

Description

A method for removing microcystins in water blooms by microbial degradation

technical field

The patent application of the present invention relates to a microbial strain (Ralstonia solanacearum bacterium CGMCC0856, referred to as: RS bacterium, preservation date December 18, 2002, depository unit, Institute of Microbiology, Chinese Academy of Sciences, preservation number: 0856) screened by us and enzyme preparation degradation The method for removing microcystins (Microcystins, referred to as: MC) is characterized in that the cultured RS bacteria and the prepared enzyme preparation are used as a fast, safe and efficient biocatalyst, and are added to the water polluted by MC in a certain proportion. Finally, a method for efficiently degrading and removing MC. Belongs to the field of biotechnology.

Background technique

Eutrophication of water body refers to the phenomenon of cyanobacteria bloom caused by excessive nitrogen, phosphorus and other nutrients in lakes, reservoirs and rivers, which changes the ecological structure and function of water bodies, leading to the abnormal reproduction and growth of algae, especially cyanobacteria. When the algae bloom is seriously polluted, a thick blue-green lake indigo is formed on the water surface, which emits an unpleasant smell, which not only destroys a healthy and balanced aquatic ecosystem, but also causes harm to humans due to the release of a variety of algal toxins after the algae cells rupture. It poses a serious threat to the safety of drinking water for animals. At present, the frequency and severity of cyanobacterial blooms in freshwater lakes in the world are showing a rapid growth trend, and the occurrence locations are all over the world. 53, 28, 48 and 41% of the lakes in Europe, Africa, North America and South America have different degrees of eutrophication respectively; 54% of the lakes in the Asia-Pacific region are in a state of eutrophication; Different degrees of eutrophication. Since the first report of animals dying from drinking water containing cyanobacteria in 1878, the deaths of aquatic animals, birds, livestock and even humans caused by algal toxins have occurred frequently at home and abroad. How to control the pollution of cyanobacteria blooms and effectively remove algae toxins is an environmental scientific problem facing our country and the world.

The main harm caused by cyanobacterial bloom pollution is the release of various types of algal toxins into the water after the rupture of toxic cyanobacterial cells. The most serious type of algal toxin. The research results show that the main target of MC is the liver, which mainly manifests as hyperemia and swelling of the liver, which can lead to liver hemorrhage and necrosis in severe cases. The mechanism of MC toxicity is to inhibit the activity of protein phosphatase in hepatocytes, induce hyperphosphorylation of cytokeratin, and cause microfilament breakdown, rupture and hemorrhage of mammalian hepatocytes. According to another survey, the presence of MC in drinking water has a great correlation with the incidence of primary liver cancer and colorectal cancer in the population.

Although microbial degradation is a very promising method to remove MC, the ring structure and spacer double bonds of MC are quite stable, and general peptide decomposing enzymes cannot decompose MC, only some special microbial strains have The ability to degrade MC is also an important reason why MC can exist in natural water for a long time. Chinese scholars used sequencing batch biofilm reactors to degrade three types of MC. It was shown that the mixed bacteria had a certain degradative ability to MC, and the degradation of MC was better under aerobic conditions than anaerobic conditions, but within 3 days the MC The removal amount is lower than 400μg/l. Japanese scholars have found that Pseudomonas aeruginosa and Sphingomonas isolated from natural water bodies have the ability to degrade 2 types of MC-LR and RR, among which Sphingomonas The degradation rates of MC-RR and LR by Sphingomonas reached 13.0 and 5.4 mg/l per day, respectively, which is the largest biodegradation rate of MC reported in the world. Australian scholars have studied the degradation pathway of MC-LR by Sphingomonas, and found that at least three microcystinases (microcystinase) are involved in the catalytic degradation of MC-LR, and the corresponding genes cloning and molecular characterization. Although there are other foreign scholars who have studied the degradability of MC by pure strains and mixed strains, there is no literature report on the efficient degradation of MC by pure strains in my country. Therefore, it is of great significance to screen out new microbial strains with my country's independent intellectual property rights that can efficiently degrade and remove MC, both in basic research and application development.

Invention content:

In the research work of biodegrading MC, we isolated a pure microbial strain capable of degrading MC, which was identified as Ralstonia solanacearum (RS bacteria) by the Institute of Microbiology, Chinese Academy of Sciences. The study found that this strain can completely degrade MC-RR and LR with an initial concentration of 50.2 and 30.1 mg/l within 3 days, and the daily average degradation rate of MC-RR and LR is as high as 16.7 and 9.4 mg/l, which is much higher than The daily degradation rates of MC-RR and LR are 13.0 and 5.4 mg/l according to the latest foreign reports. At the same time, there is no research report on the degradation of MC by RS bacteria at home and abroad. The content of the present invention is to use the screened RS bacteria and the contained enzyme as a biocatalyst to efficiently remove the MC pollution in the water body by dosing.

Description of drawings

The microbial strain used in the experiment is Ralstonia solanacearum bacterium CGMCC0856, abbreviated as: RS bacterium. The preservation date is December 18, 2002. The preservation unit is the Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is 0856.

Fig.1 The kinetic process of Ralstonia solanacearum degrading MC-RR and LR

1. The RS bacteria cells cultured in batches for 3 days were harvested by centrifugation, and after being added to 100mM phosphate buffer (pH7.0) containing MC-RR and LR, the process of RS bacteria degrading and removing MC showed that as the initial RS bacteria cells dried With the increase of heavy concentration, the speed of degradation and removal of MC is accelerated. When the initial dry weight concentration of cells is 0.05g/l and above, the initial 51.5 and 29.5mg/l MC-RR and LR can be completely degraded within 24 hours removal (Figures 2 and 3).

Figure 2 Degradation and removal of MC-RR by different cell dry weight concentrations of Ralstonia solanacearum

Fig.3 Degradation and removal of MC-LR by different cell dry weight concentrations of Ralstonia solanacearum

2. After crushing the cultured RS bacteria cells with ultrasonic vibration, after 20 minutes of high-speed centrifugation at 18,000 rpm, take the supernatant and the cell-free extract of RS bacteria (TOC=47.3mg/l) as an enzyme preparation, add into 100 mM phosphate buffer (pH 7.0) containing MC-RR and LR. The HPLC profile shows that the peak heights of MC-RR and LR can be observed to significantly reduce (Fig. 4 and 5) in only 20 minutes of reaction, and an intermediate metabolite A and C (Fig. 4 and 5) of enzyme-catalyzed degradation have been found respectively ).

Fig.4 HPLC spectrum of Ralstonia solanacearum enzyme-catalyzed degradation of MC-RR

Fig.5 HPLC spectrum of Ralstonia solanacearum enzyme-catalyzed degradation of MC-LR

The results of our research showed that both RS bacteria cells and RS bacteria cell-free extract (enzyme) had a strong ability to degrade and remove MC-RR and LR.

Detailed ways

1. First prepare the growth medium of RS bacteria, which consists of (per liter): MgSO ₄ 7H ₂ O 1.0g, KH ₂ PO ₄ 0.5g, K ₂ HPO ₄ 4.0g, NaCl 1.0g, CaCl _20.0mg , FeSO ₄ 5.0mg, ZnCl ₂ 5.0mg, MnCl ₂ 4H ₂ O 5.0mg, CuCl ₂ 0.5mg, add 100ml of cyanobacteria cell-free extract with the initial concentration of MC-RR and LR respectively 500 and 300mg/l as RS bacteria growth carbon and nitrogen sources. The initial pH of the prepared medium is about 7.5. Add 100 ml of the prepared liquid medium into a 500 ml triangular flask, seal the mouth of the bottle with absorbent cotton, and then sterilize at high temperature and pressure (124°C) for 20 minutes, and then sterilize for 20 minutes under ultraviolet radiation in a clean workbench .

2. Under sterile conditions in a clean workbench, inoculate 1 ml of RS bacteria solution into the culture medium of the Erlenmeyer flask, carry out batch culture at a temperature of 30°C and a shaker speed of 150 rpm for 3 days, and then use centrifugation (12000 rpm, 10 minutes) to harvest the RS bacteria cells by pouring off the supernatant, and finally store the harvested RS bacteria cells in 0.5% NaCl solution as the RS bacteria agent. Medium and long term storage.

3. Take 20ml of RS bacterial cell suspension with a dry weight of 1.3g/l and add it to a 50ml glass tube. After inserting the glass tube into ice water, use an ultrasonic cell disruptor to crush RS bacterial cells. The conditions are: 2 seconds, ultrasonic oscillation for 10 seconds, and crushing time of 15 minutes (5 minutes each time). After the cell disruption is complete, centrifuge the cell disruption solution at 18,000 rpm for 20 minutes, and then slowly pour out the supernatant as the cell-free extract (enzyme preparation) of RS bacteria. If it is not used temporarily, it can also be stored at -80°C Long-term storage in the refrigerator.

4. According to the concentration of MC in drinking water or other water bodies polluted by cyanobacteria blooms, the cultured and prepared RS bacteria and enzyme preparations are added in a certain proportion as a fast, safe and efficient biocatalyst to achieve rapid and efficient degradation and removal MC purpose.

5. In the biological treatment process of sewage where MC is found, RS bacteria and enzyme preparations can also be added in a certain proportion, so that the efficiency of MC removal in sewage treatment plants can be greatly improved.

6. For animals acutely poisoned by MC pollution, it is possible to explore the application of rescue and detoxification through oral RS bacteria enzyme preparations and other methods.

7. In summary, the present invention is based on screening and cultivating Ralstonia solanacearum bacteria, using RS bacteria and enzyme preparations as a biocatalyst to efficiently remove MC in cyanobacteria-contaminated water bodies. It is of great significance in degrading and removing MC pollution produced by cyanobacteria blooms.

Claims (4)

1. A method for degrading and removing microcystins (Microcystins) by using Ralstonia solanacearum bacterium CGMCC0856, which is characterized in that the cultured Ralstonia solanacearum bacterium CGMCC0856 cells or the enzyme extracted and prepared from Ralstonia solanacearum bacterium CGMCC0856 cells are added in a certain proportion After the cyanobacteria bloom pollutes the water body, it achieves a method for quickly, safely and efficiently degrading and removing microcystins. 2. In the method described in claim 1, the culture of Ralstonia solanacearum CGMCC0856 includes batch and fermentation culture methods. 3. In the method described in claim 1, the method of removing microcystins by using Ralstonia solanacearum CGMCC0856 cells or the prepared enzyme. 4. In the method described in claim 1, the Ralstonia solanacearum CG MCC0856 cells or the enzyme prepared are applied to the rescue and detoxification of microcystin poisoned animals and humans.

Images