CN110606876B - N-15 labeled microcystin and production method thereof - Google Patents
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Abstract
The invention discloses an N-15 marked microcystin and a production method thereof, wherein the production method comprises the following steps: cleaning the microcystis: selecting microcystis in logarithmic growth phase as algae seeds, performing suspension cleaning on the algae seeds by using deionized water, centrifuging, and collecting algae for at least 2 times; culturing microcystis: inoculating the cleaned microcystis into a special culture medium with N-15, culturing under light with light intensity of 2500-; extracting microcystin: and (3) cracking the microcystis cells in the culture solution, centrifuging and collecting supernatant to obtain crude microcystin extract. The production method is simple and easy to operate, the prepared microcystins have high N-15 labeling rate, the matrix effect is reduced when the microcystins are used as standard substances, the quantification is more accurate, and the method has better application prospect compared with the existing non-standard microcystins.
Description
Technical Field
The invention relates to the technical field of production methods of microcystins, in particular to an N-15 labeled microcystin and a production method thereof.
Background
Microcystis (Microcystis aeruginosa) is the dominant species for the formation of blue algae "fresh water bloom". The method has strong adaptability and high propagation speed, the produced microcystins (Microcystis) are mainly LR, RR, YR and the like, are monocyclic heptapeptides with biological activity, can be enriched in aquatic zooplankton and fish, when algae cells are cracked and die, the toxins can be directly released into a water body, and human beings can induce liver injury, even liver cancer and other diseases by drinking polluted water sources or eating aquatic animals, so that the microcystins content in the water sources and food needs to be quickly, efficiently and accurately extracted and detected to ensure the dietary health. Therefore, the production of high-purity and high-stability microcystin standard products is not only a technical problem which needs to be overcome urgently, but also has great economic significance.
In recent years, the microcystin standard products in domestic markets have few varieties, low purity and low stability, and are difficult to meet the requirement of accurate detection, but the imported standard products have high price and unbalanced supply and demand, and isotope-labeled microcystin internal standard products do not exist. In the mass spectrum detection process, the isotope internal standard can reduce the matrix effect, so that the quantification is more accurate, and meanwhile, the isotope internal standard can be used as a reference standard substance to evaluate the efficiency of the extraction and processing process and the detection process, and can not be replaced by a common standard substance. Therefore, there is a need to develop a simple, efficient and low-cost method for producing isotope-labeled microcystins, which promotes the rapid development of the industrialization of isotope internal standard products and creates higher technical and economic values.
At present, the synthesis route of microcystin molecules in microcystis is not clear, the source of C, H, O in the molecules is complex, if the isotope of the three elements is used for marking microcystin, the cost is higher, the process is uncontrollable, and the nitrogen source in a culture medium is relatively single. At present, no report about isotope-labeled microcystin internal standard products and production thereof is found.
Disclosure of Invention
Aiming at the technical problems, the embodiment of the invention provides the N-15 marked microcystin and the production method thereof, the production method of the microcystin has simple steps and controllable isotope marking, and the yield and the marking rate of the prepared N-15 marked microcystin are high.
The invention provides the following technical scheme:
a method for producing N-15 labeled microcystins, comprising the steps of:
s1, cleaning microcystis: selecting microcystis in logarithmic growth phase as algae seeds, performing suspension cleaning on the algae seeds by using deionized water, and collecting algae after centrifugation; the number of washing times is 3-5;
s2, culturing microcystis: inoculating the cleaned microcystis into a special culture medium with N-15, culturing under light with the light intensity of 2500-;
s3, extracting microcystin: and (3) cracking the microcystis cells in the culture solution, centrifuging and collecting supernatant to obtain crude microcystin extract.
Preferably, in step S2, the special culture medium includes macroelement components and trace element components at the following concentrations:
the macroelement component comprises: n-15 labeled nitrogen source 0.1-1.8g/L, KH2PO4 20-50mg/L、KCl20-40mg/L、MgSO4·7H2O 50-100mg/L、CaCl2·2H2O20-50 mg/L, citric acid 4-8mg/L, FeCl3 4-6mg/L、Na2CO3 10-30mg/L；
The trace element component comprises: h3BO3 1.5-3.0mg/L、MnCl2·4H2O 1.5-2.5mg/L、ZnSO4·7H2O0.1-0.3mg/L、Na2MoO4·2H2O 0.2-0.5mg/L、CuSO4·5H2O 0.05-0.1mg/L、CoCl2·6H2O0.03-0.06mg/L；
The balance being solvent ddH2O。
The special culture medium not only provides macronutrients such as N, P, K, Mg, Ca, Fe and the like necessary for the growth and the propagation of the microcystis and a plurality of trace elements, but also removes CH marked by N-15 in all the macroelements and the trace elements4N2O, no N element, and the only nitrogen source is marked by N-15, which is convenient for producing N-15 marked microcystin, and the isotope marking method of the nitrogen source is simpler and controllable, the culture cost is low, and the isotope marking rate of the microcystin is high.
Such as the use of C-13 for labeling C-containing compounds in the medium and C-13-labeled CO2As a carbon source of microcystis in the culture process, C-13 labeled microcystin can be cultured, but the culture process needs to strictly prevent air (or CO2 in the air) from entering a culture system, and the process is difficult to control; in addition, the culture medium contains a plurality of carbon-containing compounds, and the carbon-containing compounds are completely replaced by the C-13 marker, so that the culture cost is greatly increased; if only the macroelements are replaced by isotopes (Na)2CO3，CO2) The growth of algae cells will be affected without adding other compounds with low C content. Similar problems can occur when other elements H and O are isotopically labeled.
Preferably, the N-15 labeled nitrogen source is one or a combination of several of the following N-15 labeled components: CH (CH)4N2O 100-250μg/L、NaNO3 1.2-1.6g/L、KNO3 1.4-1.8g/L。
Further, the special culture medium also comprises other nitrogen-containing trace element components marked by N-15, preferably, the other nitrogen-containing trace element components marked by N-15 are ferric ammonium citrate 5-8mg/L, EDTANa20.8-1.2mg/L、Co(NO3)2·5H2One or more of O0.04-0.08 mg/L. Therefore, other trace elements can be supplemented, the special culture medium can be further optimized, and N-15 isotopes can be further introduced, so that the specific gravity of the N-15 isotopes in the culture medium is improved, and the labeling rate of the microcystins is improved.
Wherein CH is adopted4N2O100-250 mg/L is most preferred as the nitrogen source because of the N-15 labeled CH4N2O and N in the urea are high in percentage content and are easily absorbed and utilized by plants, so that the production cost of the isotope-labeled microcystins is effectively reduced.
More preferably, the specialized medium comprises the following concentrations of macroelement components and micronutrient components:
the macroelement component comprises: n-15 labeled Nitrogen Source CH4N2O 180-220mg/L、KH2PO4 40-50mg/L、KCl 30-40mg/L、MgSO4·7H2O 60-90mg/L、CaCl2·2H235-50mg/L of O and 5-7mg/L, FeCl of citric acid3 5-6mg/L、Na2CO3 15-25mg/L；
The trace element component comprises: h3BO3 2.0-3.0mg/L、MnCl2·4H2O 1.8-2.3mg/L、ZnSO4·7H2O0.2-0.3mg/L、Na2MoO4·2H2O 0.3-0.5mg/L、CuSO4·5H2O 0.06-0.1mg/L、CoCl2·6H2O0.04-0.06 mg/L. The balance being solvent ddH2O。
Preferably, the inoculation ratio of the microcystis to the special culture medium is 1:10-1: 30.
Preferably, during the culture process of the microcystis, the algae liquid obtained by the first transfer is continuously transferred for 3-5 times as the algae seed so as to continuously improve the concentration of microcystin in the culture product and the N-15 marking rate of the microcystin.
Preferably, the cracking method is high temperature treatment or ultrasonic treatment, wherein the conditions of the high temperature treatment are as follows: treating at 100-; the conditions of the ultrasonic treatment were: centrifuging the culture solution, collecting algae cell precipitate, adding appropriate amount of water into the precipitate, and performing ultrasonic treatment at 50-70 deg.C for 15-30 min.
Preferably, the method also comprises a purification step of the microcystins:
performing vacuum rotary evaporation on the crude microcystin extract, and performing primary purification through an adsorption column to obtain primary pure liquid;
concentrating the primary pure solution by vacuum rotary evaporation, purifying the concentrated toxin primary pure solution again by HPLC, concentrating and freeze-drying the product to obtain high-purity N-15 labeled microcystin powder.
The invention also provides N-15 marked microcystin which is prepared by the production method.
The invention has the following beneficial effects:
1. the production method is simple and easy to operate, has strong practicability, and is suitable for industrial production of products.
2. The microcystin prepared by the invention has high N-15 labeling rate and high purity, can be used as a standard substance to reduce matrix effect, has more accurate quantification, and has better application prospect compared with the existing non-standard microcystin.
3. The invention selects the nitrogen source as the isotope labeled element, and has the advantages of easy operation, easy quantification, low culture cost and no inhibition of the growth of algae cells compared with other nutrient elements.
Drawings
FIG. 1 is a HPLC-UV chromatogram after purification of microcystin-LR;
FIG. 2 is a HPLC-UV chromatogram after purification of microcystin-RR.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Experimental materials:
1. micro-capsule algae: adopting microcystis aeruginosa FACHB-315 mainly producing MC-LR and microcystis aeruginosa FACHB-912 mainly producing MC-RR as experimental algae species, which are purchased from freshwater algae seed bank of Chinese academy of sciences; the microcystin standard substance adopts the following components: the specification of the microcystin LR and the microcystin RR are both 10 mug/ml, and the microcystin LR and the microcystin RR are purchased from Beijing Wanjiayi biological science and technology limited company.
2. Culture medium:
(1) macroelement mother liquor: n-15 labeled CH4N2O 100-250g/L；KH2PO4 20-50g/L；KCl20-40g/L；MgSO4·7H2O 50-100g/L；CaCl2·2H2O 20-50g/L；Citric acid 4-8g/L；FeCl34-6g/L；Na2CO310-30 g/L; the above mother solutions are prepared separately. Sterilizing and cooling the macroelement mother liquor, and storing at 4 ℃, wherein the dosage of each macroelement mother liquor is 1 ml/L.
(2) The formula of the microelement mother solution is as follows: h3BO3 1.5-3.0g/L；MnCl2·4H2O 1.5-2.5g/L；ZnSO4·7H2O 0.1-0.3g/L；Na2MoO4·2H2O 0.2-0.5g/L；CuSO4·5H2O 0.05-0.1g/L；CoCl2·6H2O0.03-0.06 g/L. After the above microelement mother liquor is prepared, sterilizing, cooling and storing at 4 deg.C, each dosage is 1 ml/L.
EXAMPLE 1 production of N-15 labeled microcystins
1. The production method of the N-15 marked microcystin comprises the following steps:
s1, cleaning microcystis: selecting microcystis cultured in a normal BG11 culture medium in a logarithmic phase as algae seeds, performing suspension cleaning on the algae seeds by using deionized water, and collecting algae after centrifugation; cleaning for 3-5 times until removing nitrogen source and other nutrient salts in the original culture medium, and collecting microcystis.
S2, culturing microcystis: inoculating cleaned microcystis into a special culture medium with N-15, culturing under light with light intensity of about 4000LUX, shaking culture flask uniformly for 2 times per day, and culturing for 10-30 days to obtain culture solution; sampling every week to determine the microcystin concentration and the labeling rate of N-15 in the microcystin.
In this example, experimental groups a1-a6 were set, each experimental group cultured microcystis according to the above method of this example, the inoculation ratio of the special culture medium was 1:10, the culture temperature was 25 ℃, and the light intensity was 4000LUX, and the culture medium formulation of each experimental group was as follows:
(1) the formula of the special culture medium for A1 group and A4 group is as follows:
the macroelement component comprises: n-15 labeled Nitrogen Source CH4N2O 100mg/L、KH2PO4 20mg/L、KCl20mg/L、MgSO4·7H2O 50mg/L、CaCl2·2H2O20 mg/L, citric acid 4mg/L, FeCl3 4mg/L、Na2CO3 10mg/L；
The trace element component comprises: h3BO3 1.5mg/L、MnCl2·4H2O 1.5mg/L、ZnSO4·7H2O 0.1mg/L、Na2MoO4·2H2O 0.2mg/L、CuSO4·5H2O 0.05mg/L、CoCl2·6H2O 0.03mg/L。
(2) The formula of the special culture medium for A2 group and A5 group is as follows:
macroelement components: n-15 labeled CH4N2O 200mg/L、KH2PO4 45mg/L、KCl 35mg/L、MgSO4·7H2O 70mg/L、CaCl2·2H2O40 mg/L and citric acid 6mg/L, FeCl3 5mg/L、Na2CO320mg/L；
Trace element components: h3BO3 2.5mg/L、MnCl2·4H2O 2mg/L、ZnSO4·7H2O 0.2mg/L、Na2MoO4·2H2O 0.4mg/L、CuSO4·5H2O 0.08mg/L、CoCl2·6H2O 0.05mg/L。
(3) The formula of the special culture medium for A3 group and A6 group is as follows:
the macroelement component comprises: n-15 labeled Nitrogen Source CH4N2O 250mg/L、KH2PO4 50mg/L、KCl40mg/L、MgSO4·7H2O 100mg/L、CaCl2·2H2O 50mg/L, citric acid 8mg/L, FeCl3 6mg/L、Na2CO3 30mg/L；
The trace element component comprises: h3BO3 3.0mg/L、MnCl2·4H2O 2.5mg/L、ZnSO4·7H2O 0.3mg/L、Na2MoO4·2H2O 0.5mg/L、CuSO4·5H2O 0.1mg/L、CoCl2·6H2O 0.06mg/L。
The algae solution concentration and toxin concentration of the culture solution after 20 days of culture of each experimental group are measured, and the results are shown in the following table:
TABLE-Effect of different special media on the cultivation of microcystis
In other embodiments, CH may be present at the above concentrations4N2Replacing O with 1.2-1.6g/L NaNO31.4-1.8g/L KNO35-8mg/L ferric ammonium citrate, 0.8-1.2mg/L EDTANa2Or 0.04-0.08mg/L Co (NO)3)2·5H2And O. Furthermore, the above nitrogen sources may be used in combination.
S3, extracting microcystin: carrying out high-temperature treatment on the microcystis cells in the culture solution under the conditions of: treating at 105 deg.C for 30 min; centrifuging and collecting the supernatant to obtain the crude extract of microcystin.
In other embodiments, sonication may be used: collecting culture solution, centrifuging at 4000rpm for 10min, removing supernatant to obtain algae cell precipitate, and adding appropriate amount of ddH2And O, performing ultrasonic treatment at 60 ℃ for 20min, centrifuging at 6000rpm for 8min, and taking supernatant as crude extract of microcystin.
Test example 2
(1) A plurality of experimental groups were set up according to the conditions of Table two below, using the special medium A2 in example 1, crude extract of microcystin was prepared and primarily extracted according to the method of example 1, and the algae concentration and toxin concentration of the culture solution after 20 days of culture were measured, and the results are shown in Table two below.
Influence of different culture conditions on microcystins
The detection method of the MC-LR/MC-RR concentration comprises the following steps: the detection wavelength was 238nm by HPLC detection. Chromatographic conditions, the chromatographic column is Thermo company C18(150mm 2.1mm, 3um), the column temperature is 35 ℃, the sample injection amount is 25 microliter, and the mobile phase is water and methanol (40: 60).
The microcystin has specific absorption peak at 238nm wavelength, different microcystin isomers have different retention time in HPLC, and compared with the retention time of standard microcystin, the composition of the microcystin in the sample can be determined, and the content of the microcystin in the sample can be calculated according to the peak area.
The method for measuring the labeling rate of N-15 in MC-LR/MC-RR comprises the following steps: by utilizing HPLC-MS, scanning N-15 marked MC-RR and N-15 marked MC-LR by adopting ESI in a positive/negative ion scanning mode, carrying out qualitative analysis according to the characteristic mass-to-charge ratio m/z of different microcystin molecules under the condition of mass spectrum, parent ion fragments and retention time, then obtaining the total marked amount of each microcystin in a peak area summation mode, and further calculating the N-15 marking rate of each toxin.
The specific operation method refers to the entry and exit inspection and quarantine industry standard SN/T4319-2015, wherein the specific adopted qualitative ion pair and quantitative ion pair of the two isotope-labeled microcystins are shown in the following table III:
TABLE III qualitative ion pair and quantitative ion index
| Compound (I) | Parent ion (m/z) | Quantitive ion (m/z) | Qualitative ion (m/z) |
| N-15-microcystin-LR | 1006 | 135.1 | 127.2 |
| N-15-microcystin-RR | 533 | 135.1 | 103.1 |
(2) The experimental groups 11 to 16 were set according to the culture conditions of the experimental group 2 and the experimental group 7, and the algal solutions obtained by the first culture were used as algal species, and after the algal species were transferred to culture media of different times and expanded, the algal solution concentration, the toxin MC-LR/RR concentration, and the isotopic labeling rate in the crude extract were measured, and the measurement results are as follows:
TABLE IV Effect of switching times on microcystin concentration and labeling Rate
From the results in table four, it can be seen that: in the process of culturing the microcystis, the algae liquid obtained by the first culture is taken as an algae seed to be continuously transferred for a plurality of times, so that the concentration of microcystin in the culture product and the N-15 marking rate of the microcystin can be continuously improved.
Example 3 purification of microcystins
(1) Primary purification: subjecting the crude extract (original MC-LR/RR concentration of 1.89 + -0.16 ppm and 1.36 + -0.04 ppm) to vacuum rotary evaporation at 40 deg.C, and performing primary purification with adsorption column to obtain primary pure solution. In this embodiment, the crude extract is primarily purified by using D101 macroporous resin as a filler, and the steps are as follows:
A. pretreatment of the adsorption column: 1000g of macroporous adsorbent resin swollen (95% ethanol) overnight was loaded into a 100mm by 1000mm column with the bottom plugged with 6 layers of gauze;
B. rinsing the column with 1000ml of 95% ethanol for 2 times;
C. with 1000ml ddH2Rinsing the column for 4 times;
D. sampling the crude extract of the algal toxins;
E. an elution step: in turn, respectively using 1000ml of ddH2O is eluted 2 times, 1000ml 20% ethanol is eluted 2 times, 1000ml 60% ethanol is eluted 8 times.
(2) And (3) secondary purification: carrying out vacuum rotary evaporation on the primary pure solution of the microcystin obtained in the last step at the temperature of 40 ℃ for concentration; purifying the concentrated primary pure liquid by an HPLC method, wherein the purification conditions are as follows:
column temperature, room temperature; the detection wavelength is 238 nm;
flow rate 1ml/min (analytical column), 20ml/min (preparative column);
the mobile phase is water and methanol in a volume ratio of: 45:55.
The types and models of the chromatographic columns are: bondapakTM C18Preparation of column (19mm 300mm)
The resulting purification chromatograms are shown in fig. 1 and fig. 2, respectively. And collecting the target fraction, removing methanol by rotary evaporation, and freeze-drying to obtain pure solid powder, namely high-purity N-15 labeled microcystin powder.
Purity determination of the microcystin powder:
taking the microcystin powder as a sample, preparing a solution with a certain concentration after methanol redissolution, measuring the peak area under the same liquid quality condition, comparing the peak area with the peak area of a standard product, calculating the real concentration of the prepared solution, and obtaining the ratio of the real concentration to the concentration of the prepared sample as the purity of the measured microcystin. The purity of the microcystin powder marked by N-15 can reach 96-98% by comparing with a standard product of microcystin.
The full-scale marking rate of the N-15 marked microcystin powder is as follows:
the actual detection result is MC-LR: 52.34% + -2.41%; MC-RR: 62.96% + -0.03%.
It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.
Claims (4)
1. A method for producing N-15 labeled microcystins, comprising the steps of:
cleaning microcystis aeruginosa: selecting Microcystis aeruginosa in logarithmic phase as an alga seed, performing suspension cleaning on the alga seed by using deionized water, and collecting alga bodies after centrifugation; washing for at least 2 times;
culturing microcystis aeruginosa: inoculating the cleaned microcystis aeruginosa into a special culture medium with N-15, culturing under light with light intensity of 3000-; the inoculation ratio of the microcystis aeruginosa to the special culture medium is 1:10-1: 30; in the process of culturing the microcystis aeruginosa, the algae liquid obtained by the first transfer is taken as an algae seed to be continuously transferred for 3-5 times so as to improve the microcystin concentration and the marking rate of N-15 in the culture product;
extracting microcystin: cracking microcystis aeruginosa cells in the culture solution, centrifuging and collecting supernatant to obtain crude microcystin extract;
the special culture medium comprises macroelement components and trace element components with the following concentrations:
the macroelement component comprises: n-15 labeled Nitrogen Source CH4N2O 180-220mg/L、KH2PO4 40-50mg/L、KCl 30-40mg/L、MgSO4·7H2O 60-90 mg/L、CaCl2·2H235-50mg/L of O and 5-7mg/L, FeCl of citric acid3 5-6 mg/L、Na2CO3 15-25 mg/L；
The trace element component comprises: h3BO3 2.0-3.0 mg/L、MnCl2·4H2O 1.8-2.3 mg/L 、ZnSO4·7H2O 0.2-0.3 mg/L、Na2MoO4·2H2O 0.3-0.5 mg/L、CuSO4·5H2O 0.06-0.1 mg/L、CoCl2·6H2O0.04-0.06 mg/L; the balance being solvent ddH2O。
2. The production method according to claim 1, wherein the special medium further comprises an N-15 labeled additional nitrogen-containing trace element component; the N-15 marked other nitrogen-containing trace element component is 5-8mg/L, EDTANa mg of ferric ammonium citrate2 0.8-1.2 mg/L、Co(NO3)2·5H2One or more of O0.04-0.08 mg/L.
3. The production method according to claim 1, wherein the method of pyrolysis is high temperature treatment or ultrasonic treatment, wherein the conditions of high temperature treatment are as follows: treating at 100-; the conditions of the ultrasonic treatment were: centrifuging the culture solution, collecting algae cell precipitate, adding appropriate amount of water into the precipitate, and performing ultrasonic treatment at 50-70 deg.C for 15-30 min.
4. The method of claim 1, further comprising a step of purifying the microcystin:
performing vacuum rotary evaporation on the crude microcystin extract, and performing primary purification through an adsorption column to obtain primary pure liquid;
concentrating the primary pure solution by vacuum rotary evaporation, purifying the concentrated toxin primary pure solution again by HPLC, concentrating and freeze-drying the product to obtain high-purity N-15 labeled microcystin powder.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168561A (en) * | 2007-11-27 | 2008-04-30 | 江南大学 | Method for separating and purifying microcystin |
| CN104531584A (en) * | 2014-12-31 | 2015-04-22 | 山东出入境检验检疫局检验检疫技术中心 | 15 N stable isotope marked blue-green algae and biological culture method thereof |
| WO2018206715A2 (en) * | 2017-05-09 | 2018-11-15 | Cyano Biotech Gmbh | Modified microcystins and nodularins |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168561A (en) * | 2007-11-27 | 2008-04-30 | 江南大学 | Method for separating and purifying microcystin |
| CN104531584A (en) * | 2014-12-31 | 2015-04-22 | 山东出入境检验检疫局检验检疫技术中心 | 15 N stable isotope marked blue-green algae and biological culture method thereof |
| WO2018206715A2 (en) * | 2017-05-09 | 2018-11-15 | Cyano Biotech Gmbh | Modified microcystins and nodularins |
Non-Patent Citations (3)
| Title |
|---|
| 史红星 等.微囊藻毒素产生过程中氮素作用的同位素示踪研究.《科学通报》.2008,第53卷(第4期), * |
| 微囊藻毒素产生过程中氮素作用的同位素示踪研究;史红星 等;《科学通报》;20081231;第53卷(第4期);第407-412页 * |
| 微囊藻毒素的厌氧降解途径研究;韩振莲;《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》;20170215(第2017年02期);B027-991 * |
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