CN1294190A - Method for culturing nostoc - Google Patents
Method for culturing nostoc Download PDFInfo
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- CN1294190A CN1294190A CN 99120005 CN99120005A CN1294190A CN 1294190 A CN1294190 A CN 1294190A CN 99120005 CN99120005 CN 99120005 CN 99120005 A CN99120005 A CN 99120005A CN 1294190 A CN1294190 A CN 1294190A
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Abstract
A method for culturing nostoc includes preparing culture liquid by sequentally and proportionally adding 90 solutions of magnesium sulfate, calcium chloride, citric acid, trace element A5, etc to water, preparing alga seeds by washing in alcohol solution, purifying, etc praparing inoculum, culturing, real-time monitor, screening, increasing mechanical strength of spherical bodies on screen, and drying. Its advantages are no pollution, high safety, short growth period and rich nutrients.
Description
The present invention relates to biological technical field, more specifically relate to a kind of method of cultivating Nostoc.
The beads algae is the Cyanophyta spore plants, it is a thread algae " genus ", its thallus is combined into colony by the very thin algal filament of many diameter numbers micron, colony's shape is different because of " kind ", as deliver vegetables (Nostocflageliforme Kuetz), Nostoc commune (Nostoc commune), Nostoc (Nostocsphaeroides) be hair-like, auricularia auriculajudae shape or bead (pearl) shape respectively under fresh state, the change of contracting to some extent of varying degree ground, dry back.Because blue-green algae is prokaryotic organism, also is classified as bacterium.Chinese Academy of Sciences typical case culture collection center hydrobiont institute algae kind storehouse (FACHB-collection, CAS), places such as France, the U.S., Japan, Germany have all collected many beads algae algae kinds.The beads algae is not only generally taken place at nature, and is the whole food of many nationalitys since ancient times.Nostoc, deliver vegetables and three kinds of conventional food that the beads algae all is some regional people of China such as Nostoc commune, wherein, Nostoc is documented in Compendium of Material Medica, " Compendium of Materia Medica addendum ", " the rich record of edible wild herbs ", " Shaanxi herbal medicine " and ancient books such as " mansion, Wuzhou will " because of its abundant nutritive value.Nostoc has effects such as dietotherapy, health care, nourishing, and natural collection items morning session price is up to 1600 yuan of per kilogram Renminbi.Deliver vegetables and find a good sale at home and abroad, sharp contradiction has taken place in development of resources and grassland protection.The formal name used at school of Nostoc is spherical beads algae." Nostoc " be its in China the title by traditional appellation.Pass on from one to another alchemist, the Ge Hong of pharmacy man of Shanxi court, degraded in rank during the official in office when the Northwest of Hubei Province town and take to one's bed and never leave it again.Because the people have no means of livelihood at that time, the food that several nothings are allayed one's hunger fortunately produces the food of a kind of similar auricularia auriculajudae in Dang Di rice field, the mountain region, and people allay one's hunger with this.Therefore Ge Hong pulls through their difficulties, and has cured the disease of oneself.Ge Hong finds, this food to regain one's strength, regulating the qi flowing in the channels, beauty treatment, to treat some illnesss very useful.He cures the disease as medicine this food for other people, and, when he is obtained reactivating as returning by imperial court afterwards, this food being presented to the goddess cured nyctalopia, therefore people rename as this former thing that calls the field auricularia auriculajudae and make Nostoc.The natural production status of Nostoc declines, and the contradiction that demand and collection formula are produced can not be avoided.Along with mankind's activity aggravation, in recent years, the dwindling gradually vegetatively of Nostoc, output also constantly reduces.Five tons of its natural annual production less thaies, and reduce day by day, do not satisfy demand far away, along with the development of tourism resources in recent years, the over-drastic collection has brought ecology, environmental disruption and natural resources atrophy again.Nowadays the high-yield field of many Nostoc of past has been difficult to find Nostoc " celestial track ".This chemical substance with a large amount of synthetic that use of modern agriculture is relevant, changes with Global climate change, environmental aspect also to have some relations.There is significant limitation in the Nostoc producing region, though its distribution may be found in many in the world places, but actual can form group in limited area, even in a zone, its distribution also presents special selectivity, or a water every, or face each other in the mountain on the other way round, or locate the Yang Yin on mountain, all might constitute the difference that Nostoc can maybe can not be grown.This specific specificity explanation is under natural condition, and the growth of Nostoc is very strict to the selection of natural condition.The growth conditions that Nostoc requires is diversified, as mineral nutrition, and temperature, illumination, the potential of hydrogen of water, and have or not obnoxious flavour, agricultural chemicals, natural enemy etc.Global warming, environmental change and to pollute be not can change with the will of some people in a short time desires to ask the good harvest of grain, and the use of agricultural chemicals and chemical fertilizer is also inevitable.So contradiction has just been come out, be that Nostoc output descends on the one hand, and people's increase in demand; Be that reduce in the place of production on the other hand.But see the report of Nostoc artificial culture so far.
The purpose of this invention is to provide a kind of method of cultivating Nostoc, easy to implement the method, easy to operate, solved the Nostoc growth soon, output height, the problem that nutrition is good.
In order to achieve the above object, the present invention has adopted following technical measures: carry out the breed of Nostoc, the system that at first is nutrient solution is joined, nutrient solution nutritive salt utilizes commercially available crude salt, is dissolved in tap water and gets final product, and nutritive medium adopts the BG110 culture medium prescription, be beneficial to the Nostoc growth, nutritive salt is sequentially added into during preparation, reduces sedimentary generation, then the nutrient solution for preparing is sterilized.Next is that the algae kind prepares: A, original seed, and field acquisition algae kind, the laboratory separation and purification obtains the assorted algae of nothing of different strains, aseptic, easy cultivation, output height, algae strain of good quality; B, hormogon[ium (hormocystangium) are cultivated, and utilize and cultivate the spheroplast that gets, and carry out hormocystangium in the laboratory and induce in a large number, with hormocystangium seed then, cultivate in a large number; C, the expansion production of hybrid seeds, with the spheroplast fragmentation or the inductive hormocystangium enlarged culturing of original seed, the inoculum that the miniature colony of acquisition cultivates as scale.3, cultivate,, can take to cultivate step by step leaving standstill mass-producing when cultivating Nostoc because once the cycle of Pei Yanging be 9-11 days, in 10 days, be difficult to reach ideal size (the about 0.5cm of diameter) by the cultivation of small colony.This just requires constantly to shift culture to another incubator or replacing nutrient solution, increases the volume of spheroid colony step by step.4, cultivate monitoring in real time, set up monitoring condition and standard.The equipment and instrument that needs: thermometer, photometer, soda acid indicating meter, microscope (band fluorescence), microcomputer etc., and conventional laboratory equipment and technical support.All data are set up database, computer management.5, spheroplast screening results, compared with other little algae rice, the results of Nostoc will be easy to many.As seen it get the large group naked eyes, do not have a floatability, and this just collects by filtering method easily.6, improve the physical strength that sieve is gone up spheroplast, grow that often physical strength is low for fast spheroplast, after drying, can not well keep shape more attractive in appearance, can not resile after before edible, heavily absorbing water.So the strong virtue of machinery at the high spheroplast of results prerequisite just seems particularly important.7, results are with dry, with the frond that has improved physical strength dry, air-dry or dry and all can.
The present invention compared with prior art, have the following advantages and effect: equipment is simple, and is easy to operate, pollution-free, nutrition is good, and is safe, absorbing carbon dioxide in the process that Nostoc is cultivated, and in atmosphere, give off oxygen, can play the effect that purifies air, not produce harmful or toxic substance, the nutrient solution salinity is low, potential of hydrogen approaches natural water, reusable edible.Process of growth is short, and the Nostoc growth is fast, just can finish logarithmic growth in 10 days; the efficient height, the culture of an amount of inoculation, amount of dry matter can reach every cubic metre of nutrient solution of 1g/L and can gather in the crops the dried algae of 1kg after 10 days; can be used for the cultivation of all aquatic beads algaes of spherule, be widely used in the town and country mass-producing and cultivate.
Embodiment:
Its step of method of cultivating Nostoc is as follows:
1, nutrient solution preparation (1000ml): the order that the storage solution that will weigh up or prepare adds entry is: sal epsom 0.07-0.08g, calcium chloride 0.03-0.042g, citric acid 0.005-0.007g, ammonium ferric citrate 0.005-0.007g, oxalic acid Sequestrene AA 0.001-0.002g, yellow soda ash 0.03-0.05g, soil extract 2.5-3.5ml, micro-A51ml, dipotassium hydrogen phosphate 0.07-0.09g, the nutrient solution for preparing uses with sterilization under the 118-122 ℃ of hot conditions 20 minutes, cooling back.
2, the algae kind prepares: (1) original seed: the Nostoc spheroplast of field acquisition was washed for 20 seconds with 70% ethanolic soln is quick, wash 3 times in the nutrient solution of bacterium that went out then.This spheroplast is placed in the glass paste device, adds a small amount of aseptic culture fluid, slightly homogenate.1.2% agar plate of preparation aseptic culture fluid, the homogenate of above-mentioned spheroplast is coated on the agar plate, with sealing film culture dish is sealed, place 26-36 ℃ of illumination box to cultivate, after about 1 month, just can see having little spheroplast to be grown on the agar plate, Here it is nostoc sphaeroides.
(2) axenic purification of algae kind: with the spheroplast homogenate fragmentation that aforesaid method obtains, insert and contain 0.5% sucrose, the 100ug/l penbritin in the nutrient solution of 0.1% yeast extract, is put under the dark condition, cultivates 24 hours for 30-35 ℃.Centrifugal then, aseptic culture fluid washing 3 times is coated with dull and stereotyped the cultivation with frond with (1), obtains spheroplast.Be sterile culture thing (but whether microscopic inspection has bacterium).
(3) the hormocystangium production of hybrid seeds: above-mentioned isolating algae kind is inoculated in nutrient solution, aerated culture in the glass incubator.Air flow is 1-10L/ minute.After about 10 days, the spheroplast of the about 3-10mm of collection diameter with aseptic distilled water wash once is re-applied in the fresh sterile nutrient solution then, places illumination box, at 25-30 ℃, and 5-100 μ E.m
-2.s
-1Induced under the light intensity 2-3 days.Change the hormocystangium that is suspended in the nutrient solution in the fresh medium static or aerated culture, preparation algae kind.
(4) enlarge the production of hybrid seeds: with spheroplast homogenate, insert nutrient solution, according to (1) culture condition, aerated culture, preparation algae kind.
3, preparation algae kind: the key (promptly cultivating step by step) that obtains bigger ideal spheroplast is the cultivation step by step of spheroplast.Therefore the culture preparation can be divided into for 4 steps: (1) original seed preparation (seeing 1); (2) broken thing of original seed or hormocystangium are inserted nutrient solution, with 1-10L/ minute speed aerated culture, and impose illumination in the glass incubator, intensity is at 10-800 μ E.m
-2.s
-1Between.After 10-30 days, collect the bead body, aseptic culture fluid washing 3 times.This spheroplast claims the one-level culture.(3) with the one-level culture set by step (2) cultivate once again, after 10-30 days, spheroplast is grown up to about diameter 1mm, claims that this spheroplast is the secondary culture, the secondary culture after the washing just can be used as the inoculum of a large amount of cultivations.
4, cultivate
(1) (bioreactor, photobioreactor): selection daylighting is good, the water source is local easily as cultivating the place to cultivate place and incubator.Can be in the open or in the plastic greenhouse, or bioreactor is settled at glasshouse in place with good conditionsi.Light intensity is at 10 μ E.m
-2.s
-1And full sun all can between the light intensity.Bioreactor has an injection port (to add nutrient solution and inoculation, on reactor top) leakage fluid dram (the discharging nutrient solution is at reactor bottom), a ventage (at reactor bottom), and be equipped with the stainless steel sift of an about 0.5cm in aperture, be used for screening qualified spheroplast.
(2) inoculation with cultivate: the secondary culture as inoculum, is inoculated in bioreactor with the ratio of every liter of nutrient solution 0.5-2g weight in wet base.(ventilation is played and is stirred and provide CO with the ventilation of charge of air pump
2Effect) ventilate from ventage with the speed of 1-10L/min.In order to see the pollution of weak breath body to culture, ground enters before the reactor, is the concentrated sulfuric acid aqueous solution filtration of 1-3 with cotton and pH value with gas.Air flow is controlled by valve.
5, monitoring in real time
Monitoring condition and the standard set up.Temperature: 10-30 ℃; Light intensity 10-800 μ E.m
-2.s
-1: pH:8.0-8.9; Other microorganism such as bacterium, fungi and assorted algae detect: (1) is coated with flat band method: nutrient solution is coated on the minimum medium of microbial culture, after 37 ℃ of temperature were trained more than 24 hours, whether observation was occurred by bacterium colony; (2) microscopic examination: the nutrient solution that takes a morsel, add DAPI (a kind of fluorescence dye) to final concentration 1 μ g/ml dyeing 5 minutes, under fluorescent microscope, observe with ultraviolet excitation.If finding has bluish-green motion bright spot, be contaminating microorganisms.(3) whether the microscopically direct viewing has assorted algae to exist.Standard Methods for Water and Waste Water one book that the nutrient concentration detection method is published referring to the U.S..
If the value that detects not in above-mentioned scope, then needs to solve by relative measures.As: temperature can be by regulating room temp control; Avoid severe cold and heat season; Light intensity: evening that light intensity is low or cloudy day can be started set lights, take light shading method when light intensity is too high.PH regulates by acid or alkali.Then need when microbial contamination is serious thoroughly to change nutrient solution, and reactor is carried out sterilising treatment.Then inject fresh medium during under-nutrition or add nutritive salt or the like.
6, screening results: at first make the stainless steel sieve that the hole is slightly less than 0.5cm, desire is gathered in the crops culture sieve.Spheroplast about diameter 0.5cm is stayed on the sieve, and these are exactly the product (be called sieve and go up spheroplast) that we will obtain.Microcommunity of sieving and nutrient solution continue to cultivate after processing.
7, improve the physical strength that sieve is gone up spheroplast: diameter improves physical strength greater than the spheroplast of 0.5cm by following arbitrary approach after will sieving.
(1) Na of adding 1.5g/L in nutrient solution
2CO
3, spheroplast is transferred to this containing in the fresh medium, the similarity condition aerated culture during with cultivation 3-5 days, results get final product.
(2) qualified spheroplast is statically placed in the original fluid, (1-10 ℃) leaves standstill 10-15 under low temperature.And note, when static, reactor be placed to be less than 10 μ E.m
-2.s
-1Light intensity under to prevent the cracking of spheroplast.
8, drying: the spheroplast of results is if untimely drying will rot.When dry,, therefore very easily be bonded together with holding vessel because the polysaccharide that the spheroplast born of the same parents have outward has high viscosity.Therefore when drying, frond is placed on and just can avoids on silk or the silk screen cohering.The exsiccant method has: sieve is done, air-dry or oven dry.
Claims (5)
1, a kind of method of cultivating Nostoc is characterized in that:
A, nutrient solution system are joined; The order that the storage solution for preparing is added in the entry is that sal epsom, calcium chloride, citric acid, ferric ammonium citrate, oxalic acid Sequestrene AA, yellow soda ash, soil extract, micro-A5, dipotassium hydrogen phosphate are prepared in proportion;
B, algae kind prepare: at first the Nostoc spheroplast being washed fast with ethanolic soln, secondly is with algae kind axenic purification, and the 3rd is the hormocystangium production of hybrid seeds, and the 4th is to enlarge the production of hybrid seeds;
C, preparation inoculum;
D, cultivation;
E, monitoring in real time;
F, screening results;
J, raising sieve are gone up the physical strength of spheroplast;
H, drying.
2, a kind of method of cultivating Nostoc according to claim 1 is characterized in that nutrient solution system joins:
Sal epsom 0.07-0.08g;
Calcium chloride 0.03-0.042g;
Citric acid 0.005-0.007g;
Ferric ammonium citrate 0.005-0.007g;
Oxalic acid tetraethoxide disodium salt 0.001-0.002g;
Yellow soda ash 0.03-0.05g;
Soil extract 2.5-3.5ml;
Trace element A5 1ml;
Dipotassium hydrogen phosphate 0.07-0.09g.
3, a kind of method of cultivating Nostoc according to claim 1, it is characterized in that the algae kind prepares: at first the Nostoc spheroplast is washed with 70% ethanolic soln, in the nutrient solution of bacterium that went out, wash, spheroplast is placed in the glass homogenizer, add aseptic culture fluid, homogenate with spheroplast is coated on the agar plate again, with sealing film culture dish is sealed, and places 26 ℃ of illumination boxs to cultivate; Next is an algae kind axenic purification, with spheroplast homogenate fragmentation, is pressed into and contains 0.5% sucrose, the 100ug/L penbritin, in the nutrient solution of 0.1% yeast extract, put under the dark condition, cultivated 24 hours at 30-35 ℃, centrifugal, the aseptic culture fluid washing obtains spheroplast; The 3rd is the hormocystangium production of hybrid seeds, and isolating algae kind is inoculated in nutrient solution, and aerated culture in the glass incubator is collected spheroplast and washed with sterile distilled water, places illumination box, at 25-30 ℃, and 5-10 μ E.m
-2.s
-1Induced under the light intensity 2-3 days, and changed hormocystangium in the nutrient solution static or aerated culture, preparation algae kind; The 4th is to enlarge the production of hybrid seeds, with spheroplast homogenate, inserts nutrient solution, aerated culture, preparation algae kind.
4, a kind of method of cultivating Nostoc according to claim 1 is characterized in that preparing inoculum, spheroplast is cultivated step by step at first original seed preparation; Next is that broken thing of original seed or hormocystangium are inserted nutrient solution, with 1-10L/ minute aerated culture, and imposes illumination in the glass incubator, and intensity is at 10-800 μ E.m
-2.s
-1Between, collect the bead body after 10-30 days, the aseptic culture fluid washing obtains the one-level culture; The 3rd is that the one-level culture was cultivated once by second step again, and after 10-30 days, spheroplast is grown up to diameter 1mm, is the secondary culture, and the secondary culture after the washing is as the inoculum of cultivating.
5, a kind of method of cultivating Nostoc according to claim 1 is characterized in that improving the physical strength that sieve is gone up spheroplast, adds the Na of 1.5g/L in nutrient solution
2CO
3, spheroplast transferred to contains Na
2CO
3Nutrient solution in, 3-5 days results of the similarity condition aerated culture when cultivating or spheroplast is statically placed in the original fluid, (1-10 ℃) left standstill 10-15 days under low temperature.
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|---|---|---|---|
| CN99120005A CN1096499C (en) | 1999-10-22 | 1999-10-22 | Method for culturing nostoc |
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| CN99120005A CN1096499C (en) | 1999-10-22 | 1999-10-22 | Method for culturing nostoc |
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| CN1096499C CN1096499C (en) | 2002-12-18 |
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| CN99120005A Expired - Fee Related CN1096499C (en) | 1999-10-22 | 1999-10-22 | Method for culturing nostoc |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1314711C (en) * | 2004-03-27 | 2007-05-09 | 中国科学院水生生物研究所 | Method for extracting Gexianmi amylose from nostoc |
| CN103891591A (en) * | 2014-04-14 | 2014-07-02 | 湖北工业大学 | Method for preventing nostoc sphaeroides colonies from being broken and reinforcing colony hardness |
| CN104031867A (en) * | 2014-06-17 | 2014-09-10 | 常德炎帝生物科技有限公司 | Economic and environment-friendly large-scale outdoor breeding method for nostoc sphaeroides |
| CN104031866A (en) * | 2014-06-17 | 2014-09-10 | 常德炎帝生物科技有限公司 | Ecological cultivation method and ecological cultivation system of common nostoc |
| CN104789501A (en) * | 2015-04-07 | 2015-07-22 | 张懋 | Gas-phase large-scale cultivation method of nostoc sphaeoides Kutzing |
| CN106047748A (en) * | 2016-05-25 | 2016-10-26 | 苏州维淼生物工程有限公司 | Breeding method of N.Commune and method of N.Commune for preparing dietary therapy products |
| CN106434477A (en) * | 2016-11-02 | 2017-02-22 | 常德炎帝生物科技有限公司 | Nostoc sphaeroides heterocyst embedded suspension breeding method |
| CN107646693A (en) * | 2017-11-13 | 2018-02-02 | 武汉藻尚健生物科技有限公司 | The production method of golden yellow nostoc |
| CN109392693A (en) * | 2018-11-23 | 2019-03-01 | 恩施硒谷科技股份有限公司 | A method of artificial breeding selenium-rich nostoc |
| CN111040963A (en) * | 2019-12-11 | 2020-04-21 | 常德炎帝牧源农业发展有限公司 | Nostoc sphaeroids kutz algae section induction method |
| CN112662583A (en) * | 2020-12-31 | 2021-04-16 | 中国海洋大学 | Improved culture medium suitable for vegetative growth of nostoc sphaeroides and preparation method thereof |
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| CN100386422C (en) * | 2003-11-11 | 2008-05-07 | 中国科学院水生生物研究所 | Production process of nostoc sphaeroides |
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| CN1070081A (en) * | 1992-07-28 | 1993-03-24 | 中国科学院兰州沙漠所 | Nutritional agent for artificially planting flagelliforme |
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- 1999-10-22 CN CN99120005A patent/CN1096499C/en not_active Expired - Fee Related
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| CN1314711C (en) * | 2004-03-27 | 2007-05-09 | 中国科学院水生生物研究所 | Method for extracting Gexianmi amylose from nostoc |
| CN103891591A (en) * | 2014-04-14 | 2014-07-02 | 湖北工业大学 | Method for preventing nostoc sphaeroides colonies from being broken and reinforcing colony hardness |
| CN103891591B (en) * | 2014-04-14 | 2016-01-20 | 湖北工业大学 | A kind ofly prevent nostoc colony from breaking and strengthen the method for colony's hardness |
| CN104031866A (en) * | 2014-06-17 | 2014-09-10 | 常德炎帝生物科技有限公司 | Ecological cultivation method and ecological cultivation system of common nostoc |
| CN104031867B (en) * | 2014-06-17 | 2015-11-18 | 常德炎帝生物科技有限公司 | A kind of outdoor scale breeding method of Nostoc of economic environmental protection |
| CN104031867A (en) * | 2014-06-17 | 2014-09-10 | 常德炎帝生物科技有限公司 | Economic and environment-friendly large-scale outdoor breeding method for nostoc sphaeroides |
| CN104789501A (en) * | 2015-04-07 | 2015-07-22 | 张懋 | Gas-phase large-scale cultivation method of nostoc sphaeoides Kutzing |
| CN106047748A (en) * | 2016-05-25 | 2016-10-26 | 苏州维淼生物工程有限公司 | Breeding method of N.Commune and method of N.Commune for preparing dietary therapy products |
| CN106434477A (en) * | 2016-11-02 | 2017-02-22 | 常德炎帝生物科技有限公司 | Nostoc sphaeroides heterocyst embedded suspension breeding method |
| CN106434477B (en) * | 2016-11-02 | 2019-06-28 | 常德炎帝生物科技有限公司 | A kind of embedding outstanding breeding method of nostoc heterocyst |
| CN107646693A (en) * | 2017-11-13 | 2018-02-02 | 武汉藻尚健生物科技有限公司 | The production method of golden yellow nostoc |
| CN109392693A (en) * | 2018-11-23 | 2019-03-01 | 恩施硒谷科技股份有限公司 | A method of artificial breeding selenium-rich nostoc |
| CN111040963A (en) * | 2019-12-11 | 2020-04-21 | 常德炎帝牧源农业发展有限公司 | Nostoc sphaeroids kutz algae section induction method |
| CN112662583A (en) * | 2020-12-31 | 2021-04-16 | 中国海洋大学 | Improved culture medium suitable for vegetative growth of nostoc sphaeroides and preparation method thereof |
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| CN1096499C (en) | 2002-12-18 |
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