CN110257252A - A method of isolating and purifying chlamydomonas from natural water - Google Patents
A method of isolating and purifying chlamydomonas from natural water Download PDFInfo
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- CN110257252A CN110257252A CN201910651424.7A CN201910651424A CN110257252A CN 110257252 A CN110257252 A CN 110257252A CN 201910651424 A CN201910651424 A CN 201910651424A CN 110257252 A CN110257252 A CN 110257252A
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- chlamydomonas
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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Abstract
The method that the invention discloses a kind of to isolate and purify chlamydomonas from natural water, comprising the following steps: S1, acquire water sample and planktonic algae sample from natural water respectively；S2, water sample is removed into impurity through filtering with microporous membrane, sterilized, as culture solution；S3, culture solution is configured to salinity culture solution；S4, the planktonic algae sample of acquisition is inoculated into salinity culture solution, is cultivated；S5, the purifying of chlamydomonas in observation salinity culture solution, growing state directly adopt the group of the algae in natural water and water body.Relative to traditional isolation and purification method, the present invention is simpler, direct, accurate, and convincingness is stronger.The blank of the research field has been filled up in research especially for chlamydomonas as non-advantage algae.
Description
Technical field
The invention belongs to environmental field, in particular to a kind of method that chlamydomonas is isolated and purified from natural water.
Background technique
Planktonic algae refers to the small algae for seeking life of swimming in water, including Cyanophyta (Cyanophyta), Chlorophyta
(Chlorophyta), Bacillariophyta (Bacillariophyta), Chrysophyta (Chrysophyta), Xanthophyta
(Xanthophyta), Pyrrhophyta (Pyrrophyta), Cryptophyta (Cryptorhyta) and Euglenophyta (Euglenophyta) eight
The pelagic genera of a class.Planktonic algae is the primary producer in natural water, by absorb water body in nutritive salt at
Point, and the growth and breeding that organic matter promotes itself is generated using luminous energy driving photosynthesis.The life of planktonic algae in natural water
It is long to suffer from from external factors such as illumination, nutrient concentration, water temperature, Grazing Pressure, water volume flow rates and from interspecies competition
Internal factor common restriction, so that planktonic algae is there is distributional difference spatially and temporal succession difference.Due to not
Growth characteristics with microalgae and its adaptability to environment are different, can be reached by changing key environmental factors feature from nature
In water body.
Chlamydomonas is subordinate to Chlorophyta, Chlorophyceae, volvocales, chlamydomonas section.The most of types of the category move in various
In poisons in freshwater, including certain contaminated water bodys；Minority is moved in salt water and seawater, and there are also a small number of gas to be born in moist soil
On the wall of earth, pond or well head.
At present both at home and abroad in the ecological study in relation to chlamydomonas in natural water, researcher is primarily upon chlamydomonas and is swimming
Occurrence rate and dominance in structure of plant community composition.In the laboratory controlled experiments carried out using chlamydomonas, the chlamydomonas that uses
Often purchase is from domestic algae library, or the chlamydomonas in natural water is separated under the microscope, is further continued for carrying out purifying culture.
The shortcomings that this technical method be limited by chlamydomonas in natural water initial density it is very low, be easy just to will be unable at the beginning of investigation
Get the algae of chlamydomonas.In addition, the low working efficiency for leading to separate picking chlamydomonas under the microscope of the initial density of chlamydomonas is very
It is low；Such operating method, it is time-consuming and laborious, it isolates and purifies ineffective.Furthermore there is chlamydomonas flagellum can move, and increase
The difficulty of picking is separated under microscope.
It is less to the research of non-advantage algae both at home and abroad at present, on the one hand since planktonic microalgae is food chain in natural water
Basic link, individual it is small, many kinds of, the requirement to professional technician is very high, and at present practitioner it is relatively fewer；
On the other hand, non-advantage algae quantity in natural water is few, is safe from harm to environment, is less subject to the pass of researcher
Note.Therefore, carry out research in this respect, not only filled up the blank of the research field, previous research conclusion can also be played
Assist the effect of verifying.
Summary of the invention
In view of this, the technical problems to be solved by the invention, exactly propose that one kind isolates and purifies clothing from natural water
The method of algae.
In order to solve the above technical problems, the present invention is achieved by the following scheme:
A method of isolating and purifying chlamydomonas from natural water, comprising the following steps:
S1, water sample and planktonic algae sample are acquired from natural water respectively；
S2, water sample is removed into impurity through filtering with microporous membrane, sterilized, as culture solution；
S3, culture solution addition sodium chloride is configured to salinity culture solution；
S4, the planktonic algae sample of acquisition is inoculated into salinity culture solution, is cultivated；
S5, the purifying of chlamydomonas in observation salinity culture solution, growing state.After cultivation, chlamydomonas exists planktonic algae sample
Its cell density is exponentially increased under salinity culture solution, other microalgae death disappear, and is judged as that chlamydomonas purifies successfully；Otherwise, sentence
Break unsuccessful to purify.
Preferably, the chlamydomonas isolated and purified in the present invention is that percentage is less than in total population of cells abundance
5% non-advantage algae.
Preferably, in the step S1, the aperture of miillpore filter is 0.2 μm.
Preferably, in the step S3, the salinity of salinity culture solution is 25 ‰ or 30 ‰.
Preferably, in the step S4, the density that chlamydomonas is inoculated in salinity culture solution is referring to the algae in nature
Stand density in water body.
Preferably, in the step S4, the condition of culture of planktonic algae sample is to be trained in illumination box
It supports.
Preferably, in the step S4, the incubation time of planktonic algae sample is 240 hours.
Preferably, in the step S5, observation method is the purifying that chlamydomonas is periodically observed under optics inverted microscope
And growing state.
Compared with prior art, the device have the advantages that are as follows:
The method of the present invention can rapidly isolate and purify chlamydomonas common in natural water, fill up from natural water
In be directly separated purifying chlamydomonas research blank.Efficiency not only can be improved, can also be improved the accuracy isolated and purified.Relatively
In traditional isolation and purification method, the present invention is simpler, direct, accurate, and convincingness is stronger.Especially for chlamydomonas as non-optimum
The blank of the research field has been filled up in the research of gesture algae.
Detailed description of the invention
Fig. 1 is that the Electronic Speculum of the preferred embodiment of the present invention observes figure.
Specific embodiment
The present invention is got information about to allow those skilled in the art to be more clear, below in conjunction with attached drawing, to the present invention
It is further described.
A method of isolating and purifying chlamydomonas from natural water, comprising the following steps:
S1, acquire 1 L of surface layer water sample from Guangzhou Hua Dihe with hydrophore, and with the plankton net in 10 microns of aperture into
Row trawlnet acquires high density filter algae；
S2, by water sample through miillpore filter (diameter 47mm, 0.2 μm of aperture) filtering after, high pressure sterilization, as culture solution；
S3, the algae that trawlnet acquires is seeded to and fills 10ml culture solution, and salinity gradient is followed successively by 0,1 ‰, 5 ‰,
In 10 ‰, 15 ‰, 20 ‰, 25 ‰, 30 ‰ teat glass, inoculation algae group's density is 1000cells/ml (referring to former Natural Water
Density in body)；Wherein, the inoculum density of chlamydomonas is 50cells/ml (referring to the density in former natural water)；Test tube sample
It sets and cultivates in the light incubator simultaneously, condition of culture is 25 DEG C of constant temperature, intensity of illumination 2000lux, and continuous incubation time is 240
Hour.
Culture terminates, and culture solution is shaken up, and therefrom takes 0.05ml drop after on glass slide, covering coverslip in Zeiss Axio
Microscopic count is carried out under Observer A1 optics inverted microscope.As a result as shown in Figure 1, salinity item of the display chlamydomonas 25 ‰
Part lower density reaches 104Cells/ml, the Variation of Salinity Condition lower density 30 ‰ reach 105Cells/ml, and do not find that other are micro-
Algae.Illustrate that chlamydomonas isolates and purifies success.
Culture solution is flower ground river original position river water, and after taking out 0.2 micron or more impurity, high pressure sterilization is as culture solution.Culture
Nutrient concentrations are as follows in liquid:
| Parameter | Content mg/L |
| Phosphate PO4 | 0.007 |
| Total phosphorus TP | 0.173 |
| Total nitrogen TN | 5.197 |
| Nitrate nitrogen NO3 | 1.529 |
| Nitrite nitrogen NO2 | 0.529 |
| Ammonia nitrogen NH4 | 1.527 |
| Non-ionic ammonia NH3 | 0.019 |
| Silicate SiO4 | 5.032 |
| Permanganate COD | 4.600 |
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of method for isolating and purifying chlamydomonas from natural water, which comprises the following steps:
S1, water sample and planktonic algae sample are acquired from natural water respectively；
S2, water sample is removed into impurity through filtering with microporous membrane, sterilized, as culture solution；
S3, culture solution addition sodium chloride is configured to salinity culture solution；
S4, the planktonic algae sample of acquisition is inoculated into salinity culture solution, is cultivated；
S5, the purifying of chlamydomonas in observation salinity culture solution, growing state:
After cultivation, chlamydomonas its cell density under salinity culture solution is exponentially increased planktonic algae sample, other microalgaes are dead
It disappears, is judged as that chlamydomonas purifies successfully；Otherwise, it is judged as that purifying is unsuccessful.
2. the method according to claim 1, wherein the chlamydomonas isolated and purified is the institute in total population of cells abundance
Account for non-advantage algae of the percentage less than 5%.
3. the method according to claim 1, wherein the aperture of miillpore filter is 0.2 μm in the step S1.
4. the method according to claim 1, wherein in the step S3, the salinity gradient of salinity culture solution is
25 ‰ or 30 ‰.
5. the method according to claim 1, wherein chlamydomonas is inoculated in salinity culture solution in the step S4
Stand density of the density referring to the algae in natural water.
6. the method according to claim 1, wherein in the step S4, the condition of culture of planktonic algae sample
To be cultivated in illumination box.
7. the method according to claim 1, wherein in the step S4, the incubation time of planktonic algae sample
It is 240 hours.
8. the method according to claim 1, wherein observation method is that optics inversion is micro- in the step S5
Purifying and the growing state of chlamydomonas are periodically observed under mirror.
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Citations (4)
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|---|---|---|---|---|
| CN102978275A (en) * | 2012-11-14 | 2013-03-20 | 中国水产科学研究院珠江水产研究所 | Method for judging whether growth of floating algae inhibited by nutrition or not |
| CN102643751B (en) * | 2012-04-25 | 2013-10-16 | 同济大学 | Method for quickly separating and purifying chlorella |
| CN106906270A (en) * | 2017-04-12 | 2017-06-30 | 郭雨汇 | Waste water and the method for producing carotenoid, gaseous fuel and organic fertilizer are processed using microalgae |
| CN107828660A (en) * | 2017-09-30 | 2018-03-23 | 南京大学昆山创新研究院 | A kind of method of efficiently High Density Cultivation biological feed |
-
2019
- 2019-07-18 CN CN201910651424.7A patent/CN110257252A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643751B (en) * | 2012-04-25 | 2013-10-16 | 同济大学 | Method for quickly separating and purifying chlorella |
| CN102978275A (en) * | 2012-11-14 | 2013-03-20 | 中国水产科学研究院珠江水产研究所 | Method for judging whether growth of floating algae inhibited by nutrition or not |
| CN106906270A (en) * | 2017-04-12 | 2017-06-30 | 郭雨汇 | Waste water and the method for producing carotenoid, gaseous fuel and organic fertilizer are processed using microalgae |
| CN107828660A (en) * | 2017-09-30 | 2018-03-23 | 南京大学昆山创新研究院 | A kind of method of efficiently High Density Cultivation biological feed |
Non-Patent Citations (6)
| Title |
|---|
| JOHAN A. HELLEBUST等: "OSMOREGULATION", 《ANNU. REV. PLANT. PHYSIOL.》 * |
| 中国科学院海洋研究所海洋生物种质库: "中国科学院海洋研究所海洋生物种质库", 《百度文库》 * |
| 刘梅: "谷皮菱形藻的耐盐适应性", 《水生生物学报》 * |
| 林夕亚洲: "海藻的生存", 《百度文库》 * |
| 林永成: "《海洋微生物及其代谢产物》", 31 January 2003 * |
| 过世东: "《水产饲料生产学》", 31 August 2004, 中国农业出版社 * |
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