CN104046566A - Method for rapid preparation of high density and high purity algae species - Google Patents
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- CN104046566A CN104046566A CN201310082447.3A CN201310082447A CN104046566A CN 104046566 A CN104046566 A CN 104046566A CN 201310082447 A CN201310082447 A CN 201310082447A CN 104046566 A CN104046566 A CN 104046566A
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Abstract
Belonging to the technical field of algae application, the invention discloses a method for rapid preparation of high density and high purity algae species. The invention involves an efficient culture medium and a preparation method thereof. The efficient culture medium is utilized, algae undergoes shake culture in a triangular flask, air introduction culture in a cylindrical glass tube and a flat plate photobioreactor in order. According to the algae density change in the cultivation process, the light intensity (200-10000lux) is gradually increased. At the same time, the introduction amount of CO2 is regulated, and the pH of the culture solution is maintained at 6.5-8.5, so that the demands of algae for illumination and a carbon source at different periods can be met excellently, the harm of high intensity light on algae growth is avoided, and a best growth state is maintained, thereby improving the photosynthetic efficiency. In the cylindrical glass tube and the flat plate bioreactor, daily production of 1.2g and 0.65g of dry weight algae by per liter of the culture medium can be achieved, and per liter of the culture medium can achieve a final dry algae weight of over 12g and 8g respectively. The method provided by the invention can greatly shorten the large-scale culture cycle of algae and reduce the pollution probability of algae in the culture process, thus being an effective scheme for acquiring high purity algae products.
Description
Technical field
The invention belongs to algae applied technical field, particularly the cultural method of the high pure strain of a kind of quick acquisition high-density.By regulation and control light intensity and ventilation specifically; utilize different culture apparatuses; realize and obtain at short notice the high pure strain of high-density, as the inoculum of large-scale cultivation, in order to produce open-mouthed bait, functional foodstuff, high added value secondary metabolite and the biofuel etc. of fishes and shrimps shellfish.
Background technology
Algae is the simple photo-autotroph of a class formation, in the time having water and some inorganic nutrient salts to exist, can efficiently utilize light and CO
2synthesis of organic substance.Algae has very high economic worth, if chlorella is the vegetable-protein source of high-quality, Haematocoocus Pluvialls can obtain a large amount of astaxanthins after induction, spirulina is a kind of well functional food, Dunaliella salina can be used for producing in a large number carotenoid, some marine microalgae is vegetalitas EPA and DHA main source, a lot of micro-algaes can be directly as the open-mouthed bait of the aquatic products economic animal seedling such as fish, shrimp, shellfish, crab.Micro-algae can also be removed the organic and inorganic nutrient substance in sewage fast, and utilizing micro-algae production biofuel is a focus in current bioenergy research.
Compare with higher plant, algae have individual little, photosynthetic efficiency is high, cell fission is fast, the scope that conforms extensively and yield per unit high.In today of grain, the energy and land resources growing tension, algae scale is cultivated and comprehensive development and utilization has good application prospect in the problems that solve facing mankind.During algae mass-producing is cultivated, the quantity of " seed " has restricted industrial scale, and its quality and purity have determined the yield and quality of cutting.Therefore how obtaining fast at short notice a large amount of active good high pure strains is vital core technologies in the cultivation of algae scale and application.
In algal grown, two vital factors are exactly light and carbon source (CO
2).Luminous energy is under-supply, will reduce algal grown speed, and luminous energy was supplied and can be produced at most photoinhibition, and alga cells is produced to harm, serious also can cause alga cells death.In algae culture process, algae cell density constantly changes, and how the demand of light, also increasing, is changed to regulate light intensity according to algae cell density, makes it can meet demand and the unlikely generation harm of algae to light, just can obtain maximum algae bio amount.In algae culture, CO
2be not only photosynthetic important substrate, or a kind of important pH adjusting agent.Algae cell density does not add extra CO compared with Gao Shiru
2, pH can be elevated to more than 11, and the best pH of most of algae growth is 7-8, understands like this severe inhibition algal grown.Therefore, CO
2as the effect of pH adjusting agent, in the time of alga cells high-density culture, seem particularly important.While carrying out algae culture according to a conventional method, often adopt single container, single intensity of illumination, single CO
2the method of concentration, not only causes the energy and nutrition waste, also makes the output of algae on the low side, and the output of final cutting is many at 2 g (dw) L
-1below.The present invention, just for this present situation, adopts new Production Flow Chart, improves the algae output in unit time and unit volume, provides technical support for relevant algae develops.
Summary of the invention
The invention provides the method for the high pure strain of a kind of quick acquisition high-density.The algae of conservation on solid medium is inoculated in a kind of efficient culture medium, successively through in triangular flask wave and culture, in cylindricality Glass tubing and Flat photobioreactor, pass into add CO
2air cultivate.In this process, change gradual raising light intensity according to algae density; By the feedback of pH, regulate the CO passing into simultaneously
2amount, meets the illumination of algae different growing stages and carbon source demand, has avoided the harm of high-strength light to algal grown, makes it remain on optimum growh state, thereby improves algae photosynthesis efficiency, realizes the fast breeding of alga cells.Technology provided by the invention can obtain a large amount of highly purified algae kinds at short notice, prepares link be widely used in the algae kind of algae related industries.
First the present invention provides a kind of efficient liquid substratum, and it is as follows that it comprises composition: KNO
30.125-1.0 gL
-1, NaNO
30.125-1.0 gL
-1, KH
2pO
40.04-0.5 gL
-1, Na
2hPO
40.05-0.5 gL
-1, MgSO
47H
2o 0.06-0.3 gL
-1, CaCl
20.02-0.1 gL
-1, NaHCO
30.02-3 gL
-1, FeCl
23-20 mgL
-1, Na
2eDTA 1-10 mgL
-1, H
3bO
31-10 mgL
-1, MnCl
24H
2o 0.5-5 mgL
-1, ZnSO
47H
2o 0.1-0.6 mgL
-1, CuSO
45H
2o 0.02-0.2 mgL
-1, Na
2moO
42H
2o 0.01-0.8 mgL
-1, CoCl
26H
2o 0.02-4 mgL
-1.When preparation substratum, can utilize distilled water or the tap water through placement 12h.
The preferable amount of the each nutritive salt of liquid nutrient medium is as follows: KNO
30.44 gL
-1, NaNO
30.60 gL
-1, KH
2pO
40.136 gL
-1, Na
2hPO
40.142 gL
-1, MgSO
47H
2o 0.06-0.3 gL
-1, CaCl
20.03gL
-1, NaHCO
30.2 gL
-1, FeCl
27.542 mgL
-1, Na
2eDTA 5 mgL
-1, H
3bO
33.15 mgL
-1, MnCl
2h
2o 2 mgL
-1, ZnSO
47H
2o 0.222 mgL
-1, CuSO
45H
2o 0.08 mgL
-1, Na
2moO
42H
2o 0.041 mgL
-1, CoCl
26H
2o 0.04 mgL
-1.
Take for convenience, nutritive salt is divided into four mother liquors that combine different concns, and each mother liquor is as follows: KNO
3, NaNO
3, KH
2pO
4and Na
2hPO
4be dissolved in successively in distilled water and be made into 100
mother liquor 1, CaCl
2and FeCl
2be dissolved in successively in distilled water and be mixed with 1000
mother liquor 2, MgSO
47H
2o and NaHCO
3be dissolved in successively in distilled water and be made into 1000
mother liquor 3, Na
2eDTA, H
3bO
3, MnCl
24H
2o, ZnSO
47H
2o, CuSO
45H
2o, Na
2moO
42H
2o and CoCl
26H
2o is dissolved in successively in distilled water and is mixed with 1000
mother liquor 4.When preparation substratum, the sterilizing respectively of each mother liquor, gets 10ml mother liquor 1 after cooling, and 1ml mother liquor 2,1ml mother liquor 3 and 1ml solution 4 add in 700ml water successively, after fully stirring, adds another again, and control precipitation produces, and last moisturizing is to 1L.
Algal species cultivation flow process provided by the invention is as described below.
1). experiment appliance sterilizing
In experiment, the glassware such as triangular flask used and Glass tubing adopts high temperature and high pressure method sterilizing, and bioreactor, after cleaning is dried, moves into through in the culturing room of ultra-violet sterilization, sprays 75% alcohol carry out sterilizing at inwall, after drying, seals for subsequent use.Conservation, activation and while cultivating in cylindricality Glass tubing used medium adopt autoclave sterilization.In flat-plate reactor, substratum is through negative pressure filtration sterilizing, and filter membrane aperture is 0.45 μ m.
2). algae kind is preserved the solid medium containing 1.5% agar powder with the preparation of above-mentioned Optimal Medium, by algae line or be coated with dull and stereotyped after with being placed in 500lux illumination, cultivation at 20-30 DEG C.
3) algae kind activation by conservation the algae kind on solid medium transfer in the 50-500ml of sterilizing triangular flask.In triangular flask, nutrient solution volume is no more than 2/3 of its capacity.Sterilising conditions is 103.5 kPa, 121 DEG C, and 20min.Unicell green alga or blue-green algae can directly inoculate, and form compared with the algae of large group and first grind gently in the situation that not adding the subsidiaries such as quartz sand with homogenizer or the mortar of sterilizing, are prepared into unicellular or more after microcommunity, inoculate thing.After having inoculated, be placed on shaking table, culture condition is 20-30 DEG C, and 24h is for light, and light intensity is 200-1200 lux, 20-120 rmin
-1shake, rotating speed control is not to make bottle surely have obvious cell precipitation to be advisable.Just when solid medium is transferred to triangular flask, cultivating light intensity is 200 lux, after within 2-3 days, cultivating, nutrient solution is obvious green, light intensity is brought up to 600 lux, cultivate light intensity two days later and be elevated to 1200 lux, then cultivation can be inoculated in cylindricality Glass tubing two days later.Light intensity is by regulating light source power size (as fluorescent tube number) or adding filter screen shading and realize.
4) fast culture of algae kind is inoculated into algae kind good above-mentioned activation in cylindricality transparent glass culture tube by 1:5 volume ratio, and inoculum size is advisable to be no more than 75% of Glass tubing cumulative volume, to avoid algae to be gone out by gas in culturing process.The diameter of cylindricality glass culture tube is 2-5 cm, and preferably diameter is 3.5cm, and length is 30-80 cm, and bottom is U-shaped or sharp triangular pyramidal.Culture condition is: temperature is controlled at 20-30 DEG C, and 24h is for light, and light intensity is 1000-6000 lux, pH 7-12, aerated culture.Passing into gas via hole diameter is the gas of 0.45 μ m membrane filtration, and flow is every liter of substratum 0.04-2 L min
-1, be precipitated as minimum air flow so that cell does not produce.The gas passing into is for adding 0.2-2%CO
2air, CO
2can contain high concentration CO from high-pressure cylinder (99%) or other
2gas, as thermal power plant's stack gases of treated mistake.The CO passing into
2volume ratio regulates via under meter, and when adjusting, taking nutrient solution pH as reference, higher than pH8.5, enlargement discharge, reduces flow lower than pH6.5.
Initial light intensity is 1000 lux, and 2 days raise is afterwards 3000 lux, then cultivates after 2 days and be elevated to 6000 lux, then cultivates 2 days, and now algae biomass can reach 4-12g (dw) L
-1, can be transferred in dull and stereotyped photoreactor and cultivate in a large number.
5) a large amount of cultivations of algae kind proceed to according to 1:10 volume ratio the algae of cultivating in above-mentioned cylindricality Glass tubing in Flat photobioreactor, and initial inoculum is 0.4-1 g (dw) L
-1, inoculation cumulative volume is advisable to be no more than 80% of cumulative volume.The light path of Flat photobioreactor is 2-6cm, and preferred light path is 3.5cm, and the degree of depth is 50-150 cm.Culture condition is 20-30 DEG C, and 24h is for light, and light intensity is 2000-10000 lux, pH 7-12, aerated culture.The gas passing into is for adding 0.2-2%CO
2air.CO
2can contain high concentration CO from high-pressure cylinder (99%) or other
2gas, as thermal power plant's stack gases of treated mistake.The CO passing into
2volume ratio regulates via under meter, and when adjusting, taking nutrient solution pH as reference, higher than pH8.5, enlargement discharge, reduces flow lower than pH6.5.Initial light intensity in Flat photobioreactor is made as 2000 lux, cultivates and is increased to 4000 lux after 2 days, then cultivate light intensity after 2 days and be increased to 8000lux, then cultivate two days, and algae biomass can reach 4-8 g (dw) L
-1, now can gather in the crops and analyze, or be inoculated in the bio-reactor of more volume and produce.
Compared to the prior art the method that, the present invention obtains the high pure strain of high-density fast has following features:
The 1 algae density obtaining is high, and through the light autophyting growth of 6 days, algae bio amount can be up to 12 g (dw) L
-1.
In 2 culturing process, change dynamic regulation illumination and CO according to algae cell density and pH
2, make them can meet the demand of algal grown but be unlikely to produce harm, saved the energy and resource simultaneously.
3 according to the different growth phase features of algae, adopt different reactor, carry out stage cultivation, make the frustule can Fast Growth.
4 can obtain the high pure strain of q.s at short notice, inoculate for outdoor high-density semi-open or that open scale is cultivated, shorten culture cycle, and effectively the assorted algae of containment and other biological are grown, and obtain high pure growth.
Brief description of the drawings
Fig. 1 be Chlorella vulgaris (
chlorella vugaris) growth in cylindricality Glass tubing and pH change (25 DEG C, 1.5%CO
2).
Fig. 2 be Chlorella vulgaris (
chlorella vugaris) growth in Flat photobioreactor and pH change (25 DEG C, 1.5%CO
2).
Embodiment
The invention provides a kind of high pure strain method of preparation high density fast.Be exactly to utilize efficient liquid substratum provided by the invention specifically, algae is shaken successively to cultivation, in cylindricality glass culture tube and flat bioreactor, carries out aerated culture in triangular flask.At different cultivation stages, provide different intensities of illumination and different CO
2concentration, meets algal grown process for illumination and carbon source demand, avoids light to suppress and carbon restriction, makes algae remain on optimum growh state, thereby obtains maximum biomass productive rate.With specific examples, the present invention is further elaborated below.
Embodiment
Chlorella (
chlorella vugaris) purchased from Inst. of Hydrobiology, Chinese Academy of Sciences's algae kind preservation center.
Used medium is preferred culture medium (KNO
30.44 gL
-1, NaNO
30.60 gL
-1, KH
2pO
40.136 gL
-1, Na
2hPO
40.142 gL
-1, MgSO
47H
2o 0.06-0.3 gL
-1, CaCl
20.03gL
-1, NaHCO
30.2 gL
-1, FeCl
27.542 mgL
-1, Na
2eDTA 5 mgL
-1, H
3bO
33.15 mgL
-1, MnCl
2h
2o 2 mgL
-1, ZnSO
47H
2o 0.222 mgL
-1, CuSO
45H
2o 0.08 mgL
-1, Na
2moO
42H
2o 0.041 mgL
-1, CoCl
26H
2o 0.04 mgL
-1).In substratum when preparation, adopts distilled water, and substratum is at 103.5 kPa, and sterilizing 20min at 121 DEG C is cooling rear for subsequent use.
From flat board, the healthy algae of picking falls, and is inoculated in 50ml triangular flask, and being placed in rotating speed is 100 rmin
-1shaking table on, in 25 DEG C, under 24h irradiation, carry out primary activation.Initial incubation light intensity is 200 lux, and after within 2-3 days, cultivating, nutrient solution is obvious green, and light intensity is brought up to 600 lux, then cultivates light intensity two days later and be elevated to 1200 lux, then cultivates and be inoculated into diameter 3cm in 1:3 ratio two days later, in the Glass tubing of long 60cm.
When cultivation in Glass tubing, temperature is also controlled at 25 DEG C.Initial light intensity is 1000 lux, and rising in the 3rd day is 3000 lux, within the 5th day, is elevated to 6000 lux, cultivates 2 days, and now Chlorella vulgaris biomass can reach 7.32g(dw) L
-1(Figure 1A); Cultivate as continued, can reach respectively 9.86g and 10.7g in the biomass dry weight of the 8th day and the tenth day every liter of nutrient solution.
As cultivate within the 6th day, to proceed to light path according to 1:10 ratio be 3.5cm, in the Flat photobioreactor that the degree of depth is 80cm, at 25 DEG C, carry out aerated culture.Initial light intensity is made as 2000 lux, and within the 3rd day, light intensity is increased to 4000 lux, and within the 5th day, light intensity is increased to 8000 lux, and within the 6th day, algae biomass reaches 4.96 g(dw) L
-1(Fig. 2 A), can reach 6.04 g(dw on the 8th day) L
-1.
In Glass tubing and dull and stereotyped photoreactor, pass into and add 1.5%CO
2air, all nutrient solution pH can be maintained to (Figure 1B, Fig. 2 B) between 6.5-8.5.
Claims (10)
1. a method of preparing fast the high pure strain of high-density, is characterized in that: the algae kind of conservation on solid medium transferred in triangular flask, cylindricality transparent glass culture tube and the Flat photobioreactor of sterilizing successively, shake or aerated culture; Temperature is controlled at 20-30 DEG C, and 24h is for light, and light intensity is 5-100 μ mol photons m
-2s
-1, pH 7-12; Efficient liquid substratum used is that nutritive salt below adding in distilled water or the tap water through placing 12h is formulated: KNO
30.125-1.0 gL
-1, NaNO
30.125-1.0 gL
-1, KH
2pO
40.04-0.5 gL
-1, Na
2hPO
40.05-0.5 gL
-1, MgSO
47H
2o 0.06-0.3 gL
-1, CaCl
20.02-0.1 gL
-1, NaHCO
30.02-3 gL
-1, FeCl
23-20 mgL
-1, Na
2eDTA 1-10 mgL
-1, H
3bO
31-10 mgL
-1, MnCl
24H
2o 0.5-5 mgL
-1, ZnSO
47H
2o 0.1-0.6 mgL
-1, CuSO
45H
2o 0.02-0.2 mgL
-1, Na
2moO
42H
2o 0.01-0.8 mgL
-1, CoCl
26H
2o 0.02-4 mgL
-1; KNO when preparation
3, NaNO
3, KH
2pO
4and Na
2hPO
4be dissolved in successively in distilled water and be made into 100
mother liquor 1, MgSO
47H
2o, CaCl
2and FeCl
2be dissolved in successively in distilled water and be mixed with 1000
mother liquor 2, NaHCO
3be made into 1000
mother liquor 3, Na
2eDTA, H
3bO
3, MnCl
24H
2o, ZnSO
47H
2o, CuSO
45H
2o, Na
2moO
42H
2o and CoCl
26H
2o is dissolved in successively in distilled water and is mixed with 1000
mother liquor 4; When preparation substratum, get 10ml mother liquor 1,1ml mother liquor 2,1ml mother liquor 3 and 1ml solution 4 add in 600ml water successively, after fully stirring, add another again, and control precipitation produces, and last moisturizing is to 1L.
2. a kind of quick method of preparing the high pure strain of high-density according to claim 1, is characterized in that: described algae is unicell green alga or blue-green algae, or forms green alga or the blue-green algae of colony; For forming compared with the algae of large group, the grinding gently in the situation that not adding the subsidiaries such as quartz sand with the homogenizer of sterilizing or mortar, to obtain the unicellular or inoculum of microcommunity more.
3. the method for a large amount of high pure strains of a kind of quick preparation according to claim 1, it is characterized in that: the described glassware such as triangular flask and Glass tubing adopts high temperature and high pressure method sterilizing, bioreactor is after cleaning is dried, move into through in the culturing room of ultra-violet sterilization, alcohol in inwall sprinkling 75% carries out sterilizing, after drying, seal for subsequent use.
4. the method for a large amount of high pure strains of a kind of quick preparation according to claim 1, is characterized in that: described triangular flask specification is 50-500ml; The diameter of cylindricality glass culture tube is 2-5 cm, and length is 30-80 cm, and bottom is U-shaped or sharp triangular pyramidal; The light path of Flat photobioreactor is 2-6cm, and the degree of depth is 50-100 cm.
5. the method for a large amount of high pure strains of a kind of quick preparation according to claim 1, is characterized in that: liquid nutrient medium used is that nutritive salt below adding in distilled water or the tap water through placing 12h is formulated, and the preferable amount of each nutritive salt is as follows: KNO
30.44 gL
-1, NaNO
30.60 gL
-1, KH
2pO
40.136 gL
-1, Na
2hPO
40.142 gL
-1, MgSO
47H
2o 0.06-0.3 gL
-1, CaCl
20.03gL
-1, NaHCO
30.2 gL
-1, FeCl
27.542 mgL
-1, Na
2eDTA 5 mgL
-1, H
3bO
33.15 mgL
-1, MnCl
2h
2o 2 mgL
-1, ZnSO
47H
2o 0.222 mgL
-1, CuSO
45H
2o 0.08 mgL
-1, Na
2moO
42H
2o 0.041 mgL
-1, CoCl
26H
2o 0.04 mgL
-1.
6. the method for a large amount of high pure strains of a kind of quick preparation according to claim 1, is characterized in that: algae is placed on shaking table and shakes while cultivation in triangular flask, speed is 20-120 rmin
-1, in Glass tubing or bioreactor, passing into via hole diameter is the gas of 0.45 μ m membrane filtration, flow is every liter of substratum 0.04-2 L min
-1.
7. according to the method for a large amount of high pure strains of a kind of quick preparation described in claim 1 or 6, it is characterized in that: described in to pass into gas be CO
2with the mixed gas of air, wherein CO
2shared volume ratio is 0.2-5%; Volume ratio is by regulating air and CO
2flow velocity (under meter control); CO
2gas can from high-pressure cylinder (purity>=99%) or other be containing high concentration CO
2gas, as thermal power plant's stack gases of treated mistake; CO
2ratio with control pH value of solution be advisable at 6.5-8.5, lower than this scope, reduce concentration, increase concentration higher than this scope.
8. the method for a large amount of high pure strains of a kind of quick preparation according to claim 1, is characterized in that: described light intensity is triangular flask 200-1200 lux, transparent glass tube 1000-6000 lux, Flat photobioreactor 2000-10000 lux; Intensity is by regulating light source power size (as fluorescent tube number) or adding filter screen shading and realize.
9. the method for a large amount of high pure strains of a kind of quick preparation according to claim 7, it is characterized in that: Main Basis algae cell density when light intensity regulating, just when solid medium is transferred to 100ml triangular flask, cultivating light intensity is 200 lux, after within 2-3 days, cultivating, nutrient solution is obvious green, light intensity is brought up to 600 lux, cultivate light intensity two days later and be elevated to 1200 lux, cultivate and be inoculated into diameter 3cm in 1:5 ratio two days later again, in the Glass tubing of long 60cm, initial light intensity is 1000 lux, 2 days raise is afterwards 3000 lux, cultivate and be elevated to 6000 lux after 2 days, cultivate again 2 days, now algae biomass can reach 4-12g(dw) L
-1, proceed in the Flat photobioreactor that light path is 3.5cm according to 1:10 ratio, initial light intensity is made as 2000lux, cultivates and is increased to 4000lux after 2 days, cultivates light intensity after 2 days and is increased to 8000lux, then cultivate two days, and algae biomass can reach 4-8 g(dw) L
-1.
10. the algae kind obtaining by this method is cultivated the application in open-mouthed bait, functional foodstuff, high added value secondary metabolite and the bioenergy of producing fishes and shrimps shellfish in algae mass-producing.
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| CN113684134A (en) * | 2021-07-13 | 2021-11-23 | 上海师范大学 | Method and device suitable for preserving algae living bodies in field sampling period |
| CN114032176A (en) * | 2021-11-26 | 2022-02-11 | 海南绿藻世界生物科技有限公司 | Culture system and culture method for promoting growth of microalgae |
| CN114431131A (en) * | 2022-01-07 | 2022-05-06 | 中国科学院东北地理与农业生态研究所 | Aeration culture method for improving survival rate of algae |
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