CN102071143A - Culture method for efficiently inducing accumulation of nannochloropsis oculata fat - Google Patents
Culture method for efficiently inducing accumulation of nannochloropsis oculata fat Download PDFInfo
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Abstract
The invention provides a culture method for efficiently inducing accumulation of nannochloropsis oculata fat. The culture method is as follows: preparing a culture medium and daily-added nutrient liquid; carrying out biomass accumulation culture on annochloropsis oculata for 5-8 days; introducing CO2 in annochloropsis oculata culture liquid, and regulating the concentration of the daily added nutrient liquid; and carrying out ultrasonic processing on the annochloropsis oculata culture liquid one to three times at each interval of 10 seconds and for 5-60 seconds per time by using an ultrasonic instrument under a certain frequency, so that the accumulation of the nannochloropsis oculata fat is induced. The biomass of the nannochloropsis oculata cultured by the method is 1.5-2 times as much as that of the prior art, the content of oil in the nannochloropsis oculata cultured by the method is improved by 40%-65% compared with the prior art, and the growing period of the nannochloropsis oculata cultured by the method is shortened by 2-3 days; and in the invention, carbon source is supplemented in a form of introducing CO2, and a large amount of CO2 waste gases generated in the industrial production process are absorbed in the process of the large-scale production of the nannochloropsis oculata for inducing the accumulation of fat, thus the culture method has an important significance for energy conservation and emission reduction as well as environment protection.
Description
Technical field:
The present invention relates to the lipid content of the little plan ball of a kind of efficient raising algae-X57 and the method that open type high-density is cultured little plan ball algae-X57.
Background technology
Along with the continuous expansion of global energy demand, the clean energy of seeking to substitute oil is the focus of present common concern.Biofuel (fatty acid methyl ester) is a kind of fuel that has been confirmed, with it is the extensive concern that the environment-friendly fuel energy of recyclability obtains the world, biofuel has good environmental protection characteristic, compare with petrifaction diesel, the quantity discharged of toxic gas can reduce by 90%, and it is about 10% that the quantity discharged of carbon monoxide can reduce, in addition, biodiesel raw material derives from photosynthesis, CO
2Clean quantity discharged be zero, therefore production and use biofuel can not cause Greenhouse effect, in U.S.'s biofuel mainly is raw material with the soybean, other sources comprise plam oil, rapeseed oil, tallow fatty acid, Semen Maydis oil, recovery culinary art oil and Cortex jatrophae wet goods, but can only satisfy the minimum part of current demand for fuel, be easy to generate the problem of the in short supply and consequent farm crop price increase of land resources simultaneously in the planting process, special under the situation that China has a large population and a few land, these problems are especially for outstanding.
Problems in the face of plant material production biofuel; scientist do not turned to sight with agricultural and striven ground, again with the same little algae that can utilize the photosynthesis produce oil of plant; the photosynthesis transformation efficiency of algae exceeds more than 10% of higher plant; the procreation ability is strong; growth cycle is short; exceed tens times than terrestrial plant produce oil; current; many scientists are arranged in exploration discovery new algae kind and development " engineering microalgae " both at home and abroad; hope can realize large-scale cultivation; reduce cost, provide a reliable approach for obtaining oil resource.Therefore how to improve the fat accumulation of little algae self, biodiesel raw material efficiently is provided, become the research direction of world today's new energy field.
In numerous little algae kinds, little plan ball algae (Nannochloropsis.sp) is a unicellular algae, is rich in grease, and content can reach more than 18% of total fatty acids, it is rich in various albumen, polyunsaturated fatty acid and pigment in addition, is considered to the high added value biology.The big eye of little plan ball Trentepohlia Chlorophyta algae guiding principle, be the seawater eukaryote, cell sphere, cell size 2-5 μ m, one of chromatoplast, light green, adnation, eyespot light red orange, under well-grown situation, chromatoplast is very dark, no pyrenoids, starch small grain 1-3, cell walls as thin as a wafer, cell granulations is little, and little plan ball algae growth and breeding is fast, and culture condition is simple, can carry out large scale culturing, huge business development potentiality are arranged, but association area is less to the research of little plan ball algae at present, uses also bait that are limited to halobios more.
Summary of the invention
In order to solve problems of the prior art, the present invention selects for use little plan ball algae-X57 as the algae kind, according to the metabolism of ultrasonic wave to organism, change the general culture condition of prior art, provide the growth velocity of the little plan ball of a kind of efficient raising algae-X57 and fatty accumulation ability and open type high-density to culture the method for little plan ball algae-X57.The present invention is under the prerequisite of not destroying cell, adopt suitable frequency, intensity and treatment time, to improve the metabolic efficiency of little plan ball algae-X57, accelerated cell growth, the little plan ball algae-X57 cell number of turning out with the inventive method be universal method 1.5-2.0 doubly, its lipid content has improved 40%-65%.
One of the object of the invention provides the substratum of a kind of little plan ball algae-X57.
Two of the object of the invention provides the cultural method of a kind of little plan ball algae-X57.
Three of the object of the invention provides a kind of method of inducing little plan ball algae-X57 fat accumulation.
The substratum that can efficiently improve little plan ball algae-X57 lipid content provided by the invention mainly comprises nitrogenous source, phosphorus source and molysite, and trace element and water.The preferred SODIUMNITRATE of nitrogenous source wherein, phosphorus source preferably phosphoric acid sodium dihydrogen, the preferred iron(ic) chloride of molysite, micro-preferably sulfuric acid copper, zinc sulfate, cobalt chloride, Manganous chloride tetrahydrate, substratum of the present invention can also add other useful nutritive ingredients according to different purposes.
The substratum called after FN17 of little plan ball algae-X57 provided by the present invention, its prescription is:
Sodium-chlor 18.0-40.0g/L SODIUMNITRATE 120.0-220.0mg/L
Iron trichloride 2.5-6.5mg/L disodium ethylene diamine tetraacetate 1.0-6.0mg/L
SODIUM PHOSPHATE, MONOBASIC 7.0-18.0mg/L trace element 0.5-2.0ml/L
Wherein Wei Liangyuansu compound method is:
Copper sulfate 5.5-22.5mg/L zinc sulfate 14.0-56.5mg/L
Cobalt chloride 11.5-45.5mg/L Manganous chloride tetrahydrate 113.5-453.0mg/L
Adding distil water is to 1L
Little plan ball algae provided by the present invention-X57 substratum, prescription comprises preferably:
Sodium-chlor 20.0-35.0g/L SODIUMNITRATE 150.0-200.0mg/L
Iron trichloride 3.5-5.5mg/L disodium ethylene diamine tetraacetate 2.0-3.5mg/L
SODIUM PHOSPHATE, MONOBASIC 9.0-14.0mg/L trace element 0.5-2.0ml/L
Wherein Wei Liangyuansu compound method is:
Copper sulfate 9.0-15.0mg/L zinc sulfate 19.5-32.5mg/L
Cobalt chloride 14.5-28.0mg/L Manganous chloride tetrahydrate 190.5-265.0mg/L
Adding distil water is to 1L
The compound method of daily interpolation nutritive medium mother liquor:
Solution A:
SODIUMNITRATE 280-320g/L trace element 0.5-2ml/L
Wherein trace element formula is:
Copper sulfate 1-3g/L zinc sulfate 4-6g/L
Cobalt chloride 3-5g/L Manganous chloride tetrahydrate 30-50g/L
Add water and be settled to 1L.
Solution B:
SODIUM PHOSPHATE, MONOBASIC 180-220g/L
Solution C:
Iron trichloride 6-10g/L disodium ethylene diamine tetraacetate 4-9g/L
If not refer in particular to, the chemical ingredients described in the culture medium prescription of the present invention is analytically pure commercial goods.
Using FN17 substratum provided by the present invention induces the method for little plan ball algae-X57 fat accumulation to comprise the steps:
Step 1: nutritive medium A, B, the C of preparation FN17 substratum and daily interpolation;
Step 2: little plan ball algae-X57 biomass accumulation, is 1 according to little plan ball algae-X57 with the culture volume ratio: 5-1: 10 ratio is inoculated into little plan ball algae-X57 in the substratum, temperature 18-30 ℃, pH value 6-9, illumination 5000-250001ux, light application time 10-16 hour/day, need in micro algae culturing liquid, to add the mixed solution of solution A, B, C every day, solution A: B: C was according to 10: 1: 10-25: 1: 25 mixed, add-on is 0.6-2ml/L, cultivates to reach logarithmic phase latter stage in 5-8 days; Wherein little plan ball algae-X57 biomass accumulation culture condition preferably is: little plan ball algae-X57 is 1 with the culture volume ratio: 6-1: 9, culture temperature is 22-28 ℃, the pH value is 7-8, illumination is 12000-200001ux, light application time is 11-14 hour/day, solution A, B, C were according to 12: 1: 12-20: 1: 20 mixed, every day, add-on was 0.6-1.5ml/L, cultivated 6-8 days.Best culture condition is: little plan ball algae-X57 is 1: 8 with the culture volume ratio, and temperature is 26-28 ℃, and illumination is 15000-180001ux, light application time 14 hours/day, solution A: B: C was according to 18: 1: 18 mixed, and every day, add-on was 0.8ml/L, cultivated 8 days.
Step 3: induced lipolysis accumulation, use ultrasound biological to promote auxanograph to the micro algae culturing liquid that is cultured to logarithmic phase latter stage frequency with 15-35kHz, the power of 2-8w, carry out supersound process, treatment time 10-60s, handle 1-3 time, each treatment time is spaced apart 10s, and the flow feeding concentration with 0.5-2.5L/min is the CO of 2%-6% in micro algae culturing liquid then
2Solution A: B is according to 10: 1-20: 1 mixed, every day, magnitude of recruitment was 0.5-100 μ l/L, and the add-on of solution C every day is 0.5-2ml/L, at inducing culture in the time of 2 days, again with the frequency of 10-20kHz, the power of 1-5w, treatment time 5-15s handles 1-2 time, each treatment time is spaced apart 10s, and coinduction was cultivated 4 days.Induced lipolysis accumulation condition preferably wherein: ultrasonic frequency is 15-20kHz, power be 2-6w, the treatment time is 10-30s, handles 1-2 time, each timed interval is 10s, feeding concentration with the flow of 0.8-1.5L/min then is the CO of 3%-6%
2, solution A: B is according to 12: 1-18: 1 mixed, add-on are 0.5-60 μ l/L, and the add-on of solution C is 0.5-1.5ml/L, in the time of 2 days, with the frequency of 10-15kHz, the power of 1-3w is handled 5-10s, handles 1 time again at inducing culture.Best induced lipolysis accumulation condition: ultrasonic frequency 18kHz, power are 4w, and the treatment time is 15s, handle 2 times, and feeding concentration with the flow velocity of 1.2L/min then is 5% CO
2, solution A: B was according to 15: 1 mixed, and add-on is 30 μ l/L, and the add-on of solution C is 1.2ml/L, and in the time of 2 days, with the frequency of 10kHz, the power of 3w is handled 8s, handles 1 time again at inducing culture.
The method of the substratum of little plan ball algae-X57 provided by the present invention and induced lipolysis accumulation, the method of using ultrasonication and the concentration that changes carbon source, nitrogenous source, phosphorus source and ferro element in the substratum to combine, be very beneficial for the accumulation of little plan ball algae-X57 fat, its content of oil and grease has improved 40%-65% than the single induction method of nitrogenous source restriction, simultaneously can make little plan ball algae-X57 utilize the nutritive ingredient of substratum fast, promote cell to quicken division, growth cycle has shortened 2-3 days than collective media, and its biomass accumulation is 1.5-2.0 a times of collective media.Because the present invention is to feed CO
2The fat accumulation of inducing little plan ball algae-X57 of form supplementary carbon source; so the present invention can utilize industrial gaseous waste to carry out the process that large-scale little plan ball algae-X57 cultured and finished the induced lipolysis accumulation; when inducing little plan ball algae-X57 fat accumulation, finish the processing of industrial gaseous waste, energy-saving and emission-reduction, protection environment are had great importance.Face under the routed day by day weary situation of the energy in the world today in addition; the little plan ball algae-X57 that cultivates with the inventive method accumulates owing to increasing substantially its fat; therefore be suitable as the raw material of biofuel, can be the present energy dilemma of solution and make positive contribution.
Description of drawings
Fig. 1 adds the solution A of different volumes, the mixed solution of B, C and the growth curve in collective media every day for little plan ball algae-X57 in the FN17 substratum.
Fig. 2 induces the growth curve that accumulates little plan ball algae-X57 with the single condition induced lipolysis of nitrogen element for ultrasonic wave and the many conditions of culture condition
Fig. 3 is nitrogenous source and the phosphorus source that adds high density the induced lipolysis accumulation stage, the growth curve of little plan ball algae-X57 during high-intensity supersound process
Fig. 4 is nitrogenous source and the phosphorus source that adds extremely low concentration the induced lipolysis accumulation stage, the growth curve of little plan ball algae-X57 during more weak supersound process
Fig. 5 is that induction period adds the nitrogenous source and the phosphorus source of higher concentration, the growth curve of little plan ball algae during supersound process of middle intensity
Embodiment
1, the prescription of FN17 substratum is as follows:
Sodium-chlor 35g/L SODIUMNITRATE 160mg/L
Iron trichloride 4.5mg/L disodium ethylene diamine tetraacetate 2.5mg/L
SODIUM PHOSPHATE, MONOBASIC 12.0mg/L trace element 1ml/L
Wherein Wei Liangyuansu compound method is: get copper sulfate 11mg, zinc sulfate 25mg, cobalt chloride 22.5mg, Manganous chloride tetrahydrate 220mg adding distil water to 1L.
Solution A:
SODIUMNITRATE 290g/L trace element 1ml/L
Wherein trace element formula is:
Copper sulfate 1.5g/L zinc sulfate 4.5g/L
Cobalt chloride 3g/L Manganous chloride tetrahydrate 35g/L
Add water and be settled to 1L
Solution B:
SODIUM PHOSPHATE, MONOBASIC 190g/L
Solution C:
Iron trichloride 7g/L
Solution A: B: C was according to 18: 1: 18 mixed, and the add-on of every day is respectively 6ml/L, 8ml/L and 10ml/L.With the above-mentioned substance dissolving, be settled to 1L with distilled water, 121 ℃ of autoclaving 20min.
2, the collective media prescription is as follows:
At first prepare artificial seawater, filling a prescription is:
Sodium-chlor 18g/L magnesium sulfate heptahydrate 2.6g/L
Repone K 0.6g/L SODIUMNITRATE 1g/L
Calcium dichloride dihydrate 0.3g/L potassium primary phosphate 0.05g/L
Tri methylol amino methane 3.03g/L
Ammonium chloride 0.027g/L
Trace element 10ml iron storage liquid 1ml
Wherein Wei Liangyuansu prescription is:
Disodium ethylene diamine tetraacetate 1g/L boric acid 1.14g/L
Iron(III) chloride hexahydrate 0.049g/L Manganous sulfate monohydrate 0.164g/L
Zinc vitriol 0.022g/L cobalt chloride hexahydrate 0.0048g/L
Add water and be settled to 1L, refrigerator is preserved
The compound method of iron storage liquid is:
Disodium ethylene diamine tetraacetate 20g/L Iron(III) chloride hexahydrate 1.62g/L
0.1mol/L hydrochloric acid 900ml/L
Add water and be settled to 1L, refrigerator is preserved.
Based on the artificial seawater for preparing, nutritive ingredient below in artificial seawater, adding:
SODIUMNITRATE 0.15g/L two hypophosphite monohydrate sodium dihydrogen 0.011g/L
Trace element 1ml Fe EDTA 0.01g/L
Wherein micro-compound method:
Tetrahydrate manganese chloride 0.22g/L CoCL2 0.11g/L
Cupric sulfate pentahydrate 0.02g/L Zinc Sulphate Heptahydrate 0.044g/L
Sodium Molybdate Dihydrate 0.012g/L
Add water and be settled to 1L.
With the above-mentioned substance dissolving, be settled to 1L with distilled water, 121 ℃ of autoclaving 20min shake adding 100mlFN17 substratum and collective media in the bottle at 250ml, and inoculum density is 4 * 10
5Individual cells/ml, at 26 ℃ of temperature, light: dark=14: 10, light intensity 100001ux cultivates under the conditions such as shaking speed 140r/min.Cultivated through 7 days, the maximum biomass of little plan ball algae-X57 can reach 4.2 * 10
8Individual cells/ml (see figure 1).
Can learn according to above-mentioned different cultivation results, use FN17 substratum provided by the invention, daily adding solution A: B: C was according to 18: 1: 18 mixed, when add-on is 8ml/L, the accumulation of its biomass is to use 2 times of collective media, it is loaded down with trivial details to have overcome the collective media preparation simultaneously, the defective that cost is higher.
1. the many conditions of ultrasonic wave and culture condition are induced
In 300L small light reactor assembly, add the FN17 substratum, little plan ball algae-X57 and culture volume ratio are 1: 8 ratio inoculation, inoculum density is 2.1 * 10
5Individual cells/ml, culture temperature is 26-28 ℃, and illumination is 15000-180001ux, and light application time is 14 hours/day, the mixed solution 0.8ml/L (solution A: B: C according to 18: 1: 18 mixed) that adds solution A, B, C every day, cultivate after 8 days, with the frequency of 18kHz, the power of 4w, treatment time 15s, supersound process 2 times, each timed interval is 10s, feeding concentration with the flow velocity of 1.0-1.5L/min then is 5% CO
2Add the mixed solution (solution A: B according to 15: 1 mixed) of solution A, B every day, add-on is 30 μ l/L, the amount of disposable adding solution C is 1.2ml/L, and inducing culture is after 2 days, again with the frequency of 10kHz, the power of 3w, treatment time 8s, supersound process 1 time, coinduction fat accumulation 4 days.
2. the single induction method of nitrogenous source restriction
In 300L small light reactor assembly, add the FN17 substratum, little plan ball algae-X57 and culture volume ratio are 1: 8 ratio inoculation, inoculum density is 2.1 * 10
5Individual cells/ml, culture temperature are 26-28 ℃, and illumination is 15000-180001ux, light application time is 14 hours/day, adds FN17 nutrient solution stoste 0.8ml/L every day, cultivates after 8 days, adding SODIUMNITRATE content every day is the FN17 nutrient solution of 16mg/L, and add-on is 0.8ml/L, inducing culture 4 days.
The result shows, during with the little plan ball algae of many conditions inducing culture-X57, its growth is cultivated and promptly entered exponential phase of growth on the 3rd day rapidly, and cultivating accessible maximum cell density is 4.71 * 10
8Individual cells/ml (see figure 2), induced lipolysis accumulation back dry weight is 2.58g/L, exceeding nitrogen lacks single factor and induces 11% of gained dry cell weight, after many conditions such as process ultrasonic wave are induced, the percentage composition that its fat accounts for the frond dry weight can reach 37%, the output of total fat is 955mg/L, and the per-cent that its fat accounts for the frond dry weight is that nitrogen lacks 1.7 times of wall scroll spare inductive, as table 1:
Many conditions that table 1. ultrasonic wave combines with culture condition are induced with nitrogen and are lacked each index of wall scroll spare inductive relatively
In 300L small light reactor assembly, add the FN17 substratum, little plan ball algae-X57 and culture volume ratio are 1: 8 ratio inoculation, inoculum density is 2.21 * 10
5Individual cells/ml, culture temperature is 26-28 ℃, illumination is 15000-180001ux, and light application time is 14 hours/day, adds the mixed solution 0.8ml/L (solution A: B: C according to 18: 1: 18 mixed) of solution A, B, C every day, cultivate after 7 days, with the frequency of 35kHz, the power of 6w, treatment time 60s, supersound process 1 time, feeding concentration with the flow velocity of 0.5-1.0L/min then is 2% CO
2Add the mixed solution (solution A: B according to 10: 1 mixed) of solution A, B every day, add-on is 100 μ l/L, the amount of disposable adding solution C is 0.5ml/L, and inducing culture is after 2 days, the frequency of 20kHz, the power of 5w, treatment time 15s, supersound process 1 time, coinduction fat accumulation 4 days.
The result shows, cultivates little plan ball algae-X57 with this method, and its growth is cultivated and promptly entered exponential phase of growth on the 3rd day rapidly, and cultivating accessible maximum cell density is 4.45 * 10
8Individual cells/ml (see figure 3), induced lipolysis accumulation back dry weight is 2.39g/L, and through after inducing, the percentage composition that its fat accounts for dry weight can reach 32%, and the output of total fat is 765mg/L.
In 300L small light reactor assembly, add the FN17 substratum, little plan ball algae-X57 and culture volume ratio are 1: 8 ratio inoculation, inoculum density is 2.24 * 10
5Individual cells/ml, culture temperature is 26-28 ℃, and illumination is 15000-180001ux, and light application time is 14 hours/day, the mixed solution 0.8ml/L (solution A: B: C according to 18: 1: 18 mixed) that adds solution A, B, C every day, cultivate after 8 days, with the frequency of 10kHz, the power of 2w, treatment time 15s, supersound process 3 times, each timed interval is 10s, feeding concentration with the flow velocity of 1.5-2.5L/min is 6% CO
2Add the mixed solution (solution A: B according to 20: 1 mixed) of solution A, B every day, add-on is 0.5 μ l/L, and the amount of disposable adding solution C is 1.5ml/L, and inducing culture is after 2 days, again with frequency with 10kHz, the power of 1w, treatment time 5s, supersound process 2 times, each timed interval is 10s, coinduction fat accumulation 4 days.
The result shows, cultivates little plan ball algae-X57 with this method, and its growth is cultivated and promptly entered exponential phase of growth on the 3rd day rapidly, and cultivating accessible maximum cell density is 3.6 * 10
8Individual cells/ml (see figure 4), induced lipolysis accumulation back dry weight is 2.09g/L, and through after inducing, the percentage composition that its fat accounts for dry weight can reach 39%, and the output of total fat is 815mg/L.
In 300L small light reactor assembly, add the FN17 substratum, little plan ball algae-X57 and culture volume ratio are 1: 8 ratio inoculation, inoculum density is 2.27 * 10
5Individual cells/ml, culture temperature is 26-28 ℃, illumination is 15000-180001ux, and light application time is 14 hours/day, adds the mixed solution 0.8ml/L (solution A: B: C according to 18: 1: 18 mixed) of solution A, B, C every day, cultivate after 7 days, with the frequency of 25kHz, the power of 8w, treatment time 45s, supersound process 1 time, feeding concentration with the flow velocity of 1.0-1.5L/min then is 5% CO
2Add the mixed solution (solution A: B according to 20: 1 mixed) of solution A, B every day, add-on is 60 μ l/L, the amount of disposable adding solution C is 2ml/L, and inducing culture is after 2 days, again with the frequency of 15kHz, the power of 3w, treatment time 15s, supersound process 1 time, coinduction fat accumulation 4 days.
The result shows, cultivates little plan ball algae-X57 with this method, and its growth is cultivated and promptly entered exponential phase of growth on the 3rd day rapidly, and cultivating accessible maximum cell density is 3.82 * 10
8Individual cells/ml (see figure 5), induced lipolysis accumulation back dry weight is 2.28g/L, and through after inducing, the percentage composition that its fat accounts for dry weight can reach 34%, and the output of total fat is 775mg/L.The different levels of inductive condition sees Table 2 to little plan ball algae-X57 fat effect of accumulation among the embodiment 2,3,4,5
The different levels of table 2 inductive condition is to little plan ball algae-X57 fat effect of accumulation
Claims (10)
1. the substratum of a little plan ball algae-X57, its prescription is:
Sodium-chlor 18.0-40.0g/L SODIUMNITRATE 120.0-220.0mg/L
Iron trichloride 2.5-6.5mg/L disodium ethylene diamine tetraacetate 1.0-6.0mg/L
SODIUM PHOSPHATE, MONOBASIC 7.0-18.0mg/L trace element 0.5-2.0ml/L
Wherein Wei Liangyuansu compound method is:
Copper sulfate 5.5-22.5mg/L zinc sulfate 14.0-56.5mg/L
Cobalt chloride 11.5-45.5mg/L Manganous chloride tetrahydrate 113.5-453.0mg/L
Adding distil water is to 1L
The compound method of daily interpolation nutritive medium mother liquor:
Solution A:
SODIUMNITRATE 280-320g/L trace element 0.5-2ml/L
Wherein trace element formula is:
Copper sulfate 1-3g/L zinc sulfate 4-6g/L
Cobalt chloride 3-5g/L Manganous chloride tetrahydrate 30-50g/L
Add water and be settled to 1L.
Solution B:
SODIUM PHOSPHATE, MONOBASIC 180-220g/L
Solution C:
Iron trichloride 6-10g/L disodium ethylene diamine tetraacetate 4-9g/L.
2. the substratum of little plan ball algae-X57 as claimed in claim 1, it is filled a prescription preferably and is:
Sodium-chlor 20.0-35.0g/L SODIUMNITRATE 150.0-200.0mg/L
Iron trichloride 3.5-5.5mg/L disodium ethylene diamine tetraacetate 2.0-3.5mg/L
SODIUM PHOSPHATE, MONOBASIC 9.0-14.0mg/L trace element 0.5-2.0ml/L
Wherein Wei Liangyuansu compound method is:
Copper sulfate 9.0-15.0mg/L zinc sulfate 19.5-32.5mg/L
Cobalt chloride 14.5-28.0mg/L Manganous chloride tetrahydrate 190.5-265.0mg/L
Adding distil water is to 1L.
3. a method of inducing little plan ball algae as claimed in claim 1-X57 fat accumulation is characterized in that comprising the steps:
(1) nutritive medium mother liquor A, B, the C of preparation substratum and daily interpolation;
(2) little plan ball algae-X57 and substratum are pressed 1: 5-1: 10 volume ratio inoculation, in micro algae culturing liquid, add the mixed solution of nutritive medium mother liquor A, B, C every day, to cultivate 5-8 days, this process is the stage of little plan ball algae-X57 accumulation biomass;
(3) the little plan ball algae-X57 nutrient solution after using ultrasonic apparatus to biomass accumulation carries out supersound process, feeds CO then in micro algae culturing liquid
2, owing to join nutritive medium mother liquor A in little plan ball algae-X57 nutrient solution: the concentration of B need reduce, and the concentration of mother solution C need increase, and therefore add nutritive medium mother liquor A respectively: the mixed solution of B and mother solution C are carried out the cultivation of induced lipolysis accumulation.
4. method of inducing little plan ball algae-X57 fat accumulation as claimed in claim 3, it is characterized in that little plan ball algae-X57 is is 18-30 ℃ in temperature, the pH value is 6-9, illumination is 5000-250001ux, light application time is to carry out the accumulation of biomass under 10-16 hour/day the condition, solution A: B: C was according to 10: 1: 10-25: 1: 25 mixed, add-on are 0.6-2ml/L every day.
5. method of inducing little plan ball algae-X57 fat accumulation as claimed in claim 4, it is characterized in that the biomass accumulation condition is preferably: culture temperature 22-28 ℃, pH value 7-8, illumination 12000-200001ux, light application time 11-14 hour/day, nutritive medium mother liquor A, B, C ratio are 12: 1: 12-20: 1: 20, every day, add-on was 0.6-1.5ml/L, cultivated 6-8 days.
6. method of inducing little plan ball algae-X57 fat accumulation as claimed in claim 4, it is characterized in that best biomass accumulation condition is: nutritive medium mother liquor A, B, C ratio are 18: 1: 18, every day, add-on was 0.8ml/L, cultivated 8 days.
7. method of inducing little plan ball algae-X57 fat accumulation as claimed in claim 3 is characterized in that
Frequency when carrying out supersound process is 15-35kHz, and power is 2-8w, handles 1-3 time, is spaced apart 10s at every turn, and the treatment time is 10-60s/ time, feeds CO to micro algae culturing liquid again
2, and add nutritive medium mother liquor A: under the condition of B and C, inducing culture 2 days, with the frequency of 10-20kHz, the power of 1-5w is handled 1-2 time again, each 10s at interval handles 5-15s/ time, coinduction cultivation 4 days.
8. method of inducing little plan ball algae-X57 fat accumulation as claimed in claim 7, it is characterized in that fatty preferably inductive condition is: ultrasonic frequency is 15-20kHz, power be 2-6w, the treatment time is 10-30s, handle 1-2 time, each timed interval is 10s, and inducing culture is in the time of 2 days, again with the frequency of 10-15kHz, the power of 1-3w is handled 1 time, and the treatment time is 5-10s.
9. as claim 3,7 described methods of inducing little plan ball algae-X57 fat accumulation, it is characterized in that in micro algae culturing liquid, feeding CO
2Concentration be that 2%-6%, flow are 0.5-2.5L/min, nutritive medium mother liquor A: B is according to 10: 1-20: 1 mixed, every day, magnitude of recruitment was 0.5-100 μ l/L, the add-on of solution C is 0.5-2ml/L.
10. method of inducing little plan ball algae-X57 fat accumulation as claimed in claim 9 is characterized in that feeding CO in micro algae culturing liquid
2Concentration be 3%-6%, flow is 0.8-1.5L/min, nutritive medium mother liquor A: B is according to 12: 1-18: 1 mixed, add-on are 0.5-60 μ l/L, and the add-on of solution C is 0.5-1.5ml/L.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102492626A (en) * | 2011-12-16 | 2012-06-13 | 新奥科技发展有限公司 | Nannochloropsis sp and application thereof |
| CN102660464A (en) * | 2012-05-17 | 2012-09-12 | 国家海洋局第一海洋研究所 | Oil-enriched marine microalga and application thereof |
| CN103146581A (en) * | 2013-02-28 | 2013-06-12 | 大连理工大学 | Marine Nannochloropsis oceanica strain containing rich hexadecadienoic acid and culture method thereof capable of enhancing biomass and oil content |
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| CN109439650A (en) * | 2018-10-10 | 2019-03-08 | 安徽欣伯玉生物科技有限公司 | A kind of extracting method of Agrobacterium plasmid DNA |
| CN110540896A (en) * | 2019-09-29 | 2019-12-06 | 武汉轻工大学 | Preparation method of EPA-containing nannochloropsis oculata oil |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102492626A (en) * | 2011-12-16 | 2012-06-13 | 新奥科技发展有限公司 | Nannochloropsis sp and application thereof |
| CN102492626B (en) * | 2011-12-16 | 2015-11-25 | 新奥科技发展有限公司 | Intend Nannochloropsis oceanica and application thereof |
| CN102660464A (en) * | 2012-05-17 | 2012-09-12 | 国家海洋局第一海洋研究所 | Oil-enriched marine microalga and application thereof |
| CN103146581A (en) * | 2013-02-28 | 2013-06-12 | 大连理工大学 | Marine Nannochloropsis oceanica strain containing rich hexadecadienoic acid and culture method thereof capable of enhancing biomass and oil content |
| CN103146581B (en) * | 2013-02-28 | 2014-06-25 | 大连理工大学 | Marine Nannochloropsis oceanica strain containing rich hexadecadienoic acid and culture method thereof capable of enhancing biomass and oil content |
| CN105132404A (en) * | 2015-10-12 | 2015-12-09 | 山东大学 | Method for fast accumulating grease through ultrasonic stimulation micro algae |
| CN105132404B (en) * | 2015-10-12 | 2021-03-02 | 山东大学 | Method for rapidly accumulating grease by utilizing ultrasonic stimulation microalgae |
| CN109439650A (en) * | 2018-10-10 | 2019-03-08 | 安徽欣伯玉生物科技有限公司 | A kind of extracting method of Agrobacterium plasmid DNA |
| CN110540896A (en) * | 2019-09-29 | 2019-12-06 | 武汉轻工大学 | Preparation method of EPA-containing nannochloropsis oculata oil |
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