CN110229755A - A method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulates - Google Patents
A method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulates Download PDFInfo
- Publication number
- CN110229755A CN110229755A CN201910523205.0A CN201910523205A CN110229755A CN 110229755 A CN110229755 A CN 110229755A CN 201910523205 A CN201910523205 A CN 201910523205A CN 110229755 A CN110229755 A CN 110229755A
- Authority
- CN
- China
- Prior art keywords
- epiphysin
- frustule
- algae
- grease
- nitrogen stress
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 82
- 239000004519 grease Substances 0.000 title claims abstract description 67
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 35
- 241000195493 Cryptophyta Species 0.000 claims abstract description 79
- 230000006698 induction Effects 0.000 claims abstract description 35
- 239000003960 organic solvent Substances 0.000 claims abstract description 27
- 238000009825 accumulation Methods 0.000 claims abstract description 16
- 239000012452 mother liquor Substances 0.000 claims abstract description 16
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 11
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 11
- 239000008103 glucose Substances 0.000 claims abstract description 11
- 238000005286 illumination Methods 0.000 claims abstract description 11
- 230000003698 anagen phase Effects 0.000 claims abstract description 10
- 239000001963 growth medium Substances 0.000 claims abstract description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229960000935 dehydrated alcohol Drugs 0.000 claims abstract description 8
- 230000001737 promoting effect Effects 0.000 claims abstract 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical group OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 27
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 18
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 18
- 239000000843 powder Substances 0.000 claims description 17
- 238000005119 centrifugation Methods 0.000 claims description 16
- 239000006004 Quartz sand Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 9
- 239000012074 organic phase Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- 238000000638 solvent extraction Methods 0.000 claims description 9
- 241000579585 Monoraphidium sp. QLY-1 Species 0.000 claims description 4
- 238000012549 training Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims 1
- 239000002028 Biomass Substances 0.000 abstract description 20
- 230000004069 differentiation Effects 0.000 abstract description 8
- 239000000835 fiber Substances 0.000 abstract description 8
- 230000005791 algae growth Effects 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 abstract 2
- 239000007640 basal medium Substances 0.000 abstract 1
- 239000012895 dilution Substances 0.000 abstract 1
- 238000010790 dilution Methods 0.000 abstract 1
- 230000035882 stress Effects 0.000 description 34
- 230000000052 comparative effect Effects 0.000 description 21
- 150000002632 lipids Chemical class 0.000 description 10
- 239000000284 extract Substances 0.000 description 8
- 230000012010 growth Effects 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 239000004576 sand Substances 0.000 description 7
- 239000004575 stone Substances 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 4
- 230000036579 abiotic stress Effects 0.000 description 4
- 229930000044 secondary metabolite Natural products 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000003375 plant hormone Substances 0.000 description 3
- 241000238557 Decapoda Species 0.000 description 1
- 241001478792 Monoraphidium Species 0.000 description 1
- 108010053210 Phycocyanin Proteins 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000003225 biodiesel Substances 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- -1 grease Natural products 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/10—Production of fats or fatty oils from raw materials by extracting
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/16—Refining fats or fatty oils by mechanical means
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
Abstract
The present invention relates to a kind of methods for promoting bloom nitrogen stress to coerce lower microalgae grease accumulation using epiphysin, belong to technical field of microalga biology.The present invention is under the conditions of temperature is 24~26 DEG C, using 10g/L glucose as the BG-11 basal medium Heterotrophic culture single needle algae of carbon source, to single needle algae grow to the logarithmic growth phase later period collect frustule, use nitrogen stress BG-11 culture medium dilute resuspension frustule to 0.7g/L as induce algae solution；The epiphysin mother liquor that 10mmol/L is prepared with dehydrated alcohol, it is 10-80 μm of ol/L that epiphysin mother liquor, which is added dilution epiphysin concentration in induction algae solution, and being placed in temperature is 24~26 DEG C, 100~120 μm of ol m‑2s‑1Fiber differentiation under intensity of illumination；The grease in frustule is extracted using organic solvent.The method of the present invention is easy to operate, can shorten the period of frustule induction, improves fat content, guarantees micro algae growth, and can solve biomass present in microalgae industrialization Induction Process reduces the problems such as low with fat content.
Description
Technical field
The present invention relates to technical field of microalga biology, and in particular to it is a kind of using epiphysin promote bloom nitrogen stress stress under it is micro-
The method of algae oil and fat accumulation.
Background technique
Microalgae has certain tolerance to abiotic stress, can accumulate high value secondary metabolites, including grease, shrimp blueness
Element, phycocyanin etc..Again because it can carry out photosynthesis, solar energy can be efficiently used by water, CO2It has been converted into inorganic salts
Machine object.Therefore, raw material of the microalgae biomass as substitution terrestrial plant production bio-fuel.
Under the conditions of heterotrophism microalgae using the organic carbon sources such as glucose can Rapid Accumulation biomass, substantially reduce frustule
Growth cycle improves cellular biomass yield, but the frustule inferior quality cultivated under the conditions of heterotrophism, the algae generally obtained are thin
Born of the same parents' fat content is lower, therefore the heterotrophism stage is generally unsuitable for accumulating grease.And two-phase method induction microalgae is used to accumulate grease
As the hot spot studied now, Zhao etc. utilizes " heterotrophism-photochemical induction " that fat content in microalgae is greatly improved
(Zhao,Y.,Li,D.,Ding,K.,Che,R.,Xu,J.W.,Zhao,P.,Li,T.,Ma,H.,Yu,
X.2016.Production of biomass and lipids by the oleaginous microalgae
Monoraphidium sp.QLY-1through heterotrophic cultivation and photo-chemical
modulator induction.Bioresour Technol,211,669-76.)；In addition, bloom is shone, nitrogen stress is common
The mode of microalgae accumulation secondary metabolite is induced, but bloom coerces the lower growth that would generally inhibit frustule according to nitrogen stress, from
And micro algae biomass yield is reduced, and then increase the cost of microalgae production biodiesel.
Summary of the invention
For the above-mentioned problems of the prior art and deficiency, the present invention provides a kind of utilization epiphysin promotion bloom nitrogen stress
The method for coercing lower microalgae grease accumulation, the method for the present invention using can heterotrophism microalgae, the culture for carrying out algae heterotrophism keeps algae thin
Born of the same parents' fast-growth in a short time is then diluted to suitable concentration and carries out the nitrogen stress culture of light autotrophy, lures in combination with epiphysin
It leads frustule and largely accumulates grease, and extract grease in frustule using organic solvent；Operation of the present invention is simple, can shorten algae
The growth cycle of cell improves the yield of grease.
A method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulate, the specific steps are as follows:
(1) preparation of algae solution is induced: under the conditions of temperature is 24~26 DEG C, using 10g/L glucose as the BG-11 base of carbon source
Basal culture medium Heterotrophic culture single needle algae grows to logarithmic growth phase later period collection frustule to single needle algae, is trained with the BG-11 of nitrogen stress
It supports base and dilutes resuspension frustule to 0.7g/L as induction algae solution；
(2) induction frustule accumulates grease: the epiphysin mother liquor of 10mmol/L is prepared with dehydrated alcohol, by epiphysin mother liquor
It is added in the induction algae solution of step (1) and dilutes epiphysin concentration for 10~80 μm of ol/L, is placed in temperature and is lured for 24~26 DEG C
Lead culture；
(3) grease in organic solvent extraction step (2) frustule is utilized；
Further, the single needle algae is single needle phycomycete strain Monoraphidium sp.QLY-1 (NCBI:KM199735)；
Further, intensity of illumination is 100~120 μm of olm in the step (2)-2·s-1；
Further, organic solvent is chloroform-methanol in the step (3)；
Further, the volume ratio of chloroform and methanol is 1:2 in the chloroform-methanol；
Further, the method for the grease in frustule is extracted in step (3) are as follows: be centrifuged culture solution through 5000r/min
It is enriched with 5min, dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；Quartz sand is added and is ground, adds
Chloroform-methanol repeats extracting and whitens to frond, and organic phase is collected by centrifugation up to grease；Wherein the quality of quartz sand is dry algae
2 times of silty amount.
The invention has the benefit that
(1) growth of the controllable microalgae of plant hormone and secondary metabolite are metabolized and are concerned, abiotic to solve
Micro algae biomass is low under stress provides new solution；Epiphysin has the function of spectrum as a Plant Hormone, can
Regulate and control the form of plant roots, the sprouting of seed, period and a series of biology of response and abiotic stress, therefore, in the present invention
Using epiphysin, epiphysin facilitates growth and oil and fat accumulation of the microalgae under abiotic stress.
(2) process of the present invention is simple and convenient, originally less, and only need to add micro epiphysin can ensure microalgae in bloom
Biomass under the conditions of nitrogen stress, and can be further improved the accumulation of grease in microalgae, it is improved compared with control group fat content
1.35-1.47 again；
(3) present invention also further improves the lipid-producing that bloom nitrogen stress coerces lower microalgae, is adding micro epiphysin
In the case where, lipid-producing improves 1.25-1.43 times than control group, and when foreign aid adds 20 μm of ol/L epiphysins, grease is produced
1.06 times are improved under the more independent bloom nitrogen stress stress of rate；
(4) epiphysin molecular weight of the invention is small, is easily absorbed by plants, it can also be ensured that growth of the plant in adverse circumstance and
Development.In the culture of oil-producing microalgae, epiphysin can be used as exogenous plant hormones and microalgae promoted largely to accumulate secondary metabolite
With the resistance for improving microalgae.
Specific embodiment
With reference to embodiment, the invention will be further described.
Comparative example 1: a method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulate, specific steps are such as
Under:
(1) it induces the preparation of algae solution: under the conditions of temperature is 25 DEG C, being trained by the basis BG-11 of carbon source of 10g/L glucose
Base Heterotrophic culture single needle algae Monoraphidium sp.QLY-1 (NCBI:KM199735) is supported, to single needle algae Monoraphidium
Sp.QLY-1 (NCBI:KM199735) grows to logarithmic growth phase later period (biomass reaches 5g/L) collection frustule, and use is fresh
BG-11 culture medium dilute resuspension frustule to 0.7g/L as induction algae solution；
(2) induction frustule accumulates grease: the induction algae solution of step (1) is placed in temperature is 25 DEG C, intensity of illumination is 30 μ
mol m-2s-1Under the conditions of, frustule is collected by centrifugation in cold light lamp continuous light Fiber differentiation daily；
(3) grease in organic solvent extraction step (2) frustule is utilized；Wherein organic solvent is chloroform-methanol,
The volume ratio of chloroform and methanol is 1:2 in chloroform-methanol；The method that organic solvent extracts the grease in frustule are as follows: will
Culture solution is through 5000r/min centrifugal enrichment 5min, and dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；Stone is added
Sand is simultaneously ground, and is added chloroform-methanol and is repeated to extract and whiten to frond, organic phase is collected by centrifugation up to grease；
Wherein the quality of quartz sand is 2 times of dry algae powder quality.Fat content is up to 36.72% (being shown in Table 1) of dry cell weight, biology
Amount is 0.85g/L.
Comparative example 2: a method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulate, specific steps are such as
Under:
(1) it induces the preparation of algae solution: under the conditions of temperature is 25 DEG C, being trained by the basis BG-11 of carbon source of 10g/L glucose
Base Heterotrophic culture single needle algae is supported, logarithmic growth phase later period collection frustule is grown to single needle algae, is trained with the BG-11 of fresh nitrogen stress
It supports base and dilutes resuspension frustule to 0.7g/L as induction algae solution；
(2) induction frustule accumulate grease: by the induction algae solution of step (1) be placed in temperature be 25 DEG C, intensity of illumination 100
μmol·m-2·s-1Under the conditions of, frustule is collected by centrifugation in cold light lamp continuous light Fiber differentiation daily；
(3) grease in organic solvent extraction step (2) frustule is utilized；Wherein organic solvent is chloroform-methanol,
The volume ratio of chloroform and methanol is 1:2 in chloroform-methanol；The method that organic solvent extracts the grease in frustule are as follows: will
Culture solution is through 5000r/min centrifugal enrichment 5min, and dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；Stone is added
Sand is simultaneously ground, and is added chloroform-methanol and is repeated to extract and whiten to frond, organic phase is collected by centrifugation up to grease；
Wherein the quality of quartz sand is 2 times of dry algae powder quality.Fat content is up to 51.67% (being shown in Table 1) of dry cell weight, biology
Amount is 0.81g/L, and the biomass under the conditions of bloom nitrogen stress is the 95.29% of comparative example 1, and frustule fat content and grease produce
Rate has increased separately 1.41 and 1.35 times compared with comparative example 1.
Embodiment 1: a method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulate, specific steps are such as
Under:
(1) it induces the preparation of algae solution: under the conditions of temperature is 25 DEG C, being trained by the basis BG-11 of carbon source of 10g/L glucose
Base Heterotrophic culture single needle algae is supported, logarithmic growth phase later period collection frustule is grown to single needle algae, with the BG-11 culture medium of nitrogen stress
Resuspension frustule is diluted to 0.7g/L as induction algae solution；
(2) induction frustule accumulates grease: the epiphysin mother liquor of 10mmol/L is prepared with dehydrated alcohol, by epiphysin mother liquor
It is added in the induction algae solution of step (1) that dilute epiphysin concentration be 20 μm of ol/L, is placed in that temperature is 25 DEG C, intensity of illumination is
105μmol·m-2·s-1Under the conditions of, frustule is collected by centrifugation in cold light lamp continuous light Fiber differentiation daily；
(3) grease in organic solvent extraction step (2) frustule is utilized；Wherein organic solvent is chloroform-methanol,
The volume ratio of chloroform and methanol is 1:2 in chloroform-methanol；The method that organic solvent extracts the grease in frustule are as follows: will
Culture solution is through 5000r/min centrifugal enrichment 5min, and dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；Stone is added
Sand is simultaneously ground, and is added chloroform-methanol and is repeated to extract and whiten to frond, organic phase is collected by centrifugation up to grease；
Wherein the quality of quartz sand is 2 times of dry algae powder quality.The 1st day fat content is induced to reach up to dry cell weight
51.38% (being shown in Table 1), biomass 0.83g/L under bloom nitrogen stress, add the biology of 20 μm of ol/L epiphysins as known from Table 1
Amount reaches the 97.65% of comparative example 1, and biomass is 1.02 times of comparative example 2, and frustule fat content and lipid-producing relatively compare
Example 1 has increased separately 1.46 and 1.43 times, has increased separately 1.04 and 1.06 times compared with comparative example 2.
Embodiment 2: a method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulate, specific steps are such as
Under:
(1) it induces the preparation of algae solution: under the conditions of temperature is 24 DEG C, being trained by the basis BG-11 of carbon source of 10g/L glucose
Base Heterotrophic culture single needle algae is supported, logarithmic growth phase later period collection frustule is grown to single needle algae, with the BG-11 culture medium of nitrogen stress
Resuspension frustule is diluted to 0.7g/L as induction algae solution；
(2) induction frustule accumulates grease: the epiphysin mother liquor of 10mmol/L is prepared with dehydrated alcohol, by epiphysin mother liquor
It is added in the induction algae solution of step (1) that dilute epiphysin concentration be 40 μm of ol/L, is placed in that temperature is 24 DEG C, intensity of illumination is
110μmol·m-2·s-1Under the conditions of, frustule is collected by centrifugation in cold light lamp continuous light Fiber differentiation daily；
(3) grease in organic solvent extraction step (2) frustule is utilized；Wherein organic solvent is chloroform-methanol,
The volume ratio of chloroform and methanol is 1:2 in chloroform-methanol；The method that organic solvent extracts the grease in frustule are as follows: will
Culture solution is through 5000r/min centrifugal enrichment 5min, and dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；Stone is added
Sand is simultaneously ground, and is added chloroform-methanol and is repeated to extract and whiten to frond, organic phase is collected by centrifugation up to grease；
Wherein the quality of quartz sand is 2 times of dry algae powder quality.As known from Table 1, under bloom nitrogen stress, the life of 40 μm of ol/L epiphysins is added
Object amount reaches the 98.82% of comparative example 1, and biomass is 1.04 times of comparative example 2, and frustule fat content and lipid-producing are more right
Ratio 1 has increased separately 1.35 and 1.34 times, respectively the 96.05% of comparative example 2 and 99.57%.
Embodiment 3: a method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulate, specific steps are such as
Under:
(1) it induces the preparation of algae solution: under the conditions of temperature is 25 DEG C, being trained by the basis BG-11 of carbon source of 10g/L glucose
Base Heterotrophic culture single needle algae is supported, logarithmic growth phase later period collection frustule is grown to single needle algae, with the BG-11 culture medium of nitrogen stress
Resuspension frustule is diluted to 0.7g/L as induction algae solution；
(2) induction frustule accumulates grease: the epiphysin mother liquor of 10mmol/L is prepared with dehydrated alcohol, by epiphysin mother liquor
It is added in the induction algae solution of step (1) that dilute epiphysin concentration be 80 μm of ol/L, is placed in that temperature is 26 DEG C, intensity of illumination is
115μmol·m-2·s-1Under the conditions of, frustule is collected by centrifugation in cold light lamp continuous light Fiber differentiation daily；
(3) grease in organic solvent extraction step (2) frustule is utilized；Wherein organic solvent is chloroform-methanol,
The volume ratio of chloroform and methanol is 1:2 in chloroform-methanol；The method that organic solvent extracts the grease in frustule are as follows: will
Culture solution is through 5000r/min centrifugal enrichment 5min, and dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；Stone is added
Sand is simultaneously ground, and is added chloroform-methanol and is repeated to extract and whiten to frond, organic phase is collected by centrifugation up to grease；
Wherein the quality of quartz sand is 2 times of dry algae powder quality.As known from Table 1, under bloom nitrogen stress, the life of 80 μm of ol/L epiphysins is added
Object amount is only the 90.59% of comparative example 1, biomass be the 95.06% of comparative example 2, frustule fat content and lipid-producing compared with
Comparative example 1 has increased separately 1.39 and 1.25 times, respectively the 98.47% of comparative example 2 and 92.57%.
Embodiment 4: a method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulate, specific steps are such as
Under:
(1) it induces the preparation of algae solution: under the conditions of temperature is 25 DEG C, being trained by the basis BG-11 of carbon source of 10g/L glucose
Base Heterotrophic culture single needle algae is supported, logarithmic growth phase later period collection frustule is grown to single needle algae, with the BG-11 culture medium of nitrogen stress
Resuspension frustule is diluted to 0.7g/L as induction algae solution；
(2) induction frustule accumulates grease: the epiphysin mother liquor of 10mmol/L is prepared with dehydrated alcohol, by epiphysin mother liquor
It is added in the induction algae solution of step (1) that dilute epiphysin concentration be 20 μm of ol/L, is placed in that temperature is 26 DEG C, intensity of illumination is
120μmol·m-2·s-1Under the conditions of, frustule is collected by centrifugation in cold light lamp continuous light Fiber differentiation daily；
(3) grease in organic solvent extraction step (2) frustule is utilized；Wherein organic solvent is chloroform-methanol,
The volume ratio of chloroform and methanol is 1:2 in chloroform-methanol；The method that organic solvent extracts the grease in frustule are as follows: will
Culture solution is through 5000r/min centrifugal enrichment 5min, and dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；Stone is added
Sand is simultaneously ground, and is added chloroform-methanol and is repeated to extract and whiten to frond, organic phase is collected by centrifugation up to grease；
Wherein the quality of quartz sand is 2 times of dry algae powder quality.As known from Table 1, under coercing under nitrogen stress, increase illumination to 120 μm of ol
m-2·s-1, the biomass for adding 20 μm of ol/L epiphysins is the 96.47% of comparative example 1, and biomass is 1.01 times of comparative example 2,
Frustule fat content and lipid-producing have increased separately 1.47 and 1.42 times, respectively 1.04 Hes of comparative example 2 compared with comparative example 1
1.05 again.
Embodiment 5: a method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulate, specific steps are such as
Under:
(1) it induces the preparation of algae solution: under the conditions of temperature is 25 DEG C, being trained by the basis BG-11 of carbon source of 10g/L glucose
Base Heterotrophic culture single needle algae is supported, logarithmic growth phase later period collection frustule is grown to single needle algae, with the BG-11 culture medium of nitrogen stress
Resuspension frustule is diluted to 0.7g/L as induction algae solution；
(2) induction frustule accumulates grease: the epiphysin mother liquor of 10mmol/L is prepared with dehydrated alcohol, by epiphysin mother liquor
It is added in the induction algae solution of step (1) that dilute epiphysin concentration be 10 μm of ol/L, is placed in that temperature is 26 DEG C, intensity of illumination is
100μmol·m-2·s-1Under the conditions of, frustule is collected by centrifugation in cold light lamp continuous light Fiber differentiation daily；
(3) grease in organic solvent extraction step (2) frustule is utilized；Wherein organic solvent is chloroform-methanol,
The volume ratio of chloroform and methanol is 1:2 in chloroform-methanol；The method that organic solvent extracts the grease in frustule are as follows: will
Culture solution is through 5000r/min centrifugal enrichment 5min, and dry algae powder is made in freeze-drying after washing 3 times repeatedly with distilled water, weighs；Stone is added
Sand is simultaneously ground, and is added chloroform-methanol and is repeated to extract and whiten to frond, organic phase is collected by centrifugation up to grease；
Wherein the quality of quartz sand is 2 times of dry algae powder quality.As known from Table 1, under bloom nitrogen stress, the life of 10 μm of ol/L epiphysins is added
Object amount is the 97.65% of comparative example 1, and biomass is 1.02 times of comparative example 2, and frustule fat content and lipid-producing relatively compare
Example 1 has increased separately 1.4 and 1.37 times, respectively the 99.65% of comparative example 2 and 101.67%.
Micro algae biomass, Biomass yield, fat content and lipid-producing result under the different training modes of table 1
Note: MT- epiphysin
Heterotrophic culture microalgae can quickly increase micro algae biomass, and the frustule after heterotrophism is induced using light autotrophy accumulates oil
Rouge finds to can promote the accumulation of grease in frustule under the conditions of nitrogen stress, but micro algae growth is suppressed；Add the outer of appropriate concentration
Source epiphysin can further improve the accumulation of grease in frustule under abiotic stress, and maintain the growth of frustule；Pass through
The method of this induction microalgae, had both realized quick, a large amount of accumulation of grease in frustule, and had in turn ensured the stable growth of microalgae,
It is remarkably improved the lipid-producing of microalgae.But when epiphysin excessive concentration, there is certain toxic action to microalgae, it is suppressed that algae
The growth of cell.
The above is only preferable case of the invention, does not make any restrictions to the present invention, all for the present invention
Any simple modification, alteration or imitation that technology contents do the above case study on implementation belongs to the protection of technical solution of the present invention
Range.
Claims (6)
1. a kind of method for promoting bloom nitrogen stress to coerce lower microalgae grease accumulation using epiphysin, it is characterised in that: specific steps
It is as follows:
(1) it induces the preparation of algae solution: under the conditions of temperature is 24~26 DEG C, being trained by the basis BG-11 of carbon source of 10g/L glucose
Base Heterotrophic culture single needle algae is supported, logarithmic growth phase later period collection frustule is grown to single needle algae, with the BG-11 culture medium of nitrogen stress
Resuspension frustule is diluted to 0.7g/L as induction algae solution；
(2) induction frustule accumulates grease: preparing the epiphysin mother liquor of 10mmol/L with dehydrated alcohol, epiphysin mother liquor is added
It is 10-80 μm of ol/L that epiphysin concentration is diluted into the induction algae solution of step (1), and being placed in temperature is 24~26 DEG C of induction trainings
It supports；
(3) grease in organic solvent extraction step (2) frustule is utilized.
2. the method according to claim 1 for promoting bloom nitrogen stress to coerce lower microalgae grease accumulation using epiphysin, special
Sign is: single needle algae is single needle phycomycete strain Monoraphidium sp.QLY-1 (NCBI:KM199735).
3. the method according to claim 1 for promoting bloom nitrogen stress to coerce lower microalgae grease accumulation using epiphysin, special
Sign is: intensity of illumination is 100~120 μm of olm in step (2)-2·s-1。
4. the method according to claim 1 for promoting bloom nitrogen stress to coerce lower microalgae grease accumulation using epiphysin, special
Sign is: organic solvent is chloroform-methanol in step (3).
5. the method according to claim 4 for promoting bloom nitrogen stress to coerce lower microalgae grease accumulation using epiphysin, special
Sign is: the volume ratio of chloroform and methanol is 1:2 in the chloroform-methanol.
6. the method according to claim 4 for promoting bloom nitrogen stress to coerce lower microalgae grease accumulation using epiphysin, special
Sign is: the method for the grease in frustule is extracted in step (3) are as follows: by culture solution through 5000r/min centrifugal enrichment 5min, uses
Dry algae powder is made in freeze-drying after distilled water washs 3 times repeatedly, weighs；Quartz sand is added and is ground, it is molten to add chloroform-methanol
Liquid repeats extracting and whitens to frond, and organic phase is collected by centrifugation up to grease；Wherein the quality of quartz sand is the 2 of dry algae powder quality
Times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910523205.0A CN110229755A (en) | 2019-06-17 | 2019-06-17 | A method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulates |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910523205.0A CN110229755A (en) | 2019-06-17 | 2019-06-17 | A method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulates |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110229755A true CN110229755A (en) | 2019-09-13 |
Family
ID=67859954
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910523205.0A Pending CN110229755A (en) | 2019-06-17 | 2019-06-17 | A method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulates |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110229755A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607238A (en) * | 2019-10-24 | 2019-12-24 | 昆明理工大学 | Method for promoting grease accumulation of monoraphidium by combining nitrogen deficiency with strigolactone |
Citations (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004035798A2 (en) * | 2002-10-18 | 2004-04-29 | Cropdesign N.V. | Identification of e2f target genes and uses thereof |
| CN102191179A (en) * | 2011-04-21 | 2011-09-21 | 中国科学院青岛生物能源与过程研究所 | Method for culturing marine oil-producing microalgae |
| CN102268377A (en) * | 2011-07-11 | 2011-12-07 | 江南大学 | Method for improving lipid producing microalga biomass and lipid accumulation with two stage culture strategy of mixotrophic and nitrogen-rich-nitrogen-deficient culture |
| CN103289886A (en) * | 2012-03-01 | 2013-09-11 | 中国科学院青岛生物能源与过程研究所 | Microalgae half-dry solid state adherent cultivation device for alternate illumination of bright and dark light |
| CN103484372A (en) * | 2013-10-01 | 2014-01-01 | 陈意民 | Culture method for increasing biomass and grease yield of microalgae |
| CN103592283A (en) * | 2013-11-21 | 2014-02-19 | 中国科学院青岛生物能源与过程研究所 | Method for quickly detecting microalgae energy-generating process |
| CN104140929A (en) * | 2013-05-07 | 2014-11-12 | 中国科学院上海高等研究院 | Method for improving microalgae growth rate and oil yield |
| EP2942390B1 (en) * | 2014-05-07 | 2017-10-04 | Commissariat À L'Énergie Atomique Et Aux Énergies Alternatives | Green microalgae lacking a functional DYRKP-1 gene, for use for increased production of feedstock |
| CN107384802A (en) * | 2017-08-23 | 2017-11-24 | 山东大学 | A kind of method for promoting microalgae grease to accumulate and keep microalgae high-biomass |
| KR20180076607A (en) * | 2016-12-28 | 2018-07-06 | 인천대학교 산학협력단 | Cosmetic composition comprising ishige okamurae extract with sleeping induction function, and cosmetics using the same |
| CN108456700A (en) * | 2018-04-16 | 2018-08-28 | 昆明理工大学 | A method of improving grease in microalgae using give up mash and magnesium ion of molasses |
| CN108531401A (en) * | 2018-05-30 | 2018-09-14 | 昆明理工大学 | A method of utilizing the useless mash of microalgae processing molasses |
| CN108587918A (en) * | 2018-05-30 | 2018-09-28 | 昆明理工大学 | A method of it is accumulated based on simultaneous foster microalgae grease |
| CN108588136A (en) * | 2018-04-16 | 2018-09-28 | 昆明理工大学 | A method of promoting heterotrophic microalgae oil and fat accumulation using epiphysin joint nitrogen stress |
| CN108753620A (en) * | 2018-05-30 | 2018-11-06 | 昆明理工大学 | A method of improving haematococcus pluvialis biomass and content astaxanthin |
| CN109504716A (en) * | 2019-01-29 | 2019-03-22 | 西北农林科技大学 | A kind of cultural method promoting scenedesmus obliquus oil-producing |
| CN109561727A (en) * | 2016-08-04 | 2019-04-02 | 西雅图咖米公司 | Health control composition and its preparation and application |
| WO2019156583A1 (en) * | 2018-02-07 | 2019-08-15 | Вадим Борисович ГАВРИЛЮК | Method for producing an agent for treatment of wounds |
| CN110607238A (en) * | 2019-10-24 | 2019-12-24 | 昆明理工大学 | Method for promoting grease accumulation of monoraphidium by combining nitrogen deficiency with strigolactone |
| US10689662B2 (en) * | 2010-04-28 | 2020-06-23 | Evogene Ltd. | Isolated polynucleotides and polypeptides for increasing plant yield and/or agricultural characteristics |
| US10791690B2 (en) * | 2009-06-10 | 2020-10-06 | Evogene Ltd. | Isolated polynucleotides and polypeptides, and methods of using same for increasing nitrogen use efficiency, yield, growth rate, vigor, biomass, oil content, and/or abiotic stress tolerance |
-
2019
- 2019-06-17 CN CN201910523205.0A patent/CN110229755A/en active Pending
Patent Citations (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004035798A2 (en) * | 2002-10-18 | 2004-04-29 | Cropdesign N.V. | Identification of e2f target genes and uses thereof |
| US10791690B2 (en) * | 2009-06-10 | 2020-10-06 | Evogene Ltd. | Isolated polynucleotides and polypeptides, and methods of using same for increasing nitrogen use efficiency, yield, growth rate, vigor, biomass, oil content, and/or abiotic stress tolerance |
| US10689662B2 (en) * | 2010-04-28 | 2020-06-23 | Evogene Ltd. | Isolated polynucleotides and polypeptides for increasing plant yield and/or agricultural characteristics |
| CN102191179A (en) * | 2011-04-21 | 2011-09-21 | 中国科学院青岛生物能源与过程研究所 | Method for culturing marine oil-producing microalgae |
| CN102268377A (en) * | 2011-07-11 | 2011-12-07 | 江南大学 | Method for improving lipid producing microalga biomass and lipid accumulation with two stage culture strategy of mixotrophic and nitrogen-rich-nitrogen-deficient culture |
| CN103289886A (en) * | 2012-03-01 | 2013-09-11 | 中国科学院青岛生物能源与过程研究所 | Microalgae half-dry solid state adherent cultivation device for alternate illumination of bright and dark light |
| CN104140929A (en) * | 2013-05-07 | 2014-11-12 | 中国科学院上海高等研究院 | Method for improving microalgae growth rate and oil yield |
| CN103484372A (en) * | 2013-10-01 | 2014-01-01 | 陈意民 | Culture method for increasing biomass and grease yield of microalgae |
| CN103592283A (en) * | 2013-11-21 | 2014-02-19 | 中国科学院青岛生物能源与过程研究所 | Method for quickly detecting microalgae energy-generating process |
| EP2942390B1 (en) * | 2014-05-07 | 2017-10-04 | Commissariat À L'Énergie Atomique Et Aux Énergies Alternatives | Green microalgae lacking a functional DYRKP-1 gene, for use for increased production of feedstock |
| CN109561727A (en) * | 2016-08-04 | 2019-04-02 | 西雅图咖米公司 | Health control composition and its preparation and application |
| KR20180076607A (en) * | 2016-12-28 | 2018-07-06 | 인천대학교 산학협력단 | Cosmetic composition comprising ishige okamurae extract with sleeping induction function, and cosmetics using the same |
| CN107384802A (en) * | 2017-08-23 | 2017-11-24 | 山东大学 | A kind of method for promoting microalgae grease to accumulate and keep microalgae high-biomass |
| WO2019156583A1 (en) * | 2018-02-07 | 2019-08-15 | Вадим Борисович ГАВРИЛЮК | Method for producing an agent for treatment of wounds |
| CN108456700A (en) * | 2018-04-16 | 2018-08-28 | 昆明理工大学 | A method of improving grease in microalgae using give up mash and magnesium ion of molasses |
| CN108588136A (en) * | 2018-04-16 | 2018-09-28 | 昆明理工大学 | A method of promoting heterotrophic microalgae oil and fat accumulation using epiphysin joint nitrogen stress |
| CN108753620A (en) * | 2018-05-30 | 2018-11-06 | 昆明理工大学 | A method of improving haematococcus pluvialis biomass and content astaxanthin |
| CN108587918A (en) * | 2018-05-30 | 2018-09-28 | 昆明理工大学 | A method of it is accumulated based on simultaneous foster microalgae grease |
| CN108531401A (en) * | 2018-05-30 | 2018-09-14 | 昆明理工大学 | A method of utilizing the useless mash of microalgae processing molasses |
| CN109504716A (en) * | 2019-01-29 | 2019-03-22 | 西北农林科技大学 | A kind of cultural method promoting scenedesmus obliquus oil-producing |
| CN110607238A (en) * | 2019-10-24 | 2019-12-24 | 昆明理工大学 | Method for promoting grease accumulation of monoraphidium by combining nitrogen deficiency with strigolactone |
Non-Patent Citations (4)
| Title |
|---|
| XUNZAN DONG等: ""Enhancement of lipid production and nutrient removal of Monoraphidium sp. FXY-10 by combined melatonin and molasses wastewater treatment Monoraphidium sp. FXY-10 by combined melatonin and molasses wastewater treatment"", 《JOURNAL OF THE TAIWAN INSTITUTE OF CHEMICAL ENGINEERS》 * |
| 宋雪婷等: ""外源褪黑素对缺氮胁迫下单针藻Monoraphidium sp．QLY-1 中油脂合成的影响"", 《中国油脂》 * |
| 李喜明等: ""褪黑素调控缺氮胁迫下单针藻中油脂积累的影响"", 《食品与发酵工业》 * |
| 李大菲等: ""褪黑素对单针藻油脂积累的影响"", 《水生生物学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607238A (en) * | 2019-10-24 | 2019-12-24 | 昆明理工大学 | Method for promoting grease accumulation of monoraphidium by combining nitrogen deficiency with strigolactone |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105803010B (en) | A method of based on heterotrophic microalgae oil and fat accumulation | |
| CN108588136A (en) | A method of promoting heterotrophic microalgae oil and fat accumulation using epiphysin joint nitrogen stress | |
| JP6807329B2 (en) | Methods and applications for culturing ω-7 fatty acid compounds and yellow-green algae to produce the compounds | |
| CN107384802A (en) | A kind of method for promoting microalgae grease to accumulate and keep microalgae high-biomass | |
| CN107475171A (en) | Application of the epiphysin in oil-producing microalgae fat content is improved | |
| CN103266062B (en) | Fresh water Chlorella pyrenoidosa XJ01 strain and application thereof in fixing CO2 and producing microalga grease | |
| CN108587918A (en) | A method of it is accumulated based on simultaneous foster microalgae grease | |
| CN104152357A (en) | High-density culture method for improving chlorophyll and protein content of chlorella at same time | |
| CN109355322A (en) | The method for improving heterotrophic microalgae lipid-producing using epiphysin joint salt stress | |
| CN104830691A (en) | Culture method of chlorella | |
| CN104830693A (en) | Nutrient switching method for culturing oleaginous microalgae | |
| CN103695482B (en) | A kind of method utilizing oil-extracted algae slag to produce microalgae grease | |
| CN110607238A (en) | Method for promoting grease accumulation of monoraphidium by combining nitrogen deficiency with strigolactone | |
| CN103952312A (en) | Freshwater Chlorella sorokiniana GS30 and application thereof | |
| CN104561146A (en) | PH two-stage adjustment culture method for increasing micro-algal grease yield | |
| CN104357327A (en) | Method for culturing microalgae on large scale by bean product wastewater | |
| CN110229755A (en) | A method of promote bloom nitrogen stress to coerce lower microalgae grease using epiphysin and accumulates | |
| CN109355321A (en) | A method of microalgae grease yield is improved using walnut shell extract | |
| CN106164281A (en) | Produce method and the manufacture method of higher unsaturated fatty acid of lubricant component | |
| CN105969664A (en) | Method for promoting oil accumulation of microalgae by adding high-concentration organic wastewater into natural seawater | |
| CN105755091A (en) | Method for increasing chlorella oil yield through co-culture of rice Pantoea agglomerans and chlorella | |
| CN109929886A (en) | A method of utilizing walnut shell extract culture single needle algae generation diesel oil | |
| CN110093381A (en) | A method of promote microalgae grease to accumulate using xylose | |
| CN109504716A (en) | A kind of cultural method promoting scenedesmus obliquus oil-producing | |
| CN103305425A (en) | Method for improving yield of botryococcus braunii hydrocarbons by illumination regulation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190913 |