CN102796725A - Method for rapidly accumulating lipid mutant microalgae strain by mutagenesis of Chlorella vulagris through laser - Google Patents
Method for rapidly accumulating lipid mutant microalgae strain by mutagenesis of Chlorella vulagris through laser Download PDFInfo
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- CN102796725A CN102796725A CN2012102873145A CN201210287314A CN102796725A CN 102796725 A CN102796725 A CN 102796725A CN 2012102873145 A CN2012102873145 A CN 2012102873145A CN 201210287314 A CN201210287314 A CN 201210287314A CN 102796725 A CN102796725 A CN 102796725A
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Abstract
The invention provides a method for rapidly accumulating lipid mutant microalgae strain by mutagenesis of Chlorella vulagris through laser, which comprises the following steps: 1) taking a fluorescent lamp as a light source for culturing Chlorella vulagris cells to a logarithmic phase; 2) taking 20ml of vulagris liquid in the logarithmic phase and dumping into a small wide-mouth bottle, processing for 3.9-4.1 minutes through semiconductor laser irradiation, magnetically stirring during the irradiation process to obtain the mutant microalgae strain; 3) dumping the vulagris liquid after mutagenesis by irradiation in a 50ml triangular flask filled with BG11 nutritive salt, wherein the final concentration of the nutritive salt in the vulagris liquid is 1/4 times, culturing for 20 days under the conditions of temperature of 20-25 DEG C and light intensity of 1800-2000lx, then transplanting the vulagris liquid to a 300ml triangular flask, continuously culturing for 20 days, then transplanting the vulagris liquid to a 1000ml triangular flask, continuously culturing for 26 days, during the culture phase, adding the BG11 nutritive salt with 1/4 times concentration once every 7 days, and timely swing the vulagris liquid for three times every day. The method provided by the invention has the advantages of easy and simple operation, low cost and high efficiency.
Description
Technical field
The present invention provides a kind of induced by laser limnetic chlorella to accumulate the method for grease sudden change algae strain fast, belongs to biological technical field.
Background technology
Chlorella (Chlorella) is a kind of common aquatic unicellular algae, is an important genus in the green alga chlorella section, and ecological the distribution extensively all has distribution in seawater and fresh water, comprise about 10 kinds at present.Chlorella cells contains rich in protein, indispensable amino acid, polysaccharide, lipid, chlorophyll, Serlabo and VITAMINs or the like, in protective foods, aquaculture, makeup, medicine and other fields, uses extensively, and supply falls short of demand in the domestic and international market.Compare with countries such as the U.S., Japan, Israel, the chlorella large-scale industrialization cultivation level of China relatively lags behind.Limnetic chlorella is the unicellular fresh water algae of a kind of sphere, is grown in usually in the water body of eutrophication, but can the raised growth breeding under the artificial culture condition.Factors such as nutritive salt, illumination, pH value, temperature, trace element, bicarbonate of ammonia all can influence this algae growth.At present, main traditional open outdoor Da Chi training method that adopts is cultivated in the chlorella industrialization, and the culture biomass of this mode is generally lower, and has shortcomings such as growth cycle length, easy pollution.It is to carry out the photoautotrophic classical pathway of chlorella that bioreactor is cultivated, but shortcomings such as cost is high, small scale have also limited its development.
The shortage of fossil energy and serious environmental are polluted and are made the exploitation of renewable energy source become very urgent.Little algae because of be prone to cultivate, oleaginousness is high and do not strive advantage such as ground with farming is regarded as unique biodiesel raw material that might substitute fossil energy fully by domestic and international expert.At present, utilize little algae production biofuel cost also too high, comparing with fossil energy on the price does not have competitive power.Obtain the strain of good produce oil algae and be reduce utilize little algae prepare biofuel cost most critical and the most effective step.Laser is a kind of photon stream, as a kind of novel gene sudden change mutagenic compound, has that energy density height, monochromaticity are good, a high directivity, simple to operate, plurality of advantages such as aberration rate is high, speed is fast, cost is low, damage is light, pollution-free, high safety.The laser mutation breeding of China has all been obtained great successes at food crop, cash crop, aquatic products and microorganism field since starting at the beginning of the seventies in last century.Little algae is the excellent material that carries out induced by laser, and it is simple in structure, life cycle is short, photosensitivity is strong, metabolic process is very easily affected by environment, and is also to be detected easily.Domestic relevant laser is started in the nineties in last century to the Study on Mutagenic Effects of little algae, only limits to the little algaes of minority economy such as tenaculat Habenaria, Haematocoocus Pluvialls, but has all obtained more satisfactory effect.The Fujian Normal University village is intelligent has done some useful explorations in this respect like seminar since 2000; Utilize laser that little algae is carried out mutagenesis, the mutagenic fungi growth velocity that obtains, pigment, protein, soluble polysaccharide content etc. all have raising in various degree than the strain of setting out.Fujian Normal University has announced the method (200410039822) of the rich selenium porphyridium stran of a kind of laser-microwave irradiation seed selection, relates to utilizing YAG laser and microwave irradiation to combine rich selenium domestication, the method for screening rich selenium porphyridium stran.The laser that is adopted, microwave irradiation equipment are simple, operational safety, mutagenesis are effective, in the seed selection of little algae algae kind, bigger promotional value are arranged.
At present, disclosing some on the home market stimulates the related patent U.S. Patent No. technology of rapid growth of chlorella, and great majority are improved to medium component.Announced a kind of culture media composition (200610024004) that high-density high-quality ordinary chlorella is cultivated that is suitable for like East China University of Science, said substratum is basically by KNO
3(7～11 grams per liter), glucose (25～35 grams per liter) and small amounts of inorganic salt, trace element and water are formed.The substratum of this invention can make Chlorella vulgaris reach under the prerequisite of high-density culture, and the frond quality is cultivated to compare with heterotrophism and had a more substantial increase, and cultivates the level when reaching the cultivation of light autotrophy when finishing.Tsing-Hua University provides a kind of method (200610078003) of culturing heterotrophic chlorella with high density.This method is in the aerobic culture bottle, to add the substratum and a little addition of C SL that contains glucose; Fast culture chlorella under the heterotrophism condition; This technology has solved heterophytic chlorella to the inadequate problem of glucose utilization; Thereby simplified production technique, reduced production cost, helped the extensive high-concentration culturing of heterophytic chlorella, shortcoming is that the chlorella harvested biomass is on the low side.Also having some is to realize the high-density culture of chlorella through optimizing bioreactor or fermentor cultivation condition.Dilution and light autotrophy as East China University of Science provides a kind of method (200610025618) of culturing chlorella with high-density and high-quality, this cultural method to comprise bio-reactor high Density Heterotrophic, high-density algae liquid are cultivated three phases.Can make chlorella cell density in short culture cycle reach the level of high-density culture, also can make the quality of chlorella cultivate to compare and have a more substantial increase, reach the level when the light autotrophy is cultivated with heterotrophism.University of Science & Technology, Beijing provide a kind of can be unglazed according to the chlorella vulgaris and the cultural method (200510086288) of heterotrophic growth; Adopt unglazed according to heterotrophism batch and fermentation culture method; Can in 3～6 day time, turn out chlorella; Dried cell weight concentration reaches the chlorella culture of 20～50g/L, through can be used as human health food or pharmaceuticals behind centrifugal, drying and the compressing tablet.Shortcoming is that operating process is complicated, and the production cycle is long, and relates to the use of plant and instrument such as bioreactor and fermentor tank.Tsing-Hua University discloses a kind of high-density fermentation of heterotrophic chlorella production method of bio-diesel oil (200810100871) of utilizing.This method is to be raw material with the heterophytic chlorella in bio-reactor middle-high density fermentation culture; The mainly screening through high growth rates and high fat content algae kind, direct inoculation are carried out the cultivation of secondary high density fermentation to shaking in the bottle to cultivate and to be forwarded to as first order seed in the fermentor tank; Carry out condition optimizing and process control; Collect behind the frond through separating, collect and dry, extracting algae oil, prepare biofuel through transesterification again.Tsing-Hua University also disclose simultaneously a kind of from the autotrophy to the heterotrophism two steps cultivate chlorella production method of bio-diesel oil (200810112998), except the preliminary culture technique of chlorella difference slightly, all the other production technique are similar in this patent.In addition; Tsing-Hua University discloses a kind of chlorella and cultural method and its application (200910083797) in production of biodiesel recently; Screen a chlorella YJ1, be mainly used in and make up city sewage deep treatment technology, when disposing of sewage, can obtain biofuel.Though the patented technology of above-mentioned relevant promotion chlorella growth and production biofuel is advanced; But the content that does not all relate to the little algae breeding of laser irradiation mutagenicity high-yield oil; And need large-scale professional facilities such as bioreactor and fermentor tank, to equipment requirements than higher, the technical matters relative complex; And expense is high, and this has increased the difficulty of production cost and technology popularization undoubtedly.
Summary of the invention
The purpose of this invention is to provide and a kind ofly have plurality of advantages such as simple to operate, speed is fast, cost is low, damage is light, pollution-free, high safety and can short-term mutagenesis go out the method that limnetic chlorella accumulates the strain of grease sudden change algae fast.Its concrete technical scheme is:
A kind of induced by laser limnetic chlorella accumulates the method for grease sudden change algae strain fast, it is characterized in that adopting following steps:
(1) preparation algae liquid: at temperature 20-25 ℃, light intensity is that 1800-2000lx, Light To Dark Ratio are 12h/12h, cultivates the limnetic chlorella cell to logarithmic phase with the daylight lamp as light source, and acquisition will be carried out the algae liquid of laser irradiation mutagenesis;
(2) laser irradiation mutagenesis: get 20ml logarithmic phase algae liquid and fall in little wide-necked bottle, handle 3.9-4.1min, keep irradiation even with magnetic agitation in the irradiation process, obtain the strain of sudden change algae with the 808nm semiconductor laser irradiation;
(3) strain of sudden change algae is cultivated: the algae liquid behind the irradiation mutagenesis is poured in the 50ml triangular flask that BG11 nutritive salt is housed; The final concentration of nutritive salt is 1/4 times in the algae liquid; Under 20 ~ 25 ℃, light intensity 1800 ~ 2000lx condition, cultivated 20 days, then algae liquid is transferred in the 300ml triangular flask, continue under these conditions to cultivate 20 days; Again algae liquid is transferred in the 1000ml triangular flask; Continue under these conditions to cultivate 26 days, whole cultivation stage added BG 11 nutritive salt of 1/4 times of concentration in per 7 days, regularly shook algae every day 3 times.
Described induced by laser limnetic chlorella accumulates the method for grease sudden change algae strain fast, in the step (2), and the integrated light source laser apparatus of LOS-BLD-0808-12W-C that semiconductor laser adopts extra large special photoelectricity Ltd to produce, power 6w.
Described induced by laser limnetic chlorella accumulates the method for grease sudden change algae strain fast, and limnetic chlorella is derived from Inst. of Hydrobiology, Chinese Academy of Sciences, is numbered No. 9 algae strain.
The present invention compared with prior art, its advantage is:
1, the present invention's simple (laser irradiation of short period of time), cost is low, damage is light, pollution-free, high safety, and starting material limnetic chlorella cell can be cultivated by ordinary method voluntarily.
2, the mutagenesis of the present invention's ability goes out to significantly improve the limnetic chlorella mutant strain of total lipid content; The experiment proof; Equal conditions was cultivated 66 days down; The mutagenesis group mutant strain of semiconductor laser irradiation 3.9-4.1min is compared with the strain of setting out without too drastic light irradiation processing, and total fat content is brought up to 1.78-1.88 times of the strain of setting out respectively, and total lipid content has reached the 34.03-34.98% of frustule dry weight respectively.Have as the potentiality of industrial biological diesel production with the little algae algae of energy kind.
Embodiment
Limnetic chlorella all adopts No. 9 limnetic chlorellas that Inst. of Hydrobiology, Chinese Academy of Sciences preserves in following examples; The integrated light source laser apparatus of LOS-BLD-0808-12W-C that the 808nm semiconductor laser adopts extra large special photoelectricity Ltd to produce, power 6w.
Embodiment 1:
Step 1, preparation algae liquid: in 20 ℃ of temperature, light intensity is that 1800lx, Light To Dark Ratio are under the 12h/12h condition, cultivates the limnetic chlorella cell to logarithmic phase with the daylight lamp as light source, makes cell concn reach 10
8Individual/ml, acquisition will be carried out the algae liquid of laser irradiation mutagenesis;
Step 2, laser irradiation mutagenesis: get 20ml logarithmic phase algae liquid and fall in little wide-necked bottle, handle 3.9min, keep irradiation even with magnetic agitation in the irradiation process, obtain the strain of sudden change algae with the 808nm semiconductor laser irradiation;
Step 3, the strain of sudden change algae are cultivated: laser irradiation enchylema is washed in the 50ml triangular flask that BG11 nutritive salt is housed; The final concentration of nutritive salt is 1/4 times in the algae liquid, is that 1800lx, Light To Dark Ratio were cultivated 20 days under for the 12h/12h culture condition in 20 ℃ of temperature, light intensity; Then above-mentioned algae liquid is transferred in the 300ml triangular flask, continues under these conditions to cultivate 20 days; Again above-mentioned algae liquid is transferred in the 1000ml triangular flask, continues under these conditions to cultivate 26 days.Whole cultivation stage added the BG11 nutritive salt of 1/4 times of concentration in per 7 days, regularly shook algae every day 3 times.
Test result: after cultivating 66 days under the above-mentioned culture condition; The grease test result shows: semiconductor laser irradiation 3.9min mutagenesis group with compare without the strain of setting out of crossing laser treatment; Fat content is brought up to 1.82 times of the strain of setting out, reaches 34.58% of dried cell weight.
Embodiment 2:
Step 1, preparation algae liquid: in 23 ℃ of temperature, light intensity is that 1900lx, Light To Dark Ratio are under the 12h/12h condition, cultivates the limnetic chlorella cell to logarithmic phase with the daylight lamp as light source, makes cell concn reach 10
8Individual/ml, acquisition will be carried out the algae liquid of laser irradiation mutagenesis;
Step 2, laser irradiation mutagenesis are got 20ml logarithmic phase algae liquid and are fallen in little wide-necked bottle, handle 4.0min with the 808nm semiconductor laser irradiation, keep irradiation even with magnetic agitation in the irradiation process, obtain the strain of sudden change algae;
Step 3, the strain of sudden change algae are cultivated: laser irradiation enchylema is washed in the 50ml triangular flask that BG11 nutritive salt is housed; The final concentration of nutritive salt is 1/4 times in the algae liquid, is that 1900lx, Light To Dark Ratio were cultivated 20 days under for the 12h/12h culture condition in 23 ℃ of temperature, light intensity; Then above-mentioned algae liquid is transferred in the 300ml triangular flask, continues under these conditions to cultivate 20 days; Again above-mentioned algae liquid is transferred in the 1000ml triangular flask, continues under these conditions to cultivate 26 days.Whole cultivation stage added the BG11 nutritive salt of 1/4 times of concentration in per 7 days, regularly shook algae every day 3 times.
Test result: after cultivating 66 days under the above-mentioned culture condition; The grease test result shows: semiconductor laser irradiation 4.0min mutagenesis group with compare without the strain of setting out of crossing laser treatment; Fat content is brought up to 1.88 times of the strain of setting out, reaches 34.98% of dried cell weight.
Embodiment 3:
Step 1, preparation algae liquid: in 25 ℃ of temperature, light intensity is that 2000lx, Light To Dark Ratio are under the 12h/12h condition, cultivates the limnetic chlorella cell to logarithmic phase with the daylight lamp as light source, makes cell concn reach 10
8Individual/ml, acquisition will be carried out the algae liquid of laser irradiation mutagenesis;
Step 2, laser irradiation mutagenesis: get 20ml logarithmic phase algae liquid and fall in little wide-necked bottle, handle 4.1min, keep irradiation even with magnetic agitation in the irradiation process, obtain the strain of sudden change algae with the 808nm semiconductor laser irradiation;
Step 3, the strain of sudden change algae are cultivated: laser irradiation enchylema is washed in the 50ml triangular flask that BG11 nutritive salt is housed; The final concentration of nutritive salt is 1/4 times in the algae liquid, is that 2000lx, Light To Dark Ratio were cultivated 20 days under for the 12h/12h culture condition in 25 ℃ of temperature, light intensity; Then above-mentioned algae liquid is transferred in the 300ml triangular flask, continues under these conditions to cultivate 20 days; Again above-mentioned algae liquid is transferred in the 1000ml triangular flask, continues under these conditions to cultivate 26 days.Whole cultivation stage added the BG11 nutritive salt of 1/4 times of concentration in per 7 days, regularly shook algae every day 3 times.
Test result: after cultivating 66 days under the above-mentioned culture condition; The grease test result shows: semiconductor laser irradiation 4.1min mutagenesis group with compare without the strain of setting out of crossing laser treatment; Fat content has improved to 1.78 times of the strain of setting out, and reaches 34.03% of dried cell weight.
The result of embodiment tests and adopts following method, and concrete steps are: 1. chlorella algae liquid is transferred in the centrifuge tube of 50ml, and after the trim, in the low-temperature and high-speed whizzer, centrifugal 10min under 8000rpm, supernatant discarded, the reject supernatant of should trying one's best; Be used for fat content mensuration with collecting good algae mud; 2. ready algae mud is placed 100 ℃ of dried overnight of electric heating constant temperature air dry oven, measure each experimental group xeraphium mass M respectively
0, and record data.Grind the xeraphium of weighing, and add isopyknic chloroform and methyl alcohol (1:2 joins existing usefulness at present); 3. under power 40W, cycle 1min, broken wall 15s, interval 5s, 3 round-robin conditions, carry out supersonic wave wall breaking; Then that broken wall is intact algae liquid is sub-packed in two 50mL centrifuge tubes; After the trim, in the low-temperature and high-speed whizzer, centrifugal 10min under the 10000rmp condition; 4. behind centrifugal the finishing, get supernatant, (chloroform: ratio supernatant) adds chloroform and vortex mixing in 3:1.Add with the isopyknic eddies of water of chloroform more afterwards and revolve mixing, leave standstill and treat its layering.Take off a layer organic phase and change in advance (the M that weighs over to
1) the 10ml centrifuge tube in, dry up the M that weighs behind the organic solvent with Nitrogen evaporator
2And record data, the Nitrogen evaporator bath temperature is set at 61 ℃; 5. the calculation formula of total lipid content is: total lipid content per-cent=(M
2-M
1)/M
0* 100%.
Claims (3)
1. an induced by laser limnetic chlorella accumulates the method for grease sudden change algae strain fast, it is characterized in that adopting following steps:
(1) preparation algae liquid: at temperature 20-25 ℃, light intensity is that 1800-2000lx, Light To Dark Ratio are 12h/12h; Cultivate limnetic chlorella (Chlorella vulgaris) cell to logarithmic phase with the daylight lamp as light source, acquisition will be carried out the algae liquid of laser irradiation mutagenesis;
(2) laser irradiation mutagenesis: get 20ml logarithmic phase algae liquid and fall in little wide-necked bottle, handle 3.9-4.1min, keep irradiation even with magnetic agitation in the irradiation process, obtain the strain of sudden change algae with the 808nm semiconductor laser irradiation;
(3) strain of sudden change algae is cultivated: the algae liquid behind the irradiation mutagenesis is poured in the 50ml triangular flask that BG11 nutritive salt is housed; The final concentration of nutritive salt is 1/4 times in the algae liquid; At 20 ~ 25 ℃, light intensity 1800 ~ 2000lx, Light To Dark Ratio is to cultivate 20 days under the 12h/12h condition, then algae liquid is transferred in the 300ml triangular flask, continues under these conditions to cultivate 20 days; Again algae liquid is transferred in the 1000ml triangular flask; Continue under these conditions to cultivate 26 days, cultivation stage added the BG11 nutritive salt of 1/4 times of concentration in per 7 days, regularly shook algae every day 3 times.
2. induced by laser limnetic chlorella according to claim 1 accumulates the method for grease sudden change algae strain fast; It is characterized in that: in the step (2); The integrated light source laser apparatus of LOS-BLD-0808-12W-C-that semiconductor laser adopts extra large special photoelectricity Ltd to produce, power 6w.
3. induced by laser limnetic chlorella according to claim 1 accumulates the method for grease sudden change algae strain fast, and it is characterized in that: limnetic chlorella is derived from Inst. of Hydrobiology, Chinese Academy of Sciences, is numbered No. 9 algae strain.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305560A (en) * | 2013-06-06 | 2013-09-18 | 山东理工大学 | Method for inducing fresh water chlorella to fast accumulate grease through plant hormone jasmonic acid |
| CN104611229A (en) * | 2014-12-22 | 2015-05-13 | 黄河三角洲京博化工研究院有限公司 | A microalgae strain having a high grease content and a preparing method thereof |
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2012
- 2012-08-12 CN CN2012102873145A patent/CN102796725A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305560A (en) * | 2013-06-06 | 2013-09-18 | 山东理工大学 | Method for inducing fresh water chlorella to fast accumulate grease through plant hormone jasmonic acid |
| CN103305560B (en) * | 2013-06-06 | 2014-07-02 | 山东理工大学 | Method for inducing fresh water chlorella to fast accumulate grease through plant hormone jasmonic acid |
| CN104611229A (en) * | 2014-12-22 | 2015-05-13 | 黄河三角洲京博化工研究院有限公司 | A microalgae strain having a high grease content and a preparing method thereof |
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