CN103305560B - Method for inducing fresh water chlorella to fast accumulate grease through plant hormone jasmonic acid - Google Patents
Method for inducing fresh water chlorella to fast accumulate grease through plant hormone jasmonic acid Download PDFInfo
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Abstract
The invention provides a method for inducing fresh water chlorella to fast accumulate grease through plant hormone jasmonic acid. The method is characterized by comprising the following steps of: (1) preparation of chlorella solution preparation: under the conditions that the temperature is 22-24 DEG C, the light intensity is 1800-2000lx, and the light-to-dark ratio is 12h/12h, culturing fresh water chlorella to logarithmic phase to obtain chlorella solution for conducting hormone induction, wherein the fresh water chlorella adopts NO.9 fresh water chlorella or NO.31 fresh water chlorella which both come from Freshwater Algae Culture Collection at the Institute of Hydrobiology, FACHB-collection; (2) acquisition of high-grease-content chlorella: adding jasmonic acid into 200ml of chlorella solution obtained in step (1) until the final concentration is 0.49-0.51mg/l; and (3) acquisition of high-grease-content chlorella: culturing the chlorella solution subjected to hormone induction in step (2) for 15 days under the conditions that the temperature is 22-24 DEG C, the light intensity is 1800-2000lx, and the light-to-dark ratio of 12h/12h, and centrifugally collecting the frond with high grease content. The method is simple and easy, low in cost, and the grease content of the target product is high, and can be used as a raw material of industrialized biological produced oil.
Description
Technical field
The method that the invention provides a Plant Hormone jasmonic induction limnetic chlorella Rapid Accumulation grease, belongs to biological technical field.
Background technology
Chlorella (Chlorella) is a kind of spherical unicellular fresh water algae, is an important genus in green alga chlorella section, ecological widely distributed, all has distribution in seawater and fresh water, comprises at present about 10 kinds.Chlorella cells contains rich in protein, indispensable amino acid, polysaccharide, lipid, chlorophyll, carotene and VITAMIN etc., in protective foods, aquaculture, makeup, medicine and other fields, is widely used.Chlorella is fast because breeding, oleaginousness advantages of higher becomes a kind of biodiesel raw material with application and DEVELOPMENT PROSPECT.At present, the inductive condition of grasping certain promotion oil and fat accumulation is to reduce to utilize chlorella to prepare one of effective way of biofuel cost.Compared with the countries such as the U.S., Japan, Israel, the chlorella mass-producing culture technique level of China relatively lags behind.At present, the Patents technology of some stimulation rapid growth of chlorella is disclosed on domestic market, as East China University of Science has announced a kind of a kind of culture media composition (200610024004) of high-density high-quality ordinary chlorella cultivation and method (200610025618) of culturing chlorella with high-density and high-quality of being suitable for, Tsing-Hua University provides a kind of method (200610078003) of culturing heterotrophic chlorella with high density.Also there is a small amount of patent to relate to and utilize chlorella production biofuel, as Tsing-Hua University discloses a kind of method of utilizing high-density fermentation of heterotrophic chlorella production biofuel
(200810100871), a kind of from autotrophy to heterotrophism two steps cultivate method (200810112998) and a kind of chlorella and cultural method and its application (200910083797) in production of biodiesel of chlorella production biofuel.Although the patented technology that relates to chlorella cultivation and production of biodiesel above-mentioned has necessarily first carries out, but all do not relate to the research contents of induction or the high Lipid-producing of stimulation chlorella, and need the large-scale professional facility such as bioreactor and fermentor tank, higher to equipment requirements, technical matters relative complex, and expense is high, this has increased production cost and technology popularization difficulty undoubtedly.Except disclosed patent, also have a small amount of academic article to relate to the impact of plant hormone on chlorella growth, as the effect (Botany Gazette, 1999 year 6th phase) of plant hormone in chlorella heterotrophy is cultivated; As the impact (journal of YCIT natural science edition, 2006 year 2nd phase) of NAA on chlorella growth and chlorophyll and protein content; As the impact (guangdong agricultural science 2012 year 8th phase) of different plant hormones (IBA, NAA, 6-BA, 2,4-D, TDA) on original chlorella growth.
Summary of the invention
The object of this invention is to provide a kind of simple to operate, speed is fast, cost is low, the method for the plant hormone jasmonic of pollution-free, high safety induction limnetic chlorella Rapid Accumulation grease.Its concrete technical scheme is:
The method of one Plant Hormone jasmonic induction limnetic chlorella Rapid Accumulation grease, is characterized in that adopting following steps:
(1) prepare algae liquid: under the condition of 22～24 DEG C of temperature, light intensity 1800～2000lx, Light To Dark Ratio 12h/12h, cultivate limnetic chlorella to logarithmic phase, obtain and will carry out the algae liquid of hormone induction, wherein limnetic chlorella adopts No. 9 limnetic chlorellas or No. 31 limnetic chlorellas, is all derived from Inst. of Hydrobiology, Chinese Academy of Sciences's algae kind storehouse;
(2) hormone induction: jasmonic is added in the 200ml algae liquid that step (1) obtains, until final concentration is 0.49～0.51mg/l;
(3) obtain high fat content frond: the algae liquid after step (2) hormone induction is cultivated after 15 days under 22～24 DEG C of temperature, light intensity 1800～2000lx, Light To Dark Ratio 12h/12h condition, and centrifugal collection obtains the frond that fat content is high.
Compared with prior art, its advantage is in the present invention:
1, the present invention is simple, cost is low, pollution-free, high safety.
2, frond fat content increases rapidly, can be used as industrial biological produce oil raw material.
Embodiment
Embodiment 1:
Step 1, preparation algae liquid: under 22 DEG C of temperature, light intensity 1800lx, Light To Dark Ratio 12h/12h condition, cultivation is derived from No. 9 limnetic chlorella cells in Inst. of Hydrobiology, Chinese Academy of Sciences's algae kind storehouse to logarithmic phase, obtains and will carry out the algae liquid of hormone induction;
Step 2, hormone induction: the jasmonic (J2500-100MG) that SIGMA company of the U.S. is produced adds in the 200ml algae liquid that step 1 obtains, until final concentration is 0.49mg/l;
The acquisition of step 3, high fat content frond: the algae liquid after step 2 hormone induction is cultivated after 15 days under 22 DEG C of temperature, light intensity 1800lx, Light To Dark Ratio 12h/12h condition, and centrifugal collection obtains the frond that fat content is high.
Test data: the last frond fat content obtaining of step 3 of the present invention is 36.48% of frustule dry weight.For proving effect of the present invention, in being embodiment 1, also set the blank group that does not add jasmonic.Blank group except step 2, the same the present invention of all the other treating processess, the frond fat content that finally records blank group is 14.59% of frustule dry weight.
Embodiment 2:
Step 1, preparation algae liquid: under 22 DEG C of temperature, light intensity 1800lx, Light To Dark Ratio 12h/12h condition, cultivation is derived from No. 31 limnetic chlorella cells in Inst. of Hydrobiology, Chinese Academy of Sciences's algae kind storehouse to logarithmic phase, obtains and will carry out the algae liquid of hormone induction;
Step 2, hormone induction: the jasmonic (J2500-100MG) that SIGMA company of the U.S. is produced adds in the 200ml algae liquid that step 1 obtains, until final concentration is 0.49mg/l;
The acquisition of step 3, high fat content frond: the algae liquid after step 2 hormone induction is cultivated after 15 days under 22 DEG C of temperature, light intensity 1800lx, Light To Dark Ratio 12h/12h condition, and centrifugal collection obtains the frond that fat content is high.
Test data: the last frond fat content obtaining of step 3 of the present invention is 36.72% of frustule dry weight.For proving effect of the present invention, in being embodiment 2, also set the blank group that does not add jasmonic.Blank group except step 2, the same the present invention of all the other treating processess, the frond fat content that finally records blank group is 19.92% of frustule dry weight.
Embodiment 3:
Step 1, preparation algae liquid: under 23 DEG C of temperature, light intensity 1900lx, Light To Dark Ratio 12h/12h condition, cultivation is derived from No. 9 limnetic chlorella cells in Inst. of Hydrobiology, Chinese Academy of Sciences's algae kind storehouse to logarithmic phase, obtains and will carry out the algae liquid of hormone induction;
Step 2, hormone induction: the jasmonic (J2500-100MG) that SIGMA company of the U.S. is produced adds in the 200ml algae liquid that step 1 obtains, until final concentration is 0.5mg/l;
The acquisition of step 3, high fat content frond: the algae liquid after step 2 hormone induction is cultivated after 15 days under 23 DEG C of temperature, light intensity 1900lx, Light To Dark Ratio 12h/12h condition, and centrifugal collection obtains the frond that fat content is high.
Test data: the last frond fat content obtaining of step 3 of the present invention is 50.86% of frustule dry weight.For proving effect of the present invention, in being embodiment 3, also set the blank group that does not add jasmonic.Blank group except step 2, the same the present invention of all the other treating processess, the frond fat content that finally records blank group is 18.77% of frustule dry weight.
Embodiment 4:
Step 1, preparation algae liquid: under 23 DEG C of temperature, light intensity 1900lx, Light To Dark Ratio 12h/12h condition, cultivation is derived from No. 31 limnetic chlorella cells in Inst. of Hydrobiology, Chinese Academy of Sciences's algae kind storehouse to logarithmic phase, obtains and will carry out the algae liquid of hormone induction;
Step 2, hormone induction: the jasmonic (J2500-100MG) that SIGMA company of the U.S. is produced adds in the 200ml algae liquid that step 1 obtains, until final concentration is 0.5mg/l;
The acquisition of step 3, high fat content frond: the algae liquid after step 2 hormone induction is cultivated after 15 days under 23 DEG C of temperature, light intensity 1900lx, Light To Dark Ratio 12h/12h condition, and centrifugal collection obtains the frond that fat content is high.
Test data: the last frond fat content obtaining of step 3 of the present invention is 49% of frustule dry weight.For proving effect of the present invention, in being embodiment 4, also set the blank group that does not add jasmonic.Blank group except step 2, the same the present invention of all the other treating processess, the frond fat content that finally records blank group is 23.26% of frustule dry weight.
Embodiment 5:
Step 1, preparation algae liquid: under 24 DEG C of temperature, light intensity 2000lx, Light To Dark Ratio 12h/12h condition, cultivation is derived from No. 9 limnetic chlorella cells in Inst. of Hydrobiology, Chinese Academy of Sciences's algae kind storehouse to logarithmic phase, obtains and will carry out the algae liquid of hormone induction;
Step 2, hormone induction: the jasmonic (J2500-100MG) that SIGMA company of the U.S. is produced adds in the 200ml algae liquid that step 1 obtains, until final concentration is 0.51mg/l;
The acquisition of step 3, high fat content frond: the algae liquid after step 2 hormone induction is cultivated after 15 days under 24 DEG C of temperature, light intensity 2000lx, Light To Dark Ratio 12h/12h condition, and centrifugal collection obtains the frond that fat content is high.
Test data: the last frond fat content obtaining of step 3 of the present invention is 48.82% of frustule dry weight.For proving effect of the present invention, in being embodiment 5, also set the blank group that does not add jasmonic.Blank group except step 2, the same the present invention of all the other treating processess, the frond fat content that finally records blank group is 28.38% of frustule dry weight.
Embodiment 6:
Step 1, preparation algae liquid: under 24 DEG C of temperature, light intensity 2000lx, Light To Dark Ratio 12h/12h condition, cultivation is derived from No. 31 limnetic chlorella cells in Inst. of Hydrobiology, Chinese Academy of Sciences's algae kind storehouse to logarithmic phase, obtains and will carry out the algae liquid of hormone induction;
Step 2, hormone induction: the jasmonic (J2500-100MG) that SIGMA company of the U.S. is produced adds in the 200ml algae liquid that step 1 obtains, until final concentration is 0.51mg/l;
The acquisition of step 3, high fat content frond: the algae liquid after step 2 hormone induction is cultivated after 15 days under 24 DEG C of temperature, light intensity 2000lx, Light To Dark Ratio 12h/12h condition, and centrifugal collection obtains the frond that fat content is high.
Test data: the last frond fat content obtaining of step 3 of the present invention is 40% of frustule dry weight.For proving effect of the present invention, in being embodiment 6, also set the blank group that does not add jasmonic.Blank group except step 2, the same the present invention of all the other treating processess, the frond fat content that finally records blank group is 31.16% of frustule dry weight.
The fat content testing method of above-described embodiment is specifically: 1. chlorella algae liquid to be measured is transferred in the centrifuge tube of 50ml, and after trim, in low-temperature and high-speed whizzer, centrifugal 10min under 8000rpm, supernatant discarded, the reject supernatant of should trying one's best; The algae mud of having collected is measured for fat content; 2. ready algae mud is placed in to 100 DEG C of dried overnight of electric heating constant-temperature blowing drying box, measures respectively each experimental group xeraphium mass M
0, and record data.Grind the xeraphium having weighed, and add isopyknic chloroform and methyl alcohol (1:2, now with the current); 3. under the condition of power 40W, cycle 1min, broken wall 15s, interval 5s, 3 circulations, carry out supersonic wave wall breaking, then algae liquid complete broken wall is sub-packed in two 50mL centrifuge tubes, after trim, in low-temperature and high-speed whizzer, centrifugal 10min under 10000rmp condition; 4. centrifugal complete after, get supernatant liquor, in 3:1(chloroform: supernatant liquor) ratio add chloroform vortex to mix.Add again afterwards with the isopyknic eddies of water of chloroform and revolve and mix, leave standstill and treat its layering.Take off a layer organic phase and proceed in advance (the M that weighs
1) 10ml centrifuge tube in, drying up with Nitrogen evaporator the obtained data of weighing after organic solvent is M2, Nitrogen evaporator bath temperature is set as 61 DEG C; 5. the calculation formula of total lipid content is: total lipid content per-cent=(M2-M1)/M0 × 100%.
Experimental data proves, plant hormone jasmonic induction limnetic chlorella for the present invention can make its fat content fast, improve significantly, can be used as industrial biological produce oil raw material.
Claims (1)
1. the method for a Plant Hormone jasmonic induction limnetic chlorella Rapid Accumulation grease, is characterized in that adopting following steps:
(1) prepare algae liquid: under the condition of 22～24 DEG C of temperature, light intensity 1800～2000lx, Light To Dark Ratio 12h/12h, cultivate limnetic chlorella to logarithmic phase, obtain and will carry out the algae liquid of hormone induction, wherein limnetic chlorella adopts No. 9 limnetic chlorellas or No. 31 limnetic chlorellas, is all derived from Inst. of Hydrobiology, Chinese Academy of Sciences's algae kind storehouse;
(2) hormone induction: jasmonic is added in the 200ml algae liquid that step (1) obtains, until final concentration is 0.49～0.51mg/l;
(3) obtain high fat content frond: the algae liquid after step (2) hormone induction is cultivated after 15 days under 22～24 DEG C of temperature, light intensity 1800～2000lx, Light To Dark Ratio 12h/12h condition, and centrifugal collection obtains the frond that fat content is high.
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