CN102443562A - Culture medium and culture method for promoting fast propagation of microalgae - Google Patents

Culture medium and culture method for promoting fast propagation of microalgae Download PDF

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CN102443562A
CN102443562A CN2010105111905A CN201010511190A CN102443562A CN 102443562 A CN102443562 A CN 102443562A CN 2010105111905 A CN2010105111905 A CN 2010105111905A CN 201010511190 A CN201010511190 A CN 201010511190A CN 102443562 A CN102443562 A CN 102443562A
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microalgae
substratum
hydroxyl
culture
algae
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CN102443562B (en
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张霖
师文静
李晓姝
王领民
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China Petroleum and Chemical Corp
Sinopec Fushun Research Institute of Petroleum and Petrochemicals
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China Petroleum and Chemical Corp
Sinopec Fushun Research Institute of Petroleum and Petrochemicals
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Abstract

The invention discloses a culture medium and a culture method for promoting fast propagation of microalgae. The culture medium for promoting the fast propagation of microalgae is characterized in that hydroxyl-containing three-carbon organic matters are added into the conventional culture medium, the concentration of the hydroxyl-containing three-carbon organic matters in the culture medium is not greater than 1g/L, preferably, the concentration of the hydroxyl-containing three-carbon organic matters is 0.05 g/L to 0.8 g/L, and the hydroxyl-containing three-carbon organic matters comprise glycerin, 1,3-propylene glycol, 1,2-propylene glycol, propyl alcohol, isopropanol, glyceric acid and glyceraldehydes. The culture method for promoting the fast propagation of the microalgae comprises the steps that: microalgae liquid and culture media of the hydroxyl-containing three-carbon organic matters are mixed in a bioreactor according to the volume proportion of 1:15-1:30, the biomass of initial microalgae in the bioreactor is 40 mg/L to 80mg/L, the culture temperature is 20 DEG C to 37 DEG C, the stirring speed is 100 rpm to 500 rpm, and the illumination intensity is 1000 lux to 5000 lux. The culture medium has the advantages that the preparation method is simple, the cost is low, and the culture medium is suitable for industrial application; and when the culture medium is adopted for microalgae culture, the complicated CO2 supply equipment and the illumination condition are not needed, and the autotrophic metabolic efficiency of the microalgae and the CO2 fixing efficiency can be fast improved, so the problem of slow accumulation of microalgae biomass is solved.

Description

A kind of substratum and cultural method that promotes proliferation of microalgae papidly
Technical field
The present invention relates to a kind of substratum and cultural method that promotes proliferation of microalgae papidly.
Background technology
Little algae is one type and in water, grows that the of a great variety and lower plant extremely widely that distributes has photosynthesis reactive system efficiently.Little algae passes through CO 2Fixing, can luminous energy be converted into chemical energy, and with organic stores such as grease or starch in somatocyte.Along with the pressure and the environmental problem of shortage of resources are increasingly serious, utilize little algae to carry out the exploitation of biofuel and part fossil energy substitute products thereof, become the focus of present research.With respect to general autotrophic organism, little algae propagation is very fast, and the nutriment that needs is few---and mainly be sunlight, water and carbonic acid gas, can not cause contradiction with agricultural land, the competition of animal husbandry land used.In a word, the having a extensive future of little algae, its exploitation will provide a kind of new renewable resources for China.
Carrying out production of biodiesel through little algae is a complicated system engineering, contains a plurality of sport technique segments, comprises that several aspects such as produce oil fat, greasy collection and processing are cultivated and induced to the scale of screening and the cultivation of little algae algae kind, little algae.Little algae starts from the sixties in 20th century as the research of biodiesel raw material; In recent years, along with development of biology, through biogenic reworking to the algae kind; The little algae resource with high oil-producing capacity that has obtained to enrich, therefore this novel production of biodiesel pattern has application prospect very much.
Yet because little algae is a kind of photosynthetic autotrophs biology that is in the evolution least significant end, the restrictive factor on its metabolic pathway is a lot, CO in the nutrient solution 2Conditions such as concentration, dissolved oxygen, light intensity all the growth of little algae is constituted influence.In addition, the production capacity inefficiency of little algae, the algae reproduction monobasic time is longer; Be tens times of a lot of industrial microorganisms even hundred times, therefore, in industrial application; How promoting proliferation of microalgae papidly, and then realize the high-density culture of little algae, is the basic problem of the little algal biomass energy of development.
At present, aspect the optimization of the selection of micro-algae culture medium nutritive ingredient and culture condition, obtained a few thing progress.CN01134147.5 described a kind of with macro cell vat liquor as substratum, be used to carry out little algae cultured method.Preparation technology is simple for this method, and nutrition composition is abundant simultaneously, is equal to a kind of substratum of pure natural, and still, because the uncertainty of this medium component, some metabolic mechanisms are undistinct, therefore is difficult to carry out large-scale industrial application.Because little algae is a kind of photoautotrophic microorganism, production capacity level and CO 2Fixed efficiency is very low, and therefore, its living weight rate of rise is starkly lower than general change can mikrobe.Aspect the optimization of little algae culture condition, main research focus is CO 2The improvement of supply form, its objective is through improving CO 2Fixed efficiency is to quicken increasing of living weight.CN200910202971.3 discloses a kind of plenum system in little algae culturing process, and its main means are with CO 2Gas injects little algae photosynthetic reactor with the micron order bubble form, to solve CO 2The problem that utilization ratio is low.CN1109936.4 discloses a kind of microalgae cell solvation and has mended the cultural method that carbon and air supporting method are gathered and be coupled, and its main means are that CO is rich in the bottom feeding from the coupling pond 2Dissolved air water is mended carbon to algae liquid, and completion cell lysing agent benefit carbon and air supporting method are gathered in the dissolved air water gasification.These two kinds of methods have all increased CO from different perspectives 2The duration of contact of gas and nutrient solution, avoided CO 2Gas has little time to be absorbed the problem of promptly being overflowed by the frond cell.But aquatic little algae is carrying out CO 2General using CO in the fixation process 3 2-Or HCO 3 -, little algae is to CO 2Tolerance generally at a narrower scope, too much CO 2Gas feeds, and is unfavorable for the accumulation of micro algae biomass.See thus, only through changing CO 2Supply, limited to the promoter action that improves micro algae biomass accumulative total.
Summary of the invention
To the deficiency of prior art, the present invention provides a kind of substratum and cultural method that promotes proliferation of microalgae papidly.This substratum compound method is simple, cost is low, is suitable for industrial application; Adopting this substratum to carry out little algae cultivates at the CO that need not complicacy 2Under supply equipment and the illumination condition, just can improve little algae autotrophic metabolism efficient and CO fast 2Fixed efficiency, accumulate problem slowly thereby solve micro algae biomass.
A kind of substratum that promotes proliferation of microalgae papidly, three carbon organism of adding hydroxyl in conventional substratum, three carbon organism concentration in substratum of hydroxyl is not more than 1g/L, preferred 0.05g/L～0.8g/L.
Among the present invention, three carbon organism of described hydroxyl comprise glycerine, 1, ammediol, 1,2-Ucar 35, propyl alcohol, Virahol, R-Glyceric acid, Glycerose, preferably glycerine.
Among the present invention, described conventional substratum consist of 0.05g/L～0.1g/L K 2HPO 43H 2O, 0.1g/L～0.3g/L KH 2PO 4, 0.05g/L～0.1g/L MgSO 47H 2O, 0.01g/L～0.1g/LCaCl 22H 2O, 0.01g/L～0.05g/L NaCl, 0.001g/L～0.01g/L FeCl 36H 2O, 0.1g/L～0.5g/L NaNO 3Or KNO 3
A kind of cultural method that promotes proliferation of microalgae papidly; The long-pending culture volume with hydroxy-containing compounds of little algae liquid mixes in bio-reactor in 1: 15～1: 30 ratio; Initial micro algae biomass is 40mg/L～80mg/L in the bio-reactor; Culture temperature is 20 ℃～37 ℃, and stirring velocity is 100rpm～500rpm, and intensity of illumination is 1000lux～5000lux.
The present invention need not gas transportation facilities carrying out the low living weight cultivation initial stage of little algae, carries out little algae through open training method and cultivates.
Among the present invention, little algae can come from any have complete luminous energy phosphorylation system and C 3The round-robin algae, like blue-green algae, green alga etc., the ball algae in the preferred green alga.
Compared with prior art the present invention promotes the substratum of proliferation of microalgae papidly and cultural method to have following advantage:
1, the present invention promotes that the substratum of proliferation of microalgae papidly is in the substratum of routine, to add a spot of hydroxyl three carbon organism, has that compound method is simple, cost is low, is suitable for advantages such as industrial application.
Can know from prior art that 2, in the culturing process of little algae, glucose is a kind of good carbon source; The same terms uses glucose the fastest as the speed of growth of the little algae of carbon source down; But can find out that from embodiments of the invention and comparative example along with the increase of incubation time, the speed of growth that contains the chlorella in the substratum of glucose is along with the consumption of glucose is obviously slowed down; The chlorella growth curve is S-type; And use the quick growth of chlorella in the organic substratum of hydroxyl three carbon to have persistence, before reaching stationary phase, growth curve is almost linear; Therefore three carbon organism of these hydroxyls such as glycerine, propyl alcohol, Ucar 35, Glycerose are not as carbon source on the traditional sense, and the effect and the glucose of three carbon organism of hydroxyl in the pathways metabolism of carbonic acid gas has obvious difference.
3, adopt substratum of the present invention to carry out little algae cultivation and can improve little algae autotrophic metabolism efficient and CO fast 2Fixed efficiency, be higher than the substratum of the glucose that contains same concentrations, accumulate problem slowly thereby solve micro algae biomass.Because the little algae of photoautotrophy is with C 3The round-robin form is carried out CO 2Fixed, i.e. CO 2At ribulose-1,5-bisphosphate, 5-di-phosphate carboxylase/oxygenase (ribulose-1,5-bisphosphate carboxylase/oxygenase; Rubisco) under the effect, with ribulose-1,5-bisphosphate, 5-di-phosphate carboxylation is 2-carboxylic-3-ketone-ribitol-1; The 5-di-phosphate; 2-carboxylic-3-ketone-ribitol-1 then, the 5-di-phosphate is cracked into two 3-phoshoglyceric acids, from then on gets into the endocellular metabolism approach.In conventional substratum, added hydroxyl three carbon organism, like glycerine, propyl alcohol, Ucar 35, Glycerose etc., as a kind of inductor, promptly cultivation initial stage of little algae as C 3Substrate analogue in the cyclic metabolism process; Its adding can be induced the undue expression of some relevant enzymes (like phosphoglyceric kinase, phosphoglyceraldehy-de dehydrogenase etc.) in the metabolic process; Because bio-metabolic process is a Kettenreaktion system; Therefore rate-limiting reaction affects the efficient of whole metabolic chain, has directly introduced the growth circulation that three carbon organism get into little algae, has promoted C 3Cyclic metabolism efficient has promptly improved CO 2Fixed efficiency.
4, among the present invention, mainly according to photoautotrophic microorganism oxidative phosphorylation process and CO 2The fixed study metabolic pathways has found that hydroxyl three carbon organism are being connected whole C O 2Carboxylation and five carbon cpd regenerative processes, the interpolation that is directed against has therefore been arranged in the substratum process for preparation three carbon organism and substrate analogues thereof of hydroxyl through the substrate for induction effect, promote the microalgae cell photosynthetic response, thereby have improved CO 2Transformation efficiency, promoted whole C O 2Fixed efficiency can promote little algae to be able to quick growth in the culturing process in early stage.With traditional various forms that passes through, simple dependence increases CO 2Air flow, to improve CO 2Transformation efficiency realizes that the method for proliferation of microalgae papidly is compared, and not only specific aim is stronger, effect is more obvious for present method, and lower to the culture condition requirement, and equipment is simple, and the energy consumption demand still less realizes in scale operation more easily.
Description of drawings
Fig. 1 is a chlorella biomass accumulation change curve in time under the different glycerol concentrations among the embodiment.
Fig. 2 is a chlorella biomass accumulation change curve in time under the different glucose concn in the comparative example.
Embodiment
Further specify effect of the present invention below in conjunction with embodiment, but be not construed as limiting the invention.
Among the present invention, be scanning wavelength, set up the typical curve between OD value and the living weight with 689nm; Through bacterium liquid OD value is measured; Thereby confirm to calculate the concentration of the living weight in the reaction system, simultaneously, in actual culturing process, can utilize spectrophotometer; Regularly measure the OD value of reaction system, to confirm the accumulation of micro algae biomass.
Embodiment 1
(1) add glycerine in the conventional substratum, making the glycerol concentration in the substratum is 0.1g/L, conventional substratum specifically consist of 0.075g/L K 2HPO 43H 2O, 0.175g/L KH 2PO 4, 0.075g/LMgSO 47H 2O, 0.025g/L CaCl 22H 2O, 0.025g/L NaCl, 0.005g/L FeCl 36H 2O, 0.25g/LNaNO 3
(2) get a kind of ball algae (chlorella vulgaris) 10mL of liquid preservation, concentration is 800mg/L, by 1: 19 volume ratio, adds the substratum that contains glycerine of the new preparation of 190mL, and little phycomycete bulk concentration is the 40mg/L level in the nutrient solution.With ventilative bacteriological filtration film the triangle bottleneck is sealed processing, be beneficial to the airiness of culture environment.
(3) select the vibration shaking table to cultivate, the vibration rotating speed is set to 140rpm.Utilize the lighting system of shaking table for culture system a lasting illumination condition to be provided, intensity of illumination is the 3000lux level.The temperature of whole culture systems is set to 25 ℃.Timing sampling shakes OD value in the bottle with the spectrophotometer analysis, thereby calculates frond concentration.
Embodiment 2
(1) add glycerine in the conventional substratum, making the glycerol concentration in the substratum is 0.25g/L, conventional substratum specifically consist of 0.075g/L K 2HPO 43H 2O, 0.175g/L KH 2PO 4, 0.075g/LMgSO 47H 2O, 0.025g/L CaCl 22H 2O, 0.025g/L NaCl, 0.005g/L FeCl 36H 2O, 0.25g/LNaNO 3
(2) get a kind of ball algae (chlorella vulgaris) 10mL of liquid preservation, concentration is about 1200mg/L, by 1: 19 volume ratio, adds the substratum that contains glycerine of the new preparation of 190mL, and little phycomycete bulk concentration is about the 60mg/L level in the nutrient solution.With ventilative bacteriological filtration film the triangle bottleneck is sealed processing, be beneficial to the airiness of culture environment.
(3) select the vibration shaking table to cultivate, the vibration rotating speed is set to 120rpm.Utilize the lighting system of shaking table for culture system a lasting illumination condition to be provided, intensity of illumination is the 3000lux level.The temperature of whole culture systems is set to 27 ℃.Timing sampling shakes OD value in the bottle with the spectrophotometer analysis, thereby calculates frond concentration.
Embodiment 3
(1) add glycerine in the conventional substratum, making the glycerol concentration in the substratum is 0.5g/L, conventional substratum specifically consist of 0.075g/L K 2HPO 43H 2O, 0.175g/L KH 2PO 4, 0.075g/LMgSO 47H 2O, 0.025g/L CaCl 22H 2O, 0.025g/L NaCl, 0.005g/L FeCl 36H 2O, 0.25g/LNaNO 3
(2) get a kind of ball algae (chlorella vulgaris) 10mL of liquid preservation, concentration is about 1600mg/L, by 1: 19 volume ratio, adds the substratum that contains glycerine of the new preparation of 190mL, and little phycomycete bulk concentration is about the 80mg/L level in the nutrient solution.With ventilative bacteriological filtration film the triangle bottleneck is sealed processing, be beneficial to the airiness of culture environment.
(3) select the vibration shaking table to cultivate, the vibration rotating speed is set to 160rpm.Utilize the lighting system of shaking table for culture system a lasting illumination condition to be provided, intensity of illumination is the 4000lux level.The temperature of whole culture systems is set to 25 ℃.Timing sampling shakes OD value in the bottle with the spectrophotometer analysis, thereby calculates frond concentration.
Comparative example
Add glucose in the conventional substratum, the glucose concn in the preparing culture medium is respectively 0.1g/L, three kinds of substratum that contain glucose of 0.25/L, 0.5g/L, conventional substratum specifically consist of 0.075g/LK 2HPO 43H 2O, 0.175g/L KH 2PO 4, 0.075g/L MgSO 47H 2O, 0.025g/L CaCl 22H 2O, 0.025g/L NaCl, 0.005g/L FeCl 36H 2O, 0.25g/L NaNO 3All the other conditions are with embodiment 1 to 3, and timing sampling shakes OD value in the bottle with the spectrophotometer analysis, thereby calculate frond concentration.
Training method according to embodiment 1～3; Logical cultivation in a few days, the accumulation of micro algae biomass is seen Fig. 1, can find out the glycerine that in substratum, adds an amount of concentration; The initial stage of cultivating; Because the adding of glycerine makes little algae algae kind spend lag phase very soon, more helps the accumulation of micro algae biomass.
Training method according to comparative example 1～3; Logical cultivation in a few days, the accumulation of living weight is seen Fig. 2, can find out; Because glucose is a kind of wide spectrum carbon source; Therefore adding glucose can cause that the ball algae carries out heterotrophic growth, and under the lower situation of glucose concn, the accumulating rate of living weight is directly proportional with the concentration of glucose in the substratum.
From embodiment and comparative example analysis, glycerine and glucose can both play promoter action to the accumulation of chlorella living weight, but both mechanism is different fully, and especially in low strength range, the effect of glycerine is better significantly.On the one hand; The adding of carbon sources such as glucose; Directly caused little algae heterotrophic growth, its speed of growth reduces along with the consumption of carbon source gradually, thereby on its growth curve, shows as the S type; But C3compounds such as glycerine are to work as the inductor form fully, in nutrient solution, exist an optimal concentration to get final product persistent generation effect.On the other hand, when carrying out the heterotrophism cultivation, general carbon source should be held in a higher concentration, therefore through relatively finding out that the organic inducing action effect of hydroxyl three carbon obviously is superior to the effect of lower concentration carbon source.

Claims (7)

1. substratum that promotes proliferation of microalgae papidly is characterized in that: described substratum is the three carbon organism that in conventional substratum, add hydroxyl, and three carbon organism concentration in substratum of hydroxyl is not higher than 1g/L.
2. according to the described substratum of claim 1, it is characterized in that: three carbon organism concentration in substratum of hydroxyl is 0.05g/L～0.8g/L.
3. according to claim 1 or 2 described substratum, it is characterized in that: three carbon organism of described hydroxyl comprise glycerine, 1, ammediol, 1,2-Ucar 35, propyl alcohol, Virahol, R-Glyceric acid, Glycerose.
4. according to the described substratum of claim 1, it is characterized in that: conventional substratum consist of 0.05g/L～0.1g/L K 2HPO 43H 2O, 0.1g/L～0.3g/L KH 2PO 4, 0.05g/L～0.1g/LMgSO 47H 2O, 0.01g/L～0.1g/L CaCl 22H 2O, 0.01g/L～0.05g/L NaCl, 0.001g/L～0.01g/L FeCl 36H 2O, 0.1g/L～0.5g/L NaNO 3Or KNO 3
5. cultural method that promotes proliferation of microalgae papidly; It is characterized in that: little algae liquid is long-pending to be mixed in bio-reactor in 1: 15～1: 30 ratio with the organic culture volume of described hydroxyl three carbon of claim 1; Initial micro algae biomass is 40mg/L～80mg/L in the bio-reactor; Culture temperature is 20 ℃～37 ℃, and stirring velocity is 100rpm～500rpm, and intensity of illumination is 1000lux～5000lux.
6. according to the described cultural method of claim 5, it is characterized in that: little algae is for having complete luminous energy phosphorylation system and C 3The round-robin algae.
7. according to claim 5 or 6 described cultural methods, it is characterized in that: little algae is the ball algae in the green alga.
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CN103352006A (en) * 2013-07-31 2013-10-16 宁波大学 Culture method for promoting autotrophy microalgae neutral lipid accumulation
CN104232559B (en) * 2013-06-09 2017-07-25 中国石油化工股份有限公司 The method of cultivating microalgae and the method for producing grease
CN110923185A (en) * 2018-09-19 2020-03-27 中国科学院青岛生物能源与过程研究所 Culture method for improving oil content of microalgae cells
US10927339B2 (en) 2017-03-17 2021-02-23 Industrial Technology Research Institute Mutant of Bacillus thuringiensis and application thereof

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Publication number Priority date Publication date Assignee Title
CN104232559B (en) * 2013-06-09 2017-07-25 中国石油化工股份有限公司 The method of cultivating microalgae and the method for producing grease
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CN103352006B (en) * 2013-07-31 2015-02-04 宁波大学 Culture method for promoting autotrophy microalgae neutral lipid accumulation
US10927339B2 (en) 2017-03-17 2021-02-23 Industrial Technology Research Institute Mutant of Bacillus thuringiensis and application thereof
CN110923185A (en) * 2018-09-19 2020-03-27 中国科学院青岛生物能源与过程研究所 Culture method for improving oil content of microalgae cells
CN110923185B (en) * 2018-09-19 2021-08-27 中国科学院青岛生物能源与过程研究所 Culture method for improving oil content of microalgae cells

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