CN102443562B - A kind of substratum and cultural method promoting proliferation of microalgae papidly - Google Patents
A kind of substratum and cultural method promoting proliferation of microalgae papidly Download PDFInfo
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- CN102443562B CN102443562B CN201010511190.5A CN201010511190A CN102443562B CN 102443562 B CN102443562 B CN 102443562B CN 201010511190 A CN201010511190 A CN 201010511190A CN 102443562 B CN102443562 B CN 102443562B
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Abstract
The present invention discloses a kind of substratum and the cultural method that promote proliferation of microalgae papidly, the substratum of described promotion proliferation of microalgae papidly is the three carbon organism adding hydroxyl in conventional medium, three carbon organism of hydroxyl in the medium concentration are not more than 1g/L, preferred 0.05g/L ~ 0.8g/L, three carbon organism of hydroxyl comprise glycerine, 1,3-PD, 1,2-propylene glycol, propyl alcohol, Virahol, R-Glyceric acid, Glycerose.A kind of cultural method promoting proliferation of microalgae papidly, the organic culture volume of micro-algae liquid sum hydroxyl three carbon mixes in bio-reactor in 1: 15 ~ 1: 30 ratios, in bio-reactor, initial micro algae biomass is 40mg/L ~ 80mg/L, culture temperature is 20 DEG C ~ 37 DEG C, stirring velocity is 100rpm ~ 500rpm, and intensity of illumination is 1000lux ~ 5000lux.Substratum compound method of the present invention is simple, cost is low, is suitable for industrial application; This substratum is adopted to carry out microdisk electrode at the CO without the need to complexity
2under supply equipment and illumination condition, just can improve micro-algae autotrophic metabolism efficiency and CO fast
2fixed efficiency, thus solve micro algae biomass accumulate problem slowly.
Description
Technical field
The present invention relates to a kind of substratum and the cultural method that promote proliferation of microalgae papidly.
Background technology
Micro-algae is that a class grows in water, of a great variety and distribution lower plant extremely widely, has efficient photosynthesis reactive system.Micro-algae passes through CO
2fixing, can be chemical energy by light energy conversion, and be stored in somatocyte with the organic form such as grease or starch.Along with the pressure of shortage of resources and environmental problem increasingly serious, utilize micro-algae to carry out the exploitation of biofuel and part fossil energy substitution product thereof, become the focus of research at present.Relative to general autotrophic organism, micro-algae propagation is very fast, and the nutriment needed is few---and mainly sunlight, water and carbonic acid gas, can not cause the contradiction of competing with agricultural land, animal husbandry land used.In a word, having a extensive future of micro-algae, its exploitation will provide a kind of new renewable resources for China.
Carrying out production of biodiesel by micro-algae is a complicated systems engineering, contains multiple sport technique segment, comprises several aspects such as the screening of micro-algae algae kind and cultivation, the scale evaluation of micro-algae and induction Lipid-producing, the collection of grease and processing.Micro-algae starts from the sixties in 20th century as the research of biodiesel raw material, in recent years, along with the development of biotechnology, by the biogenic reworking to algae kind, obtained abundant micro-algae resource with high oil-producing capacity, therefore this novel production of biodiesel pattern very has application prospect.
But because micro-algae is a kind of photosynthetic autotrophs biology being in evolution least significant end, the restrictive factor on its metabolic pathway is a lot, CO in nutrient solution
2the condition such as concentration, dissolved oxygen, light intensity all impact is formed on the growth of micro-algae.In addition, the capacity efficiency of micro-algae is low, and the time of an algae reproduction generation is longer, a lot of industrial microorganism tens doubly even hundred times, therefore, in industrial application, how promoting proliferation of microalgae papidly, and then realize the high-density culture of micro-algae, is the basic problem of the development microalgae biomass energy.
At present, in the selection of micro-algae culture medium nutritive ingredient and the optimization of culture condition, achieve a few thing progress.CN01134147.5 describes one using macro cell vat liquor as substratum, for carrying out the method for microdisk electrode.The method preparation technology is simple, and nutrition composition enriches simultaneously, be equal to a kind of substratum of pure natural, but due to the uncertainty of this medium component, some metabolic mechanisms is undistinct, are therefore difficult to carry out large-scale industrial application.Because micro-algae is a kind of photoautotrophic microorganism, production capacity level and CO
2fixed efficiency is very low, and therefore, its biomass rate of rise is starkly lower than general change energy microorganism.In the optimization of microdisk electrode condition, main study hotspot is CO
2the improvement of supply form, its objective is by improving CO
2fixed efficiency, to accelerate increasing of biomass.CN200910202971.3 discloses a kind of plenum system in microdisk electrode process, and its Main Means is by CO
2gas injects micro-algae photosynthetic reactor, to solve CO with microbubble form
2the problem that utilization ratio is low.CN1109936.4 discloses a kind of microalgae cell solvation and mends carbon and By Bubble-floating Method and to gather the cultural method be coupled, and its Main Means passes into be rich in CO bottom the pond that is coupled
2dissolved air water, carries out benefit carbon to algae liquid, completes cell lysing agentization and mend carbon and By Bubble-floating Method and gather in dissolved air water gasification.These two kinds of methods add CO all from different perspectives
2the duration of contact of gas and nutrient solution, avoid CO
2gas has little time by frond Cell uptake namely by the problem of overflowing.But aquatic micro-algae is carrying out CO
2general CO in fixing process
3 2-or HCO
3 -, micro-algae is to CO
2tolerance generally a narrower scope, too much CO
2gas passes into, and is unfavorable for the accumulation of micro algae biomass.See thus, only by change CO
2supply, limited to the promoter action improving micro algae biomass accumulative.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of substratum and the cultural method that promote proliferation of microalgae papidly.This substratum compound method is simple, cost is low, is suitable for industrial application; This substratum is adopted to carry out microdisk electrode at the CO without the need to complexity
2under supply equipment and illumination condition, just can improve micro-algae autotrophic metabolism efficiency and CO fast
2fixed efficiency, thus solve micro algae biomass accumulate problem slowly.
Promote a substratum for proliferation of microalgae papidly, add three carbon organism of hydroxyl in conventional medium, three carbon organism of hydroxyl in the medium concentration are not more than 1g/L, preferred 0.05g/L ~ 0.8g/L.
In the present invention, three carbon organism of described hydroxyl comprise glycerine, 1,3-PD, 1,2-propylene glycol, propyl alcohol, Virahol, R-Glyceric acid, Glycerose, preferably glycerine.
In the present invention, described conventional medium consist of 0.05g/L ~ 0.1g/LK
2hPO
43H
2o, 0.1g/L ~ 0.3g/LKH
2pO
4, 0.05g/L ~ 0.1g/LMgSO
47H
2o, 0.01g/L ~ 0.1g/LCaCl
22H
2o, 0.01g/L ~ 0.05g/LNaCl, 0.001g/L ~ 0.01g/LFeCl
36H
2o, 0.1g/L ~ 0.5g/LNaNO
3or KNO
3.
A kind of cultural method promoting proliferation of microalgae papidly, the culture volume of micro-algae liquid sum hydroxy-containing compounds mixes in bio-reactor in 1: 15 ~ 1: 30 ratios, in bio-reactor, initial micro algae biomass is 40mg/L ~ 80mg/L, culture temperature is 20 DEG C ~ 37 DEG C, stirring velocity is 100rpm ~ 500rpm, and intensity of illumination is 1000lux ~ 5000lux.
The present invention, carrying out the low biomass Initial stage of culture of micro-algae without the need to gas transportation facilities, carries out microdisk electrode by open training method.
In the present invention, micro-algae can come from and anyly has complete luminous energy phosphorylation system and C
3the algae of circulation, as blue-green algae, green alga etc., the ball algae in preferred green alga.
Compared with prior art the present invention promotes that the substratum of proliferation of microalgae papidly and cultural method tool have the following advantages:
1, the present invention promotes that the substratum of proliferation of microalgae papidly is in the substratum of routine, add a small amount of hydroxyl three carbon organism, has that compound method is simple, cost is low, is suitable for the advantages such as industrial application.
2, from prior art, in the culturing process of micro-algae, glucose is a kind of excellent carbon source, use glucose the fastest as the speed of growth of the micro-algae of carbon source under the same terms, but as can be seen from examples and comparative examples of the present invention, along with the increase of incubation time, the speed of growth containing the chlorella in the substratum of glucose obviously slows down along with the consumption of glucose, chlorella growth curve is S-type, and use the quick growth of hydroxyl three carbon organic medium ball algae to have persistence, before reaching stationary phase, growth curve is almost linear, therefore glycerine, propyl alcohol, propylene glycol, three carbon organism of these hydroxyls such as Glycerose are not as carbon source in traditional sense, the effect in the pathways metabolism of carbonic acid gas of three carbon organism of hydroxyl and glucose have obvious difference.
3, adopt substratum of the present invention to carry out microdisk electrode and can improve micro-algae autotrophic metabolism efficiency and CO fast
2fixed efficiency, higher than the substratum of the glucose containing same concentrations, thus solve micro algae biomass accumulate problem slowly.Because the micro-algae of photoautotrophy is with C
3the form of circulation carries out CO
2fix, i.e. CO
2at ribulose-1,5-bisphosphate, 5-bisphosphate Carboxylase/oxygenase (ribulose-1,5-bisphosphatecarboxylase/oxygenase, Rubisco) under effect, by ribulose-1,5-bisphosphate, 5-bisphosphate carboxylation is 2-carboxylic-3-ketone-ribitol-1,5-bisphosphate, then 2-carboxylic-3-ketone-ribitol-1,5-bisphosphate is cracked into two 3-phoshoglyceric acids, from then on enters endocellular metabolism approach.In conventional medium, add hydroxyl three carbon organism, as glycerine, propyl alcohol, propylene glycol, Glycerose etc., as a kind of inductor, namely at the Initial stage of culture of micro-algae as C
3substrate analogue in cyclic metabolism process, adding of it can the undue expression of some relevant enzymes (as phosphoglyceric kinase, phosphoglyceraldehy-de dehydrogenase etc.) in inducible metabolism process, because bio-metabolic process is a chain reaction system, rate-limiting reaction affects the efficiency of whole metabolic chain, therefore directly introduce the growth circulation that three carbon organism enter micro-algae, improve C
3cyclic metabolism efficiency, namely improves CO
2fixed efficiency.
4, in the present invention, mainly according to photoautotrophic microorganism oxidative phosphorylation process and CO
2fixing study metabolic pathways, has found that hydroxyl three carbon organism is connected whole CO
2carboxylation and five carbon compound regenerative processes, therefore have in substratum process for preparation for three carbon organism and the substrate analogues thereof that with the addition of hydroxyl, by substrate for induction effect, promote microalgae cell photosynthetic response, thus improve CO
2transformation efficiency, improve whole CO
2fixed efficiency, can promote that micro-algae is able to quick growth in early stage in culturing process.Pass through various forms with traditional, simple dependence increases CO
2air flow, to improve CO
2transformation efficiency, the method realizing proliferation of microalgae papidly is compared, and not only specific aim is stronger, effect is more obvious for present method, and requires lower to culture condition, and equipment is simple, and energy requirements is less, more easily realizes in scale operation.
Accompanying drawing explanation
Fig. 1 is chlorella biomass accumulation change curve in time under different glycerol concentration in embodiment.
Fig. 2 is chlorella biomass accumulation change curve in time under different glucose concn in comparative example.
Embodiment
Further illustrate effect of the present invention below in conjunction with embodiment, but be not construed as limiting the invention.
In the present invention, take 689nm as scanning wavelength, establish the typical curve between OD value and biomass, by measuring bacterium liquid OD value, thus determine the concentration of the biomass calculated in reaction system, meanwhile, can spectrophotometer be utilized in actual culturing process, the OD value of Timing measurement reaction system, to determine the accumulation of micro algae biomass.
Embodiment 1
(1) add glycerine in conventional medium, make the glycerol concentration in substratum be 0.1g/L, conventional medium specifically consist of 0.075g/LK
2hPO
43H
2o, 0.175g/LKH
2pO
4, 0.075g/LMgSO
47H
2o, 0.025g/LCaCl
22H
2o, 0.025g/LNaCl, 0.005g/LFeCl
36H
2o, 0.25g/LNaNO
3.
(2) get a kind of ball algae (chlorellavulgaris) 10mL of liquid preservation, concentration is 800mg/L, and by 1: 19 volume ratio, add the substratum containing glycerine that 190mL newly prepares, in nutrient solution, micro-phycomycete bulk concentration is 40mg/L level.With ventilative bacteriological filtration film, sealing process is carried out to triangle bottleneck, be beneficial to the air circulation of culture environment.
(3) select vibration shaking table to cultivate, vibration rotating speed is set to 140rpm.Utilize the lighting system of shaking table to provide a lasting illumination condition for culture system, intensity of illumination is 3000lux level.The temperature of whole culture systems is set to 25 DEG C.Timing sampling with OD value in spectrophotometer analysis shaking flask, thus calculates frond concentration.
Embodiment 2
(1) add glycerine in conventional medium, make the glycerol concentration in substratum be 0.25g/L, conventional medium specifically consist of 0.075g/LK
2hPO
43H
2o, 0.175g/LKH
2pO
4, 0.075g/LMgSO
47H
2o, 0.025g/LCaCl
22H
2o, 0.025g/LNaCl, 0.005g/LFeCl
36H
2o, 0.25g/LNaNO
3.
(2) a kind of ball algae (chlorellavulgaris) 10mL of liquid preservation is got, concentration is about 1200mg/L, by 1: 19 volume ratio, add the substratum containing glycerine that 190mL newly prepares, in nutrient solution, micro-phycomycete bulk concentration is about 60mg/L level.With ventilative bacteriological filtration film, sealing process is carried out to triangle bottleneck, be beneficial to the air circulation of culture environment.
(3) select vibration shaking table to cultivate, vibration rotating speed is set to 120rpm.Utilize the lighting system of shaking table to provide a lasting illumination condition for culture system, intensity of illumination is 3000lux level.The temperature of whole culture systems is set to 27 DEG C.Timing sampling with OD value in spectrophotometer analysis shaking flask, thus calculates frond concentration.
Embodiment 3
(1) add glycerine in conventional medium, make the glycerol concentration in substratum be 0.5g/L, conventional medium specifically consist of 0.075g/LK
2hPO
43H
2o, 0.175g/LKH
2pO
4, 0.075g/LMgSO
47H
2o, 0.025g/LCaCl
22H
2o, 0.025g/LNaCl, 0.005g/LFeCl
36H
2o, 0.25g/LNaNO
3.
(2) a kind of ball algae (chlorellavulgaris) 10mL of liquid preservation is got, concentration is about 1600mg/L, by 1: 19 volume ratio, add the substratum containing glycerine that 190mL newly prepares, in nutrient solution, micro-phycomycete bulk concentration is about 80mg/L level.With ventilative bacteriological filtration film, sealing process is carried out to triangle bottleneck, be beneficial to the air circulation of culture environment.
(3) select vibration shaking table to cultivate, vibration rotating speed is set to 160rpm.Utilize the lighting system of shaking table to provide a lasting illumination condition for culture system, intensity of illumination is 4000lux level.The temperature of whole culture systems is set to 25 DEG C.Timing sampling with OD value in spectrophotometer analysis shaking flask, thus calculates frond concentration.
Comparative example
Add glucose in conventional medium, the glucose concn in preparation substratum is respectively 0.1g/L, 0.25/L, 0.5g/L the three kinds substratum containing glucose, conventional medium specifically consist of 0.075g/LK
2hPO
43H
2o, 0.175g/LKH
2pO
4, 0.075g/LMgSO
47H
2o, 0.025g/LCaCl
22H
2o, 0.025g/LNaCl, 0.005g/LFeCl
36H
2o, 0.25g/LNaNO
3.All the other conditions are with embodiment 1 to 3, and timing sampling with OD value in spectrophotometer analysis shaking flask, thus calculates frond concentration.
According to the training method of embodiment 1 ~ 3, logical cultivation in a few days, the accumulation of micro algae biomass is shown in Fig. 1, the glycerine adding appropriate concentration in the medium can be found out, the initial stage of cultivating, due to adding of glycerine, make micro-algae algae kind spend lag phase very soon, be more conducive to the accumulation of micro algae biomass.
According to the training method of comparative example 1 ~ 3, logical cultivation in a few days, the accumulation of biomass is shown in Fig. 2, can find out, because glucose is a kind of wide spectrum carbon source, therefore adding glucose can cause ball algae to carry out heterotrophic growth, and when glucose concn is lower, the accumulating rate of biomass is directly proportional to the concentration of glucose in substratum.
From embodiment and comparative example analysis, glycerine and glucose can play promoter action to the accumulation of chlorella biomass, but both mechanism is completely different, and especially in low strength range, the effect of glycerine is significantly good.On the one hand, adding of the carbon sources such as glucose, directly cause micro-algae heterotrophic growth, its speed of growth reduces gradually along with the consumption of carbon source, thus S type is shown as on its growth curve, but the C3compounds such as glycerine are work as inductor form completely, get final product lasting generation effect when there is an optimal concentration in nutrient solution.On the other hand, when carrying out Heterotrophic culture, general carbon source should be held in a higher concentration, and therefore by relatively finding out, the organic inducing action successful of hydroxyl three carbon is better than the effect of lower concentration carbon source.
Claims (1)
1. one kind promotes the cultural method of proliferation of microalgae papidly, it is characterized in that: micro-algae liquid sum culture volume mixes in bio-reactor in 1: 15 ~ 1: 30 ratios, in bio-reactor, initial micro algae biomass is 40mg/L ~ 80mg/L, culture temperature is 20 DEG C ~ 37 DEG C, stirring velocity is 100rpm ~ 500rpm, intensity of illumination is 1000lux ~ 5000lux, described substratum is the three carbon organism adding hydroxyl in conventional medium, three carbon organism of described hydroxyl are glycerine, three carbon organism of hydroxyl in the medium concentration are 0.05g/L ~ 0.8g/L, micro-algae is the ball algae in green alga, described conventional medium consist of 0.05g/L ~ 0.1g/LK
2hPO
43H
2o, 0.1g/L ~ 0.3g/LKH
2pO
4, 0.05g/L ~ 0.1g/LMgSO
47H
2o, 0.01g/L ~ 0.1g/LCaCl
22H
2o, 0.01g/L ~ 0.05g/LNaCl, 0.001g/L ~ 0.01g/LFeCl
36H
2o, 0.1g/L ~ 0.5g/LNaNO
3or KNO
3.
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| 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 |
| 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 |
| 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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Non-Patent Citations (2)
| Title |
|---|
| Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions;Yanna Liang;《Biotechnol Lett》;20091231;第31卷;第1044–1045页以及附图3 * |
| 搅拌式光生物反应器培养紫球藻的条件优化;陈必链;《福建师范大学学报(自然科学版)》;20040630;第20 卷(第2 期);摘要以及92页 * |
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