CN103045478B - Method for culturing oil-producing microalgae with high density and high lipid production rate - Google Patents
Method for culturing oil-producing microalgae with high density and high lipid production rate Download PDFInfo
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- CN103045478B CN103045478B CN201110318772.6A CN201110318772A CN103045478B CN 103045478 B CN103045478 B CN 103045478B CN 201110318772 A CN201110318772 A CN 201110318772A CN 103045478 B CN103045478 B CN 103045478B
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Abstract
The invention belongs to the technical field of unicellular oil and biodiesel, and in particular relates to a method for synchronously improving biomass and lipid production rate of the oil-producing microalgae. The method comprises the following steps of: culturing the oil-producing microalgae in a logarithmic phase into a liquid culture medium of sterile addictive solution into which carbon sources and/or nitrogen sources are added; and culturing a mixed solution for 6-40 days in a swing bed which is continuously irradiated at the temperature of 18-35 DEG C, the rotation speed of 100-200rpm and the illumination intensity of 0-150 mu mol m-2s＜-1＞, wherein the sterile addictive solution is added into the liquid culture medium of the addictive solution into which carbon sources and/or nitrogen sources are added during culture. By the method, the growth process and the lipid production process of the oil-producing microalgae can be technically integrated through an alternating and circular control mode, so that the concentration of algae cells can be improved, the lipid can be effectively produced at the same time without replacement of culture medium compositions, batch supply of full-nutrition nourishment materials and real-time calculation of acceleration rate of nourishment material flow, and therefore, the method is easy to operate, and greatly reduces the production cost.
Description
Technical field
The invention belongs to Unicell Oils and Fats and production of biodiesel technical field, improve oil-producing particularly to a kind of synchronous mode
The Biomass of microalgae and the method for Gum yield.
Background technology
Oil-producing microalgae aboundresources, can grow under multiple condition of culture, and photosynthetic efficiency is high, and Biomass is high, growth
Breeding is fast, and growth cycle is short, and the ability of itself synthetic ester oil is strong, thus is considered to prepare Unicell Oils and Fats and life by many scholars
One of optimum feed stock of thing diesel oil.Microalgae grease can produce biodiesel by carrying out transesterification with low-carbon alcohols, using micro-
Algae produces biodiesel, has huge potentiality to the energy shortage and environmental pollution alleviating facing mankind, gives birth to reducing simultaneously
Produce in living to the dependence of fossil energy, ensure that national energy security has far reaching significance.Microalgae grease is by entering with higher alcohols
Row ester exchange reaction, can be used for producing biodegradable lubricating oil, solvent naphtha, paint and printing ink solvent, surfactant, glues
Connect the products such as agent, high-valued potentiality are big.Part microalgae grease contains polyunsaturated fatty acid, is to produce that to have high added value slender
The important source material of born of the same parents' oils and fatss.
But using microalgae produce Unicell Oils and Fats and biodiesel there is a problem of one larger be exactly in incubation
In, frustule growth is asynchronous with product fat.Competitive relation is there is, the general fat microalgae that produces is just between frond propagation and oil synthesis
Under the conditions of normal growing environment, the less lipid of cell inner accumulation.Under the environmental condition being unfavorable for growth and breeding, just enter and produce
Oily pattern, the more lipid of cell inner accumulation and other degeneration-resistant active substances, to maintain population to survive.Ask to solve these
Topic, realizes the controlled modulation of frustule growth pattern and oil-producing pattern, is used for nutriment and supplies culture technique or more in batches
The method changing culture media composition.However, these technology and method still have limitation, control complicated nutrition supply very
Bother and easily malfunction.
Content of the invention
Present invention aim at providing a kind of synchronous mode simple to operate to improve the side of oil-producing microalgae Biomass and Gum yield
Method.For achieving the above object, the technical solution used in the present invention is：
Take the logarithm the oil-producing microalgae of trophophase, cultivate to the liquid training of the aseptic annex solution being added with carbon source and/or nitrogen source
In foster base, it is 18-35 DEG C in temperature, rotating speed is 100-200rpm, intensity of illumination is 0-150 μm of ol m-2s-1The shaking of continuous illumination
Shaken cultivation 6-40 days in bed, up in algae solution, other nutrients such as phosphorus, sulfur, magnesium etc. exhaust and (take culture fluid to adopt ion color
Spectrometry measures nutrient residual quantity), culture terminates；Wherein, it is added with the fluid medium of the annex solution of carbon source and/or nitrogen source
Add aseptic annex solution again in middle incubation.
Described add annex solution or the carbon source annex solution both of which adding carbon source and nitrogen source during aseptic annex solution again, two
Kind of interpolation pattern alternately, until cultivate to add again carbon and nitrogen sources to improve Biomass and Gum yield effect inconspicuous when
Terminate to add.
Described every kind of interpolation pattern be spaced apart 2-5 days.
Described carbon source is inorganic carbon source and/or organic carbon source；Described inorganic carbon source is carbon dioxide, carbonate and/or carbonic acid
Hydrogen salt；Organic carbon source is glucose, sucrose, Fructose, glycerol, ethanol and/or acetate.
Described nitrogen source is inorganic nitrogen-sourced and/or organic nitrogen source；Wherein inorganic nitrogen-sourced for nitrate, ammonium salt and/or ammonia；Machine
Nitrogen source is carbamide, yeast powder and/or peptone.
Described oil-producing microalgae reaches the microalgae the cultivated strain of dry cell weight more than 8% for fat content.
The principle of the invention is：Competitive relation is there is, the general fat microalgae that produces is in growth between frond propagation and oil synthesis
When nitrogen exhausts and has excessive carbon to synthesize for lipid in culture medium, more lipid could be accumulated in the cell.Need
Cut off the supply of nutrient substance nitrogen in suitable trophophase, algae thus can be controlled to enter oil-producing pattern.But, this is also simultaneously
Slow down the growth of frustule, even result in death, need to produce the fat phase and reopen for business the supply of material nitrogen suitable, as long as looking for
This amount accurate, control algae strain be in batch (-type) nitrogen lack and continue carbon supply status so that it may realize the growth of oil-producing microalgae with
Produce fat synchronously to carry out.
Advantage for present invention：The present invention by the growth course of oil-producing microalgae and produces fat process by replacing, circulating
Control model carry out Integration ofTechnology, frustule concentration can be improved, again can efficiently produce fat simultaneously, compared with prior art, this
Operation is simple and feasible for the method that invention provides, and need not change culture media composition, need not entirely supply nutrition nutriment in batches, need not be real
When calculate adjustment nutriment stream rate of acceleration, annex solution preparation with low cost is simple, thus greatly reducing production cost.
Brief description
Fig. 1 is the technical solution of the present invention schematic flow sheet that the embodiment of the present invention is provided.
The object of the invention, function and advantage will be described further in conjunction with the embodiments referring to the drawings.
Specific embodiment
As shown in figure 1, technical scheme schematic flow sheet provided in an embodiment of the present invention.
Specifically, the invention provides a kind of method of culture oil-producing microalgae is it is characterised in that with simple to operation
Flow process is realized synchronous mode and is improved oil-producing microalgae Biomass and Gum yield, comprises the following steps：
(1) choose suitable carbon source and nitrogen source, be respectively prepared annex solution, high pressure steam sterilization or filtration sterilization；Carbon source was both
Including inorganic carbon source such as carbon dioxide, carbonate and/or bicarbonate, also include organic carbon source for example glucose, sucrose, Fructose,
Glycerol, ethanol and/or acetate.Nitrogen source had both included inorganic nitrogen-sourced such as nitrate, ammonium salt and/or ammonia, also included organic nitrogen source
As carbamide, yeast powder and/or peptone.
(2) take culture in oil-producing microalgae liquid medium within, by adding the carbon annex solution described in step (1) in algae solution
With nitrogen annex solution, frustule is controlled to enter growth pattern；By adding the carbon annex solution described in step (1) in algae solution, control
Frustule enters oil-producing pattern；Described adds carbon, nitrogen annex solution in algae solution, and addition manner is disposable interpolation；Between interpolation
Every being advisable with 2-5 days, addition can completely be consumed by frustule in interpolation interim and is advisable with the carbon source adding and nitrogen source,
Add number of times to convert divided by each addition and obtain with the carbon of frustule, the actual total requirement of nitrogen.
(3) two kinds described in step (2) add patterns alternately, until other nutrients such as phosphorus, sulfur, magnesium in algae solution
Etc. exhausting, culture terminates, and harvesting frustule breaking cellular wall carries fat.Maximum biomass and maximum Gum yield now can be obtained simultaneously.Described
Other nutrients to algae solution such as phosphorus, sulfur, magnesium etc. when exhausting culture terminate, refer to add carbon and nitrogen sources more biological to improving
Amount and Gum yield effect are inconspicuous, reach additional carbon in highest and culture fluid in algae cell density and terminate when being totally consumed
Culture.
Embodiment 1
(1) choose glucose as carbon source, potassium nitrate, as nitrogen source, is configured to the aseptic annex solution of high concentration.
(2) take dark Heterotrophic culture in oil-producing microalgae chlorella (Chlorella kessleri) liquid medium within, culture
Base is Kuhl culture medium, formula：10g/L glucose, 1g/L potassium nitrate, 89mg/L disodium hydrogen phosphate, 621mg/L bis- water
Sodium dihydrogen phosphate, 246mg/L Magnesium sulfate heptahydrate, 9.3mg/L EDTA, 6.9mg/L ferrous sulfate heptahydrate, 14.7mg/L bis- water chlorine
Change calcium, 0.29mg/L zinc sulphate heptahydrate, 0.17mg/L manganese sulfate monohydrate, 0.06mg/L boric acid, 0.002mg/L copper sulphate pentahydrate,
0.012mg/L tetra- water ammonium molybdate, pH 6.5.Algae kind presses 10% (volume ratio) inoculum concentration, is inoculated into containing 100ml aseptic Kuhl culture
In the 250ml triangular flask of base, it is 25 C in temperature, continuous darkness, shaken cultivation in the shaking table for 150rpm for the rotating speed.
(3) after cultivating 4 days, aseptically, add glucose to final concentration 20g/L into culture fluid, continue culture 5
After it, add glucose into culture fluid to final concentration 20g/L, potassium nitrate extremely final concentration of 1g/L, after being cultivated for 5 days,
Culture terminates, and Biomass and Gum yield are measured by sampling.Biomass adopts dry weight method to measure and (takes 5ml algae suspension in room temperature 3000rpm
Centrifugation 5min, abandoning supernatant.The distillation washing 2 times of frustule precipitation, is then filled on Whatman GF/C filter paper, puts 80
Weigh after drying to constant weight in DEG C baking oven), record biomass concentration and be up to 18g/L；Fat content measures (color by gas chromatogram (GC)
Spectral condition is：DB-23 capillary gas chromatographic column 30m × 0.25mm × 0.25 μm；Nitrogen is carrier gas；Using shunt mode, enter
Sample volume is 1 μ l；Injector temperature is 250 DEG C；Fid detector temperature is 260 DEG C；The temperature of column oven is with 2.5 DEG C/min
Heating rate rise to 240 DEG C from 140 DEG C), measure fat content be up to dry cell weight 48%.
Embodiment 2
(1) choose glucose as carbon source, potassium nitrate, as nitrogen source, is configured to the aseptic annex solution of high concentration.
(2) take light Heterotrophic culture in oil-producing microalgae chlorella (Chlorella kessleri) liquid medium within, culture
Base is Kuhl culture medium, formula：10g/L glucose, 1g/L potassium nitrate, 89mg/L disodium hydrogen phosphate, 621mg/L bis- water
Sodium dihydrogen phosphate, 246mg/L Magnesium sulfate heptahydrate, 9.3mg/L EDTA, 6.9mg/L ferrous sulfate heptahydrate, 14.7mg/L bis- water chlorine
Change calcium, 0.29mg/L zinc sulphate heptahydrate, 0.17mg/L manganese sulfate monohydrate, 0.06mg/L boric acid, 0.002mg/L copper sulphate pentahydrate,
0.012mg/L tetra- water ammonium molybdate, pH 6.5.Algae kind presses 10% inoculum concentration, is inoculated into containing 100ml aseptic Kuhl culture medium
In 250ml triangular flask, it is 25 C in temperature, rotating speed is 150rpm, intensity of illumination is 100 μm of ol m-2s-1The shaking of continuous illumination
Shaken cultivation in bed.
(3) after cultivating 2 days, aseptically, add glucose to final concentration 10g/L into culture fluid, continue culture 2
After it, add glucose to final concentration 10g/L, potassium nitrate to final concentration of 0.5g/L into culture fluid, be cultivated for 3 days
Afterwards, add glucose into culture fluid to final concentration 10g/L, after continuing culture 3 days, add glucose into culture fluid to dense eventually
Degree 10g/L, potassium nitrate to final concentration of 0.5g/L, after continuing culture 4 days, culture terminates, and Biomass and Gum yield are measured by sampling.
Biomass adopts dry weight method to measure and (takes 5ml algae suspension in room temperature 3000rpm centrifugation 5min, abandoning supernatant.Frustule precipitation is used
Distillation washing 2 times, is then filled on Whatman GF/C filter paper, puts and weigh after drying to constant weight in 80 DEG C of baking ovens), record biology
Amount is at concentrations up to 19g/L；Fat content measures by gas chromatogram (GC) that (chromatographic condition is：DB-23 capillary gas chromatographic column
30m×0.25mm×0.25μm；Nitrogen is carrier gas；Using shunt mode, sampling volume is 1 μ l；Injector temperature is 250 DEG C；
Fid detector temperature is 260 DEG C；The temperature of column oven is to rise to 240 DEG C with the heating rate of 2.5 DEG C/min from 140 DEG C), survey
Determine the 47% of fat content up to dry cell weight.
Embodiment 3
(1) choose sucrose as carbon source, carbamide, as nitrogen source, is configured to the aseptic annex solution of high concentration.
(2) take light Heterotrophic culture in oil-producing microalgae chlorella (Chlorella zofingiensis) liquid medium within,
Culture medium is Kuhl culture medium, formula：10g/L sucrose, 0.3g/L carbamide, 89mg/L disodium hydrogen phosphate, 621mg/L bis-
Water sodium dihydrogen phosphate, 246mg/L Magnesium sulfate heptahydrate, 9.3mg/L EDTA, 6.9mg/L ferrous sulfate heptahydrate, 14.7mg/L bis- water
Calcium chloride, 0.29mg/L zinc sulphate heptahydrate, 0.17mg/L manganese sulfate monohydrate, 0.06mg/L boric acid, 0.002mg/L five water sulphuric acid
Copper, 0.012mg/L tetra- water ammonium molybdate, pH 6.5.Algae kind presses 10% inoculum concentration, is inoculated into containing 100ml aseptic Kuhl culture medium
In 250ml triangular flask, it is 25 DEG C in temperature, rotating speed is 150rpm, intensity of illumination is 100 μm of ol m-2s-1The shaking table of continuous illumination
Middle shaken cultivation.
(3) after cultivating 4 days, aseptically, add sucrose to final concentration 10g/L into culture fluid, continue culture 4 days
Afterwards, add sucrose to final concentration 10g/L, carbamide to final concentration of 0.3g/L into culture fluid, after being cultivated for 4 days, cultivate
Terminate, Biomass and Gum yield are measured by sampling.Biomass adopts dry weight method to measure and (takes 5ml algae suspension in the centrifugation of room temperature 3000rpm
5min, abandoning supernatant.The distillation washing 2 times of frustule precipitation, is then filled on Whatman GF/C filter paper, puts 80 DEG C of bakings
Weigh after drying to constant weight in case), record biomass concentration and be up to 15g/L；Fat content measures (chromatostrip by gas chromatogram (GC)
Part is：DB-23 capillary gas chromatographic column 30m × 0.25mm × 0.25 μm；Nitrogen is carrier gas；Using shunt mode, sample introduction body
Amass as 1 μ l；Injector temperature is 250 DEG C；Fid detector temperature is 260 DEG C；The temperature of column oven is with the liter of 2.5 DEG C/min
Warm speed rises to 240 DEG C from 140 DEG C), measure the 47% of fat content up to dry cell weight.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art in the technical scope of present disclosure, technology according to the present invention scheme and its
Inventive concept equivalent or change in addition, all should be included within the scope of the present invention.
Claims (1)
1. a kind of high density High yielding rate cultivate oil-producing microalgae method it is characterised in that：Take the logarithm the oil-producing microalgae of trophophase
Chlorella Chlorella kessleri, cultivates to the fluid medium of the aseptic annex solution being added with carbon source and nitrogen source,
Temperature is 18-35 DEG C, and rotating speed is 100-200rpm, and intensity of illumination is 0-150 μm of ol m-2s-1Vibrate in the shaking table of continuous illumination
Culture 6-40 days；
Wherein, add aseptic annex solution in incubation in the fluid medium of the annex solution being added with carbon source and nitrogen source again；
Add the annex solution both of which adding carbon source annex solution or carbon source and nitrogen source during aseptic annex solution, two kinds of interpolation moulds again
Alternately, up in algae solution, nutrient phosphorus, sulfur, magnesium exhaust formula；
Every kind of interpolation pattern be spaced apart 2-5 days；
The addition manner of the described annex solution adding carbon source annex solution or carbon source and nitrogen source is disposable interpolation；
Described carbon source is inorganic carbon source and/or organic carbon source；Described inorganic carbon source is carbon dioxide, carbonate and/or bicarbonate
Salt；Organic carbon source is glucose, sucrose, Fructose, glycerol, ethanol and/or acetate；
Described nitrogen source is inorganic nitrogen-sourced and/or organic nitrogen source；Wherein inorganic nitrogen-sourced for nitrate, ammonium salt and/or ammonia；Organic nitrogen
Source is carbamide, yeast powder and/or peptone.
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| WO2018173051A1 (en) * | 2017-03-20 | 2018-09-27 | Algalife Ltd. | Composition comprising cultivated microalgae for use in coloring processes |
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| CN104131046B (en) * | 2014-08-14 | 2018-01-02 | 新奥科技发展有限公司 | A kind of oil-producing method of microalgae Immobilized culture |
| CN106434356B (en) * | 2016-12-12 | 2020-01-17 | 国投生物科技投资有限公司 | Microalgae culture method |
| CN110195019A (en) * | 2019-05-10 | 2019-09-03 | 中国科学院水生生物研究所 | A kind of cultural method of High yield proteid chlorella |
| CN114015493B (en) * | 2021-09-26 | 2024-02-20 | 上海师范大学 | Method for extracting grease from algae |
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| CN101230364A (en) * | 2008-02-25 | 2008-07-30 | 清华大学 | Method for producing biodiesel by high-density fermentation of heterotrophic chlorella |
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| CN101230364A (en) * | 2008-02-25 | 2008-07-30 | 清华大学 | Method for producing biodiesel by high-density fermentation of heterotrophic chlorella |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018173051A1 (en) * | 2017-03-20 | 2018-09-27 | Algalife Ltd. | Composition comprising cultivated microalgae for use in coloring processes |
| IL269406B (en) * | 2017-03-20 | 2022-11-01 | Algalife Ltd | Composition comprising cultivated microalgae for use in coloring processes |
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