CN103773689B - A kind of open type cultivates the method for oil-containing microorganism - Google Patents
A kind of open type cultivates the method for oil-containing microorganism Download PDFInfo
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Abstract
The invention discloses a kind of method that open type cultivates oil-containing microorganism, including: (1) cultivates saccharomyces olei seed liquor；(2) autotrophy microalgae seed liquor is cultivated；(3) meromict cultivation is carried out in saccharomyces olei seed liquor and autotrophy microalgae seed liquor are accessed the open type bioreactor of laid inside membrane separation device, bioreactor is divided into upper and lower two regions by membrane separation device, saccharomyces olei is cultivated at membrane separation device lower area, autotrophy microalgae carries out photosynthesis growth at membrane separation device upper area, the culture medium composition of lower regions is consistent with condition of culture, supplements the organic carbon source needed for saccharomyces olei growth metabolism in incubation.This method has raising autotrophy microalgae and saccharomyces olei cell harvesting amount, improves inorganic carbon source (CO2) utilization rate, improve microbial grease content, simplified culture device, improve the advantage such as autotrophy micro-algae large-scale culture efficiency.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method cultivating oil-containing microorganism, relate in particular to a kind of meromict and cultivate autotrophy microalgae and saccharomyces olei to obtain high oil-containing microbial cell harvest yield, the method simultaneously improving oil-containing microbial cell fat content.
Background technology
Along with socio-economic development, the supply of fossil resource is the most in short supply, oil product imbalance between supply and demand is the most outstanding day by day, and corresponding problem of environmental pollution is the most prominent, develops renewable resources by all kinds of means and becomes inevitable.With the biodiesel that the Renewable resources such as microbial grease are source, there is the performances such as energy density is high, sulfur content is low, burning is abundant, lubricity is good, also there is the features such as renewable, environmental friendliness, easily biological-degradable, storing and transporting security, the capability of antidetonance are good, can be as the succedaneum of fossil energy.
Document shows, under appropriate conditions, the oils and fats that certain micro-organisms produces and stores accounts for more than the 20% of its biological total amount, and this quasi-microorganism is referred to as oil-containing microorganism, it is known that have the microorganism producing oils and fats in antibacterial, yeast, mycete, algae.Microbial grease is also known as Unicell Oils and Fats, and its fatty acid composition is similar to general Vegetable oil lipoprotein, and with C16, C18 system fatty acid, as saturated in Palmic acid, stearic acid, oleic acid and linoleic acid etc. and undersaturated fatty acid is main.Fermentable utilizes substrate spectrum wider; glucose, fructose, sucrose, molasses, starch and cellulosic hydrolysate etc. can be directly utilized, new grease production method not only can be provided, and cheap abandoned biomass can be utilized; reduce grease production cost, protect environment.Therefore, microbial grease is potential animal and plant fat alternate resources, has broad application prospects.
The preparation of microbial grease typically uses yeast to be fermented bacterium, carries out biomass accumulation through conventional microorganism culturing, obtains oil-containing microbial cells, and then process this thalline, obtain microbial grease.CN200610113582.X discloses a kind of method utilizing fermentable oils and fats to prepare biodiesel, i.e. with yeast for microorganism Lipid-producing strain, by accessing this yeast microbes in Lipid-producing bacterium culture medium, production of lipids by microbial fermentation is carried out under certain condition of culture, the most by centrifugation, separate, extract and obtain fermentable oils and fats, with this microbial grease as raw material, under catalyst action, carry out transesterification, obtaining biodiesel, by-product is glycerol.This technical process uses single microorganism fermentation culture process, and its bio-oil is accumulated in intracellular, and cell harvesting amount is the key factor of restriction oils and fats harvest yield.
In addition to saccharomyces olei, microalgae is an other class oil-containing microorganism, and microalgae, rich in nutritional labelings (such as spirulina etc.) such as unsaturated fatty acid, starch and protein, can be used as the field raw materials such as the energy, medicine and food；By conditional regulatory, microalgae cell can accumulate fatty acid, some microalgae such as chlorella in a large number, and its body fat acid content can account for the 30%～60% of dry cell weight.Utilize and cultivate microalgae and accumulate oil resource, have become as and develop renewable resources the most popular research field currently with solar energy and fixing carbon dioxide, not only there is huge market potential, and there is prominent social value.
The growth pattern of microalgae cell is generally light autotrophy and carbon source heterotrophism two kinds, light autotrophy process CO to be consumed2, CO2Effectively utilize absorption, be the key realizing preferable culture effect, exist simultaneously and supplement CO2O is produced with photosynthesis2Desorbing, the problem of discharge.CN200410020978.0 and CN03128138.9 all uses and adds a kind of installation method in photo-bioreactor system, realizes CO2Supply, also can realize the parsing effect of certain oxygen simultaneously.Carbon source heterotrophism process is to utilize organic carbon source to substitute the CO during autotrophy for substrate2Carrying out the accumulation of microalgae biomass, vitro growth rates is very fast, but intracellular oil and fat accumulation level is the lowest.
Have involved although prior art produces microbial grease to saccharomyces olei and autotrophy microalgae, but being all that oil-containing microorganism each carries out single culture, the research that training method collaborative for the mixing between different types of oil-containing microorganism realizes the aspects such as high-efficiency grease results is less.Generally there is the deficiencies such as cost is high, microbial cell accumulation is slow, fat content is low in incubation.CN200910237936.5 discloses a kind of method being mixed yeast and algae production oils and fats, and the method utilizes industrial wastewater for culture medium, is mixed yeast and algae, produces microbial grease.But, owing to the mixed culture of yeast and algae is all Heterotrophic culture, intracellular oil and fat accumulation level is relatively low；And two kinds of cells are gathered in the crops together and extract oils and fats, the method extracted due to oils and fats is different, and breaking cellular wall requires difference, is mixed in one piece of poor effect.
Summary of the invention
For the deficiencies in the prior art, the present invention provides a kind of method that open type cultivates oil-containing microorganism.Autotrophy microalgae and saccharomyces olei are carried out meromict cultivation in open type bioreactor by this method, have raising autotrophy microalgae and saccharomyces olei cell harvesting amount, improve inorganic carbon source (CO2) utilization rate, improve microbial grease content, simplified culture device, improve the advantage such as autotrophy micro-algae large-scale culture efficiency.
Open type of the present invention cultivates the method for oil-containing microorganism, including following content:
(1) saccharomyces olei seed liquor is cultivated；
(2) autotrophy microalgae seed liquor is cultivated；
(3) meromict cultivation is carried out in saccharomyces olei seed liquor and autotrophy microalgae seed liquor are accessed the open type bioreactor of laid inside membrane separation device, bioreactor is divided into upper and lower two regions by membrane separation device, saccharomyces olei is cultivated at membrane separation device lower area, autotrophy microalgae carries out photosynthesis growth at membrane separation device upper area, the culture medium composition of lower regions is consistent with condition of culture, supplements the organic carbon source needed for saccharomyces olei growth metabolism in incubation.
In the present invention, membrane separation device can use the various film device with membrance separation function, it is preferred to use ceramic membrane separation device, and film hole diameter is 0.05 μm～0.5 μm, is separated with autotrophy microalgae cell by saccharomyces olei cell.It is 1:1～5:1 that bioreactor is divided into the volume ratio in upper and lower two regions by membrane separation device.
In the present invention, described bioreactor also includes distribution device, is laid on the bottom in reactor lower part region, and the gas of distribution can be in membrane separation device enters whole bioreactor.
In the present invention, saccharomyces olei include saccharomyces cerevisiae (S.cerevisiae), rhodotorula glutinis (R.glutinis), trichosporon cutaneum (Trichosporoncutaneum) etc., organic carbon source is glucose, fructose, starch, cellulosic hydrolysate etc..It is method well known to those skilled in the art that saccharomyces olei seed liquor is cultivated, and as used stirring type bioreactor, adds culture medium and saccharomyces olei, cultivates under appropriate conditions.
In the present invention, autotrophy microalgae includes chlorella, Wild Vitis species, little ring algae, diatom etc., and it is method well known to those skilled in the art that autotrophy microalgae seed liquor is cultivated, as used conventional airlift photobioreactor to carry out seed liquor cultivation.
In the present invention, in meromict incubation, saccharomyces olei seed liquor is 1:1～1:10 with the volume ratio that is initially accessed of autotrophy microalgae seed liquor.The initial medium that meromict is cultivated uses the culture medium needed for saccharomyces olei, adds the inorganic salt needed for autotrophy micro algae growth and trace element (as added related substances by the composition of SE culture medium) etc. simultaneously.In meromict incubation, with batch or organic carbon source needed for continuously etc. mode supplements saccharomyces olei growth course.
In the present invention, meromict condition of culture typically uses the condition of the conditional likelihood with saccharomyces olei growth course, and as temperature is generally 20 DEG C～37 DEG C, pH value is generally 6～9, preferably 6.5～7.5 etc..
Saccharomyces olei and autotrophy microalgae are carried out meromict cultivation by the inventive method, saccharomyces olei and autotrophy micro algae growth condition and required carbon source different, complement each other and promote.Autotrophy microalgae is discarded carbon source (CO after utilizing saccharomyces olei fermentation2) it is own growth carbon source, maintain system more stable osmotic pressure condition (pH value) simultaneously, ensure that saccharomyces olei constantly grows by supplementary organic carbon source.Saccharomyces olei is kept apart by membrane separation device with autotrophy microalgae, it is achieved the most individually growing space.Culture medium composition is then to realize being blended and intercommunication, the carbon source that sweat is used by microalgae has toleration, do not affect the growth of microalgae and the accumulation of oils and fats, and be there is not impact in the sweat of saccharomyces olei, required saccharomyces olei cell can be obtained simultaneously, thus realize saccharomyces olei and autotrophy microalgae meromict incubation in bioreactor.
The inventive method selects saccharomyces olei and the autotrophy microalgae of suitable kind of suitable kind, by controlling saccharomyces olei and the incubation of autotrophy microalgae, both are made to form stable meromict cultivating system, and set up different iuntercellular and jointly utilize environmental nutrient condition and growth relationship that iuntercellular does not interferes with each other, achieve the efficient growth process of saccharomyces olei and autotrophy microalgae, improve the accumulative effect of oils and fats, and the oils and fats harvest yield that improve in single microorganism incubation in unit fermentation system, thus the preparation for microbial grease is laid a good foundation.Further, the method extracted due to oils and fats is different, and breaking cellular wall requires difference, and oils and fats is gathered in the crops and extracted to two kinds of cells respectively, better.
Accompanying drawing explanation
Fig. 1 is one concrete technology schematic flow sheet of the present invention.
Wherein: 1-saccharomyces olei seed, 2-autotrophy microalgae seed, 3-saccharomyces olei seed liquor cultivates reactor, 4-autotrophy microalgae seed liquor cultivates reactor, 5-saccharomyces olei seed liquor inoculation pipeline, 6-autotrophy microalgae seed liquor inoculation pipeline, 7-membrane separation device, 8-open type bioreactor, 9-mixed culture medium and acid-base neutralization agent enter pipeline, 10-autotrophy micro algae growth district, 11-saccharomyces olei vitellarium, 12-distribution device, 13-offgas outlet.
Detailed description of the invention
Open type of the present invention cultivates the method for oil-containing microorganism, specifically include following content: before meromict is cultivated, two kinds of oil-containing microorganisms individually carry out seed liquor cultivation, stirring type bioreactor is used to carry out saccharomyces olei seed liquor cultivation, use bioreactor to carry out autotrophy microalgae seed liquor to cultivate, include saccharomyces olei and saccharomyces olei fermentation medium in stirring type bioreactor, in bioreactor, include autotrophy microalgae and autotrophy SE culture medium.After seed liquor cultivation is qualified, transfer to cultivate the lower area of the open type bioreactor of the laid inside membrane separation device of saccharomyces olei and cultivate the upper area of bioreactor of autotrophy microalgae respectively through respective inoculation pipeline, and add mixed culture medium nutritional labeling and acid-base neutralization agent regulation to the meromict through aseptic process and cultivate required growth conditions.Wherein the organic carbon source in mixed culture medium grows for saccharomyces olei, and the produced CO of saccharomyces olei growth2For autotrophy micro algae growth.Saccharomyces olei seed liquor is 1:1～1:10 with autotrophy microalgae seed liquor inoculation volume ratio.Inoculate that later half co-culture system is controlled by normal pH, temperature controls and ventilation etc. controls incubation.When wherein meromict is cultivated, organic carbon source preferably employs fed-batch mode and carries out, and it is utilized growth by saccharomyces olei, produces more CO after saccharomyces olei growth2, discharge extracellular and enter in culture systems.The CO discharged due to saccharomyces olei2It is in dissolved state, therefore can be delivered to, by membrane pores, the autotrophy microalgae utilization that the upper area of open type bioreactor is cultured in based system immediately, as the carbon source supply of microalgae cell growth.
The detailed process process of the present invention is further illustrated below by accompanying drawing.As it is shown in figure 1, saccharomyces olei seed 1 is cultivated reactor 3 cultivate growth to reach Biomass OD value through saccharomyces olei seed liquor is 5.0～15.0；Autotrophy microalgae seed 2 is cultivated reactor 4 cultivate growth to reach Biomass OD value through autotrophy microalgae seed liquor is 2.0～10.0.By cultured saccharomyces olei seed liquor by inoculating the lower area that pipeline 5 is transferred to the open type bioreactor 8 of laid inside membrane separation device 7, i.e. saccharomyces olei vitellarium 11；By autotrophy microalgae seed liquor by inoculating pipeline 6 and be transferred to the upper area of this bioreactor 8, i.e. autotrophy micro algae growth district 10 carries out meromict cultivation；And mixed culture medium and acid-base neutralization agent 9 added in bioreactor mix, after mixing is analyzed in detection, in culture systems, pH value regulates the pH to 6.5～7.5 of mixed-culture medium simultaneously.Gas is entered in reaction system by distribution device 12, it is passed through gas and arrives autotrophy micro algae growth district 10 through saccharomyces olei vitellarium 11, thus realizing the needs of supply in saccharomyces olei and open type bioreactor, the gas unification being passed through is discharged whole reaction system by offgas outlet 13.
In the present invention, saccharomyces olei realizes fast-growth under the conditions of organic carbon source, and saccharomyces olei consumes organic carbon source, cellulation biomass and various metabolite, cellular biomass is rejected by the lower area at bioreactor owing to the bodily form is relatively big, and fermentation simultaneously produces CO2Be delivered to the upper area of open type bioreactor behind gas discharge extracellular through membrane pores, in making cultivating system, inorganic carbon source content increases, and autotrophy microalgae utilizes these inorganic carbon sources to carry out photosynthesis, it is thus achieved that the growth of microalgae self.Meanwhile, saccharomyces olei fermentation makes the pH value of system decline, and along with the growth of microalgae, utilizes the CO dissolved2, make the pH value of system increase, both act on each other, and the pH of regulation system is in optimum range.
In the present invention, bioreactor can be the open type reactors such as board-like, tubular type, airlift agitation formula, the nitrogen that can be passed through or carbon dioxide containing gas.Other operating condition of bioreactor yeast routinely and microdisk electrode condition control.The determinator of cultivating system carbon dioxide and dissolved oxygen content can be set, adjust ventilation as required, to obtain good effect.There is provided pH electrode detection, in order to add control sour, that alkali realizes system pH by external source simultaneously.
In the present invention, it is HCl, H that pH controls acid neutralizing agent2SO4、HNO3Deng conventional mineral acid, alkali nertralizer is NaOH, NaHCO3, KOH, Ca(OH)2, inorganic base that ammonia etc. is conventional.
In the present invention, meromict incubation temperature controls as internal coil pipe mode of heating.
In the present invention, can be passed through nitrogen or carbonated gas in the lower area and upper area of the bioreactor laying membrane separation device, gas is by being entered upper area by film space after lower area, and then draws whole reactor；Being passed through culture fluid change in volume that gas flow cultivates with open type meromict and change, being passed through gas volume speed and amassing ratio with training system bulk cargo liquid is the unit-gas amount that 0.1vvm～1.0vvm(unit liquid volumes per minute clock is passed through).
In the present invention, mixed culture medium needed for culture medium is saccharomyces olei and autotrophy microalgae in bioreactor, saccharomyces olei can be provided to grow desired nutritional material, also provide for autotrophy microalgae photosynthetic growth desired nutritional composition, two kinds of cells are separated by membrane separation device, realizing the most individually growing space, weaken different iuntercellular ecology influence, the most also the results for respective cell provide conveniently.Saccharomyces olei utilizes the culture medium that it is suitable, and wherein carbon source is organic carbon source, grows, it is thus achieved that the growth of Biomass, and part organic carbon source is breathed by sweat and generated CO2, CO that saccharomyces olei is generated2In cultivating system, carry out photosynthetic carbon source as autotrophy microalgae, thus obtain the growth of microalgae cell.
In the present invention, organic carbon source needed for saccharomyces olei can be with glucose, fructose, starch, cellulosic hydrolysate etc., the addition employing stream of this organic carbon source adds arbitrary way to be carried out, analyze according to culture fluid sample component, the organic carbon source concentration level of detecting system at any time, carries out stream by the organic carbon source mother solution prepared by depletion rate and adds.
In the present invention, the initial organic carbon source concentration (with Organic substance quality densitometer, lower same) of meromict incubation culture medium is 1.0%～5.0%, is consumed by saccharomyces olei growth in incubation, organic carbon source is constantly added through adding pump, maintains organic carbon source concentration 1.0%～2.0%.
In the present invention, the culture medium of saccharomyces olei seed liquor uses its saccharomyces olei culture medium being suitable for, and the culture medium of autotrophy microalgae seed liquor uses microalgae SE culture medium.Meromict incubation uses saccharomyces olei culture medium and microalgae SE culture medium to combine, and the composition such as including organic carbon source, inorganic salt and trace element becomes mixed culture medium.Preferably condition of culture is: seed liquor inoculum concentration (accounts for bioreactor volume): 5%～20%, temperature: 25 DEG C～30 DEG C, ventilation: 0.1vvm～1.0vvm, speed of agitator: 100rpm～400rpm, time: 24h～120h.
Scheme 1(comparative example)
Being cultivated in 300mL shaking flask by rhodotorula glutinis (offer of Chinese microorganism strain preservation center), used medium is rhodotorula glutinis PDA culture medium, obtains required seed liquor after cultivating 20h, and OD value is about 12.0.The rhodotorula glutinis seed liquor of cultivation accesses the lower area of the 20L open type bioreactor of the ceramic membrane separation device of the laid inside that the inventive method is mentioned, and film hole diameter is 0.1 μm, and the volume ratio in upper and lower two regions of bioreactor is 1:1.Distribution device is laid, it is possible to achieve the back-mixing of culture fluid bottom bioreactor；Bioreactor is vitreous body, has temperature control disk pipe, and pH, O in reactor2And CO2Sensor.
PDA culture medium prescription (in terms of every liter): glucose 20.0g, Rhizoma Solani tuber osi liquid 1000mL, pH are natural.The wherein making of potato nutritional liquid: boil after 200g Rhizoma Solani tuber osi is cut into small pieces 20～40 minutes, multilamellar filtered through gauze, be settled to 1000mL.
SE culture medium prescription (in terms of every liter):
| NaNO3 | 0.20g |
| K2HPO4·3H2O | 0.07g |
| MgSO4·7H2O | 0.07g |
| CaCl2·2H2O | 0.03g |
| KH2PO4 | 0.18g |
| NaCl | 0.03 |
| Soil extract (soil extract) | 40mL |
| FeCl3·6H2O | 0.01 |
| Fe-EDTA | 1mL |
Condition of culture is, use mixed culture medium (based on PDA culture medium, add appropriate substances by SE culture medium composition simultaneously): inoculum concentration (accounts for bioreactor volume, lower same) it is 10%, temperature is 30 DEG C, ventilation (nitrogen) 0.4vvm, and speed of agitator is 200rpm, time be 120h, pH be 7.0.
Terminate after cultivating 5 days cultivating, collect saccharomyces olei cell, analyze its dry weight and fat content, draw the harvest yield of unit fermentation system oil-containing microbial grease.
Scheme 2(comparative example)
Chlorella (purchased from aquatic institute of Chinese Academy of Sciences algae kind storehouse 1#) is carried out in shaking flask light autotrophy cultivation, and used medium is SE culture medium, obtains required seed liquor after cultivating 2 days, and OD value is about 8.0.Cultured chlorella seed liquor is seeded in 20L open type bioreactor by the inoculum concentration by 10%, and culture medium is SE culture medium.Bioreactor is airlift agitation formula, it is possible to achieve the back-mixing of culture fluid；Reactor is vitreous body, arranges daylight lamp source, automatically controls the switch time setting light, forms brightness process conversion in chlorella incubation.Temperature control disk pipe, and pH, O is had in reactor2And CO2Sensor.Condition of culture is with comparative example scheme 1, and ventilation uses air compressor compressed nitrogen to be passed through, ventilation 0.4vvm.Algae kind is chlorella vulgaris, and the culture medium of chlorella incubation is with the mixed culture medium of scheme 1.
Culture fluid (presses the ratio-dependent of 12:12 hour every day) under set light dark period, terminates cultivating, collect autotrophy microalgae cell, analyze its dry weight and fat content, draw the harvest yield of unit fermentation system oil-containing microbial grease after cultivating 5 days.
Scheme 3(comparative example)
Rhodotorula glutinis seed liquor cultural method is with comparative example scheme 1, and rhodotorula glutinis and chlorella, with comparative example scheme 2, are used respective culture medium to carry out single culture in shaking flask, cultivate and obtain required seed liquor by chlorella seed liquor condition of culture.Form seed mixture liquid after cultured rhodotorula glutinis seed liquor and chlorella seed liquor being mixed with the volume ratio of 1:4, be seeded in the 20L open type bioreactor containing mixed culture medium by the inoculum concentration of 10%.Bioreactor is airlift agitation formula, it is possible to achieve the back-mixing of culture fluid；Reactor is vitreous body, arranges fluorescent light source, automatically controls the switch time setting light, forms brightness process conversion in chlorella incubation.Temperature control disk pipe, and pH, O is had in reactor2And CO2Sensor.Synchronized mixes is cultivated ventilation and is used gas compressor compressed nitrogen to be passed through, ventilation 0.4vvm.Mixed culture medium, with the mixed culture medium of scheme 1, supplements organic carbon source by scheme 1 simultaneously.
Culture fluid under set light dark period (in the ratio of 12:12 hour every day), terminate after cultivating 5 days cultivating, collect saccharomyces olei cell and the autotrophy microalgae cell of mixing, analyze its dry weight and fat content, draw the harvest yield of unit fermentation system oil-containing microbial grease.
Scheme 4(comparative example)
Rhodotorula glutinis seed liquor cultural method is with comparative example scheme 1, Wild Vitis species (purchased from aquatic institute of Chinese Academy of Sciences algae kind storehouse 357#) seed liquor condition of culture is with comparative example scheme 2, rhodotorula glutinis and Wild Vitis species use in shaking flask respective culture medium carry out single culture, cultivates and obtain required seed liquor.Form seed mixture liquid after cultured rhodotorula glutinis seed liquor and Wild Vitis species seed liquor being mixed with the volume ratio of 1:4, be then seeded in the 20L open type bioreactor containing mixed culture medium by the inoculum concentration of 10%.Bioreactor is airlift agitation formula, it is possible to achieve the back-mixing of culture fluid；Reactor is vitreous body, arranges fluorescent light source, automatically controls the switch time setting light, forms brightness process conversion in Wild Vitis species incubation.Temperature control disk pipe, and pH, O is had in reactor2And CO2Sensor.Synchronized mixes is cultivated ventilation and is used gas compressor compressed nitrogen to be passed through, ventilation 0.4vvm.Mixed culture medium, with scheme 1 mixed culture medium, supplements organic carbon source by scheme 1 simultaneously.
Culture fluid under set light dark period (in the ratio of 12:12 hour every day), terminate after cultivating 5 days cultivating, collect saccharomyces olei cell and the autotrophy microalgae cell of mixing, analyze its dry weight and fat content, draw the harvest yield of unit fermentation system oil-containing microbial grease.
Scheme 5(embodiment 1)
Rhodotorula glutinis seed liquor cultural method is with comparative example scheme 1, and rhodotorula glutinis and chlorella, with comparative example scheme 2, are used respective culture medium to carry out single culture in shaking flask, cultivate and obtain required seed liquor by chlorella seed liquor condition of culture.Cultured rhodotorula glutinis seed liquor and chlorella seed liquor are seeded to respectively lower area and the upper area of the 20L open type bioreactor of laid inside ceramic membrane separation device containing mixed culture medium, total inoculum concentration is the 10% of bioreactor volume, and wherein the inoculation volume ratio of rhodotorula glutinis seed liquor and chlorella seed liquor is 1:4.The volume ratio in upper and lower two regions of bioreactor is 1:1.The film hole diameter of ceramic membrane separation device is 0.1 μm.Distribution device is laid, it is possible to achieve the back-mixing of culture fluid bottom bioreactor；Reactor is vitreous body, arranges fluorescent light source, automatically controls the switch time setting light, forms brightness process conversion in chlorella incubation.Temperature control disk pipe, and pH, O is had in reactor2And CO2Sensor.Meromict is cultivated ventilation and is used air compressor compressed nitrogen to be passed through, ventilation 0.4vvm.Mixed culture medium, with the mixed culture medium of scheme 1, supplements organic carbon source by scheme 1 simultaneously.
Culture fluid under set light dark period (in the ratio of 12:12 hour every day), terminate after cultivating 5 days cultivating, collect saccharomyces olei cell and autotrophy microalgae cell respectively, analyze dry cell weight and fat content, draw the harvest yield of unit fermentation system oil-containing microbial grease.
Scheme 6(embodiment 2)
Rhodotorula glutinis seed liquor cultural method is with comparative example scheme 1, Wild Vitis species (purchased from aquatic institute of Chinese Academy of Sciences algae kind storehouse 357#) seed liquor condition of culture is with comparative example scheme 2, use respective culture medium to carry out single culture in shaking flask rhodotorula glutinis and Wild Vitis species, obtain required seed liquor.Cultured rhodotorula glutinis seed liquor and Wild Vitis species seed liquor are inoculated respectively lower area and the upper area of the 20L open type bioreactor of laid inside ceramic membrane separation device, total inoculum concentration is the 10% of bioreactor volume, and wherein the inoculation volume ratio of rhodotorula glutinis seed liquor and Wild Vitis species seed liquor is 1:4.The volume ratio in upper and lower two regions of bioreactor is 1:1.The film hole diameter of ceramic membrane separation device is 0.1 μm.Distribution device is laid, it is possible to achieve the back-mixing of culture fluid bottom bioreactor；Reactor is vitreous body, arranges fluorescent light source, automatically controls the switch time setting light, forms brightness process conversion in chlorella incubation.Temperature control disk pipe, and pH, O is had in reactor2And CO2Sensor.The ventilation of meromict incubation uses gas compressor compressed nitrogen to be passed through, ventilation 0.4vvm.Mixed culture medium, with scheme 1 mixed culture medium, supplements organic carbon source by scheme 1 simultaneously.
Culture fluid under set light dark period (in the ratio of 12:12 hour every day), terminate after cultivating 5 days cultivating, collect saccharomyces olei cell and autotrophy microalgae cell respectively, analyze dry cell weight and fat content, draw the harvest yield of unit fermentation system oil-containing microbial grease.
Above-described embodiment experimental result such as table 1 below:
The cultivation results of each embodiment of table 1
| Scheme | Microorganism dry weight, g/L | Fat content, % | Oils and fats harvest yield, g/L |
| 1 | 8.20 | 5.4 | 0.44 |
| 2 | 5.21 | 7.4 | 0.39 |
| 3 | 12.60 | 31.8 | 4.01 |
| 4 | 13.26 | 32.9 | 4.36 |
| 5 | 22.37 | 33.4 | 7.47 |
| 6 | 23.45 | 35.2 | 8.25 |
From the data of table 1 it can be seen that the inventive method (scheme 5 and 6) drastically increases the harvest yield of oil-containing microbial cell, fat content have also been obtained raising.Under identical condition of culture, compared with cultivating through synchronized mixes with microorganism single culture and two kinds of microorganisms, the present invention uses membrane separation device, in open type bioreactor, saccharomyces olei and autotrophy microalgae are separately carried out meromict cultivation, the reactant liquor system used realizes back-mixing, gather in the crops the most respectively and mention oils and fats, obtained fat content has obtained bigger raising, and in unit fermentation system, oils and fats harvest yield is improved significantly.The inventive method can realize the high efficiently multiplying of oil-containing microorganism, and can improve unit volume microbial grease harvest yield.
Claims (10)
1. the method that open type cultivates oil-containing microorganism, including following content:
(1) saccharomyces olei seed liquor is cultivated；
(2) autotrophy microalgae seed liquor is cultivated；
(3) meromict cultivation is carried out in saccharomyces olei seed liquor and autotrophy microalgae seed liquor are accessed the open type bioreactor of laid inside membrane separation device, bioreactor is divided into upper and lower two regions by membrane separation device, saccharomyces olei is cultivated at membrane separation device lower area, autotrophy microalgae carries out photosynthesis growth at membrane separation device upper area, the culture medium composition of lower regions is consistent with condition of culture, supplements the organic carbon source needed for saccharomyces olei growth metabolism in incubation.
The most in accordance with the method for claim 1, it is characterised in that: membrane separation device uses ceramic membrane separation device, and film hole diameter is 0.05 μm～0.5 μm.
3. according to the method described in claim 1 or 2, it is characterised in that: it is 1:1～5:1 that bioreactor is divided into the volume ratio in upper and lower two regions by membrane separation device.
The most in accordance with the method for claim 1, it is characterised in that: described bioreactor also includes distribution device, is laid on the bottom in reactor lower part region, and the gas of distribution can be in membrane separation device enters whole bioreactor.
The most in accordance with the method for claim 1, it is characterised in that: saccharomyces olei include saccharomyces cerevisiae (S.cerevisiae), rhodotorula glutinis (R.glutinis) or trichosporon cutaneum (Trichosporoncutaneum), organic carbon source is glucose, fructose, starch or cellulosic hydrolysate.
The most in accordance with the method for claim 1, it is characterised in that: autotrophy microalgae includes chlorella, Wild Vitis species, little ring algae or diatom.
7. according to the method described in claim 1,5 or 6, it is characterised in that: in meromict incubation, saccharomyces olei seed liquor is 1:1～1:10 with the volume ratio that is initially accessed of autotrophy microalgae seed liquor.
The most in accordance with the method for claim 1, it is characterised in that: the initial medium that meromict is cultivated uses the culture medium needed for saccharomyces olei, adds the inorganic salt needed for autotrophy micro algae growth and trace element simultaneously.
9. according to the method described in claim 1,5 or 8, it is characterised in that: in meromict incubation, supplement organic carbon source needed for saccharomyces olei growth course with batch or continuation mode.
The most in accordance with the method for claim 1, it is characterised in that: meromict condition of culture is: temperature is 20 DEG C～37 DEG C, and pH value is 6～9.
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| 不同生物量的乳酸杆菌对对虾养殖池3种常见优势微藻的影响;王昊, 等;《台湾海峡》;20090531;第28卷(第2期);第223页摘要 * |
| 地衣芽孢杆菌与3种微藻生长的相互影响;李卓佳, 等;《农业环境科学学报》;20091231;第28卷(第4期);第840页第1.2.3、1.2.4、2.1节、第843页右栏第1段 * |
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