CN103773691A - Enclosed rapid culture method of microalgae - Google Patents
Enclosed rapid culture method of microalgae Download PDFInfo
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- CN103773691A CN103773691A CN201210404148.2A CN201210404148A CN103773691A CN 103773691 A CN103773691 A CN 103773691A CN 201210404148 A CN201210404148 A CN 201210404148A CN 103773691 A CN103773691 A CN 103773691A
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Abstract
The invention discloses an enclosed rapid culture method of microalgae. The method includes the steps of: (1) cultivating a facultative anaerobic bacteria seed liquid; (2) cultivating an autotrophic microalgae seed liquid; and (3) inoculating the facultative anaerobic bacteria seed liquid and the autotrophic microalgae seed liquid into a closed photobioreactor with a built-in membrane separation device to carry out semi-mixing culture, fermenting the anaerobic bacteria in the membrane separation device, and subjecting the autotrophic microalgae to photosynthesis growth in the photobioreactor outside the membrane separation device, wherein the medium composition and culture conditions are the same in the two reactors, and supplementing organic carbon source required by the growth and metabolism of the facultative anaerobic bacteria in the culture process. The photobioreactor also comprises a guide shell, the membrane separation device is arranged in the guide shell, and a reflective membrane is pasted on the outer surface of the guide shell. The method has the advantages of increasing the yield of autotrophic microalgae, the utilization rate of an inorganic carbon source (CO2), and oil content of the microorganisms, and simplifying the culture device.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method of cultivating micro-algae, relate in particular to a kind of closed meromict fast culture autotrophy oil-producing microalgae and facultative anaerobic bacteria and obtain high microalgae cell harvest yield, improve the method for microalgae cell fat content simultaneously.
Background technology
Along with socio-economic development, the supply of fossil resource is day by day in short supply, oil product imbalance between supply and demand is also outstanding day by day, and corresponding problem of environmental pollution is also more outstanding, and developing renewable resources by all kinds of means, it is inevitable to become.Take renewable resourcess such as microbial oils as the biofuel in source has that energy density is high, sulphur content is low, the performance such as sufficient combustion, oilness be good, also there is the features such as renewable, environmental friendliness, readily biodegradable, storing and transporting security, the capability of antidetonance be good, can be used as the substitute of fossil energy.
Micro-algae is one of oil-containing microorganism, and micro-algae is rich in the nutritive ingredients such as unsaturated fatty acids, starch and protein (as spirulina etc.), can be used as the field raw materials such as the energy, medicine and food; Regulate and control by condition, microalgae cell can accumulate lipid acid in a large number, has that algae is as chlorella slightly, and its body fat acid content can account for 30%～60% of dry cell weight.Utilize and cultivate micro-algae and accumulate oil resource, become and utilized at present sun power and the stabilizing carbon dioxide the most popular research field of developing renewable resources, not only there is huge market potential, and there is outstanding social value.
Microalgae cell growth pattern is generally divided into two kinds of light autotrophy and carbon source heterotrophism, and light autotrophy process will consume CO
2, CO
2photosynthesis by microalgae cell is efficiently fixed as Cell Component, is the key of culture effect of realizing ideal, and exists simultaneously and supplements CO
2produce O with photosynthesis
2desorb, the problem of discharge.CN200410020978.0 and CN03128138.9 all adopt and in photo-bioreactor system, add a kind of installation method, realize CO
2supply, also can realize certain oxygen simultaneously and resolve effect.Carbon source heterotrophism process is to utilize organic carbon source for the CO in the alternative autotrophy process of substrate
2carry out the accumulation of micro-algal biomass, Growth of Cells speed, but in cell, oil and fat accumulation level is lower.
Utilize other non-alga microbial and micro-algae to carry out the research of mixed culture also many, CN200910038908.0 discloses a kind of method of genus bacillus regulation and control planktonic microalgae mixed culture, discovery when the method is used genus bacillus to carry out mixed culture to each micro-algae in co-culture system, each micro algae growth equilibrium, good stability, can avoid the species diversity of algae phase in co-culture system single, avoid the quantity of certain micro-algae to occur extreme advantage or inferior position, therefore the method is mainly used in controlling certain micro-algae spreading unchecked in water body.CN200910038910.8 discloses a kind of lactobacillus and has regulated and controled the method for micro-algae mixed culture and synergistic purification cultivation discharge water, the method is by the direct or indirect effect of lactobacillus, regulate and control the quantitative proportion of various micro-algaes, make each algae growth balanced, good stability, reaches the object of denitrification dephosphorization to cultivation waste discharge by the synergy between bacterium, algae.Above-mentioned prior art is to control the growth equilibrium of algae in water body by thalline, is not the oil and fat accumulation promoting in growth and the microalgae cell of algae.
Summary of the invention
For the deficiencies in the prior art, the invention provides the method for the micro-algae of a kind of closed fast culture.Autotrophy oil-producing microalgae and facultative anaerobic bacteria in closed photo bioreactor, are realized meromict fast culture by this method, has the autotrophy of raising microalgae cell harvest yield, improves inorganic carbon source (CO
2) utilization ratio, improve microbial oil content, simplify the advantages such as culture apparatus; Do not affect the fermenting process of facultative anaerobic bacteria simultaneously, also can be by the anaerobically fermenting product in separation and Culture liquid in results oil-containing micro-algae cell, realizing bacterium anaerobically fermenting production of chemicals, is a kind of high-efficient culture method that grease and biochemicals are produced simultaneously.
The method of the micro-algae of the closed fast culture of the present invention, comprises following content:
(1) cultivate facultative anaerobic bacteria seed liquor;
(2) cultivate the micro-algae seed liquor of autotrophy;
(3) facultative anaerobic bacteria seed liquor and the access of the micro-algae seed liquor of autotrophy are had in the closed photo bioreactor of built-in membrane separation unit and carry out meromict cultivation, facultative anaerobic bacteria is in the inner fermentation of membrane separation unit, microalgae cell carries out photosynthesis growth in the bioreactor beyond membrane separation unit, in two reactors, substratum composition is consistent with culture condition, supplements the required organic carbon source of facultative anaerobic bacteria growth metabolism in culturing process; Described bioreactor also comprises guide shell, and membrane separation unit is built in guide shell, and outlet is concordant with bioreactor outlet; Described bioreactor has tapetum lucidum, and tapetum lucidum is attached to guide shell outside surface.
In the inventive method, membrane separation unit preferably adopts ceramic membrane separation device, and film hole diameter is 0.05 μ m～0.5 μ m.
In the inventive method, tapetum lucidum preferably adopts 3M tapetum lucidum, and 3M tapetum lucidum is a kind of granulated glass sphere filled type tapetum lucidum, and the back side scribbles pressure sensitive adhesive and easily peels off backing paper, even under complete moisture state or under wide-angle, also can keep its good reflective function.Tapetum lucidum can partly or entirely cover the outside surface of guide shell, and the light of external light source is injected bioreactor and arrived guide shell place and realize reflectively through tapetum lucidum, and light turns back to light source place, thereby realizes two light path environment.
In the inventive method, facultative anaerobic bacteria comprises genus bacillus, fusiform bacilarmature, bifidus bacillus, Bacterium lacticum or klebsiella etc., organic carbon source is glucose, glycerine, fructose, starch, cellulosic hydrolysate etc., the meta-bolites of facultative anaerobic bacteria, be that facultative anaerobic bacteria tunning is generally 1, ammediol and/or organic acid etc., the tunning that different facultative anaerobic bacterias obtains is different, different and different according to the kind of facultative anaerobic bacteria.It is method well known to those skilled in the art that facultative anaerobic bacteria seed liquor is cultivated, and as adopted stirring type bioreactor, adds substratum and facultative anaerobic bacteria, under suitable condition, cultivates.
In the inventive method, the micro-algae of autotrophy comprises chlorella, grape algae, little ring algae, diatom etc., and it is method well known to those skilled in the art that the micro-algae seed liquor of autotrophy is cultivated, as adopts conventional airlift photobioreactor to carry out seed liquor cultivation.
In the inventive method, in closed meromict culturing process, the initial access volume ratio of facultative anaerobic bacteria seed liquor and micro-algae seed liquor is 1:1～1:10.The initial medium that meromict is cultivated adopts facultative anaerobic bacteria required substratum, adds microalgae cell grow required inorganic salt and trace element (as the composition interpolation related substances by SE substratum) etc. simultaneously.Meromict culturing process with batch or the mode such as continuous supplement the required organic carbon source of facultative anaerobic bacteria fermenting process.
In the inventive method, closed meromict culture condition is general to be adopted and the condition of the conditional likelihood of facultative anaerobic bacteria fermenting process, and as temperature is generally 20 ℃～37 ℃, pH value is generally 6～9, is preferably 6.5～7.5 etc.
The inventive method utilizes membrane separation unit that facultative anaerobic bacteria and microalgae cell are carried out to closed meromict cultivation, and facultative anaerobic bacteria is different from microalgae cell growth conditions and required carbon source, complements each other and promotes.Microalgae cell is discarded carbon source (CO after utilizing facultative anaerobic bacteria fermentation
2) for self growth carbon source, maintain system osmotic pressure condition (pH value) more stably simultaneously, constantly grow by supplementing organic carbon source assurance facultative anaerobic bacteria.Meanwhile, bioreactor has tapetum lucidum, posts tapetum lucidum at bioreactor guide shell outside surface, makes external light source realize return, increases intensity of photosynthesis.By photosynthesis and under tapetum lucidum effect, culture system is under two light path environment and makes microalgae cell Fast Growth.
The inventive method facultative anaerobic bacteria and microalgae cell are realized independent separately growing space by membrane separation unit, and substratum composition is to realize blend and intercommunication, thereby realizes the meromict culturing process of facultative anaerobic bacteria and microalgae cell.Select suitable kind facultative anaerobic bacteria and suitable kind microalgae cell, by controlling the culturing process of facultative anaerobic bacteria and microalgae cell, make both form stable meromict culture system, and set up that different iuntercellulars utilize environmental nutrient condition jointly and growth relationship that iuntercellular does not interfere with each other, and microalgae cell is under two light path environment, realize the Fast Growth process of oil-containing micro-algae cell, and improve the accumulative effect of grease, improve the grease harvest yield in unit fermentation system in single oil-containing micro-algae culturing process, thereby for the preparation of microbial oil is laid a good foundation.Be there is not to impact in the fermenting process of facultative anaerobic bacteria simultaneously, can obtain required tunning simultaneously.Carbon source and tunning that micro-algae is used fermenting process all have tolerance, do not affect the growth of micro-algae and the accumulation of grease.Compared with two kinds of microbial synchronous mixed cultivation process, micro-algal biomass results simply, are avoided gathering in the crops together with bacterial cell, and affect the usefulness of algae mud grease Extraction parts.
Accompanying drawing explanation
Fig. 1 is a kind of concrete technology schematic flow sheet of the present invention.
Wherein: 1-facultative anaerobic bacteria seed, the micro-algae seed of 2-, 3-facultative anaerobic bacteria seed liquor is cultivated reactor, the micro-algae seed liquor of 4-is cultivated reactor, 5-facultative anaerobic bacteria seed liquor inoculation pipeline, the micro-algae seed liquor inoculation of 6-pipeline, 7-membrane separation unit, 8-closed photo bioreactor, 9-mixed culture medium and acid-base neutralisation agent enter pipeline, 10-guide shell, 11-tapetum lucidum, 12-inlet mouth, the outlet of 13-tail gas.
Embodiment
The method of the micro-algae of the closed fast culture of the present invention, specifically comprise following content: before meromict is cultivated, two kinds of microorganisms carry out separately respectively seed liquor cultivation, adopt stirring type bioreactor to carry out the cultivation of facultative anaerobic bacteria seed liquor, adopting bioreactor to carry out the micro-algae seed liquor of autotrophy cultivates, in stirring type bioreactor, comprise facultative anaerobic bacteria, anaerobic bacterium fermention medium, in bioreactor, comprise micro-algae algae kind and autotrophy SE substratum.After seed liquor cultivation is qualified, transfer in the closed photo bioreactor with tapetum lucidum of the built-in micro-algae of membrane separation unit neutral incubation of cultivating facultative anaerobic bacteria through inoculation pipeline separately respectively, and add and be adjusted to meromict through the mixed culture medium nutritive ingredient of aseptically process and acid-base neutralisation agent and cultivate required growth conditions.Wherein the organic carbon source in mixed culture medium is for facultative anaerobic bacteria growth, and the facultative anaerobic bacteria CO producing that grows
2for micro algae growth.Facultative anaerobic bacteria seed liquor and microalgae cell seed liquor inoculation volume ratio are 1:1～1:10.Inoculate later half co-culture system by normal pH control, temperature control and air flow etc. control culturing process.When wherein meromict is cultivated, organic carbon source most preferably adopts fed-batch mode to carry out, and it is grown by facultative anaerobic bacteria utilization, after facultative anaerobic bacteria fermentation, produces more CO
2, discharge extracellular and enter in culture systems.Due to the CO of facultative anaerobic bacteria cell discharge
2in dissolved state, therefore can be delivered to the microalgae cell utilization in mixed culture medium system in bioreactor by membrane pores immediately, as the carbon source supply of microalgae cell growth.
Further illustrate detailed process process of the present invention below by accompanying drawing.As shown in Figure 1, facultative anaerobic bacteria seed 1 is cultivated reactor 3 incubation growth through facultative anaerobic bacteria seed liquor to reach biomass OD value is 5.0～15.0; It is 2.0～10.0 that micro-algae seed 2 reaches biomass OD value through micro-algae seed liquor cultivation reactor 4 incubation growth.Cultured anaerobic bacterium seed liquor is transferred to respectively in built-in membrane separation unit 7 and in closed photo bioreactor 8 and carries out meromict cultivation by inoculating pipeline 6 by inoculating pipeline 5, microalgae cell seed liquor, and mixed culture medium and acid-base neutralisation agent 9 are added in membrane separation unit and bioreactor and are mixed, simultaneously through detection analyze mix after in culture systems pH value regulate pH to 6.5～7.5 of mixed-culture medium.Tapetum lucidum 11 is attached to the outside surface of bioreactor guide shell 10, realizes two light path effects of external light source.Gas is entered in reaction system by inlet mouth 12, utilize threeway to pass into respectively membrane separation unit 7 and guide shell 10 inner sides, need thereby realize to air feed in facultative anaerobic bacteria in membrane separation unit and bioreactor, the gas unification passing into is discharged whole reaction system by tail gas outlet 13, control exhaust valve opening, making reactor internal pressure is 0.01 ~ 0.05MPa.
In the inventive method, facultative anaerobic bacteria is realized Fast Growth under organic carbon source condition, and facultative anaerobic bacteria consumes organic carbon source, founder cell biomass and various meta-bolites, cellular biomass is because the bodily form is compared with large and tunicle is trapped in membrane separation unit inside, and fermentation simultaneously produces CO
2gas is delivered in whole mixed culture based system through membrane pores after discharging extracellular, inorganic carbon source content in culture system is increased, microalgae cell utilizes under these inorganic carbon sources and reflective film formed pair of light path environment and carries out photosynthesis, obtains the Fast Growth of microalgae cell self.Simultaneously facultative anaerobic bacteria fermentation declines the pH value of system, and along with the growth of microalgae cell, utilizes the CO of dissolving
2, make the pH value of system increase, both act on each other, and the pH of regulation system is in subject range.
In the inventive method, bioreactor can be the closed reactors such as board-like, tubular type, airlift agitation formula, the nitrogen that can pass into or carbon dioxide containing gas and guide shell effect form back-mixing state, paste 3M tapetum lucidum realize two light path environment at guide shell outside surface.Other operational condition of bioreactor bacterium and control of micro-algae culture condition routinely.The determinator of culture system carbonic acid gas and dissolved oxygen content can be set, adjust as required air flow, to obtain good effect.Provide pH electrode to detect, to add acid, alkali to realize the control of system pH by external source simultaneously.
In the inventive method, it is HCl, H that pH controls acid neutralizing agent
2sO
4, HNO
3etc. conventional mineral acid, alkali neutralizing agent is NaOH, NaHCO
3, KOH, Ca(OH)
2, the conventional mineral alkali such as ammoniacal liquor.
In the inventive method, the control of meromict culturing process temperature is inner coil pipe type of heating.
In the inventive method, meromict can pass into nitrogen or carbonated gas in cultivating in membrane separation unit and bioreactor.Ventilation line exists with threeway form after entering bioreactor, and wherein a branch road passes in membrane separation unit, and the envrionment conditions of bacterium anaerobically fermenting is provided, and whole reactor is drawn in outlet; All the other two gas circuits pass into respectively the guide shell medial region of both sides, realize ventilation guide functions, and reaction solution in the bioreactor outside membrane separation unit can be mixed.Pass into gas volume and change with nutrient solution volume change, passing into gas volume speed and culture systems dress liquid volume ratio is the unit gas volume that 0.1vvm～1.0vvm(unit liquid volume per minute passes into).
In the inventive method, substratum in bioreactor is facultative anaerobic bacteria and the required mixed culture medium of microalgae cell, the fermentation growth desired nutritional material of facultative anaerobic bacteria can be provided, also providing is the photosynthetic growth desired nutritional of microalgae cell composition, two kinds of cells are separated and are come by membrane separation unit, realize growth separately, weaken different iuntercellular ecological impacts, simultaneously also for the results of microalgae cell provide convenience.Facultative anaerobic bacteria utilizes its suitable substratum, and wherein carbon source is organic carbon source, grows, and obtains the growth of biomass, and part organic carbon source is breathed and generated CO by fermenting process
2, the CO that facultative anaerobic bacteria generates
2in culture system, carry out photosynthetic carbon source as micro-algae, thereby obtain the growth of microalgae cell.
In the inventive method, the required organic carbon source of facultative anaerobic bacteria can be glycerine, glucose, fructose, starch, cellulosic hydrolysate etc., adding of this organic carbon source adopts fed-batch mode to carry out, according to nutrient solution sample analysis of components, the organic carbon source concentration level of detection system at any time, carries out stream by the organic carbon source mother liquor preparing by spending rate and adds.
In the inventive method, the initial organic carbon source concentration of meromict culturing process substratum is (with organism quality densitometer, lower same) be 1.0%～5.0%, in culturing process by the anaerobic bacterium consumption of growing, organic carbon source is constantly added through adding pump, and maintaining organic carbon source concentration is 1.0%～2.0%.
In the inventive method, the substratum of facultative anaerobic bacteria seed liquor adopts its applicable facultative anaerobic bacteria substratum, and the substratum of the micro-algae seed liquor of autotrophy adopts micro-algae SE substratum.Meromict culturing process adopts facultative anaerobic bacteria substratum and micro-algae culture medium to combine, and comprises the compositions such as organic carbon source, inorganic salt and trace element, becomes mixed culture medium.Preferred culture condition is: seed liquor inoculum size (accounting for bioreactor volume, V/V): 5%～20%; Temperature: 25 ℃～30 ℃; Air flow: 0.1vvm～1.0vvm, mixing speed: 100rpm～400rpm, time: 24h～120h.
Scheme 1(comparative example)
Klebsiella (Chinese microorganism strain preservation center C GMCC No.0798) is cultivated in 300mL shaking flask, and used medium is facultative anaerobic bacteria glycerin medium, after cultivation 20h, obtains required seed liquor, and OD value is 12.0 left and right.By the facultative anaerobic bacteria seed liquor access of cultivating, containing in 10L ceramic membrane separation device built-in in the 20L closed photo bioreactor of mixed culture medium, film hole diameter is 0.1 μ m.Bioreactor is airlift agitation formula, can realize the back-mixing of nutrient solution; Reactor is vitreum, has temperature control coil pipe in reactor, and pH, O
2and CO
2sensor.
Facultative anaerobic bacteria is Cray Bai Shi pneumobacillus, and its substratum is glycerin medium.
Glycerin medium formula (in every liter): NH
4cl 5.35 g, KCl 0.75 g, NaH
2pO
41.38 g, Na
2sO
40.28 g, MgCl
26H
2o 0.26 g, CaCl
2h
2o 0.02 g, yeast extract 1.0 g, glycerine 40 g.
SE culture medium prescription (in every liter):
| NaNO 3 | 0.20g |
| K 2HPO 4·3H 2O | 0.07g |
| MgSO 4·7H 2O | 0.07g |
| CaCl 2·2H 2O | 0.03g |
| KH 2PO 4 | 0.18g |
| NaCl | 0.03 |
| Soil extract (soil extract) | 40mL |
| FeCl 3·6H 2O | 0.01 |
| Fe-EDTA | 1mL |
Culture condition is, adopt mixed culture medium (take glycerin medium as basis, add suitable material by SE substratum composition simultaneously): inoculum size (accounts for bioreactor volume, V/V, lower same) be 10%, temperature is 30 ℃, air flow (nitrogen) 0.4vvm, mixing speed is 200rpm, and the time is 120h, and pH value is 7.0.
Cultivate and within 5 days, stop afterwards cultivating, collect microorganism cells, survey dry weight and fat content, the harvest yield of the unit's of drawing fermentation system microbial oil.Wherein initial glycerin medium organic carbon source (glycerine) mass concentration is 40.0g/L, and process is added organic carbon source, and to maintain content be 15.0g/L left and right, and cultivating residual glycerol content while end is 5.0g/L.Utilize after the centrifugal collection thalline of liquid-phase chromatographic analysis fermented liquid primary product 1,3-PD concentration in clear liquid to determine the fermentation level of facultative anaerobe.
Scheme 2(comparative example)
Chlorella (purchased from the algae kind storehouse 1# of aquatic institute of the Chinese Academy of Sciences) is carried out to the cultivation of light autotrophy in shaking flask, and used medium is SE substratum, cultivates and obtains required seed liquor after 2 days, and OD value is 8.0 left and right.By 10%(V/V) inoculum size cultured chlorella seed liquid is seeded in 20L closed photo bioreactor, substratum is SE substratum.Bioreactor is airlift agitation formula, can realize the back-mixing of nutrient solution; Reactor is vitreum, and fluorescent lamp source is set, and automatically controls the switching time of setting light, forms the dark process conversion of light in chlorella culturing process.In reactor, there is temperature control coil pipe, and pH, O
2and CO
2sensor.Culture condition is with comparative example scheme 1, and ventilation adopts air compressor compressed nitrogen to pass into, air flow 0.4vvm.Algae kind is Chlorella vulgaris, and the substratum of chlorella culturing process is with the mixed culture medium of scheme 1.
Nutrient solution (is pressed the ratio-dependent of 12:12 hour every day) under set light dark period, cultivates and within 5 days, stops afterwards cultivating, and collects microorganism cells, surveys dry weight and fat content, the grease harvest yield of the unit's of obtaining fermentation system.
Scheme 3(comparative example)
Cray Bai Shi pneumobacillus seed liquor cultural method is with comparative example scheme 1, and chlorella seed liquid culture condition, with comparative example scheme 2, uses substratum separately to carry out single culture Cray Bai Shi pneumobacillus and chlorella in shaking flask, cultivates and obtains required seed liquor.After cultured Cray Bai Shi pneumobacillus seed liquor and chlorella cells seed liquor are mixed with the volume ratio of 1:4, form mixed seeds liquid, by 10%(V/V) inoculum size be seeded to together in the 20L closed photo bioreactor containing mixed culture medium.Bioreactor is airlift agitation formula, can realize the back-mixing of nutrient solution; Reactor is vitreum, and fluorescent light source is set, and automatically controls the switching time of setting light, forms the dark process conversion of light in chlorella culturing process.In reactor, there is temperature control coil pipe, and pH, O
2and CO
2sensor.Mixed culture medium, with the mixed culture medium of scheme 1, supplements organic carbon source by scheme 1 simultaneously.
Synchronized mixes is cultivated ventilation and is adopted gas compressor compressed nitrogen to pass into, air flow 0.4vvm.Nutrient solution is (in the ratio of 12:12 hour every day) under set light dark period, cultivates and within 5 days, stops afterwards cultivating, and collects microorganism cells, surveys dry weight and fat content, the grease harvest yield of the unit's of obtaining fermentation system.Utilize after the centrifugal collection thalline of liquid-phase chromatographic analysis fermented liquid product 1,3-PD concentration in clear liquid to determine the fermentation level of facultative anaerobe.
Scheme 4(comparative example)
Cray Bai Shi pneumobacillus seed liquor cultural method is with comparative example scheme 1, grape algae (purchased from the algae kind storehouse 357# of aquatic institute of the Chinese Academy of Sciences) seed liquor culture condition is with comparative example scheme 2, in shaking flask, use substratum separately to carry out single culture Cray Bai Shi pneumobacillus and grape algae, cultivate and obtain required seed liquor.After cultured Cray Bai Shi pneumobacillus seed liquor and grape frustule seed liquor are mixed with the volume ratio of 1:4, form mixed seeds liquid, then by 10%(V/V) inoculum size be seeded to together in the 20L closed photo bioreactor containing mixed culture medium.Bioreactor is airlift agitation formula, can realize the back-mixing of nutrient solution; Reactor is vitreum, and fluorescent light source is set, and automatically controls the switching time of setting light, forms the dark process conversion of light in grape algae culturing process.In reactor, there is temperature control coil pipe, and pH, O
2and CO
2sensor.Mixed culture medium, with the mixed culture medium of scheme 1, supplements organic carbon source by scheme 1 simultaneously.
Synchronized mixes is cultivated ventilation and is adopted gas compressor compressed nitrogen to pass into, air flow 0.4vvm.Nutrient solution is (in the ratio of 12:12 hour every day) under set light dark period, cultivates and within 5 days, stops afterwards cultivating, and collects microorganism cells, surveys dry weight and fat content, the grease harvest yield of the unit's of obtaining fermentation system.Utilize after the centrifugal collection thalline of liquid-phase chromatographic analysis fermented liquid product 1,3-PD concentration in clear liquid to determine the fermentation level of facultative anaerobe.
Scheme 5(embodiment 1)
Cray Bai Shi pneumobacillus seed liquor cultural method is with comparative example scheme 1, and chlorella seed liquid culture condition, with comparative example scheme 2, uses substratum separately to carry out single culture Cray Bai Shi pneumobacillus and chlorella in shaking flask, cultivates and obtains required seed liquor.Cultured Cray Bai Shi pneumobacillus seed liquor and chlorella seed liquid are seeded to respectively in the built-in 10L ceramic membrane separation device and 20L closed photo bioreactor containing mixed culture medium, inoculum size is 10%(V/V), the inoculative proportion of Cray Bai Shi pneumobacillus seed liquor and chlorella seed liquid is 1:4.Ceramic membrane separation device and bioreactor are airlift agitation formula, can realize the back-mixing of nutrient solution; Bioreactor is vitreum, and the outer fluorescent light source that arranges is controlled the switching time of setting light automatically, forms the dark process conversion of light in chlorella culturing process.Because reactor guide shell outside surface posts tapetum lucidum, make the light of external light source form reflection at tapetum lucidum place, realize region between light source and guide shell and, for two light path states, improve light source utilization ratio.In reactor, there is temperature control coil pipe, and pH, O
2and CO
2sensor.Mixed culture medium, with the mixed culture medium of scheme 1, supplements organic carbon source by scheme 1 simultaneously.
Meromict is cultivated ventilation and is adopted gas compressor compressed nitrogen to pass into, air flow 0.4vvm.Nutrient solution is (in the ratio of 12:12 hour every day) under set light dark period, cultivates and within 5 days, stops afterwards cultivating, and collects the microalgae cell in bioreactor, analyzes microalgae cell dry weight and fat content, the grease harvest yield of the unit's of obtaining fermentation system.Utilize after the centrifugal collection thalline of liquid-phase chromatographic analysis mixed fermentation liquid product 1,3-PD concentration in clear liquid to determine the fermentation level of facultative anaerobe.
Scheme 6(embodiment 2)
Cray Bai Shi pneumobacillus seed liquor cultural method is with comparative example scheme 1, and grape algae seed liquor culture condition, with comparative example scheme 2, uses substratum separately to carry out single culture Cray Bai Shi pneumobacillus and grape algae in shaking flask, obtains required seed liquor.Cultured Cray Bai Shi pneumobacillus seed liquor and grape algae seed liquor are seeded to respectively in the built-in 10L ceramic membrane separation device and 20L closed photo bioreactor containing mixed culture medium, inoculum size is 10%(V/V), the inoculative proportion of Cray Bai Shi pneumobacillus seed liquor and grape algae seed liquor is 1:4.Ceramic membrane separation device is airlift agitation formula, can realize nutrient solution back-mixing; Exterior light bio-reactor is vitreum, and the outer fluorescent light source that arranges is controlled the switching time of setting light automatically, forms the dark process conversion of light in chlorella culturing process.Same scheme, due to the existence of guide shell outside surface tapetum lucidum, makes bioreactor space in two light path states, improves system light intensity.In reactor, there is temperature control coil pipe, and pH, O
2and CO
2sensor.Mixed culture medium, with the mixed culture medium of scheme 1, supplements organic carbon source by scheme 1 simultaneously.
The ventilation of meromict culturing process adopts gas compressor compressed nitrogen to pass into, air flow 0.4vvm.Nutrient solution is (in the ratio of 12:12 hour every day) under set light dark period, cultivate and within 5 days, stop afterwards cultivating, collect the micro-algae microorganism cells in bioreactor, analyze microalgae cell dry weight and fat content, the grease harvest yield of the unit's of obtaining fermentation system.Utilize after the centrifugal collection thalline of liquid-phase chromatographic analysis fermented liquid product 1,3-PD concentration in clear liquid to determine the fermentation level of facultative anaerobe.
Above-described embodiment experimental result is as shown in table 1:
The cultivation results of the each embodiment of table 1
| Scheme | Microorganism dry weight, g/L | Fat content, % | Grease harvest yield, g/L | Facultative anaerobe fermentation level, g/ |
| 1 | 7.02 | 2.2 | 0.15 | 54.2 |
| 2 | 5.21 | 7.4 | 0.39 | - |
| 3 | 10.40 | 32.6 | 3.90 | 52.8 |
| 4 | 11.06 | 33.7 | 3.73 | 56.2 |
| 5 | 17.33 | 33.9 | 5.87 | 62.1 |
| 6 | 19.15 | 36.4 | 6.97 | 67.0 |
Can find out from above-mentioned data, the inventive method (scheme 5 and 6) has greatly improved the harvest yield of microorganism cells, and fat content is also improved.Therefore adopt the inventive method, under identical culture condition, compared with cultivating through synchronized mixes with microorganism single culture method and two kinds of microorganisms, present method adopts membrane separation unit that facultative anaerobic bacteria and microalgae cell are separated to cultivation, adopt tapetum lucidum to form the two light path states of photo-bioreactor system simultaneously, improved phototranstormation efficiency, the reaction solution system adopting realizes back-mixing, and the grease harvest yield obtaining has obtained larger raising.In the unit fermentation system that this meromict is cultivated, grease harvest yield is improved significantly.Can find out by the fermentation level correlation data of facultative anaerobic bacteria simultaneously, mutually isolate cultivate in the situation that at facultative anaerobic bacteria bacterium and microalgae cell, klebsiella carries out anaerobically fermenting and produces the level of 1,3-PD and have a little and improve.This inventive method is described, in realizing biomass collection and then obtaining bio-oil, can also realizes fermentation using bacteria and produce other target products, thereby improve the utilising efficiency of microorganism.
Claims (12)
1. a method for the micro-algae of closed fast culture, comprises following content:
(1) cultivate facultative anaerobic bacteria seed liquor;
(2) cultivate the micro-algae seed liquor of autotrophy;
(3) facultative anaerobic bacteria seed liquor and the micro-algae seed liquor of autotrophy are accessed respectively and in the closed photo bioreactor with built-in membrane separation unit, carry out meromict cultivation, anaerobic bacterium is in the inner fermentation of membrane separation unit, microalgae cell carries out photosynthesis growth in the bioreactor beyond membrane separation unit, in two reactors, substratum composition is consistent with culture condition, supplements the required organic carbon source of facultative anaerobic bacteria growth metabolism in culturing process; Described bioreactor also comprises guide shell, and membrane separation unit is built in guide shell, and outlet is concordant with bioreactor outlet; Described bioreactor has tapetum lucidum, and tapetum lucidum is attached to guide shell outside surface.
2. in accordance with the method for claim 1, it is characterized in that: membrane separation unit adopts ceramic membrane separation device, and film hole diameter is 0.05 μ m～0.5 μ m.
3. in accordance with the method for claim 1, it is characterized in that: tapetum lucidum adopts 3M tapetum lucidum, partly or entirely cover the outside surface of guide shell.
4. in accordance with the method for claim 1, it is characterized in that: facultative anaerobic bacteria comprises genus bacillus, fusiform bacilarmature, bifidus bacillus, Bacterium lacticum or klebsiella, organic carbon source is glucose, glycerine, fructose, starch or cellulosic hydrolysate.
5. in accordance with the method for claim 1, it is characterized in that: facultative anaerobic bacteria seed liquor is cultivated and adopted stirring type bioreactor, the micro-algae seed liquor of autotrophy is cultivated and is adopted airlift photobioreactor.
6. it is characterized in that in accordance with the method for claim 1: the micro-algae of autotrophy comprises chlorella, grape algae, little ring algae or diatom.
7. according to the method described in claim 1,4 or 6, it is characterized in that: in meromict culturing process, the initial access volume ratio of facultative anaerobic bacteria seed liquor and micro-algae seed liquor is 1:1～1:10.
8. in accordance with the method for claim 1, it is characterized in that: the initial medium that meromict is cultivated adopts facultative anaerobic bacteria required substratum, add microalgae cell grow required inorganic salt and trace element simultaneously.
9. according to the method described in claim 1,4 or 8, it is characterized in that: meromict culturing process with batch or continuous mode supplement the required organic carbon source of facultative anaerobic bacteria fermenting process.
10. in accordance with the method for claim 9, it is characterized in that: the initial organic carbon source concentration of facultative anaerobic bacteria substratum is take organism quality densitometer as 1.0%～5.0%, and meromict culturing process maintains organic carbon source concentration take organism quality densitometer as 1.0%～2.0%.
11. in accordance with the method for claim 1, it is characterized in that: the condition that meromict is cultivated is 20 ℃～37 ℃ of temperature, pH value 6～9.
12. in accordance with the method for claim 1, it is characterized in that: it is 5.0～15.0 that facultative anaerobic bacteria seed liquor is cultured to biomass OD value, and it is 2.0～10.0 that micro-algae seed liquor is cultured to biomass OD value.
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