CN106635782A - Oleaginous microalgae integrated culture reactor and culture method - Google Patents
Oleaginous microalgae integrated culture reactor and culture method Download PDFInfo
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Abstract
The invention relates to an oleaginous microalgae integrated culture reactor, which comprises a first incubator, a second incubator, a first stirring motor, a second stirring motor, a circulating water pump, a microalgae harvesting device, a first stirring paddle and a second stirring paddle, wherein the first stirring paddle is arranged in the first incubator and is driven to rotate by virtue of the first stirring motor; the second stirring paddle is arranged in the second incubator and is driven to rotate by virtue of the second stirring motor; a seed inlet is formed at the upper side of the second incubator and a medium inlet is formed in the upper side of the first incubator; the first incubator and the second incubator communicate; and the microalgae harvesting device, which is arranged under the first incubator, is used for receiving microalgae generated in the first incubator. The reactor provided by the invention has the various characteristics of being small in volume, strong in flexibility, easy for transformation and the like. The invention also relates to a culture method; and with the application of the culture method, the microalgae, which is high in oil yield and high in growth speed, can be cultivated.
Description
Technical field
The present invention relates to regenerated biological energy technical field, more particularly to a kind of oil-producing microalgae integral type culture reaction
Device, further relates to a kind of cultural method.
Background technology
Biodiesel refers to the long-chain fat dialkylaminobenzoic acid list that animal and plant or microbial grease are obtained Jing after esterification
Ester.Because with cleaning, renewable, environment amenable characteristic, biodiesel has been increasingly subject to America and Europe and some energy consumptions of Asia
The attention of big country simultaneously obtains significant progress.
With the improvement of people's living standards, the consumption to the energy increasingly increases, in the year two thousand fifty, the energy-output ratio in the whole world
It is up to twice now.Fossil energy consumption totally in the case of, people have invested bioenergy sight one after another, biological
The energy is the key for solving energy problem.It is the representative of clean energy resource, not only there is fabulous recyclability, and environment friend
It is good, green cleaning, the exploitation of bioenergy, using effectively providing power for social development, and drive other related products
Industry, the such as manufacturing industry of transport, generating and correlation, the microalgae diesel oil in bioenergy has also therefore obtained again people's
Pay attention to.
From the point of view of current technical merit both domestic and external, the industrialization that microalgae is refined oil is realized, also need to solve following technology
A difficult problem：
1. seed selection oil productivity is high, the microalgae of fast growth；
2. the inexpensive process units of microalgae is cultivated in exploitation industrialization.
The content of the invention
The invention aims to solve above-mentioned technical problem, and then provide a kind of oil-producing microalgae integral type culture reaction
Device, small volume, it is adaptable to the oil-producing microalgae of the culture of scale ground and collection high-quality, can provide for scale of mass production from now on and most may be used
The data leaned on, possesses flexibility strong, it is easy to the various characteristic such as transformation.
Technical scheme：
A kind of oil-producing microalgae integral type cultivation reactor, including：First incubator, the second incubator, the first stirring motor,
Second stirring motor, water circulating pump, microalgae recovery device, the first stirrer paddle, the second stirrer paddle；First stirrer paddle sets
It is placed in first incubator, and drives rotation, second stirrer paddle to be arranged at described by first stirring motor
In second incubator, and rotation is driven by second stirring motor；Seed charging is provided with above second incubator
Mouthful, it is provided with medium feed mouth above first incubator；First incubator is connected with the second incubator, described
Microalgae recovery device is arranged on the first incubator lower section, for receiving the microalgae produced in first incubator.
Further, the first heating water bath set is provided with the outside of first incubator, outside second incubator
Side is provided with the second heating water bath set, the first heating water bath set, the second heating water bath set by first circulation waterpipe and
Second circulation waterpipe is connected, and between the first circulation waterpipe, second circulation waterpipe the water circulating pump is provided with.
Further, dissolved oxygen meter and PH detection meters are respectively arranged with first incubator, the second incubator.
Further, it is provided with electrically operated valve on the pipeline that first incubator and the second incubator are connected.
Further, the first thief hatch, microalgae outlet, second incubator are provided with below first incubator
Lower section be provided with the second thief hatch.
The invention has the advantages that：Oil-producing microalgae integral type cultivation reactor disclosed by the invention, is first shut off
The each electrically operated valve of reactor, culture medium and seed are squeezed in the second cultivation reactor, by adjusting the second cultivation reactor
Temperature, by thermometer the second cultivation reactor temperature is determined, and is made the second cultivation reactor reach High Density Cultivation and is most preferably cultivated
Temperature.Air is passed through in by giving the second cultivation reactor, and monitors dissolved oxygen amount in culture medium, made by the second stirrer paddle
The uniform abundant dissolved oxygen of microalgae.After certain hour is cultivated, open electrically operated valve and sampled, adopted sample and closed motor-driven valve
Door, determination sample absorbance, it is determined that reaching enough microalgae density.Electrically operated valve is opened, the microalgae Jing after High Density Cultivation is made
Continuation culture is carried out into the first cultivation reactor.After certain hour is cultivated, open electrically operated valve and sampled from thief hatch,
Sample is adopted and has closed electrically operated valve, determination sample absorbance has determined that microalgae grease enrichment reaches to a certain degree, opened motor-driven valve
Door, the microalgae after grease is enriched with is entered in microalgae recovery device.
The present invention also provides a kind of cultural method, comprises the steps：
The first step, preparing experiment equipment and material；
Second step, the enrichment culture of microalgae cultivates the algae source required for follow-up oil accumulation experiment；
3rd step, oil and fat accumulation；
4th step, micro algae biomass and grease are determined.
Further, the experimental facilities include illumination box, ultra-low temp, freeze drier, centrifuge, point
Light photometer, superclean bench, steam sterilization pan, electric drying oven with forced convection, temperature automatically controlled shaking table.
Further, including illumination box culture and heterotrophism no light culture, the light dark period in illumination box
For 15h:9h.
Further, the culture medium that the oil and fat accumulation is adopted is for BG culture mediums.
Using the cultural method of the present invention, flexibly, wide accommodation can flexibly convert the nutrition supplying in two sections to technique
Give and ambient As, these flexible modes are adapted to different types of microalgae and other microorganisms, bioenergy
Production and useless source resource combine, while biodiesel is generated to water process in many not degradable small molecules have
Machine thing product is degraded, and for water process facility is provided.
Beneficial effects of the present invention will be become readily apparent from by the description of following specific embodiment.
Description of the drawings
Fig. 1 is the structural representation of the embodiment of the present invention；
Fig. 2 is the growth curve chart of the embodiment of the present invention；
The incubators of 1- first in figure；The incubators of 2- second；The stirring motors of 3- first；The stirring motors of 4- second；5- recirculated waters
Pump；6- microalgae recovery devices；The stirrer paddles of 7- first；The stirrer paddles of 8- second；9- the first heating water bath sets；The water-baths of 10- second add
Hot jacket；11- first circulation waterpipes；12- second circulation waterpipes；13- dissolved oxygen meters；14-PH detection meters；15- electrically operated valves；
The thief hatch of 16- first；17- microalgaes are exported；The thief hatch of 18- second.
Specific embodiment
Oil-producing microalgae integral type cultivation reactor disclosed in the present embodiment, including
First incubator 1, the second incubator 2, the first stirring motor 3, the second stirring motor 4, water circulating pump 5, microalgae are adopted
Receive device 6, the first stirrer paddle 7, the second stirrer paddle 8；First stirrer paddle 7 is arranged in the first incubator 1, and is stirred by first
Mix motor 3 and drive rotation, the second stirrer paddle 8 is arranged in the second incubator 2, and drives rotation by the second stirring motor 4；The
The top of two incubators 2 is provided with seed charging aperture, and the top of the first incubator 1 is provided with medium feed mouth；First culture
Device 1 is connected with the second incubator 2, and microalgae recovery device 6 is arranged on the lower section of the first incubator 1, is produced in the first incubator 1 for receiving
Raw microalgae.
Preferably, the outside of the first incubator 1 is provided with the first heating water bath set 9, the outside of the second incubator 2 is arranged
There is the second heating water bath set 10, the first heating water bath covers the 9, second heating water bath set 10 and passes through first circulation waterpipe 11 and second
Circulating water pipeline 12 is connected, and water circulating pump 5 is provided between first circulation waterpipe 11, second circulation waterpipe 12.
Further, dissolved oxygen meter 13 and PH detections meter 14 are respectively arranged with the first incubator 1, the second incubator 2.
Specifically, it is provided with electrically operated valve 15 on the pipeline that the first incubator 1 is connected with the second incubator 2.
More specifically, the lower section of the first incubator 1 is provided with the first thief hatch 16, microalgae outlet 17, the second incubator 2
Lower section be provided with the second thief hatch 18.
The operation principle of the embodiment of the present invention is as follows：
All electrically operated valves 15 are first shut off, will be trained by the medium feed mouth of the second incubator 2 with medium feed pump
Foster base is squeezed in the second incubator 2, then oil-producing microalgae seed is squeezed into into the second training by seed charging aperture with seed feed pump
In foster device 2.Circulating hot water covers 10 imports and enters by the second heating water bath, and by the second heating water bath set 10 water outlet is exported, from
And the temperature of the second incubator of increasing high density 2, the temperature of the second incubator 2 is determined by thermometer, reach the second incubator 2
High Density Cultivation optimum culturing temperature.Given in the second incubator 2 by aeration peristaltic pump and aeration head and be passed through air, and passed through
Dissolved oxygen amount in the monitoring culture medium of dissolved oxygen meter 13, by the second stirrer paddle 8 the uniform abundant dissolved oxygen of microalgae is made, and is counted by pH detections
14 monitoring culture medium acid-base values.After certain hour is cultivated, open electrically operated valve 15 and sampled from thief hatch, adopt sample pass
Electrically operated valve 15, determination sample absorbance are closed, it is determined that reaching enough microalgae density.Electrically operated valve 15 is opened, Jing high density is made
Microalgae after culture carries out continuation culture into the first incubator 1.After certain hour is cultivated, electrically operated valve 15 is opened from sampling
Mouth is sampled, and has been adopted sample and has been closed electrically operated valve 15, and determination sample absorbance determines that microalgae grease enrichment reaches certain journey
Degree, opens electrically operated valve 15, and the microalgae after grease is enriched with is entered in microalgae recovery device 6.
The cultural method of the present invention comprises the steps：
The first step, preparing experiment equipment and material；
Second step, the enrichment culture of microalgae cultivates the algae source required for follow-up oil accumulation experiment；
3rd step, oil and fat accumulation；
4th step, micro algae biomass and grease are determined.
Specifically, the experimental facilities of the present embodiment includes 150C type digital display illumination boxs；- 50 DEG C of DW-50W255 types surpass
Cord blood case；LGJ-10D type freeze driers；TDL-40B type centrifuges；T6 type ultraviolet/visible light spectrophotometers；SW-
CJ-1G type superclean benches；BXM-30R type vertical pressure steam sterilization pans；101-2AB type electric drying oven with forced convections；ATL-
The temperature automatically controlled shaking table of 032LR types；PHS-2C types PH are counted.
Experiment material includes sodium nitrate；Dipotassium hydrogen phosphate；Magnesium sulfate；Calcium chloride；Citric acid；Ferric citrate；Boric acid；Chlorine
Change manganese；Zinc sulfate；Sodium molybdate；Copper sulphate；Cobalt nitrate；Glucose；Peptone；Soluble starch；Maltose.
Second step, the enrichment culture of microalgae
The enrichment work of microalgae is the algae source required in order to cultivate follow-up oil accumulation experiment.It is divided into according to experiment demand
Two kinds of forms of illumination box culture and the culture of heterotrophism no light.Light dark period in illumination box is 15h:9h, daily
Sooner or later flask need to be shaken up.Light culture case does not then need any illumination.In order to prevent all kinds of germ contaminations, culture from using instrument device
Ware will enter high-temperature sterilization.Periodically with micro- sem observation algae kind situation, upgrowth situation is determined and with the presence or absence of pollution.Algae kind
Renewed vaccination be, in order to keep the freshness of algae kind, for oil accumulation test fixed young algae source is provided.Lead to during inoculation
Cross measurement absorbance and blood counting chamber determines inoculum density, generally use absorbance in the range of 1-2, the algae source of late log phase
Inoculation.Inoculation volume is the 15% of target nutrient solution volume.
3rd step, oil and fat accumulation
In oil accumulation experiment, different condition of culture has different operating parameters.Culture medium used is still in experiment
For BG culture mediums, the configuration standard of culture medium follows strictly the formula such as table 1 that the aquatic institute algae place in Chinese Academy of Sciences Wuhan is provided
It is shown.
Table 1BG culture medium prescriptions
Meanwhile, adjust pH value by way of addition 1M HCl or 1M NaOH solutions so that the nutrient solution pH in test
Concentration is between 5 to 10.Temperature range is then set between 10 DEG C to 45 DEG C.The setting range of intensity of illumination from 0lux to
5000lux.Microalgae in experiment sample detecting after 72 hours.
Used carbon source has glucose and the useless carbon source of simulation in oil accumulation experiment.
4th step, micro algae biomass and grease assay method
The biomass estimation of microalgae and the key that the measure of oil content is test.To the grasp of each details and ripe in test
Experienced operation may insure the accurate of data.
(1) harvesting of micro algae biomass
Centrifugal process microalgae is employed in test, will grow into research needs the algae solution in stage to be transferred in centrifuge tube,
Centrifuge is put into, 5min is centrifuged under the rotating speed of 4000rpm, abandon supernatant, lower floor's concentration algae mud is put into into refrigerator freezing
Proceed to after 30min in freeze drier and be dried 24h.It is stored in after taking-up standby in 4 DEG C of refrigerator.
(2) assay method of micro algae biomass
The measure of biomass mainly determines biomass by using spectrophotometric nephelometry, and cooperation is weighed and inhaled
Shading value finds optimal increment matched curve.
A. dry cell weight method
Take 4mL algae solutions to be placed in the 5mL centrifuge tubes weighed, 5min is centrifuged under 5000-7000rpm rotating speeds, remove supernatant
Liquid.And be put in drying baker and dried to constant weight at 65 DEG C.Three Duplicate Samples are measured simultaneously, are averaged.
B. nephelometry
Absorbance (OD540) of the microalgae solution in the case where wavelength is 540nm is measured using UV, visible light spectrophotometer.Spend
Ionized water measures the absorbance in 540nm as reference.The growth curve of microalgae is drawn by the absorbance for measuring daily.
And the growing state to microalgae makes analysis.
C. the determination of growth curve
Growth curve is the single batch micro algae growth shape of description obtained by microalgae solution absorbance and dry weight combine
The line of condition.By growth curve, we can be directly related in algae solution containing the dry weight of algae amount from the size of absorbance, be easy to examination
The progress tested.
The measuring method of dry cell weight is the most direct method that biomass is calculated, during need to be related to centrifugation, washing
With the several steps of drying.Because there is preferable positive correlation between cell concentration and dry weight, we can set up
A set of calibration curve, by the measurement to absorbance the size of biomass in sample is obtained indirectly.This computational methods are also exactly
Usual means in most unicellular microalgae research.
In BG culture mediums, pH value nature, intensity of illumination 2000lux, inoculum concentration 1:5th, train under conditions of 25 DEG C of cultivation temperature
Support, obtain growth curve as shown in Figure 2.Unicellular alga needs certain temperature range during photosynthesis.Temperature
The change of degree for example heats up and can have certain promotion or inhibitory action when either cooling to photosynthesis.There is research to point out
Photosynthesis and respiratory intensity all can be affected by temperature, and this Two Mainstays is had with the growth metabolism of microalgae
Close relation.And microalgae belongs to ecological heat type microorganism, they must obtain thermal source from environment, and temperature is significantly
Change can cause growth rate, metabolic efficiency and intracellular biochemical reaction speed great change.Therefore temperature during culture is
Affect one of key factor of unicellular micro algae growth.The temperature that microalgae is adapted to is often between 15 DEG C -40 DEG C.And not
The scope that congener microalgae is adapted to is also different.The optimal temperature upper limit of single celled green alga microalgae is at 36 DEG C or so
And most suitable temperature should be between 25 DEG C～32 DEG C.
This group experiment purpose be it is determined that unglazed cultivation conditions under, in the stable condition of other factors, search out
The cultivation temperature of most energy-conservation and efficiency highest.Temperature change in experiment be divided into 20 DEG C, 25 DEG C, 30 DEG C, 27 DEG C, 32 DEG C it is several
Individual standard.Illumination is 0lux；Initial pH is 6.8；Speed is shaken for 160rpm；Throughput is 50mL/d；Culture medium is BG culture mediums；Portugal
Grape sugar carbon source concentration is 1g/L, and under the conditions of initial absorbance identical 72h is cultivated.
Experimental result is as shown in table 2 below
The growing state of microalgae under the different temperatures of table 2
By shown in upper table, it can be seen that when cultivation temperature is at 30 DEG C, absorbance (540nm) is maximum, that is, frond
Concentration is maximum, so microalgae optimum growth temperature is 30 DEG C.
Initial pH value in culture medium can affect one of many biochemical reactions such as micro algae growth and metabolism it is important because
Element, pH can interfere the usability of carbon dioxide in photosynthesis, while microalgae will be affected to utilize organic carbon in respiration
Source efficiency.And because pH can affect the permeance property of cell membrane, so as to cell can be caused to the nutrient ions in nutrient solution
Absorption and using being affected by certain.Recycling simultaneously to metabolic intermediate product and algae is endotoxic also has
It is certain to affect.Identical with temperature and illumination, the pH for being best suitable for algal grown has nothing in common with each other in different algae inter-species.There is research to point out,
The pH of micro algae growth is best suitable between 5 to 8, pH7 is most suitable.Also research is pointed out, because microalgae meeting in growth course
Because the absorption of carbon dioxide raises pH, optimal initial pH should be 6 or so.So our research will be in pH5
The growing state of C.protothecoides 3 is investigated in 10 this scope.
So the purpose of this experiment searches out one is best suitable for the growths of C.protothecoides 3 and oil accumulation
PH value.PH changes in experiment are divided into 5.5,6.5,7.5,8.5,9.5.Illumination is 0lux；Temperature is 25 DEG C；Shaking speed is
160rpm；Throughput is 50mL/d；Culture medium is BG culture mediums；Glucose carbon source concentration be 1g/L, inoculum concentration 1:5, it is initial to inhale
72h is cultivated under the conditions of luminosity identical.Experimental result is as shown in table 3 below
Growing state under the microalgae different pH condition of table 3
By shown in upper table 4, it can be seen that when initial pH value is 5.5, absorbance (540nm) is maximum, that is, frond
Concentration is maximum, so the initial pH value of microalgae the most suitable growth is 5.5.
Initial inoculation affects the reproduction speed of algae kind, in BG culture mediums, initial pH natures, intensity of illumination 0lux, culture temperature
Incubation time 72h under the conditions of 25 DEG C of degree, initial absorbance identical.
Experimental result is as shown in table 4 below
Growing state of the microalgae of table 4 in the case of different vaccination amount
By shown in upper table, it can be seen that when inoculum concentration is 1:When 5, absorbance (540nm) is maximum, that is, frond concentration
Maximum, so the inoculum concentration of microalgae the most suitable growth is 1:5.
The concentration of organic carbon source has important impact to algae kind growth rate, in BG culture mediums, pH value nature, intensity of illumination
0lux, inoculum concentration 1:5th, incubation time 72h under the conditions of 25 DEG C of cultivation temperature, initial absorbance identical.
Experimental result is as shown in table 5 below
Growing state of the microalgae of table 5 under the conditions of different culture media
By shown in upper table, it can be seen that when culture medium is BG+2g/LC6H12O6, absorbance (540nm) is maximum,
It is exactly that frond concentration is maximum, so the culture medium of microalgae the most suitable growth is BG+2g/LC6H12O6.Understand to inhale by growth curve
Luminosity is 1.637, and its frond dry weight has reached more than 0.5.
In sum, microalgae optimum growing condition is inoculum concentration 1:5th, 30 DEG C and BG+2g/ of initial pH value 5.5, cultivation temperature
LC6H12O6 culture mediums.
Using the cultural method of the present invention, flexibly, wide accommodation can flexibly convert the nutrition supplying in two sections to technique
Give and ambient As, these flexible modes are adapted to different types of microalgae and other microorganisms, bioenergy
Production and useless source resource combine, while biodiesel is generated to water process in many not degradable small molecules have
Machine thing product is degraded, and for water process facility is provided.
Above example is the exemplary illustration to this patent, does not limit its protection domain, people in the art
Member can also locally be changed to it, as long as no the Spirit Essence beyond this patent, all in the protection domain of this patent.
Claims (9)
1. a kind of oil-producing microalgae integral type cultivation reactor, it is characterised in that include：First incubator (1), the second incubator
(2), the first stirring motor (3), the second stirring motor (4), water circulating pump (5), microalgae recovery device (6), the first stirrer paddle
(7), the second stirrer paddle (8)；First stirrer paddle (7) is arranged in first incubator (1), and by described first
Stirring motor (3) drives rotation, second stirrer paddle (8) to be arranged in second incubator (2), and by described second
Stirring motor (4) drives rotation；Seed charging aperture, first incubator (1) are provided with above second incubator (2)
Top be provided with medium feed mouth；First incubator (1) connects with the second incubator (2), the microalgae recovery device
(6) the first incubator (1) lower section is arranged on, for receiving the microalgae produced in first incubator (1).
2. oil-producing microalgae integral type cultivation reactor according to claim 1, it is characterised in that first incubator
(1) the first heating water bath set (9) is provided with the outside of, on the outside of second incubator (2) the second heating water bath set is provided with
(10), the first heating water bath set (9), the second heating water bath set (10) are by first circulation waterpipe (11) and second circulation
Waterpipe (12) is connected, and the first circulation waterpipe (11), second circulation waterpipe are provided with the recirculated water between (12)
Pump (5).
3. oil-producing microalgae integral type cultivation reactor according to claim 2, it is characterised in that first incubator
(1) dissolved oxygen meter (13) and PH detections meter (14), are respectively arranged with the second incubator (2).
4. oil-producing microalgae integral type cultivation reactor according to claim 3, it is characterised in that in first incubator
(1) it is provided with electrically operated valve (15) on the pipeline connected with the second incubator (2).
5. oil-producing microalgae integral type cultivation reactor according to claim 4 and cultural method, it is characterised in that described
The first thief hatch (16), microalgae outlet (17) are provided with below one incubator (1), are set below second incubator (2)
It is equipped with the second thief hatch (18).
6. a kind of cultural method based on bioreactor culture microalgae described in claim 1-5 any one, it is characterised in that include
Following steps：
The first step, preparing experiment equipment and material；
Second step, the enrichment culture of microalgae cultivates the algae source required for follow-up oil accumulation experiment；
3rd step, oil and fat accumulation；
4th step, micro algae biomass and grease are determined.
7. cultural method according to claim 6, it is characterised in that the experimental facilities includes illumination box, ultralow
Warm storage box, freeze drier, centrifuge, spectrophotometer, superclean bench, steam sterilization pan, electric drying oven with forced convection, from
Dynamic temperature control shaking table.
8. cultural method according to claim 7, it is characterised in that including illumination box culture and the training of heterotrophism no light
Support, the light dark period in illumination box is 15h:9h.
9. cultural method according to claim 8, it is characterised in that the culture medium that the oil and fat accumulation is adopted is BG cultures
Base.
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| CN110396470A (en) * | 2019-08-27 | 2019-11-01 | 济宁学院 | Microalgae fixes flue gas CO2The device and method of conversion of biomass |
| CN113355216A (en) * | 2021-05-25 | 2021-09-07 | 江苏海洋大学 | Controllable light-controlled temperature LED closed alga culture device and culture method thereof |
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| CN103627620A (en) * | 2012-08-28 | 2014-03-12 | 西门子公司 | Method and apparatus for producing lipids through microalgae culture |
| CN103981089A (en) * | 2014-06-09 | 2014-08-13 | 山东省科学院中日友好生物技术研究中心 | Solar microalgae biological reaction system |
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| CN103589644A (en) * | 2013-11-28 | 2014-02-19 | 黑龙江省能源环境研究院 | Method for culturing oil-producing microalgae by using brewery sewage |
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| CN113355216B (en) * | 2021-05-25 | 2023-06-20 | 江苏海洋大学 | Light-controlled temperature-controlled LED closed algae culture device and culture method thereof |
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