CN104789476B - A kind of cell inoculation method - Google Patents
A kind of cell inoculation method Download PDFInfo
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- CN104789476B CN104789476B CN201510190308.1A CN201510190308A CN104789476B CN 104789476 B CN104789476 B CN 104789476B CN 201510190308 A CN201510190308 A CN 201510190308A CN 104789476 B CN104789476 B CN 104789476B
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- cell
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
Abstract
The invention provides a kind of cell inoculation method, belong to field of microalgae cultivation, can effectively solve the problem that cellular layer bubble problem in microalgae immobilization breeding process, effectively facilitate transmission of the nutrient solution between cell, so as to improve the cultured output of microalgae.Methods described includes：Prepare the solution containing microalgae cell；Preset cultivation carrier；The solution containing microalgae cell is attached on the carrier with the microporous substances with micropore character.The cell seeded process that the present invention is cultivated available for microalgae immobilization.
Description
Technical field
The present invention relates to field of microalgae cultivation, more particularly to a kind of cell inoculation method.
Background technology
Microalgae immobilization cultivation refers to free microalgae cell being fixed on carrier, cell is in the shape of geo-stationary
State, and nutrient solution has a kind of breeding way of relative flow state.However, common problem is at present, microalgae cell
It is attached on carrier and is cultivated, microalgae cell is released in growth course along with due to photosynthesis absorbing carbon dioxide
Oxygen, but due between cell apposition growth can form close contact, the oxygen of generation can not be discharged in environment in time, because
There is situation about bubbling in this cellular layer in immobilization breeding process.Due to the formation of bubble so that nutrient solution is between cell
Transmission it is slower, so as to cause mass transfer effect of the cell in terms of nutrition poor, so cause cell due to lack nutrition and by
Degradation is died, and the cultured output of microalgae declines.
The content of the invention
The invention provides a kind of cell inoculation method, it can effectively solve the problem that cellular layer rises in microalgae immobilization breeding process
Bubble problem, effectively facilitates transmission of the nutrient solution between cell, so as to improve the cultured output of microalgae.
To reach above-mentioned purpose, the present invention is adopted the following technical scheme that：
The present invention provides a kind of cell inoculation method, and methods described includes：
Prepare the solution containing microalgae cell；
Preset cultivation carrier；
The solution containing microalgae cell is attached on the carrier with the microporous substances with micropore character.
Specifically, it is described that the solution containing microalgae cell is attached to the load with the microporous substances with micropore character
Include on body：The solution containing microalgae cell and microporous substances formation mixed solution, the mixed solution is attached to
On the carrier.
Wherein, the consumption of microporous substances is in the mixed solution, the volume of the solution containing microalgae cell with it is described
The ratio between stacking volume of microporous substances is 20:1-1:5.
Preferably, it is described the mixed solution is attached on the carrier after, methods described also includes：
Solution containing microalgae cell is attached to and has been attached with the carrier of the mixed solution.
Specifically, it is described that the solution containing microalgae cell is attached to the load with the microporous substances with micropore character
Include on body：The solution containing microalgae cell and the microporous substances are in turn attached on the carrier.
Preferably, the described solution containing microalgae cell and the microporous substances are in turn attached on the carrier is wrapped
Include：First the microporous substances are attached on the carrier, then the solution containing microalgae cell is attached to and has been attached with
On the carrier of the microporous substances.
Alternatively, the solution containing microalgae cell and the microporous substances are in turn attached on the carrier described
Before, methods described also includes：Stickum is applied on the carrier.
Specifically, the microporous substances include calcium silicates, activated carbon, silica, micropore ceramics, filamentous algae.
Preferably, the ratio between the volume of single microporous substances and the volume of single microalgae cell are 10-100：1.
Preferably, the concentration of the solution containing microalgae cell is 0.1-100g/L
The invention provides a kind of cell inoculation method, by the solution containing microalgae cell and the micropore thing with micropore character
Matter has all been attached on cultivation carrier, so, and the micropore of microporous substances can be provided in gas passage, breeding process for gas circulation
The oxygen that microalgae is produced can be discharged in environment by the gas passage in time, so as to avoid hindering because causing cellular layer to bubble
Transmission of the nutrient solution between cell, and then ensure that cytotrophy is sufficient, improve the cultured output of microalgae.
Brief description of the drawings
Fig. 1 is a kind of flow chart of cell inoculation method provided in an embodiment of the present invention；
Fig. 2 is the distribution schematic diagram that cell provided in an embodiment of the present invention is attached to microporous substances on cultivation carrier.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole embodiments.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
As shown in figure 1, the embodiment of the present invention provides a kind of cell inoculation method, methods described can specifically include：
Prepare the solution containing microalgae cell；
Preset cultivation carrier；
The solution containing microalgae cell is attached on the carrier with the microporous substances with micropore character.
The embodiment of the present invention provides a kind of cell inoculation method, by the solution containing microalgae cell with having the micro- of micropore character
Hole material has all been attached on cultivation carrier, so, and microporous substances can be provided in gas passage, breeding process for gas circulation
The oxygen that microalgae is produced can be discharged in environment by the gas passage in time, so as to avoid hindering because causing cellular layer to bubble
Transmission of the nutrient solution between cell, and then ensure that cytotrophy is sufficient, improve the cultured output of microalgae.
Before inoculation, microalgae cell to be seeded is first typically configured to the certain density solution containing microalgae cell, is
For the sake of simplicity, hereafter the solution containing microalgae cell can be referred to as frustule liquid.The concentration of frustule liquid typically can be
0.1-100g/L, preferably 10-50g/L.Concentration is too big, and cell can be made to contact even closer each other in growth course；
Concentration is too small, then can cause the waste to resources such as the light provided in breeding process, nutrient solutions, and provided by the present invention this is dense
Degree both provides suitable microalgae cell inoculum concentration, and the resources such as light, nutrient solution can be made full use of again.
In seeded process, frustule liquid and the microporous substances with micropore character, which are attached on carrier, can be included such as
Lower two kinds of concrete modes：
1) frustule liquid and microporous substances can be pre-mixed, is then attached on carrier, is specifically as follows simultaneously：First
By above-mentioned frustule liquid and microporous substances formation mixed solution, then the mixed solution is attached on the carrier.
2) frustule liquid and microporous substances are in turn attached on the carrier.
For mode 1), this inoculation method can be seen as to " frustule liquid+microporous substances " inoculation pattern, such as Fig. 2
Shown, principle shows the distribution of microalgae cell and microporous substances on the carrier after inoculation, and microporous substances 1 and frustule 2 are all
It has been uniformly adhered on carrier, what frustule and microporous substances were formed on carrier is the mixed layer of frustule and microporous substances
3, microporous substances are separated by cell to a certain extent, it is to avoid frustule all flocks together, and are formed and are in close contact, in this base
On plinth, the oxygen that the micropore of microporous substances can also produce for microalgae provides gas passage, oxygen is discharged to by micropore in environment,
Eliminate cellular layer bubble problem caused by can not being discharged in time due to gas.
Wherein, microporous substances can be the solid particle with micropore character, for example calcium silicates, activated carbon, silica,
Micropore ceramics etc., its surface and inside have substantial amounts of Minute pores, and these stomatas can provide the passage of gaseous fluid；Micropore
Material can also be filamentous algae, the frond of filamentous algae be generally by cylindric cell be connected it is single-row, branch is not thread
Body, is that will not form dense contact between cell in a class more special microalgae, growth course, can leave more loose
Space, thus the gas produced in breeding process can discharge in space.
The consumption of microporous substances is appropriate, should can provide enough gas passages, also to ensure connecing for microalgae cell
Kind of amount, specifically, by volume, the consumption of microporous substances is in mixed solution, the volume of frustule liquid and microporous substances
The ratio between stacking volume is 20:1-1:5, for example can be 20:1、15:1、10:1、5:1、5:2、1:1、1:3、1:5 etc., that is,
The consumption of microporous substances is determined with the stacking volume of the volume of frustule liquid and microporous substances, is so easy to weigh micropore thing
Matter, in actual applications directly, conveniently.It should be noted that the stacking volume of microporous substances can be understood as being weighed with container
Microporous substances occupied spatial volume in a reservoir, is also the scale value of container during microporous substances.
The volume of single microporous substances also wants suitable size, big to increase the thickness of seed layer, and small can not have
The gas that effect ground produces microalgae is discharged.In a preferred embodiment of the invention, the volume of single microporous substances and single microalgae are thin
The ratio between volume of born of the same parents can be 10-100：1, such as 10:1、15:1、20:1、50:1、70:1.
Further, in another preferred embodiment of the invention, after mixed solution is attached on the carrier, may be used also
It has been attached with so that the solution containing microalgae cell is attached on the carrier of the mixed solution.
This inoculation method can be seen as to " (microporous substances+cell)-cell " inoculation pattern.In this mode, first
The frustule liquid and microporous substances formation mixed solution of certain volume are taken, mixed solution is then formed one layer on carrier first
The combined inoculation layer of microporous substances and frustule liquid, and then take the frustule liquid of certain volume to be inoculated in the combined inoculation layer
On, that is, the cellular layer after being inoculated with can be divided into upper and lower two aspects, and aspect below is to be mixed using microporous substances with frustule
Close, form the mixed uniformly mixed layer of cell-microporous substances, this layer is with cellular layer by this confluent monolayer cells and above due to photosynthetic
Oxygen produced by effect is conducted by the micro channel of this layer, can prevent the blister formation of cell surface layer.Upper strata it is thin
Born of the same parents' layer avoids microporous substances from influenceing the reception total amount of sunshine exposed to top layer, is further promoting each cell confluent monolayer cells just
It is frequently grown.
For mode 2), frustule liquid and microporous substances, which are in turn attached on carrier, can specifically include：First by micropore
Material is attached on carrier, and then frustule liquid is attached on the carrier for being attached with microporous substances.Can be by this inoculation
Method, which is seen as, is initially formed one layer of microporous substances layer on " microporous substances-cell " inoculation pattern, carrier, then in the base of this layer
One layer of cells layer is formed on plinth, microporous substances layer is by cellular layer because the oxygen produced by photosynthesis is led to by the micropore of this layer
Road is conducted, and can prevent the blister formation of cell surface layer.
It is understood that mode 1) in can equally be well applied to mode 2 for the description and restriction of microporous substances) in.
In order to improve adhesive strength of the microporous substances on carrier, can before attaching it on carrier, especially when
When microporous substances selection is solid particle, first stickum is applied on carrier, i.e. " stickum-microporous substances-cell "
Inoculation pattern；First stickum can certainly be mixed with microporous substances, be then attached to together on carrier, form goo
The mixed layer of matter and microporous substances, i.e. " (stickum+microporous substances)-cell " are inoculated with pattern.
Specifically, stickum can be any cell growth it is non-toxic and do not reacted with carrier, with viscous
The material of property, such as some binding agents or microalgae cell.Here, microalgae cell is due to itself having certain viscosity, thus
Stickum is may also serve as, now, inoculation method of the present invention is：Minimal amount of frustule liquid is first attached to carrier
On, then adhere to microporous substances, frustule liquid successively again, this inoculation method can be regarded as " cell-microporous substances-cell "
Inoculation pattern, the main purpose for the frustule liquid being attached to first on carrier is to function similarly to the effect of stickum, is improved
The adhesive strength of microporous substances and carrier.
It is understood that the vaccination ways such as conventional spraying, suction filtration, coating, immersion are suitable for side of the present invention
Above-mentioned frustule liquid, such as by taking suction filtration as an example, can be added in Suction filtration device by method, and the suction filtration that then pressurizes is attached by frustule liquid
On carrier, complete inoculation.The present invention is not further qualified herein.
Cell inoculation method provided in an embodiment of the present invention is further described below by specific embodiment.
Microalgae cell to be seeded is by taking chlorella as an example, and it is 1g/L containing microalgae cell that concentration is prepared into before inoculation
Solution (frustule liquid) 50ml.
Comparative example：
50ml frustule liquid is inoculated on diameter 47mm acetate fiber filter membrane (105 DEG C using the mode of negative pressure leaching
Dry to constant weight, record quality), filter membrane is fixed on liner plate and cultivated again, artificial light source 150umol/m2/s, process control algae
Cell liquid pH value is in the range of 7-9, and temperature control is at 23-27 DEG C, by the culture of 5 days, by frustule and acetate fiber filter membrane
Together place 105 DEG C of baking ovens to dry to constant weight, calculate the increased weight of frustule using differential technique, the light-receiving area yield of microalgae is
12.0g/m2/d。
Embodiment 1：
Microporous substances use silica dioxide granule.
By the volume that the volume of frustule liquid is 50ml, frustule liquid：The stacking volume of silica is 5:1 measures dioxy
Silicon carbide particle.
Silica dioxide granule is attached on diameter 47mm acetate fiber filter membrane first with the mode of negative pressure leaching, then
Reuse the mode of negative pressure leaching and frustule liquid is inoculated on the acetate fiber filter membrane to (105 DEG C dry to constant weight, and record matter
Amount), filter membrane is fixed on liner plate and cultivated again, artificial light source 150umol/m2/s, and process control frustule liquid pH value is in 7-9
In the range of, frustule and acetate fiber filter membrane, by the culture of 5 days, are together placed 105 DEG C of bakings by temperature control at 23-27 DEG C
Case is dried to constant weight, and the increased weight of frustule is calculated using differential technique, and the light-receiving area yield of microalgae is 13.8g/m2/d, and right
Ratio is compared to improve 15%.
Embodiment 2：
Microporous substances use activated carbon granule.
By the volume that the volume of frustule liquid is 50ml, frustule liquid：The stacking volume of activated carbon is 8:3 take activated carbon
Grain.
Mixed solution is formed after activated carbon granule and frustule liquid are mixed, then this is mixed using the mode of negative pressure leaching
Solution inoculum is closed to diameter 47mm acetate fiber filter membrane (105 DEG C dry to constant weight, and record quality), filter membrane is fixed on liner plate again
It is upper to be cultivated, artificial light source 150umol/m2/s, process control frustule liquid pH value is in the range of 7-9, and temperature control is in 23-
27 DEG C, by the culture of 5 days, frustule and acetate fiber filter membrane are together placed into 105 DEG C of baking ovens and dried to constant weight, differential technique is utilized
The increased weight of frustule is calculated, the light-receiving area yield of microalgae is 14.3g/m2/d, and 19.2% is improved compared with comparative example.
Embodiment 3：
Microporous substances use silica dioxide granule.
By the volume that the volume of frustule liquid is 35ml, frustule liquid：The stacking volume of silica is 10:1 measures two
Silicon oxide particle.
Mixed solution is formed after silica dioxide granule and frustule liquid are mixed, is inoculated with using the mode of negative pressure leaching
On to diameter 47mm acetate fiber filter membrane (105 DEG C dry to constant weight, and record quality), the frustule layer containing particle is formed, then
Using suction filtration mode by the remaining uniform suction filtration of 15ml frustules liquid to its surface on the basis of this layer, then to be fixed on liner plate enterprising
Row culture, artificial light source 150umol/m2/s, process control frustule liquid pH value is in the range of 7-9, and temperature control is in 23-27
DEG C, by the culture of 5 days, frustule and acetate fiber filter membrane are together placed into 105 DEG C of baking ovens and dried to constant weight, differential technique meter is utilized
The increased weight of frustule is calculated, the light-receiving area yield of microalgae is 15.5g/m2/d, and 29% is improved compared with comparative example.
Embodiment 4：
Microporous substances use filamentous algae.
By the volume that the volume of frustule liquid is 35ml, frustule liquid：The stacking volume of filamentous algae is 1:1 measure it is thread
Algae.
Mixed solution is formed after filamentous algae and frustule liquid are mixed, diameter is inoculated into using the mode of negative pressure leaching
On 47mm acetate fiber filter membrane (105 DEG C dry to constant weight, and record quality), the frustule layer containing particle is formed, then in this layer
On the basis of using suction filtration mode by the remaining uniform suction filtration of 15ml frustules liquid to its surface, then be fixed on liner plate and trained
Support, artificial light source 150umol/m2/s, process control frustule liquid pH value is in the range of 7-9, and temperature control is at 23-27 DEG C, warp
The culture for spending 5 days, the frustule on acetate fiber filter membrane is washed till in beaker using culture medium, in order using 200,300 mesh
Bolting silk removes the thread frustule in frustule liquid, forms chlorella algae cell liquid to the carry out filter 23 of frustule liquid.Again
Chlorella algae cell liquid is subjected to suction filtration using the acetate fiber filter membrane (with before drying to constant weight, recording quality) used in culture, will
Frustule and acetate fiber filter membrane are together placed 105 DEG C of baking ovens and dried to constant weight, and the increased weight of frustule is calculated using differential technique,
Light-receiving area yield is 15.0g/m2/d, and 25% is improved compared with comparative example.
From the above results, compared with comparative example, embodiment 1-4 uses cell inoculation method of the present invention, will be micro-
Hole material and chlorella algae cell liquid is all attached on carrier, effectively excludes the gas that breeding process is produced, it is to avoid bubble shows
As, it is ensured that the normal growth of cell；Compared with embodiment 1,2, embodiment 3,4 is employed " microporous substances+cell-cell "
Inoculation pattern, upper cell layer covers microporous substances, improves microalgae cell to the reception of illumination to promote microalgae cell
Growth, improve cultured output.
Obviously, above-described embodiment is only intended to clearly illustrate example, and the not restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or
Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or
Change the protection domain still in the invention.
Claims (9)
1. a kind of cell inoculation method, it is characterised in that methods described includes：
Prepare the solution containing microalgae cell；
Preset cultivation carrier；
The solution containing microalgae cell is attached on the carrier with the microporous substances with micropore character, the micropore thing
Matter includes calcium silicates, activated carbon, silica, micropore ceramics, filamentous algae.
2. cell inoculation method according to claim 1, it is characterised in that
Described be attached to the solution containing microalgae cell on the carrier with the microporous substances with micropore character includes：
The solution containing microalgae cell and microporous substances formation mixed solution, the load is attached to by the mixed solution
On body.
3. cell inoculation method according to claim 2, it is characterised in that the consumption of the microporous substances is, described to contain
The ratio between stacking volume of the volume of the solution of microalgae cell and the microporous substances is 20:1-1:5.
4. cell inoculation method according to claim 2, it is characterised in that the mixed solution is attached into institute described
After stating on carrier, methods described also includes：
Solution containing microalgae cell is attached to and has been attached with the carrier of the mixed solution.
5. cell inoculation method according to claim 1, it is characterised in that
Described be attached to the solution containing microalgae cell on the carrier with the microporous substances with micropore character includes：
The solution containing microalgae cell and the microporous substances are in turn attached on the carrier.
6. cell inoculation method according to claim 5, it is characterised in that it is described by the solution containing microalgae cell with
The microporous substances, which are in turn attached on the carrier, to be included：
First the microporous substances are attached on the carrier, then the solution containing microalgae cell is attached to and has been attached with
On the carrier of the microporous substances.
7. cell inoculation method according to claim 5, it is characterised in that described by the solution containing microalgae cell
Before being in turn attached to the microporous substances on the carrier, methods described also includes：
Stickum is applied on the carrier.
8. the cell inoculation method according to claim any one of 1-7, it is characterised in that the body of the single microporous substances
The ratio between product and the volume of single microalgae cell are 10-100：1.
9. the cell inoculation method according to claim any one of 1-7, it is characterised in that the solution containing microalgae cell
Concentration be 0.1-100g/L.
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