CN106497792A - A kind of cultural method of photo bio cell - Google Patents
A kind of cultural method of photo bio cell Download PDFInfo
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- CN106497792A CN106497792A CN201610996229.4A CN201610996229A CN106497792A CN 106497792 A CN106497792 A CN 106497792A CN 201610996229 A CN201610996229 A CN 201610996229A CN 106497792 A CN106497792 A CN 106497792A
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- 238000000034 method Methods 0.000 title claims abstract description 55
- 239000012531 culture fluid Substances 0.000 claims abstract description 114
- 239000007788 liquid Substances 0.000 claims abstract description 107
- 239000004744 fabric Substances 0.000 claims abstract description 96
- 230000001413 cellular effect Effects 0.000 claims abstract description 65
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 230000008569 process Effects 0.000 claims description 13
- 238000009826 distribution Methods 0.000 claims description 8
- 239000003086 colorant Substances 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 5
- 230000033228 biological regulation Effects 0.000 claims description 3
- 239000013530 defoamer Substances 0.000 claims description 3
- 230000008030 elimination Effects 0.000 claims description 3
- 238000003379 elimination reaction Methods 0.000 claims description 3
- 230000003247 decreasing effect Effects 0.000 claims description 2
- 238000007689 inspection Methods 0.000 claims description 2
- 230000001850 reproductive effect Effects 0.000 abstract description 2
- 239000010410 layer Substances 0.000 description 227
- 210000004027 cell Anatomy 0.000 description 74
- 229920005479 Lucite® Polymers 0.000 description 6
- 239000004926 polymethyl methacrylate Substances 0.000 description 6
- 241000195493 Cryptophyta Species 0.000 description 5
- 238000009395 breeding Methods 0.000 description 5
- 230000001488 breeding effect Effects 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 235000016425 Arthrospira platensis Nutrition 0.000 description 4
- 240000002900 Arthrospira platensis Species 0.000 description 4
- 229940082787 spirulina Drugs 0.000 description 4
- 238000010030 laminating Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000195663 Scenedesmus Species 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 229920002301 cellulose acetate Polymers 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 235000002470 Asclepias syriaca Nutrition 0.000 description 1
- 244000000594 Asclepias syriaca Species 0.000 description 1
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 1
- 241000180279 Chlorococcum Species 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 241000168517 Haematococcus lacustris Species 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000011229 interlayer Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010408 sweeping Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
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- 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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Abstract
A kind of cultural method of photo bio cell is embodiments provided, is related to the culture field of photo bio cell, can convenient, effectively be eliminated the bubble in photo bio cell attachment culture, further improve its reproductive efficiency.The method includes, step (1), judges that bubble whether there is between carrier layer and cloth liquid layer, or is present between carrier layer and cellular layer；Step (2), the position occurred according to bubble, adjust the default quantity delivered of culture fluid, until bubble is fully drained；Including when bubble is present between carrier layer and cloth liquid layer, increasing the quantity delivered of culture fluid, until bubble is fully drained；When bubble is present between carrier layer and cellular layer, stop, or stop the supply of culture fluid repeatedly, until bubble is fully drained；Step (3), according to culture fluid default quantity delivered, continue culture photo bio cell.Culture for photo bio cell.
Description
Technical field
A kind of the present invention relates to culture field of photo bio cell, more particularly to cultural method of photo bio cell.
Background technology
Extensive, the high yielding culture of photo bio cell (such as microalgae) is the bottleneck for restricting its industrialized development, inexpensive, high
The exploitation of effect training method is the key of large-scale production.Existing training method is specially being opened based on " water body culture "
Put in formula raceway pond, closing or semi-enclosed reactor and photo bio cell and nutrient, water are mixed, and provide it suitable
Suitable illumination so as to cell division breeding nature growth.
But find in actual applications, when being cultivated to photo bio cell using water body training method, light utilization ratio
Room for promotion limited, prevent above-mentioned training method from meeting the technical need of current photo bio cell large-scale culture.
In order to preferably improve the light utilization efficiency of photo bio cell, the mode of " attachment culture " is applied to photo bio cell
Culture.As shown in figure 1, attachment culture specifically arranges one layer with the cloth liquid layer for holding liquid energy power in support layer surface, by light
Biological cell is fixed or is attached on the culture carrier (carrier layer i.e. in figure) on cloth liquid layer surface, or surface and inside, is led to
Cross to cell sustainable supply culture fluid and make carrying layer surface schizogamy of the cell on cloth liquid layer surface grow to form cellular layer.
However, finding using during said method culture photo bio cell, illumination is good, temperature high in the case of
(such as noon), the carbon dioxide gass cognition dissolved in culture fluid discharge, and photo bio cellular metabolism can also produce oxygen, therefore
Cause between carrier layer and cellular layer, between cloth liquid layer and carrier layer, easily form bubble.Bubble can cause cellular layer obtain
Obtain the culture fluid from cloth liquid layer with carrier layer to supply, cell is in turn resulted in as nutrition supply is limited and make the division of its own
Breeding is limited.
Therefore, a kind of debubbling method convenient, effective during photo bio cell attachment culture is developed to photo bio
The attachment culture of cell is significant.
Content of the invention
In consideration of it, for solving problem of the prior art, embodiments of the invention provide a kind of culture side of photo bio cell
Method, can convenient, effectively eliminate the bubble in photo bio cell attachment culture, further improve its reproductive efficiency.
For reaching above-mentioned purpose, embodiments of the invention are adopted the following technical scheme that：
A kind of cultural method of photo bio cell is embodiments provided, methods described includes, step (1), judgement
Bubble whether there is between carrier layer and cloth liquid layer, or be present between the carrier layer and cellular layer；Step (2), basis
The position that the bubble occurs, adjusts the default quantity delivered of culture fluid, until the bubble is fully drained；Including when described
When bubble is present between the carrier layer and the cloth liquid layer, increase the quantity delivered of the culture fluid, until the bubble quilt
It is completely exhausted out；When the bubble is present between the carrier layer and the cellular layer, stops, or stop repeatedly the culture
The supply of liquid, until the bubble is fully drained；Step (3), according to the culture fluid described default quantity delivered, continue training
Support the photo bio cell.
Optionally, step (1) specifically includes, when cell layer surface occurs raised, if described raised in individual point
The photo bio cellular colours of the photo bio cell on cloth, and the projection in drying regime, and/or in the projection
Change, it is determined that the bubble is present between carrier layer and cloth liquid layer；When cell layer surface occurs raised, if described
It is in moisture state, and/or the light in the projection that projection is in the photo bio cell in population distribution, and the projection
Biological cell color does not change, it is determined that the bubble is present between carrier layer and cellular layer.
Optionally, described when the bubble is present between the carrier layer and the cloth liquid layer, increase the culture
The quantity delivered of liquid, until the bubble is fully drained, specifically includes, when the bubble is present in the carrier layer with the cloth
When between liquid layer, increase the quantity delivered of the culture fluid, make the culture fluid on the cloth liquid layer through 1～20 flow process,
So that the bubble is fully drained；Wherein, the flow definition be with the culture fluid from the top of cloth liquid layer stream to
The process of bottom.
Optionally, described when the bubble is present between the carrier layer and the cellular layer, stop, or stop repeatedly
The only supply of the culture fluid, until the bubble is fully drained, specifically includes, and step (2.1), is present in when the bubble
When between the carrier layer and the cellular layer, stop the supply of the culture fluid；Step (2.2), observe the carrier layer with
The elimination situation of the bubble between the cellular layer, if not observing the presence of the bubble, illustrates the bubble quilt
It is completely exhausted out；Step (2.3) if yet suffering from the bubble not being eliminated between the carrier layer and the cellular layer,
Restart to supply the culture fluid until the continuous drop for having the culture fluid flows out from the cloth liquid layer, stop confession again
Give the culture fluid；Step (2.4), circulation execute the step (2.2) and the step (2.3) successively, until the bubble
It is fully drained.
Optionally, described when the bubble is present between the carrier layer and the cellular layer, stop, or stop repeatedly
The only supply of the culture fluid, until the bubble is fully drained, also includes, the bubble of discharge is moved to the carrier
Between layer and the cloth liquid layer；Increase the quantity delivered of the culture fluid, until the bubble is fully drained.
Optionally, increase the quantity delivered of the culture fluid to not sweeping away the photo bio cell in the carrier layer
Maximum stream flow.
Optionally, the described default quantity delivered according to the culture fluid, continues the culture photo bio cell, bag
Include, defoamer is added in the culture fluid, or removes the light being dissolved in the culture fluid using selectivity through film
The cell secreta of biological cell.
Optionally, before step (3) are executed, methods described also includes, checks the carrier layer and the cloth liquid
Between layer, whether the bubble between the carrier layer and the cellular layer thoroughly exclude；If the carrier layer and the cloth
Between liquid layer, and/or between the carrier layer and the cellular layer, still suffer from the bubble then repeating said steps (2).
It is further preferred that between the inspection carrier layer and the cloth liquid layer, the carrier layer and the cell
Whether the bubble between layer thoroughly excludes, if between the carrier layer and the cloth liquid layer, and/or the carrier layer and institute
State, specifically include, gradually decrease the supply of the culture fluid
Until stopping completely, observe whether the carrier layer is fitted completely with the cloth liquid layer, the carrier layer with the cellular layer is
No fit completely；If the carrier layer is not exclusively fitted with the cloth liquid layer, and/or the carrier layer is not complete with the cellular layer
Full laminating, then illustrate between the carrier layer and the cloth liquid layer, and/or still suffer between the carrier layer and the cellular layer
The bubble, repeating said steps (2).
Optionally, described according to the default quantity delivered, continue the culture photo bio cell, including when the bubble
When being present between the carrier layer and the cloth liquid layer, the quantity delivered of the culture fluid is gradually lowered to the default supply
Amount, continues the culture photo bio cell；When the bubble is present between the carrier layer and the cellular layer, gradually increase
Plus the quantity delivered of the culture fluid is to the default quantity delivered, continue the culture photo bio cell.
Optionally, before step (1) is executed, methods described also includes, the photo bio cell is being trained
At the beginning of supporting, the bubble produced due to adhering to the operation of culture between carrier layer and cloth liquid layer is removed.
It is further preferred that described at the beginning of cultivating to the photo bio cell, remove carrier layer and cloth liquid layer it
Between the operation due to attachment culture and the bubble that produces, specifically include, by corresponding with cloth liquid layer for a line of carrier layer side
Contact；To contact while as starting point, the carrier layer and the cloth liquid layer is made to relative with the starting point side another
Bar side slowly sequentially contacts；And/or, carrier layer is placed on behind cloth liquid layer surface, the quantity delivered of culture fluid and/or water is adjusted
More than the default quantity delivered of the culture fluid, to remove the behaviour due to attachment culture between the carrier layer and the cloth liquid layer
The bubble that makees and produce；Stop the supply of the culture fluid and/or the water, so that the carrier layer is tight with the cloth liquid layer
Laminating；Gradually increase the quantity delivered of the culture fluid until the default quantity delivered.
It is further preferred that default quantity delivered of the quantity delivered of the regulation culture fluid and/or water more than the culture fluid
To the maximum stream flow not swept away the photo bio cell in the carrier layer.
This is based on, by embodiments providing a kind of cultural method of photo bio cell, when carrier layer and cloth liquid
After there is bubble between layer, by increasing the quantity delivered of culture fluid so that bubble follows the flowing of culture fluid to be expelled to load together
Outside body layer and cloth liquid layer, so as to remove the bubble between two-layer；After occurring bubble between carrier layer and cellular layer, by stopping
Only, or repeatedly stop the supply of culture fluid so that be present in the bubble between carrier layer and cellular layer by atmospheric pressure and culture
The collective effect of the vacuum force that liquid is formed between cloth liquid layer and carrier layer, by being moved between carrier layer and cellular layer or
Drain between carrier layer and cloth liquid layer, can flow out with the culture fluid of increase flow afterwards, so as to remove carrier layer and cellular layer it
Between bubble.The effect of the convenient, bubble effectively eliminated in photo bio cell attachment culture is reached, which is further increased numerous
Grow efficiency.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
Accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is the principle schematic of current photo bio cell attachment culture；
Fig. 2 is a kind of cultural method schematic flow sheet of photo bio cell provided in an embodiment of the present invention；
Fig. 3 is the operation chart that carrier layer is contacted with cloth liquid layer in the embodiment of the present invention.
Specific embodiment
Accompanying drawing in below in conjunction with the embodiment of the present invention, to the embodiment of the present invention in technical scheme carry 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 embodiment.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 Fig. 2 embodiments providing a kind of cultural method of photo bio cell, the method includes,
Step (1), judge that bubble whether there is between carrier layer and cloth liquid layer, or be present in carrier layer and cellular layer it
Between；
Step (2), the position occurred according to bubble, adjust the default quantity delivered of culture fluid, until bubble is fully drained；
Including when bubble is present between carrier layer and cloth liquid layer, increasing the quantity delivered of culture fluid, until bubble is fully drained；
When bubble is present between carrier layer and cellular layer, stop, or stop the supply of culture fluid repeatedly, until bubble is arranged completely
Go out；
Step (3), according to culture fluid default quantity delivered, continue culture photo bio cell.
It should be noted that above-mentioned photo bio cell can be spherical algae, filamentous algae, arc algae, spirulina, triangle
Any one in algae, polygon algae, scenedesmus, chlorella, Chlorococcum, Haematococcus Pluvialis, silkweed and filamentous cyanobacteria.
In embodiments of the present invention, cloth liquid layer by have hold liquid energy power, such as porous material or water wetted material constitute, with true
Protect culture fluid can sufficiently be absorbed by cell during being permeated along the direction of cell bottom to top layer.
Then illustrate to occur in that bubble in the system of above-mentioned attachment culture when photo bio cell surface occurs raised, specifically
Observation process can be completed by human eye, also can be by setting up high-definition camera being monitored to cultivating system.
Original quantity delivered in above-mentioned steps (2), when the default quantity delivered of culture fluid refers to that normal breeding is cultivated.Above-mentioned
The supply of culture fluid " repeatedly stop " referring to and stop culture fluid supply, open culture fluid supply, stop the anti-of culture fluid supply
Multiple process.
This is based on, by embodiments providing a kind of cultural method of photo bio cell, when carrier layer and cloth liquid
After there is bubble between layer, by increasing the quantity delivered of culture fluid so that bubble follows the flowing of culture fluid to be expelled to load together
Outside body layer and cloth liquid layer, so as to remove the bubble between two-layer；After occurring bubble between carrier layer and cellular layer, by stopping
Only, or repeatedly stop the supply of culture fluid so that be present in the bubble between carrier layer and cellular layer by atmospheric pressure and culture
The collective effect of the vacuum force that liquid is formed in cloth liquid layer and carrier interlayer, by being moved between carrier layer and cellular layer or arrange
To between carrier layer and cloth liquid layer, can flow out with the culture fluid of increase flow afterwards, so as to remove between carrier layer and cellular layer
Bubble.The effect of the convenient, bubble effectively eliminated in photo bio cell attachment culture is reached, which is further increased and is bred
Efficiency.
The following detailed description of each step and its concrete operations in the embodiment of the present invention：
Preliminary step
Before execution step (1), above-mentioned cultural method provided in an embodiment of the present invention may also include, thin to photo bio
At the beginning of born of the same parents are cultivated, the bubble produced due to adhering to the operation of culture between carrier layer and cloth liquid layer is removed.
Here, above-mentioned " at the beginning of culture " refers to, after photo bio cell is seeded in carrier layer, culture fluid supply is opened
Before beginning normally to cultivate.
As shown in figure 3, above-mentioned steps specifically can be entered using the combination of any one or two kinds of modes in following two modes
OK, i.e.,：
(1) mode one
Corresponding with cloth liquid layer for a line of carrier layer side is contacted；(the labelling in figure contacting while as starting point
For a), making carrier layer slowly sequentially contact to the another a line (being labeled as b in figure) relative with starting point side with cloth liquid layer, with to the greatest extent
Amount has bubble to press from both sides between the two layers during avoiding this.
(2) mode two is placed on carrier layer behind cloth liquid layer surface, adjusts the quantity delivered of culture fluid and/or water more than culture
The default quantity delivered of liquid, the bubble produced with removing the operation due to attachment culture between carrier layer and cloth liquid layer；Stop
Culture fluid and/or the supply of water, so that carrier layer is brought into close contact with cloth liquid layer.
Here, the quantity delivered for adjusting culture fluid and/or water is more than the default quantity delivered of culture fluid to not by carrier layer
The maximum stream flow that photo bio cell sweeps away, in order to avoid the photo bio cell in inoculation is washed away.
I.e. according between the two layers still with the presence of bubble, then mode two can be continued executing with after the operation of aforesaid way one
Operation.
The quantity delivered of culture fluid can gradually be increased after aforesaid way one and/or mode two has been carried out until default supply
Amount.
Step (1)
Further, above-mentioned steps (1) specifically may include following process：
When there is projection in cell layer surface, if raised in individual distribution, and the photo bio cell in projection is in dry shape
Photo bio cellular colours in state, and/or projection change, for example, can be that cellular colours turn yellow, it is determined that bubble is present
Between carrier layer and cloth liquid layer；
When cell layer surface occurs raised, if it is in moistening shape that projection is in the photo bio cell in population distribution, and projection
Photo bio cellular colours in state, and/or projection do not change, it is determined that bubble is present between carrier layer and cellular layer.
Wherein, above-mentioned " individual distribution " refers to that the negligible amounts of bubble, individuality are larger, and multiple bubbling polymerizations form, are in
The lamellar bubble of protuberance；Above-mentioned " population distribution " refers to that the quantity of bubble is more, individual less, how in hemispherical or most
Spherical a group.
Step (2)
When bubble is present between carrier layer and cloth liquid layer, increase the quantity delivered of culture fluid, until bubble is arranged completely
The step of going out specifically includes：
When bubble is present between carrier layer and cloth liquid layer, increase the quantity delivered of culture fluid, culture fluid is made in cloth liquid layer
On through 1～20 flow process so that bubble is fully drained；Wherein, flow definition is to be flowed from the top of cloth liquid layer with culture fluid
Process to bottom.
Here, consider production efficiency, culture fluid is preferably on cloth liquid layer through 5～10 flow processs.Also, increase
The quantity delivered of culture fluid is to the maximum stream flow not swept away the photo bio cell in carrier layer, in order to avoid affect follow-up normal breeding
Culture.
When bubble is present between carrier layer and cellular layer, stop, or stop the supply of culture fluid repeatedly, until bubble
The step of being fully drained specifically includes：
Step (2.1), the supply for when bubble is present between carrier layer and cellular layer, stopping culture fluid；Here, cultivate
After liquid stops supply, the culture fluid that iuntercellular is filled gradually is lost in, and can now be clearly observed cellular layer by moistening shape
State is changed into drying regime, can't see iuntercellular culture fluid liquid reflective.
The elimination situation of the bubble between step (2.2), observation carrier layer and cellular layer, if do not observe depositing for bubble
Then illustrating that bubble is fully drained；Above-mentioned step (3) can be proceeded after this.
Step (2.3) restarts supply training if yet suffering from the bubble not being eliminated between carrier layer and cellular layer
Nutrient solution is flowed out from cloth liquid layer up to the continuous drop for having culture fluid, stops supply culture fluid again；Now, cell is by aforementioned step
Suddenly the drying regime in (2.1) is changed into moisture state, and is finally changed into drying regime again.
Step (2.4), circulation execution step (2.2) and step (2.3) successively, are fully drained up to bubble, here it
Above-mentioned step (3) can be proceeded afterwards.
During above-mentioned steps (2.1)～step (2.4) is carried out, the bubble of discharge can be moved to carrier layer and cloth liquid
Between layer.The step of can perform the bubble between above-mentioned removal carrier layer and cloth liquid layer afterwards.
Wherein, during the bubble between carrier layer and cloth liquid layer is removed, increase the quantity delivered of culture fluid to not by load
The maximum stream flow that photo bio cell on body layer sweeps away, in order to avoid affect follow-up normal breeding culture.
Checking step
Further, before execution step (3), said method also includes：
Check between carrier layer and cloth liquid layer, whether the bubble between carrier layer and cellular layer thoroughly excludes；If bubble is thorough
Bottom excludes and then can proceed with follow-up step (3).
If between carrier layer and cloth liquid layer, and/or still suffering from bubble then repeat step (2) between carrier layer and cellular layer.
Above-mentioned checking process specifically includes following steps：
The supply of culture fluid is gradually decreased until stopping completely, whether observation carrier layer is fitted completely with cloth liquid layer, carrier
Whether layer is fitted completely with cellular layer；
If carrier layer is not exclusively fitted with cloth liquid layer, and/or carrier layer is not exclusively fitted with cellular layer, carrier layer is described
Between cloth liquid layer, and/or bubble between carrier layer and cellular layer, is still suffered from, repeat step (2).
Here it is possible to the whether complete of laminating judged with the presence or absence of projection by observation of cell surface.
Step (3)
After having executed above-mentioned steps (2), if bubble is present between carrier layer and cloth liquid layer, culture can be gradually lowered
The quantity delivered of liquid continues culture photo bio cell to default quantity delivered；If bubble is present between carrier layer and cellular layer,
The quantity delivered of culture fluid can gradually be increased to default quantity delivered, continue culture photo bio cell.
Further, as the cell secreta of photo bio cell is easily bubbled, therefore " the continuation training of above-mentioned steps (3)
Support " also include, defoamer is added in culture fluid, or it is thin to remove the photo bio being dissolved in culture fluid using selectivity through film
The cell secreta of born of the same parents, to alleviate the bubble problem in subsequent attachment culture.
Wherein, selectivity is specifically as follows the membrance separation facility such as ultrafilter membrane, micro-filtration membrane through film.
Two specific embodiments are provided below to describe said method provided in an embodiment of the present invention in detail.
Embodiment 1
With composite fibre film as carrier layer in the present embodiment, with canvas as cloth liquid layer, using lucite tube to cloth liquid layer
Supply culture fluid；In the collecting tank (volume is 1m × 2m × 0.2m) that culture fluid is stored in, the high 0.08m of liquid level.By small-sized latent
Water pump supplies the culture fluid in collecting tank to lucite tube, on pipeline of UNICOM's pump with lucite tube, is provided with stream
Adjustable valve.
Concrete incubation step is as described below：
Bottle green scenedesmus are seeded on composite fibre film, after cultivating 2 days, projection occurs in cell layer surface, it is observed that sending out
Existing：Raised (quantity totally 3), floor space minimum in irregular shape, raised of being distributed in individuality is about 2cm2, be about 10cm2
(explanation is raised individual larger), in yellowish-brown, convex surfaces cell judges that bubble is located between carrier layer and cloth liquid layer；
Increase about 15 flow processs of culture fluid flow control valve (having no during this that cell comes off from carrier layer), by flow
Regulating valve is recalled to default quantity delivered (i.e. the initial cultivation flow of culture fluid).Observation of cell layer surface illustrates bubble without projection
Excluded completely；
2 are instilled in culture fluid and drips polyethers foam inhibitor, proceed " attachment culture ".
Embodiment 2
With cellulose acetate film as carrier layer in the present embodiment, with sponge as cloth liquid layer, using lucite tube to cloth liquid layer
Supply culture fluid；In the collecting tank (volume is 1m × 2m × 0.2m) that culture fluid is stored in, the high 0.10m of liquid level.By small-sized latent
Water pump supplies the culture fluid in collecting tank to lucite tube, on pipeline of UNICOM's pump with lucite tube, is provided with stream
Adjustable valve.
Concrete incubation step is as described below：
Aeruginouss spirulina is seeded on cellulose acetate film, after cultivating 1 day, projection occurs in cell layer surface, it is observed that sending out
Existing：Raised is in that population distribution (quantity totally 135), shape mostly dome-type, raised floor space minimum are about 0.2cm2, the largest of about
For 2cm2(explanation is raised individual less), in aeruginouss, convex surfaces cell judges that bubble is located between cellular layer and carrier layer；
Close culture fluid flow control valve to be lost in spirulina surface cell culture, i.e., cell is thin in shape, observation is dried
Cellular surface is raised to eliminate about 70%～80%, and turn-on flow rate regulating valve (has no during this that cell comes off from carrier layer).When
After having observed that the continuous drop of culture fluid is flowed out from cloth liquid layer, flow control valve is again switched off to spirulina superficial cell in dry
After dry shape, it has been observed that cell surface is raised all eliminating, illustrate that bubble is excluded completely；
The culture fluid in collecting tank is filtered one time using ultrafiltration apparatuss, to remove cell secreta therein, solute
And after impurity, proceed " attachment culture ".
It should be noted that one of ordinary skill in the art will appreciate that：Realize the whole or portion of said method embodiment
Can be completed by the related hardware of programmed instruction step by step, aforesaid program can be stored in embodied on computer readable storage
In medium, the program upon execution, executes the step of including said method embodiment；And aforesaid storage medium can include：
ROM (Read-Only Memory, the abbreviation of read only memory), RAM (Random Access Memory, random access memory
The abbreviation of device) magnetic disc or CD etc. are various can be with the medium of store program codes.
The above, the only specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, any
Those familiar with the art the invention discloses technical scope in, change or replacement can be readily occurred in, should all be contained
Cover within protection scope of the present invention.Therefore, protection scope of the present invention should be defined by the scope of the claims.
Claims (13)
1. a kind of cultural method of photo bio cell, it is characterised in that methods described includes,
Step (1), judge that bubble whether there is between carrier layer and cloth liquid layer, or be present in the carrier layer and cellular layer it
Between；
Step (2), the position occurred according to the bubble, adjust the default quantity delivered of culture fluid, until the bubble is complete
Discharge；Including when the bubble is present between the carrier layer and the cloth liquid layer, increasing the supply of the culture fluid
Amount, until the bubble is fully drained；When the bubble is present between the carrier layer and the cellular layer, stop,
Or stop repeatedly the supply of the culture fluid, until the bubble is fully drained；
Step (3), according to the culture fluid described default quantity delivered, continue the culture photo bio cell.
2. cultural method according to claim 1, it is characterised in that step (1) specifically includes,
When cell layer surface occurs raised, if the raised photo bio cell in individual distribution, and the projection
The photo bio cellular colours in drying regime, and/or in the projection change, it is determined that the bubble is present in load
Between body layer and cloth liquid layer；
When cell layer surface occurs raised, if described raised in population distribution, and the photo bio cell in the projection
The photo bio cellular colours in moisture state, and/or in the projection do not change, it is determined that the bubble is present in
Between carrier layer and cellular layer.
3. cultural method according to claim 1, it is characterised in that described when the bubble be present in the carrier layer with
When between the cloth liquid layer, increase the quantity delivered of the culture fluid, until the bubble is fully drained, specifically include,
When the bubble is present between the carrier layer and the cloth liquid layer, increase the quantity delivered of the culture fluid, make institute
Culture fluid is stated on the cloth liquid layer through 1～20 flow process, so that the bubble is fully drained；Wherein, the flow process is fixed
Justice is the process with the culture fluid from the top stream of the cloth liquid layer to bottom.
4. cultural method according to claim 1, it is characterised in that described when the bubble be present in the carrier layer with
When between the cellular layer, stop, or stop repeatedly the supply of the culture fluid, until the bubble is fully drained, specifically
Including,
Step (2.1), the confession for when the bubble is present between the carrier layer and the cellular layer, stopping the culture fluid
Give；
Step (2.2), the elimination situation of the bubble that observes between the carrier layer and the cellular layer, if do not observe institute
The presence of bubble is stated, then illustrates that the bubble is fully drained；
Step (2.3) is opened again if yet suffering from the bubble not being eliminated between the carrier layer and the cellular layer
The beginning supply culture fluid is flowed out from the cloth liquid layer up to the continuous drop for having the culture fluid, is stopped described in supply again
Culture fluid；
Step (2.4), circulation execute the step (2.2) and the step (2.3) successively, until the bubble is arranged completely
Go out.
5. cultural method according to claim 1, it is characterised in that described when the bubble be present in the carrier layer with
When between the cellular layer, stop, or stop repeatedly the supply of the culture fluid, until the bubble is fully drained, also wrap
Include,
The bubble that discharges is moved between the carrier layer and the cloth liquid layer；
Increase the quantity delivered of the culture fluid, until the bubble is fully drained.
6. cultural method according to claim 1 or 5, it is characterised in that increase the quantity delivered of the culture fluid to
The maximum stream flow that the photo bio cell in the carrier layer sweeps away.
7. cultural method according to claim 1, it is characterised in that the described default supply according to the culture fluid
Amount, continues the culture photo bio cell, including,
Defoamer is added in the culture fluid, or removes the light being dissolved in the culture fluid using selectivity through film
The cell secreta of biological cell.
8. cultural method according to claim 1, it is characterised in that before step (3) are executed, methods described is also
Including,
Check whether the bubble between the carrier layer and the cloth liquid layer, between the carrier layer and the cellular layer is thorough
Bottom excludes；
If between the carrier layer and the cloth liquid layer, and/or still suffering from the gas between the carrier layer and the cellular layer
Steep then repeating said steps (2).
9. cultural method according to claim 8, it is characterised in that the inspection carrier layer and the cloth liquid layer it
Between, the bubble between the carrier layer and the cellular layer whether thoroughly exclude, if the carrier layer and the cloth liquid layer
Between, and/or the bubble then repeating said steps (2) between the carrier layer and the cellular layer, are still suffered from, specifically include,
The supply of the culture fluid is gradually decreased until stopping completely, observes whether the carrier layer is pasted completely with the cloth liquid layer
Close, whether the carrier layer is fitted completely with the cellular layer；
If the carrier layer is not exclusively fitted with the cloth liquid layer, and/or the carrier layer is not exclusively fitted with the cellular layer,
Then illustrate to still suffer from the gas between the carrier layer and the cloth liquid layer, and/or between the carrier layer and the cellular layer
Bubble, repeating said steps (2).
10. cultural method according to claim 1, it is characterised in that described according to the default quantity delivered, continues culture
The photo bio cell, including,
When the bubble is present between the carrier layer and the cloth liquid layer, the quantity delivered of the culture fluid is gradually lowered extremely
The default quantity delivered, continues the culture photo bio cell；
When the bubble is present between the carrier layer and the cellular layer, gradually increase the quantity delivered of the culture fluid extremely
The default quantity delivered, continues the culture photo bio cell.
11. cultural methods according to claim 1, it is characterised in that before step (1) is executed, methods described
Also include,
At the beginning of cultivating to the photo bio cell, the operation due to attachment culture between carrier layer and cloth liquid layer is removed
And the bubble for producing.
12. cultural methods according to claim 11, it is characterised in that described the photo bio cell is being cultivated
At the beginning of, the bubble produced due to adhering to the operation of culture between carrier layer and cloth liquid layer is removed, is specifically included,
Corresponding with cloth liquid layer for a line of carrier layer side is contacted；To contact while as starting point, the carrier layer is made
Slowly sequentially contact to the another a line relative with the starting point side with the cloth liquid layer；And/or, carrier layer is placed on cloth
Behind liquid layer surface, the quantity delivered of regulation culture fluid and/or water is more than the default quantity delivered of the culture fluid, to remove the carrier
The bubble produced due to adhering to the operation of culture between layer and the cloth liquid layer；Stop the culture fluid and/or the water
Supply so that the carrier layer is brought into close contact with the cloth liquid layer；
Gradually increase the quantity delivered of the culture fluid until the default quantity delivered.
13. cultural methods according to claim 12, it is characterised in that the quantity delivered of the regulation culture fluid and/or water
It is more than the default quantity delivered of the culture fluid to the maximum stream flow not swept away the photo bio cell in the carrier layer.
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| US6348347B1 (en) * | 1998-03-31 | 2002-02-19 | Micro Gaia Co., Ltd. | Fine algae culture device |
| US7897379B2 (en) * | 2007-02-26 | 2011-03-01 | Corning Incorporated | Device and method for reducing bubble formation in cell culture |
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