CN106635805A - A culture method for monoraphidium - Google Patents
A culture method for monoraphidium Download PDFInfo
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- CN106635805A CN106635805A CN201510438771.3A CN201510438771A CN106635805A CN 106635805 A CN106635805 A CN 106635805A CN 201510438771 A CN201510438771 A CN 201510438771A CN 106635805 A CN106635805 A CN 106635805A
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Abstract
The invention relates to the field of microalgae biology, and discloses a culture method for monoraphidium. The method includes (1) subjecting the monoraphidium to culture of a biomass increasing stage; (2) when the OD680 value of an alga solution is 0.01-10, separating the alga solution to obtain a separated clear liquid I and monoraphidium mud, and returning the separated clear liquid I to the step (1) for reuse; (3) subjecting the monoraphidium mud obtained in the step (2) to culture of an oil and grease accumulating stage, with the culture manner being nitrogen starvation stress culture; (4) separating after 5-10 days of stress culture to obtain a separated clear liquid II and monoraphidium with accumulated oil and grease, and returning the separated clear liquid II to the step (3) for reuse; and (5) drying the monoraphidium with accumulated oil and grease to obtain a monoraphidium product. According to the method, operation of a culture process is stable, quality control is easy, and all culture liquids in the culture process can be recovered and reused so that the culture cost is low and no environment pollution is generated.
Description
Technical field
The present invention relates to microalgae biological field, in particular it relates to a kind of cultural method of single needle algae.
Background technology
Single needle algae (Monoraphidium) is a kind of monoplast green alga, with the irregular spindle bodily form
Shape, cell is long 12-26 μm, wide 4-8 μm, it is easy to breed in fresh water, while biomass is accumulated,
A certain amount of carbon dioxide is consumed, the purification for being conducive to environment is protected.Additionally, the biomass in single needle algae
Rich in grease, fat content, can be used as the potential next of bioenergy up to 30% or so of dry cell weight
Source.Simultaneously also containing abundant class beta carotene, vitamin and carbohydrate, tool in single needle algae
There is very high added value.Therefore, the large-scale farming tool of single needle algae has very important significance.But, it is single
The large-scale culture of pin algae and the research of production technology are but also little.
Wherein, patent application CN103555584A and CN103540533A provides two kinds of single needle algae algaes
Plant, and the cultural method of provided algae kind is provided, single needle algae fat content highest can reach 30.4%.
Open cultivation is carried out using the culture medium specified, by adding watery hydrochloric acid to adjust pH value and adding wadding
Solidifying agent flocculation harvesting.But add the separation clear liquid of algae solution after flocculant to be difficult to reuse, aquaculture cost compared with
It is high.Meanwhile, discharge of wastewater is big, there is certain environmental pollution.
Patent application CN104073437A discloses a kind of cultural method of single needle algae, is supported using open
Grow, it is possible to use the mixed gas of content 0.03-35% carbon dioxide, need using flocculant and by adjusting
Section pH value causes single needle algae precipitation to acid, and then realizes the centrifugation of single needle algae.But the method is same
Due to adding flocculant to cause after harvesting, separating liquid is difficult to reuse sample, aquaculture cost is higher and can make
Into certain environmental pollution.
The content of the invention
The invention aims to overcome drawbacks described above of the prior art, there is provided a kind of single needle algae support
Method is grown, the method can realize the continuous cultivation of single needle algae, breeding process stable operation, quality control
Easily, single needle algae fat content is high, and all nutrient solutions can be recycled in breeding process, supports
Grow low cost and the problem of discharging of waste liquid and environmental pollution will not be produced.
To achieve these goals, the invention provides a kind of cultural method of single needle algae, the method includes：
(1) single needle algae is carried out the culture of biomass build phase；
(2) as the OD of culture to algae solution680Be worth for 0.01-10 when, algae solution is separated, divided
From clear liquid I and single needle algae algae mud, separate clear liquid I and be back to reuse in step (1)；
(3) the single needle algae algae mud for obtaining step (2) carries out the culture in oil and fat accumulation stage, the training
Foster mode is nitrogen hunger coercing cultivation；
(4) separated after coercing cultivation 5-10 days, obtain separating the single needle of clear liquid I I and oil and fat accumulation
Algae, separates clear liquid I I and is back to reuse in step (3)；
(5) the single needle algae of oil and fat accumulation is dried to obtain into single needle algae product.
The method of the present invention, is that a kind of single needle algae biomass increases the continuous life cultivated with oil and fat accumulation culture
The method of product, can realize the continuous cultivation of single needle algae, and breeding process stable operation, quality control is easy,
The fat content of the single needle algae of the inventive method cultivation is high, and all nutrient solutions can in breeding process
Recycle, aquaculture cost is low and will not produce the problem of discharging of waste liquid and environmental pollution.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Specific embodiment
The specific embodiment of the present invention is described in detail below.It should be appreciated that this place is retouched
The specific embodiment stated is merely to illustrate and explains the present invention, is not limited to the present invention.
The invention provides a kind of cultural method of single needle algae, methods described includes：
(1) single needle algae is carried out the culture of biomass build phase；
(2) as the OD of culture to algae solution680Be worth for 0.01-10 when, algae solution is separated, divided
From clear liquid I and single needle algae algae mud, separate clear liquid I and be back to reuse in step (1)；
(3) the single needle algae algae mud for obtaining step (2) carries out the culture in oil and fat accumulation stage, the training
Foster mode is nitrogen hunger coercing cultivation；
(4) separated after coercing cultivation 5-10 days, obtain separating the single needle of clear liquid I I and oil and fat accumulation
Algae, separates clear liquid I I and is back to reuse in step (3)；
(5) the single needle algae of oil and fat accumulation is dried to obtain into single needle algae product.
In the inventive method, under preferable case, step (1) is carried out in biomass increases breeding device,
It is Race-way photobioreactor or closed photo bioreactor that biomass increases breeding device.For opening
There is no particular limitation for formula bioreactor and closed photo bioreactor, can be respectively this area
Conventional various Race-way photobioreactors and closed photo bioreactor.Such as open photo-biological
Reactor can be track optical biological reactor or box-type bioreactor；Open photo-biological is anti-
The material for answering device can be metal, cement, unorganic glass, lucite or plastics, and plastics can include
Polyethylene, polypropylene, polyvinyl chloride, Merlon etc..Preferably, Race-way photobioreactor is
Transparent polypropylene casing or cement track pond.Closed photo bioreactor can be anti-for bubble type photo-biological
Answer device, airlift photobioreactor, stirred photobioreactor etc.；Closed photo bioreactor
Material can be unorganic glass, lucite or transparent plastic, transparent plastic can include polyethylene,
Polypropylene, polyvinyl chloride, Merlon etc..Preferably, closed photo bioreactor is self-cleaning
Bubble type polyvinyl chloride bioreactor.
In the inventive method, for single needle algae algae kind and nutrient solution, there is no particular limitation, can be respectively
Various single needle algae algae kinds commonly used in the art and nutrient solution, this is known to those skilled in the art, here
Repeat no more.
In the inventive method, it will be understood by those skilled in the art that cultivating in actual single needle algae
Cheng Zhong, the pH value of cultivation temperature, intensity of illumination and nutrient solution is often uncontrollable in an accurate point
Value, but fluctuated within the scope of one.Under preferable case, in step (1), the condition of culture
Including：Temperature is 15-35 DEG C, and intensity of illumination is 1000-15000Lux, and pH value is 5-14, further
Preferably 8-12.Wherein, the condition of culture also includes：The carbon source being passed through in incubation is containing two
The gas of carbonoxide, the content of carbon dioxide in gas is 0.03-30 weight %, preferably 1-25 weight
%, more preferably 5-15 weight %.When illumination is carried out, light source can be natural light, it is also possible to
For artificial light sources (such as LED/light source), preferably natural light.Gas containing carbon dioxide can be height
Pure carbon dioxide or the gas containing carbon dioxide of industrial process discharge, what industrial process was discharged contains two
The gas of carbonoxide can include the gas containing carbon dioxide of power plant and refinery.Preferably, contain
The gas for having carbon dioxide is high-purity carbon dioxide or refinery hydrogen preparing tail gas.Those skilled in the art should
It is understood by, the gas containing carbon dioxide needs to carry out purification removing admittedly before single needle algae breeding device is entered into
Body particulate matter is simultaneously cooled down.
In the inventive method, it will be understood by those skilled in the art that biomass increases the number of breeding device
Mesh should be with the growth cycle of single needle algae, harvesting cycle, harvesting quantity, single needle algae in oil and fat accumulation breeding device
The growth cycle of accumulation grease matches, wherein, it can be 1 that single needle algae biomass increases breeding device,
Can also be multiple parallel operations.
In the inventive method, under preferable case, in step (2), cultivate to the OD of algae solution680It is worth and is
0.05-5, more preferably 0.1-5, more preferably 0.1-3.
In the inventive method, under preferable case, in step (2), by whole algae solutions be transferred to separation with
Separated in storage and transportation apparatus.
In the inventive method, in step (2), for by algae solution be transferred to separation with storage and transportation apparatus in
There is no particular limitation for mode, can be the mode of various transfers commonly used in the art.Wherein, transfer
Mode is preferably the conveying transfer of force of gravity, overflow transfer, siphon transfer or pressure, further preferably
Shift for pressure conveying, pressure conveying transfer is such as carried out with peristaltic pump.
In the inventive method, under preferable case, in step (2), detached mode be centrifugation,
It is separated by filtration or gravity settling separation, is more preferably separated by filtration.The mode being separated by filtration can be with
Separate for natural filtration, pressure filtration separation and vacuum filtration are separated, preferably vacuum filtration is separated.
In the inventive method, under preferable case, separation separates void tower with storage and transportation apparatus for cylinder, described
It is 1-10 that cylinder separates the ratio of height to diameter of void tower:1, more preferably 1.5-5:1, and the cylinder point
Bottom from void tower is provided with filter medium.For filter medium, there is no particular limitation, can be ability
The conventional various filter mediums in domain, under preferable case, filter medium is glass sand, filter cloth or filter paper, enters one
Step is preferably glass sand；The aperture of filter medium is 1-60 μm, more preferably 10-30 μm.
In the inventive method, under preferable case, cylinder separates void tower and is provided with agitator, further excellent
Selection of land, agitator be anchor agitator, gate stirrer or helix(ribbon type) agitator, still more preferably for
Gate stirrer.
In the inventive method, it will be understood by those skilled in the art that separating the number with storage and transportation apparatus
Can be 1 depending on the volume that biomass increases the nutrient solution in breeding device and oil and fat accumulation breeding device,
Can also be multiple parallel operations.
In the inventive method, under preferable case, clear liquid I will be separated and be back to reuse in step (1)
Mode includes：Return after (fresh) nutrient solution for separating clear liquid I with supplement is mixed in medium tank
Increase breeding device to biomass.
In the inventive method, under preferable case, step (3) is carried out in oil and fat accumulation breeding device, oil
Fat accumulation breeding device is Race-way photobioreactor or closed photo bioreactor, preferably open
Bioreactor.For Race-way photobioreactor and closed photo bioreactor are without special
Limit, various Race-way photobioreactors commonly used in the art can be respectively and closed photo thing is anti-
Answer device.For example Race-way photobioreactor can be track optical biological reactor or box-type photo-biological
Reactor；The material of Race-way photobioreactor can be metal, cement, unorganic glass, organic glass
Glass or plastics, plastics can include polyethylene, polypropylene, polyvinyl chloride, Merlon etc..Preferably,
Race-way photobioreactor is transparent polypropylene casing or cement track pond.Closed photo bioreactor
Can be bubble type bioreactor, airlift photobioreactor, stirred photobioreactor etc.；
The material of closed photo bioreactor can be unorganic glass, lucite or transparent plastic, transparent modeling
Material can include polyethylene, polypropylene, polyvinyl chloride, Merlon etc..
In the inventive method, under preferable case, in step (3), single needle algae algae mud with separate clear liquid I I
Separate with mix in storage and transportation apparatus after be transferred in oil and fat accumulation breeding device and carry out the oil and fat accumulation stage
Culture.It is further preferred that the mode being transferred in oil and fat accumulation breeding device is siphon transfer, vacuum turn
Move or pressure conveying transfer, still more preferably shift for pressure conveying, such as carry out pressure with peristaltic pump
Conveying transfer.
In the inventive method, under preferable case, in step (3), by single needle algae algae mud with separate it is clear
Liquid II is in separation and before mixing in storage and transportation apparatus, is washed with deionized single needle algae algae mud, and will washing
Liquid is back to reuse in step (1).For washing concrete mode without restriction, as long as can be right
Separate and washed with the single needle algae algae mud in storage and transportation apparatus, it is preferable that the number of times of washing is 1-5
It is secondary, more preferably 3-4 time；Every time the volume of washing deionized water used is the single needle algae of washing
1-5 times of algae mud volume, preferably 3-4 times.
In the inventive method, under preferable case, cleaning solution is back to into the mode of reuse in step (1)
Including：Biomass is back to after cleaning solution is mixed with (fresh) nutrient solution for supplementing in medium tank
Increase breeding device.
In the inventive method, for the mode of stress single needle algae accumulation grease, there is no particular limitation, can be with
For various modes commonly used in the art.In step (3), nitrogen hunger coercing cultivation can be by reducing oil
Nitrogen content is realizing in the nutrient solution in fat accumulation stage, it is preferable that nitrogen in the nutrient solution in oil and fat accumulation stage
Content for biomass build phase nutrient solution in nitrogen content 0-99%, more preferably 0-30%,
Still more preferably it is 0.Nitrogen in the nutrient solution is preferably sodium nitrate.Under preferable case, except nitric acid
Beyond sodium, the culture that the nutrient solution that the culture of oil and fat accumulation stage is used is used with biomass build phase culture
Liquid phase is same.
In the inventive method, under preferable case, the condition of culture includes：PH value is 5-12, and temperature is
24-30 DEG C, intensity of illumination is 1000-15000Lux.
In the inventive method, it will be understood by those skilled in the art that the number of oil and fat accumulation breeding device
The cultivation volume and growth cycle that single needle algae in breeding device should be increased with biomass matches, and can be 1,
Can also be multiple parallel operations.
In the inventive method, under preferable case, in step (4), detached mode be centrifugation,
It is separated by filtration or gravity settling separation, is more preferably separated by filtration.
In the inventive method, under preferable case, clear liquid I I will be separated and be back to reuse in step (3)
Mode includes：Clear liquid I I will be separated to separate and storage and transportation apparatus by being pumped into, with the single needle algae isolated
Algae mud is well mixed.It is further preferred that separating clear liquid I I by peristaltic pump conveying.People in the art
Member is it is understood that the reuse of separation clear liquid I I for convenience, can separate clear liquid I I and first store
It is standby in Clear liquid tank.
In the inventive method, in step (5), first the single needle algae of oil and fat accumulation can be delivered to into concentration tank
In be dried again.For dry mode, there is no particular limitation, can be commonly used in the art various
Drying mode, under preferable case, dry mode is entered to spontaneously dry, being vacuum dried or be spray-dried
One step is preferably whirlwind spray drying.
Embodiment
Hereinafter will be described the present invention by embodiment, but the present invention is not limited only to this.
In embodiment and comparative example, single needle algae is purchased from Wuhan aquatile research institute of the Chinese Academy of Sciences.
The OD value (OD values) of algae solution is determined：OD value spectrophotometric determination, to distill
Water is compared, and light absorption value of the algae solution at wavelength 680nm is determined, as the index of algae solution concentration.
The composition of BG11 culture mediums is shown in Table 1- tables 2 during culture, the NaNO in BG11 culture mediums3For nitrogen
Source.
The BG11 culture mediums of table 1
| Component | Content, mg/L |
| K2HPO4·3H2O | 40 |
| NaNO3 | 1500 |
| Na2CO3 | 20 |
| MgSO4·7H2O | 75 |
| CaCl2·2H2O | 36 |
| Citric acid | 6 |
| Ferric citrate | 6 |
| EDTA-2Na | 1 |
| Micro- A5 | 1 |
The trace element A5 of table 2
| Component | Composition, mg/L |
| H3BO3 | 2860 |
| MnCl2·4H2O | 1810 |
| ZnSO4·7H2O | 222 |
| CuSO4·5H2O | 79 |
| NaMoO4·5H2O | 390 |
| Co(NO3)2·6H2O | 50 |
Refinery hydrogen preparing tail gas for Shijiazhuang refinery branch company of Sinopec company refinery hydrogen preparing tail gas, dioxy
The content for changing carbon is 15-20 weight %, removes solid particulate matter and is cooled down using purification is front carried out.
Closed photo bioreactor be polyvinyl chloride film bag, a diameter of 220mm, a height of 1500mm,
Volume is 35L, without inner draft tube.
Open raceway pond bioreactor is cement track pond, and light-receiving area is 50m2。
Cultivation speed (the g/m of single needle algae2/ d)=single needle algae dry weight/single needle algae biomass build phase receives
The culturing time of light area/single needle algae biomass build phase.
Separate and lucite cylinder of the bottom with glass sand filter medium that storage and transportation apparatus are a 100L
Shape separates void tower, and ratio of height to diameter is 2:1, frame type stirring, with the delivery pipe that bottom is inserted into from top,
And be connected with oil and fat accumulation breeding device, a diameter of 10mm of delivery pipe, the specification of glass sand is G3, aperture
For 15-30 μm.
Embodiment 1
The present embodiment is used for the method that explanation cultivates single needle algae using closed photo bioreactor.
In the present embodiment, the culture and the culture in oil and fat accumulation stage of single needle algae biomass build phase exist
Carry out in closed photo bioreactor.Wherein, the closed photo thing of biomass build phase culture is anti-
Device is answered for 1, the closed photo bioreactor of oil and fat accumulation stage culture is 1, is separated and accumulating
Device is 1.
Nutrient solution is constituted by sodium nitrate, sodium acid carbonate and BG11 culture mediums are compound, nitre in every liter of nutrient solution
The content of sour sodium is 0.02mol, and the content of sodium acid carbonate is 0.1mol.
(1) the one-level culture of single needle algae
Single needle algae algae kind is inoculated in the 2000mL triangular flasks equipped with 1000mL nutrient solutions, inoculation is single
The OD of nutrient solution after pin algae algae kind680It is worth for 0.015.20-30 DEG C, 1800-2200Lux, pH value
Algae solution is cultivated under 10-12 to OD680It is worth for 3.3, it is standby.PH value is by high-purity titanium dioxide in incubation
Carbon is adjusted.
(2) culture of single needle algae biomass build phase is carried out in 1 closed photo bioreactor：
The single needle algae algae kind of 28L nutrient solutions and one-level culture, training are added after closed photo bioreactor sterilization
The initial OD of nutrient solution680It is worth for 0.1.Refinery hydrogen preparing tail gas passes through from closed photo bioreactor bottom
The dispersion of gas stone produces agitation, mixing with the entrance of bubbling form, and charge flow rate is 200L/h.In 20-24
DEG C, cultivate under intensity of illumination 1800-5000Lux, pH value 8-10, pH value passes through refinery hydrogen preparing tail gas
Charge flow rate control.
(3) single needle algae separates and transfer：The OD of nutrient solution after cultivating 15 days680Value reaches 3.1, leads to
The mode for crossing the conveying transfer of wriggling pump pressure is anti-from the closed photo thing of step (2) by single needle algae algae solution
Answer and be fully transferred in device separation and storage and transportation apparatus.Filter through glass sand, obtain separating clear liquid I and single needle
Algae algae mud, separates clear liquid I and enters in nutrient solution medium tank, is then washed with deionized isolated
Single needle algae algae mud 3 times, uses every time water 5L, cleaning solution to enter in nutrient solution medium tank, and supplements BG11
The corresponding content of culture medium and sodium nitrate to aforementioned nutrient solution, now the OD values of mixed liquor are 1.1.So
Afterwards by mixed liquor in the closed photo bioreactor that peristaltic pump enters back into step (2), continue single
The culture of pin algae biomass build phase.28L liquid is pumped up into separation and storage and transportation apparatus by Clear liquid tank,
Mix with the single needle algae algae mud being filtrated to get, after being well mixed, mixed liquor is by peristaltic pump Jing line pressures
Conveying transitions into single needle algae oil and fat accumulation breeding device.
(4) in 1 oil and fat accumulation breeding device, i.e., single needle algae oil is carried out in closed photo bioreactor
The accumulation of fat：The conveying of mixed liquor pressure is transferred to after closed photo bioreactor, not contain sodium nitrate
Nutrient solution is cultivated to accumulate grease.Temperature is 28-32 DEG C during culture, and intensity of illumination is
1000-5000Lux, pH value is 8-12.Refinery hydrogen preparing tail gas is logical from closed photo bioreactor bottom
The dispersion of gas stone is crossed with the entrance of bubbling form, and produces agitation, mixing, the air inflow of refinery hydrogen preparing tail gas is
200L/h, pH value is controlled by the charge flow rate of refinery hydrogen preparing tail gas.
Culture stops air inlet after 5 days, standing separation obtains separating the single needle algae of clear liquid I I and oil and fat accumulation,
Separate clear liquid I I and enter Clear liquid tank, the single needle algae of oil and fat accumulation enters concentration tank and obtains after vacuum drying
To single needle algae powder.By the total volume meter of single needle algae biomass build phase nutrient solution, single needle algae cultivation speed
Rate is 12g/m2/ d, fat content is 36.6% in the single needle algae for obtaining.
Embodiment 2
The present embodiment is used for the method that explanation cultivates single needle algae using Race-way photobioreactor.
In the present embodiment, the culture and the culture in oil and fat accumulation stage of single needle algae biomass build phase exist
Carry out in open raceway pond bioreactor.Wherein, the open race of biomass build phase culture
Road pond bioreactor is 1, the open raceway pond bioreactor of oil and fat accumulation stage culture
For 1, it is 1 to separate with storage and transportation apparatus.
Nutrient solution is constituted by sodium nitrate, sodium acid carbonate and BG11 culture mediums are compound, nitre in every liter of nutrient solution
The content of sour sodium is 0.02mol, and the content of sodium acid carbonate is 0.1mol.
(1) the one-level culture of single needle algae
Single needle algae algae kind is inoculated in the 2000mL triangular flasks equipped with 1000mL nutrient solutions, inoculation is single
The OD of nutrient solution after pin algae algae kind680It is worth for 0.015.20-30 DEG C, 1800-2200Lux, pH value
Algae solution is cultivated under 10-12 to OD680It is worth for 3.3, it is standby.PH value is by high-purity titanium dioxide in incubation
Carbon is adjusted.
(2) single needle algae biomass build phase is carried out in 1 open raceway pond bioreactor
Culture：10000L nutrient solutions and one-level culture are added in open raceway pond bioreactor
Single needle algae algae kind, the initial OD of nutrient solution680It is worth for 0.07.Refinery hydrogen preparing tail gas enters from above culture
In liquid, raceway pond is entered by disperser bubbling form, charge flow rate is 2000L/h.22-25 DEG C,
Cultivate under intensity of illumination 1800-5000Lux, pH value 10-12, pH value is entered by refinery hydrogen preparing tail gas
Throughput is controlled.
(3) single needle algae separates and transfer：The OD of nutrient solution after cultivating 15 days680Value reaches 1.6, leads to
Cross peristaltic pump all to turn single needle algae algae solution from the open raceway pond bioreactor of step (2)
Move to separation and storage and transportation apparatus.Filter through glass sand, obtain separating clear liquid I and single needle algae algae mud, separate
Clear liquid I is entered in nutrient solution medium tank, is then washed with deionized isolated single needle algae algae mud 4
It is secondary, use water 100L, cleaning solution to enter in nutrient solution medium tank every time, and supplement BG11 culture mediums and nitre
The corresponding content of sour sodium to aforementioned nutrient solution, now the OD values of mixed liquor are 0.53.Then by mixed liquor
In the open raceway pond bioreactor that peristaltic pump enters back into step (2), continue single needle algae
The culture of biomass build phase.80L liquid is pumped up into separation and storage and transportation apparatus by Clear liquid tank, with mistake
The single needle algae algae mud mixing that filter is obtained, after being well mixed, mixed liquor is supported through delivery pipe into oil and fat accumulation
Device is grown, while supplementing clear liquid in Clear liquid tank to 10000L.
(4) in 1 oil and fat accumulation breeding device, i.e., list is carried out in open raceway pond bioreactor
The accumulation of pin algae oil fat：Mixed liquor siphon is transferred to after open raceway pond bioreactor, not contain
The nutrient solution of sodium nitrate is cultivated to accumulate grease.Temperature is 22-25 DEG C during culture, and intensity of illumination is
1800-5000Lux, pH value is 10-12.Refinery hydrogen preparing tail gas is entered by disperser bubbling form and opened
Formula raceway pond bioreactor, and agitation, mixing are produced, the air inflow of refinery hydrogen preparing tail gas is
600L/h, pH value is controlled by the charge flow rate of refinery hydrogen preparing tail gas.
Culture stops air inlet after 6 days, centrifugation obtains separating the single needle algae of clear liquid I I and oil and fat accumulation,
Separate clear liquid I I and enter Clear liquid tank, the single needle algae of oil and fat accumulation enters concentration tank and is spray-dried through whirlwind
After obtain single needle algae powder.By the total volume meter of single needle algae biomass build phase nutrient solution, single needle algae
Cultivation speed is 10.5g/m2/ d, the content of grease is 31.7% in the single needle algae for obtaining.
Embodiment 3
The present embodiment is used for the method that explanation cultivates single needle algae using combined type bioreactor.
In the present embodiment, the culture of single needle algae biomass build phase is entered in closed photo bioreactor
OK, the culture in oil and fat accumulation stage is carried out in open raceway pond bioreactor.Wherein, it is biological
The closed photo bioreactor of amount build phase culture is 10, the opening of oil and fat accumulation stage culture
Formula raceway pond bioreactor is 1, and it is 1 to separate with storage and transportation apparatus.
Nutrient solution is constituted by sodium nitrate, sodium acid carbonate and BG11 culture mediums are compound, nitre in every liter of nutrient solution
The content of sour sodium is 0.02mol, and the content of sodium acid carbonate is 0.1mol.
(1) the one-level culture of single needle algae
Single needle algae algae kind is inoculated in the 2000mL triangular flasks equipped with 1000mL nutrient solutions, inoculation is single
The OD of nutrient solution after pin algae algae kind680It is worth for 0.015.20-30 DEG C, 1800-2200Lux, pH value
Algae solution is cultivated under 10-12 to OD680It is worth for 3.3, it is standby.PH value is by high-purity titanium dioxide in incubation
Carbon is adjusted.
(2) single needle algae biomass build phase is carried out in the closed photo bioreactor of 10 parallel connections
Culture：The single needle of 28L nutrient solutions and one-level culture is added in each closed photo bioreactor
Algae algae kind, the initial OD of nutrient solution680It is worth for 0.12.Refinery hydrogen preparing tail gas reacts from closed photo thing
The bottom of device is disperseed with the entrance of bubbling form by gas stone, and produces agitation, mixing, and charge flow rate is
200L/h.Cultivate under 20-25 DEG C, intensity of illumination 1800-5000Lux, pH value 9-11, pH value is led to
Cross the charge flow rate control of refinery hydrogen preparing tail gas.
(3) single needle algae separates and transfer：Culture after cultivating 15 days in each closed photo bioreactor
The OD of liquid680Value reaches 3-4, by single needle algae algae solution from the 10 of step (2) by way of siphon is shifted
It is fully transferred to separate and storage and transportation apparatus in individual closed photo bioreactor.Filter through glass sand, obtain
Clear liquid I and single needle algae algae mud are separated, clear liquid I is separated and is entered in nutrient solution medium tank, then use deionization
The isolated single needle algae algae mud of water washing 3 times, use every time water 50L, and cleaning solution is entered cultivates liquid medium
In tank, and the corresponding content of BG11 culture mediums and sodium nitrate to aforementioned nutrient solution is supplemented, now mixed liquor
OD values be 0.75.Then mixed liquor is respectively enterd again 10 envelopes of step (2) through peristaltic pump
In enclosed bioreactor, continue the culture of single needle algae biomass build phase.80L liquid is by clear liquid
Tank is pumped up into separation and storage and transportation apparatus, is mixed with the single needle algae algae mud being filtrated to get, after being well mixed,
Mixed liquor transitions into oil and fat accumulation breeding device by the conveying of peristaltic pump Jing line pressures, while supplementing clear liquid
Clear liquid in tank is to 300L.
(4) in 1 oil and fat accumulation breeding device, i.e., list is carried out in open raceway pond bioreactor
The accumulation of pin algae oil fat：The conveying of mixed liquor pressure is transferred to after open raceway pond bioreactor, with
Nutrient solution without sodium nitrate is cultivated to accumulate grease.Temperature is 20-25 DEG C during culture, and illumination is strong
Spend for 1800-5000Lux, pH value is 10-12.Refinery hydrogen preparing tail gas is entered by disperser bubbling form
Open raceway pond bioreactor, and agitation, mixing are produced, the air inflow of refinery hydrogen preparing tail gas is
600L/h, pH value is controlled by the charge flow rate of refinery hydrogen preparing tail gas.
Culture stops air inlet after 5 days, and glass sand is separated by filtration, and obtains separating the list of clear liquid I I and oil and fat accumulation
Pin algae, separates clear liquid I I and enters Clear liquid tank, and the single needle algae of oil and fat accumulation enters concentration tank and through whirlwind spray
Mist obtains single needle algae powder after being dried.It is single by the total volume meter of single needle algae biomass build phase nutrient solution
The cultivation speed of pin algae is 11g/m2/ d, the content of grease is 30.8% in the single needle algae for obtaining.
Comparative example 1
This comparative example is used for the method that explanation cultivates single needle algae using single Race-way photobioreactor.
Nutrient solution is constituted by sodium nitrate, sodium acid carbonate and BG11 culture mediums are compound, nitre in every liter of nutrient solution
The content of sour sodium is 0.02mol, and the content of sodium acid carbonate is 0.1mol.
(1) the one-level culture of single needle algae
Single needle algae algae kind is inoculated in the 2000mL triangular flasks equipped with 1000mL nutrient solutions, inoculation is single
The OD of nutrient solution after pin algae algae kind680It is worth for 0.015.20-30 DEG C, 1800-2200Lux, pH value
Algae solution is cultivated under 10-12 to OD680It is worth for 3.3, it is standby.PH value is by high-purity titanium dioxide in incubation
Carbon is adjusted.
(2) 10000L nutrient solutions and one-level culture are added in open raceway pond bioreactor
Single needle algae algae kind, nutrient solution initial OD values are 0.07.Refinery hydrogen preparing tail gas is entered from above in nutrient solution,
Raceway pond, charge flow rate 2000L/h are entered by disperser bubbling form.In 22-25 DEG C, intensity of illumination
Cultivate under 1800-5000Lux, pH value 8-12, pH value is controlled by the charge flow rate of refinery hydrogen preparing tail gas.
The OD of nutrient solution after cultivating 18 days680Value reaches 1.2, by peristaltic pump conveying centrifugation harvesting, obtain from
Heart liquid and single needle algae algae mud, single needle algae algae mud obtains single needle algae powder after whirlwind spray drying.Centrifugation
Liquid enters nutrient solution medium tank, and supplements BG11 culture mediums and sodium nitrate accordingly containing to aforementioned nutrient solution
Amount, now the OD values of mixed liquor are 0.1.Then mixed liquor is pumped back to into open race through wriggling
In the bioreactor of road pond, continue the cultivation of single needle algae.By the stereometer of nutrient solution, the cultivation of single needle algae
Speed is 0.9g/m2/ d, the content of grease is 22% in the single needle algae for obtaining.
The cultural method of the single needle algae of the present invention, can realize the continuous cultivation of single needle algae, breeding process behaviour
Stablize, easily, single needle algae fat content is high for quality control, and all nutrient solutions are all in breeding process
Can recycle, aquaculture cost is low and will not produce the problem of discharging of waste liquid and environmental pollution.
The preferred embodiment of the present invention described in detail above, but, the present invention is not limited to above-mentioned reality
The detail in mode is applied, in the range of the technology design of the present invention, can be to the technical side of the present invention
Case carries out various simple variants, and these simple variants belong to protection scope of the present invention.
It is further to note that each particular technique described in above-mentioned specific embodiment is special
Levy, in the case of reconcilable, can be combined by any suitable means, in order to avoid need not
The repetition wanted, the present invention is no longer separately illustrated to various possible combinations.
Additionally, can also be combined between a variety of embodiments of the present invention, as long as its
Without prejudice to the thought of the present invention, it should equally be considered as content disclosed in this invention.
Claims (18)
1. a kind of cultural method of single needle algae, it is characterised in that methods described includes：
(1) single needle algae is carried out the culture of biomass build phase；
(2) as the OD of culture to algae solution680Be worth for 0.01-10 when, algae solution is separated, divided
From clear liquid I and single needle algae algae mud, separate clear liquid I and be back to reuse in step (1)；
(3) the single needle algae algae mud for obtaining step (2) carries out the culture in oil and fat accumulation stage, the training
Foster mode is nitrogen hunger coercing cultivation；
(4) separated after coercing cultivation 5-10 days, obtain separating the single needle of clear liquid I I and oil and fat accumulation
Algae, separates clear liquid I I and is back to reuse in step (3)；
(5) the single needle algae of oil and fat accumulation is dried to obtain into single needle algae product.
2. method according to claim 1, wherein, in step (1), the condition of the culture
Including：Temperature is 15-35 DEG C, and intensity of illumination is 1000-15000Lux, and pH value is 5-14, preferably
8-12。
3. method according to claim 1, wherein, in step (2), cultivate to algae solution
OD680It is worth for 0.05-5, preferably 0.1-3.
4. method according to claim 1, wherein, in step (2), by the transfer of whole algae solutions
Separated with storage and transportation apparatus to separating.
5. method according to claim 4, wherein, in step (2), the mode of the transfer
Shift for the conveying of force of gravity, overflow transfer, siphon transfer or pressure, preferably pressure conveying transfer.
6. the method according to claim 1 or 4, wherein, it is described detached in step (2)
Mode is centrifugation, is separated by filtration or gravity settling separation, is preferably separated by filtration.
7. method according to claim 4, wherein, the separation is cylinder with storage and transportation apparatus
Void tower is separated, it is 1-10 that the cylinder separates the ratio of height to diameter of void tower:1, preferably 1.5-5:1, and it is described
Cylinder separates the bottom of void tower and is provided with filter medium.
8. method according to claim 7, wherein, the filter medium be glass sand, filter cloth or
Filter paper, preferably glass sand；The aperture of the filter medium is 1-60 μm, preferably 10-30 μm.
9. method according to claim 7, wherein, the cylinder separates void tower and is provided with to be stirred
Device is mixed, the agitator is preferably anchor agitator, gate stirrer or helix(ribbon type) agitator, further
Preferably gate stirrer.
10. method according to claim 4, wherein, in step (3), single needle algae algae mud with
Separate clear liquid I I the separation with mix in storage and transportation apparatus after be transferred in oil and fat accumulation breeding device and carry out
The culture in oil and fat accumulation stage.
11. methods according to claim 10, wherein, in being transferred to oil and fat accumulation breeding device
Mode is siphon transfer, vacuum transfer or pressure conveying transfer, and preferably pressure conveys transfer.
12. methods according to claim 10, wherein, in step (3), by single needle algae algae
Mud is separated and before mixing in storage and transportation apparatus, is washed with deionized single needle algae with clear liquid I I is separated described
Algae mud, and cleaning solution is back to into reuse in step (1).
13. methods according to claim 12, wherein, the number of times of washing is 1-5 time, preferably
For 3-4 time；Every time the volume of washing deionized water used is the 1-5 of the single needle algae algae mud volume of washing
Times, preferably 3-4 times.
14. methods according to claim 1, wherein, in step (3), the oil and fat accumulation stage
Nutrient solution in nitrogen content for nitrogen content in the nutrient solution of biomass build phase 0-99%, it is further excellent
Elect 0-30% as, be still more preferably 0.
15. methods according to claim 14, wherein, the condition of the culture includes：PH value
For 5-12, temperature is 24-30 DEG C, and intensity of illumination is 1000-15000Lux.
16. methods according to claim 1, wherein, in step (4), the detached side
Formula is centrifugation, is separated by filtration or gravity settling separation.
17. methods according to claim 1, wherein, in step (5), the side of the drying
To spontaneously dry, being vacuum dried or be spray-dried, preferably whirlwind is spray-dried formula.
18. methods according to claim 1, wherein, step (1) increases cultivation in biomass
Carry out in device, it is that Race-way photobioreactor or closed photo thing are anti-that the biomass increases breeding device
Answer device；Step (3) is carried out in oil and fat accumulation breeding device, and the oil and fat accumulation breeding device is open
Bioreactor or closed photo bioreactor.
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