Continuous gradient supplementation method and device applied to efficient expanding propagation of haematococcus pluvialis

A kind of Haematococcus pluvialis, gradient technology, applied in the biological field, can solve the problems of low cell biomass astaxanthin yield, low cell photosynthetic activity, great influence of seasonal changes, etc., to shorten the culture period, easy and efficient operation, The effect of avoiding pollution

Pending Publication Date: 2019-08-30
INST OF AQUATIC LIFE ACAD SINICA
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Problems solved by technology

In the first stage of cell culture, there are three culture models in the existing literature reports. One is the photoautotrophic model, which uses light energy for photosynthesis. The model that has successfully achieved large-scale production in the world is mainly based on photosynthesis. The two-stage model of autotrophy, the traditional two-stage photoautotrophy has problems such as difficult expansion, low yield, large land occupation, great influence from seasonal changes, and low efficiency.
In addition, there are few reports on heterotro

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  • Continuous gradient supplementation method and device applied to efficient expanding propagation of haematococcus pluvialis
  • Continuous gradient supplementation method and device applied to efficient expanding propagation of haematococcus pluvialis
  • Continuous gradient supplementation method and device applied to efficient expanding propagation of haematococcus pluvialis

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Experimental program
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Example Embodiment

[0032] Example 1:

[0033] Taking Haematococcus pluvialis FACHB-797 as an example, it does not constitute a limitation to the present invention. This method can be applied to all Haematococcus pluvialis (heterotrophic).

[0034] A continuous gradient feeding method for efficient propagation and propagation of Haematococcus pluvialis species, the steps are:

[0035] A. The preparation of different gradient feed media:

[0036] The medium in the tank used in the case is modified kobayashi’s basal medium, in which the yeast extract is replaced with sodium nitrate, and the rest of the composition is as follows: 1.2g sodium acetate (CH 3 COONa), 0.405g (L-asparagine (L-asparagine), 0.5g sodium nitrate (NaNO 3 ), 0.2g magnesium chloride hexahydrate (MgCl 2 ·6H 2 O), 0.01g ferrous sulfate heptahydrate (FeSO 4 ·7H 2 O), 0.02g calcium chloride dihydrate (CaCl 2 ·2H 2 O) and 1000mL deionized water; sterilize the prepared whole medium at a high temperature of 121°C for about 30 minutes; the conti...

Example Embodiment

[0047] Example 2:

[0048] according to figure 1 , figure 2 , image 3 It can be seen that a continuous gradient feeding method is applied to the device for efficient propagation of Haematococcus pluvialis species source. It consists of fermentation tank 1, air inlet 2, air outlet 3, inlet 4, outlet 5, supplement Feed port 6, pH control unit 7, dissolved oxygen control unit 8, temperature control unit 9, main control unit 10, continuous gradient feeding unit 11, air input unit 12, continuous carbon and nitrogen concentration feeding unit 13, trace element concentration feeding unit The feed unit 14, the water bottle 15, the feed tube 16, the ventilation tube 17, the peristaltic pump 18, the valve 19, the feed tube 20, the multi-channel liquid splitting device 21, and the carbon and nitrogen concentration feed bottle 22 are composed of: fermentation Tank 1 is respectively connected with main control unit 10, continuous gradient feeding unit 11, and air input unit 12 to form the m...

Example Embodiment

[0050] Example 3:

[0051] A continuous gradient feeding method for efficient propagation and propagation of Haematococcus pluvialis species, the process is:

[0052] The immobilized cells obtained by the continuous gradient feeding method were photosynthetically induced to produce astaxanthin under indoor conditions. The cells from the above sources were diluted 10 times with sterile tap water, and the light conditions were 100 μmol photons·m -2 ·S -1 , The temperature is controlled at 24 or 25 or 26 ℃, cultured for 8 or 9 or 10 days, the final astaxanthin yield is 15.45 mg L -1 d -1 , The average biomass yield is 0.50g L -1 d -1 .

[0053] The effect is shown in Table 1. Table 1 is a comparison between the device and the reported "two-step method" for culturing Haematococcus pluvialis to produce astaxanthin. The results show that the continuous gradient feeding method and device are used for efficient propagation of Haematococcus pluvialis. A large number of imine spores can be ob...

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Abstract

The invention discloses a continuous gradient supplementation method and device applied to efficient expanding propagation of haematococcus pluvialis. The continuous gradient supplementation method comprises the steps of A, compounding different gradient supplementation culture mediums; B, performing activation and species expanding on the haematococcus pluvialis: transferring sterilized culture mediums into a conical flask on a superclean working table, and performing inoculation with haematococcus pluvialis algae species; C, performing inoculation of the haematococcus pluvialis into a fermenter: detecting the quantity of cells of algae liquid after species expanding, performing thorough cleaning with fresh sterilized culture mediums, performing resuspending, and performing inoculation into the fermenter; D, monitoring culturing parameters/performing pH control: monitoring the algae liquid through a main control unit; and E, performing directional adjustment of C/N ratio of the culture mediums. The device consists of the fermenter, an air inlet/outlet, a sample inlet/out, a supplementation opening, a pH/temperature/dissolved oxygen control unit and a continuous gradient supplementation unit. Through the adoption of the method, infection with bacteria in the transforming and culture process can be avoided, and the utilization efficiency of substrates is improved; produced cellsare used for subsequent astaxanthin induction, and the survival rate is high; and the yield of biomass in the stage is as high as 0.30g L<-1>d<-1>, and the yield of the astaxanthin in the stage is ashigh as 15.45mg L<-1>d<-1>.

Description

technical field [0001] The invention relates to the field of biological technology, more specifically to a continuous gradient feeding method applied to the high-efficiency propagation of Haematococcus pluvialis, and also relates to a continuous gradient feeding device, which is especially suitable for Haematococcus pluvialls. The stage of efficient multiplication of provenance in large-scale cultivation. Background technique [0002] Haematococcus pluvialis is a freshwater unicellular green alga with double flagella, belonging to the Chlorophyta, Volvox, and Haematococceae. Under stress conditions, such as strong light, nitrogen deficiency and other conditions, cells can accumulate carotenoids, of which more than 85% are astaxanthin and astaxanthin esters. [0003] Astaxanthin is a kind of keto-type secondary carotenoid which is a source of vitamin A. Its chemical name is 3,3'-dihydroxy-4,4'-diketo-β,β'-carotene. It is worth noting that the astaxanthin molecule contains l...

Claims

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Application Information

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IPC IPC(8): C12N1/12C12M1/38C12M1/36C12M1/04C12M1/00C12R1/89
CPCC12N1/12C12M21/02C12M29/00C12M29/06C12M29/20C12M41/26C12M41/32C12M41/12Y02A40/80
Inventor 宋立荣卢哲郑凌凌李林李天丽
Owner INST OF AQUATIC LIFE ACAD SINICA
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