CN109810904B

CN109810904B - Method for concentrating and collecting isochrysis galbana by using ethanol and realizing semi-continuous culture

Info

Publication number: CN109810904B
Application number: CN201910258186.3A
Authority: CN
Inventors: 黄旭雄; 薛毓才
Original assignee: Shanghai Ocean University
Current assignee: Shanghai Ocean University
Priority date: 2019-04-01
Filing date: 2019-04-01
Publication date: 2021-11-09
Anticipated expiration: 2039-04-01
Also published as: CN109810904A

Abstract

The invention relates to the technical field of microalgae biology, in particular to a method for concentrating and collecting Isochrysis galbana by using ethanol and realizing semi-continuous culture, wherein the ethanol is added into a dinoflagellate solution of Isochrysis galbana to be collected after culture, standing for 18-24 hours, then a siphoning method is used for collecting dinoflagellate cells of Isochrysis galbana precipitated at the bottom of a container, and meanwhile, a new culture solution is added into the rest dinoflagellate solution for re-aeration culture, so that the semi-continuous culture of Isochrysis galbana is realized. The method is characterized in that the isochrysis galbana is treated by using ethanol with extremely low concentration to enable the isochrysis galbana to lose the movement capability, so that the purpose that most of isochrysis galbana cells in the algae liquid are collected by using a precipitation method can be realized, the recovery rate is up to more than 70%, the added ethanol has low concentration and is extremely easy to volatilize, the collected microalgae cells are safe and non-toxic, the resuspension performance is good, the ethanol cost is very low, the residual isochrysis galbana cells in the algae liquid after the siphoning collection of the precipitated algae cells can still continue to proliferate after supplementing a new culture solution under the aeration condition, and the semi-continuous culture of the isochrysis galbana is realized.

Description

Method for concentrating and collecting isochrysis galbana by using ethanol and realizing semi-continuous culture

Technical Field

The invention relates to the technical field of microalgae biology, in particular to a method for collecting dinoflagellates such as globulina by using ethanol concentration and realizing semi-continuous culture.

Background

Isochrysis galbana (Isochrysis galbana), Dinophyceae, Isochrysis. The single-cell sports individual has changeable shape, is oval, has no cell wall and has two flagella with equal length. The microalgae is small in size, generally 4-7 microns in length, 2.7-4.4 microns in width and 2.4-3.0 microns in thickness, free of cell walls, easy to digest, rich in polysaccharide, carotene and high-energy lipid substances, and is widely used as bait in aquaculture (particularly shellfish culture) all over the world. The dinoflagellates such as the ball and the like are also natural treasury of physiologically active substances, wherein the beta-carotene, EPA, DHA and other high unsaturated fatty acids have the functions of resisting aging, stress, promoting development of nervous system and the like. Recent studies have shown that specially induced dinoflagellates such as dinoflagellates can also accumulate high levels of oil and fat in cells, and thus, dinoflagellates such as dinoflagellates are also a candidate species for biodiesel production.

The large-scale culture and the efficient collection of the dinoflagellates such as the dinoflagellates are involved in the feeding of the dinoflagellates such as the dinoflagellates as bait microalgae, the production of biodiesel or the extraction of bioactive substances by utilizing the dinoflagellates such as the dinoflagellates. However, the cell culture density of the dinoflagellates such as the cocci is relatively low under the autotrophic condition, generally not more than 1000 ten thousand cells/ml, and the cells are tiny, so that the efficient collection of the dinoflagellates such as the cocci is an important technical link for restricting the industrial production and application of the dinoflagellates such as the cocci. The existing commonly used microalgae harvesting methods mainly comprise a centrifugation method, a membrane filtration method, a flocculant method and a natural precipitation method. The energy consumption for collecting algae cells by adopting the traditional centrifugation method is large, the algae cells collected by the action of the centrifugal force cannot be well restored to a suspended state, and the algae cells are inconvenient to feed as bait microalgae subsequently. Although the natural precipitation method is low in cost, the characteristic that the dinoflagellates such as the cocci have flagella and can move is difficult to be effectively implemented. The flocculation of the dinoflagellate cells such as the globule and the like by using a flocculant method usually has the problems that the flocculant remains and the collected cells are not easy to resuspend. The membrane filtration method also has the problems of expensive productive equipment and low efficiency. In conclusion, the traditional microalgae harvesting method cannot be effectively applied to the efficient harvesting of the isochrysis galbana, and a high-efficiency and low-cost method is urgently needed for the harvesting of the isochrysis galbana.

The experiment of concentrating dinoflagellates such as the Strongylocentrotus sp by using the ultrafiltration technology is carried out in the research on the unicellular algae ultrafiltration concentration technology (Zhouwei et al, aquatic science, No. 27 of 2008, Vol. 5, No. 5), and the result shows that the ultrafiltration technology is completely suitable for the concentration of unicellular algae, and the proper operation pressure is preferably selected in order to achieve good concentration effect and not influence the activity of the algae. In the literature, "flocculation of isochrysis galbana by three flocculants" (gaowei et al, journal of ecology, vol. 31, No. 10, 2012), the influence of 3 flocculants on the sedimentation of isochrysis galbana was performed, and the harvesting effect and influence of different flocculants on isochrysis galbana on algal bodies were analyzed.

However, there is no report on a method of using ethanol for precipitation, harvesting dinoflagellates such as cocci, and realizing semi-continuous culture.

Disclosure of Invention

The invention aims to solve the problems of high cost, complicated operation (a centrifugal method and a membrane filtration method), flocculant residue and flocculant toxicity (a flocculant method) in the traditional method for collecting the dinoflagellates such as the cocci and the like, and provides a method for collecting the dinoflagellates such as the cocci by using ethanol and realizing semi-continuous culture on the basis of repeatedly testing and screening various substances.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for concentrating and harvesting dinoflagellates such as Strongylocentrotus with ethanol and realizing semi-continuous culture, which comprises the following steps: adding ethanol into the alga solution of the isochrysis galbana which is to be collected after culture, uniformly mixing, standing for 18-24 hours, collecting the alga cells of the isochrysis galbana which are precipitated at the bottom of the container by a siphonage method, and simultaneously adding new culture solution into the rest alga solution for aeration culture again to realize the semi-continuous culture of the isochrysis galbana.

As a preferable technical scheme of the invention, the adding concentration of the ethanol is 0.02-0.04% (v/v), namely 200-400 mul of ethanol is added into each liter of algae liquid.

As a preferable technical scheme of the invention, the standing is lightproof standing.

In a preferred embodiment of the present invention, the composition of the new culture medium is the same as that of the original culture medium.

As a preferable technical scheme, the volume ratio of the new culture solution to the residual culture solution after harvesting is 1: 2-5.

The invention has the advantages that:

1. by adopting an ethanol precipitation method, the recovery ratio is high and can reach more than 70 percent of the total amount of algae cells in the algae liquid.

2. The ethanol has low cost, the precipitation method is simple to collect and easy to operate, and the high-efficiency collection of the dinoflagellate such as the coccobacillus by the precipitation method is realized after the ethanol is used for treatment.

3. The added ethanol has low content and is volatile, the harvested algae cells basically do not contain ethanol, the harvested algae cells are not poisoned, and the microalgae fed by the method can be used as bait and do not produce any poison to the fed organisms.

4. The upper layer algae liquid contains a small amount of isochrysis galbana cells, ethanol is completely volatilized after aeration, the growth of the residual chrysophytes is not influenced, the algae cells can still continue to grow after nutrient salts are added again, and the semi-continuous culture of the isochrysis galbana is realized.

Drawings

FIG. 1 shows the settling rates of Isochrysis galbana after different concentrations of ethanol have been treated for different periods of time.

FIG. 2 is a graph showing the re-cultivation growth of supernatant algal fluid after ethanol precipitation of Isochrysis galbana cells; wherein, A is normal non-aerated culture, B is normal aerated culture, and C is aeration culture of upper layer algae liquid after ethanol precipitation of algae cells.

FIG. 3 is a growth curve of re-culture of Isochrysis galbana cells harvested with ethanol.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention; furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Examples

Culturing Isochrysis galbana with F/2 culture solution to relative growth reduction period, and collecting.

Wherein the F/2 culture solution for culturing the dinoflagellates such as the cocci is as follows: adding 74.8mgNaNO per liter seawater₃、4.4mg NaH₂PO₄、12mg Na₂SiO₃·9H₂O, 1mL of trace element solution and 1mL of vitamin solution. Solution of trace elements: 23mg of ZnSO₄·4H₂O，7.3mg Na₂MoO₄·2H₂O，17.8mg MnCl₂·4H₂O，12mg CoCl₂·6H₂O，10mg CuSO₄·5H₂O，4.35g Na₂EDTA，3.9g FeC₆H₅O₇·5H₂O,1000mL of distilled water. Vitamin solution: 0.5mg vitamin B₁₂0.5mg biotin, 100mg vitamin B₁1000mL of distilled water.

Wherein the inoculation density of Isochrysis galbana is 1 × 10⁶cell/mL at 25 deg.C with illumination intensity of 70 μmol/m²S, illumination period 16L: culturing for 6 days under 8D condition, and the cell density of algae reaches 6.0 × 10⁶cell/mL, ready for harvest.

50mL of the algae solution to be harvested is put into a sterilized 50mL conical centrifuge tube, 10 mu L of absolute ethyl alcohol is added, namely the concentration of the ethyl alcohol is 0.02% (v/v), and the mixture is uniformly mixed. Standing for 24 hours under dark condition, and directly collecting the dinoflagellate cells such as the cocci precipitated at the bottom by using a siphon method.

Wherein the residual algae solution after siphoning is transferred to a sterilized 500mL conical flask, and a new culture solution is added, the volume of the new culture solution F/2 and the volume of the upper layer algae solution are in accordance with a ratio of 1:5, and the density of the algae solution is 6 × 10⁵cell/mL at a temperature of 25 ℃ and an illumination intensity of 70 mu mol/m²S, illumination period 16L: and (5) continuing the culture under the condition of 8D by aeration.

The implementation effect is as follows:

after 24 hours, the cell density of the isochrysis galbana in the supernatant algae liquid is from 6.0X 10⁶The cell/mL is reduced to 1.2X 10⁶cell/mL, the algae cell precipitated at the bottom reaches 80 percent, namely more than 70 percent of algae cells can be collected by using a siphon method.

The upper layer algae liquid can be cultured continuously, and the density of the aerated culture reaches 5.3 multiplied by 10 days⁶cell/mL (see C in FIG. 2).

Meanwhile, the collected cells of the dinoflagellate such as the coccobata are cultured again, and the collected cells are inoculated into a 250mL conical flask filled with new F/2 culture solution, wherein the inoculation density is 8 multiplied by 10⁵cell/mL. The culture conditions are that the temperature is 25 ℃, and the illumination intensity is 70 mu mol/m²S, illumination period 16L: and 8D. The harvested algae cells still survive, and after culturing for 10 days, the density reaches 5.2 multiplied by 10⁶cell/mL (see FIG. 3).

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

1. A method for concentrating and harvesting dinoflagellates such as a bulb by using ethanol and realizing semi-continuous culture is characterized by comprising the following steps: adding ethanol into the alga solution of the isochrysis galbana which is to be collected after culture, uniformly mixing, standing for 18-24 hours, collecting the alga cells of the isochrysis galbana which are precipitated at the bottom of the container by a siphonage method, and simultaneously adding new culture solution into the rest alga solution for aeration culture again to realize semi-continuous culture of the isochrysis galbana; the adding concentration of the ethanol is 0.02-0.04% (v/v), namely 200-400 mul of ethanol is added into each liter of algae liquid.

2. The method of claim 1, wherein the resting is a light-protected resting.

3. The method of claim 1, wherein the composition of the new culture fluid is the same as the original culture fluid.

4. The method according to claim 1, wherein the volume ratio of the new culture solution to the culture solution remaining after harvesting is 1: 2-5.