CN110819536B

CN110819536B - Culture medium containing branched chain amino acid and application of culture medium in culture of phaeodactylum tricornutum

Info

Publication number: CN110819536B
Application number: CN201911233076.8A
Authority: CN
Inventors: 胡晗华; 潘玉芳; 于晨
Original assignee: Institute of Hydrobiology of CAS
Current assignee: Institute of Hydrobiology of CAS
Priority date: 2019-12-05
Filing date: 2019-12-05
Publication date: 2020-10-27
Anticipated expiration: 2039-12-05
Also published as: CN110819536A

Abstract

The invention relates to an application of branched chain amino acid in culture of Phaeodactylum tricornutum, and also relates to a culture medium and a culture method for culturing Phaeodactylum tricornutum. The culture medium can accelerate the growth speed of the phaeodactylum tricornutum and prolong the logarithmic phase, thereby obtaining higher biomass when the culture is finished. Meanwhile, the phaeodactylum tricornutum cultured by the culture medium has obviously higher neutral lipid content.

Description

Culture medium containing branched chain amino acid and application of culture medium in culture of phaeodactylum tricornutum

Technical Field

The invention relates to the field of economic microalgae culture, in particular to application of branched chain amino acid in culture of phaeodactylum tricornutum, and also relates to a culture medium and a culture method for the phaeodactylum tricornutum.

Background

Phaeodactylum tricornutum is a marine unicellular microalgae belonging to the phylum Diatom, class Luciformes, order Phaeodactylum, genus Phaeodactylum, and is a primary productivity algae of common marine ecosystem. The phaeodactylum tricornutum has the characteristics of easy culture, perennial growth and easy regulation, and contains abundant neutral lipid, fucoxanthin and other substances, so that the culture of the phaeodactylum tricornutum for producing neutral lipid and fucoxanthin or serving as a bait has great advantages.

The currently common culture medium for the phaeodactylum tricornutum algae is f/2 seawater culture, the culture medium takes nitrate as a nitrogen source, the concentration is about 900 mu M, and the culture medium can ensure that the phaeodactylum tricornutum algae can grow well, but is not beneficial to the accumulation of neutral lipid in algae cells. Researchers have attempted to improve the medium formulation to increase the neutral lipid content of Phaeodactylum tricornutum cells and have found that when the N concentration is reduced to about 500. mu.M, the neutral lipid content of algal cells can be greatly increased. However, the reduction of the N content in the medium makes it apparent that the proliferation of Phaeodactylum tricornutum is limited. This results in a pair of contradictions, increasing nitrogen content, fast growth, large biomass, but low neutral lipid content; reduce nitrogen content, high neutral lipid content, slow growth and little biomass.

Disclosure of Invention

In the process of researching the culture conditions of the phaeodactylum tricornutum, the inventor finds that when the phaeodactylum tricornutum is cultured by using branched chain amino acid as a unique nitrogen source in a culture medium, the growth speed of algae cells is accelerated, the logarithmic phase is prolonged, and the neutral lipid content of the cultured algae cells is improved.

Based on the above findings, the present invention provides the use of branched chain amino acids in the cultivation of Phaeodactylum tricornutum.

The invention also provides a culture medium for culturing phaeodactylum tricornutum, which comprises branched chain amino acid as a nitrogen source.

In a preferred embodiment, the branched chain amino acids are one or more combinations of leucine, valine, and isoleucine.

In a preferred embodiment, the branched-chain amino acid serves as the sole nitrogen source and is present in an amount such that the N concentration in the medium is from 500. mu.M to 900. mu.M.

In a preferred embodiment, the medium is a variant of the f/2 artificial seawater medium, the nitrogen source in the f/2 artificial seawater medium being replaced by nitrate to the branched-chain amino acids.

The present invention also provides a method for culturing phaeodactylum tricornutum, comprising the step of culturing phaeodactylum tricornutum using the medium according to any one of claims 2 to 5.

In a preferred embodiment, the cultivation time is 8 days to 14 days.

The culture medium can accelerate the growth speed of the phaeodactylum tricornutum and prolong the logarithmic phase, thereby obtaining higher biomass when the culture is finished. Meanwhile, the phaeodactylum tricornutum cultured by the culture medium has obviously higher neutral lipid content.

Drawings

FIG. 1 is a photograph of a Phaeodactylum tricornutum culture in a triangular flask after 6 days of culture in different media;

FIG. 2 shows the cell density of Phaeodactylum tricornutum PT1 cultured in different culture media with the time;

FIG. 3 shows the change of the fluorescence intensity of the cells of the algae after nile red staining during the culture of Phaeodactylum tricornutum PT1 in different culture media with the time of culture;

FIG. 4 shows the results of thin layer chromatography analysis after neutral lipids were extracted from the Phaeodactylum tricornutum cultures cultured at

days

10 and 12.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention. The term is defined as:

1. preparation of culture Medium

In the embodiment, f/2 artificial seawater is used as a basic culture medium for improvement. The following is a preparation method of the f/2 artificial seawater culture medium:

and respectively dissolving the solution A and the solution B, then mixing, fixing the volume to 1L, adjusting the pH to about 8.0, and sterilizing at high temperature and high pressure to obtain the artificial seawater.

Then f/2Medium is added into each liter of artificial seawater, and the following components and dosage of the f/2Medium are as follows:

wherein, the components of the f/2 Trace Metal Solution are shown in Table 1, and the components of the f/2 Vitamin Solution are shown in Table 2.

TABLE 1 composition of f/2 Trace Metal Solution

FeCl₃·6H₂O	3.15g
		Na₂EDTA·2H₂O	4.8g
CuSO₄·5H₂O(9.8g/L dH₂O)	1.0ml
		Na₂MoO₄·2H₂O(6.3g/L dH₂O)	1.0ml
ZnSO₄·7H₂O(22.0g/L dH₂O)	1.0ml
		CoCl₂·6H₂O(10.0g/L dH₂O)	1.0ml
MnCl₂·4H₂O(180.0g/L dH₂O)	1.0ml
		Adding water to	1.0L

TABLE 2 f/2 composition of Vitamin Solution

Vitamin B₁₂(1.0g/L dH₂O)	1.0ml
		Biotin(0.1g/L dH₂O)	10.0ml
Thiamine HCl	200.0mg
		Distilled water to	1.0L

In the following experiments, the medium used was the same as the f/2 artificial seawater in other components except that the NaNO was replaced by a branched chain amino acid₃As a nitrogen source, and the final concentration of the nitrogen source was reduced to 500 μ M. The nitrogen sources in the examples are shown in Table 3.

Table 3 nitrogen sources added in the examples

2. Culture of Phaeodactylum tricornutum and analysis of oil content of algal cells

Using the medium examples and comparative examples above, the wild type Phaeodactylum tricornutum PT1 was cultured, respectively. 120mL of medium was added to each 250mL Erlenmeyer flask, and 3 replicates of each medium were set up. Shaking culture in constant temperature illumination shaking culture box with the same illumination intensity of 100 mu mol photons m^-2s^-1Continuous light irradiation, 100rpm shaking table rotation speed, initial inoculation concentration 2 x 10⁵Individual cells/mL.

Cell densities were measured and collected at

day

0, 2, 4, 6, 8, 10 and 12 of culture at 1.2 x 10⁷The cells were analyzed for neutral lipid content in algal cells by Nile Red staining, and further, 6X 10 cells were collected on

days

10 and 12⁷Individual cells were used for total lipid extraction and thin layer chromatography analysis.

FIG. 1 shows photographs of Phaeodactylum tricornutum cultured in different media on day 6. It can be seen that the color of the phaeodactylum tricornutum culture obtained by using nitrate (levo-first) as a nitrogen source was relatively light, while the color of the phaeodactylum tricornutum culture obtained by using branched-chain amino acids (leucine, isoleucine, and valine in order from levo-second to right) as a nitrogen source was relatively dark, indicating a high pigment accumulation. This difference continued until the end of the culture.

The growth of Phaeodactylum tricornutum is shown in FIG. 2, the Phaeodactylum tricornutum cultured with sodium nitrate as the only nitrogen source reaches the plateau stage about 6 days of culture, the Phaeodactylum tricornutum cultured with branched chain amino acid as the only nitrogen source reaches the plateau stage 10-12 days, and the cell density of the algae at 12 days is increased by 67% -80%. Wherein the culture density of Ile as the only nitrogen source is slightly higher than that of Leu and Val as the only nitrogen source.

Nile Red staining analysis showed (FIG. 3) that sodium nitrate as a nitrogen source was slightly higher in the accumulation of neutral lipids in the cells of Phaeodactylum tricornutum cultured than branched chain amino acids as a nitrogen source on days 4-6 of culture. However, from day 8 onwards, the accumulation of neutral lipids in the cells of phaeodactylum tricornutum cultured with branched-chain amino acids as a nitrogen source gradually exceeded that of phaeodactylum tricornutum cultured with sodium nitrate as a nitrogen source. When the culture is carried out till the 12 th day, the accumulation of neutral lipid of the algal cells taking the branched-chain amino acid as the only nitrogen source is not reduced, even is obviously increased by 18 to 32 percent, wherein the accumulation of the neutral lipid of the algal cells taking Leu and Val as the only nitrogen source is higher. The thin layer chromatography (FIG. 4) and Nile Red staining analysis showed consistent results, with branched-chain amino acids as the sole nitrogen source and the ratio of Triglyceride (TAG) content to NO in algal cells₃ ^-The content of TAG in algae cells is slightly higher as the only nitrogen source.

From the above experiments, it can be seen that the use of branched chain amino acids to culture phaeodactylum tricornutum can not only accelerate the growth of phaeodactylum tricornutum, but also increase the accumulation of neutral lipid in algal cells.

The use of f/2 artificial seawater as the basal medium for the improvement of the basal medium in the present application is only one embodiment of the present invention, and one skilled in the art can use any other medium formulation as long as its nutrient components are sufficient for the growth of phaeodactylum tricornutum and the nitrogen source is replaced by branched chain amino acids.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A medium for culturing phaeodactylum tricornutum, comprising a branched-chain amino acid as a nitrogen source, the branched-chain amino acid being one or a combination of leucine, valine and isoleucine, the branched-chain amino acid serving as the only nitrogen source, and being in an amount such that the N concentration in the medium is 500 μ M, the medium being a modification of f/2 artificial seawater medium, the nitrogen source in the f/2 artificial seawater medium being replaced with a nitrate salt for the branched-chain amino acid.

2. A method for culturing Phaeodactylum tricornutum, comprising the step of culturing Phaeodactylum tricornutum using the medium of claim 1.

3. The method of claim 2, wherein the culturing period is 8 days to 14 days.