CN114164116A

CN114164116A - Chlorella culture method

Info

Publication number: CN114164116A
Application number: CN202111537927.5A
Authority: CN
Inventors: 路延笃; 陈玉婷; 顾佳华
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2021-12-15
Filing date: 2021-12-15
Publication date: 2022-03-11
Anticipated expiration: 2041-12-15
Also published as: CN114164116B

Abstract

The invention relates to the technical field of microalgae culture, in particular to a culture method of chlorella. The golden pomfret cooking liquor is used as a main nutrient substance for culturing the chlorella, the growth of the chlorella is obviously promoted, the promotion effect is more obvious after acetic acid, sea salt and sodium nitrate are added, and meanwhile, the content of polyunsaturated fatty acid in the chlorella is greatly improved, so that the cultured chlorella has higher nutritional value, and the golden pomfret cooking liquor can be widely used for preparing health-care products and foods.

Description

Chlorella culture method

Technical Field

The invention relates to the technical field of microorganisms, in particular to a culture method of chlorella.

Background

Chlorella is a unicellular algae widely distributed in ponds, lakes, oceans in Chlorophyta (Chlorophyta) and Chlorella (Chlorella), and the cells are spherical or oval in shape, vary in size depending on the species, and are usually 3-8 μm in diameter. The chlorella is rich in protein, fat, carbohydrate, various vitamins and other bioactive substances, can be used as human food or animal feed, and has a wide development and utilization prospect. The chlorella is rich in protein, and the essential amino acids forming the chlorella protein account for 42 percent of the total amino acids; the content of polysaccharide in chlorella is only second to protein, which accounts for about 20% of dry weight, and the polysaccharide has the function of regulating the immunity of the organism; chlorella can accumulate a large amount of fatty acids under nitrogen deficiency and high light conditions, wherein the content of long-chain unsaturated fatty acids such as linoleic acid and linolenic acid is rich, and the long-chain unsaturated fatty acids are essential unsaturated fatty acids for human and animals.

The nutrition modes of chlorella include autotrophy, heterotrophy and mixotrophy, so the culture modes are also diversified and can be divided into autotrophy culture, heterotrophy culture and mixed culture, and scientists can adopt different culture modes according to different experimental purposes. At present, the culture method of chlorella is complicated, is influenced by various conditions, and has an unsatisfactory growth rate, so that the culture method capable of obviously promoting the growth of chlorella and simultaneously improving the content of polyunsaturated fatty acid is needed to be provided, and the chlorella obtained by culture is widely used for preparing health-care products and foods.

Disclosure of Invention

In view of the above, the present invention provides a method for culturing chlorella, which can significantly promote the growth of chlorella and increase the content of polyunsaturated fatty acids in chlorella.

A culture method of chlorella comprises the following steps:

collecting cooking liquor of golden pomfret, and pretreating to obtain culture solution;

diluting the culture solution, adjusting the pH value to 7-9, collecting chlorella in the middle logarithmic phase, centrifuging, inoculating the precipitated chlorella into the diluted culture solution, and initially culturing to obtain OD₇₅₀And culturing for 5-8 days, wherein the culture time is 0.5.

The pretreatment comprises the following steps:

filtering the cooking liquor of the golden pomfret by using gauze, centrifuging and taking supernate;

collecting supernatant, filtering, treating with 121 deg.C high pressure steam for 20min, and cooling to room temperature.

In some embodiments, the dilution factor is 4-8, preferably 8.

The culture method provided by the invention further comprises the step of adding acetic acid into the diluted culture solution.

In some embodiments, the acetic acid is added in an amount of 0.25 to 2g/L based on the concentration of acetic acid in the culture medium. In some embodiments, it may be 2g/l, 1g/l, 0.5g/l, 0.25 g/l.

In some embodiments, the temperature of the culture is 15-35 ℃, preferably 23 ℃.

In some embodiments, the lighting conditions of the culture are: 24 hours illumination, the illumination intensity is 50-250 mu mol. phosns. m^-2·s^-1The culture period was periodically shaken 3 times manually per day. Among them, the light intensity is preferably 250. mu. mol. phos. m^-2·s^-1。

In some embodiments, the present invention provides a cultivation method further comprising the step of adding sea salt and sodium nitrate simultaneously with the addition of the acetic acid.

Wherein the addition amount of the sea salt is 17.5-70 per mill of salinity in the culture solution, and preferably 52.5 per mill; the addition amount of the sodium nitrate is that the ratio of N: p is 10-25: 1, preferably 15: 1.

Experiments show that the golden pomfret cooking liquor is used as a main nutrient substance for culturing the chlorella, and acetic acid and sea salt are properly added, so that the growth of the chlorella is obviously promoted, the content of polyunsaturated fatty acid in the chlorella is greatly increased, the cultured chlorella has higher nutritional value, and the golden pomfret cooking liquor can be widely used for preparing health-care products and foods.

Drawings

FIG. 1 is a graph of the change in dry weight of Chlorella in different wastewater dilution concentrations;

FIG. 2 is a graph showing the change of phosphate in Chlorella under different wastewater dilution concentrations;

FIG. 3 is a graph showing dry weight change of Chlorella at different temperatures, wherein 3-a is a graph showing dry weight change at 15 deg.C, 3-b is a graph showing dry weight change at 25 deg.C, and 3-c is a graph showing dry weight change at 35 deg.C;

FIG. 4 is a graph showing the change of dry weight of Chlorella under different light intensities, wherein 4-a is 50. mu. mol. phos. m^-2·s^-1As a result of the culture, 4-b was 150. mu. mol. phos. m^-2·s^-1As a result of the culture, 4-c was 250. mu. mol. phos. m^-2·s^-1The result of the culture of (3);

FIG. 5 is a graph of the dry weight change of Chlorella at different pH;

FIG. 6 is a graph showing the change of cell number of Chlorella under different nitrogen-phosphorus ratios;

FIG. 7 is a graph showing the cell number change of Chlorella in different salinity;

FIG. 8 is a graph showing the change in the cell number of Chlorella under different acetic acid concentrations;

FIG. 9 shows the results of the cell count, phosphate (9-c), and chlorophyll change (9-b) of Chlorella in example 5 under different culture conditions.

Detailed Description

The invention discloses a culture method of chlorella, which can be realized by appropriately improving process parameters by referring to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention is further illustrated by the following examples:

example 1 Effect of dilution factor, culture temperature, pH on Chlorella growth

(1) Collecting the golden pomfret cooking liquor, filtering by using a warp cloth to remove coarse impurities, centrifuging, and collecting supernatant to remove suspended impurities;

(2) filtering with filter paper with different pore diameters, treating with high pressure steam at 121 deg.C for 20min, and cooling to room temperature to obtain culture solution;

(3) the culture broth was diluted with sterile water to 5 gradients: 2 times, 4 times, 6 times, 8 times, 10 times (respectively noted as 2, 4, 6, 8, 10 times), inoculating chlorella having initial OD of about 0.5, and inoculating chlorella at 25 deg.C and 50. mu. mol. photos. m^-2·s^-1And culturing for 8 days in a constant-temperature incubator with 24-hour illumination, measuring the dry weight and pH, Fv/Fm and water quality (phosphate content) of the culture medium every day, and culturing in an F2 culture medium to serve as a control group, wherein the results are shown in the figure 1-2. Selecting the optimal culture solution dilution concentration according to the chlorella dry weight and the nutrient salt removal rate, wherein when the chlorella is diluted 8 times in the culture solution, the chlorella grows adaptively in the culture solution, and the biomass is(dry weight 1.35g/l) is the highest, phosphate removal rate (31.70%) is the best, three levels of culture solution dilution concentration factors with dilution concentration of 4 times, 6 times and 8 times as the response surface are selected for carrying out response surface experiments.

(4) Inoculating Chlorella into the diluted culture solution, culturing at 15 deg.C, 25 deg.C and 35 deg.C, measuring dry weight and pH, and measuring Fv/Fm daily, wherein the detection result is shown in FIG. 3. Selecting the optimal growth temperature according to the dry weight of the chlorella, wherein the chlorella can grow adaptively under the three temperature gradients, and the biomass reaches 0.82g/l at 35 ℃, which is the optimal temperature for the growth of the chlorella.

(5) Inoculating Chlorella in optimum wastewater dilution concentration, and culturing under light intensity of 50, 150, 250 μmol. photons. m^-2·s^-1The dry weight and pH, Fv/Fm, were measured daily in the environment of (1), and the results are shown in FIG. 4. Selecting optimum growing light intensity according to chlorella dry weight, and selecting light intensity of 250 μmol. phos. m^-2·s^-1In the environment of (2), the dry weight of the diluted concentration of each wastewater reaches the highest, which is the optimal light intensity for the growth of chlorella.

(6) The chlorella is inoculated in the optimal wastewater dilution concentration, the initial pH value of the wastewater is adjusted to be 4, 5, 6, 7, 8, 9 and 10, the dry weight and the pH value, Fv/Fm, of the wastewater are measured every two days, and the detection result is shown in figure 5. The optimum growth pH value is selected according to the dry weight of the chlorella, the chlorella can grow well in neutral and alkaline environment, the growth is inhibited when the pH is 4, the biomass reaches the maximum (the dry weight is 1.44g/l) when the pH is 9, and three levels of

pH

7, 8 and 9 are selected as pH factors of response surface experiments for further experiments.

Taking 3 levels of the 3 factors (

pH

7, 8 and 9;

temperature

15, 25 and 35, dilution concentration 4 times, 6 times and 8 times), taking the dry weight as a response value to perform a response surface experiment, and screening the chlorella in the golden pompano cooking liquor under the optimal environmental conditions.

Table 1 shows the results of the Chlorella response surface experiment

The optimum environmental conditions (8-fold dilution concentration, pH 8-8.5, temperature 23 ℃) were selected based on the results of the response surface.

Example 2

Setting up the experimental group and the control group:

experimental group (8), the culture step included:

(3) diluting the culture solution by 8 times with sterile water, adjusting the pH to 8-8.5, and inoculating to the initial OD₇₅₀0.1 of Chlorella, irradiating at 23 deg.C for 24 hr with light at intensity of 250 μmol. photons. m^-2·s^-1The culture was carried out in a constant temperature incubator for 8 days while shaking manually 3 times at regular intervals every day.

Control group (labeled F2): f2 is used as a culture medium (pH 8-8.5) to inoculate initial OD₇₅₀0.1 of Chlorella, irradiating at 23 deg.C for 24 hr with light at intensity of 250 μmol. photons. m^-2·s^-1The culture was carried out in a constant temperature incubator for 8 days while shaking manually 3 times at regular intervals every day.

Example 3

The difference from example 2 is that: diluting the culture solution by 8 times, and adding sea salt, sodium nitrate and acetic acid, wherein the acetic acid is added until the concentration of the acetic acid in the culture solution is 1g/l, the sea salt is added until the salinity is 52.5 per mill, and the sodium nitrate is added until the salinity is N: and P is 15:1, and other steps are under the same conditions. The detection proves that the growth amount of the chlorella reaches 0.825g/l, the removal rate of phosphate reaches 97.36 percent, and the results are shown in table 2 and figure 9.

Example 4

The difference from example 2 is that: after diluting the culture solution by 8 times, acetic acid was added to the culture solution so that the concentration of acetic acid was 1g/l, and the conditions were the same in all the other steps. Through detection, the growth amount of chlorella reaches 0.94g/L, the removal rate of phosphate reaches 89.38%, and the results are shown in table 2 and figure 9.

Example 5

In the example, the influence of acetic acid addition and acetic acid non-addition on the growth of chlorella is considered, and the experimental groups are as follows:

experimental groups:

experimental group 8 in example 2 (labeled a-8 in this example); example 3 group: m-8 — (15:1,52.5 ‰); example 4 group: m-8 group.

Control group:

control group 1: A-F2, control F2 in example 2;

control group 2: a-8 (15:1, 52.5%), the control group added sea salt to a salinity of 52.5% on the basis of test group 8 of example 2, sodium nitrate to N: the other steps were the same as in example 2, except that P was 15: 1.

Control group 3: M-F2 was added acetic acid (concentration: 1g/l) to the control F2 of example 2, and the other culture conditions were changed to the control F2.

The results of the cell count, dry weight, chlorophyll, and water quality (phosphate, total phosphorus, total nitrogen content) measurements of chlorella cultured according to the methods of the above control group and experimental group are shown in table 2 and fig. 9.

TABLE 2

The results show that the chlorella growth amount, the chlorophyll content and the phosphate removal rate of the treatment groups M-8, M-F2 and M-8- (15:1,52.5 permillage) added with acetic acid are obviously improved compared with the treatment groups A-8, A-F2 and A-8- (15:1,52.5 permillage) added with no acetic acid, respectively, and the results show that the growth of the chlorella can be obviously promoted after the acetic acid is added, and the phosphate removal rate of the chlorella can be improved.

The biomass of the experimental group A-8 is obviously higher than that of A-F2, and the cell number reaches 1.65 x 10^⁷cells/ml, dry weight up to 0.491g/l, phosphate removal rate up to 79.43%. Watch (A)Obviously, the chlorella can obviously improve the biomass of the chlorella by taking the golden pompano cooking liquor as a culture medium, and simultaneously remove the phosphate of the cooking liquor.

Wherein, in the group M-8 × (15:1,52.5 ‰), namely the culture method of adding sea salt, sodium nitrate and acetic acid in example 3, the growth amount of chlorella reaches 0.825g/l, the removal rate of phosphate reaches 97.36%, the growth of chlorella can be obviously promoted, the removal rate of phosphate by chlorella can be obviously improved, and the effect is better.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for culturing chlorella, comprising:

2. The culture method according to claim 1, wherein the pretreatment comprises:

3. The culture method according to claim 1, wherein the dilution is 4 to 8 times.

4. The culture method according to claim 1, further comprising the step of adding acetic acid to the diluted culture solution.

5. The culture method according to claim 4, wherein the amount of acetic acid added is 0.25 to 2g/L based on the concentration of acetic acid in the culture medium.

6. The method according to claim 1, wherein the temperature of the culture is 15 to 35 ℃.

7. The culture method according to claim 1, wherein the culture is performed under the following conditions: 24 hours illumination, the illumination intensity is 50-250 mu mol. phosns. m^-2·s^-1(ii) a The incubation period was periodically shaken manually 3 times per day.

8. The culture method according to any one of claims 1 to 7, further comprising a step of adding sea salt and sodium nitrate together with the addition of the acetic acid.

9. The culture method according to claim 8, wherein the salt is added in an amount of 17.5 to 70% o by salinity in the culture medium.

10. The culture method according to claim 8, wherein the amount of sodium nitrate added is such that the ratio of N: p is 10-25: 1.