CN114164116B

CN114164116B - Chlorella culture method

Info

Publication number: CN114164116B
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: 2023-10-24
Anticipated expiration: 2041-12-15
Also published as: CN114164116A

Abstract

The invention relates to the technical field of microalgae culture, in particular to a method for culturing chlorella. According to the invention, the golden pomfret cooking liquid is used as a main nutrient substance for cultivating chlorella, so that 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 cultivated chlorella has higher nutritive value, and the golden pomfret cooking liquid 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 method for culturing chlorella.

Background

Chlorella is a single-cell algae widely distributed in ponds, lakes, and oceans in Chlorophyta (Chlorophyta) and Chlorella, and the cells are spherical or widely elliptic, and the sizes of the cells are different according to the types, and the diameter is usually 3-8 microns. The chlorella is rich in proteins, fat, carbohydrates, multiple vitamins and other bioactive substances, can be used as human food or animal feed, and has wide development and utilization prospects. The chlorella not only has rich protein content, but also has the necessary amino acid for forming the chlorella protein accounting for 42 percent of the total amino acid; the content of polysaccharide in Chlorella is only inferior to protein, about 20% of dry weight, and the polysaccharide has the function of regulating organism immunity; chlorella is capable of accumulating fatty acids in large amounts under nitrogen-deficient, high-light conditions, and is rich in long-chain unsaturated fatty acids such as linoleic acid and linolenic acid, which are essential unsaturated fatty acids for humans and animals.

The nutrition modes of the chlorella comprise autotrophy, heterotrophy and mixotrophic, so that the culture modes of the chlorella are also diversified, and the chlorella can be divided into autotrophy culture, heterotrophy culture and mixed culture, so that scientists can adopt different culture modes according to different experimental purposes. At present, the culture method of the chlorella is complicated and is influenced by various conditions, and the growth rate is not ideal, so the culture method capable of obviously promoting the growth of the chlorella and improving the polyunsaturated fatty acid content 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 invention provides a method for culturing chlorella, which can obviously promote the growth of the chlorella and increase the content of polyunsaturated fatty acids in the chlorella.

A method of culturing chlorella comprising:

collecting the cooking liquid of golden pomfret, and preprocessing to obtain a culture liquid;

diluting the culture solution, regulating pH to 7-9, collecting Chlorella in mid-log phase, centrifuging, inoculating the precipitated algae into the diluted culture solution, and initially culturing OD ₇₅₀ Culturing for 5-8 days at 0.5.

The pretreatment comprises the following steps:

filtering the boiled liquid of golden pomfret with gauze, centrifuging and taking supernatant;

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

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

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 from 0.25 to 2g/L to the concentration of acetic acid in the culture broth. In some embodiments it may be specifically 2g/l, 1g/l, 0.5g/l, 0.25g/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 of illumination with the intensity of 50-250 mu mol/photons·m ^-2 ·s ^-1 Manual shaking was performed 3 times daily for the incubation period. Of these, the illumination intensity is preferably 250. Mu. Mol. Photons.m ^-2 ·s ^-1 。

In some embodiments, the culture method provided by the invention further comprises the step of adding sea salt and sodium nitrate simultaneously with the acetic acid.

Wherein the adding amount of the sea salt is 17.5-70 per mill, preferably 52.5 per mill of the salinity of the culture solution; the addition amount of the sodium nitrate is as follows: p=10 to 25:1, preferably 15:1.

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

Drawings

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

FIG. 2 is a graph of phosphate change of Chlorella at various wastewater dilution concentrations;

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

FIG. 4 is a graph showing the change in dry weight of Chlorella under different light intensities, wherein 4-a is 50μmol.photons.m ^-2 ·s ^-1 As a result of the culture of (2), 4-b was 150. Mu. Mol. Photons.m ^-2 ·s ^-1 The culture result of (C) was 250. Mu. Mol. Photons.m in 4-c ^-2 ·s ^-1 Is a culture result of (a);

FIG. 5 is a graph showing changes in dry weight of Chlorella at different pH;

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

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

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

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

Detailed Description

The invention discloses a method for culturing chlorella, which can be realized by appropriately improving process parameters by a person skilled in the art by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

The invention is further illustrated by the following examples:

example 1 influence of dilution factors, culture temperature, pH on Chlorella growth

(1) Collecting golden pomfret cooking liquid, filtering by using warp cloth, removing 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 121deg.C for 20min, and cooling to room temperature to obtain culture solution;

(3) The culture was diluted to 5 gradients with sterile water: 2-fold, 4-fold, 6-fold, 8-fold, 10-fold (2, 4, 6, 8, 10 respectively), and initial od=0.5 of chlorella at 25 ℃,50 μmol·photons·m ^-2 ·s ^-1 The cells were incubated in a 24-hour incubator for 8 days, and the dry weight and pH, fv/Fm, water quality (phosphate content) and F2 medium were measured daily as a control group, and the results are shown in FIGS. 1 to 2. The optimal dilution concentration of the culture solution is selected according to the dry weight and the nutrient salt removal rate of the chlorella, when the chlorella is diluted 8 times in the culture solution, the chlorella grows adaptively in the culture solution, the biomass (dry weight 1.35 g/l) is the highest, the phosphate removal rate (31.70%) is the best, and three levels of dilution concentration factors of the culture solution with 4 times, 6 times and 8 times of dilution concentration are selected to serve as the response surface for response surface experiments.

(4) Chlorella is inoculated in the optimal dilution concentration of culture solution, cultured at 15 deg.C, 25 deg.C and 35 deg.C, and its dry weight and pH are measured daily, fv/Fm and detection results are shown in figure 3. The optimal growth temperature is selected according to the dry weight of the chlorella, 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 optimal diluted concentration of wastewater, culturing at 50, 150, 250 μmol·photons·m ^-2 ·s ^-1 The dry weight and pH, fv/Fm, and the results of the measurements are shown in FIG. 4. The optimal growth light intensity is selected according to the dry weight of the chlorella, and the light intensity is 250 mu mol. Photons.m ^-2 ·s ^-1 In the environment of (2), the dry weight of each diluted concentration of wastewater reaches the highest, and the diluted concentration is the optimal light intensity for the growth of chlorella.

(6) Chlorella was inoculated in the optimal wastewater dilution concentration, and the initial pH value of the wastewater was adjusted to=4, 5,6,7,8,9, 10, and the dry weight and pH, fv/Fm, were measured every two days, and the detection results are shown in FIG. 5. The optimal growth pH was chosen according to the dry weight of chlorella, which grew well in neutral and alkaline environments, inhibited at ph=4, and the highest biomass (1.44 g/l dry weight) at ph=9, with ph=7, 8,9 being chosen as three levels of pH factor for the response surface experiments for the next step.

Taking 3 levels of the 3 factors (pH=7, 8,9; the temperature is 15, 25, 35, the dilution concentration is 4 times, 6 times, 8 times), taking dry weight as a response value as a response surface experiment, and screening the optimal environmental condition of the chlorella in the golden pomfret cooking liquid.

Table 1 shows the experimental results of chlorella response surface

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

Example 2

Setting an experimental group and a control group:

experimental group (noted 8 x), the culture steps included:

(3) Diluting the culture solution by 8 times with sterile water, regulating pH to 8-8.5, inoculating initial OD ₇₅₀ Chlorella with the illuminance of 250 μmol/photons/m at 23deg.C for 24 hr ^-2 ·s ^-1 Is cultured in a constant temperature incubator for 8 days, and is manually shaken 3 times a day at regular time.

Control group (labeled F2): f2 is used as a culture medium (pH is 8-8.5), and the initial OD is inoculated ₇₅₀ Chlorella with the illuminance of 250 μmol/photons/m at 23deg.C for 24 hr ^-2 ·s ^-1 Is cultured in a constant temperature incubator for 8 days, and is manually shaken 3 times a day at regular time.

Example 3

The difference from example 2 is that: after diluting the culture solution by 8 times, adding sea salt, sodium nitrate and acetic acid, wherein the concentration of the acetic acid in the culture solution is 1g/l, the sea salt is added to the salinity of 52.5 per mill, and the sodium nitrate is added to N: p=15:1, other steps are the same. Through detection, the growth amount of the chlorella reaches 0.825g/l, the phosphate removal rate reaches 97.36%, and the results are shown in Table 2 and FIG. 9.

Example 4

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

Example 5

In this example, the effect of adding acetic acid and not adding acetic acid on chlorella growth was examined, and the experimental group was set as follows:

experimental group:

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

Control group:

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

control group 2: a-8%o (15:1, 52.5%o), the control group was added with sea salt to a salinity of 52.5%o based on 8%o of the test group of example 2, and sodium nitrate to N: p=15:1, other steps are the same as in the experimental group of example 2.

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

Chlorella was cultured according to the methods of the control group and the experimental group, the cell number was measured every two days, the dry weight was measured on day 8, and the results of the measurement of chlorophyll, water quality (phosphate, total phosphorus, total nitrogen content) are shown in Table 2 and FIG. 9.

TABLE 2

The results show that compared with the treatment groups A-8, A-F2 and A-8 (15:1, 52.5 mill) without acetic acid, the treatment groups M-8, M-F2 and M-8 (15:1, 52.5 mill) with acetic acid are obviously improved in growth amount, chlorophyll content and phosphate removal rate, and the chlorella growth can be obviously promoted after acetic acid is added, so that the removal rate of chlorella to phosphate is improved.

The biomass of experimental group A-8 is significantly higher than that of A-F2, and its cell number is up to 1.65X10) ⁷ The dry weight of cells/ml reaches 0.491g/l, and the phosphate removal rate reaches 79.43%. The method shows that the chlorella can obviously improve the biomass of the chlorella by taking the golden pomfret cooking liquid as a culture medium and remove the phosphate of the cooking liquid.

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

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A method for culturing chlorella, comprising:

diluting the culture solution, regulating the pH value to 7-9, collecting chlorella in mid-log phase, centrifuging, inoculating precipitated algae into the diluted culture solution, and initially culturing OD ₇₅₀ Culturing for 5-8 days at 0.5;

the pretreatment comprises the following steps:

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

the dilution multiple is 4-8 times;

the culture method further comprises the step of adding acetic acid into the diluted culture solution;

the addition amount of the acetic acid is 0.25-2 g/L of the concentration of the acetic acid in the culture solution;

the temperature of the culture is 15-35 ℃;

the illumination conditions of the culture are as follows: 24-hour illumination, wherein the illumination intensity is 50-250 mu mol/photons/m ^-2 ·s ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Manually shaking 3 times a day in the culture period;

the step of adding sea salt and sodium nitrate is also included while adding the acetic acid;

the adding amount of the sea salt is 17.5-70 per mill of the salinity of the culture solution;

the addition amount of the sodium nitrate is as follows: p=10 to 25:1.