CN110699258B

CN110699258B - Culture method for improving chlorella cell biomass

Info

Publication number: CN110699258B
Application number: CN201911127852.6A
Authority: CN
Inventors: 朱至; 蒋继宏
Original assignee: Jiangsu Normal University
Current assignee: Jiangsu Normal University
Priority date: 2019-11-18
Filing date: 2019-11-18
Publication date: 2021-09-24
Anticipated expiration: 2039-11-18
Also published as: CN110699258A

Abstract

A culture method for increasing cell biomass of Chlorella comprises adding culture medium into photoreactor, sterilizing at high temperature, and inoculating Chlorella pyrenoidosa; inoculating bacillus subtilis natto after a period of time; introducing compressed air under a certain light intensity and temperature condition for culture; obtaining chlorella and natto bacillus subtilis biomass. The culture method provided by the invention promotes the improvement of the biomass yield of the chlorella by constructing a dual-bacterium collaborative co-culture system, and overcomes the problem of high culture cost caused by low biomass in the chlorella culture process. The bacillus subtilis natto is applied to the algae culture process, the bacillus subtilis natto is a well-known safe probiotic, the chlorella and the bacillus subtilis natto biomass obtained by the double-bacteria synergistic co-culture process belong to safe and non-toxic products, and the further processing can be carried out according to different requirements of commercial application.

Description

Culture method for improving chlorella cell biomass

Technical Field

The invention relates to the technical field of algae cultivation, in particular to a cultivation method for improving algae cell biomass in a chlorella cultivation process.

Background

Photosynthetic algae have been widely used in the fields of human health food, medicine and high-value animal feed, and in addition, environmental pollution and potential energy crisis worldwide have made increasing attention to the development of biofuels from algal biomass as a raw material (Zhang, w., p.zhang, h.sun, m.chen, s.lu and p.li.effects of vacuum organic carbon source on the growing and biochemical composition of Chlorella pyridoidosa. bioreduction Technol,2014,173: 52-58). Chlorella is a kind of universal unicellular green algae. The chlorella algae cells are rich in various high-value compounds, about 40 percent of protein, and the amino acids in the protein are complete in variety, comprise eight essential amino acids for human bodies, and have balanced composition; about 25% lipids, and unsaturated fatty acid content higher than many plants; about 20% of saccharides including active polysaccharides having algal cell characteristics, plant growth factors, and the like; about 5% cellulose; about 10% of vitamins and trace elements, wherein the vitamins include VB1, VB2, VB6, VB12, VE, VC, nicotinic acid, folic acid, pantothenic acid, biotin and the like; the trace elements include K, Na, Ca, P, Mg, Fe, etc. The chlorella has comprehensive and balanced nutritional value, so that the chlorella has higher application value in the field of functional foods, is listed as a green nutritional source health food for human in the 21 st century by the food and agricultural organization of the United nations, and has various physiological effects of resisting tumors, preventing and treating arteriosclerosis, resisting radiation and the like (Yinhua, Liyan group, Chua Qizhen, and the like, the recycling of a culture solution of the chlorella pyrenoidosa, the institute of Guangdong ocean university, 2019, volume 39, phase 4, pages 49-55) proved by intracellular bioactive substances proved by the people who have improved immunity of the organism. Meanwhile, the oil content in the chlorella cells can be remarkably increased to 50-70% under the condition of nitrogen stress, so that the chlorella cells become one of biological liquid fuel sources with huge potential. In conclusion, the development and utilization of chlorella have extremely wide market prospects. And the large-scale culture and the acquisition of chlorella biomass are the precondition for developing various application researches. Therefore, the development of a culture process for improving the biomass of chlorella aiming at a culture system of chlorella is urgently needed, and the improvement of the biomass also has positive practical significance for controlling the cost of the whole chlorella industry.

Disclosure of Invention

The invention aims to provide a culture method for improving the cell biomass of chlorella to solve the problem of low cell concentration of chlorella generally existing in the process of culturing chlorella.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

a culture method for increasing the cell biomass of chlorella algae comprises the following steps:

s1: adding a culture medium into a photoreactor, sterilizing at high temperature, and inoculating chlorella pyrenoidosa;

s2: inoculating bacillus subtilis natto after a period of time;

s3: introducing compressed air under a certain light intensity and temperature condition for culture;

s4: obtaining chlorella cell biomass.

Furthermore, the inoculation number ratio of the chlorella in S1 and S2 to bacillus subtilis natto is 1-5: 10-1.

Further, the step S2 specifically includes: inoculating bacillus subtilis natto after 0-72 hours for double-bacterium co-culture;

further, the step S3 specifically includes: at a light intensity of 50 μ Em^-2s^-1And introducing 0.1vvm of compressed air at the temperature of 30 ℃ for culture.

Further, the culture medium is BG11 culture medium.

Compared with the prior art, the invention has the beneficial effects that:

according to the culture method, the double-bacterium collaborative co-culture system is constructed, so that the improvement of the biomass yield of the chlorella is promoted, and the problem of high culture cost caused by low biomass in the chlorella culture process is solved;

according to the culture method, the bacillus natto is applied to the algae culture process, the bacillus natto is recognized safe probiotics, the chlorella obtained by the double-bacteria collaborative co-culture process and the bacillus natto biomass belong to safe and non-toxic products, and the further processing can be carried out according to different requirements of commercial application.

Drawings

FIG. 1 is a graph showing the change of chlorophyll content of chlorella in a culture system under coculture conditions of chlorella alone culture and inoculation of Bacillus subtilis natto and chlorella in an amount of 1:10 in a glass photoreactor according to example 1 of the present invention, with inoculation being performed for 0 hour at the beginning of chlorella culture;

FIG. 2 is a graph showing the change of chlorophyll content of chlorella in a culture system under coculture conditions of chlorella alone culture in a glass photoreactor and inoculation of Bacillus subtilis natto and chlorella in an inoculation amount of 1:1 and inoculation after culturing chlorella for 24 hours in example 2 of the present invention;

FIG. 3 is a graph showing the change of chlorophyll content of chlorella in a culture system under coculture conditions of chlorella alone culture in a glass photoreactor and inoculation of Bacillus subtilis natto and chlorella at an inoculation amount of 5:1 and inoculation after culturing chlorella for 72 hours in example 3 of the present invention.

The specific implementation mode is as follows:

the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The chlorella pyrenoidosa used in the invention is preserved in freshwater algae seed bank of Chinese academy of sciences with the number of FACHB-5; the bacillus subtilis natto is preserved by China industrial microorganism strain preservation management center with the number CICC 10262.

Example 1

And (3) inoculating the bacillus natto into a culture system according to the inoculation ratio of the bacillus natto to chlorella cells of 1:10 when the chlorella is cultured for 0 hour initially.

The specific method is that 500mL of photoreactor is added with BG11 culture medium, and 4X 10 is inoculated after high temperature sterilization⁷Inoculating 4 × 10 Chlorella pyrenoidosa simultaneously⁶Bacillus subtilis natto with light intensity of 50 μ Em^-2s^-1And introducing 0.1vvm of compressed air at the temperature of 30 ℃ for culture.

FIG. 1 shows the trend of the biomass of Chlorella pyrenoidosa, characterized by the total chlorophyll content, in the individual culture and co-culture systems. As can be seen from figure 1, the biomass of chlorella is obviously increased in the whole culture process by adopting the co-culture process, 30-36mg/L chlorophyll content can be stably obtained by singly culturing the chlorella, 42-48mg/L chlorophyll content can be stably obtained by co-culture, and the biomass can be increased by 33% by respectively taking the highest value for calculation.

Example 2

Culturing chlorella for 24 hours, and inoculating the bacillus natto into the culture system according to the inoculation ratio of the bacillus natto to chlorella cells of 1: 1.

The specific method is that 500mL of photoreactor is added with BG11 culture medium, and 4X 10 is inoculated after high temperature sterilization⁷Culturing Chlorella pyrenoidosa for 24 hr, inoculating into the culture medium 4 × 10⁷Bacillus subtilis natto with light intensity of 50 μ Em^-2s^-1And introducing 0.1vvm of compressed air at the temperature of 30 ℃ for culture.

As shown in the attached figure 2, the chlorophyll content of 33-41mg/L can be stably obtained by comparison with the culture process of the chlorella in the single culture, while the chlorophyll content of 82-85mg/L can be stably obtained by co-culture, and the biomass can be increased by 107% by respectively taking the highest value for calculation.

Example 3

Culturing the chlorella for 72 hours, and inoculating the bacillus natto into the culture system according to the inoculation ratio of the bacillus natto to the chlorella cells of 5: 1.

The specific method is that 500mL of photoreactor is added with BG11 culture medium, and 4X 10 is inoculated after high temperature sterilization⁷Culturing Chlorella pyrenoidosa for 72 hr, inoculating into 2 × 10⁸Bacillus subtilis natto with light intensity of 50 μ Em^-2s^-1And introducing 0.1vvm of compressed air at the temperature of 30 ℃ for culture.

As shown in figure 3, in this example, 30-35mg/L chlorophyll content can be stably obtained by comparison with the culture process of Chlorella vulgaris in single culture, and 43-45mg/L chlorophyll content can be stably obtained by co-culture, and the biomass can be increased by 28% by taking the highest value respectively. .

Claims

1. A culture method for increasing the cell biomass of chlorella is characterized by comprising the following steps:

s1: adding culture medium into photoreactor, sterilizing at high temperature, and inoculating Chlorella pyrenoidosa (C. pyrenoidosa)Chlorella pyrenoidosa ）；

S2: inoculating Bacillus subtilis natto after 0-72 hoursBacillus natto) Performing double-bacterium co-culture, wherein the inoculation number ratio of chlorella in S1 and S2 to bacillus subtilis natto is 1-5: 10-1;

s3: at a light intensity of 50 μ Em^-2s^-1Introducing 0.1vvm of compressed air at 30 ℃ for culture;

s4: obtaining chlorella cell biomass.

2. The culture method according to claim 1, wherein the medium is BG11 medium.