JPS5933353B2 - Chlorella cultivation method - Google Patents

Description

[Detailed description of the invention] The present invention relates to an effective method for culturing Chlorella.

Normally, chlorella is cultivated in a large culture tank in a medium supplemented with nutrients and exposed to sunlight, but chlorella grows extremely slowly and its productivity is low.

However, recently there has been an extremely strong demand for chlorella as a natural nutrient, and efficient production of chlorella is desired.

The present inventors conducted research to find an effective method for propagating Chlorella, and found that the addition of grain syrup significantly increases the propagation efficiency.

When chlorella is cultured by adding grain syrup to the medium, it is possible to obtain a 20% to 100% increase in bacterial weight compared to when culturing without adding chlorella.

Chlorella used in the present invention is Chlor-el
la ellipsoidea Gerneck IA
M 0-27, Chlorella ellipsoi
dea Gerneck IAM (1!-102, Ch.
lorella ellipsoidea ()e
rneckT, 1. (generally distributed strain by Tokuyo Research Institute).

There are various basic media for chlorella culture, but the following experimental medium is Myer 5-4NA5.
Medium (hereinafter referred to as N4N) is suitable.

Myers-4NA 5 medium composition (culture solution lt
Medium) KNO3...0.05M...1.
25. ! li! Mg5o, 7H20...0.
OIM・・・・・・1.25. ! i'KI(2PO4・
...0.009M...1.25gFeS
O4・7H20...10-6M...0
．． 02gB ・-・-0,5ppIn-・---H8
BO3-...-2,857nfIMn...
0.5 ppTn"・・Mn01.・4) L, 0-
・1.81 '119Zo・・・0. O5ppm
...4 mouths S0,7H20...0.22
7IlfIOu...0.02ppIIl...
...C! u So 4・5H20・・・・・・0.
0782η Mo----・0.01ppIn・----・3 (
NH4)20-7MO03-4H20...0.
171■ Of course, all conventionally used inorganic and organic culture solutions can be used for culturing Chlorella in the present invention.

In the present invention, a culture medium is prepared by adding only grain syrup to water, or by adding grain syrup to a conventionally used culture medium.

The grain syrup used here is commonly called Daystiller's Soluble, which is the waste liquid from distillation of whisky.

It is sufficient that the amount of grain syrup added is about 1 to 10% based on the culture solution.

The optimal amount for addition differs depending on whether grain syrup is added alone or in various media, so it is necessary to determine the amount to be added by measuring the maximum growth amount in each case.

Seed culture is performed prior to main culture.

For seed culture, a loopful of Chlorella 1 was inoculated into a basic medium such as M4N, and cultured under light irradiation at 25° Ctt'c with continuous ventilation of air containing about 1% carbon dioxide gas for about 5 days. A seed culture solution is obtained.

The seed culture is added to a large volume of grain syrup supplemented culture and incubated under sunlight.

The culture may be either standing still or aeration with agitation.

Cultivation is carried out for 4 to 8 days, and chlorella is obtained by filtering the culture solution.

The chlorella culture with grain syrup addition of the present invention can increase the yield of chlorella by 20-100%.

Next, test examples and examples of the present invention will be shown.

Test Example I M4N507rLl was placed in a small flat flask, and Ohlorella ellipsoidea Ger
neck IAMo-27, IAM O-102 and T
, I was inoculated with one platinum loopful of each of 9 to IOK l.
The cells were cultured at 25°C for 5 days under continuous aeration of air containing 1% monocarbon dioxide under UX light irradiation.

Collect 5 ml of algae liquid from the obtained seed culture solution and add 4
5ml of each test medium was inoculated.

The medium was 1.0.57711 grain syrup.
A mixture of 0% water suspension and 45 wLl of M4H (G.

S, 0.1% addition group), a mixture of 2.5 ml of 10% water suspension of grain syrup and 40 ml of M4N (G,
Three types were prepared: 8.1% addition group) and 3.45 ml of M4N (control group).

Each of these media was inoculated with 5 ml of algal liquid, and cultured in a small flat flask at 25° C. for 2 days under irradiation with light of 9 to 10 Klux while continuously aerating air containing 1% carbon dioxide gas.

The effect of adding grain syrup was at the start of culture and at culture 4.
Cell number and total cell volume (packed) of algae liquid at 8 hours
cell volume) were measured and compared.

The results are shown in Table 1.

As a result of Table 1, G, S, IAM C-102 and T,
1. It has been revealed that it has a proliferation-promoting effect on.

That is, the cell volume increase rate during 2 days of culture was higher than that of the control.
The increase in the 8.1% addition group was more than double, and the rate of increase in cell number was also nearly double.

Test example 2 0hlorella ellipsoidea Ge
Preculture was carried out in the same manner as in Test Example 1 using rne-ck T-I-, and main culture was carried out in the same manner with the addition of 2% and 5% grain syrup (G, S,) 10% suspensions.

The conditions are shown in Table 2 below.

In Table 2, especially for the G, 8.5% addition area,
In order to examine whether G and S can be used as energy sources, the cells were cultured in the dark.

At that time, as in Test Example 1, the number of cells and total cell volume were measured.

The culture results are shown in Table 3 below.

In Table 3, when comparing the proliferation when G and S were diluted only in water, it was found that after 2 days of culture, the cell volume in the water-only control (test group 1) was 3.3 times that at the beginning, and the cell number was 3.3 times that at the beginning. 4.7 times as much, whereas in the G, 8.2% added area (test area 3), the cell volume was 16.8 times, and the number of cells was 7.1 times, clearly G,
Growth was promoted by S.

G. Comparing the 8.2% addition (diluted in water) group (test group 3) with the control (test group 2) medium group, the increase in the number of cells during the 2-day culture was small; The increase in volume is approximately 1
．． At 7 times, the growth was promoted more than the medium.

From previous experiments, it has been found that when M4N'''N is cultured, the cell volume does not increase at all under light shielding.

In Table 3, G. Added S, under light shielding (test group 7)
When cultured in (8) and (8), the cell volume was approximately doubled.It is thought that some of G, S, and S are also used as energy sources.

However, since the increase was considerably greater under light irradiation (compared test plots 5 to 8), it is thought that G and S act more strongly as growth promoting factors than as energy sources.

The reason why the growth in the 8.5% G and 8.5% added plots (comparing test plots 3 to 6) was smaller than that in the 2% added plot is because G and S are colored substances that absorb irradiated light, which may affect growth. I think this is because not enough light was provided.

Normally, when cultured with M4N, the algae concentration reaches saturation after 6 to 7 days of culture.

The cell volumes on the 6th day of culture were compared.

As a result, as is clear from the table, G. The S-added group showed a volume increase rate 2 to 3 times that of the control medium group.

In this case, the color of G and S diluted in water increased to a yellowish tinge.

Test Example 3 A 10% water suspension of grain syrup was centrifuged (i, o
Prepare the supernatant solution (o ox, y, to minutes) and make a culture medium by adding it to water at a concentration of 5%, 2%, 1%, 0.5%, 0% (control). , and also % concentration of M4N
Culture media were prepared at concentrations of 5%, 2%, 1%, 0.5%, and 0% (control).

These media were packed into test tubes, and then Chlorella
llipsoidea Gerneck T, 1. The stock solution was seed cultured in the same manner as in Test Example 1, and the OD of 660 nm was
The value is approximately 0.1, then 90 per minute.
Shaking back and forth.

The cells were cultured with shaking at 25°C under 2 Klux irradiation.

For each, the OD value at 660 nm (OD660) was measured over time during the culture.

The results are shown in FIGS. 1 and 2.

Figure 1 shows the case where G, S, and supernatant liquid are added to water.
A is 5%, B is 2%, C is 1%, D is 0.5%, E is 0
% (control) is shown respectively.

Figure 2 shows the case where G, S, and supernatant liquid are added to M4N at a concentration of %, F is 5%, G is 2%, H is 1%, ■ is 0.5%, and J is 0%. (control) are shown respectively.

As is clear from the figure, even when cultured in water, the concentration of M
When cultured in 4N, growth promotion increased as the concentration of G and S added increased.

Example 1 Pour 530 ml of water into a 1000 ml transparent plastic cylindrical culture tank.
and then add 10% grain syrup suspension to 170%
to adjust the culture medium.

This includes Chlorella ellipsoidea
5 of Gerneck IAMO-27 seed culture solution was added and cultured at 15 to 25°C (natural conditions) for 7 days under natural conditions of irradiating sunlight only during the daytime with air aeration.

The number of cells obtained was 8 x 1010ce11s/l.

5.6X1010cells/l for culture with water only
Met.

Example 2 Similar to Example 1, water was replaced with M4N and 0hlo
rella ellipsoidea Gerneck
IAMC-27 was cultured.

After 7 days of culture, the number of cells obtained was II'Xl010c
e11s/l, but the volume increase rate was 17 times.

On the other hand, in the case of only M4N, the number of cells is 12 and 010 ce l.
I s /l, but the volume increase rate was only 11 times.

[Brief explanation of the drawing]

Figure 1 shows the culture curves for each aqueous medium in Test Example 3, with A being 5%, B being 2%, C being 1%, D being 0.5%,
E indicates 0%, Figure 2 shows the culture curve in each M4N medium in Test Example 3, F indicates 5%, and G indicates 2%.
%, ■ indicates 1%, ■ indicates 0.5%, and J indicates 0%.

Claims (1)

[Claims] 1. A method for culturing chlorella, which is characterized in that 1 to 10% of grain syrup is added to the medium when culturing chlorella.