JPS5832590B2 - Diatom algae growth method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION As part of the method for effectively utilizing solar energy, the present invention aims to investigate the effects of diatoms, a single-celled plant known by microscopic observation to contain a large amount of oil droplets and which is autotrophic with respect to carbon and nitrogen sources. The purpose is to propagate them and extract useful oil from them to use as an energy source or resource.

The diatom taken up in this invention is the resistant diatom Phaeod.
actylum tricornutum.

At the beginning of the research, we selected a Haxo-8weeny culture solution from a large number of diatom culture solutions, poured 31 culture solutions into 51 turnip flasks, sterilized them, and then added 15 to 181 rL of diatoms for transplantation. inoculate,
The number of individuals is approximately 50,000/ml, and the air is aerated (513/ym).
By doing so, CO2 contained in the air is supplied, and a weak light of approximately 8,000 lux is supplied mainly from the bottom surface. 7 in a bright room (air bath) at approximately 20°C under continuous irradiation with artificial light (fluorescent lamp + incandescent lamp)
Culture was performed for ~8 days.

During this period, a certain amount of the culture solution was removed aseptically every day and the increase in the number of diatoms was measured using a Coulter counter and a hemocytometer. After the culture was completed, all the diatoms were collected by centrifugation and the water was removed. We measured the amount of compressed algae and its dry weight, and decided to collect diatoms based on the dry weight.

The composition of the Haxosinii medium is as follows.

The yield obtained from the culture solution of approximately 2.711) at the time of 31 measurements by the above method is as follows.

During this time, we researched the amount of soil exudate added to the culture solution, and found that 2%
The addition was revised to 5%.

In addition, the N source was changed after confirming that the effect remained the same even if cheaper NaNO3 was used instead of KNO3.

Since the amount of NaNO3tK2HPo4 added was relatively small, it seemed better to use a slightly larger amount for the purpose of large-scale culture of diatoms, so we attempted an experiment to double the amount. However, the yield actually decreased.

Subsequent research has increased the number of culture days from 7 to 8 days to 10 to 13 days.
It was found that the number of individuals hardly increased when extended to a day, but the amount of compaction and dry weight increased.

This seems to mean that each individual diatom is growing larger, and based on this finding, the number of days for subsequent culture was set at about 10 to 14 days.

Also, after changing the number of culture days from 10 to 14, Na
No 3 z K2 As a result of considering increasing the amount of HPO4 added, the result obtained earlier was obtained, and NaNO3 was doubled (N
2) It was found that the best combination of 2HPO4 was 3 times (P-3).

Furthermore, in line with conventional research on mass culture of diatoms, all culture solutions were sterilized using a pressure cooker and cultured aseptically at the beginning of the research.

It was predicted that this sterilization operation would be the most difficult point in the future when mass production is carried out outdoors in the open.

Try 2x NaN03(N 2)t 3x 2H
When culturing was carried out without sterilization using PO4 (P-3) and 5% soil exudate (S, E,) medium (only aeration was performed with sterilization), microbial contamination (
contamination) did not occur at all.

This seems to be due to the condition of the culture solution, which is considered to be oligotrophic even in this state.

This allowed subsequent experiments to be carried out without sterilization, simplifying the procedure and guaranteeing a large number of possibilities for the future.

As a result of the improvements in the above points, the production volume has far exceeded that at the beginning of the research, as shown below. The soil leachate (S, E,) that has been used in the past is heated by heating 1 kg of soil with 11 parts of water. 171 with the extracted liquid! It contains only about 100 cubic centimeters of solid matter, and only a very small amount of active ingredients.

This substance is thought to contain trace amounts of elements (hormones, etc.) necessary for the growth of diatoms, and the identification of this hormone-like substance is an important objective of research.

For this purpose, for example, large amounts of extraction treatment using various organic solvents are required, but soil inherently has characteristics that make it unsuitable for such methods.

Therefore, as an attempt, rice straw was composted using only water, and compost leachates (F, E,) were prepared by heating and extracting 1 kg of compost with 11 water.

The compost leachate (F, E,) contains about 750 solids per liter, and when used as the compost leachate (F, E,), which has the same amount of solids as the soil leachate (S, E,), the results are extremely As a result, soil leachate (S
, E,) was found to improve the collection rate.

Compost can also be extracted with methanol, and in this case,
It is considered suitable for researching active ingredients because it is easy to analyze.

Twice as much NaN0s (N2), three times as much as 2HPO4
(P-3) and a culture solution containing compost leachate (F, E,), the yield became even better as shown below.

Also, 2 times NaNOs (N 2 ), 3 times 2H
PO4 (P-3), 5% compost leachate (F, E,) culture solution (hereinafter referred to as modified Haxosinii culture solution),
211 flat culture bottles (thickness approx. 4
crrt) with 1.5β of the culture solution and 20.
When culturing was performed using artificial light of 000, 15,000, and 8,0001 ux, results were obtained that completely contradicted conventional theory, and it was found that the yield was much higher with strong light.

This can be considered as follows.

In other words, the composition of the culture solution used conventionally was not optimal for the growth of diatoms, and something was lacking, so the so-called "Liebing's law of least amount" determined that the missing ingredient was rate-limiting. Therefore, no matter how strong the light was, the proliferation in proportion to the amount of light did not increase, but twice as much NaN
03 (N-2), 3x 2HPO, (P-3) 5% compost leachate (F, E,) culture solution contains this deficient component (probably Mormon-like contained in the compost leachate (F, E,) material)
It is thought that the composition of the culture solution was close to perfect because it contained sufficient amount of light, and light became rate-limiting.

Under this light condition of 20,000 lux, the yield was as follows.

The value obtained when culturing 1.51 of the culture solution in 21 flat culture bottles was doubled and converted to 31 of the culture solution.

This result shows that diatoms can grow from sunlight outside (even on cloudy winter days).
This indicates that the prediction that growth would be poor under strong light (1,000 lux) indicates that this is just a culture, and this guarantees the possibility of mass production in the future. It has been thought that the growth rate is slowed down because they produce substances that inhibit their growth, but no research has been conducted to prove whether this is true or not.

Focusing on this point, in the middle of culturing (7 pewter) activated carbon was added using 50η
As a result of repeated attempts to add this to 31 culture solutions, in rare cases the yield of the added plots may be lower.

By adding activated carbon, an increase in dry weight of about 17.6% on average was observed, and by adding this treatment, the yield increased dramatically, as shown below.

In parallel to culturing using only artificial light in the laboratory, culturing using sunlight was experimentally carried out using 51 turnip flasks in an acrylic aquarium (20°C) installed in a greenhouse.

Six 54-cubic flasks were placed in the acrylic water tank, and four of them were illuminated with a fluorescent light from the bottom at night (including during the day when the illuminance was 10,000 lux or less).

Therefore, two lights were illuminated by sunlight during the day, and the remaining four lights were illuminated by sunlight during the day and artificial light at night.

After installing this device and performing several trials, the following combination of experiments was conducted twice.

Chlorella is Myers-, which is said to be the best for chlorella culture.
4NA5 medium was used.

However, since the optimum temperature for growing chlorella is 25℃,
℃ is considered to be quite low.

From the above results, the total cumulative amount of sunlight is 7 in the case of completely clear skies.
Although it is about 1.4 times the amount of rainy days and 7 days of cloudy weather, there is almost no difference in the yield of diatoms, and artificial light irradiation at night does not have much effect on the increase in yield.

In other words, it can be said that it is possible to expect a considerable yield by cultivating diatoms in an extensive manner and using only daytime sunlight, regardless of whether it is rain or shine.

Regarding the effects of S and E, there is a theory that it is based on vitamin Bl (V Bl ) or vitamin B12 (VBI In order to examine the effect of adding VB1□, the following culture test was conducted.

(I) Various concentrations of VB and VB, 2 effect-modified Hakeso Sini medium (does not contain F, E, etc.) 〇 Six-chain culture device (light source, incandescent lamp 20.0OO1ux) 10-day culture, no activated carbon Addition.

Conventionally, the amount of VBl and VB1□ added to diatom culture solution as growth promoting elements is 0.5 ppm each.
, O,OO2 ppm, which agrees with the above results.

Therefore, further research was carried out using VB1 and VBl2 at these concentrations.

(II) F, E, (5%) 2 V Bl (0,5p
pm) j Effects of VBl2 (0,002 ppm) alone or in combination Whether or not F, E, includes vBl, ■B12 must go through the results of the analysis of F, E (currently under consideration), but the above The result is F at least under strong light conditions.
, E, added plot has better yield than VBl, VB1□ single addition plot, and VB1+F, E, plot and VVB1+VB1□
+F, E, indicates that the highest yield is obtained in the ward; F;
This is considered to indicate that there is a component different from VBl or vB12 among the active ingredients contained in E.

Therefore, vitamin B1. It is also an aspect of the present invention to perform culture by adding vitamin B1□ or a substance containing vitamin B1□.

Currently, wastewater from sewage treatment plants is promising as a "substance containing this substance," and since it also contains nitrogen bodies and phosphorus bodies, it is presumed to be suitable for main culture.

(I [) Finally, using a six-chain culture device, using an incandescent lamp equivalent to 20.000 lux as a light source, and adding activated carbon (same method as above), the cells were cultured for 10 days (under strong light). The following results were obtained, and the dry amount per 31 culture fluids was actually 5.52. ! 9, VBl, VB, 2.
The combination of F, E, and activated carbon gave the highest yield.

Claims (1)

[Claims] 1. Compost leachate is used instead of soil leachate in the culture solution of Haxosinii, and twice the amount of NaN is used instead of KNO3.
Using Os, and using a modified Hatasosinii culture solution containing three times the amount of 2HPO4 instead of 2HPO4, supplying CO2 to this, and culturing diatoms under irradiation with artificial light or sunlight. A method for growing diatoms characterized by 2. The method according to claim 1, wherein the illuminance of the irradiation is strong, such as about 20,000 lux or more. 3. The method according to claim 1, wherein the culturing is carried out for a long period of about 10 to 14 days. 4. The method according to claim 1, in which the CO2 is supplied with sterilized air. 5. The method according to claim 1, in which the culture is performed without sterilization, except that the CO2 is supplied with sterilized air. Method described. 6. Compost leachate was used instead of soil leachate in the Haxosinii culture solution, and twice the amount of NaN was used instead of KNO3.
O3 was used, and activated carbon was added to a modified Haxosinii culture solution containing three times the amount of 2HPO4 instead of 2HPO4, and CO2 was supplied to this, and the culture was incubated under irradiation with artificial light or sunlight. A method for growing diatoms, which is characterized by culturing diatoms. 7. The method according to claim 6, wherein the illuminance of the irradiation is strong, such as about 20,000 lux or more. 8. The method according to claim 6, wherein the culturing is carried out for a long period of time, about 10 to 14 days. 9. The method according to claim 6, wherein the CO2 is supplied using sterilized air. 10. The method according to claim 6, wherein the culture is carried out without sterilization, except that the CO2 is supplied with sterilized air. 11 Use compost exudate instead of soil exudate in the culture solution of Haxosinii, and double the amount instead of KNO3 (7)
Using N a NOa, and adding vitamin B1, vitamin B12, or a substance containing them to a modified Haxosinii culture solution containing three times the amount of 2HPO4 instead of 2HPO4. , and CO2
A method for growing diatom algae, which comprises supplying diatom and culturing diatom algae under irradiation with artificial light or sunlight.