JPS5926266B2 - How to grow diatoms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of resistant diatoms (Phae odac ty
The present invention relates to a method for growing diatoms such as lumtricornutum.

Diatoms can be grown by exposing them to light in a culture solution mainly composed of NaNO3 and 2HPO4, and some substances, such as soil exudate, or a plant hormone called cytokinin, can be grown in this culture solution. It is known that growth is promoted by adding .

However, in the case of soil leachate, the growth promoting effect is insufficient, and plant hormones can have a promoting effect or, conversely, a growth suppressing effect depending on conditions such as the amount added, so reliable effects are not expected. The drawback was that it couldn't be done.

An object of the present invention is to provide a method for growing diatoms that consistently provides good and stable growth effects.

According to the results of numerous experiments conducted by the present inventors, common soil leachate obtained from substances such as soil, compost, and sludge through conventional hot water extraction can be added to the culture medium in its sludge form. Although the growth-promoting effect when added is not significant, repeated filtration using molecular sieves
It has been found that it is possible to separate a fraction that has a remarkable growth-promoting effect and a fraction that shows a growth-suppressing effect.

That is, it is clear that the soil infiltration liquid contains both a component that has a promoting effect on the proliferation of diatoms and a component that suppresses the growth of diatoms.

As a result of investigating what kind of substance is the active ingredient in the fraction that has a proliferation-promoting effect, the present inventors discovered that one of the main active ingredients is 2'-deoxyadenosine. It was completed.

The outline of this experiment is as follows.

Search for growth-promoting substances A boiling water extraction concentrate of fully ripened rice straw compost was selected as the standard soil infiltration solution, and 144 m9 of solid matter was obtained by freeze-drying this.
(equivalent to 220 g of compost) was suspended in 1.5 ml of distilled water, and the insoluble matter (25 mI?) was centrifuged (100 mI?).
00 rpm, 30 m1n).

The obtained supernatant was subjected to gel filtration chromatography (carrier: Sephadex Gl 5 , 16×847 mm, elution solvent: 0.03 M ammonium acetate aqueous solution, pH 6,7).

The eluate is ■Fuji 2F 3F 50 Then, each fraction was lyophilized to dryness.

As a result of the above purification operation, growth promoting activity was observed in the 9th fraction of gel filtration chromatography.

This ninth fraction was then subjected to high performance liquid chromatography (HPLC) in order to further purify the active substance.
The detected peak of 254 m1n showed that
It was divided into fractions (A-M).

Details of HPLC are as follows.

Column: Micro Honda Pack CI8 l Separation: 5% acetonitrile-ammonium acetate aqueous solution (0, OLM, pH 4,0) Detection method: Results of ultraviolet absorption biological test at 254 mm and 2807 ILr/L, remarkable in fractions C and E. Proliferation promoting activity was observed.

Fractions C and E are almost single compounds based on HPLC characteristics (hereinafter referred to as "compounds C and E")
Compounds C and E were extracted from a large amount of compost in order to isolate them in amounts that would allow structure determination.

Isolation of Growth Promoting Substances 18.2 g of solid matter obtained by freeze-drying 1.671 g of boiling water extract of 22.5 ky of compost was suspended in 120 ml of distilled water and dissolved in cine by centrifugation (10,000 rpm, 40 ml). (4.10 g) was removed.

The supernatant was divided into two portions and subjected to ion exchange chromatography (carrier: SP Sephadex, 26 mrn x 917 mm).

First, ammonium acetate aqueous solution (0, OLM, pn4.
0) 720m1. The impurities in the lower part were eluted and removed.

Next, ammonium acetate aqueous solution 1 (0.01M, pH 6,
5) Among the fractions eluted at 720 ral, fractions containing compounds C and E were combined.

The weight of this combined fraction is 37% of the compost after freeze-drying.
It was 79■ per kg.

Furthermore, for the purpose of purification, gel filtration chromatography (carrier: Sephadex G15, 10 mm x 10 mm x 570
Solvent: 0.03M 77M acetate aqueous solution, pH 6,
5), and after removing 68 ml of the initial eluate, 12 ml containing compounds C and E were dried to obtain a little less than 1 ml of compound material.

To finally isolate compounds C and E, HPLC
(Column: Radial Pack C18, eluent: 8% acetonitrile-ammonium acetate aqueous solution (0, OLM, p
H4,0), detection method: same as above).

As a result, compounds C and E, which were single in HPLC, could be obtained.

Physicochemical data of compound C 1) Ultraviolet absorption spectrum 2) Mass spectrometry spectrum + rn/e: 267 (M), 250.237.
178 +164.148,136.135 (Base heating L 119,108 3) Nuclear magnetic resonance spectrum (270MHz, DMSO-d6.δ) 3.56
(LH2da t 5'a ) 3.67 (LH, dd
, 5'b) 3.96 (LH, d'd, 4') 4.14 (tH, t, 3') 4.60 (LH, t, 2') 5.87 (LH, t, 1' ) 8.15 (LH,'S, 2or8) 8.37 (LH2S 58or2) J5t, , 5b=12.2H2 J4', 5'8-J4', 5'1) -3゜6J3,4-
41 J2', 3'-54 J1'+2'-5,5 Physicochemical data of compound E (1) Ultraviolet absorption spectrum (2) Mass spectrometry Svek 1 Herm m/e: 251 (M+), 234.221 ,1
64°162.136,135 (base peak).

119.108 (3) Nuclear magnetic resonance spectrum (270I117IE (Z, DMSO-d6.δ)2.
25 (IH, m, 2'a) 2.72 (LH, m, 2'b) 3.50 (II (+ dd, 5'a) 3.61
(LH, dd, 5'b)3.88 (IHtrn
,”) 4.40 (LH, m, 3') 6.33 (LH+rn, ]-') 8.12 (LH, S, 2or8) 8.32 (LH, S, 8or2) J5'a+5'l) = 1.19) (Z J4', 5'a = J4'+ 5'l) = 4
．． 0J3', 4'-2,6 J2 a+3 = 2.8 J2'b, 3'-5,6 J2 a+2 b = 13.3 Jl, 2a-6,3 Jtt21) = 7.6 Compound C and Compound E The structural compounds C and E were estimated to be adenosine and 2'-deoxyadenosine, respectively, from the above-mentioned physicochemical data and chromatographic behavior.

Therefore, we compared the physicochemical data with standard products of adenosine and 2'-thioxyadenosine, and found good agreement between the two.

Also in HPLC, Compound C and Compound E each showed the same retention time as the standard product.

It should be noted that Compound C and Compound E isolated from 37 kg of compost could not be weighed due to their trace amounts, but they were purified by HP using standard products adenosine and 2'-deoxyadenosine.
When quantified by LC, each was about 0. ITnI? Met.

From the above results, it has been chemically determined that the growth promoting substance in the soil leachate is 2'-deoxyadenosine, which is represented by the following structural formula, in addition to adenosine.

The present inventor confirmed that the growth-promoting substance in soil leachate is adenosine, and filed an application for a method for growing diatoms in a culture solution to which adenosine was added [7].
7-29991).

Therefore, this invention provides 41 that diatoms are grown in a culture solution to which 2'-deoxyadenosine, which has been newly confirmed to be contained in soil leachate, is added.

Also, in the case of 2-deoxyadenosine, as in the case of adenosine, the growth promoting effect varies greatly depending on the amount of 2'-deoxyadenosine added.

According to the experimental results, when the amount of 2'-deoxyadenosine added is small, a remarkable growth-promoting effect is obtained, but when the amount added exceeds a certain limit, a growth-suppressing effect appears, and this limit is approximately 0. It was confirmed that it was OOlppm.

Note that whether 2'-deoxyadenosine is obtained by enzymatic or pressure hydrolysis of yeast nucleic acid or synthesized, its growth-promoting effect remains the same.

A preferred culture solution used in the method of this invention has, for example, the following basic composition.

Basic composition of culture solution NaNO3320mg K2HPO485 ■ FeC130,78 ~ MnC120,06m9 EDTA 8 ■ Seawater 600m1 Pure water 200m1 In addition to this basic composition, about 0.000 lppm or less 2
By growing diatoms according to a conventional method using a culture solution containing 7-thioxyadenosine,
Although there was no significant difference in the rate of increase in the number of individuals, a significant increase in dry weight was confirmed.

Example 1 Culture bottles each containing 800 ml of culture solution having the above-mentioned basic composition were prepared, and a predetermined amount of 2'-deoxyadenosine was added to each bottle.
A predetermined amount of Eodactylum tricornutum) was inoculated and cultured.

This culture is continuously irradiated with artificial light of approximately 18,000 lux while air is pumped approximately 1.2 lux for CO2 supply.
It was introduced at a flow rate of 51.7 ml and carried out at 20° C. for 10 days.

After the cultivation was completed, the population, compaction amount, and dry weight of the diatoms were measured.

In addition, cultivation was carried out under the same conditions without adding □ 2'-deoxyadenosine and in which 4 ml of the soil infiltration solution was added to the 2-seed deoxyadenosine substitute.

These results are summarized in Table 1.

In addition, the number of individuals is the number of individuals per rnl of culture solution.
It is expressed as r Counter value.

Example 2 A culture experiment similar to Example 1 was conducted, and the results shown in Table 2 were obtained.

Example 3 The culture experiment was repeated under the same conditions as in Example 1, and the results shown in Table 3 were obtained.

Based on the results obtained in Examples 1 to 3 above, the dry weight of diatoms obtained using a culture solution with only the basic composition was set as 100, and the dry weight in the case of other culture solutions was calculated. The results are shown in Table 4.

As is clear from the above results, approximately 0.00
When 2'-deoxyadenosine is added at a rate of 0.01 ppm or less, the basic composition alone, the soil exudate is added, and 2'-deoxyadenosine is added at a rate of 0.01 ppm or less.
It can be seen that the dry weight is significantly increased compared to the case where it is added at a ratio of 0,001 or more.

Claims (1)

[Claims] 1. A method for growing diatoms, which comprises growing diatoms in a culture solution to which 27-deoxyadenosine has been added at a rate of about 0.000 lppm or less.