JPS5847159B2 - Method for producing flocculating active substances using microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a flocculant using microorganisms, and more specifically to a method for producing a flocculant using a microorganism, and more specifically, a method for producing a flocculant using a microorganism, which is a flocculant producing bacterium belonging to the genus Dematium in the family Dematiaceae. The present invention relates to a method for culturing A4257 bacteria to produce an agglutinating active substance that has agglutinating activity not only against proteins but also against organic substances, inorganic substances, minerals, biological cells, and the like.

Conventionally, methods for producing protein aggregation active substances using microorganisms have been proposed, for example, in JP-A-51-86189 and JP-A-51-115993, but the present invention is based on methods that are different from the microorganisms disclosed in these publications. Using microorganisms, flocculation activity has excellent flocculation activity not only for proteins but also for a wide range of colloidal substances such as organic substances, inorganic substances, minerals, biological cells, etc. suspended, dispersed, and floating in liquids. It provides a method for producing substances.

The microorganism used in the method of the present invention is microorganism microorganism number i425.
No. 7, and the bacterium of the present invention belongs to the family Black Mycoceae (Demaceae).
tiaceae) of the genus Dematium (Demat iu)
It is a flocculating active substance producing bacterium belonging to the group M), and its mycological characteristics are explained below.

Mycological Characteristics of the Isolated Bacteria The colony initially develops as a smooth, off-white, slimy, glossy, oil drop-like (fat-like) yeast-like structure, and filamentous fungal bodies grow radially from the periphery, giving it a curly appearance. It is filamentous and has dendritic growth.

These filamentous fungi grow well not only on the surface of the medium but also in the soil.

After a while, light brown spots appear on the surface of the colony, which gradually turn into black spots until the entire surface becomes dark black.

A large number of pale brown oval or oval conidia are attached to this filamentous fungal body.

These conidia easily break apart.

On the other hand, conidia are also attached to the surface of the oil droplet-shaped colony.

The culture solution containing sugar becomes very viscous and produces thick colonies of leathery black moss on the surface of the solution.

The optimum growth temperature is 20 to 25°C, at which alcohols and organic acids are produced from sugars such as glucose and sucrose, and they also have a unique aroma.

■Culture characteristics* (Note) ■Solid medium: Potato, on glucose agar medium. Initially, the colony has a smooth surface, is transparent, has a shiny oil drop shape, and is viscous, grayish-white, yeast-like, and curls radially from the periphery of the colony. Filament-like, just dendritic, fungal cells grow, and these filament-like fungi grow well not only on the surface of the medium but also inside the medium.

Eventually, some parts of the tree-like branches turn blackish brown.

After 3 to 4 days of culturing, light brown spots appear on the surface of the colony, which gradually becomes dark black and spreads over the entire surface, and finally becomes black as a whole (after 7 days of culture).

Although growth is slow on Zapek agar medium, the culture characteristics are as described above. It takes about 3 weeks for the colony surface to become entirely black.

◎ Liquid culture: Bacterial cells grow in a floating state in the potato/glucose medium (3 days of culture), the number of colonies gradually increases, and eventually (7 days of culture) the liquid is filled with viscous colonies.

A dark black thatch appears on the tube wall and gradually forms on the liquid surface (15 days of culture).

This fungal lid is thick and gelatinous.

Although it grows in the same way in Zuapek's medium, it is very slow and the number of bacterial cells is small, and a considerable black thatch forms on the liquid surface after about 3 weeks of culture.

2. Morphological characteristics The young cells are transparent, filamentous, curly, and dendritic, and the fungal cells (filaform-like) have black, oval, spore-like cells clustered here and there.

In addition, the oil drop-shaped colonies have black spore-like substances attached to them.

This will fall apart when subjected to impact. 3. Physiological Characteristics The optimal growth temperature is 20-25°C. It has a unique aroma, producing viscous substances from glucose, sucrose, etc., and producing alcohols and organic acids from sugars such as glucose.

Note) *References include Applied Dutch Studies Guidelines (An 1) by George SMITH.
introduction to industry
lmycocogy) (P68-97) Based on Applied Microbiology (P83-87).

Isolation of bacteria and detection of flocculation A 5-part solution of raw sugar was prepared as a bacterial isolation medium, sterilized using a conventional method, and dispensed into 100ml Erlenmeyer flasks at 20rn1.
After adjusting H to be slightly acidic, sterilize it again, add 1 ml of the stock solution described below to this liquid medium, and let it stand at room temperature (25 ml).
The cells were cultured at ~30°C) and sampled every - day to examine aggregation.

That is, a solution of kaolin (special grade reagent manufactured by Chiyoryaku Co., Ltd.), which is normally used in agglutination tests, was prepared, and the pH was made slightly acidic to prepare a test solution.

The above stock solution is granulated sugar, a dilute solution of raw sugar that was left in a beaker for a long time during the separation and analysis of the polymeric polysaccharide contained in raw sugar (a solution dialyzed with a cellulose membrane of 24 people (diameter)) After noticing that the viscosity of the drug had increased, he decided to undergo dialysis again.

During this operation, when a small amount of diatomaceous earth or activated carbon is added, this solution exhibits extremely flocculating properties, i.e., the diatomaceous earth or activated carbon instantly coagulates at the bottom of the beaker, which is not seen with other commercially available flocculants. I discovered that there is power.

Furthermore, various substances, substances mainly composed of aluminum silicate such as kaolin and bentonite, or inorganic substances, are added to this solution.
That is, it was qualitatively confirmed that significant aggregation occurs when neutral salts such as calcium carbonate, barum sulfate, silver chloride, and organic substances are added.

For this agglutination test, add 25 liters of this sample solution to a 50 TL test tube.
ml, add 1 ml of culture solution to it, stir it up and down 10 times, stand still for 3 minutes, and measure the turbidity of the supernatant using a photoelectric colorimeter at 720 "phl 0 mi/cell". did.

In addition, the amount of kaolin remaining in the supernatant was measured gravimetrically to examine the flocculation properties.

The results are shown in FIGS. 1 to 9.

The aggregation property is remarkable from the culture solution at the initial culture time.

This suggests that this aggregating active substance metabolized in the medium has an aggregating effect even in minute amounts.

Note that as the culture time progressed (96 hours), the odor of acetone and butanol became stronger.

Therefore, we set the culture time to 72 hours, repeated this culture 10 times, and selected a culture solution with strong cohesion, no acetone or butanol odor, and a unique aroma (rose flower aroma) of the main culture solution. Pure herb was isolated from the liquid.

Pure separation of the herb - Make a 5-factor solution of raw sugar or sucrose ← sucrose, adjust the pH to 5-6, add 0.2% powdered yeast extract (manufactured by Chiyo Yakuhin), and add agar (0.16-factor). The mixture was heat sterilized by a conventional method and dispensed into chalets to serve as a separation medium.

The culture solution was diluted 100 times, 200 times, and 500 times with sterilized water, and 1 ml was dispensed into a petri dish and cultured at 30°C.

As a result, three types of colonies were detected, and slant culture and liquid culture were repeated to isolate pure three types of bacteria.

All colonies at the initial stage of culture (around 48 hours of culture) are cream-colored, but as time passes, the center of the surface of the colony becomes black, and as time passes, black hyphae appear on the back of the colony, centered around the colony. The bacteria that extends into the medium in the form of seed roots is designated as isolated bacteria I.

A bacterium whose cream-colored colony does not change color over time and swells up is called an isolated bacterium (■), and a bacterium whose cream color turns brown with the passage of time is called an isolated bacterium (■).

The three types of isolated bacteria were statically cultured in a culture solution with the composition described in the text, and the cohesive force of the culture solution was examined using a kaolin 1 solution.

The results showed that only isolated bacteria 11, that is, bacteria whose colony surface turned black over time and black hyphae extended like roots on the back, had flocculating properties, and the apparent viscosity of the main culture solution increased with culture time. Furthermore, we detected an aroma unique to this herb (the aroma of rose flowers).

Other isolates ■. No aggregation was detected in the culture solution of (2), and an acetic acid odor was detected for the isolate (■), and a butyric acid odor was detected for the isolate (■).

The above isolate I is the subject of the present invention, the family Melanobacteriaceae (Demminaceae).
ematium of dematium of at 1aceae)
) (hereinafter simply referred to as the present invention bacterium).

Explanation of micrographs of pure isolated bacterial isolate I, ~Reference photos 1 to 12 are photographs of pure isolated bacterial isolate I that was slant cultured in a medium with the following composition and then liquid stationary cultured in this medium composition.
Shown below.

Medium composition Czapeks = Glucose 5%~Su'
crose 5% Potato, extract -Glucose
5%~5ucrose 5% Yeast, extract = Glucose 5
%~5ucrose 5 (i) Koji, Wasser=Glucose 5%~5
ucrose 5% Reference photos 1 to 5 show 51 ant of isolate I in this culture composition.
The surface is black in the color photograph taken by culture (slope culture).

Reference photos 6 to 11 are 51ant, Culture (
The photograph shows that the viscosity of each culture solution has increased significantly when the bacteria were cultured in liquid (stationary method) using the same medium composition.

In addition, the aroma peculiar to this plant was detected in all the media.

Reference photo 12 shows a colony in which one spore was cultured in this medium, and the colony surface is black.

The micrograph below shows the bacteria isolated from the colony in Photo 12.

Microscopic photographs in place of drawings, Figures 10 to 18, are 60x, 150x, and 600x photographs of this isolated bacterium ■, showing the hyphae, grown spores, septa, and the viscosity that adheres to the hyphae and spore surfaces. Substances can be seen.

Cultivation and production of flocculant using the isolate I isolated by the present inventor As described in the text, the pure isolate I (Dematium genus of Demat 1aceae)
The results of the production of aggregated substances using and culture conditions are as follows.

Hexoses such as glycose, fructose, and galactose, trisaccharides such as sucrose, and polysaccharides such as starch were used as carbon sources, and 0.2% yeast extract was added to this and cultured by a static method. After one week, the culture solution was Aggregation properties were investigated, and in all cases, culture solutions with aggregation properties were obtained.

Further, the production amount, pH change, and residual sugar relative to the substrate are shown in Figure 2.

Furthermore, by simply adding glucose or the like as a carbon source to a commonly used synthetic medium such as Czapeke medium, flocculating substances can be produced in the culture solution.

As mentioned above, it has been found that the bacteria of the present invention can produce aggregated substances in the culture solution with a simple medium composition mainly consisting of carbohydrates.

For example, if raw sugar is used as a carbon source, there is no need to add other nutrients (N source, inorganic substances, etc.).

Figure 3 shows the results of adult flocculation of aggregated substances when cultured using only raw sugar as the culture composition.

As a result of culturing at a carbon source concentration of 5 to 20, it was found that as the concentration increased, the amount of aggregated substances produced relative to the substrate decreased, and that around 5% was preferable.

That is, since the viscosity of this flocculating substance is extremely high, it was determined that the growth of bacteria would be physically inhibited when this substance reached a certain concentration.

The pH does not require particularly strict adjustment if it is initially adjusted to be slightly acidic.

It becomes slightly acidic during the course of culture.

Although both static and shaking culture produce aggregated substances, it was found that shaking culture produced aggregated substances faster.

The yield of this flocculating substance based on the substrate (carbon source) is 10% or more and is inversely proportional to the substrate concentration.

As an example, using glucose, sucrose, fructose, and primrose as carbon sources, 50 TrL of a medium was placed in a 200 ml Erlenmeyer flask, and static culture was performed. The results are shown in FIG.

Culture conditions, medium composition Carbon source Glucose Concentration: 5 Fructose 5: Granulated sugar 5: Raw sugar 5: N source Powdered yeast was added to the culture other than raw sugar 02 was added to the mouth pH 5.0 Adjusted with HCl Temperature 28-30 'C The inoculum is a culture solution obtained by culturing this isolate ~I with shaking for 7 days.
TLl was added.

Separation and purification method of flocculated substances produced by isolated bacteria I ■ Isolated bacteria I (Dematium genus of the Black fungus family) was inoculated into the culture medium under the above culture conditions. Precipitation treatment at 3.00 Orpl/min j to remove bacterial cells, filtration and separation to remove bacterial cells, and add ethanol to the separated solution to a concentration of 30 to 40 parts (acetone or methanol may also be used). A thin film forms on the surface of the culture solution, and when stirred, fibrous or cotton-like substances instantly aggregate in one place.

Separate the aggregated substances by centrifugation or by allowing them to adhere to a stirring rod,
Dissolve it in water again, add ethanol to re-agglomerate it, separate it and dry it under reduced pressure to obtain an aggregated substance.

The separated aggregated substance is grayish white and can be easily powdered.

Drawing substitute photographs No. 19 to 22 show electron scanning micrographs of separated aggregated substances.

It is inferred that this substance is a homogeneous high-molecular-weight substance because it coagulates instantaneously with low concentration of ethyl alcohol.

Separation and purification method■ It was discovered that the flocculating substances in the future culture solution are significantly flocculated when aluminum ions are added in acidic conditions, and that they are flocculated by calcium ions in alkaline conditions.

The aluminum ion to be added is aluminum sulfate or its polymer, and the calcium ion is a chloride, lime, etc.

The results of aggregation of this substance by specific inorganic ions are shown in Figure 5.

Based on this property, we established a method to separate aggregated substances in culture fluid.

That is, the culture solution is heated and embedded (100° C. for 15 minutes) to remove microbial cells by filtration or centrifugation, and the solution from which the microbial cells have been removed is mixed with 0.05-0.
When an inorganic ion of 10φ, that is, an aluminum compound when the liquid is made acidic, and a calcium compound when it is alkaline, is added and stirred, the flocculating substance is completely agglomerated.

This can be separated by filtration or centrifugation, and dried to obtain a solid powdery agglomerated substance.

Separation and purification method■ After heating the culture solution (100℃ 15 minutes), remove the bacterial cells,
Concentrate the liquid containing no bacterial cells and use 10% flocculant as a flocculant.

When used industrially as a flocculant, it is very rational to handle it as a liquid for both use and production, as it is a very stable substance and does not require dissolution during use.

The table below summarizes the separation and purification methods for this substance (agglomerated substance).

Separation of this substance by addition of inorganic salt, Separation method (2) involves quantifying the concentration of this substance in the culture solution and adding the calculated amount of inorganic salt, since this substance and inorganic salt react quantitatively.

Since the residual liquid from the separated Shibukawa still contains some carbon sources, it is adjusted to a pH suitable for culture and used repeatedly.

The added inorganic ions such as aluminum do not act as a culture inhibitor for the bacteria of the present invention.

Use and utilization as a flocculant - Isolated bacteria I, D of the Black Mycoceae family
It was obtained by culturing the genus ematium and using the separation method invented by the inventors.

The amount of liquid, solid or solid aggregates containing inorganic ions is very small, i.e. from 0.1 ppm to 3.0 ppm based on the amount of liquid. Op-
It has recently been discovered that by adding it, it has the property of completely coagulating and precipitating organic and inorganic substances and biological microorganisms that are dispersed, suspended, colloidal, or floating in water or liquids containing water.

It can be said that the flocculating effect of this substance is significantly stronger than that of existing commercially available flocculants (inorganic and organic).

Furthermore, since it is a metabolite of microorganisms, it has the advantage of not having the problem of secondary contamination caused by flocculants.

The conditions during aggregation are: (1) The optimum pH range is acidic to slightly acidic, and no cohesive force is shown in alkaline conditions.

FIG. 6 shows the results of the cohesive force of this flocculating substance due to changes in pH.

■Reaction temperature ranges from room temperature to high temperature, regardless of cohesive force.

■After adding this flocculating substance, gentle stirring is required.

■The amount used as a flocculant is o, i ppm ~ 3, Op.
I)m may be used, and the amount used is not related to substances to be aggregated except for special substances.

As an example, FIG. 7 shows the cohesive properties of this substance, which were tested using a kaolin aqueous solution.

On the acidic side, for substances that do not agglomerate with this aggregating active substance, such as cellulose powder, starch particles, etc., after adding and stirring this aggregating active substance, 1/30 to 1/30 of the added aggregating active substance.
It was discovered that when 1/40th the amount of aluminum ions was added and stirred, cellulose powder, starch particles, etc. coagulated and precipitated instantly.

As a result, all organic and inorganic substances that are suspended, dispersed, floating, or colloidal in water on the acidic side were able to coagulate and precipitate.

The results of an agglutination test between this substance and aluminum ions are shown in Figure 8.

Although the cohesive force of this flocculating active substance on the alkaline side is very weak, it was discovered that by adding calcium ions, the cohesive force becomes similar to the cohesive force of this substance on the acidic side.

Table 9 shows the test results of calcium ion addition on the alkaline side.

The amount of calcium ions added is greater than the amount of aluminum ions added on the acidic side, and is required to be 20 to 30 times the amount of the main flocculating substance added, that is, 40 to 8 Qppm.

The results of this experiment showed that in all pH ranges, this flocculating active substance was able to flocculate and precipitate organic and inorganic substances that were suspended, dispersed, floating, or colloidal in water even in small amounts. .

The aggregation effect of this substance is due to its extremely uniform high molecular weight and extremely high affinity for water (this inference can be made from the fact that it coagulates instantly with low concentration of alcohol), as if it were a fine-grained net in water. They are uniformly packed and hydrated with water, but when charged particles or inorganic substances enter this, the dispersed net loses its electrical balance and clumps together, just like catching fish with a net. It is inferred that matter is captured by

This becomes clear by observing aggregation when a small amount of aluminum ions are added to an aqueous solution of the present aggregation active substance.

Furthermore, when ethanol is added to the solution of the flocculating active substance, a thin film is formed on the interface, similar to when a collodion film is formed, and it can be observed that many such films are piled up.

The aggregating active substance is used as an aqueous solution.

In addition, the aluminum-containing flocculating substance and calcium-containing flocculating active substance obtained by the purification method are used as an alkaline or acidic aqueous solution.

Physicochemical properties of this active substance - This flocculated active substance separated and purified with ethanol is soluble in water, and the specific viscosity of its 0.1 aqueous solution is 4 to 5, which is equivalent to the viscosity of a 40 coefficient solution of sugar. .

The anthrone, Molisch, and Biuret reactions of this flocculating active substance are all the same, and the qualitative reaction of one COOH group by the carbazole reaction is also shown in ■.If the quantitative value is shown in terms of galacturonic acid, the content is in the range of 10 to 15. be.

Furthermore, undecomposed substances remained when hydrolyzed with N H2SO4 for 24 hours, and sugars such as glucose, galactose, and mannose were detected using paper chromatography to determine the sugar composition of the hydrolyzed solution.

Furthermore, the results of infrared chromatography of this aggregation active substance are shown in FIG.

In this infrared ray, absorption of -COOH was confirmed, but absorption of amide groups etc. was not clear.

This aggregation active substance is estimated to be a high molecular weight substance containing an organic acid and having glucose, galactose, etc. as its main components.

Viscosity of the flocculating active substance purified and separated from the culture solution: Solubility in ethyl alcohol (alcohol): 40 or less Soluble: 40 to 45 Insoluble, becomes albumen-like.

Water retention: 45 or higher It becomes albumen-like or film-like (collodion film), but when agitated, it becomes a flocculent aggregate.

Odor of purified and separated flocculating active substance Odorless taste Tasteless hygroscopicity Weak (room temperature) Color Grayish brown fibrous substance A rare solution of the substance (agglomerating active substance) (concentration 1 to 1010)
Divalent or trivalent inorganic ions or heavy metal ions such as Ca++, A7---, Mg, Zn, Pd, etc. are added to 0pp (equivalent amount or X).

(below), this substance will completely aggregate and become fibrous.

This reaction with inorganic ions is quantitative.

[Brief explanation of the drawing]

Figures 1 to 9 are charts for testing the culture yield, aggregation properties, etc. of the agglutinating active substance produced by the method of the present invention.
Figures 0 to 18 are microscopic photographs of microorganisms used in the method of the present invention, and are photographs of spores, hyphae, septa, or sticky substances adhering to spores and hyphae. Figures 19 to 22 are electron scanning micrographs of separated and purified aggregation active substances, 100x, 700x, 1.0x.
It is 00 times bigger. FIG. 23 is an infrared absorption spectrum of the separated aggregation active substance (KBr method).

Claims (1)

[Scope of Claims] 1. A method for producing an aggregating active substance by a microorganism characterized by culturing an aggregating active substance producing bacterium of the microorganism microorganism number M4257 belonging to the genus Dematium of the family Dematylaceae. Manufacturing method. 2. A method for producing an agglutinating active substance using a microorganism, characterized in that the culture according to claim 1 is carried out by inoculating a medium containing carbohydrates as a main carbon source. 3. Claim No. 3 characterized in that the flocculating active substance has flocculating activity against organic matter, inorganic matter, mineral matter, biological bacteria, etc. that are suspended, dispersed, or floating in a liquid and exist in colloidal form. A method for producing an agglutinating active substance using a microorganism according to item 1 or 2.