JPS6147512B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a flocculant using microorganisms, and more specifically, by culturing flocculant-active substance-producing bacteria belonging to the genus Dematium of the family Dematiaceae, it is possible to produce a flocculant that not only targets proteins but also organic matter, inorganic matter, mineral matter, and living organisms. The present invention relates to a method for producing an agglutinating substance having agglutinating activity against bacterial 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.
The present invention uses microorganisms different from the microorganisms disclosed in these publications, and is applicable not only to proteins, but also to a wide range of substances such as organic substances, inorganic substances, mineral substances, biological cells, etc. suspended, dispersed, or floating in liquids. The present invention provides a method for producing a flocculating active substance having excellent flocculating activity against colloidal substances. The present inventor previously applied for a method for producing an agglutinating active substance using a microorganism characterized by culturing an agglutinating active substance producing bacterium belonging to the Dematium genus of the Dematiaceae family (patent no.
No. 1223494 (Japanese Patent Publication No. 58-47159) (hereinafter referred to as the original invention), the present invention is a further improvement and expansion of this original invention. In the detailed description of the invention, the original invention states that the concentration of the carbon source in the medium is preferably around 5% (i.e.,
The flocculating active substance produced by this isolate (Dematium of Dematiaceae), Microtechnical Laboratory Bacteria No. 4257, shows that as the substrate concentration increases, the amount of flocculating substance produced relative to the substrate decreases). However, the inventor further increased the concentration of carbon source in the medium to 5%.
As a result of culturing as follows (results of static culture or tank culture using fermentation tank 3), the production amount of flocculating active substance per unit or solution per unit or solution is higher when the medium concentration is around 1% than when it is 5%. This discovery led to the present invention. In this way, the fact that the maximum yield of the target product is shown at a dilute medium concentration of around 1% means that, for example, glucose, sucrose, etc. can be obtained from wastewater from various agricultural industries, livestock industries, food processing industries, etc. This means that even a dilute carbon source of around 1% is suitable as a culture medium for the bacteria of the present invention, and this also leads to effective treatment and utilization of these wastewaters, and also allows for the use of bacteria that produce the carbon sources. The coexistence of the present invention also makes it possible to culture the bacteria of the present invention and obtain the desired agglutinating active substance in high yields, which has great industrial and economical value. The microorganisms used in the method of the present invention are
It is an agglutinating active substance producing bacterium belonging to the genus Dematium of the family Dematiaceae, including the bacterium No. 4257 (hereinafter simply referred to as the example bacterium), and its mycological characteristics are described in the example above. The explanation based on bacteria is as follows. A. Mycological characteristics of the isolated bacteria Colonies initially have a smooth surface, are grayish white, and have a mucous,
It grows like a shiny oil droplet (fat-like) yeast,
Filament-like fungal cells grow radially from the periphery, forming a curly, filamentous structure that resembles a tree. 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 light brown oval or oval conidia are lateral 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 colonies. The culture solution containing sugar becomes very viscous,
Produces leathery black moss in thick colonies on the liquid surface. The optimal growth temperature is 20 to 25°C, at which it produces alcohols and organic acids from sugars such as glucose and sucrose, and has a unique aroma. 1 Culture characteristics * (Note) ○A Solid medium: Potato, 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 in the medium. Eventually, some parts of the tree-like branches turn blackish brown. After 3 to 4 days of culture, light and dark brown spots appear on the surface of the colony, but after that it gradually becomes light and dark black and spreads over the entire surface, and finally turns black as a whole (after 7 days of culture). Growth is slow on the top, but the culture characteristics are as described above.It takes about 3 weeks for the colony surface to completely turn black. ○B Liquid culture: Bacterial cells grow in suspension in the potato and glucose medium (Culture 3)
Days), the number of colonies gradually increases, and eventually (7 days after culture), the liquid is filled with viscous colonies. Then, dark brown fungal moss appeared on the tube wall,
It gradually appears on the liquid surface (15 days of culture). This fungal lid is thick and gelatinous. Although it grows in the same way in Czapetz medium, it is very slow and the number of bacterial cells is small, and after about 3 weeks of culturing, a considerable amount of black fungal moss is formed on the liquid surface. 2. Morphological characteristics Young cells are transparent, filamentous, curly, and arborescent, and the fungus (filament-like) has black, oval, spore-like cells growing laterally from here and there. In addition, the oil drop-shaped colonies have black spore-like substances attached to them. This will break apart if you hit a point of impact. 3. Physiological characteristics The optimal growth temperature is 20-25°C. Viscous substances are produced from glucose, sucrose, etc., and alcohols and organic acids are produced from sugars such as glucose. *(Note) References include Guidelines for Applied Mycology (An
introduction to industrial mythology)
(P68-97) Based on Applied Microbiology (P83-87). B Isolation of bacteria and detection of flocculation Make a 1% solution of raw sugar as a bacterial isolation medium, sterilize it by the usual method, and add 20
After dispensing ml and adjusting the pH to slightly acidic, sterilize again, add 1 ml of the stock solution described below to this liquid medium, culture by standing method (room temperature 25-30℃), and sample every day. Cohesion was investigated. That is, a 1% solution of kaolin (special grade reagent manufactured by Takeda Pharmaceutical Co., Ltd.), which is normally used in agglutination tests, was prepared, and the pH was made slightly acidic to use as a test solution. The above stock solution is granulated sugar, a dilute solution of raw sugar (24Å (diameter)) that was left in a beaker for a long time during analysis.
I noticed that the viscosity of the solution (dialysed using a cellulose membrane) had increased, so I decided to repeat the dialysis. When a small amount of diatomaceous earth or activated carbon is added during the operation, this solution has a very remarkable flocculating property, i.e., the diatomaceous earth or activated carbon instantly solidifies at the bottom of the beaker, which is not seen with other commercially available flocculants. I discovered that there is a cohesive force. Furthermore, various substances, substances mainly composed of aluminum silicate such as kaolin and pentonite, inorganic substances such as neutral salts such as calcium carbonate, barium sulfate, and silver chloride, and organic substances are added to this solution. It was qualitatively confirmed that significant aggregation occurred. For this agglutination test, add 25ml of this sample solution to a 50ml test tube.
1 ml of culture solution was added to this, stirred up and down 10 times, and then allowed to rest for 3 minutes. The turbidity of the supernatant was measured using a photoelectric colorimeter at 720 mph/10 mm/cell. In addition, the amount of kaolin remaining in the supernatant was measured gravimetrically to examine the flocculation, and it was found that the flocculation was significant 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. As the culture time progressed (96 hours), the odor of acetone and butanol became stronger. Therefore, the culture time was set to 72 hours, and this culture was
Repeat 10 times and add acetone to the culture solution with strong cohesive force.
A culture solution with no butanol odor and a scent unique to the main culture solution (rose flower aroma) was selected, and the main bacteria was isolated from this solution. Pure isolation of this bacterium ~ Make a 1% solution of raw sugar, sucrose, or glucose, pH 5-6
and powdered yeast extract (manufactured by Takeda Pharmaceutical).
Agar (0.16%) was added at 0.2%, heat sterilized using a conventional method, and the mixture was dispensed into a shear dish to prepare 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 shear 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. Colonies at the initial stage of culture (culture time 48
(Before and after time) All of them are cream colored, but as time passes, the center of the surface of the colony becomes black, and as time passes, black hyphae grow on the back of the colony in the form of roots in the medium centering on the colony. The bacteria are isolated. Bacteria whose cream-colored colonies do not change color and swell over time are considered isolated bacteria, and bacteria whose cream color turns brown with the passage of culture time are considered isolated bacteria. 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 1% kaolin solution. The results showed that only the isolated bacteria, that is, the bacteria whose colony surface turned black over time and black hyphae extended in a root-like manner on the back, had flocculating properties, and the apparent viscosity of the main culture solution increased with culture time. In addition, an aroma (rose flower aroma) unique to this fungus was detected. other isolates,
No aggregation was detected in the culture solution, but an acetone odor was detected in the isolated bacteria, and a butyric acid odor was detected in the isolated bacteria. The above-mentioned isolated bacteria belong to the genus Dematium of the family Dematiaceae, which is the subject of the present invention.
This is an agglutinating active substance producing bacterium (hereinafter simply referred to as the present invention bacterium) belonging to . C. Description of the pure isolated bacteria The pure isolated bacteria were slant cultured in a medium with the following composition, and further subjected to liquid stationary culture in this medium composition. Medium composition: Czapek's ~ Glucose 1% ~ Sucrose 1% Potat. extract〜Glucose1%〜Sucrose1% Yeast.extract〜Glucose1%〜Sucrose1% Koji.Wasser〜Glucose1%〜Sucrose1% D Cultivation using isolated bacteria and production of aggregated substances Purely isolated bacteria (Dematiaceae)
Production of flocculants using Dematium spp.
The results of the culture conditions are as follows. 5 Glucose, fructose,
Using disaccharides such as hexoses such as galactose or polysaccharides such as starch, 1% yeast extract was added to this (medium 3), and cultured by the static method (air volume is equal to that of the medium/min). , initial PH
5.0) After one week, the aggregation properties of the culture solutions were examined, and in all cases, culture solutions with aggregation properties were obtained. The production amount, PH change, and residual sugar relative to the substrate at that time are shown in Figure 1. It has also been found that flocculating substances can be produced in a culture solution simply by adding glucose or the like as a carbon source to a commonly used synthetic medium such as Czapeke medium. As mentioned above, it was 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, sucrose, glucose, etc. are used as a carbon source, it is not necessary to add other nutrients (N source minerals, etc.). When we investigated the results of flocculant production when culturing with only raw sugar as the medium composition, we found that when culturing with a carbon source concentration of 5% or less, the production of flocculent substances relative to the substrate increased as the concentration decreased. Yes, 1%
It was found that the maximum yield was given before and after. If the pH is adjusted to slightly acidic initially, there is no need for particularly strict adjustment. 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 faster aggregated substances. The yield of this flocculating substance relative to the substrate (carbon source) is approximately 10% to 50%, and is inversely proportional to the substrate concentration. That is, as is clear from the relationship between culture time and aggregation active substance/substrate x 100 (yield 10%) in Figure 2, the yield is about 50% of the maximum value at a substrate (raw sugar) concentration of 1%, and from this Regardless of whether the concentration was increased or decreased, the yield tended to decrease. Substrate (raw sugar) concentration 5% or 0.5%
The yield was about 10%. If the substrate (raw sugar) concentration exceeds 5%, the yield will be drastically reduced, making it uneconomical. A significant decrease in yield is also observed at concentrations closer to 0.5%. E Separation and purification method of agglutinating active substance produced by isolated bacteria Separation and purification method The culture solution obtained by inoculating the isolated bacteria (Dematium sp. minute), centrifugation treatment 3000rpm/mi
When the bacterial cells are removed by filtration and separation, and ethanol is added to the separated solution to a concentration of 30 to 40% (acetone or methanol may be used), a thin film is formed on the surface of the ethanol and culture solution. occurs,
When stirred, fibrous or cotton-like substances instantly aggregate in one place. The aggregated substance is separated by centrifugation or attached to a stirring rod, dissolved in water again, ethanol is added to cause re-agglomeration, and after separation, the substance is dried under reduced pressure to obtain an aggregated active substance. The separated aggregated active substance is grayish white and can be easily made into powder.
It is inferred that this agglutinating active substance is a homogeneous high-molecular-weight substance because it coagulates instantaneously with ethyl alcohol at a low concentration. Separation and purification method It was discovered that this agglutinating active substance in the culture solution shows significant aggregation when aluminum is added in acidic conditions, and agglomerates due to calcium ions in alkaline conditions. The aluminum ion to be added is aluminum sulfate or its polymer, and the calcium ion is added as a chloride.
Or lime etc. We established a method to separate the agglutinating active substance in the culture solution from the results of aggregation of the agglutinating active substance using specific inorganic ions. That is, after heat-treating the culture solution (100℃/5 minutes), remove the bacterial cells by centrifugation, and add 0.05 to 0.10% to the solution from which the bacterial cells were removed.
When the inorganic ions, that is, an aluminum compound when the liquid is made acidic, and a calcium compound when the liquid is alkaline, are added and stirred, the flocculating active substance is completely agglomerated. This can be separated by filtration or centrifugation, and then dried to obtain the agglomerated active substance in the form of a solid powder. Separation and Purification Method After heating the culture solution (100°C/5 minutes), remove the bacterial cells, and concentrate the liquid that does not contain bacterial cells (approximately 10%) to use 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 agglutinating active substance.

[Table] F Separation of this flocculating active substance by adding inorganic salts In the separation method, the flocculating active substance and inorganic salt react quantitatively, so the concentration of this flocculating active substance in the culture solution is quantified and the calculated amount is calculated. Add inorganic salts.
Since the residual liquid from the separation method still contains some carbon source, it must be 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. G. Use as a flocculant The isolated bacterium, ie, the genus Dematium of the family Black Mycoceae, was cultured, and the present flocculating active substance was obtained by the above-mentioned separation method. These liquids, solids, or solid agglomerated active substances containing inorganic ions can be dispersed, suspended, or colloided in water or a liquid containing water by adding very small amounts, that is, 0.1 ppm to 3.0 ppm to the liquid amount. It was discovered that it has the property of completely coagulating and precipitating floating organic and inorganic substances and biological bacteria. The flocculating action of this flocculating active substance is similar to that of existing commercial flocculants (inorganic,
It can be said that it is extremely strong compared to organic (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 optimal pH range for aggregation is acidic to slightly acidic, and the desired cohesive force will not be achieved under alkaline conditions.
The reaction temperature ranges from room temperature to high temperature and has no relation to cohesive force. It is advantageous to carry out gentle stirring after addition of the flocculent. The amount used as a flocculant is
It may be 0.1 ppm to 3.0 ppm, and the amount used is not related to substances to be aggregated except for special substances. Substances that do not aggregate with this aggregation active substance on the acidic side,
For example, in an aqueous solution of cellulose powder, starch particles, etc., after adding and stirring the present flocculating active substance, adding aluminum ions in an amount of 1/30 to 1/40 of the added flocculating active substance and stirring, cellulose powder, starch particles, etc. It was discovered that the particles coagulate and precipitate in an instant. 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. Although the cohesive force of this agglomerating 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. . The amount of calcium ions added is greater than the amount of aluminum ions added on the acidic side, and is 20 to 30 times the amount of the main flocculation substance added, i.e. 40 to 80 ppm.
Good to have. From the results of this experiment, it was confirmed that in all pH ranges, this flocculating substance can coagulate and precipitate a small amount of organic and inorganic substances that are suspended, dispersed, floating, or colloidal in water. The flocculation effect of this flocculating substance is because this flocculating substance has an extremely uniform high molecular weight and has a very high affinity for water (this inference can be made from the fact that it flocculates instantly with low concentration of alcohol), so it is as if the flocculating effect is caused by fine grains in water. The fine net is uniformly packed and hydrated with water, but when charged particles or inorganic substances enter the net, the dispersed net loses electrical balance and clumps together, as if catching fish with a net. It is inferred that it captures matter as it captures it. This becomes clear by observing the aggregation when a small amount of aluminum ion is 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 at the interface, similar to when a collodion film is formed, and it can be observed that many such films overlap. The aggregating active substance is used as an aqueous solution. Furthermore, 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. H Physical and chemical properties of the flocculating active substance The flocculating active substance separated and purified with ethanol is soluble in water, and its 0.1% aqueous solution has a specific viscosity of 4 to 5, which corresponds to the viscosity of a 40% sugar solution. This agglutinating active substance undergoes all of the Anthrone, Moritzsch, and Buillet reactions, as well as the qualitative reaction of the -COOH group by the carbazole reaction.If the quantitative value is expressed in terms of galactulonic acid, the content is 10 to 15%. . Also INH ₂ SO ₄
When hydrolyzed for 24 hours, undecomposed substances remained, and the sugar composition of the hydrolyzed solution was determined using paper chromatography, and sugars such as glucose, galactose, and mannose were detected. Furthermore, the results of infrared chromatography (KBr method) of this aggregation active substance are shown in FIG. In this infrared ray, absorption of -COOH was confirmed, but the 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 as a main component such as glucose or galactose.

【table】 Elemental analysis of purified and separated aggregated active substances H 6.52% C 41.04 N 0.14 O 51.74 Ash 0.56 (hygroscopicity) Molecular weight: 1 million or more (estimated) qualitative reaction Anthrone reaction Galbazole reaction Beuretz reaction ninhydrin reaction

[Table] Solubility in ethyl alcohol Concentration (alcohol)% 40% or less Soluble 40-45% Insoluble, becomes albumen-like. High water retention: 45% or more It becomes albumen-like or film-like (collodion film), but when agitated, it becomes a flocculent aggregate. Odor of the purified and separated flocculating active substance Odorless Taste Tasteless Hygroscopicity Weak (room temperature) Color Grayish brown fibrous substance Dilute solution of this flocculating active substance (concentration 1~
If divalent or trivalent inorganic ions or heavy metal ions such as Ca ⁺⁺ , Al ^--- , Mg, Zn, Pd, etc. are added (equivalent amount or (1/10 or less)) to 100ppm), this substance will be completely This reaction with inorganic ions is quantitative. Additional relations Original invention (Patent No. 1228494 (Special Publication No.
(No.)) is an essential part of its invention, which describes ``a method for producing a flocculating active substance using a microorganism characterized by culturing a flocculating active substance-producing bacterium belonging to the genus Dematium of the family Dematiaceae.'' However, the present invention is an invention in which all of the matters essential to the composition of the invention of the original invention are the main parts of the composition, and the present invention has the same purpose as the original invention. Therefore, it satisfies the requirements for an additional patent under Article 31, Item 1 of the Patent Act.

[Brief explanation of the drawing]

Figure 1 is a chart showing the relationship between pH, agglutination active substance yield %, substrate consumption % sugar, and culture time when the bacteria of the present invention was cultured at a substrate concentration (raw sugar) of 1%. The figure is a chart showing the relationship between the yield of the agglutinating active substance and the culture time when the substrate concentration (raw sugar) is 1% and 5%. It is a figure of an infrared absorption spectrum.

Claims (1)

[Scope of Claims] 1. A flocculating activity by a microorganism characterized by culturing a flocculating active substance-producing bacterium belonging to the genus Dematium of the family Dematiaceae under a medium concentration of carbon source of 0.5 to 5%. A method of manufacturing a substance. 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 glucose or other carbohydrates as the main carbon source. 3 Organic, inorganic, and mineral substances in which agglomerated active substances are suspended, dispersed, or floated in a liquid and exist in colloidal form;
3. A method for producing an agglutinating active substance using a microorganism according to claim 1 or 2, characterized in that biological cells or the like have an agglutinating activity.