JP3044284B2 - High aggregation activity mutant - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a wastewater treatment system, and more particularly, to a food factory wastewater such as a distillation wastewater which contains a high concentration of organic substances and is difficult to filter.
The present invention relates to a method for treating a waste liquid, which comprises aggregating solids in wastewater with newly bred yeast having a high aggregation activity, and collecting and removing the solids. The present invention also relates to a novel yeast mutant having a high agglutinating activity, which has been successfully bred newly, and an industrial culture method thereof.

[0002]

2. Description of the Related Art As a method for treating liquor distillation waste liquid, cultured cells of yeast Trichosporon sp M111 strain isolated from nature,
Further, a method has been proposed in which solids in a shochu distillation waste liquid are aggregated by adding vegetable fibers to perform solid-liquid separation.

[0003] However, solid-liquid separation of waste liquid is facilitated by the flocculating action of the present yeast, and filterability is greatly improved. However, it is necessary to add cells to a concentration of 2 × 10 ⁷ cells / ml. However, there is a disadvantage that large-scale cultivation must be performed for practical use of this yeast, and it is not yet sufficient.

[0004]

SUMMARY OF THE INVENTION As described above, yeast
The effective use amount of the Trichosporon sp M111 strain needs to be 1 to 2 × 10 ⁷ cells / ml or more as the final concentration in the waste liquid.
However, even with the most efficient cultivation method at present for obtaining the present yeast by cultivation, the final bacterial concentration is 1-2 × 10 ⁸ cells / ml in a culturing time of 48 hours. Therefore, a 1/10 amount of the yeast culture solution is required for the treatment amount of the distillation waste liquid. For example, in order to treat a 10 ton / day amount of distillation waste liquid, the culturing amount of the yeast M111 strain must be 1 ton / day, and there is a problem that the equipment cost for culturing becomes high. Therefore, the present inventors have newly set as a new problem the development of a method for reducing the number of culture days and increasing the amount of yeast growth as a method for solving this problem.

[0005]

Means for Solving the Problems In order to solve the above-mentioned problems, a mutant strain having a growth rate higher than that of a parent strain and a final growth amount higher than that of a parent strain was determined from a currently used yeast strain Trichosporon sp M111. The research was conducted for the purpose of separating and breeding. As a result, the target mutant (Trichospor
on sp M111-HAG-No 3) could be isolated. The isolation of the mutant strain increased the cultivation efficiency of the yeast, and made it possible to reduce the cost of the yeast cultivation equipment. further,
The yeast M111 strain has a morphologically dimorphic form of a mycelium and a yeast (single cell), but the agglutinating activity is higher in the yeast form. In this regard, the strain is also suitable for the aggregation treatment.

The present invention has been completed based on these new findings and as a result of further studies. Hereinafter, the present invention will be described in detail.

[0007] The mutant having high agglutinating activity according to the present invention is obtained by using a fibrous agglutinating yeast strain M111 (Trichosporonsp M111) as a parent strain, mutagenizing the parent strain, and isolating a target strain from the mutant strains. Can be

[0008] Mutation treatment is performed according to a conventional method,
For example, ultraviolet irradiation, X-ray irradiation, treatment with N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine, ethyl methanesulfonate (EMS) or the like is performed alone or in an appropriate combination, and is repeated as necessary. .

More specifically, the high agglutinating activity mutant according to the present invention can be isolated as follows. That is, the parent strain M111 was irradiated with ultraviolet rays or EMS.
After the treatment, colonies are formed on a YPD agar medium containing 0.3% citric acid, and strains having different colony shapes from the parent strain are isolated. These strains were cultivated in a baffled Erlenmeyer flask at 130 rpm using a YPD liquid medium by rotary shaking, and the morphology of the obtained cells and cellulose fibers (KC Floc)
Is assayed by the dilution method. Then, the mutant strain is compared with the parent strain with respect to the cohesiveness to the shochu distillation waste liquid, the solid-liquid separation effect, and the culture characteristics when the waste liquid filtrate is used as a medium.

[0010] As a result, several mutants morphologically different from the parent strain were isolated on the YPD citrate agar medium. A strain exhibiting agglutinating activity with was able to be isolated. M111
The strain exhibits mycelial and yeast-like dimorphism, and the agglutinating activity is higher in the case of yeast. The mutant strain exhibited a yeast-like morphology in shaking culture, had a smaller cell size than the parent strain, rapidly proliferated, reached steady-state growth in a short time, and increased the final yeast count. However, the final cell weight was not different from that of the parent strain. The agglutinating activity with the cellulose fiber per unit of culture solution or per dry weight unit was higher than that of the parent strain, but no difference was observed in the aggregating activity per unit of bacterial cell.

From the above, it was presumed that the effect of the mutant strain was that the number of yeasts per unit of culture solution increased and the agglutinating activity was superior to that of the parent strain due to the reduced cell size. It should be noted that there was no particular difference between the parent strain and the basic mycological properties of the mutant strain, the fiber coagulation activity per number of bacteria, and the distillation waste liquid treatment capacity.

The present invention further encompasses the use of the excellent properties of the novel mutant strain, and examples thereof include solid-liquid separation of liquor distillation waste liquid and use of a waste water treatment system.

[0013] Unlike ordinary waste liquids, in particular, for purifying a solution containing solids together with high-concentration organic substances, such as liquor distillation waste liquids, first, the removal of solids is an essential pretreatment step. However, a satisfactory method for this pretreatment has not yet been developed. Therefore, the present inventors have made it an issue to develop a method for quickly coagulating solids contained in distillation waste liquid and the like, and to easily carry out solid-liquid separation. As a result, in the technology to remove solids, a process that is completely contrary to the common general knowledge of adding a solid content called plant fiber to a distillation waste liquid having a high solid content is performed, and the tricosporone genus that was successfully separated this time The solid-liquid separation of high-concentration effluents has been successfully carried out very efficiently by using microbial treatment in combination.

The present invention has been made based on such new findings, and its basic technical idea is that a new strain belonging to Trichosporone together with a plant fiber or a mixture containing a plant fiber as a main component is obtained by removing solids The solid content is quickly added to the food wastewater containing the solid content to quickly coagulate, and the solid content is easily separated and removed.

In the wastewater treatment system according to the present invention, Trichosporon sp. M111-HAG-No3, a novel yeast mutant belonging to the genus Trichosporon, which has been successfully newly isolated this time, is used.
AG-No 3) (FERM P-14997) is used. This yeast mutant includes, in addition to the isolated yeast mutant itself and its spores, a wet cake, a culture solution, a culture, its concentration or All yeast-containing substances such as pastes and diluents can be used, and it is also possible to return and use at least a part of yeast-containing substances obtained after treating waste liquid.

In the present invention, it is necessary to add plant fibers and / or their contents to the waste liquid to be treated in treating the microorganisms. The amount of addition varies depending on the type of plant fiber used, the type of waste liquid to be treated, and the like, but at least about 0.1% of dry solid content relative to the amount of waste liquid is required. Preferably 0.1 to 30%, more preferably 0.5
About 20%.

As vegetable fiber, KC-Floc W-5
0 (products of Takeda Pharmaceutical Co., Ltd.); okara, bagasse, beet lees, brassica, bran, threshing lees, starch production lees, and other agricultural production lees; citric acid and other organic acid fermented lees, soy sauce lees, Fermented lees such as alcohol-produced lees; wood or fruit pulp; drainage sludge from this plant, and the like.

Examples of the liquor distillation waste liquid include a wide variety of liquor liquor distillation waste liquid produced from wheat, rice, buckwheat and the like, and / or waste liquid produced as a by-product during the distillation of various liquors such as alcohol distillation waste liquid. The above-mentioned plant fiber (the content thereof) is added to and mixed with the liquor distillation waste liquid of these (these), and the Trichosporon mutant strain isolated this time (the culture, the content and / or the processed product thereof) is added. Inoculate and incubate, and solid matter aggregates and precipitates immediately after addition.

The liquid portion thus separated by solid-liquid separation is discharged to a river or the like as it is when the degree of contamination is low. The treatment may be carried out using various microorganisms, physical treatment, chemical treatment or a combination thereof, and the treatment is carried out very efficiently.

On the other hand, the solid part separated from the liquid can be effectively used as feed, fertilizer, soil improving material, artificial soil, etc. as it is, or after dehydration to form a paste, or further drying. If necessary, it can be reused in the present invention as a plant fiber. If desired, it can be incinerated or returned to the soil. Since the solid portion has no problem in safety, there is no possibility of causing secondary pollution even if this treatment is performed.

Hereinafter, embodiments of the present invention will be described.

[0022]

Example 1 Trichosporon sp M111 strain (parent strain) was transformed into YPD
After shaking culture in a medium (yeast extract, peptone, glucose-containing medium) for 30 hours, the cells are collected by centrifugation,
1 × 10 ⁸ cels in 50 mM citrate buffer (pH 4.1)
It was suspended at a concentration of ls / ml and irradiated under a 15 W ultraviolet lamp at a distance of 15 cm for 3 minutes. The viable cell rate after irradiation was 15%. The irradiated cells were applied to a YPD agar medium containing 0.3% citric acid so that about 400 colonies were formed per petri dish. All of the above operations were performed aseptically. From the obtained 50,000 colonies, 7 strains having colony forms different from the parent strain were selected. The seven strains (M111-No1 to M111-No7) thus isolated were cultured in a baffled Erlenmeyer flask at 130 rpm using a YPD liquid medium. The number of proliferating bacteria after 24 hours and 48 hours of culture is shown in Table 1 below.

[0023]

[Table 1]

As is clear from the above results, M out of 7 strains
Two strains, 111-No3 strain and M111-No6 strain, showed better growth than the parent strain.

[0025]

Example 2 The two mutant strains (M111-No3 and M111-No6), each of which demonstrated excellent growth properties in the above example, were subjected to an agglutination activity test with cellulose fibers.

A culture solution containing the cells after culturing the above two mutant strains (and the parent strain as a control) for 48 hours was used as a stock solution and 1 /
The solution diluted to 2, 1/4, 1/8, and 1/16 was added to 0.5 ml of a 0.5% aqueous solution of vegetable fiber KC-Floc W-50 (manufactured by Takeda Pharmaceutical Co., Ltd.) in an amount of 25 μl each.
1 was added and the agglutinating activity was assayed. The resulting,
Table 2 below shows the results of a comparison test of the agglutination activity between the cell culture solution and the cellulose fiber.

[0027]

[Table 2]

As is clear from the above results, it was confirmed that the two mutant strains having high proliferative properties had higher aggregation properties than the parent strain. Among these two mutant strains, the M111-No3 strain showing particularly excellent growth and aggregability was confirmed by microscopic observation to have a higher percentage of yeast cells than the parent strain and a smaller size. Therefore, this strain was identified as a new mutant strain, and this strain was identified as Trichosporone species M111-
It was newly named as HAG-No3 (Trichosporon sp M111-HAG-No3), and deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology as FERM P-14997.

[0029]

Example 3 The suitability test of the distillation waste liquid as a culture medium was carried out by centrifuging the barley shochu vacuum distillation waste liquid at 3000 rpm for 15 minutes, and diluting the supernatant No5A filter paper filtrate in several stages as a medium. Was. YPD medium was used as a control. Table 3 shows the results. The supernatant of the centrifuged supernatant of the vacuum distillation waste liquid was viscous in the undiluted solution and was not sufficiently stirred, so that the growth of the bacterium was poor. However, the dilution two-fold showed the same growth as the YPD medium. Further, no difference was observed in the cohesiveness of the cultured cells with the KC floc.

[0030]

[Table 3]

From the above results, it was shown that the 2-fold diluted supernatant after solid-liquid separation of the waste liquid of barley shochu distillation can be used for cultivation, and the culturing cost can be reduced. Thus, M11
In the cultivation of one strain, the liquid part itself after the waste liquid treatment or the diluted part is intact (generally, it is not necessary to add a nutrient, etc., but if desired, the addition of a nutrient, etc. is of course possible. There is a point that can be used as a medium
This is particularly advantageous from an industrial point of view in that the waste liquid to be treated can be recycled and used without using an expensive medium substrate for culture.

[0032]

Example 4 To 100 g of a waste liquid of barley shochu under reduced pressure distillation, vegetable fibers and a cell suspension were added in this order, and the improvement of filterability for solid-liquid separation was evaluated. KC- as a vegetable fiber
1% of Flock W-50 (product of Takeda Pharmaceutical Co., Ltd.)
Trichosporon sp. M111-
HAG-No 3 (FERM P-14997) cell suspension was added to a final concentration of 2 × 1
0 ⁷ cells / ml was added to make a total volume of 110 ml.

(Filtration conditions) Kiriyama funnel S-60 (product of Kiriyama Seisakusho Co., Ltd.) (diameter 60 mm) was laid with stainless steel mesh (330 mesh, 45 μm pore diameter) on the bottom surface, and coagulated waste liquid was poured on it. The solution was subjected to suction filtration at 0.6 KPa. The test was performed at room temperature (20 ° C.), and the amount of filtrate obtained in a predetermined time was measured, and the result is shown in FIG.

(Results) As is apparent from the above results, according to the present invention (KC floc + cells), excellent solid-liquid separation is achieved in both the barley shochu distillation waste liquid and the buckwheat shochu distillation waste liquid. It was confirmed that these waste liquid treatments were performed extremely efficiently.

[0035]

According to the present invention, a novel yeast mutant has been isolated and created. This mutant has a growth rate of about 2 times that of the parent strain.
Times (grows to the same number as conventional strains in just 24 hours)
Thus, it has an excellent characteristic that the growth rate is extremely fast and the final growth amount is larger than that of the parent strain. In addition, this mutant strain is not a mycelium but a yeast-like (single-cell-like) morphology having high agglutinating activity from the early stage of culture, and is a strain suitable for agglutination treatment.

According to the present invention, this useful novel mutant is isolated,
In addition to the remarkable effect of the invention, we have also developed an efficient culture method using distilled effluents, and also used the useful properties of these mutant strains to treat wastewater, especially alcoholic beverages that have been extremely difficult to treat. There is also a remarkable effect that a practical or industrial waste liquid treatment can be economically performed, that is, the treatment of the distillation waste liquid efficiently.

Therefore, according to the present invention, by employing a completely novel constitution in which a microorganism treatment using the novel mutant strain and a plant fiber treatment are used in combination, a high concentration of solids and a high viscosity can be obtained. In addition to simply enabling solid-liquid separation of waste liquids, such as distillation waste liquid, for which solid-liquid separation is very difficult because waste liquid treatment is extremely difficult, it is necessary to enable it in a very short time and efficiently. It was the first successful one, especially the first successful commercialization or industrialization.

As a result, highly viscous waste liquid containing high concentration solids, such as various shochu distillation waste liquids made from wheat, rice, buckwheat, etc., and liquor distillation waste liquids such as alcohol production waste liquids can be efficiently removed in a very short time. Solid-liquid separation can be performed in a short time, and waste liquid treatment, which conventionally required a long time, can be performed in a short time, and practical use or industrialization has been achieved for the first time.

[Brief description of the drawings]

FIG. 1 shows the flocculation treatment of barley shochu waste liquid with M111-No3 bacteria.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Jin Shimoi 8511-14 Misono, Saijo-cho, Higashihiroshima City, Hiroshima Pref.National Institute of Brewing (72) Inventor Riki Fujii 8511-14, Misono, Saijocho, Higashihiroshima-shi, Hiroshima NTS Brewery Research Laboratory (72) Inventor Yujiro Iwashita 11808 Komiyachi, Akamachi, Amakusa-gun, Kumamoto Affiliated company Amakusa Sake Brewery (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 1/16 C02F 3/34 WPI ( DIALOG) BIOSIS (DIALOG) JICST file (JOIS)

Claims (7)

(57) [Claims] 1. A yeast mutant strain having a high agglutinating activity, Trichosporone species M111-
HAG-No3 (Trichosporon sp M111-HAG-No3). 2. A method for solid-liquid separation of a waste liquor, comprising treating the liquor distillation waste liquor with vegetable fibers and / or a substance thereof and the yeast mutant according to claim 1. 3. The method according to claim 2, wherein the liquor distillation waste liquid is a shochu distillation waste liquid and / or an alcohol distillation waste liquid made from wheat, rice, buckwheat or the like. 4. The plant fiber according to claim 2, wherein the plant fiber is plant fiber products, agricultural production residue, fermentation residue, wood or fruit pulp, and / or wastewater sludge from a plant of the plant. The method according to claim 1. 5. A method for treating alcoholic liquor waste, comprising adding the yeast mutant strain according to claim 1 together with vegetable fibers and / or its contents to the alcoholic liquor waste liquor to cause the solids in the liquor to agglomerate. Method. 6. After the solid-liquid separation by the method according to any one of claims 2 to 4, the liquid part is further treated with microorganisms.
A method for treating a liquor distillation waste liquid, which comprises performing a physical treatment and / or a chemical treatment. 7. A method of using a liquid part of a liquor distillation waste liquid and / or a dilution thereof, characterized by using Trichosporon sp. M111-HAG-No3 (Trichosporon sp.).
Culture method of M111-HAG-No3).