JPS5985287A - Novel microorganism - Google Patents

Abstract

PURPOSE:A novel yeast lytic microorganism YLM-1 capable of growing on live yeast cells as a sole carbon source and nitrogen source. CONSTITUTION:A yeast lytic microorganism YLM-1 (FERM-P No.6781) strain capable of growing on live yeast cells, e.g. Endomyces geotrichum IFO 9541 or Saccharomyces cerevisiae IFO 0203, isolated from an activated sludge in the Brewery Research Institute of the National Tax Administrative Agency as a sole carbon source and nitrogen source. The strain has the following characteristics; (1) Gram-negative. (2) cellular form; bacillus. (3) Motility. (4) Aerobe. (5) Capable of growing on live yeast cells as a sole carbon source and nitrogen source.

Description

[Detailed description of the invention] The present invention relates to a novel microorganism.

The novel microorganism YLM-/ of the present invention is characterized in that it grows using live yeast cells as a single carbon source and nitrogen source.

In recent years, efforts have been made to search for yeast-lytic microorganisms for the purpose of preparing yeast protoplast dogs for genetic manipulation and cell fusion, and for use in studying the structure of yeast cell walls.

The inventors have developed a new wastewater treatment method [Yoshizawa: Noka, JJ, 7θj~7// (ypg/)] in which highly concentrated food manufacturing wastewater is treated with yeast and then subjected to biological treatment such as activated sludge.
``In J, the yeast grown in the yeast tank is grown in the activated sludge tank,
In order to investigate how yeast-dissolving microorganisms disappear, a culture medium for yeast-dissolving microorganisms was developed12, and yeast treatment was carried out using the medium1-As a result of searching for yeast-dissolving microorganisms in activated sludge tanks, the National Tax Agency Isolation of a new microorganism YLM-/ (hereinafter referred to as [this microorganism-1]) that grows in the carbon and nitrogen sources of fermentation from the activated sludge of the brewing laboratory.
~, Deposited as Fiber Science and Technology Research Institute No. 1.7g1 1~, The present invention was completed/8 There have been many reports on microorganisms that lyse yeast cell walls [Funatsu and Tsuru eds. These microorganisms secrete cell wall-soluble MNf to the outside of the cells, thereby lysing only dead cells or both dead and viable cells of the heat-treated yeast. It grows easily in meat juice medium, medium containing yeast extract and/or peptone, etc.

Although the morphology of this microorganism is similar to that of ordinary bacteria, it does not lyse dead yeast cells and only lyses live cells.
The lysis method is also that unlike known microorganisms, yeast cell wall lytic enzymes are not secreted outside the bacterial cells, and the microorganism grows while attached to viable yeast cells without causing yeast lysis. It is a very specific microorganism, as it does not grow in meat juice medium, medium containing yeast extract and/or peptone, etc.

According to the present invention, viable yeast cells can be obtained by directly inoculating the present microorganism into a yeast suspension and culturing it in an aerobic environment without using complicated means such as purifying yeast lytic enzymes. It can be dissolved, and yeast extract can be efficiently produced from the culture solution. In addition, yeast cells that proliferate through yeast treatment of food manufacturing wastewater are dissolved by this microorganism, and this microorganism facilitates wastewater treatment in activated sludge tanks.This microorganism does not exhibit the following mycological properties.8 (Morphological findings) ■Cell shape and size: 03-0 g microns x 0.9-/9 g microns■ Motility: Reeds■ Epiphytic status of flagella: 1 It has a book flagellum.

■ Durham staining: negative (growth condition) ■ Yeast Hansenula anomala (H
Ansenula anomala) and the present microorganism are simultaneously smear-cultured: A puff-colored, translucent growth zone of the present microorganism is marked in the center of the white, opaque growth zone of the yeast.

■ Meat juice agar medium: No growth.

■ Proteose Pegton (manufactured by Tifco) O! Person in charge,
Ferment fFJ: extract 0/%, agar medium: no growth.

■ Meat: r-#su 0.3%, tryptone) 05%,
D-glucose 07%, agar medium: No growth.

■ Tryptecase soy broth (manufactured by BB”L)
Agar medium: No growth.

■ Kasiton O. ．． ff%, yeast extract, glycerin 0
5%, agar medium: no growth.

(Physiological properties a) Cl) Catalase activity: + ■ Attitude towards oxygen: Aerobic (■ Growth pH: pH 6~/θ ■ Growth temperature: /2.J”C~3J℃ (lysis of viable yeast cells) ■ Representative examples of yeast that are lysed by the present microorganism (the present microorganism grows at the same time as lysis) are shown below.

EndorITyces geotrichum
E+accharomyces cerevisi (E+accharomyces cerevisi)
aθ) IFO 0,! θ3 Schwanniomyce o(:ciden
talis ) 1FO /gll/ ebaryomyces hansenii ( ) ebaryomyces hanseni. i
) I F 0 0 0 3 +2 Pichj.a membranaefaciens
q) I F O θ/62g Hansenu-'La murakji > I
F O 8g97Citeromyces matrjJens
js ) I F O 0 9 3 y Kluyveromyces 1act.is
Engineering FO/. :p67 Dekkera intermelia I
F○ /bu9/Saccharo]1ycopeis 1'il
+u]igera) 7 F O θ/θ3 Lipomyces Starkey (:ee Starkeyj.)
'LP' 0/, 2 g 9 HarlSenlaJ'lpOra valb
'I'orulopeis candida.
) ■ F Cl θ Otsu. ?
3 Brettanomyces intermediu
s ) IFO/3; f 7 Candicla krueei IFO/
395 Trichosporon fermentans
) I FO//99 ■ A typical example of yeast fl that is not dissolved by this microorganism (no non-microbial growth occurs) is the convex Schizosaccharomyces pombe shown below.
e) IFOθ3cmyces salmonico
lor) IFO/θdgcr'yptococcus albidue
I F Oθ3'7g Trichosporon cutaneum (Trj, chosporon cutaneum)
Engineering FO//9g Rhodotoru Fist Rubra (Rhodotoru]-a, rubra) I
'F○ θ to 7θ Rhodosporidium toruloides (Rhodosporidium toruloj, d
es ) I F Oθ5S nleucosporidium
Leucoeporidi+im 5COttii
) IFO/, 77 microorganisms were isolated by a specific isolation procedure from activated sludge at the National Tax Agency's Brewing Research Laboratory, where yeast treatment is carried out. That is, the activated sludge was appropriately diluted with sterilized physiological saline, and 5θ μl of the diluted solution and 1 rne of live yeast cell suspension were dissolved immediately before use. Quickly add to the following agar medium/8'rrte kept at 95℃,
After mixing well, the mixture was poured into a sterilized dish and solidified. After the agar was made to the same size, it was cultured at 36°C for 5 days.

Composition of agar medium for biological separation of this machine (total volume 7θme) Activated sludge extract 3θme '/15 phosphate buffer (pH 7θ) 5θme pH adjustment (sodium hydroxide solution) pH 2θ agar (Dicots Bact Agar) θ72 (agar medium is /) θ℃
Sterilization was performed for /θ minutes. ) In addition, live yeast cell suspension I'
Hansenula,
a, normala, )Y-/(Feikoken Bacteria No. 3S) was added to YM medium (containing glucose, yeast extract θ3%,
Malt extract 03%, peptone θj%)/θθml was inoculated with a platinum loop, shaken and cultured at 56°C for 2 days. Approximately 1-11q of washed bacterial cells are washed once with sterilized water! Prepare by suspending in sterilized water so that ×/θ9/me 1. Ta.

In addition, the activated sludge extract is obtained by washing the activated sludge with distilled water.
Put the /S ml into a Braun crushing bottle with a volume of qθml, add glass beads Q07 with a diameter of 5 to 05 mm, and process with a Braun cell crusher until the cells in the activated sludge are completely crushed. (It took about 5 minutes.) After soaking, wash the crushed liquid and the crushed activated sludge that arrived at the glass beads with distilled water.
The total volume of the supernatant obtained by centrifugation at ptn/θ for 11 JIP+- was prepared with distilled water and jOme.

When this microorganism grows, a pre-lytic state occurs around the area, so collect the center of the pre-lytic area, dilute it again with sterilized physiological saline, and add the above culture to the bacterial colonies other than the pre-lytic area. The process was repeated from step 1 until it was no longer detected for purification.

Preservation of this microorganism after purification was carried out using yeast Hansenula anomaloma on a broth agar slant medium.
ala) Y-/(Feikoken Bacteria Bssnq No.) and non-microorganisms were smeared at the same time and cultured at 56°C for 2 to 3 days.Puff-colored translucent in the center of the white opaque yeast growth zone. A good method is to confirm the growth zone of non-microorganisms and store at 2°C.

In order to culture this microorganism, viable yeast cells are necessary, and if a heat-sterilized yeast suspension is used instead of a live yeast cell suspension in the above isolation procedure, the cells of this microorganism may be isolated. It is impossible to separate.

Inorganic salts essential for the growth of this microorganism are phosphates and magnesium salts, and activated sludge extract is preferred over magnesium salts. The effect of the activated sludge extract on the growth of non-microorganisms is due to the growth-promoting effect of an unknown Buddha compound in addition to magnesium contained therein.

PL+ of the medium is 6 or more, good size (2 or 7 to 95, culture temperature g!, is i, :i, s °C ~, 3;'C, preferably 1) 7 °C to 33'l''' and aerobically for 1 to q days (2 to S days on solid medium).

A method for separating the microorganism thus cultured from the culture solution and/or slightly remaining yeast;
Usual solid-liquid separation means such as centrifugation, filtration, coagulation precipitation, isoelectric precipitation, etc. can be used, but the simplest method and 1 to 3. Most of the yeast can be removed as a residue by centrifugation for θθθrpm/θ minutes. Depending on the purpose, the supernatant liquid can be used as a suspension of this microorganism, but in order to completely remove yeast, the supernatant liquid must be further diluted with
．． By filtering through a membrane filter with a pore size of 2 microns or less and θg microns or more, the fermentation gradient can be completely removed from the filtrate.

To obtain the present microorganism cells from the filtrate through the membrane filter, /θθθθry+m, :L! It is preferable to collect the bacterial cells as a precipitate obtained by centrifugation for θ minutes or as a filtration residue using a membrane filter of 0.5 microns or less.

The purity of non-microorganisms obtained by each of the above methods can be verified by performing the above-mentioned in-mold operation [1] for an appropriately diluted solution with sterile physiological saline. Apply 1~. The diluted solution was applied to a broth agar plate (12) and incubated at 3θ℃ for 2 to 7 days.
Each can be assayed by culturing for days.

The culture solution obtained by culturing non-organic microorganisms using viable yeast cells contains nucleic acids and proteins and becomes a good yeast extract.

In addition, when yeast treatment is adopted in wastewater treatment, glucose:peptone:yeast is used as a model wastewater at a COD ratio of /θ:/:5, and the sludge load θθS is combined with COD/7.
If activated sludge treatment is performed with MLSS, yeast will be smoothly digested in the activated sludge tank, but if the yeast exceeds this ratio, the predation effect on yeast by protozoa in the activated sludge will be extremely reduced. The yeast flows into the effluent water undigested. In such a case, if the yeast cells are once dissolved by the present microorganism and then transferred to activated sludge, the present microorganism cells will become food for the filler organisms in the activated sludge.
This will enable smooth wastewater treatment.

Next, reference examples of the present invention will be shown.

Reference example/ 7730 to make antimicrobials 7×/θ7/ff1e
Molar phosphate buffer, magnesium sulfate = 2 ppm, yeast Hansenula, ano
mala) y −/ (9:L?iJl fungal infection No. 33 tag number) ix10'7tne, p117θ medium 100m
g, and cultured with shaking in a Sakaro flask for 3θ°C. As a result, the number of yeast bacteria was d no x/θ6/m/! (9
(dissolved in steamed rice), became non-microbial and became V no × / θ nme. Culture solution /θθθθr7nom,! Centrifugation was performed for θ minutes, and the resulting soil was filtered using an ultraviolet absorption method [Biochemistry Experimental Research Group, ed. "Biochemistry Experimental Methods", page 76].
Using B [Kyoshi Sugawara, Masami Soejima, “Protein Determination Method”]
/, ff, page 5] As a result of quantifying protein 1~,
7/ppm (ribonucleic acid; -te) of nucleic acids and 6S
A yeast extract containing proteins of ppnt (1- with stamp albumin) was obtained.

Reference Example: Shaking culture in rice washing wastewater for 3θ℃ for 1 to 2 days) Yeast) S7
Hasenula anoma,
a) Y-7 (Feikoken Bibori No. 3529) to 3.0
The microorganisms were collected by centrifugation for 1-00 rptnla minutes, and 48M phosphate buffer and activated sludge extract were added to the microorganisms so that the activated sludge dry matter concentration was 1-Xθθθppm, and the microorganisms were adjusted to /x/θ77' trte. Add so that
Shaking culture for 7 days at 30°C. As a result, no
/θR7me existed yeast at the end of culture /×/θ6/
ml; 7, while non-microorganisms are 2. , SX/θ'
/me increased.

Add activated sludge to this culture solution with M'LSS.
, θθθppm and aerated at 1.25℃ for 1 day.
rat! What was present was 7×/θ6/ after activated sludge treatment.
mQ? It decreased to . In other words, the organic matter in the rice washing wastewater passed through yeast and was finally preyed on by protozoa in the activated sludge and converted into the microorganisms of Yazumoto.

Claims (1)

[Claims] (1) A novel microorganism strain YLM-/ (Feikoken Bacterial Serial No. 67g/) characterized by having the following mycological properties. B. Gram-negative mouth Cell shape is rod-like Bacteria C. Motile B. Aerobic Bacteria E. Propagation of live yeast cells with a single carbon source and a nitrogen source.