JPS61115485A - Novel pseudomonas vesicularis var. povalolyticus - Google Patents

Abstract

NEW MATERIAL:Pseudomonas vesicularis var. povalolyticus capable of assimilating polyvinyl alcohol. USE:Separation of PVA used in textile sizing agent, adhesive, synthetic starch, etc. PREPARATION:Natural environment was searched widely by using a medium satisfying a certain kind of nutrient requirement and prepared by adding a yeast extract to a medium composed of PVA and an inorganic salt, and a novel bacterial strain separated from the activated sludge for municipal sewage treatment was cultured to obtain Pseudomonas vesicularis var. povalolyticus PH strain (FERM P-7922). When the cultured bacterial cells are cultured in contact with PVA, the cell number (CELL) increases and the PVA concentration decreases with cultivation period.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to a novel Pseudomonas bacterium isolated from nature and a method for decomposing polyvinyl alcohol using the bacterium.

(Prior Art) Polyvinyl alcohol (PVA), which is used in large quantities in fiber sizing agents, adhesives 9, synthetic sizing agents, etc., is a persistent substance that does not easily decompose when released into the natural world.

In processing this, it is necessary to devise measures such as acclimating microorganisms that have a strong ability to assimilate and decompose PVA to a high concentration. but.

Acclimatization of microorganisms requires a long period of time and a great deal of effort. On the other hand, bacteria that have the ability to assimilate and decompose PVA have already been
(Although there have been some reports.

In either case, it is simply decomposition due to symbiosis or mixed culture. When assimilating and decomposing PVA through symbiosis or mixed culture, it is difficult to determine optimal culture conditions because it is rare for two or more types of bacteria to have exactly the same optimal culture conditions. In these cases, culture conditions are determined mainly by focusing on bacteria involved in PVA decomposition and maintaining the optimum conditions. However, the optimal conditions do not necessarily match the optimal conditions of other bacteria, which ultimately leads to a decrease in culture efficiency. Therefore, it is strongly desired that PVA be degraded by a single pure bacterium.

(Object of the invention) An object of the present invention is to provide a novel Pseudomonas vesicularis varietus povaloliticus. Another object of the present invention is to provide a method for assimilating and decomposing polyvinyl alcohol using the novel Pseudomonas vesicularis varietus povaloliticus.

(Structure of the Invention) The novel strain of the present invention - Pseudomonas vesicularis varietus povaloliticus
vesicularis var, povaloly
ticus) has the ability to assimilate polyvinyl alcohol, thereby achieving the above objective. This new microorganism is a PVA (polymerization degree: 500-1700) shown in Table 1.
After adding yeast extract to a medium consisting of inorganic salts and saponification degree (regardless of whether it is completely or partially saponified) and satisfying certain nutritional requirements, we searched widely in nature using this, and found that urban sewage It was separated and collected from activated sludge for treatment. Specifically, Pseudomonas vesicularis varietus povalolyticus PH strain (Pseudomonas
monas vesicularis var, po
valolyticusP) I) (Accession number: FERMP-7922).

Based on the properties described below, this new strain was
y's Manual of Determinati
veBacteriology, the 8th e
It was identified with reference to the bacterial classification book of ditor+ 1974J.

Table 1 PVA synthetic medium* PVA: NH4N0z = 10: 1
Added so that ff November 1! Table 2 shows the mycological properties of the Pseudomonas vesicularis varietus bohalolyticus PH strain of the present invention. In my home country, growth is extremely poor in broth liquid culture, broth agar plate culture, broth agar slant culture, and broth gelatin puncture culture, so Bacto Tryptone 1 is not included in the description of growth conditions.
%, Bacto yeast extract 0.1% (pH 7.2, abbreviated as TY medium) medium, and the growth conditions were established by TY liquid culture, TY agar slant culture, TY agar plate culture, and TY gelatin puncture culture. Here, the agar used was dry powdered agar that had been washed with distilled water for two weeks or more. In addition, for the use of carbon sources, 1 carbon source is added to peptone water with 0.05% yeast extract added.
% was added and examined.

(Left below) The strain of the present invention is different from Pseudomonas vesicularis in that it has the ability to assimilate polyvinyl alcohol and produces nitrous acid from the auxotrophic substance nitric acid.

and fructose availability, all other characteristics consistent with this. Therefore, one domestic strain is Pseudomonas
Although it is a near-green strain of B. vesicularis, it is considered to be a new species.
It was identified as Pseudomonas vesicularis varietus povaloliticus because of its poval resolution.

The strain of the present invention, Pseudomonas sp, PII, was obtained from Nishizawa et al.
As shown in Table 3, the bacterial strain Pseudomonas sp, VS25B (Japanese Patent Publication No. 57-49194) is capable of denitrification, citric acid utilization, production of pigments, production of acids and alkalis from sugars, and nutritional requirements. They differ in their physiological properties related to sex. Therefore, both are different strains.

(The following is a blank space) Table 3 The bacterial strains of the present invention are also described by Shiso et al.
2) strain Pseudomonas sp, No. Z-
301 and the same No. Z-302, as shown in Table 4, are oxidase, catalase, hydrogen sulfide generation,
They differ in physiological properties regarding reduction of vP Tesro nitrate and production of pigment, as well as growth temperature (whether or not they grow at 41°C is an important identification factor for Shademonas). Therefore, the native strain is also different from the strain of Igaku et al.

The strain of the present invention is also used by Suzuki et al.
It differs from the polyvinyl alcohol-degrading strain Pseudomonas C-3 of Publication No. 4) in growth state 1, production of pigment, gelatin liquefaction, etc. In particular, the strain of Suzuki et al. has characteristics of the genus Flavobacterium in that it produces a water-insoluble yellow pigment. In this way, the strain of the present invention is also different from the strain of Suzuki et al.

Culture conditions It doesn't have to be anything special as a medium, just meat extract.

Organic nutritional sources such as malt extract, peptone, and phosphate, magnesium, sodium, and potassium.

A normal medium containing an appropriate inorganic nutrient source such as iron may be used, but it must contain yeast extract or vitamin B1 and leucine. As yeast extract.

Usually o,oot%~0.1% preferably o,oo
It is used in a range of s% to 0.1%.

As the carbon source, various P' substances, polyvinyl alcohol, etc., which served as substrates in the polyvinyl alcohol assimilation ability test, are used. As the nitrogen source, inorganic nitrogen such as nitrates and ammonium salts, organic nitrogen such as amine groups, etc. are used. The culture temperature is 15°C to 35°C, preferably 25°C to 35°C.
It is 0°C. Culture pl+ is 6-9. Preferably, it is 7 to 8, and the bacterial strain is cultivated for 3 days to 1 week with aerobic stirring or shaking. yeast extract, PVA, etc. as shown in Table 1.
By culturing aerobically in an inorganic salt medium, the medium contains 0
．． PVA contained in a proportion of 1% to 1.0% is almost completely decomposed. Instead of yeast extract.

Vitamins (e.g. calcium pantothenate).

Inositol, niacin, para-aminobenzoic acid.

Even when using pyridoxine, thiamine, biotin, vitamin 81□, etc.) and casamino acids, PVA is almost completely degraded.

In addition to this, PVA decomposition in the present invention includes yeast extract, vitamins, and casamino acids.

Or, grow this strain in PVA or an appropriate carbon source or TY medium to which a substance showing the same effect is added,
This can be carried out by appropriately selecting at least one of the following: resting bacterial cells, bacterial cell extract (crude enzyme solution), purified enzyme, immobilized bacterial cells, and immobilized enzyme state. The reaction system at that time is shown in Table 5.

The amount of bacterial cells or extract used in Table 5 is that the final concentration as a bacterial cell suspension is 0.1 or more and the absorbance at 600 nm is usually 0.1 or more.

It is in the range of 0.5-30. The bacterial cell extract is obtained by processing the bacterial cells using an ultrasonic crusher or a French press.

Absorbance at 280 nm is o, i or more 1 Usually, 0.5 to 5
．． Used in the i range of 0. Reaction pH is 6~
It should be in the range of 9. Preferably, it is adjusted to a range of 7 to 8.

The concentration of PVA is selected from 0.05% to 1.0%, preferably from 0.1% to 1.0%. A 0.1 mol phosphate buffer is used as the buffer, but is not limited thereto. The reaction temperature may be within the range of 15°C to 35°C. Preferably it is 25°C to 30°C. When using resting bacterial cells, P can be obtained without significant growth of bacterial cells.
VA can be almost completely decomposed. Therefore, when PVA is degraded using immobilized microbial cells, maintenance and management of the one-product decomposition system is easy because overgrowth of the microbial cells does not occur. The PVA-degrading enzyme produced by this strain is contained in large amounts in the cultured cells and culture solution, and it can be easily concentrated and recovered by the usual salting-out method using ammonium sulfate.
It can be subjected to VA decomposition. Its PVA degradability is extremely stable.

The amount of PVA in the system is determined by, for example, a coloring method using iodine.

(Example) The present invention will be described below with reference to Examples.

Example 1 50” 100 m7! of culture medium with the composition shown in Table 6 was prepared at 300I
II! ! It was placed in an Erlenmeyer flask and sterilized. The native strain was grown in advance on an agar slant medium of TY or PVA/yeast extract, inoculated into a medium in an Erlenmeyer flask, and cultured with shaking at 30°C for 5 days. The results are shown in the figure. Bacterial cell concentration is 60
The absorbance at 0 nm and PVA11 were determined by the color method with iodine. As is clear from the figure, PVA in the medium was almost completely decomposed after about 5 days of culture.

Table 6 Example 2 The native strain was grown in a medium having the composition shown in Table 6, and the cells were collected by a conventional method such as centrifugation.

PVA was added to a phosphate buffer solution at pH 7.2 containing a final concentration of 0.1% so that the final concentration was 5.0 based on the absorbance at 600 nm, and the mixture was shaken at 30°C for 24 hours. PVA was almost completely decomposed.

The native culture was grown in a medium with the composition shown in Table 6, and the bacterial cell extract was added to the PVA-containing buffer solution of Example 2 to a final concentration of 2.
When mixed so that the absorbance at 80 nm was 1.0 and reacted at 30° C. for 20 minutes, about 50% of PVA was decomposed.

(Effects of the Invention) The new strain of the present invention thus has the ability to assimilate and decompose PVA. By using this strain, PVA in the culture medium can be reduced.
is effectively decomposed. PVA can be effectively decomposed using resting cells of this strain, corpuscular extract, purified enzyme, and the like.

[Brief explanation of the drawing]

The figure is a graph showing the PVA assimilation and decomposition ability of the strain of the present invention.

Claims (1)

[Claims] 1. Pseudomonas vesicularis varietus povalolyticus having the ability to assimilate polyvinyl alcohol. 2. Pseudomonas vesicularis varietus povalolyticus according to claim 1, which is Pseudomonas vesicularis varietus povalolyticus PH strain. 3. Cultivating Pseudomonas vesicularis varietus povalolyticus having the ability to assimilate polyvinyl alcohol in a polyvinyl-containing medium, or contacting the cultured cells of Pseudomonas vesicularis varietus povalolyticus or its culture filtrate with polyvinyl alcohol. How to decompose polyvinyl alcohol.