JP2005278639A - Pva-degrading bacterium and method for degrading pva - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a PVA (polyvinyl alcohol)-degrading bacterium which singly has an excellent degrading ability and an assimilating ability also against various PVA derivatives, to provide a method for degrading and assimilating PVA with the bacterium, and to provide a method for degrading and assimilating PVA, wherein a microorganism group containing the bacterium exhibits a high PVA-degrading activity even in an environment in which a nutrition source capable of be easily assimilated with microorganisms, such as gelatin or starch, coexists. <P>SOLUTION: The method for degrading polyvinyl alcohol or a polyvinyl alcohol derivative comprises bringing a bacterial strain belonging to Sphingomonas aromaticivorans sp., a microorganismic group containing the bacterial strain, or the cell ingredients of the bacterial strain into contact with the polyvinyl alcohol or the polyvinyl alcohol derivative. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to the decomposition and utilization of polyvinyl alcohol (hereinafter referred to as “PVA”).

  PVA is widely used industrially as a water-soluble synthetic polymer, and is widely used for raw materials of synthetic fibers or films, fiber processing agents, paper processing agents, adhesives, inorganic binders, vinyl chloride resin polymerization stabilizers, etc. Has been.

  In recent years, interest in environmental problems on a global scale has increased, and research on biodegradable polymers that are degraded by microorganisms and enzymes in the natural environment has been actively conducted. In addition, a technology that reduces the burden on the environment by decomposing various compounds before being released to the environment has been attracting attention. Conventionally, PVA waste liquid has been considered as a cause of COD increase in wastewater, and microbial treatment with activated sludge has been studied, and the search for degrading bacteria has been promoted. Pseudomonas bacteria, Enterobacter bacteria, Acinetobacter bacteria, Corynebacterium bacteria, Rhodococcus bacteria, Caseobacter bacteria, Sphingomonas bacteria, etc. are known as microorganisms having a resolution against PVA. (Patent Documents 1 to 6, etc.) The microorganisms that have been reported so far are bacteria having a PVA-decomposing ability and symbiotic bacteria.

Japanese Patent Publication No.55-1791 Japanese Patent Publication No.57-27713 Japanese Patent Publication No.57-27714 JP-A-8-140667 Japanese Unexamined Patent Publication No. 7-108297 JP 2003-250527 A

  The present invention provides a PVA-degrading bacterium having an excellent ability to decompose and assimilate various PVA derivatives alone, a method for degrading and assimilating PVA using the bacterium, and microorganisms such as gelatin and starch It is an object of the present invention to provide a method for degrading and assimilating PVA in which a microorganism group containing the bacterium exhibits high PVA degrading activity even in a nutrient source coexisting environment that is easy to assimilate.

  The present inventors searched for microorganisms widely from the natural world such as rivers and soils and activated sludge, and as a result, Sphingomonas aromaticivorans isolated from the activated sludge of Fuji Photo Film Co., Ltd. Fujinomiya Plant and the bacteria It has been found that a microorganism group containing Pha has excellent decomposition and assimilation ability with respect to PVA and its derivatives, and by using this, it is possible to satisfactorily purify effluents containing various types of PVA. In addition, the present inventors have found that even if nutrient sources that are easily assimilated by microorganisms such as gelatin and starch are present in the drainage liquid, the PVA decomposition can be satisfactorily performed without delay.

  That is, according to the present invention, a strain belonging to the species Sphingomonas aromaticivorans, a microorganism group containing the strain, or a cell component of the strain is brought into contact with polyvinyl alcohol or a polyvinyl alcohol derivative. A method for decomposing polyvinyl alcohol or a polyvinyl alcohol derivative is provided.

According to another aspect of the present invention, a strain belonging to the species of Sphingomonas aromaticivorans, a microorganism group containing the strain, or a cell component of the strain containing polyvinyl alcohol or a polyvinyl alcohol derivative There is provided a method for purifying drainage, drainage or soil, comprising contacting the drainage, drainage or soil.
Preferably, the strain belonging to the species Sphingomonas aromaticivorans is Sphingomonas aromaticivorans UF-7 (accession number FERM BP-10187).

  According to another aspect of the present invention, there is provided a polyvinyl alcohol-degrading bacterium deposited as Sphingomonas aromaticivorans UF-7 (Accession No. FERM BP-10187).

According to still another aspect of the present invention, a microorganism group containing Sphingomonas aromaticivorans UF-7 (Accession No. FERM BP-10187) is immobilized on a carrier, and is converted into polyvinyl alcohol or a polyvinyl alcohol derivative. There is provided a method for decomposing polyvinyl alcohol or a polyvinyl alcohol derivative, which comprises contacting.
According to still another aspect of the present invention, a microorganism group containing Sphingomonas aromaticivorans UF-7 (Accession No. FERM BP-10187) is immobilized on a carrier, and polyvinyl alcohol or a polyvinyl alcohol derivative is obtained. There is provided a method for purifying drainage, drainage or soil, which comprises contacting the wastewater, drainage or soil contained therein.
Preferably, the immobilization carrier is activated carbon or a crosslinked polymer.

  The PVA decomposition method of the present invention is useful for decomposing and assimilating PVA in various waste water, drainage, soil purification treatment, and compost treatment.

  As PVA in the present invention, in addition to PVA obtained by saponifying polyvinyl acetate, PVA derivatives imparted with functionality such as anionic, cationic, silane-reactive, terminal hydrophobic PVA, and the like can be mentioned. By using the bacterium of the present invention, it is possible to decompose and assimilate regardless of polymerization degree, complete saponification, or partial saponification of PVA, but as PVA excellent in degradability, the polymerization degree is 300 to 3500, The saponification degree is 86 to 99%, and examples of the PVA derivative include anionic or terminal hydrophobic PVA.

  The bacterium having the ability to decompose and assimilate polyvinyl alcohol according to the present invention is a strain belonging to the species of Sphingomonas aromaticivorans.

  This Sphingomonas aromaticibolans species having the ability to decompose and assimilate polyvinyl alcohol has been widely searched from the natural world. For example, it can be separated as follows.

  The Sphingomonas aromaticibolans species having the ability to decompose and assimilate the polyvinyl alcohol of the present invention is isolated, for example, as follows. That is, samples of rivers, activated sludge, soil, seawater, lake water, etc. are filtered with a membrane filter (pore size 0.1-0.5 μm), and the membrane filter is immersed in a PVA liquid medium and cultured with shaking to decompose PVA. Determine the presence of bacteria. Thereafter, the culture solution is spread on an appropriate medium to form colonies. The obtained colonies may be inoculated into a low nutrient medium containing PVA, and bacteria having PVA resolution may be selected and separated.

  An example of a bacterium having the ability to degrade and assimilate PVA and its derivatives according to the present invention is Sphingomonas aromaticivorans UF-7 strain. The Sphingomonas aromaticivorans UF-7 strain is registered with the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1st, 1st, Higashi 1-chome, Tsukuba City, Ibaraki, Japan) on February 4, 2004. Deposited as -19664 and transferred to International Deposit under the Budapest Treaty as of December 21, 2004 as deposit number FERM BP-10187. The mycological properties and genetic analysis results of Sphingomonas aromaticivorans UF-7 strain are described below.

(Mycological properties)
Medium properties: LB agar (Sambrook, J., EF Fritsch, and T. Maniatis. (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, NY), yellow, low convex To form circular colonies.

(Genetic analysis)
Analysis of 16S rDNA
UF-7 strain is inoculated into LB agar medium, DNA is extracted from the culture at 30 ° C, amplified by PCR, purified PCR product, BigDye ^TM Terminator v.3.1 (Appried Biosystem) The sequence of 16SrDNA is determined using a cycle sequence reaction, and the 16SrDNA analysis is performed using the FASTA program of DNA Data Bank of Japan (http://www.ddbj.nig.ac.jp/) on the web. Went. As a result, 16S rDNA of the UF-7 strain of the present invention was 98.2% of that of Sphingomonas aromaticivorans (M. Sakamoto et al., DDBJ / EMBL / GenBank databases direct submittion. Accession No. AB025012). It had homology.

  In principle, the culture of the bacteria is based on, for example, the culture of microorganisms belonging to the genus Sphingomonas, and is preferably performed under aerobic conditions such as a shaking culture method by liquid culture and an aeration and agitation culture method. It is.

  As a medium used for the culture, any of various synthetic media, a semi-synthetic medium, a natural medium and the like for aerobic bacteria can be used. For example, meat extract or peptone may be used. The bacterium uses methionine as an essential nutrient.

  The pH of the medium is preferably 6-8. The culture temperature is preferably maintained at a temperature at which Sphingomonas aromaticibolans grows well, usually about 15 to 42 ° C, particularly preferably about 25 to 38 ° C. In general, the culture time can be aerobically about 2 days to 1 week in both cases of liquid shaking culture and aeration and agitation culture. In addition, said culture conditions can be suitably changed according to the kind and characteristic of microorganisms to be used, external conditions, etc.

  The bacterium of the present invention can satisfactorily degrade and assimilate PVA and PVA derivatives as shown in Examples below. Accordingly, a strain belonging to the species of Sphingomonas aromaticivorans, a microorganism group containing the strain, or a cell component of the strain, for example, industrial waste such as domestic wastewater, industrial wastewater, drainage, This can be purified by bringing it into contact with a PVA-containing material such as soil. Here, the bacterial cell components include cultured bacterial cells belonging to the genus Sphingomonas, immobilized bacterial cells, bacterial cell extracts, immobilized bacterial cell extracts, bacterial cell extracts, culture solutions, and culture broths Etc. are included. Examples of the carrier that can be used for fixing bacteria include activated carbon and a crosslinked polymer.

As a method for decomposing PVA using the microorganism of the present invention, for example, in the case of wastewater purification, the bacterium Sphingomonas aromaticivorans of the present invention or a group of microorganisms containing the bacterium is immobilized on an individual source. Means for attaching the treatment device, means for adding the bacterium of the present invention or a group of microorganisms containing the bacterium in an aeration tank, etc. Means etc. are mentioned. In the case of environmental purification, means for applying the bacterium of the present invention or a group of microorganisms containing the bacterium to rivers and lakes in the natural environment can be employed. Such PVA decomposition treatment is performed at pH 4 to 10, preferably pH 5 to 8, treatment temperature 5 to 40 ° C., preferably 15 to 35 ° C., usually 0.5 hours to 14 days, preferably 1 hour to 7 days. Good to do.
The following examples further illustrate the present invention in detail but are not to be construed to limit the scope thereof.

Example 1: Isolation of Sphingomonas aromaticibolans UF-7 strain
100 ml of PVA medium (Table 1) was prepared, and in order to remove impurities such as acetate contained in the raw material PVA, E. coli wild strain MG1655 without PVA resolution was inoculated and cultured overnight at 30 ° C. with shaking. The culture solution was sterilized by filtration with a membrane filter (pore size: 0.2 μm), 50 μl of the PVA waste solution-treated sludge suspension was inoculated into the filtered PVA medium, and cultured with shaking at 30 ° C. for 10 days. After applying a part of the culture solution to LB agar medium and culturing at 30 ° C for 1 week, select a single colony from the plate where growth was observed, perform PVA resolution confirmation and identification test, Sphingomonas Aromaticibolans UF-7 strain was obtained.

Example 2: Decomposition and assimilation of PVA using Sphingomonas aromaticibolans UF-7 strain As a preculture, UF-7 strain grown on LB agar medium was inoculated into 4 ml of LB liquid medium with a platinum loop, Shaking culture was performed at 30 ° C. for about 4 days. The cells were separated from the culture solution, washed several times with a modified PVA medium (Table 2), inoculated into a new modified PVA medium, and cultured with shaking at 30 ° C. A portion of the culture solution was extracted over time, and the turbidity (OD600) and PVA concentration were measured (Kim, BC, CK Sohn, SK Lim, JW Lee, and W. park. (2003) Degradation of polyvinyl alcohol by Sphingomonas sp. SA3 and its symbiote. J. Ind. Microbiol. Biotechnol., 30, 70-74). The results are shown in FIG. As can be seen from the results in FIG. 1, the UF-7 strain did not require the addition of PQQ, and was shown to efficiently degrade PVA alone.

Example 3: Decomposition and assimilation of PVA using microorganism group containing Sphingomonas aromaticivorans UF-7 strain Microorganism group culture solution containing Sphingomonas aromaticivorans UF-7 strain accumulated from activated sludge 1 ml was added to 20 ml of a medium containing various PVA (Table 3), cultured with shaking at 28 ° C. and 140 rpm, a part of the culture solution was taken out over time in the same manner as in Example 2, and the PVA concentration was measured. The results are shown in FIG.

  As can be seen from the results in FIG. 2, this group of microorganisms showed a high PVA degrading activity independent of the PVA species. There was no delay in PVA degradation even in the presence of easily assimilated gelatin.

Example 4: Decomposition and assimilation of PVA using a carrier on which a microorganism group containing Sphingomonas aromaticivorans UF-7 was immobilized Sphingomonas aromaticibolans UF-7 accumulated from activated sludge The microorganism group culture solution was immobilized on 35 g of Kuraray carrier (Kragale), 100 ml of the medium shown in Table 4 was added, and the mixture was cultured with shaking at 28 ° C. and 140 rpm. As in Example 2, a part of the culture solution was extracted over time, and the PVA concentration was measured. The results are shown in FIG. As can be seen from the results in FIG. 3, it was confirmed that PVA can be processed in a short time.

Example 5: PVA decomposition treatment with accumulated sludge immobilized on an activated carbon carrier Sphingomonas aromaticivorans A group of microorganisms including UF-7 strain together with activated carbon carrier BCP (Denka Engineering) for water treatment The cells were cultured for about a week in a PVA-containing medium to immobilize the cells. The following experiment was conducted using this carrier.
20 ml of a medium containing 0.5% PVA and 0.1% yeast extract was added to 6 ml of microorganism-immobilized BCP, and cultured with shaking at 28 ° C. and 140 pm. The culture medium was sampled and centrifuged, and the PVA concentration in the supernatant was quantified by the Finley method (KI / I ₂ -H ₃ BO ₃ , 690 nm). The results are shown in FIG. 4 below. The PVA concentration drops to 1/10 or less in 3 days, and the effect of increasing the density of the cells by immobilizing the carrier appears.

Example 6: Amino acid requirement study 20 standard amino acids (0.1% of each amino acid, 0.20% in total) were added to the modified PVA medium, and when the UF-7 strain was cultured alone, cell growth was observed. About 0.12% of PVA was confirmed to be almost completely degraded in 5 days (FIG. 5). Therefore, it was revealed that the components required by the UF-7 strain are present in 20 standard amino acids.

  Furthermore, the role of standard amino acids was investigated. Two glucose media without dry yeast extract were prepared, and 20 standard amino acids were added in appropriate amounts to one of them. The UF-7 strain was cultured alone for 1 week in these two types of media, and the growth of the bacteria was observed. As a result, growth of the bacteria was confirmed in the medium to which the amino acid was added, but no growth was observed in the medium to which the amino acid was not added. From these results, it is considered that the 20 standard amino acids are not limited to PVA degradation but are necessary as nutrients for the growth of the UF-7 strain.

  Next, essential amino acids of UF-7 strain were examined from 20 standard amino acids. First, prepare two modified PVA media, add appropriate amounts of each amino acid mixture divided into two groups of 10 to the medium, and culture the UF-7 strain for 1 week. It was measured. The essential amino acids were narrowed down and identified from the group of amino acids showing cell growth and PVA degradation activity by the same operation. As a result, it was confirmed that the bacteria grew and PVA was decomposed only by adding only methionine (FIG. 6). The above results revealed that the essential nutrient for UF-7 strain is methionine.

Modified PVA medium
PVA 1.0g
Solution I (below) 50ml
Solution II (below) 50ml
Trace metal (below) 2ml
Prepared to 1.0 L with H ₂ O

Solution I
(NH ₄ ) ₂ SO ₄ 20 g / l
MgSO ₄ 19.6 g / l
NaCl 2.0 g / l

Solution II
KH ₂ PO ₄ 8.0 g / l
K ₂ HPO ₄ 64 g / l

Trace metal
CaCl ₂ 250 μM
(NH ₄ ) ₆ Mo ₇ O ₂₄ 1.5 μM
H ₃ BO ₃ 200 μM
CoCl ₂ 15 μM
CuSO ₄ 5 μM
MnCl ₂ 40 μM
ZnSO ₄ 5 μM
FeSO ₄ 5 mM

FIG. 1 shows the results of measuring the degradation and utilization of PVA using Sphingomonas aromaticibolans UF-7 strain. FIG. 2 shows the results of measuring the degradation and utilization of PVA using a microorganism group containing Sphingomonas aromaticibolans UF-7 strain. FIG. 3 shows the results of measuring the degradation and utilization of PVA using a carrier on which a microorganism group including Sphingomonas aromaticibolans UF-7 strain is immobilized. FIG. 4 shows the result of measuring PVA decomposition by accumulated sludge immobilized on an activated carbon carrier. FIG. 5 shows the growth and PVA resolution of the UF-7 strain in an amino acid-added PVA medium. FIG. 6 shows the PVA resolution of the UF-7 strain in PVA medium supplemented with amino acids and methionine.

Claims (8)

Polyvinyl alcohol or polyvinyl alcohol comprising contacting a strain belonging to the species of Sphingomonas aromaticivorans, a microorganism group containing the strain, or a cell component of the strain with polyvinyl alcohol or a polyvinyl alcohol derivative Degradation method of derivatives. The polyvinyl alcohol according to claim 1, wherein the strain belonging to the species Sphingomonas aromaticivorans is Sphingomonas aromaticivorans UF-7 (accession number FERM BP-10187). Alternatively, a method for decomposing a polyvinyl alcohol derivative. A strain belonging to the species of Sphingomonas aromaticivorans, a microorganism group containing the strain, or a cell component of the strain is brought into contact with waste water, drainage or soil containing polyvinyl alcohol or a polyvinyl alcohol derivative. Waste water, drainage, or soil purification method. The drainage according to claim 3, wherein the strain belonging to the species Sphingomonas aromaticivorans is Sphingomonas aromaticivorans UF-7 (accession number FERM BP-10187), Wastewater or soil purification method. Polyvinyl alcohol-degrading bacterium deposited as Sphingomonas aromaticivorans UF-7 (Accession No. FERM BP-10187). Polyvinyl alcohol or polyvinyl alcohol comprising immobilizing a microorganism group including Sphingomonas aromaticivorans UF-7 (Accession No. FERM BP-10187) on a carrier and contacting with polyvinyl alcohol or a polyvinyl alcohol derivative Degradation method of derivatives. A microorganism group including Sphingomonas aromaticivorans UF-7 (Accession No. FERM BP-10187) is immobilized on a carrier and brought into contact with waste water, drainage or soil containing polyvinyl alcohol or a polyvinyl alcohol derivative. Waste water, drainage, or soil purification method. The method according to claim 6 or 7, wherein the immobilization carrier is activated carbon or a crosslinked polymer.

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