JP2002125660A - New microorganism belonging to bacillus thuringiensis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new microorganism which has a high α-amylase activity performance and can degrade starch contained in organic waste water and organic/biological sludge. SOLUTION: (1) A new microorganism Bacillus thuringiensis X-1 belonging to Bacillus thuringiensis and having a performance for degrading starch contained in organic waste fluids and/or biological sludge. (2) A new microorganism Bacillus thuringiensis X-1 belonging to the above-described Bacillus thuringiensis and deposited as accession number FERM P-18014.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel microorganism,
More specifically, the present invention relates to a novel microorganism Bacillus thuringiensis X-1 belonging to Bacillus thuringiensis which can degrade starch contained in organic waste liquid and / or biological sludge.

[0002]

2. Description of the Related Art Conventionally, the decomposition of organic matter by activated sludge has been performed by a group of microorganisms living in the sludge. There is no.

[0003]

In recent years, many types of bacteria exhibiting amylase activity have been isolated from biological sludge discharged from human waste treatment plants that do not generate sludge. It is expected to decompose starchy components contained in wastewater, organic and / or biological sludge, and to suppress the amount of generated sludge. It is an object of the present invention to provide a novel microorganism having a high α-amylase activity capable of degrading starch contained in organic wastewater and organic / biological sludge.

[0004]

Means for Solving the Problems The present inventors have conducted intensive studies to search for microorganisms having a function of efficiently decomposing starchy components contained in organic waste liquid and biological sludge. The present inventors have completed the present invention by isolating bacteria having desired characteristics and identifying their species. That is, the invention claimed in the present application is as follows.

(1) A new microorganism belonging to Bacillus schuligensis X-1 having the ability to degrade starch contained in organic waste liquid and / or biological sludge. (2) A novel microorganism belonging to Bacillus schuligensis X-1 according to (1), which is deposited under accession number FERM P-18014.

[0006] (3) The novel microorganism belonging to Bacillus schuligensis according to (1) or (2), wherein the novel microorganism has the following mycological properties A to D. X-1. A. Morphological properties (1) Cell shape: bacilli, (2) Motility: +, (3) Spores: +, (4) Gram stain: + B. Growth state in medium (1) Standard agar culture: + C. Physiological properties (1) Gram stain: +, (2) Nitrate reducing ability: +, (3) Denitrification:-, (4) VP test:
+, (5) Indole formation: +, (6) Starch hydrolysis: +, (7) Soybean oil degradability: +, (8) Use of inorganic nitrogen source: +, (9) Oxidase: +, ( 10) Catalase: +,
(11) Range of growth: temperature 13-40 ° C., (12) Attitude to oxygen: aerobic, (13) OF test: glucose ++, (1
4) Utilization of ammonia: +, (15) Decomposition of NaHS: + Genetic properties (1) G + C content: 35 mol%, (2)
Based on genomic DNA analysis (base 5 to base 1540) of 16S ribosomal RNA, homology to Bacillus schulgensis: 99.22%, (3) base sequence: SEQ ID NO: 1.

In order to determine the biological characteristics of the novel microorganism obtained according to the present invention, the mycological properties of this microorganism, namely A. Morphological properties, B. Growth state in medium, C.I. Physiological properties and D. Tests were performed for genetic properties. The results are shown below. A. Morphological properties (1) Cell shape and size: 0.8% Nutrient broth (OxidCM-1), 0.8% glucose, 0.02% dried yeast extract and 0.6% salt as medium
The culture was carried out at 32 ° C. on an agar medium consisting of 1.5% (all W / V%) and 1.4 W / V% agar.
When cultured for 24 hours, the bacterium was a 1.5 × 5 μm gram-positive bacillus, and further cultured at room temperature for 5 days.
Many on long chains were observed.

(2) Existence of motility: The motility can be confirmed in the suspension specimen, and the motility was pericytic. (3) Presence or absence of spores: Spores are formed, oval in shape, and expanded from the cells. (4) Gram stain: Stainability was shown in the initial sampling of colony formation. The cells at this point are vegetative cells,
No sporulation was observed. B. Growth state in culture medium (1) Standard agar medium: 32 ° C 2
The morphology of the colonies cultured for 4 hours was a milky wrinkled colony.

C. Physiological properties (1) Gram staining: +, (2) Nitrate reducing ability: +, (3)
Denitrification:-, (4) VP test: +, (5) Indole formation: +, (6) Starch hydrolysis: +, (7) Soybean oil degradability: +, (8) Inorganic nitrogen source Use: +, (9) Oxidase: +, (10) Catalase: +, (11) Range of growth: temperature 13-40 ° C.
The temperature was set in 1 ° C increments up to ° C, and the growth was observed for 24 hours. The high temperature side was set at 40, 45, 50, and 55 ° C., and the growth in a liquid medium and a slant medium was observed for 24 hours. (12) Attitude to oxygen: aerobic, (13) OF test: glucose ++, (14) availability of ammonia: +, (15) Na
Degradability of HS: +

D. Genetic properties The G + C content was 35 mol%, and the literature value of Bacillus thuringiensis (Holt JGBergey's Manual of Systematic B
^{acteriology, 9 th ed., Vol} ., ed. By PHA Sneath,
Williams ＄ Wilkins, Baltimore, 1986, pp.1104-113
9).

The measuring method is MgSO_Four・ 4-5
H_TwoO and MnSO_Four1200mg / l each using
Adjust the solution and aseptically add Mg to the sterilized medium.⁺4mg as
/ L, Mn²⁺And 1.5 mg / l
Culture was performed at 7 ° C. for 4-5 days until just before spore formation. Centrifugation
The cells obtained by the separation were 0.1 M EDTA / 0.15 M Na
Washed with Cl and stored at -20 ° C. Follow the general method
To extract DNA. DNA GC analysis for DNA degradation
Perform at 50 ° C for 1 hour using a kit (Yamasa Shoyu)
Was. The G + C content was analyzed by HPLC (LC-LC manufactured by Shimadzu Corporation).
9A and SDPD-6A) at 270 nm
And the column was AQ-312 (manufactured by YSC).
0x150 mm) and 10 mM H_ThreePO_Four-10m
MH _TwoPO_Four(PH 3.5 ± 0.01), flow rate 1.8
Elution was performed at ml / min.

As a genetic analysis method, analysis of 16S ribosomal RNA genomic DNA was employed. Analysis was performed using PE Applied DNA collected from the bacteria collected by a commonly used method.
Using a kit manufactured by Biosystems, genomic DNA of 16S rRNA was amplified according to this protocol.
The primers used were 5f, 338f, 515
f, 776f, 1087f, 1174f and 357
r, 531r, 810r, 1104r, 1193r, 1
540r. PCR temperature conditions are 95 ° C for 30 seconds, 6
30 cycles were performed at 0 ° C. for 30 seconds and at 72 ° C. for 45 seconds. Sequences were analyzed using ABIPRISM 310. The obtained sequence data is PE Applied Biosy
When the homology to Bacillus schulgensis was calculated using the MicroSeq database of stems, 9
9.22%. The obtained novel microorganism had a base sequence of 5 base to 1540 base of SEQ ID NO: 1.

The properties of the novel microorganism described above are
Bacillus thuringis X-1 (Bacillus thuring)
iensis X-1). The obtained Bacillus schuligensis X-1 was obtained by the applicant on September 4, 2000, 1-3-1 Higashi-cho, Tsukuba-shi, Ibaraki, Japan. Deposited at the Technical Research Institute and given the accession number FERM P-18014. The present invention includes the deposited microorganism as well as its mutants and progeny having the above-mentioned abilities.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The novel microorganism Bacillus schuligensis X-1 of the present invention was isolated as follows. 0.1 ml of a sludge suspension obtained by dispersing the collected sludge in a blender for 10 seconds was diluted with sterilized 0.6% saline to 10 ² , 1.
0 ^4, was diluted 10 ⁶ times. 0.1 ml of each diluent was used for A
The cells were spread on an agar plate composed of a medium and cultured at 32 ° C. Bacterial identification was based on colony shape, microscopic observation of cells, and biochemical tests. Also, use Bacillu on flat or sloped surfaces.
Since s-form bacteria form spores, the presence or absence of spore formation of each colony strain was determined 2 to 5 days after the appearance of colonies. When a plurality of strains were mixed in the colony, they were further separated by a dilution method. Gram-negative bacteria and Bacillus
If the genus bacteria could not be isolated by the dilution method, spores of Bacillus genus produced by culturing on the slope consisting of the following medium A were suspended in sterilized 0.6% saline and heated at 85 ° C. for 10 minutes. The gram-negative bacteria which cannot be separated were isolated. Composition of medium A Nutrient broth (Oxioid CM-1) 8 g Glucose 8 g NaCl 6 g Dry yeast extract (Difco) 0.2 g Agar 14 g DW 1000 ml

A starch degradation test was performed on the obtained colonies. Starch degradation test was potato starch 5
g, 4 g of nutrient broth, 8 g of glucose, and 15 g of agar were dissolved in 1000 ml of distilled water, sterilized, sterilized, and then dispensed in 20 ml portions into a 9 cm diameter petri dish to prepare an agar medium. After culturing for 3 days, the diameter of the halo formed by applying the I ₂ -KI solution was measured,
++, ++ for 5 mm or more, + for less than 5 mm, and-for no halo. These tests were performed, and the strain having the highest starch degradability was designated as X-1. Similarly, a strain (X-2) showing starch degradability was selected as a comparative strain.

Bacillus schuligensis X-1 and X-
The results of a starch degradability test with No. 2 were as follows. From the above results, Bacillus schulgensis X-1 strain was
It was determined that the strain had a very high α-amylase activity. The base sequence of the analyzed 16S rRNA of the Bacillus juligensis X-1 strain has SEQ ID NO: 1.

[0017]

According to the novel microorganism Bacillus schuligensis X-1 of the present invention, since it has high performance of α-amylase activity, starch contained in organic waste liquid and / or biological sludge can be decomposed. In addition, the amount of generated sludge can be significantly reduced.

[0018]

[Sequence list] <160> 1 <210> 1 <211> 1543 <212> DNA <213> Bacillus thuringiensis <400> 1 ggagttgatc ctggctcagg attaacgccg gcggcgtgcc taatacatgc aagtcgagccat gagttag gagttag gagttag gacctgctcgagcc gagttc gagcag 120g gaaccgcatg gttcgaaatt gaaaggcggc ttcggctgtc acttatggat ggacccgcgt 240 cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag ccgacctgag 300 agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta 360 gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt gatgaaggct 420 ttcgggtcgt aaaactctgt tgttagggaa gaacaagtgc tagttgaata agctggcacc 480 ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacct 540 aggtggcaag cgttatccgg aattattggg cgtaaagcgc gcgcaggtgg tttcttaagt 600 ctgatgtgaa agcccacggc tcaaccgtgg agggtcattg gaaactggga gacttgagtg 660 cagaagagga aagtggaatt ccatgtgtag cggtgaaatg cgtagagata tggaggaaca 720 ccagtggcga aggcgacttt ctggtctgta actgacactg aggcgggagc780g aaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta agtgttagag 840 ggtttccgcc ctttagtgct gaagttaacg cattaagcac tccgcctggg gagtacggcc 900 gcaaggctga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt 960 aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgaaaa ccctagagat 1020 agggcttctc cttcgggagc agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg 1080 tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg ccatcattaa 1140 gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa 1200 tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacggta caaagagctg 1260 caagaccgcg aggtggagct aatctcataa aaccgttctc agttcggatt gtaggctgca 1320 actcgcctac atgaagctgg aatcgctagt aatcgcggat cagcatgccg cggtgaatac 1380 gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg 1440 gtggggtaac ctttttggag ccagccgcct aaggtgggac agatgattgg ggtgaagtcg 1500 taacaaggta gccgtatcgg aaggtgcggc tggatcacct cct 1543

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12R 1:07) C12R 1:07) (72) Inventor Tatsuo Nakatani 1 Yawata Kaigan-dori Mitsui, Ichihara (72) Inventor Ryozo Irie Misuzu 7448-66 Misuzu, Ina-shi, Nagano F-term (reference) 4B065 AA20X AC20 BB18 BB40 CA32 CA55 4D040 DD03 DD11 DD24

Claims (3)

[Claims] 1. A new microorganism belonging to Bacillus schuligensis X-1 having the ability to degrade starch contained in organic waste liquid and / or biological sludge. 2. The new microorganism belonging to Bacillus schuligensis X-1 according to claim 1, which has been deposited under accession number FERM P-18014. 3. The novel microorganism belonging to Bacillus schuligensis X-1 according to claim 1 or 2, wherein the novel microorganism has the following mycological properties A to D. . A. Morphological properties (1) Cell shape: bacilli, (2) Motility: +, (3) Spores: +, (4) Gram stain: + B. Growth state in medium (1) Standard agar culture: + C. Physiological properties (1) Gram stain: +, (2) Nitrate reducing ability: +, (3) Denitrification:-, (4) VP test:
+, (5) Indole formation: +, (6) Starch hydrolysis: +, (7) Soybean oil degradability: +, (8) Use of inorganic nitrogen source: +, (9) Oxidase: +, ( 10) Catalase: +,
(11) Range of growth: temperature 13-40 ° C., (12) Attitude to oxygen: aerobic, (13) OF test: glucose ++, (1
4) Utilization of ammonia: +, (15) Decomposition of NaHS: + Genetic properties (1) G + C content: 35 mol%, (2)
Based on genomic DNA analysis (base 5 to base 1540) of 16S ribosomal RNA, homology to Bacillus schulgensis: 99.22%, (3) base sequence: SEQ ID NO: 1.