JP2000270845A - Microorganism having oil and fat-decomposing activity and treatment of waste water - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a microorganism belonging to the genus Pseudomonas capable of decomposing oils and fats in a prescribed temperature range, maintaining a high oil and fat-decomposing capability even in a low temperature range and performing a treatment of an oil-containing waste water. SOLUTION: This microorganism can decompose oils and fats such as a salad oil at 10-37 deg.C, preferably at 10-22 deg.C and more preferably at 10-15 deg.C. Further, the microorganism belonging to the genus Pseudomonas is Pseudomonas sp-TI-C strain (FERM P-17292) or Pseudomonas sp-TI-E strain (FERM P-17293). It is preferable to add the above microorganism to the wastewater containing oils and fats and treate at 10-37 deg.C temperature range to decompose the oils and fats.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microorganism having a fat / oil decomposability in a low temperature range and a method for treating wastewater.

[0002]

2. Description of the Related Art Conventionally, oil-containing wastewater has been treated by a coagulation pressure flotation method, which is a physicochemical method. However, this method has been pointed out with a problem that a large amount of excess sludge or odor is generated. For this reason, in recent years, an oil-containing wastewater treatment method using a microorganism having an oil / fat decomposition ability has been proposed and put into practical use (Japanese Patent Application Laid-Open No. 6-153922). However, despite the fact that the wastewater temperature may drop to around 15 ° C in actual treatment facilities, at the laboratory level, the measurement of the fat and oil decomposition capacity of microorganisms is performed at around 22 to 30 ° C. Almost no studies have been made on the ability of microorganisms to decompose fats and oils in a low temperature range. Usually, when the temperature of the treated water is lowered, not only the activity of decomposing microorganisms is reduced, but also the fats and oils are easily solidified, so that it is expected that the performance of the oil-containing wastewater treatment using microorganisms will be significantly reduced. For this reason, when treating oil-containing wastewater in a low temperature range, measures such as increasing the capacity of the treatment water tank or providing an oil / fat separation tank in front of the aeration tank are required. Water quality can also be significantly reduced.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a microorganism and a wastewater treatment method having a fat / oil decomposability in a low temperature range.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, succeeded in isolating a microorganism having an oil and fat decomposability in a low temperature region from activated sludge acclimated by oil and fat. Then, the present invention has been completed. That is, the present invention is a microorganism belonging to the genus Pseudomonas, which can decompose oils and fats at 10 to 37 ° C. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas sp. TI-C and Pseudomonas sp. TI-E. Furthermore, the present invention is a method for treating wastewater, wherein the microorganism is added to wastewater containing fats and oils. The temperature of the drainage is, for example, 10 to 37
° C, preferably 10 to 22 ° C. Hereinafter, the present invention will be described in detail.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism of the present invention is isolated by selecting a microorganism having an oil and fat decomposing ability from activated sludge acclimated by oil and fat, and measuring the oil and fat decomposing ability at 15 ° C. for 24 hours. Is a new bacterium. The microorganism of the present invention is capable of decomposing fats and oils in a wide temperature range, and is not only a medium temperature range (for example, 22 to 37 ° C) but also a low temperature range (for example, 10 to 22 ° C, preferably 10 to 15 ° C). However, they have decomposition activity.

[0006] 1. Mycological properties The microorganisms isolated as described above were converted to BIOLOG M
Physiological properties were measured using icrostation System and API 20E manufactured by BioMerieux Biotech Japan, and identification was performed using each database. further,
Determine a part of the base sequence of DNA encoding 16s rRNA,
CBI program BLAST (http://www.ncbi.nlm.nih.gov/
Identification was performed by BLAST /). As a result, it was found that the microorganism of the present invention belongs to the genus Pseudomonas and corresponds to Pseudomonas sp. However, the microorganisms are different from known strains belonging to Pseudomonas sp. In terms of the rate of fat and oil decomposition in a low-temperature region, and thus are recognized as new strains, and the microorganisms are identified as Pseudomonas sp. TI-C (Pseudomonas sp. TI. -C strain) and Pseudomonas sp. TI-E strain (Pseudomonas sp. TI-E strain).

[0007] Pseudomonas sp. TI-C strain (hereinafter referred to as "TI-C strain") and Pseudomonas sp. TI-C
E strain (hereinafter referred to as “TI-E strain”) was deposited on March 10, 1999 with the Institute of Biotechnology, Institute of Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki Prefecture). The accession number is FERM P-17292 for the TI-C strain (identification: Pseudomonas sp. TI-C) and the TI-E strain (identification: Pseudomo
nas sp. TI-E) is FERM P-17293.

A. Morphological properties (1) Cell shape and size: TI-C strain Bacillus (1.4-1.7 μm long, 0.8-1.1 μm wide) TI-E strain Bacillus (1.2-2.2 μm long, 0.6-1.0 μm wide) (2) Cell polymorphism: No TI-C strain No TI-E strain

B. Cultural properties CGY medium (Casciton 5 g / L, glycerin 5 g / L, yeast extract 1 g / L: pH = 7.2) TI-C strain TI-E strain・ Colony color Light yellow Light yellow ・ Colony transparency None None ・ Colony・ Surface ・ Smooth ・ Smooth ・ Protruding state

C. Physiological properties (+) indicate false positives. TI-C strain TI-E strain (1) Gram stainability--(2) Utilization of citric acid + + (3) H ₂ S production--(4) Indole production--(5) Acetone production + + ( 6) Gelatinase + + (7) NO ₂ production + + (8) Reduction to N ₂ gas + + (9) Oxidase + + (10) β-galactosidase--(11) Arginine dihydrolase + + ( 12) Lysine decarboxylase--(13) Ornithine decarboxylase--(14) Urease--(15) Tryptone fan deaminase (+)-(16) Utilizable rhamnose--Amygdalin--α-cyclodextrin-- Dextrin-(+) glycogen (+) (+) tween 40 + + tween 80 + + N-acetyl-D-galactosamine-(+) N-acetyl-D-glucosamine (+) + adoritol + + L-arabinose (+ ) (+) D-arabitol + + cellobiose--i-erythritol + + D-fructose + + L-fucose +-D-galactose + + gentiobi Aose--α-D-glucose (+) (+) m-inositol (+) (+) α-D-lactose--lactulose--maltose-+ D-mannitol (+) (+) D-mannose + + D-Meribiose (+) (+) β-methyl D-glucoside--D-psicose (+) + D-raffinose--L-mannose--D-sorbitol--Sucrose-(+) Trehalose + + Turanose --Xylitol + + Methyl pyruvate + + Monomethylsuccinic acid + + Acetic acid + + Cis-aconitic acid + + Citric acid + + Formic acid + + D-galacturonic acid lactone + + D-galacturonic acid--D-gluconic acid + + D-glucosamic acid + + D-glucuronic acid--α-hydroxybutyric acid + + β-hydroxybutyric acid + + γ-hydroxybutyric acid--P-hydroxyphenylacetic acid + + itaconic acid + + α-ketobutyric acid + + α-ketoglutar Acid + + α-ketovaleric acid--DL-lactic acid + + malonic acid + + Propionic acid (+) + quinic acid + + D-ascchanic acid--sebacic acid (+)-succinic acid + + bromosuccinic acid + + succinamic acid + + glucuronamide--alaninamide + + D-alanine + + L-alanine + + L-alanyl-glycine + + L-asparagine + + L-aspartic acid + + L-glutamic acid + + glycyl-L-aspartic acid--glycyl-L-glutamic acid + + L-histidine + + hydroxy L-proline + + L-leucine + + L-omithine + + L-phenylalanine--L-proline + + L-pyroglutamic acid + + D-serine + + L-serine + + L-threonine + + glycyl-L-aspartate + + Glutamic acid + + urocanic acid + + inosine + + uridine + + thymidine--phenylethylamine--putrescine-(+) 2-aminoethanol + + 2,3-butanediol-(+) glycerol + + D, L-α- Glycerol phosphate + + glucose 1-phosphate - (+) glucose-6-phosphate - (+)

Next, a method for determining a part of the DNA base sequence encoding 16s rRNA and the base sequence will be described. ○ How to determine the nucleotide sequence
Biology (eds.) (Greene Publishing Associates and W
iley-Interscience, NY (1987)).
The following primers are used, and the DNA sequence is a
utomated DNA sequencer (ABI 377; Perkin Elmer, In
c., USA). ○ Primer Forward: (PA) 5'-AGAGTTTGATCCTGGCTCAG-3 '(20me
r) (SEQ ID NO: 1) Reverse: (1) 5'-GTATTACCGCGGCTGCTG-3 '(18mer)
(SEQ ID NO: 2)

Nucleotide sequence The nucleotide sequence of the TI-C strain is shown in SEQ ID NO: 3, and the nucleotide sequence of the TI-E strain is shown in SEQ ID NO: 4. In the present invention, the above TI-C strain and
In addition to the TI-E strain, mutants obtained by mutating by natural or artificial means and having the above-mentioned ability are all included in the present invention. In order to grow the present microorganism, a usual culture method can be used. The cultivation is preferably performed under aerobic conditions, and includes a CGY medium containing an organic substance, an inorganic salt, a nitrogen source, and other nutrient sources, an L medium (peptone 10 g / L,
The microorganism of the present invention is inoculated into yeast extract 5 g / L, NaCl 5 g / L: pH = 7.2) and the like, and cultured by, for example, a shaking culture method, aeration stirring culture method, or the like. The temperature conditions in the above culture are set within the range of the growth temperature of the microorganism used, preferably within the range of the optimum growth temperature. For example, 22 to 37 ° C, preferably 30 ° C
Can be set to The pH of the medium was 7.0 to 7.5.
May be set in the range.

Substances added to the medium as inorganic salts include phosphates, magnesium salts, calcium salts, iron salts,
In addition, trace metal salts may be used as necessary. In addition, as the nitrogen source, it is sufficient that the test bacteria can assimilate,
For example, peptone, cacitone, urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, various amino acids and the like can be mentioned. One of these nitrogen sources may be used, or two or more thereof may be appropriately combined. Furthermore, vitamins, yeast extracts, malt extracts, and the like may be added in appropriate amounts as nutrients for promoting the growth of the microorganism of the present invention.

The cultivation time varies depending on the amount and type of nutrient, but is usually 1 day or more, preferably 2 to 3 days. In the present invention, the microorganism is previously cultivated in a culture solution containing a carbon source that the microorganism can assimilate, for example, acetic acid, lactic acid, glutamic acid, etc. And culturing may be performed.

2. Wastewater purification treatment method As described above, the microorganism of the present invention not only has a fat and oil decomposition activity even in a temperature range of 10 to 37 ° C, but also has a low temperature range (10 to 22 ° C).
C., preferably 10 to 15 C.). Therefore, the microorganism of the present invention can be used for treating (purifying) wastewater in a low-temperature region. In the present invention, the fats and oils to be subjected to the decomposition treatment contain glycerin triester of a fatty acid as a main component. For example,
Salad oil, olive oil, rapeseed oil, sesame oil, tallow, triolein and the like. However, in the present invention, complex lipids (eg, phospholipids such as lecithin and kephalin),
Free fatty acids, long-chain alcohols, sterols, fat-soluble vitamins (eg, vitamins A, D, E) are also included in the fats and oils to be decomposed. The microorganism of the present invention is added to wastewater containing the fats and oils (domestic wastewater, kitchen wastewater, factory wastewater, etc.).

Batch treatment method The amount to be added can be appropriately determined depending on the concentration of fats and oils, and is not particularly limited, but is preferably about 50 mg / L in dry cell weight per 250 mg / L fats and oils concentration. . The temperature of the waste water is desirably 15 to 30 ° C. After addition of the microorganism, the cells are cultured under aerobic conditions for 0.5 to 1 day.

Continuous treatment method Similar to the ordinary activated sludge method, a flow control tank, an aeration tank, a sedimentation tank and a return sludge pipe are provided, and the oil-containing wastewater to be treated is continuously passed through. The aeration tank is set under aerobic conditions using a blower, a diffuser or the like. Next, the microorganism of the present invention is added to an aeration tank or a flow control tank. The amount to be added can be appropriately determined depending on the concentration of the fat and oil, and is not particularly limited, but is preferably about 50 to 200 mg / L. It is desirable that the temperature of the waste water is 15 to 30 ° C. After the cultivation, whether or not the fats and oils have been decomposed is measured. The measurement of the fat and oil decomposition rate
The test can be performed by the following test methods.

○ Method for Decomposing Oils and Fats An appropriate amount of cells cultured at 30 ° C. for 48 hours in a CGY medium is added to the medium shown in Table 1. Next, for example, rotation shaking cultivation is performed for 24 hours in a thermostat in which temperature is appropriately set to decompose fats and oils. The fat and oil decomposition rate is measured by measuring the fat and oil (n-HEX extract substance concentration) remaining in the medium.

[0019]

[Table 1]

[0020]

The present invention will be described more specifically with reference to the following examples. However, the technical scope of the present invention is not limited to these examples. (Example 1) Decomposition test of fats and oils The fat and oil-degrading bacteria TI-C strain and TI-E strain of the present invention were added to the medium of Table 1 to which fats and oils were added at a concentration of 250 mg / L so as to be equivalent to 50 mg / L. The cells were cultured for 24 hours in a thermostat at an appropriate temperature. After the cultivation, the fat and oil decomposition rate was measured by measuring the concentration of the n-HEX extract.

The test was carried out in the same manner as the decomposition test of the fat-decomposing bacteria, using a commercially available microbial preparation for decomposing fats and oils and activated sludge (MLSS = 2000 mg / L). The results are shown in FIG. In FIG. 1, in the activated sludge, the fat and oil decomposition rate sharply decreases as the water temperature decreases, whereas the microorganism of the present invention maintains the decomposition activity at 10 to 37 ° C. (22% under the conditions of this example). 97-99% at 10 ° C), high fat and oil decomposition rate even at 10 ° C (65% for TI-C strain, 79% for TI-E strain)
It was found to have Table 2 also shows that the microorganism of the present invention has high fat and oil decomposing ability also in beef tallow and olive oil.

[0022]

[Table 2]

Example 2 In this example, an oil / fat decomposition test was conducted when the microorganism of the present invention was added to activated sludge at a water temperature of 15 ° C. Salad oil 250m in 2000mg / L activated sludge
The medium of Table 1 containing g / L and the fat-and-oil-degrading bacteria TI-C strain and TI-E strain of the present invention were each added at 50 mg / L, and the fat and oil degradation rate was measured in the same manner as in Example 1. In addition, using a commercially available microbial preparation and activated sludge (2000 mg / L) as a target, a test was performed in the same manner as the decomposition test of the fat and oil decomposing bacteria. The results are shown in FIG.
As is clear from FIG. 2, it was found that the addition of the microorganism of the present invention to the activated sludge greatly improved the fat and oil decomposition rate.

[0024]

According to the present invention, there is provided a method for treating microorganisms and wastewater having a fat and oil decomposability in a low temperature range. Since the microorganisms of the present invention can maintain high oil and fat resolution even in a low temperature range, it is possible to efficiently perform oil-containing wastewater treatment.

[0025]

[Sequence List] SEQUENCE LISTING <110> TAISEI CORPORATION <120> MICROORGANISMS CAPABLE OF DEGRADING OILS AND FATS <130> P98-0742 <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Synthetic oligonucleotide <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220><223> Synthetic oligonucleotide <400> 2 gtattaccgc ggctgctg 18 <210> 3 <211> 377 <212> DNA <213> Pseudomonas sp. <400> 3 cgctggcggc aggcctaaca catgcaagtc gagcggtaga gagaagcttg cttctcttga 60 gagcggcgga cgggtgagta atgcctagga atctgcctgg tagtggggga taacgttcgg 120 aaacggacgc taataccgca tacgtcctac gggagaaagc aggggacctt cgggccttgc 180 gctatcagat gagcctaggt cggattagct agttggtggg gtaatggctc accaaggcga 240 cgatccgtaa ctggtctgag aggatgatca gtcacactgg aactgagaca cggtccagac 300 tcctacggga ggcagcagtg gggaatattg gacantgggc gaaagcctga tccagccatg 360 ccgcgtgt11 gaaag 377 <210> 4 <1> t <1> t <1> t <1> 4 <1> t <1> t <1> t <1> t <1> t <1> t <1> 4 t1 <t> gc tcagattgaa cgctggcggc agg
cctaaca catgcaagtc gagcggtaga 60 gagaagctttg cttctctttga gagcggcggga cgg
gtgagta atgcctaggga atctgcctgg 120 tagtggggga taacgttcgg aaaacggacgc taa
taccgca tacgtcctac gggagaaaagc 180 aggggaccttt cgggcctttgc gctatcatgat gag
cctaggt cggatttagct aggttggtgggg 240 gtaatggctc accagaggcga cgatccgtaa ctg
gtctgag aggatgatca gtcactactgg 300 aactgagaca cggtccagac tcctacggga ggc
agcagtg gggaatattg gacaatggggc 360 gaagagcctga tccagccatg ccgcgtgttgt gaa
gaaggtc ttcggattgt aaagcacttt 420 aagttgggag gaagggttgt agattataact tct
gcaattt tgacgttacc gacagaataa 480 gcaccgggcta a
491

[0026]

[Sequence List Free Text]

 SEQ ID NO: 1: Synthetic oligonucleotide. SEQ ID NO: 2: Synthetic oligonucleotide.

[Brief description of the drawings]

FIG. 1 is a graph showing fat and oil decomposition characteristics of a microorganism of the present invention in a low temperature range.

FIG. 2 is a graph showing the effect of improving the fat and oil decomposition rate when the microorganism of the present invention is added to activated sludge.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) C12R 1:38) (C12N 15/09 ZNA C12R 1:38) F term (reference) 4B024 AA11 BA80 CA03 DA05 HA19 4B065 AA41X AC12 AC20 BA22 CA55 4D040 DD03

Claims (4)

[Claims] 1. A microorganism belonging to the genus Pseudomonas, which is capable of decomposing fats and oils at 10 to 37 ° C. 2. The microorganism according to claim 1, wherein the microorganism belonging to the genus Pseudomonas is Pseudomonas sp. TI-C or Pseudomonas sp. TI-E. 3. The wastewater containing fats and oils according to claim 1 or 2,
A method for treating wastewater, comprising adding the microorganism described in the above. 4. The treatment method according to claim 3, wherein the waste water is at a temperature of 10 to 37 ° C.