JPH10276771A - Oligotrophic microorganism and clean-up using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To clean up the natural environment in a state thereof no nutrient salt added thereto and carry out the cleaning treatment of oligotrophic wastewater from an oil refinery, a chemical factory, etc., by treating contaminants containing a carbon source with a microorganism growable under conditions of <=5 ppm phosphorus and <=50 ppm nitrogen. SOLUTION: Soil contaminated with petroleum, wastewater from an oil refinery, etc., are treated with a microorganism growable under conditions of a high concentration of organic carbons, <=5 ppm phosphorus concentration and <=50 ppm nitrogen concentration. The microorganism is a phenol- assimilating microorganism and, e.g. Burkholderia cepacia 1A strain (FERM P-15566) has assimilating properties for phenol, benzene, toluene and xylene (an isomeric mixture).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a microorganism capable of decomposing environmental pollutants, growing under low nutrient conditions in which only the concentration of nutrients such as phosphorus and nitrogen is low, and purifying the environment using the microorganism, especially aromatic compounds, It relates to phenol wastewater treatment technology.

[0002]

2. Description of the Related Art The natural environment is generally considered to be under nutrient conditions, and wastewater from refineries and chemical factories is poorly nutrient wastewater that is poor in phosphorus and nitrogen. Cannot grow. With conventional environmental purification technology,
(NH ₄ ) ₂ SO ₄ , NH ₄ Cl, Na ₃ PO ₄ ,
A method of increasing the activity of microorganisms by adding nutrients such as Na ₂ HPO ₄ has been performed [(DA Graves et al.,
Applied Biochemistry and
Biotechnology, vol. 28, P813
(1991): Shigeaki Harayama, "Small creatures protecting the earth", Gihodo Shuppan, P109 (1995): Nishimura
Seed, soil and microorganisms, No. 46, P19 (1995): C.I.
H. Nelson et al., "Hydrocarbon Bi.
oremedation ", CRC Press, P
125 (1994))]. In wastewater treatment, it is considered necessary to control the ratio of carbon: phosphorus: nitrogen in order to maintain the growth and activity of microorganisms. carbon:
To control the phosphorus: nitrogen ratio, it is common to add nutrients to wastewater ["Wastewater Treatment Technical Guide", Petroleum Association of Japan, P129 (1975): "Pollution Prevention Techniques and Regulations" , Pollution Prevention Association, P215
(1992)]. Further, JP-A-6-500495 describes a method for efficiently and continuously providing nutrients to a polluted environment. JP-A-7-507208 discloses that a phosphate ester is added to simultaneously supply nutrient substances. A method for emulsifying contaminants is described.

[0003]

As described above, it is necessary to increase the activity of microorganisms by adding nutrients for natural and environmental purification and treatment of low-nutrient wastewater. However, nutrients added to the polluted environment and wastewater are one of the costs for environmental purification and wastewater treatment. In particular, when the polluted place to be purified is in a wide range or when the amount of wastewater to be treated is large, the cost of the added nutrients becomes enormous. Further, excessively added nutrients cause secondary pollution such as nitrate pollution of groundwater and eutrophication of the environment, and therefore, it is important to control the method and amount of addition.

That is, the nitrogen source is oxidized to a nitrate by the action of soil microorganisms. Therefore, when an excessive nitrogen source is provided, the concentration of nitrate ions in soil and groundwater increases. Nitrate ions have been reported to be carcinogenic, and contamination with drinking water has become a problem (Michiji Tsurumaki, “Present State of Groundwater and Soil Contamination and Purification Measures”, Japan Industrial Technology Association, P98
(1993)). In addition, when excessively added nutrients, particularly phosphorus and nitrogen sources, flow into rivers and lakes through rainwater and groundwater, they cause eutrophication of the environment. Since eutrophication of the environment causes abnormal occurrence of blue-green algae and moldy smell of drinking water, many methods for removing phosphorus and nitrogen in domestic wastewater have been proposed [Kazunori Nakamura, "Microbial Ecology 17", Academic Publishing Center, P31 (1991): Tetsuro Fukase,
"Small creatures protecting the earth", Gihodo Publishing, P1
33 (1995)]. P. Morgan et al. Point out that the addition of nutrients to the soil environment inhibits the activity of native microorganisms to decompose organic substances (Wat. Sci.
Tech. , Vol. 6, P63 (1990)). To avoid secondary contamination by added nutrients, it is necessary to add nutrients while controlling them. However, installing such a control device in a wastewater treatment facility makes the facility more expensive and increases wastewater treatment costs. Furthermore, since polluted environments such as contaminated soil and groundwater are open systems, it is necessary to close the polluted environment by some method in order to add nutrients efficiently while controlling them. However, closing the polluted environment by civil engineering requires a large amount of money, and is technically difficult, especially when the polluted environment is large. Therefore, it cannot be said that it is practically impossible to prevent secondary contamination by added nutrients in a polluted environment.

[0005] The existence of undernutrient microorganisms has been known for a long time, and much research has been conducted. Until now, low-vegetative microorganisms have been discussed only in terms of the amount of organic carbon in the growth conditions, and microorganisms that can grow at a concentration of 1 to 15 ppm or less of organic carbon have been studied under the name of low-vegetative microorganisms.
[(Hiroshi Unemoto, "Skillful Lifestyle of Bacteria Living in a Special Environment", Kyoritsu Shuppan, P7 (1993): Mitsuru Eguchi,
"Marine Microorganisms and Biotechnology", Gihodo Publishing, P
68 (1991): Yuichi Suwa et al., "Methods for Separating Microorganisms", R & D Planning, P545 (1990)].
However, wastewater from a naturally polluted environment or a refinery or a chemical factory has a high concentration of organic carbon as a pollutant and a low concentration of other nutrients such as phosphorus and nitrogen. Microorganisms that grow under such a condition that the concentration of organic carbon is high and only other nutrients such as phosphorus and nitrogen are low and decompose pollutants are not applicable to the conventional concept of low-nutrition microorganisms, There is no report so far.

SUMMARY OF THE INVENTION An object of the present invention is to use microorganisms useful for purification of a natural environment under low nutrient conditions or treatment of low nutrient wastewater from a refinery or a chemical factory without adding nutrients. It is to provide an improved purification technology.

[0007]

Means for Solving the Problems The present inventors provide a technique for purifying the natural environment under low nutritional conditions or treating low nutrient wastewater in oil refineries and chemical factories without adding nutrients. A number of experiments were performed with the aim of doing so. Although the normal natural environment is considered to be undernutrition, the present inventors focused on phosphorus and nitrogen, which are particularly important for the growth of microorganisms, and measured the concentrations of phosphorus and nitrogen in the natural environment. And underground water have a nitrogen concentration of about 60p as shown in Table 1 below.
pm or less, and the phosphorus concentration is about 3 ppm or less. In the ocean, nutrient concentrations are even lower, with nitrogen concentrations of about 1.0 ppm or less and phosphorus concentrations of about 0.1 ppm or less. Based on the measured values of the concentrations of phosphorus and nitrogen in the natural environment, the concentration of the target organic carbon is high, the other phosphorus and nitrogen grow only under low nutrient concentrations, and decompose pollutants. The medium for obtaining the resulting microorganism has a phosphorus concentration of about 5 pp.
m and a nitrogen concentration of about 50 ppm or less, and completed the present invention. Therefore,
The first of the features of the present invention is that the content of phosphorus is 5 ppm or less and the content of nitrogen
The present invention relates to a microorganism capable of growing under 0 ppm or less. A second feature of the present invention relates to microorganisms useful for purifying the natural environment or treating low-nutrient wastewater in refineries and chemical factories, and a purification technique using the microorganisms.

[0008]

[Table 1] 1 Based on measurement by the inventor 2 Microbial ecology 12, P10, from the Society Press Center (1989) 3 Minoru Nishimura, Soil and microorganisms, No. 46, from P19 (1995) 4 Ecology of microorganisms 3, P4, from the Society Press Center (1987) In addition, wastewater from refineries and chemical factories has a high concentration of contaminant organic carbon, and other phosphorus and nitrogen. And only the nutrient concentration is low. In contrast, medium components commonly used in microbial research are under high nutritional conditions as shown in Table 2 below. Therefore, when using microorganisms obtained in the culture medium of previous research for environmental purification and wastewater treatment, these environmental pollutants cannot be decomposed, so nutrient sources, especially phosphorus sources and nitrogen, are required for the growth of microorganisms. You need to give a source.

[0009]

[Table 2] 1 Measured by the inventor 4 Microbial ecology 3, P4, from Gakkai Shuppan Center (1987) * Far East Pharmaceutical Co., Ltd. ** Nippon Pharmaceutical Co., Ltd.

Microorganisms that grow under conditions where the concentration of organic carbon is high and only the concentration of nutrients such as phosphorus and nitrogen is low and decompose pollutants can be obtained by using a medium having a phosphorus concentration of 5 ppm or less and a nitrogen concentration of 50 ppm or less. For example, phenol 10
It can be obtained by searching under the condition of 0 to 1000 ppm as a carbon source. The carbon source can be any organic substance that is going to be decomposed, such as phenol, benzene, toluene,
Examples include aromatic compounds such as xylene and cresol, organic chlorine compounds such as trichloroethylene, and polycyclic aromatic compounds such as naphthalene and biphenyl. The phosphorus source may be substantially any substance that can be used by microorganisms as a phosphorus source, and may be monoammonium phosphate, monosodium phosphate, monopotassium phosphate, ammonium potassium phosphate, disodium hydrogenphosphate, phosphate Inorganic phosphates such as dipotassium hydrogen, ammonium dihydrogen phosphate, sodium dihydrogen phosphate and potassium dihydrogen phosphate and hydrates of these salts, yeast extracts, natural products containing phosphorus such as gravy extract, Organic phosphorus compounds such as phosphoric acid esters can be used. Similarly, the nitrogen source may be substantially any substance that can be used by microorganisms as a nitrogen source.
Ammonium salts such as ammonium sulfate, monoammonium phosphate, ammonium nitrate, ammonium potassium hydrogen phosphate, ammonium chloride and hydrates of these salts;
Examples include nitrates such as ammonium nitrate, sodium nitrate and potassium nitrate, nitrites such as sodium nitrite, urea, amino acids, yeast extract, broth extract, nitrogen-containing natural products such as peptone, and organic nitrogen-containing compounds such as amide compounds. it can.

Since the microorganism of the present invention can grow under low nutrient conditions of 5 ppm or less of phosphorus and 50 ppm or less of nitrogen, it can be applied to environmentally friendly environmental purification and wastewater treatment without adding nutrients. It is possible. However, the microorganism of the present invention has a phosphorus content of 5 ppm or less and a nitrogen content of 50 ppm.
Having the property of being able to grow under low nutrient conditions of not more than ppm does not mean that the microorganism cannot grow under the condition of 5 ppm or more of phosphorus or 50 ppm or more of nitrogen. Include obligately undernutrient microorganisms that can grow only under low nutrient conditions and facultative undernutrition microorganisms that can grow under both low and high nutrient conditions. The microorganism of the present invention includes prokaryotic microorganisms including eubacteria (protobacteria) and archaebacteria (archaea) and eukaryotic microorganisms including yeasts and molds. Or a mixture of several types. The use form of the microorganism of the present invention may be used in a suspended state, but it can also be used by immobilizing it on a suitable carrier in order to increase the cell concentration. As a carrier for immobilization, inorganic substances such as activated carbon and glass beads, resin carriers such as polystyrene and polypropylene,
Organic substances such as alginate gel and polyvinyl alcohol gel can be used.

[0012]

【Example】

Example 1 Search for phenol assimilating bacteria growing under low phosphorus concentration and low nitrogen concentration conditions (natural environment) (Preparation of search medium) As a search medium, a medium shown in Table 3 below (hereinafter referred to as an E-medium) ) Diluted to 1/10 (1 / 10E-medium: about 5 ppm phosphorus, about 50 pp nitrogen)
m), and those diluted 1/100 (1 / 100E
-Medium: Two types of about 0.5 ppm of phosphorus and about 5 ppm of nitrogen) were used. In addition, phenol 1,00 as a carbon source
0 ppm was added. The pH of each medium was 7.0.

[0013]

[Table 3]

(Method A for Obtaining Degradative Bacteria)
Using about 480 types of soil around Tsukuba City, only microorganisms were extracted and added to the medium by the following method. 30 ml of sterilized water was placed in a 300 ml Erlenmeyer flask, and 5 g of a soil sample was added thereto. This was shaken for one day to extract bacteria. After extraction, centrifugation (2,000 rpm, 5m
in) to precipitate the soil, and the supernatant was filtered through a filter paper and a glass filter to remove soil fine particles. The resulting bacterial solution was filtered through a nitrocellulose filter (0.2 μm) to collect microorganisms. This filter was appropriately cut, added to the medium, and searched. The search was performed by the following method. 1 / 10E- in a 300 ml Erlenmeyer flask
30 ml of the medium or 1 / 100E-medium was added thereto, and the nitrocellulose filter described above was added thereto.
Shaking culture was performed for one day. For those in which growth was observed, 1 / 10E-medium or 1 / 100E-medium 8m
of 100 μl in a large test tube having an inner diameter of 24 mm and cultured for 5 days. This was repeated twice to isolate the bacteria. For isolation of the bacterium, 1.2% agarose [aga] was added to the aforementioned 1 / 10E-medium or 1 / 100E-medium.
rose (ultra-pure grad for electrophoresis
e)]. The above-mentioned culture solution was appropriately diluted and applied thereto, and cultured at 30 ° C. for 5 days to isolate a growing colony.

In both the 1 / 10E-medium and the 1 / 100E-medium, bacterial growth was observed in a large number of samples. As a result, four types of colonies having different shapes were observed, and 2-3 colonies were obtained for each of the colonies. Of the 14 candidate strains obtained, 5 strains which did not lose their separation ability during storage (4 obtained from 1 / 10E-medium)
(1 strain obtained from 1 / 100E-medium) was used for the subsequent studies. All these strains are 1/10 medium and broth medium [nutrient broth].
(NB)]. When a search was carried out on a 1 / 10E-medium, the growth of the bacteria was observed in all soil (extracted bacterial cells) samples. Therefore, the bacteria growing under such nutrient conditions (about 5 ppm of phosphorus and about 50 ppm of nitrogen) were observed. Seems to be quite a lot. In addition, there was no difference in the bacterial species between the bacteria obtained on the 1 / 10E-medium and the bacteria obtained on the 1 / 100E-medium, as long as the morphology of the colony was observed. Further, since the bacteria obtained on the 1 / 100E-medium can grow on the 1 / 10E-medium, it seems that they are the same bacteria or at least taxonomically related.

(Method B for Obtaining Degradative Bacteria)
Unlike the method A for obtaining the degrading bacteria, about 20 types of soil around Atsugi city were used, and the soil sample was directly added to the 1 / 10E-medium without being filtered using a filter. That is, about 1 g of the soil sample is 50 ml.
Was added to a 500 ml Erlenmeyer flask containing the above 1 / 10E-medium, and cultured at 30 ° C. for about 1 week.
After culturing in a 00 ml Erlenmeyer flask for about one week, colonies were isolated using a plate.

Example 2 (Identification of Degrading Bacteria Obtained in Example 1) All the phenol-assimilating bacteria obtained by the method A for obtaining the degrading bacteria are Gram-negative, have motility, and have oxidase,
Both catalase were positive. Also, the OF test showed oxidation. From the results of other various physiological tests, all five strains obtained were Burkholderia cepaci.
a. However, since the results of the nitric acid reduction ability, the generation of acid from glucose, the decomposition test of escalin, etc. are different, the same Burkholderia. cep
Asia is also divided into the following four groups.

The phenol-assimilating bacterium obtained by the method B for obtaining a degrading bacterium is one of the obtained strains, which is gram-negative and has motility, is positive for oxidase and catalase, and has an OF test. Shows that Ps shows oxidation and produces a fluorescent dye in King's B medium.
eudomonas putida9-1A1 (FER
MP-15571). In the other type, the morphology of the rod is coryneform, which is gram-positive and non-motile, has meso-diaminopimelic acid as a cell wall diamino acid, and has a linear saturated type and 10
From the test results of having a branched fatty acid having a methyl group at the position, Rhodococcus sp. 10-1
A1 (FERMP-15572) was fixed.
The following table shows the characteristics of the phenol assimilating bacteria.

[0019]

[Table 4]

Characteristics of phenol assimilating bacterium 1A (2)

[Table 5]

[0021]

[Table 6]

Characteristics of phenol assimilating bacterium 1C (2)

[Table 7]

[0023]

[Table 8]

[0024]

[Table 9]

[0025]

[Table 10]

[0026]

[Table 11]

[0027]

[Table 12]

[0028]

[Table 13]

[0029]

[Table 14]

[0030]

[Table 15]

[0031]

[Table 16]

Example 3 Search for Phenol-Utilizing Bacteria Growing under Low Phosphorus Concentration and Low Nitrogen Concentration Conditions (Conditions for Refinery Drainage) (Preparation of Search Medium) Since information on the analysis results was obtained, based on this information, in this example, a new search medium was prepared as shown in Table 18.
(Hereinafter, F-medium) diluted to 1/100 (1 / 100F-medium: about 1 ppm of phosphorus and about 20 ppm of nitrogen) was used. To this, 100 ppm of phenol was added as a carbon source. The pH of the medium was 7.0. In addition, trace metal (trace meta)
The composition of l) is as shown in Table 19 below.

[0033]

[Table 17]

[0034]

[Table 18]

[0035]

[Table 19]

(Method C for Obtaining Decomposed Bacteria)
Using about 480 types of soil around Tsukuba City, only microorganisms were extracted and added to the medium by the following method. 30 ml of sterilized water was placed in a 300 ml Erlenmeyer flask, and 5 g of a soil sample was added thereto. This was shaken for one day to extract bacteria. After extraction, centrifugation (2,000 rpm, 5m
in) to precipitate the soil, and the supernatant was filtered through a filter paper and a glass filter to remove soil fine particles. The resulting bacterial solution was filtered through a nitrocellulose filter (0.2 μm) to collect microorganisms. This filter was appropriately cut, added to the medium, and searched. The search was performed by the following method. 1 / 100F in 300ml Erlenmeyer flask
-30 ml of medium was added, and the above-mentioned nitrocellulose filter was added thereto, followed by shaking culture at 30 ° C for 5 days. As for the growth, 100 μl was placed in a large test tube having an inner diameter of 24 mm containing 8 ml of 1 / 100F-medium.
l Subculture was carried out for 5 days. This was repeated twice to isolate the bacteria. For isolation of the bacterium, 1.2% agarose [agarose (u
[tra-pure grade)] was used. The above-mentioned culture solution was appropriately diluted and applied thereto, and cultured at 30 ° C. for 5 days to isolate a growing colony. Since the growth of the bacteria was observed in a large number of samples of the 1 / 100F-medium, isolation of the bacteria was attempted. As a result, colonies having different shapes were recognized, and thus colonies were obtained. All these strains are in broth medium [nutri
ent broth (NB)].

Example 4 (Identification of Degrading Bacteria Obtained in Example 3) One of the phenol-utilizing bacteria obtained was a gram-positive bacterium, one was a yeast, and three were gram-negative bacteria. Was. Arthrobacter atrocyaneus
3A strain (FERM P-16016) Cryptococcus albidus 3H strain (FERM P-16015) Alcaligenes denitrificans
subsp. denitrificans K2 strain (FERM P-16012) Alcaligenes denitrificans
subsp. denitrificans K3 strain (FERM P-16013) Alcaligenes denitrificans
subsp. denitrificans K19 strain (FERM P-16014) The properties of the phenol assimilating bacteria are shown in Tables 20 to 28 below.

[0038]

[Table 20]

[0039]

[Table 21]

[0040]

[Table 22]

[0041]

[Table 23]

[0042]

[Table 24]

[0043]

[Table 25]

[0044]

[Table 26]

[0045]

[Table 27]

[0046]

[Table 28]

Example 5 Search for bacteria capable of assimilating aromatic compounds growing under low phosphorus concentration and low nitrogen concentration conditions (natural environment) (Preparation of medium for search)
100 dilution (1 / 100F-medium: about 1
ppm, nitrogen about 20 ppm). To this, benzene, toluene and xylene were added as carbon sources in amounts of 10 respectively.
0 ppm was added. The pH of the medium was 7.0.

(Method D for Obtaining Decomposed Bacteria)
Using about 150 types of soil around Tsukuba City, only microorganisms were extracted and added to the medium by the following method. 30 ml of sterilized water was placed in a 300 ml Erlenmeyer flask, and 5 g of a soil sample was added thereto. This was shaken for one day to extract bacteria. After extraction, centrifugation (2,000 rpm, 5m
in) to precipitate the soil, and the supernatant was filtered through a filter paper and a glass filter to remove soil fine particles. The resulting bacterial solution was filtered through a nitrocellulose filter (0.2 μm) to collect microorganisms. This filter was appropriately cut, added to the medium, and searched. The search was performed by the following method. 1 / 100F in 300ml Erlenmeyer flask
-30 ml of medium was added, and the above-mentioned nitrocellulose filter was added thereto, followed by shaking culture at 30 ° C for 5 days. As for the growth, 100 μl was placed in a large test tube having an inner diameter of 24 mm containing 8 ml of 1 / 100F-medium.
l Subculture was carried out for 5 days. This was repeated twice to isolate the bacteria. For isolation of the bacterium, 1.2% agarose [agarose (u
[tra-pure grade)] was used. The above-mentioned culture solution was appropriately diluted and applied thereto, and cultured at 30 ° C. for 5 days to isolate a growing colony. Since the growth of the bacteria was observed in a large number of samples of the 1 / 100F-medium, isolation of the bacteria was attempted. As a result, colonies having different shapes were recognized, and thus colonies were obtained. All these strains are in broth medium [nutri
ent broth (NB)].

Example 6 (Identification of Degrading Bacteria Obtained in Example 5) The two types of the assimilating bacteria of the aromatic compounds obtained were all Gram-negative bacteria. Burkholderia pickettttii
K11 strain (FERMP-16207) Burkholderia pickettttii
K37 strain (FERMP-16208) The characteristics of the aromatic compound assimilating bacteria are shown in Tables 29 to 30 below.

[0050]

[Table 29]

[Table 30]

Example 7 Decomposition of phenol by the decomposing bacteria obtained in Example 1 under low phosphorus and low nitrogen conditions (Decomposition method) The test strain was placed on a 1/10 E-plate medium (1,000 ppm phenol). The cells were inoculated and cultured at 30 ° C. The grown cells were inoculated into a plastic tube (confirmed that phenol does not adsorb to the tube) containing 2 ml of 1 / 10E-medium, and a shaking incubator (MBSS) was used.
-10, Marubishi), and the amount of phenol degraded after the culture was quantified using gas chromatography. The initial phenol concentration was 100 ppm and 1000 ppm, and sampling was performed 3 and 6 days later, respectively. The conditions of gas chromatography are shown below. Column (Column): UnisoleF-200 Injection temperature (Inj.temp): 180 ° C. Column temperature (Col. temp): 150 ° C. Detector (Detector): FID Injection amount (inj.size): 2 μl Internal standard substance (IS) : Diethylene glycol Table 31 shows the results of the decomposition test. All four strains used in the experiment had phenol-degrading ability.

[0052]

[Table 31]

(Comparison experiment of phenol degradability between strain obtained in Example 1 and existing strain) Example 8 As test strains, isolates 1A, 1C and 1 obtained in Example 1 were used.
Five strains E, 1H and 2D were used. For comparison, ATCC (American Type Culture) which has been reported as a phenol-degrading bacterium has been reported.
e Culture Collection) stock strain was also used. The ATCC-preserved phenol-degrading bacteria are the following four strains.・ Pseudomonas putida ATCC 11172 ・ Rhodococcus rhodochrous ATCC 14347 ・ Vibrio cycloteses ATCC 14635 ・ Rhodococcus zopfii ATCC 51349
Inoculate in a plastic tube containing 2 ml of a medium in which the phosphorus and nitrogen concentrations of the F-medium have been increased to about 500 ppm, and use a shaking incubator (MBSS-10, Marubishi).
C. for 4 days. The amount of phenol degradation after culturing
It was quantified using gas chromatography.

(Results and Discussion) The results of the decomposition test are shown in Table 32 below.
To 33. The degraded bacteria obtained were 1/100 for all 5 strains
It grew well on the F-medium, and most of the added 100 ppm phenol was completely decomposed within 4 days.
In addition, even if the concentration of phosphorus and nitrogen in the medium was increased, no inhibition was observed in both growth and phenol degradation. On the other hand, AT
In the CC stock, all strains did not grow at all in the basal medium. The slight decrease in phenol in the table is probably due to adsorption to the inoculated cells. In addition, the concentration of phosphorus and nitrogen in the medium was 500 p, respectively.
When a medium with an increased pm was used, as shown in Table 33, growth was observed in three strains other than the ATCC 14635 strain, and phenol degradation was observed. From these results, the five strains of phenol-degrading bacteria are novel bacteria having good phenol-degrading ability, which grow under low-phosphorus-low-nitrogen conditions under which no known phenol-degrading bacteria can grow. it is conceivable that.

[0055]

[Table 32]

[0056]

[Table 33]

Example 9 Decomposition of Various Aromatic Compounds Using Phenol-Assimilating Bacteria Grown under Low Phosphorus and Low Nitrogen Concentrations As a test strain, the isolate 1A (Bruk) obtained in Example 1 was used.
(Holderia cepacia 1A) was used to confirm the assimilation of phenol, benzene, toluene, and xylene (mixture of isomers). The amount of various aromatic compounds as substrates was 100 ppm, and the medium was cultured at 30 ° C. for 4 days using a 1 / 100F-medium. The results are shown in Table 34 below.
Shown in

[0058]

[Table 34] From the results in the preceding table, it was confirmed that the growth of the microorganism was higher than in the case where none of the substrates (carbon sources) had a carbon source.

Example 10 Degradation Characteristics of Aromatic Compounds under Low Phosphorus Concentration and Low Nitrogen Concentration Conditions by Decomposing Bacteria Obtained in Example 3 (Degradation Method)
A (Arthrobacter atrocyaneu)
s3A) and K2 (Alcaligenes den)
itrificans subs. denitrific
cansK2) was used to confirm the assimilation of phenol. The amount of phenol as a substrate was set to 100 ppm, and the phenol assimilation was confirmed using a medium in which the concentration of phosphorus or nitrogen was changed to 5 ppm as a basic medium and a medium in which the concentration of phosphorus or nitrogen was changed. . That is, the test strains 3A and K2 are inoculated into the medium,
The cultivation was performed at 30 ° C. for 3 days under a rotational shake of 600 rpm. The degree of growth of the bacterial cells is determined by the absorbance at 580 nm (A580).
The phenol concentration was measured by HPLC.

Example 11 Example 5 Decomposition property of the obtained decomposed bacteria against various aromatic compounds under low phosphorus concentration and low nitrogen concentration conditions (Decomposition method) The K1 grown on a 1 / 100NB plate
1 and K37 were suspended in 1 / 100F medium.
00 μl of 155 ml vial (30 ml of medium)
Inoculation. Thereafter, 100 ppm of each of various aromatic compounds (benzene, toluene, xylene) as substrates were added (a total of 300 ppm). However, xylene used was one in which o-, m-, and p- were previously mixed at a ratio of 1: 2: 1. Seal the vial with a Teflon-coated butyl rubber stopper and an aluminum seal.
Shaking culture was performed at 0 ° C. The experiment was performed in duplicate. At regular intervals, 50 μl of the gas phase was withdrawn with a gas tight syringe and quantified by GC. Further, 0.5 ml of the culture solution was withdrawn with a sterilized 1 ml syringe, and the growth amount was measured.

(Results and Discussion) The results of the decomposition test are shown in FIG.
-4 are shown. From the results shown in FIGS. 1 and 2, it can be seen that the nitrogen concentration in the medium is about 5 ppm and the phosphorus concentration in the medium is about 5 ppm for the strain 3A to degrade 100 ppm of phenol. From the results shown in FIGS. 3 and 4, it can be seen that the K2 strain is sufficient to decompose 100 ppm of phenol if the nitrogen concentration in the medium is about 0.5 ppm and the phosphorus concentration is about 1 ppm. Also,
From the results shown in FIGS. 5 and 6, the growth of the bacteria was observed,
Since benzene, toluene, and xylene are decomposed along with this, it can be seen that the K11 strain and the K37 strain grow by assimilating benzene, toluene, and xylene.

Hereinafter, embodiments of the present invention will be described. 1. A microorganism capable of growing under the conditions of 5 ppm or less of phosphorus and 50 ppm or less of nitrogen. 2. The microorganism according to the above 1, wherein the microorganism belongs to the genus Pseudomonas. 3. 2. The microorganism according to the above 2, wherein the microorganism belongs to Pseudomonas putida. 4. The microorganism is Pseudomonas putida9
The microorganism according to 1, 2, or 3, which is a -1A1 strain (FERM P-15571).

5. 2. The microorganism according to the above 1, wherein the microorganism belongs to the genus Burkholderia. 6. The microorganism is Burkholderia cepaci
The microorganism according to the above 5, which belongs to a. 7. The microorganism is Burkholderia cepaci
The microorganism according to the above 6, which is a 1A strain (FERM P-15566). 8. The microorganism is Burkholderia cepaci
The microorganism according to the above 6, which is a 1C strain (FERM P-15567). 9. The microorganism is Burkholderia cepaci
The microorganism according to the above 6, which is a 1E strain (FERM P-15568). 10. The microorganism is Burkholderia cepac
The microorganism of 6 above, which is an ia 1H strain (FERM P-15569). 11. The microorganism is Burkholderia cepac
The microorganism of 6 above, which is ia 2D strain (FERM P-15570).

12. Microorganisms Burkholderia
The microorganism according to the above 5, which belongs to pickettii. 13. Microorganisms Burkholderia picke
13. The microorganism according to the above 12, which belongs to the strain ttiii K11 (FERM P-16207). 14． Microorganisms Burkholderia picke
13. The microorganism according to the above 12, which belongs to ttii K37 strain (FERM P-16208).

15. The microorganism of 1 above, wherein the microorganism belongs to the genus Rhodococcus. 16. The microorganism is Rhodococcus sp. 10-
The above 15 which is 1A1 strain (FERMP-15572).
Microorganisms.

17. The microorganism is Arthrobacter
The microorganism of 1 above, which belongs to the genus. 18. The microorganism is Arthrobacter atroc
The microorganism according to the above 17, which belongs to Yaneus. 19. The microorganism is Arthrobacter atroc
The microorganism according to the above 15, which is a strain of Yaneus 3A (FERM P-16016).

20. The microorganism is Cryptococcus
The microorganism of 1 above, which belongs to the genus. 21. The microorganism is Cryptococcus albid
20. The microorganism according to the above 20, wherein the microorganism belongs to us. 22. The microorganism is Cryptococcus albid
2 which is a us3H strain (FERMP-16015).
1 microorganism.

23. The microorganism of 1 above, wherein the microorganism belongs to the genus Alcaligenes. 24. The microorganism is Alcaligenes denitr
23. The microorganism according to the above 23, which belongs to ifnicans. 25. The microorganism is Alcaligenes denitr
ificans subsp. denitrifica
24. The microorganism according to the above 24, which belongs to ns. 26. The microorganism is Alcaligenes denitr
ificans subsp. denitrifica
The microorganism according to the above 23, which is an nsK2 strain (FERM P-16012). 27. The microorganism is Alcaligenes denitr
ificans subsp. denitrifica
The microorganism according to the above 26, which is an ns K3 strain (FERM P-16013). 28. The microorganism is Alcaligenes denitr
ificans subsp. denitrifica
The microorganism of 22 above, which is ns K19 strain (FERM P-16014).

29. The microorganism according to any one of 1 to 28, wherein the aromatic compound is phenol. 30. 30. The microorganism according to 1 to 29, wherein the aromatic compound is at least one selected from the group consisting of benzene, toluene, and xylene. 31. A method for purifying a petroleum pollutant, comprising treating the petroleum pollutant with the microorganism according to 29 or 30. 32. 31. A method for purifying 31 petroleum pollutants in which the petroleum pollutant is oil-polluted soil or refinery wastewater. 33. A method for purifying an aromatic compound contaminant, comprising treating the aromatic compound contaminant with the microorganism according to 29 or 30. 34. 33. The method for purifying an aromatic compound contaminant according to the above item 33, wherein the aromatic compound contaminant is aromatic compound contaminated soil or wastewater containing an aromatic compound. 35. When aromatic contaminants are phenol, benzene,
3. The contaminant which is contaminated with at least one selected from the group consisting of toluene and xylene.
The method for purifying an aromatic compound contaminant according to 3 or 34.

[0070]

[Effect] Low-cost environmental purification or wastewater treatment without worrying about secondary pollution without adding nutrients such as phosphorus source and nitrogen source to natural pollution environment and low nutrient wastewater from refineries and chemical factories. The present invention provides a microorganism which can be used and an environmental purification or wastewater treatment method using the microorganism.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of phenol degradation when the nitrogen concentration in the medium of phenol-assimilating bacterium 3A was changed.

FIG. 2 is a diagram showing the results of phenol degradation when the phosphorus concentration in the medium of phenol-assimilating bacterium 3A was changed.

FIG. 3 is a graph showing the results of phenol degradation when the concentration of nitrogen in the medium of phenol-assimilating bacterium K2 was changed.

FIG. 4 is a graph showing the results of phenol degradation when the concentration of phosphorus in the medium of phenol-assimilating bacterium K2 was changed.

FIG. 5 is a diagram showing a change over time when aromatic hydrocarbon assimilating bacteria K11 decompose and assimilate benzene, toluene, and xylene.

FIG. 6 is a diagram showing a change over time when aromatic hydrocarbon assimilating bacteria K37 assimilate benzene, toluene, and xylene.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12N 1/00 C12N 1/16 G 1/16 B09B 3/00 ZABE // (C12N 1/20 C12R 1:01) (C12N 1 / 20 C12R 1:40) (C12N 1/20 C12R 1:06) (C12N 1/20 C12R 1:05) (C12N 1/16 C12R 1: 645)

Claims (21)

[Claims] Claims: 1. A phosphorous content of 5 ppm or less and a nitrogen content of 50 ppm.
A microorganism that can grow under the following conditions. 2. The microorganism according to claim 1, wherein the microorganism belongs to the genus Pseudomonas. 3. The method according to claim 1, wherein the microorganism is Pseudomonas pu.
The microorganism according to claim 2, which belongs to Tida. 4. The microorganism according to claim 1, wherein the microorganism belongs to the genus Burkholderia. 5. The microorganism is Burkholderia c.
5. The microorganism according to claim 4, which belongs to Epacia. 6. The method according to claim 6, wherein the microorganism is Burkholderia p.
The microorganism according to claim 4, which belongs to ickettii. 7. The microorganism according to claim 1, wherein the microorganism belongs to the genus Rhodococcus. 8. The microorganism according to claim 1, wherein the microorganism belongs to the genus Arthrobacter. 9. The method according to claim 9, wherein the microorganism is Arthrobacter a.
The microorganism according to claim 8, wherein the microorganism belongs to Trocyaneus. 10. The microorganism according to claim 1, wherein the microorganism belongs to the genus Cryptococcus. 11. The method according to claim 11, wherein the microorganism is Cryptococcus.
The microorganism according to claim 10, which belongs to albidus. 12. The microorganism according to claim 1, wherein the microorganism belongs to the genus Alcaligenes. 13. The method according to claim 12, wherein the microorganism is Alcaligenes d.
2. It belongs to E. enrichificans.
2. The microorganism according to 2. 14. The method according to claim 1, wherein the microorganism is capable of decomposing an aromatic compound.
The microorganism according to 7, 8, 9, 10, 11, 12, or 13. 15. The microorganism according to claim 14, wherein the aromatic compound is phenol. 16. The microorganism according to claim 14, wherein the aromatic compound contains at least one member selected from the group consisting of benzene, toluene, and xylene. 17. A method for purifying a petroleum pollutant, comprising treating the petroleum pollutant with the microorganism according to claim 14, 15, or 16. 18. The method according to claim 17, wherein the petroleum pollutant is petroleum contaminated soil or refinery wastewater. 19. A method for purifying an aromatic compound contaminant, comprising treating the aromatic compound contaminant with the microorganism according to claim 14, 15, or 16. 20. The method for purifying an aromatic compound contaminant according to claim 18, wherein the aromatic compound contaminant is aromatic compound contaminated soil or wastewater containing an aromatic compound. 21. The aromatic compound contaminant is phenol,
21. The method for purifying an aromatic compound pollutant according to claim 19, wherein the pollutant is a pollutant contaminated with at least one selected from the group consisting of benzene, toluene, and xylene.