JPH08197086A - Treatment of waste water containing normalhexane extract - Google Patents

Abstract

PURPOSE: To efficiently degrade and remove normalhexane extracts in waste water without subjecting the waste water to oil-water separation by a physicochemical method. CONSTITUTION: Various normalhexane extracts contained in waste water are degraded using Pseudomonas sp. ER-B1 strain (FERM P-14699) belonging to Pseudomonas Migula and having ability to degrade normalhexane extracts.

Description

Detailed Description of the Invention

[0001]

This invention relates to Pseudomonas (Pseudom).
new Pseudomonas sp. (Pse) belonging to the genus onas and having high resolution for various n-hexane extracts.
udomonas sp.) ER-B1 strain (FERM P-146
Hereinafter, the present invention simply relates to a method of biologically decomposing and removing an n-hexane extract contained in wastewater using ER-B1 strain).

[0002]

2. Description of the Related Art Because of its properties, n-hexane extract in wastewater has been hitherto physicochemically processed by a natural separation method, a pressure floating separation method, or an adsorption method using various oil adsorbents. Have been removed by. Among them, the pressure floating separation method in which a flocculant is used together is effective for removing the n-hexane extract, and has been applied to many processing facilities so far. However, the pressure-floating separation method using a coagulant together with a large amount of sludge and a shortage of disposal sites have become a problem, and nowadays more and more establishments are refraining from this method. Further, the odor of the separated sludge containing the n-hexane extract is also a problem. In other physicochemical methods such as grease traps, in the case of emulsified n-hexane extract rich wastewater,
It is often difficult to stably remove up to the discharge standard value.

The n-hexane extraction substance means a substance which is extracted by n-hexane and does not volatilize by drying at 80 ± 5 ° C. for 30 minutes. Among them, hydrocarbons and their derivatives, various animal and vegetable oils and fats, mineral oils, sterols and the like are included. Among them, animal and vegetable oils and fats contained in kitchen wastewater can be decomposed and removed to some extent by biological treatment methods such as the activated sludge method, but when the oil and fat load increases, the oils and fats become sludge. Attached to the surface of flocs and biofilms,
In some cases, the permeability of oxygen and the substrate is adversely affected, the processability gradually deteriorates, and eventually the process becomes impossible. Apart from such a method, JP-A-5-146798 discloses that the fats and oils in the wastewater are hydrolyzed into fatty acids and glycerin in advance by using lipase to make them easier to assimilate by microorganisms, and then the activity of the latter stage is described. A method of treating with sludge has been proposed. However, in this method, it is necessary to maintain the pH at 6.5 or more by adding an alkaline agent in order to prevent insolubilization of the fatty acid which is a hydrolyzate, and it is mainly obtained as an extracellular enzyme. Since lipase is an organic substance containing protein as a main component, it is considered that the effect of lipase treatment may be reduced due to microbial decomposition, adsorption to SS components and the like.
Furthermore, since the target of this method is specified to the wastewater containing animal and vegetable fats and oils that are substrates for lipase, it cannot be applied to the wastewater containing other n-hexane extracts such as mineral oil.

[0004] Further, JP-A-5-346036, JP-A-5-245489 and JP-A-4-179745.
JP-A-3-254893 and JP-A-3-254893 describe the use of microorganisms that assimilate and decompose fats and oils or oils. However, the above-mentioned document makes little disclosure about the microorganisms used. JP-A-3-270781 and JP-A-3-2751
In Japanese Patent Publication No. 95, a microorganism that assimilates and decomposes an oil component is specifically exemplified by its species name. However, the oil component assimilation / decomposition ability of the microorganisms exemplified in these documents is not disclosed in detail. JP-A-3-94898
The publication discloses the use of aerobic lipid-utilizing bacteria and their lipid-utilizing / degrading ability. However, the aerobic lipid-assimilating bacterium described in this document is used to assimilate even mineral oil.
It could not be disassembled.

Of course, there are microorganisms that can utilize not only animal and plant lipids but also mineral oils. However, conventionally both seed oils are usually treated as separate oils. In order to collectively purify wastewater in which animal and vegetable oils and mineral oils may be mixed, for example, bacteria that secrete lipase that decomposes animal and vegetable oils and bacteria that eat mineral oil may be used individually or in a mixture. However, when different bacteria are mixed, there is a problem that the n-hexane extract is not decomposed properly due to differences in optimal growth conditions and competition in growth. In addition, it is institutional problem to operate them separately.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above problems of the prior art.
Using a novel n-hexane extract degrading bacterium, it has been difficult to remove various n-hexane extracts which have been difficult until now (including, for example, the case where animal and vegetable fats and oils are mixed). It is to provide a method of efficiently decomposing.

[0007]

[Means for Solving the Problems] In order to achieve the above object, the inventors of the present invention attempted to separate a microorganism having a high n-hexane extract decomposing ability from the natural world, and used soil or activated sludge as a separation source. As a result, the present invention was completed by isolating Pseudomonas sp. ER-B1 strain having high n-hexane extract decomposing ability from activated sludge. That is, the present invention belongs to the genus Pseudomonas and has Pseudomonas sp.
as sp.) ER-B1 strain is used for decomposing various n-hexane extracts contained in the wastewater, and is a method for treating n-hexane extract-containing wastewater. As an embodiment of this method, ER is contained in activated sludge or various biofilms.
-It is attempted to grow the B1 strain and treat the wastewater containing the n-hexane extract.

When the culture of the ER-B1 strain is incorporated into a biological wastewater treatment method, the dry matter equivalent weight is 1%.
It is preferable to add 0.01 to 10 parts by weight to 00 parts by weight of sludge. Incorporation of the bacterial cells is carried out by an appropriate method such as injection of a culture solution, spraying, addition as a powder preparation, or the like. Further, the assembling timing can be set to be continuous or intermittent so as to suit the situation such as operation and load. The dry matter equivalent weight refers to the amount of slurry-like sludge corresponding to the weight when the sludge in the form of a slurry is dried, and is also called sludge dry weight. If it is less than 0.01 part by weight, a sufficient amount of ER-B1 strain is not observed to be colonized.
-The hexane extract is not decomposed properly. Again 10
If it exceeds the weight part, the cost becomes high, and the BOD value derived from the culture solution, for example, may increase and the waste water treatment device may be burdened, which is not preferable. The ER-B1 strain culture to be added usually has a bacterial cell concentration of 0.01 to 1
The stationary phase culture medium is used at 00 × 10 ¹⁰ cells / ml.

[0009] Below, Pseud
omonas sp.) ER-B1 strain (1) isolation method, (2)
The mycological properties, (3) culture method, (4) storage method, (5) properties of lipase produced by the same strain, and (6) addition method will be described. The test and classification method of the mycological properties were performed based on the following documents. Yabuuchi Eiko et al .; Nane Shuppan "Bacterial identification method that accompanies new taxonomy"
(1987), Takayuki Ezaki et al .; Journal of Japanese Bacteriology, 45, 8
51 (1990), Bar Jay's Manual of
Systematic Bacteriology (Bergey's Manual
of Systematic Bacterio1ogy) (1984) and Barjay's Manual of Determinative B
acterio1ogy) (1994).

(1) Separation method A medium containing 1% lard, 0.1% yeast extract, 0.1% ammonium phosphate, 0.02% potassium chloride, 0.02% magnesium sulfate, and 0.5% calcium carbonate. (PH
After sterilizing 7.6) in an autoclave at 121 ° C for 15 minutes, add soil (7 samples) and activated sludge (6 samples) appropriately diluted with sterilized water, and shake at culture temperature of 28 ° C for 3 days. did. Then, the operation of substituting 1% of the culture solution into the medium was repeated twice. On the other hand, 1.5% of agar was added to the above medium, and an agar medium in which lard was emulsified and dispersed by a homogenizer was prepared, and 0.2 ml of a diluted solution of the above culture medium was smeared to remove bacteria forming a clear zone. 18 strains were isolated. Next, in the above-mentioned medium, a liquid medium in which the amount of lard added was 0.1% and a liquid medium in which 0.1% of an emulsion-type water-soluble cutting oil mainly containing mineral oil was added instead of lard, respectively. 5% of the suspension of the above-mentioned isolated bacteria prepared and prepared to have a constant turbidity (OD ₆₆₀ 1.0) was added, and the reduction rate of lard and cutting oil after 24 hours was examined. . As a result, as shown in Table 1, the ER-B1 strain separated from the activated sludge showed the highest decomposition rate in both lard and cutting oil.

[0011]

[Table 1]

(2) Mycological properties 1) Morphology (a) Cell shape and size: length about 2 microns, width about 1
Micron bacillus, (b) Motility: Yes, (c) Flagella: Extremely monohair, (d) Spore: No, (e) Gram stainability: Negative 2) Growth state (a) Meat agar plate culture: circular, The surface is smooth and glossy. Does not produce a characteristic colony pigment. (b) Broth agar slant culture: filamentous, smooth and glossy surface. Does not produce a characteristic colony pigment. (c) Broth liquid culture: normal growth, suspension, no pigment formation. (d) Broth gelatin stab culture: liquefy gelatin.

3) Physiological properties (a) Attitude toward oxygen: aerobic, (b) oxidase: positive, (c) catalase: positive, (d) OF test: aerobically produced acid, (e) PHB Accumulation: negative, (f) indole production: negative, (g) esculin degradation: negative, (h) water-soluble dye formation: positive, (i) fluorescent dye formation: positive, (j) arginine dihydrase: Positive, (k) Growth at 41 ° C: Positive, (l) Levan production from sucrose: Negative, (m) Denitrification reaction: Negative, (n) Starch degradation: Negative, (o) Assimilation: Glucose , Geraniol, L-valine,
β-alanine, DL-arginine and citric acid are assimilated, but trehalose, 2-ketogluconic acid and meso-inositol are not assimilated. 4) Other properties (a) Quinone type: Q-9, (b) GC content: 66 (mol%;
HPLC method) (c) Acyl amidase: negative

As a result of searching based on the above-mentioned mycological properties and the classification method of the above-mentioned literature, this strain is a gram-negative bacillus,
Since it has polar flagella and its quinone system is Q-9, it was identified as a bacterium belonging to Pseudomonas. In addition, the flagella are extremely monochorionic and produce fluorescent dyes,
Further, it grew at 41 ° C. and showed a property similar to Pseudomonas aeruginosa such as high GC content. However, Pseudomonas aeruginosa's major denitrifying ability and acylamidase activity are negative in this strain, and 2-ketogluconic acid cannot be assimilated, and Pseudomonas aeruginosa
(uginosa) and other characteristics, so it was considered that it may be another bacterial species. Therefore, four types of Pseudomonas (Pseudom) similar to the above physiological and morphological characteristics were
onas) bacterium (Pseudomonas pu
tida), Pseudomonas medocina (Pseudomonas me
ndocina), Pseudomonas pseudoalcaligenes
Subspice Pseudomonas
pseudoalcaligenes subsp. pseudoalcaligenes), and Pseudomonas aerugin
osa)) and Pseudomonas sp.
As sp.) ER-B1 strain was subjected to a DNA homology test.
As a result, as shown in Table 2, no high homology was obtained with any of the bacterial species.

[0015]

[Table 2]

From the above findings, in terms of morphology and physiological characteristics, Pseudomonas aeruginos
Although it is identified as a strain related to (a), its genetic properties are completely different, so this strain is Pseudomonas.
It was determined that the strain was a new strain belonging to s) and was named Pseudomonas sp. ER-B1 strain. This strain was deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, on December 12, 1994. The microorganism accession number is FERM P-14699.

(3) Culture method Pseudomonas sp. Of the present invention
The ER-B1 strain can be cultivated in an ordinary medium in which various inorganic salts such as carbon source, nitrogen source, and phosphoric acid, and trace nutrient sources such as vitamins and amino acids are prepared. Preferably, peptone, meat extract, tryptone, etc. are used as the carbon source and nitrogen source, and ammonium sulfate, dipotassium diphosphate, magnesium sulfate, etc. are used as the inorganic salt, and the medium pH is 5 to 5.
10, preferably pH 6-8, culture temperature 15-45 ℃,
It is desirable to perform aerobic culture under the condition of 25 to 30 ° C. Further, the same strain produces lipase extracellularly, in which case, when defatted soybean flour is used as a carbon source and a nitrogen source, a culture with high lipase activity can be obtained, and lipase activity is further increased by adding lecithin. It gets even higher. It should be noted that in the broth medium, the viability of the cells remains unchanged, but lipase is hardly produced. Further, the culture temperature is preferably 30 ° C. or lower. At 30 ° C. or higher, the lipase productivity is reduced, and at 40 ° C. or higher, it is not produced. A suitable culture time is 24-48 hours, during which
The pH of the culture solution rises to about 8.5, but it is not necessary to control the pH. In addition, when performing aeration culture, it is necessary to add an antifoaming agent to suppress foaming, but a silicon-based antifoaming agent is effective, and the addition of about 0.5% increases the lipase activity. To do.

(4) Storage method Cultured cells can be stored by various drying methods such as a vacuum drying method and a freeze vacuum drying method. After removing the culture supernatant, physiological saline or phosphate buffer is used. It is also possible to suspend the cells in a solution containing no organic substance such as a liquid and store the liquid in a refrigerator for a long period of time. It is most convenient to store by the freezing method. In that case, when storing at -20 ° C, a cryoprotective agent such as glycerol is required, but at -60 ° C or lower, no protective agent is added. By freezing the culture as it is, it can be stably stored for a long period of time.

(5) Properties of lipase The method for measuring lipase activity is as follows: Yamada-Machida method (Journal of the Japanese Society of Agricultural Chemistry, Vol. 36, pages 860-864, 1962).
Was used as a substrate, and the amount of the enzyme that liberates 1 μmol of fatty acid in 1 minute at a reaction temperature of 37 ° C. was 1 unit (unit: abbreviated as U). The lipase of Pseudomonas sp. ER-B1 strain used in the test was the culture medium that was shake-cultured for 48 hours in a 500 ml shaking flask (medium amount 100 ml) using the medium of Table 3. The supernatant was obtained by removing the bacterial cells with a lipase activity of 42.4 U / ml (the activity of the culture solution was 70.5 U / ml).

[0020]

[Table 3]

Table 4 shows Pseud.
omonas sp.) The properties of the lipase produced by the ER-B1 strain are shown.

[0022]

[Table 4]

(6) Addition method Pseudomonas sp. Obtained by the above method
When the (Pseudomonas sp.) ER-B1 strain culture solution is used for treating oil-containing wastewater, the addition method may be either a continuous injection method or an intermittent injection method. Depending on the type of wastewater and operating conditions, Pseud
If the omonas sp.) ER-B1 strain has poor colonization, continuous injection is preferable, and a concentrated culture can be added separately. When injecting continuously,
It depends on the type of wastewater, the concentration of n-hexane extract, the coagulation / sedimentation of activated sludge, the state of biofilm, the degree of feeding by protozoa, etc. Addition of 0.01 to 1% (w / w), preferably 0.05% or more per day is effective for reducing the concentration of the n-hexane extract in the treated water, and also for Pseudomonas sp. .) The ER-B1 strain competes ecologically with other microorganisms, and the addition amount is necessary in order to exist in a certain amount in the biological treatment system. On the other hand, in the case of intermittent injection, the sludge dry weight was applied during the first injection,
0.1-10% (w / w), preferably 1% or more may be added, and thereafter, it may be added at any time depending on the quality of the treated water, but once every 3 days to once a month, preferably once a week. 1
It is advisable to add 0.1% (w / w) per dry weight of sludge. Regardless of the injection method, Pseudomonas sp. (Epseudomonas sp.) E is produced when the activated sludge has a good coagulation property or when a biofilm is formed on the surface of various carriers.
When the culture solution of the R-B1 strain was added, its fixability was good,
Subsequent processing is performed well, and the amount of culture solution injected can be reduced. Note that known techniques such as adding sludge containing the ER-B1 strain that has flowed out of the biological treatment tank to the biological treatment tank as return sludge can also be used in combination.

Pseudomonas sp. ER-B fixed on activated sludge or biofilm.
When the number of bacteria in one strain is to be counted, King B medium (2% proteose peptone, 1% glycerol, 0.15% dipotassium phosphate, magnesium sulfate) supplemented with 50 mg / liter of the antibiotic ampicillin is used. By culturing with 15%, agar 1.5%, pH 7.2) at a culturing temperature of 41 ° C. for 24 hours, colonies of the same strain can be selectively counted.

[0025]

EXAMPLES Next, examples of the present invention will be described. (Example 1) A 200-liter fermentor was charged with 120 liters of the medium shown in Table 3, and sterilized at 121 ° C for 30 minutes.
After the medium was sufficiently cooled, a culture solution of Pseudomonas sp. ER-B1 strain that had been pre-cultured was inoculated and inoculated at 25 ° C., a stirring blade rotation speed of 200 rpm, and an aeration rate of 120 liters / min. It was cultured under aerobic conditions for 30 hours. The culture broth thus obtained was subjected to ultrafiltration membrane 2
After doubling concentration, it was frozen and stored at -80 ° C. The culture concentrate obtained had a lipase activity of 120 U / ml and a viable cell count of 1.5 × 10 ¹¹ cells / ml. The solid content of the culture solution was 1.6%. As a result of examining the change over time in the residual lipase activity and the viable cell count of this frozen culture, the viable cell count decreased to 6.6 × 10 ¹⁰ cells / ml after 7 days,
After that, no decrease in the number of bacteria was observed (measured until the 180th day). On the other hand, the lipase activity maintained the activity before freezing, and no decrease in enzyme activity was observed.

Example 2 Meat extract 40 mg / liter, peptone 40 mg / liter, sodium hydrogen carbonate 250 mg / liter, potassium dibasic phosphate 10 mg / liter
Liter, magnesium sulfate 20 mg / liter, pH
100 ml of artificial wastewater prepared to 7.0, olive oil,
Emulsion-type water-soluble cutting oil (hereinafter also simply referred to as cutting oil) mainly composed of soybean oil, lard, and mineral oil, light oil, and heavy oil A were added to each to a concentration of 1,000 mg / liter, and Pseudomonas SP (Pse
udomonas sp.) ER-B1 strain culture solution was examined for degradability. Reaction temperature 25 ° C., reaction time 12 hours, culture medium solid concentration 160 mg / liter (concentrated culture medium 1% (v /
v) addition). Reaction container is 500 ml
A shake flask was used. The Pseudomonas sp. ER-B1 strain culture medium was prepared by thawing the frozen culture medium prepared in Example 1 in a water bath at 30 ° C. The experimental results are as shown in Table 5, 70 to 90% for animal and vegetable oils and 50 to 70% for mineral oils.
Was the decomposition rate.

[0027]

[Table 5]

Example 3 To thawed Pseudomonas sp. ER-B1 strain culture solution was added to artificial wastewater (the same as in Example 2) in which the concentration of added olive oil was changed, to decompose olive oil. I checked. Experimental conditions and methods were the same as in Example 2. The experimental results are shown in Table 6. When the initial olive oil concentration is 400 mg / liter or less, the removal rate is 90% or more.
Even in the case of 00 mg / liter, the removal rate was 68%.

[0029]

[Table 6]

(Example 4) Pseudomonas SP (P
The thawed culture solution of the seudomonas sp.) ER-B1 strain was added to the cafeteria wastewater containing about 150 mg / liter of the n-hexane extract substance, and the effect was compared with that of activated sludge. 500 m
l Shake the flask with the concentrated sludge (MLS) from the sewage treatment plant.
S7,800 mg / liter) 25 ml and cafeteria wastewater 75
ml was added. In a flask prepared in the same manner, the thawed culture solution was added with 0.01%, 0.1%, and
1.0% was added and shaken for 12 hours under the temperature condition of 25 ° C. The experimental results are shown in Table 7. By adding the Pseudomonas sp. ER-B1 strain culture solution, the removal rate of the n-hexane extract substance was increased.

[0031]

[Table 7]

(Example 5) The cutting oil used in Example 2 was added to the canteen waste water of Example 4 to a final concentration of 50 mg / liter, and Pseudom sp.
Onas sp.) ER-B1 strain culture medium was added and the effect was compared with activated sludge. The experiment method and conditions were the same as in Example 4, and the culture solution was added to a 500 ml shaking flask containing activated sludge and waste water at 0.01% per sludge dry weight of 0.
1% and 1.0% were added respectively. The experimental results are shown in Table 8. Pseudomonas sp. ER
-By adding the B1 strain culture solution, the removal rate of the n-hexane extract substance was increased.

[0033]

[Table 8]

(Example 6) Pseudomonas sp. ER prepared in Example 1 was prepared by setting up two series of activated sludge treatment experimental devices each consisting of an aeration tank and a sedimentation tank.
The removal property of the n-hexane extract and the colonization property of the same strain when the thawed culture solution of the B1 strain was added were examined. The two series of experimental systems are control groups in which RUN1 does not add the culture solution,
RUN2 is 1.0% (w / w) of culture liquid per dry weight of sludge
It is the experimental section added. Activated sludge concentration is 2,000mg
/ Liter, and the wastewater used was the cafeteria wastewater used in Example 4. The aeration tank used had an effective volume of 10 liters, and the canteen wastewater was continuously passed at a flow rate of 30 liters / day. The experimental results are shown in Table 9. RUN2 supplemented with culture medium
Is lower in concentration of n-hexane extract in the treated water than RUN1 (control), and King B added with ampicillin.
Pseudomonas sp.
As a result of counting the viable cell count of the sp.) ER-B1 strain, the cell count of 2.2 × 10 ¹⁰ cells / ml was maintained even 28 days after the addition of the culture solution.

[0035]

[Table 9]

(Embodiment 7) 20% (v / v) of polyether sponge molded into a 10 mm square per volume of the reaction tank
Three series of filled processing devices were provided, and Pseudomonas sp. ER-B1 was used as in Example 6.
The ability of the strain to remove the n-hexane extract and the colonization of the strain were examined. The three series of experimental systems were control groups in which RUN1 did not add the culture solution, RUN2 was an experimental group in which the culture solution was added at 0.1% (w / w) per dry weight of sludge attached to the sponge, and RUN3 was , 1.0% (w / w) was added. Concentration of activated sludge attached to sponge is 6,800
mg / liter-sponge (per 1 liter of sponge volume), and the wastewater used was the canteen wastewater used in Example 4. The aeration tank used had an effective volume of 20 liters, and the canteen wastewater was continuously passed at a flow rate of 60 liters / day. The experimental results are shown in Table 10. RUN2,3 supplemented with culture medium
Had a lower n-hexane extract concentration in the treated water than RUN1 (control), but RUN2 had a culture solution addition of 3
After a day, the removal rate of the n-hexane extract decreased. On the other hand, after adding the culture solution, RUN3 had a lower concentration of n-hexane extract in the treated water than that of RUN1 and was stably treated. In addition, Pseudomonas sp. (Pseudomonas sp.) Was prepared using King B medium supplemented with ampicillin.
As a result of counting the viable cell count of the p.) ER-B1 strain, RUN2 showed a correlation with the removal rate of the n-hexane extract, and the cell count decreased 3 days after the start of the experiment. In contrast, RU
In N3, the number of bacteria was 10 ¹⁰ cells / liter-sponge or more during the experiment.

[0037]

[Table 10]

[0038]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, the waste water containing the n-hexane extract is separated into n- in the wastewater by the biological treatment of the strain ER-B1 without oil-water separation by the physicochemical method. It is possible to efficiently decompose and remove the xanthate extract.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:38) (72) Inventor Nobukazu Kawata 4-2-1 Motofujisawa, Fujisawa-shi, Kanagawa Inside the EBARA Research Institute

Claims (4)

[Claims] 1. A variety of n-hexane extracts contained in wastewater are obtained by using Pseudomonas sp. ER-B1 strains belonging to the genus Pseudomonas and having the ability to decompose n-hexane extract. A method for treating wastewater containing an n-hexane extract, which comprises decomposing. 2. A method for treating wastewater using activated sludge, wherein pseudomonas is added to a group of microorganisms constituting the activated sludge.
The method for treating wastewater containing an n-hexane extract according to claim 1, wherein a culture of Pseudomonas sp. ER-B1 strain is added. 3. A method for treating wastewater using a biofilm, wherein the group of microorganisms constituting the biofilm is Pseudomonas.
The method for treating wastewater containing an n-hexane extract according to claim 1, wherein a culture of Pseudomonas sp. ER-B1 strain is added. 4. Pseudomonas sp. (Pseudomonas sp.) Against 100 parts by weight of the dry matter is added to the microorganism group.
As sp.) The ER-B1 strain culture is added in an amount of 0.01 to 10 parts by weight.
A method for treating wastewater containing hexane extract.