WO1999036506A1 - Microorganisms and preparation for disposing of organic wastewater - Google Patents

Microorganisms and preparation for disposing of organic wastewater Download PDF

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Publication number
WO1999036506A1
WO1999036506A1 PCT/KR1999/000028 KR9900028W WO9936506A1 WO 1999036506 A1 WO1999036506 A1 WO 1999036506A1 KR 9900028 W KR9900028 W KR 9900028W WO 9936506 A1 WO9936506 A1 WO 9936506A1
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Prior art keywords
wastewater
synthetic
microorganism
microorganisms
pseudomonas
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PCT/KR1999/000028
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French (fr)
Inventor
Hyun Soo Kim
Kwang Keun Oh
Cheol Woo Lee
Jeong Hwan Kim
Geun Woo Park
Yeong Joong Jeon
Jae Heung Lee
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Cheil Jedang Corporation
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Priority to AU20775/99A priority Critical patent/AU2077599A/en
Publication of WO1999036506A1 publication Critical patent/WO1999036506A1/en

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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/02—Aerobic processes
    • C02F3/10—Packings; Fillings; Grids
    • C02F3/105—Characterized by the chemical composition
    • C02F3/108—Immobilising gels, polymers or the like
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

Definitions

  • the present invention relates to a biological disposal of wastewater. Specifically, it relates to novel microorganisms useful in a biological disposal of organic wastewater and to a microorganism preparation that is effective in disposing of organic composite wastewater such as food and stock wastewater.
  • Rhodococcus was reported in Gennadi et al., Appl. Environ, Microbiol. 61, 4191:1995.
  • a method for decomposing iprodine by Arthrobacter was also reported in Danielle et al., Appl. Environ. Microbiol. 61, 3216:1995.
  • Studies to screen a microorganism for disintegrating non-decomposable materials are actively progressing. Techniques to dispose of non-decomposable materials by immobilization of microorganisms are also being studied.
  • the principle of the biological disposal of wastewater rests on the conversion by microorganisms of organic substances present in wastewater into inorganic substances.
  • the disposal process consists of an adsorption step in which nutritional substances (organic substances) make contact with microbial cells, a synthesis step in which the nutritional substances absorbed on the microbial cells are decomposed by various enzymes originating within the cells while some substances are assimilated into the cells, and a sedimentation step in which the cells form a floe so that it can easily precipitate.
  • the feature of each step is the acceleration of agglutination, growth, decomposition and sedimentation. This requires the use of microorganismswhich are very agglutinative, and which promote the decomposition of various organic substances, and have strong growth properties. Therefore, such microorganisms must be enriched in order to maintain these properties.
  • the preparation must feature an excellent maintenance of microorganisms which have good agglutinative, decompositive and growth properties. This preparation would be very significant in that it could be used in industrial factories and field wastewaters.
  • microorganisms capable of treating organic composite wastewater in industrial factories.
  • Microorganisms were isolated from wastewater originating from various industrial factories and were cultured in organic composite wastewater. Their growth and efficacy in removing organic substances were measured.
  • the inventors found several novel microorganisms which have an excellent ability to dispose of organic composite wastewater.
  • the present invention thus results from the formulation of novel microorganisms which have proved to act most effectively on organic wastewater.
  • the inventors confirmed that the agglutinative, decompositive, growth and sedimentation properties of the several novel microorganisms are enhanced when the microorganisms are used together in the disposal of organic wastewater.
  • the present invention provides several novel microorganisms, Pseudomonas sp. CJ-B25, Lactobacillus sp. CJ-E30, Micrococcus sp. CJ-C14, Pseudomonas sp.
  • the present invention also provides a microorganism preparation for disposing of wastewater which comprises purple non-sulfur bacteria (for example, ATCC 11166), Pseudomonas sp. CJ-B25, Lactobacillus sp. CJ-E30, Micrococcus sp. CJ-C14, Pseudomonas sp. CJ-F31, Erwinia sp. CJ-D17, Cellulomonas sp. CJ-G22 and Bacillus sp. (for example, ATCC 21770) in combination with organic and inorganic carriers, nutrients and minerals.
  • purple non-sulfur bacteria for example, ATCC 11166
  • Pseudomonas sp. CJ-B25 Lactobacillus sp. CJ-E30
  • Micrococcus sp. CJ-C14 Pseudomonas sp. CJ-F31
  • the present invention provides a method for producing a microorganism preparation for disposing of wastewater which comprises absorbing purple non-sulfur bacteria, Pseudomonas sp. CJ-B25, Lactobacillus sp. CJ-E30, Micrococcus sp. CJ-C14, Pseudomonas sp. CJ-F31, Erwinia sp. CJ-D17, Cellulomonas sp. C J-G22 and Bacillus sp.
  • first mixing and enriching culture process culturing the resulting absorbent
  • second mixing and aging culture process mixing the culture with inorganic carrier, nutrients and minerals to maintain and promote the activity of the microorganisms
  • drying and screening process drying and screening the mixture to yield powder that has an appropriate water content and particle size
  • Figure 1 shows the efficacy of the first screened microorganisms B07, B12, B17, B25, and B35 to remove BOD in beverage wastewater.
  • Figure 2 shows the efficacy of Pseudomonas sp. CJ-B25 to remove BOD in synthetic wastewaters.
  • Figure3 shows the efficacy of the first screened microorganisms C08, C14, C19, C28, and C30 to remove BOD in meat processing wastewater.
  • Figure 4 shows the efficacy of Micrococcus sp. CJ-C14 to remove BOD in synthetic wastewaters.
  • Figure 5 shows the efficacy of the first screened microorganisms D02, D 15,
  • Figure 6 shows the efficacy of Erwinia sp. CJ-D17 to remove BOD in synthetic wastewaters.
  • Figure 7 shows the efficacy of the first screened microorganisms F18, F26, F31, and F33 to remove BOD in food fermentation wastewater.
  • Figure 8 shows the efficacy of Pseudomonas sp. CJ-F31 to remove BOD in synthetic wastewaters.
  • Figure 9 shows the efficacy of the first screened microorganisms El 6, E27, E30, and E47 to remove BOD in edible oil wastewater.
  • Figure 10 shows the efficacy of Lactobacillus sp. CJ-E30 to remove BOD in synthetic wastewater.
  • Figure 11 shows the efficacy of the first screened microorganisms G07, G09, G16, G22 and G25 to remove BOD in sugar processing wastewater.
  • Figure 12 shows the efficacy of Cellulomonas sp. CJ-G22 to remove BOD in synthetic wastewaters.
  • Figure 13 shows disposal and sedimentation resulting from the input of the microorganism preparation according to the present invention in synthetic wastewaters.
  • Figure 14 shows the disposal efficacy of the microorganism preparation according to the present invention in organic composite field wastewaters (saccharide wastewater, protein wastewater, lipid wastewater and amixture thereof).
  • microorganisms of the present invention were deposited at the permanent collection of the Korean Culture Center of Microorganisms, Seoul,
  • the microorganism preparation yielded by this method can comprise Staphylococcus sp., Flavobacterium sp., Sphaerotilus sp., Zoogloea sp., and Nitrosomonas sp., in addition to the above-mentioned specific strains.
  • Pseudomonas sp. CJ-B25 forms round and flattened colonies on a composite medium. Tables 2, 3 and 4 below set forth its characteristics.
  • Lactobacillus sp. CJ-E30 forms round and convex colonies on a composite medium.
  • Tables 5, 6 and 7 set forth its characteristics.
  • Micrococcus sp. CJ-C14 forms round and protruding colonies on a composite medium. Tables 8, 9 and 10 below set forth its characteristics. Table 8. Morphological and Cultural Characteristics
  • Pseudomonas sp. CJ-F31 forms round and protruding colonies on a composite medium. Tables 11, 12 and 13 below set forth its characteristics.
  • a method for producing a microorganism preparation for purifying wastewater is as follows:
  • the above-mentioned strains possessing a high capability of decomposing organic substances are cultured in a liquid medium and are absorbed on an organic carrier.
  • the organic carrier include rice bran, wheat sheath, starch, soy bean and sawdust.
  • a crushed agricultural waste such as rice bran and wheat sheath is preferred in view of waste reuse.
  • the crushed rice bran is evenly mixed with a high liquid culture of the above strains (10% to 15%). Water is added to the mixture until the moisture content is adjusted to a range of 40% to 60%. This moisture content is critical because microorganisms grow well under conditions of appropriate moisture. A moisture content of 45% to 55% is preferable.
  • the organic carrier is additionally supplemented with nutritional ingredients such as nitrogen, phosphorous and minerals.
  • the mixture is cultured in a cylindrical roll cultivator at 25°C to 40°C for 1 to 2 days.
  • the temperature and oxygen supply are periodically adjusted during cultivation. This process is referred to as "first mixing and enriching culture”. Following the enriching culture, the second mixing is earned out.
  • an inorganic carrier is mixed with nutritional ingredients and minerals.
  • the inorganic carrier selected should not affect the functioning of the wastewater-disposal equipment.
  • examples of the inorganic carrier include silica, bentonite, zeolite and white clay.
  • a synthetic hydrolysis-silica exhibits especially good adsorption, sedimentation and diffusion. Synthetic hydrolysis-silica can be prepared by physico-chemical sedimentation in a liquid medium. It is a nontoxic white powder. The properties of the synthetic hydrolysis-silica are shown in Table 20 below.
  • the hydrolysis-silica Because of its large surface area and great adsorptiveness, the hydrolysis-silica absorbs and immobilizes microorganisms and thereby promotes their growth. In addition, the hydrolysis-silica has the advantage of preserving microorganisms because it so moisture retentive. Moreover, owing to such properties, the hydrolysis-silica generates sludge and floe well, and thus promotes sedimentation. The inventors were able to enhance the preservation of microorganisms by using the synthetic hydrolysis-silica, thus solving problems presented by prior microorganism preparations created to preserve microorganisms. In addition, an agglutinating agent or sedimenting agent was not required if the hydrolysis-silica was to be used to field disposal system.
  • the synthetic hydrolysis-silica was added in ratios of 15% to 30%, and was well mixed. Nutrients and minerals were additionally mixed in and the mixture was aged in a cylindrical roll cultivator at 25°C to 40°C for 1 day. The temperature and oxygen supply was periodically adjusted. This process is referred to as the second mixing and aging culture.
  • the synthetic hydrolysis-silica creates various effects as mentioned above. However, for the desired effect and for economic efficiency, it is best to add the synthetic hydrolysis-silica in ratios of 10% to 30%.
  • the nitrogen and phosphorous essential for the growth of microorganisms are included as nutrient ingredients.
  • an ammonium phosphate was used in ratios of 5% to 10%, and appropriate amounts of vitamins and trace elements such as minerals were used.
  • a moisture content is maintained in ratios of 35% to 40%.
  • the basic microorganism preparation was thereby obtained.
  • the moisture content was appropriately adjusted and a drying and screening process was conducted to remove masses or large particles.
  • the properties of the microorganism preparation of the present invention for purifying wastewater were analyzed.
  • the number of the viable cells was at least 3.0 x 10 9 , the moisture content was between 35% and 38%, and the apparent density was about 0.45 g/ml.
  • A. Isolation of the Strain Wastewater specimens were collected from beverage factories and adapted for an appropriate period.
  • the adapted microorganisms were suspended in a sterile saline solution and were smeared on a plate medium prepared by dissolving Luria-Bertani medium (LB medium; tryptone 0.1 g, yeast extract 0.05 g, sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g) in 1 L of beverage wastewater.
  • the smeared petri dishes were incubated at 25°C to 30°C for 1 to 3 days. Twenty (20) single colony forming species well grown on the plate medium were isolated.
  • the isolated microorganisms were inoculated by a platinum loop in culture tubes, each containing 5 ml of LB medium without agar. They were cultured in an agitation cultivator at 25°C to 30°C for 24 hours. The liquid culture was centrifuged at 10,000 rpm in VS15000CFN (Vision) for 5 minutes and the microorganisms were recovered. Some of the recovered microorganisms were used for subsequent experiments and the remainder was lyophilized for storage.
  • microorganism species were suspended in sterile distilled water. Each microorganism was inoculated by l%(v/v) in beverage wastewater adjusted to 100 ppm to 2,000 ppm (BOD), and was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and centrifuged.
  • BOD ppm to 2,000 ppm
  • BOD of the supernatant was measured to compare the organic substances-removing capability.
  • efficacy to remove BOD in beverage wastewater adjusted to 1,000 ppm was measured after 24 hours, five species (B07, B12, B17, B25, B35) showing a removal efficacy of at least 50% were first screened. The results are shown in Figure 1. Additionally, the growth of the first screened five species and their capability for removing organic substances in synthetic wastewater was compared.
  • the following synthetic wastewaters were prepared and used: a saccharide wastewater containing starch, glucose and sugar (synthetic wastewater A); a glycoprotein wastewater containing glucose (synthetic wastewater B); a glycolipid wastewater containing glucose (synthetic wastewater C); and a mixture of the synthetic wastewaters A, B and C (synthetic wastewater D).
  • the synthetic wastewater comprises the basic ingredients of glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1 and calcium chloride 50 mg/1.
  • the synthetic wastewater A additionally contains starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1.
  • the synthetic wastewater B additionally contains peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1.
  • the synthetic wastewater C additionally contains rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1.
  • the synthetic wastewater D was prepared by mixing the synthetic wastewaters A, B and C. Each synthetic wastewater was diluted to an appropriate concentration and then used.
  • the strains recovered by the above centrifugation were suspended in sterile distilled water and were inoculated by l%(v/v) in synthetic wastewaters A, B, C and D adjusted to BOD of 1,000 ppm.
  • the inoculated wastewater was cultured in an agitation cultivator at 25°C to 30°C.
  • a liquid culture was aseptically collected and appropriately diluted.
  • the absorbency of the dilution was measured by a spectrometer to observe the growth of the microorganisms.
  • the BOD of the supernatant obtained by centrifugation was measured to evaluate the capability of the microorganism to dispose of organic substances.
  • the results are shown in Figure 2.
  • the B25 strain was well grown in the synthetic wastewaters A, B, C and D and the BOD-removing efficacies of the B25 strain in the synthetic wastewaters A, B, C and D were 74%, 48%, 72% and 54%, respectively.
  • Wastewater specimens were collected from edible oil factories and adapted for an appropriate period.
  • the adapted microorganisms were suspended in a sterile saline solution and were smeared on a plate medium prepared by dissolving
  • Luria-Bertani medium (LB medium; tryptone 0.1 g, yeast extract 0.05 g, sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g) in 1 L of edible oil wastewater (filtered on a sterile 0.2 ⁇ m filter).
  • the smeared petri dishes were incubated at 25°C to 30°C for 1 to 3 days. Twenty (20) single colony forming species well grown on the plate medium were isolated.
  • the isolated microorganisms were inoculated by a platinum loop in culture tubes each containing 5 ml of LB medium without agar and were cultured in an agitation cultivator at 25°C to 30°C for 24 hours.
  • the liquid culture was centrifuged at 10,000 rpm in VS15000CFN (Vision) for 5 minutes and the microorganisms were recovered. Some of the recovered microorganisms were used for subsequent experiments and the remainder was lyophilized for storage.
  • microorganism species were suspended in sterile distilled water. Each microorganism was inoculated by l%(v/v) in edible oil wastewater adjusted to 200 ppm to 800 ppm (BOD) and was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and was centrifuged. The BOD of the supernatant was measured to compare the organic substances-removing capability. When the efficacy to remove BOD in an edible oil wastewater adjusted to 500 ppm was measured after 24 hours, five species (C08, C14, C19, C28, C30) showing a removal efficacy of at least 50% were first screened. The results are shown in Figure 3.
  • the synthetic wastewater comprises the basic ingredients of glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1 and calcium chloride 50 mg/1.
  • Synthetic wastewater A additionally contains starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1.
  • Synthetic wastewater B additionally contains peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1.
  • Synthetic wastewater C additionally contains rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1.
  • Synthetic wastewater D was prepared by mixing the synthetic wastewaters A, B and
  • Wastewater specimens were collected from meat processing factories and adapted for an appropriate period.
  • the adapted microorganisms were suspended in a sterile saline solution and were smeared on a plate medium prepared by dissolving Luria-Bertani medium (LB medium; tryptone 0.1 g, yeast extract 0.05 g, sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g) in 1 L of meat processing wastewater (filtered on a sterile 0.2 ⁇ m filter).
  • the smeared petri dishes were incubated at 25°C to 30°C for 1 to 3 days. Twenty (20) single colony forming species well grown on the plate medium were isolated.
  • the isolated microorganisms were inoculated by a platinum loop in culture tubes each containing 5 ml of LB medium without agar and were cultured in an agitation cultivator at 25°C to 30°C for 24 hours.
  • the liquid culture was centrifuged at 10,000 rpm in VS 15000CFN (Vision) for 5 minutes and the microorganisms were recovered. Some of the recovered microorganisms were used for subsequent experiments and the remainder was lyophilized for storage.
  • microorganism species were suspended in sterile distilled water. Each microorganism was inoculated by l%(v/v) in meat processing wastewater adjusted to 100 ppm to 500 ppm (BOD) and was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and was centrifuged. The BOD of the supernatant was measured to compare the organic substances-removing capability. When the efficacy to remove BOD in meat processing wastewater adjusted to 500 ppm were measured after 24 hours, five species (D02, D 15 , D 17, D26, D29) showing a removal efficacy of at least 50% were first screened. The results are shown in Figure 5.
  • the growth of the first screened five species and their capability for removing organic substances in a synthetic wastewater was compared.
  • the following synthetic wastewaters were prepared and used; a saccharide wastewater containing starch, glucose and sugar (synthetic wastewater A); a glycoprotein wastewater containing glucose (synthetic wastewaterB); a glycolipidwastewater containing glucose (synthetic wastewater C); and a mixture of the synthetic wastewaters A, B and C (synthetic wastewater D) .
  • the synthetic wastewater comprises basic ingredients of glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1 and calcium chloride 50 mg/1.
  • Synthetic wastewater A additionally contains starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1.
  • Synthetic wastewater B additionally contains peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1.
  • Synthetic wastewater C additionally contains rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1.
  • Synthetic wastewater D was prepared by mixing synthetic wastewaters A, B and C. Each synthetic wastewater was diluted to appropriate concentrations and then used. The strains recovered by the above centrifugation were suspended in sterile distilled water and inoculated by l%(v/v) in synthetic wastewaters A, B, C and D adjusted to BOD of 1,000 ppm. The inoculated wastewater was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and appropriately diluted.
  • the absorbency of the dilution was measured by a spectrometer to observe the growth of the microorganisms.
  • the BOD of the supernatant obtained by centrifugation was measured to evaluate the capability of the microorganism to dispose of organic substances. The results are shown in Figure 6.
  • the D17 strain was well grown in synthetic wastewaters A, B, C and D and the BOD-removing efficacy of the D17 strain in synthetic wastewaters A, B, C and D were 52%, 74%, 62% and 70%, respectively.
  • Wastewater specimens were collected from food fermentation factories and adapted for an appropriate period.
  • the adapted microorganisms were suspended in a sterile saline solution and were smeared on a plate medium prepared by dissolving Luria-Bertani medium (LB medium; tryptone 0.1 g, yeast extract 0.05 g, sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g) in 1 L of food fermentation wastewater (filtered on a sterile 0.2 ⁇ m filter).
  • the smeared petri dishes were incubated at 25°C to 30°C for 1 to 3 days. Twenty (20) single colony forming species well grown on the plate medium were isolated.
  • the isolated microorganisms were inoculated by a platinum loop in culture tubes each containing 5 ml of LB medium without agar and were cultured in an agitation cultivator at 25°C to 30°C for 24 hours.
  • the liquid culture was centrifuged at 10,000 rpm in VS15000CFN (Vision) for 5 minutes and the microorganisms were recovered. Some of the recovered microorganisms were used for subsequent experiments and the remainder was lyophilized for storage.
  • microorganism species were suspended in sterile distilled water. Each microorganism was inoculated by l%(v/v) in food fermentation wastewater adjusted to 700 ppm to 2,000 ppm (BOD) and was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and was centrifuged. The BOD of the supernatants was measured to compare the organic substances-removing capability. When the efficacy to remove BOD in a beverage wastewater adjusted to 1 ,000 ppm were measured after 24 hours, five species (F 18, F26, F31 , F33, F36) showing a removal efficacy of at least 50% were first screened.
  • the synthetic wastewater comprises the basic ingredients of glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1 and calcium chloride 50 mg/1.
  • Synthetic wastewater A additionally contains starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1.
  • Synthetic wastewater B additionally contains peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1.
  • Synthetic wastewater C additionally contains rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1.
  • Synthetic wastewater D was prepared by mixing synthetic wastewaters A, B and C. Each synthetic wastewater was diluted to an appropriate concentration and then used.
  • the strains recovered by the above centrifugation were suspended in a sterile distilled water and were inoculated by 1 %(v/v) in synthetic wastewaters A, B, C and D adjusted to BOD of 1,000 ppm.
  • the inoculated wastewater was cultured in an agitation cultivator at 25°C to 30°C.
  • a liquid culture was aseptically collected and appropriately diluted.
  • the absorbency of the dilution was measured by a spectrometer to observe the growth of the microorganisms.
  • the BOD of the supernatant obtained by centrifugation was measured to evaluate the capability of the microorganism to dispose of organic substances. The results are shown in Figure 8.
  • the F31 strain was well grown in synthetic wastewaters A, B, C and D and the
  • BOD-removing efficacy of the F31 strain in synthetic wastewaters A, B, C and D were 45%, 52%, 48% and 62%, respectively.
  • Waste water specimens were collected from frozen food factories and adapted for an appropriate period.
  • the adapted microorganisms were suspended in a sterile saline solution and smeared on a plate medium preparedby dissolving Luria-Bertani medium (LB medium; tryptone 0.1 g, yeast extract 0.05 g, sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g) in 1 L of edible oil wastewater (filtered on a sterile 0.2 ⁇ m filter).
  • LB medium Luria-Bertani medium
  • yeast extract 0.05 g sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g
  • the smeared petri dishes were incubated at 25°C to 30°C for 1 to 3 days. Twenty (20) single colony-forming species well grown on the plate medium were isolated.
  • the isolated microorganisms were inoculated by a platinum loop in culture tubes each containing 5 ml of LB medium without agar, and were cultured in an agitation cultivator at 25°C to 30°C for 24 hours.
  • the liquid culture was centrifuged at 10,000 rpm in VS 15000CFN (Vision) for 5 minutes and the microorganisms were recovered. Some of the recovered microorganisms were used for subsequent experiments and the remainder was lyophilized for storage.
  • 1,500 ppm were measured after 24 hours, five species (E01, E16, E27, E30, E47) showing a removal efficacy of at least 50% were first screened.
  • the results are shown in Figure 9. Additionally, the growth of the first screened five species and their capability for removing organic substances in synthetic wastewater.
  • the following synthetic wastewaters were prepared and used: a saccharide wastewater containing starch, glucose and sugar (synthetic wastewater A); a glycoprotein wastewater containing glucose (synthetic wastewater B); a glycolipid wastewater containing glucose (synthetic wastewater C); and a mixture of the synthetic wastewaters A, B and C (synthetic wastewater D).
  • the synthetic wastewater comprises the basic ingredients of glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1 and calcium chloride 50 mg/1.
  • Synthetic wastewater A additionally contains starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1.
  • Synthetic wastewater B additionally contains peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1.
  • Synthetic wastewater C additionally contains rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1.
  • Synthetic wastewater D was prepared by mixing synthetic wastewaters A, B and C. Each synthetic wastewater was diluted to an appropriate concentration and then used. The strains recovered by the above centrifugation were suspended in sterile distilled water and inoculated by l%(v/v) in synthetic wastewaters A, B, C and D adjusted to BOD of 1,000 ppm. The inoculated wastewater was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and appropriately diluted.
  • the absorbency of the dilution was measured by a spectrometer to observe the growth of the microorganisms.
  • the BOD of the supernatant obtained by centrifugation was measured to evaluate the capability of the microorganism to dispose of organic substances.
  • the results are shown in Figure 10.
  • the E30 strain was well grown in synthetic wastewaters A, B, C and D and the BOD-removing efficacy of the E30 strain in synthetic wastewaters A, B, C and D were 60%, 55%, 49%, and 56%, respectively.
  • Example 6 Cellulomonas sp. CJ-G22
  • Wastewater specimens were collected from sugar factories and adapted for an appropriate period.
  • the adapted microorganisms were suspended in a sterile saline solution and smeared on a plate medium prepared by dissolving Luria-Bertani medium (LB medium; tryptone 0.1 g, yeast extract 0.05 g, sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g) in 1 L of sugar processing wastewater (filtered on a sterile 0.2 ⁇ m filter).
  • LB medium Luria-Bertani medium
  • tryptone 0.1 g tryptone 0.1 g
  • yeast extract 0.05 g sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g
  • the smeared petri dishes were incubated at 25°C to 30°C for 1 to 3 days. Twenty (20) single colony-forming species well grown on the plate medium were isolated.
  • the isolated microorganisms were inoculated by a platinum loop in culture tubes each containing 5 ml of LB medium without agar, and were cultured in an agitation cultivator at 25°C to 30°C for 24 hours.
  • the liquid culture was centrifuged at 10,000 rpm in VS15000CFN (Vision) for 5 minutes and the microorganisms were recovered. Some of the recovered microorganisms were used for subsequent experiments and the remainder was lyophilized for storage.
  • microorganism species were suspended in sterile distilled water. Each microorganism was inoculated by l%(v/v) in a sugar processing oil wastewater adjusted to 200 ppm to 800 ppm (BOD) and was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and was centrifuged. The BOD of the supernatant was measured to compare their organic substance-removing capability. When their efficacy for removing BOD in a sugar processing wastewater adjusted to 500 ppm were measured after 24 hours, five species (G07, G09, Gl 6, G22, G25) showing a removal efficacy of at least 50% were first screened. The results are shown in Figure 16.
  • the following synthetic wastewaters were prepared and used: a saccharide wastewater containing starch, glucose and sugar (synthetic wastewater A); a glycoprotein wastewater containing glucose (synthetic wastewater B); a glycolipidwastewatercontaining glucose (synthetic wastewater C); and a mixture of the synthetic wastewaters A, B and C (synthetic wastewater D).
  • the synthetic wastewater comprises the basic ingredients of glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1 and calcium chloride 50 mg/1.
  • Synthetic wastewater A additionally contains starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1.
  • Synthetic wastewater B additionally contains peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1.
  • Synthetic wastewater C additionally contains rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1.
  • Synthetic wastewater D was prepared by mixing the synthetic wastewaters A, B and C. Each synthetic wastewater was diluted to an appropriate concentration and then used. The strains recovered by the above centrifugation were suspended in sterile distilled water and inoculated by l%(v/v) in synthetic wastewaters A, B, C and D adjusted to BOD of 1,000 ppm. The inoculated wastewater was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and appropriately diluted.
  • the absorbency of the dilution was measured by a spectrometer to observe the growth of the microorganisms.
  • the BOD of the supernatant obtained by centrifugation was measured to evaluate the capability of the microorganism to dispose of organic substances. The results are shown in Figure 12.
  • the G22 strain was well grown in synthetic wastewaters A, B, C and D and the BOD-removing efficacy of the G22 strain in synthetic wastewaters A, B, C and D were 63%, 45%, 88% and 69%, respectively.
  • 650 ml of a liquid culture was mixed with 180 L of water. The mixture was added to 500 Kg of crushed rice bran and they were mixed. 180 L of water were added and the moisture content was adjusted to 50%. 1,386 g of the nutrient ingredients was added for the supplement of nitrogen, phosphorous and trace elements.
  • the nutrient ingredients consisted of CH 3 COONa 600 g, (NH 4 ) 2 SO 4 60 g, MgSO 4 7H 2 O 40 g, NaCl 20 g, FeCl 3 6H 2 O 1 g, CaCl 2 2H 2 O 10 g, KH 2 PO 4 100 g and yeast extract 10 g.
  • the mixture was cultured in a cylindrical cultivator at the temperature of 25 °C to 40°C for 1 to 2 days. The temperature was periodically adjusted and oxygen was supplied. After cultivation, the number of viable cells was counted. 5 x 10 10 to 3 x 10 u cells per gram of culture were found to be viable.
  • an inorganic carrier In the second mixing process, an inorganic carrier, nutrient ingredients and trace elements were added.
  • the inorganic carrier selected should be one that has not effect on a disposal system.
  • Synthetic hydrolysis-silica is a good inorganic carrier, as it results in excellent sedimentation and diffusion. 120 Kg of synthetic hydrolysis-silica was added and well mixed. Then, nutritional ingredients and trace elements were added and the mixture was aged in a cylindrical cultivator at 25°C to
  • the basic microorganism preparation for purifying wastewater was thereby developed. Finally, the basic preparation was dried and screened to create the appropriate moisture content. Masses and large particles were removed. Powders with moisture content of 35% to 38% and with particles of 75 to 100 meshes were obtained. The properties of the powdered microorganism preparation were analyzed. The number of viable cells was at least 3.0 x 10 9 /g, and the apparent density was about 0.45 g/ml. After this preparation was smeared on a plate medium, a kind of microorganism was investigated. The above six strains of the present invention were found to exist in the preparation.
  • the synthetic composite wastewater basically contains glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1, and calcium chloride 50 ml/1.
  • synthetic wastewater A consisting of starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1
  • synthetic wastewater B consisting of peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1
  • synthetic wastewater C consisting of rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1.
  • the synthetic composite wastewater was diluted to an appropriate concentration before it was used.
  • the BOD of the above synthetic composite wastewater was adjusted to 2,000 ppm and was placed in a 5L reactor. After the microorganism preparation of the present invention was added to 100 ppm, the disposal and sedimentation were measured. The activated sludge collected from a running disposal facility was used as a control. The results are shown in Figure 13.
  • the microorganism preparation of the present invention results in reductions of BOD and COD, an elevation of sedimentation and a decrease in the turbidity of the disposed water.
  • wastewaters generated from a factory manufacturing medicament from a food fermentation factory, from factories processing milk, meat, marine product, leather and livestock, and from factories producing soy and bean pastes were used.
  • a saccharide wastewater wastewater generated from factories manufacturing sugar, noodles or beverage and from a fruit processing factory were used.
  • a lipid wastewater wastewater generated from an edible oil manufacturing factory, a oil and fat processing factory, a glycerol producing factory, a frozen food factory, and a meat processing factory were used.

Abstract

A microorganism preparation for disposing of organic composite wastewater which comprises purple non-sulfur bacteria, Pseudomonas sp. CJ-B25, Lactobacillus sp. CJ-E30, Micrococcus sp. CJ-C14, Pseudomonas sp. CJ-F31, Erwinia sp. CJ-D17, Cellulomonas sp. CJ-G22 and Bacillus sp. in combination with organic and inorganic carriers, nutrients and minerals is disclosed. In addition, a method for producing said microorganism preparation and said novel microorganisms used therefor are disclosed.

Description

MICROORGANISMS AND PREPARATION FOR DISPOSING OF ORGANIC WASTEWATER
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention relates to a biological disposal of wastewater. Specifically, it relates to novel microorganisms useful in a biological disposal of organic wastewater and to a microorganism preparation that is effective in disposing of organic composite wastewater such as food and stock wastewater.
DESCRIPTION OF THE PRIOR ART
A method for decomposing aromatic halogen compounds and phenol by
Rhodococcus was reported in Gennadi et al., Appl. Environ, Microbiol. 61, 4191:1995. A method for decomposing iprodine by Arthrobacter was also reported in Danielle et al., Appl. Environ. Microbiol. 61, 3216:1995. Studies to screen a microorganism for disintegrating non-decomposable materials are actively progressing. Techniques to dispose of non-decomposable materials by immobilization of microorganisms are also being studied. For example, there is a report on methods for the continuous or semi-continuous decomposition of phenol by immobilizing Pseudomonas putida on activated carbon (Ehrhardt et al., Appl. Microbiol. Biotechnol. 30, 312:1989). Nevertheless, there are few examples to the screened microorganisms able to decompose industrial field wastewater and of their successful use in factories.
The principle of the biological disposal of wastewater rests on the conversion by microorganisms of organic substances present in wastewater into inorganic substances. The disposal process consists of an adsorption step in which nutritional substances (organic substances) make contact with microbial cells, a synthesis step in which the nutritional substances absorbed on the microbial cells are decomposed by various enzymes originating within the cells while some substances are assimilated into the cells, and a sedimentation step in which the cells form a floe so that it can easily precipitate. The feature of each step is the acceleration of agglutination, growth, decomposition and sedimentation. This requires the use of microorganismswhich are very agglutinative, and which promote the decomposition of various organic substances, and have strong growth properties. Therefore, such microorganisms must be enriched in order to maintain these properties.
It appears that prior microorganism preparations have contained unsuitable microorganisms and unbalanced nutrients. The result has been poor floe formation and slow organic substance decomposition. Consequently, the reduction times for BOD and COD are prolonged. Especially, this is true when an unbalance of nitrogen and/or phosphorus causes a division of sludge under overflow conditions in the aeration tank, resulting in an extreme decrease in wastewater disposal. Moreover, earlier microorganism preparations were weak in maintaining the microorganisms that were viable, and made the microorganisms self-lysed. As a result, earlier microorganism preparations were ill-suited for use in the disposal of field wastewaters.
Accordingly, there is a need to develop a new microorganism preparation for the purification of wastewater. The preparation must feature an excellent maintenance of microorganisms which have good agglutinative, decompositive and growth properties. This preparation would be very significant in that it could be used in industrial factories and field wastewaters.
It is impossible to apply a germ-free system and pure culture to a reaction tank for treating various contaminates. Instead, a composite system is applied. As such, it is necessary to isolate and identify very active microorganisms. To apply a purely isolated microorganism to an environmental industry, it requires a technique that makes a purely isolated microorganism the dominant species in the composite system reaction tank. In establishing such a technique, the following points should be considered in isolating and identifying the microorganism. First, specimens must be collected under conditions identical to those of the field to which the isolated microorganisms will be applied. Second, the isolated microorganisms should be adapted to the substances to be decomposed for a period sufficient to maintain the activity of the microorganism. Third, the conditions making the microorganism dominantin field wastewater should be found out. Finally, the useful microorganism must be maintained through continuous monitoring.
With these requirements in mind, the inventors have conducted a lengthy investigation to screen microorganisms capable of treating organic composite wastewater in industrial factories. Microorganisms were isolated from wastewater originating from various industrial factories and were cultured in organic composite wastewater. Their growth and efficacy in removing organic substances were measured. As a result, the inventors found several novel microorganisms which have an excellent ability to dispose of organic composite wastewater. The present invention thus results from the formulation of novel microorganisms which have proved to act most effectively on organic wastewater.
The inventors confirmed that the agglutinative, decompositive, growth and sedimentation properties of the several novel microorganisms are enhanced when the microorganisms are used together in the disposal of organic wastewater. In addition, they confirmed that mixing these microorganisms, which have been adsorbed on organic or inorganic carriers, with nutrients and minerals maintains or promotes the agglutinativeness, decompositiveness, growth and sedimentation of the microorganisms and results in the vigorous disposal of organic wastewater.
SUMMARY OF THE INVENTION
The present inventionprovides several novel microorganisms, Pseudomonas sp. CJ-B25, Lactobacillus sp. CJ-E30, Micrococcus sp. CJ-C14, Pseudomonas sp.
CJ-F31, Erwinia sp. CJ-D17, and Cellulomonas sp. CJ-G22. These strains display good growth under aerobic conditions and can excellently dispose of organic composite wastewater.
The present invention also provides a microorganism preparation for disposing of wastewater which comprises purple non-sulfur bacteria (for example, ATCC 11166), Pseudomonas sp. CJ-B25, Lactobacillus sp. CJ-E30, Micrococcus sp. CJ-C14, Pseudomonas sp. CJ-F31, Erwinia sp. CJ-D17, Cellulomonas sp. CJ-G22 and Bacillus sp. (for example, ATCC 21770) in combination with organic and inorganic carriers, nutrients and minerals. In addition, the present invention provides a method for producing a microorganism preparation for disposing of wastewater which comprises absorbing purple non-sulfur bacteria, Pseudomonas sp. CJ-B25, Lactobacillus sp. CJ-E30, Micrococcus sp. CJ-C14, Pseudomonas sp. CJ-F31, Erwinia sp. CJ-D17, Cellulomonas sp. C J-G22 and Bacillus sp. on organic carrier, culturing the resulting absorbent (hereinafter, "first mixing and enriching culture process"), mixing the culture with inorganic carrier, nutrients and minerals to maintain and promote the activity of the microorganisms (hereinafter, "second mixing and aging culture process"), and drying and screening the mixture to yield powder that has an appropriate water content and particle size (hereinafter, "drying and screening process").
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying drawings as follows:
Figure 1 shows the efficacy of the first screened microorganisms B07, B12, B17, B25, and B35 to remove BOD in beverage wastewater.
Figure 2 shows the efficacy of Pseudomonas sp. CJ-B25 to remove BOD in synthetic wastewaters.
Figure3 shows the efficacy of the first screened microorganisms C08, C14, C19, C28, and C30 to remove BOD in meat processing wastewater.
Figure 4 shows the efficacy of Micrococcus sp. CJ-C14 to remove BOD in synthetic wastewaters.
Figure 5 shows the efficacy of the first screened microorganisms D02, D 15,
D17, D26, and D29 to remove BOD in frozen food wastewater.
Figure 6 shows the efficacy of Erwinia sp. CJ-D17 to remove BOD in synthetic wastewaters. Figure 7 shows the efficacy of the first screened microorganisms F18, F26, F31, and F33 to remove BOD in food fermentation wastewater.
Figure 8 shows the efficacy of Pseudomonas sp. CJ-F31 to remove BOD in synthetic wastewaters.
Figure 9 shows the efficacy of the first screened microorganisms El 6, E27, E30, and E47 to remove BOD in edible oil wastewater.
Figure 10 shows the efficacy of Lactobacillus sp. CJ-E30 to remove BOD in synthetic wastewater.
Figure 11 shows the efficacy of the first screened microorganisms G07, G09, G16, G22 and G25 to remove BOD in sugar processing wastewater.
Figure 12 shows the efficacy of Cellulomonas sp. CJ-G22 to remove BOD in synthetic wastewaters.
Figure 13 shows disposal and sedimentation resulting from the input of the microorganism preparation according to the present invention in synthetic wastewaters.
Figure 14 shows the disposal efficacy of the microorganism preparation according to the present invention in organic composite field wastewaters (saccharide wastewater, protein wastewater, lipid wastewater and amixture thereof).
DETAILED DESCRIPTION OF THE INVENTION
The microorganisms of the present invention were deposited at the permanent collection of the Korean Culture Center of Microorganisms, Seoul,
Korea, on January 11, 1999 under the Budapest Treaty of the international recognition of the deposit of microorganisms for the purpose of patent procedure, and subcultures thereof can be obtained from the depository under the following accession numbers (Table 1). Table 1. Deposit of Instant Microorganisms
Strains Accession Nos.
Pseudomonas sp. CJ-B25 KCCM- 11044 Lactobacillus sp. CJ-E30 KCCM- 11045 Micrococcus sp. CJ-C14 KCCM- 11056 Pseudomonas sp. CJ-F31 KCCM- 11057 Erwinia sp. CJ-D17 KCCM-11058 Cellulomonas sp. CJ-G22 KCCM- 11059
The microorganism preparation yielded by this method can comprise Staphylococcus sp., Flavobacterium sp., Sphaerotilus sp., Zoogloea sp., and Nitrosomonas sp., in addition to the above-mentioned specific strains.
The characterization of the strains according to the present invention is as follows:
Pseudomonas sp. CJ-B25 forms round and flattened colonies on a composite medium. Tables 2, 3 and 4 below set forth its characteristics.
Table 2. Morphological and Cultural Characteristics
Items Feature
Gram Staining Shape Rod
Motility +
Table 3. Physiological and Biochemical Characteristics
Items Feature
Haemolysis -
Oxidase +
Catalase +
Oxygen Requirement Aerobic Indole Formation Reduction of Nitric Acid To Nitrous Acid +
Urease Formation +
Methyl Red Test Starch Hydrolysis
Gelatin Hydrolysis O/F Test O+F
Phenylalanine Diaminase +
Table 4. Carbon Utility
Items Feature
Glucose +
Fructose + Acetate
Tartrate
Sorbitol +
Mannitol +
Ethanol + Arginine m-Inositol +
Lactobacillus sp. CJ-E30 forms round and convex colonies on a composite medium. Tables 5, 6 and 7 set forth its characteristics.
Table 5. Morphological and Cultural Characteristics
Items Feature
Gram Staining +
Shape Rod
Motility +
Sporogenesis - Table 6. Physiological and Biochemical Characteristics
Items Feature
Haemolysis -
Oxidase -
Catalase -
Oxygen Requirement Anaerobic
Indole Formation -
Reduction of Nitric Acid
To Nitrous Acid -
Urease Formation -
Methyl Red Test -
Starch Hydrolysis -
Gelatin Hydrolysis -
O/F Test O+F
Citrate Test +
Table 7. Carbon Utility
Items Feature
Glucose +
Fructose
Acetate
Tartrate
Sorbitol
Mannitol +
Ethanol
Arginine
Gluconate
Micrococcus sp. CJ-C14 forms round and protruding colonies on a composite medium. Tables 8, 9 and 10 below set forth its characteristics. Table 8. Morphological and Cultural Characteristics
Items Feature
Gram Staining + Shape Tetrastreptococcus Motility + Sporogenesis
Table 9. Physiological and Biochemical Characteristics
Items Feature
Haemolysis -
Oxidase -
Catalase +
Oxygen Requirement Conditii
Indole Formation -
Reduction of Nitric Acid
To Nitrous Acid -
Urease Formation +
Methyl Red Test -
Starch Hydrolysis -
Gelatin Hydrolysis +
O/F Test O+F
VP Test -
Table 10. Carbon Utility
Items Feature
Glucose
Fructose
Acetate +
Tartrate +
Citrate +
Mannitol
Ethanol Arginine m-Inositol
Pseudomonas sp. CJ-F31 forms round and protruding colonies on a composite medium. Tables 11, 12 and 13 below set forth its characteristics.
Table 11. Morphological and Cultural Characteristics
Items Feature
Gram Staining -
Shape Rod
Motility +
Table 12. Physiological and Biochemical Characteristics
Items Feature
Haemolysis -
Oxidase -
Catalase +
Oxygen Requirement Conditii
Indole Formation -
Reduction of Nitric Acid
To Nitrous Acid +
Urease Formation -
Methyl Red Test +
Starch Hydrolysis -
Gelatin Hydrolysis +
O/F Test O+F
Citrate Test -
VP Test -
Table 13. Carbon Utility
Items Feature
Glucose + Fructose + m-Inositol +
Tartrate
Sorbitol +
Mannitol + Ethanol
Arginine Gluconate
Erwinia sp. CJ-D17 forms round and convex colonies on a composite medium. Tables 14, 15 and 16 below set forth its characteristics.
Table 14. Morphological and Cultural Characteristics
Items Feature Gram Staining
Shape Rod
Motility
Table 15. Physiological and Biochemical Characteristics
Items Feature
Haemolysis -
Oxidase -
Catalase +
Oxygen Requirement Conditional Anaerobic
Indole Formation -
Reduction of Nitric Acid
To Nitrous Acid +
Urease Formation +
Methyl Red Test -
Starch Hydrolysis -
Gelatin Hydrolysis +
O/F Test O+F
Citrate + Table 16. Carbon Utility
Items Feature
Glucose +
Fructose + Acetate
Tartrate Sorbitol
Mannitol +
Ethanol Arginine +
Gluconate
Cellulomonas sp. CJ-G22 forms round and convex colonies on a composite medium. Tables 17, 18 and 19 below set forth its characteristics.
Table 17. Morphological and Cultural Characteristics
Items Feature
Gram Staining +
Shape Rod
Motility +
Sporogenesis -
Table 18. Physiological and Biochemical Characteristics
Items Feature
Haemolysis -
Oxidase -
Catalase +
Oxygen Requirement Aerobic
Indole Formation -
Reduction of Nitric Acid
To Nitrous Acid +
Urease Formation - Methyl Red Test +
Starch Hydrolysis -
Gelatin Hydrolysis +
O/F Test O+F
VP Test -
Table 19. Carbon Utility
Items Feature
Glucose + Fructose
Acetate
Tartrate
Sorbitol
Mannitol Ethanol
Arginine
Citrate
A method for producing a microorganism preparation for purifying wastewater is as follows:
The above-mentioned strains possessing a high capability of decomposing organic substances are cultured in a liquid medium and are absorbed on an organic carrier. Examples of the organic carrier include rice bran, wheat sheath, starch, soy bean and sawdust. A crushed agricultural waste such as rice bran and wheat sheath is preferred in view of waste reuse. The crushed rice bran is evenly mixed with a high liquid culture of the above strains (10% to 15%). Water is added to the mixture until the moisture content is adjusted to a range of 40% to 60%. This moisture content is critical because microorganisms grow well under conditions of appropriate moisture. A moisture content of 45% to 55% is preferable. The organic carrier is additionally supplemented with nutritional ingredients such as nitrogen, phosphorous and minerals. The mixture is cultured in a cylindrical roll cultivator at 25°C to 40°C for 1 to 2 days. The temperature and oxygen supply are periodically adjusted during cultivation. This process is referred to as "first mixing and enriching culture". Following the enriching culture, the second mixing is earned out.
In the second mixing, an inorganic carrier is mixed with nutritional ingredients and minerals. The inorganic carrier selected should not affect the functioning of the wastewater-disposal equipment. Examples of the inorganic carrier include silica, bentonite, zeolite and white clay. A synthetic hydrolysis-silica exhibits especially good adsorption, sedimentation and diffusion. Synthetic hydrolysis-silica can be prepared by physico-chemical sedimentation in a liquid medium. It is a nontoxic white powder. The properties of the synthetic hydrolysis-silica are shown in Table 20 below.
Table 20. The Properties of Synthetic Hydrolysis-Silica
Items Values Reference
Apparent Density (g/ml) 0.14 to 0.20
Surface Area (m2/g) 200 to 300
Specific Gravity 1.95 to 2.05 pH 6.0 to 7.0 5% Solution
Index of Refraction 1.45
Dryness Loss (%) 7 to 9 105°C, 2 hours
Batch Ratio (%) 11 to 13 900°C
Because of its large surface area and great adsorptiveness, the hydrolysis-silica absorbs and immobilizes microorganisms and thereby promotes their growth. In addition, the hydrolysis-silica has the advantage of preserving microorganisms because it so moisture retentive. Moreover, owing to such properties, the hydrolysis-silica generates sludge and floe well, and thus promotes sedimentation. The inventors were able to enhance the preservation of microorganisms by using the synthetic hydrolysis-silica, thus solving problems presented by prior microorganism preparations created to preserve microorganisms. In addition, an agglutinating agent or sedimenting agent was not required if the hydrolysis-silica was to be used to field disposal system.
After the first mixing and enriching culture was finished, the synthetic hydrolysis-silica was added in ratios of 15% to 30%, and was well mixed. Nutrients and minerals were additionally mixed in and the mixture was aged in a cylindrical roll cultivator at 25°C to 40°C for 1 day. The temperature and oxygen supply was periodically adjusted. This process is referred to as the second mixing and aging culture. The synthetic hydrolysis-silica creates various effects as mentioned above. However, for the desired effect and for economic efficiency, it is best to add the synthetic hydrolysis-silica in ratios of 10% to 30%. The nitrogen and phosphorous essential for the growth of microorganisms are included as nutrient ingredients.
As nutrient ingredients, an ammonium phosphate was used in ratios of 5% to 10%, and appropriate amounts of vitamins and trace elements such as minerals were used. Following the second mixing and aging culture, a moisture content is maintained in ratios of 35% to 40%. The basic microorganism preparation was thereby obtained. Finally, the moisture content was appropriately adjusted and a drying and screening process was conducted to remove masses or large particles.
The properties of the microorganism preparation of the present invention for purifying wastewater were analyzed. The number of the viable cells was at least 3.0 x 109, the moisture content was between 35% and 38%, and the apparent density was about 0.45 g/ml.
The ability of this microorganism preparation to remove organic substances in synthetic wastewater and natural wastewater was tested, compared to the commercially available Bacterial Program (ATHEA, United States) and Polybac (POLYBAC, United States). The sedimentation thereof was also tested. The microorganism preparation of the present invention was found to be superior to the controls.
The following examples are given merely as illustrations of the present invention and demonstration of the preferred embodiments of the present invention, and are not to be considered as limiting.
Example 1 Pseudomonas sp. C J-B25
A. Isolation of the Strain Wastewater specimens were collected from beverage factories and adapted for an appropriate period. The adapted microorganisms were suspended in a sterile saline solution and were smeared on a plate medium prepared by dissolving Luria-Bertani medium (LB medium; tryptone 0.1 g, yeast extract 0.05 g, sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g) in 1 L of beverage wastewater. The smeared petri dishes were incubated at 25°C to 30°C for 1 to 3 days. Twenty (20) single colony forming species well grown on the plate medium were isolated. The isolated microorganisms were inoculated by a platinum loop in culture tubes, each containing 5 ml of LB medium without agar. They were cultured in an agitation cultivator at 25°C to 30°C for 24 hours. The liquid culture was centrifuged at 10,000 rpm in VS15000CFN (Vision) for 5 minutes and the microorganisms were recovered. Some of the recovered microorganisms were used for subsequent experiments and the remainder was lyophilized for storage.
B. Screening of Strains Capable of Disposing of Composite Wastewater
The above 20 microorganism species were suspended in sterile distilled water. Each microorganism was inoculated by l%(v/v) in beverage wastewater adjusted to 100 ppm to 2,000 ppm (BOD), and was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and centrifuged. The
BOD of the supernatant was measured to compare the organic substances-removing capability. When the efficacy to remove BOD in beverage wastewater adjusted to 1,000 ppm was measured after 24 hours, five species (B07, B12, B17, B25, B35) showing a removal efficacy of at least 50% were first screened. The results are shown in Figure 1. Additionally, the growth of the first screened five species and their capability for removing organic substances in synthetic wastewater was compared. The following synthetic wastewaters were prepared and used: a saccharide wastewater containing starch, glucose and sugar (synthetic wastewater A); a glycoprotein wastewater containing glucose (synthetic wastewater B); a glycolipid wastewater containing glucose (synthetic wastewater C); and a mixture of the synthetic wastewaters A, B and C (synthetic wastewater D). The synthetic wastewater comprises the basic ingredients of glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1 and calcium chloride 50 mg/1. The synthetic wastewater A additionally contains starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1. The synthetic wastewater B additionally contains peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1. The synthetic wastewater C additionally contains rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1. The synthetic wastewater D was prepared by mixing the synthetic wastewaters A, B and C. Each synthetic wastewater was diluted to an appropriate concentration and then used. The strains recovered by the above centrifugation were suspended in sterile distilled water and were inoculated by l%(v/v) in synthetic wastewaters A, B, C and D adjusted to BOD of 1,000 ppm. The inoculated wastewater was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and appropriately diluted. The absorbency of the dilution was measured by a spectrometer to observe the growth of the microorganisms. The BOD of the supernatant obtained by centrifugation was measured to evaluate the capability of the microorganism to dispose of organic substances. The results are shown in Figure 2. The B25 strain was well grown in the synthetic wastewaters A, B, C and D and the BOD-removing efficacies of the B25 strain in the synthetic wastewaters A, B, C and D were 74%, 48%, 72% and 54%, respectively.
Example 2 Micrococcus sp. CJ-C14
A. Isolation of the Strain
Wastewater specimens were collected from edible oil factories and adapted for an appropriate period. The adapted microorganisms were suspended in a sterile saline solution and were smeared on a plate medium prepared by dissolving
Luria-Bertani medium (LB medium; tryptone 0.1 g, yeast extract 0.05 g, sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g) in 1 L of edible oil wastewater (filtered on a sterile 0.2 μm filter). The smeared petri dishes were incubated at 25°C to 30°C for 1 to 3 days. Twenty (20) single colony forming species well grown on the plate medium were isolated. The isolated microorganisms were inoculated by a platinum loop in culture tubes each containing 5 ml of LB medium without agar and were cultured in an agitation cultivator at 25°C to 30°C for 24 hours. The liquid culture was centrifuged at 10,000 rpm in VS15000CFN (Vision) for 5 minutes and the microorganisms were recovered. Some of the recovered microorganisms were used for subsequent experiments and the remainder was lyophilized for storage.
B. Screening of Strains Capable of Disposing of Composite Wastewater
The above 20 microorganism species were suspended in sterile distilled water. Each microorganism was inoculated by l%(v/v) in edible oil wastewater adjusted to 200 ppm to 800 ppm (BOD) and was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and was centrifuged. The BOD of the supernatant was measured to compare the organic substances-removing capability. When the efficacy to remove BOD in an edible oil wastewater adjusted to 500 ppm was measured after 24 hours, five species (C08, C14, C19, C28, C30) showing a removal efficacy of at least 50% were first screened. The results are shown in Figure 3. Additionally, the growth of the first screened five species and their capability for removing organic substances in a synthetic wastewater was compared. The following synthetic wastewater were prepared and used: a saccharide wastewater containing starch, glucose and sugar (synthetic wastewater A); a glycoprotein wastewater containing glucose (synthetic wastewater B); a glycolipid wastewater containing glucose (synthetic wastewater C); and a mixture of the synthetic wastewaters A, B and C (synthetic wastewater D). The synthetic wastewater comprises the basic ingredients of glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1 and calcium chloride 50 mg/1. Synthetic wastewater A additionally contains starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1. Synthetic wastewater B additionally contains peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1. Synthetic wastewater C additionally contains rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1.
Synthetic wastewater D was prepared by mixing the synthetic wastewaters A, B and
C. Each synthetic wastewater was diluted to an appropriate concentration and then used. The strains recovered by the above centrifugation were suspended in a sterile distilled water and were inoculated by 1 %(v/v) in synthetic wastewaters A, B, C and D adjusted to BOD of 1,000 ppm. The inoculated wastewater was cultured in an agitation cultivator at the temperature of 25°C to 30°C. A liquid culture was aseptically collected and appropriately diluted. The absorbency of the dilution was measured by a spectrometer to observe the growth of the microorganisms. The BOD of the supernatant obtained by centrifugation was measured to evaluate the capability of the microorganism to dispose of organic substances. The results are shown in Figure 4. The C14 strain was well grown in the synthetic wastewaters A, B, C and D and the BOD-removing efficacy of the E30 strain in the synthetic wastewaters A, B, C and D were 61%, 53%, 85% and 66%, respectively.
Example 3
Erwinia sp. CJ-D17
A. Isolation of the Strain
Wastewater specimens were collected from meat processing factories and adapted for an appropriate period. The adapted microorganisms were suspended in a sterile saline solution and were smeared on a plate medium prepared by dissolving Luria-Bertani medium (LB medium; tryptone 0.1 g, yeast extract 0.05 g, sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g) in 1 L of meat processing wastewater (filtered on a sterile 0.2 μm filter). The smeared petri dishes were incubated at 25°C to 30°C for 1 to 3 days. Twenty (20) single colony forming species well grown on the plate medium were isolated. The isolated microorganisms were inoculated by a platinum loop in culture tubes each containing 5 ml of LB medium without agar and were cultured in an agitation cultivator at 25°C to 30°C for 24 hours. The liquid culture was centrifuged at 10,000 rpm in VS 15000CFN (Vision) for 5 minutes and the microorganisms were recovered. Some of the recovered microorganisms were used for subsequent experiments and the remainder was lyophilized for storage.
B. Screening of Strains Capable of Disposing of Composite Wastewater
The above 20 microorganism species were suspended in sterile distilled water. Each microorganism was inoculated by l%(v/v) in meat processing wastewater adjusted to 100 ppm to 500 ppm (BOD) and was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and was centrifuged. The BOD of the supernatant was measured to compare the organic substances-removing capability. When the efficacy to remove BOD in meat processing wastewater adjusted to 500 ppm were measured after 24 hours, five species (D02, D 15 , D 17, D26, D29) showing a removal efficacy of at least 50% were first screened. The results are shown in Figure 5. Additionally, the growth of the first screened five species and their capability for removing organic substances in a synthetic wastewater was compared. The following synthetic wastewaters were prepared and used; a saccharide wastewater containing starch, glucose and sugar (synthetic wastewater A); a glycoprotein wastewater containing glucose (synthetic wastewaterB); a glycolipidwastewater containing glucose (synthetic wastewater C); and a mixture of the synthetic wastewaters A, B and C (synthetic wastewater D) . The synthetic wastewater comprises basic ingredients of glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1 and calcium chloride 50 mg/1. Synthetic wastewater A additionally contains starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1. Synthetic wastewater B additionally contains peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1. Synthetic wastewater C additionally contains rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1. Synthetic wastewater D was prepared by mixing synthetic wastewaters A, B and C. Each synthetic wastewater was diluted to appropriate concentrations and then used. The strains recovered by the above centrifugation were suspended in sterile distilled water and inoculated by l%(v/v) in synthetic wastewaters A, B, C and D adjusted to BOD of 1,000 ppm. The inoculated wastewater was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and appropriately diluted. The absorbency of the dilution was measured by a spectrometer to observe the growth of the microorganisms. The BOD of the supernatant obtained by centrifugation was measured to evaluate the capability of the microorganism to dispose of organic substances. The results are shown in Figure 6. The D17 strain was well grown in synthetic wastewaters A, B, C and D and the BOD-removing efficacy of the D17 strain in synthetic wastewaters A, B, C and D were 52%, 74%, 62% and 70%, respectively.
Example 4 Pseudomonas sp. CJ-F31
A. Isolation of the Strain
Wastewater specimens were collected from food fermentation factories and adapted for an appropriate period. The adapted microorganisms were suspended in a sterile saline solution and were smeared on a plate medium prepared by dissolving Luria-Bertani medium (LB medium; tryptone 0.1 g, yeast extract 0.05 g, sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g) in 1 L of food fermentation wastewater (filtered on a sterile 0.2 μm filter). The smeared petri dishes were incubated at 25°C to 30°C for 1 to 3 days. Twenty (20) single colony forming species well grown on the plate medium were isolated. The isolated microorganisms were inoculated by a platinum loop in culture tubes each containing 5 ml of LB medium without agar and were cultured in an agitation cultivator at 25°C to 30°C for 24 hours. The liquid culture was centrifuged at 10,000 rpm in VS15000CFN (Vision) for 5 minutes and the microorganisms were recovered. Some of the recovered microorganisms were used for subsequent experiments and the remainder was lyophilized for storage.
B. Screening of Strains Capable of Disposing of Composite Wastewater
The above 20 microorganism species were suspended in sterile distilled water. Each microorganism was inoculated by l%(v/v) in food fermentation wastewater adjusted to 700 ppm to 2,000 ppm (BOD) and was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and was centrifuged. The BOD of the supernatants was measured to compare the organic substances-removing capability. When the efficacy to remove BOD in a beverage wastewater adjusted to 1 ,000 ppm were measured after 24 hours, five species (F 18, F26, F31 , F33, F36) showing a removal efficacy of at least 50% were first screened.
The results are shown in Figure 10. Additionally, the growth of the first screened five species and their capability for removing organic substances in a synthetic wastewater was compared. The following synthetic wastewaters were prepared and used: a saccharide wastewater containing starch, glucose and sugar (synthetic wastewater A); a glycoprotein wastewater containing glucose (synthetic wastewater
B); a glycolipid wastewater containing glucose (synthetic wastewater C); and a mixture of the synthetic wastewaters A, B and C (synthetic wastewater D). The synthetic wastewater comprises the basic ingredients of glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1 and calcium chloride 50 mg/1.
Synthetic wastewater A additionally contains starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1. Synthetic wastewater B additionally contains peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1. Synthetic wastewater C additionally contains rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1. Synthetic wastewater D was prepared by mixing synthetic wastewaters A, B and C. Each synthetic wastewater was diluted to an appropriate concentration and then used. The strains recovered by the above centrifugation were suspended in a sterile distilled water and were inoculated by 1 %(v/v) in synthetic wastewaters A, B, C and D adjusted to BOD of 1,000 ppm. The inoculated wastewater was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and appropriately diluted. The absorbency of the dilution was measured by a spectrometer to observe the growth of the microorganisms. The BOD of the supernatant obtained by centrifugation was measured to evaluate the capability of the microorganism to dispose of organic substances. The results are shown in Figure 8. The F31 strain was well grown in synthetic wastewaters A, B, C and D and the
BOD-removing efficacy of the F31 strain in synthetic wastewaters A, B, C and D were 45%, 52%, 48% and 62%, respectively.
Example 5 Lactobacillus sp. CJ-E30
A. Isolation of the Strain
Waste water specimens were collected from frozen food factories and adapted for an appropriate period. The adapted microorganisms were suspended in a sterile saline solution and smeared on a plate medium preparedby dissolving Luria-Bertani medium (LB medium; tryptone 0.1 g, yeast extract 0.05 g, sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g) in 1 L of edible oil wastewater (filtered on a sterile 0.2 μm filter). The smeared petri dishes were incubated at 25°C to 30°C for 1 to 3 days. Twenty (20) single colony-forming species well grown on the plate medium were isolated. The isolated microorganisms were inoculated by a platinum loop in culture tubes each containing 5 ml of LB medium without agar, and were cultured in an agitation cultivator at 25°C to 30°C for 24 hours. The liquid culture was centrifuged at 10,000 rpm in VS 15000CFN (Vision) for 5 minutes and the microorganisms were recovered. Some of the recovered microorganisms were used for subsequent experiments and the remainder was lyophilized for storage.
B. Screening of Strains Capable of Disposing of Composite Wastewater The above 20 microorganism species were suspended in sterile distilled water. Each microorganism was inoculated by l%(v/v) in frozen food wastewater adjusted to 1,000 ppm to 2,000 ppm (BOD) and cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and was centrifuged. The BOD of the supernatant was measured to compare the organic substances-removing capability. When the efficacy removing BOD in a beverage wastewater adjusted to
1,500 ppm were measured after 24 hours, five species (E01, E16, E27, E30, E47) showing a removal efficacy of at least 50% were first screened. The results are shown in Figure 9. Additionally, the growth of the first screened five species and their capability for removing organic substances in synthetic wastewater. The following synthetic wastewaters were prepared and used: a saccharide wastewater containing starch, glucose and sugar (synthetic wastewater A); a glycoprotein wastewater containing glucose (synthetic wastewater B); a glycolipid wastewater containing glucose (synthetic wastewater C); and a mixture of the synthetic wastewaters A, B and C (synthetic wastewater D). The synthetic wastewater comprises the basic ingredients of glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1 and calcium chloride 50 mg/1. Synthetic wastewater A additionally contains starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1. Synthetic wastewater B additionally contains peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1. Synthetic wastewater C additionally contains rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1. Synthetic wastewater D was prepared by mixing synthetic wastewaters A, B and C. Each synthetic wastewater was diluted to an appropriate concentration and then used. The strains recovered by the above centrifugation were suspended in sterile distilled water and inoculated by l%(v/v) in synthetic wastewaters A, B, C and D adjusted to BOD of 1,000 ppm. The inoculated wastewater was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and appropriately diluted. The absorbency of the dilution was measured by a spectrometer to observe the growth of the microorganisms. The BOD of the supernatant obtained by centrifugation was measured to evaluate the capability of the microorganism to dispose of organic substances. The results are shown in Figure 10. The E30 strain was well grown in synthetic wastewaters A, B, C and D and the BOD-removing efficacy of the E30 strain in synthetic wastewaters A, B, C and D were 60%, 55%, 49%, and 56%, respectively. Example 6 Cellulomonas sp. CJ-G22
A. Isolation of the Strain
Wastewater specimens were collected from sugar factories and adapted for an appropriate period. The adapted microorganisms were suspended in a sterile saline solution and smeared on a plate medium prepared by dissolving Luria-Bertani medium (LB medium; tryptone 0.1 g, yeast extract 0.05 g, sodium chloride 0.05 g, glucose 0.01 g, agar 0.2 g) in 1 L of sugar processing wastewater (filtered on a sterile 0.2 μm filter). The smeared petri dishes were incubated at 25°C to 30°C for 1 to 3 days. Twenty (20) single colony-forming species well grown on the plate medium were isolated. The isolated microorganisms were inoculated by a platinum loop in culture tubes each containing 5 ml of LB medium without agar, and were cultured in an agitation cultivator at 25°C to 30°C for 24 hours. The liquid culture was centrifuged at 10,000 rpm in VS15000CFN (Vision) for 5 minutes and the microorganisms were recovered. Some of the recovered microorganisms were used for subsequent experiments and the remainder was lyophilized for storage.
B. Screening of Strains Capable of Disposing of Composite Wastewater.
The above 20 microorganism species were suspended in sterile distilled water. Each microorganism was inoculated by l%(v/v) in a sugar processing oil wastewater adjusted to 200 ppm to 800 ppm (BOD) and was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and was centrifuged. The BOD of the supernatant was measured to compare their organic substance-removing capability. When their efficacy for removing BOD in a sugar processing wastewater adjusted to 500 ppm were measured after 24 hours, five species (G07, G09, Gl 6, G22, G25) showing a removal efficacy of at least 50% were first screened. The results are shown in Figure 16. Additionally, the growth of the first screened five species and their capability for removing organic substances in a synthetic wastewater was compared. The following synthetic wastewaters were prepared and used: a saccharide wastewater containing starch, glucose and sugar (synthetic wastewater A); a glycoprotein wastewater containing glucose (synthetic wastewater B); a glycolipidwastewatercontaining glucose (synthetic wastewater C); and a mixture of the synthetic wastewaters A, B and C (synthetic wastewater D). The synthetic wastewater comprises the basic ingredients of glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1 and calcium chloride 50 mg/1. Synthetic wastewater A additionally contains starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1. Synthetic wastewater B additionally contains peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1. Synthetic wastewater C additionally contains rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1. Synthetic wastewater D was prepared by mixing the synthetic wastewaters A, B and C. Each synthetic wastewater was diluted to an appropriate concentration and then used. The strains recovered by the above centrifugation were suspended in sterile distilled water and inoculated by l%(v/v) in synthetic wastewaters A, B, C and D adjusted to BOD of 1,000 ppm. The inoculated wastewater was cultured in an agitation cultivator at 25°C to 30°C. A liquid culture was aseptically collected and appropriately diluted. The absorbency of the dilution was measured by a spectrometer to observe the growth of the microorganisms. The BOD of the supernatant obtained by centrifugation was measured to evaluate the capability of the microorganism to dispose of organic substances. The results are shown in Figure 12. The G22 strain was well grown in synthetic wastewaters A, B, C and D and the BOD-removing efficacy of the G22 strain in synthetic wastewaters A, B, C and D were 63%, 45%, 88% and 69%, respectively.
Example 7
A. Culture of Microorganisms
50 ml of a sterile medium consisting of tryptone 10 g/1, yeast extract 5 g/1, sodium chloride 5 g/1 and glucose 1 g/1 was placed in a IL Erlenmyer flask. This flask was inoculated with purple non-sulfur Rhodobacter sp. ATCC 11166, Pseudomonas sp. CJ-B25, Micrococcus sp. CJ-C14, Erwnia sp. CJ-D17, Lactobacillus sp. CJ-E30, Pseudomonas sp. CJ-F31, Cellulomonas sp. CJ-G22 or
Bacillus sp. ATCC 21770 and cultivated at 25°C to 30°C, 130 rpm, for 24 hours. B. The First Mixing and Enriching Culture
650 ml of a liquid culture was mixed with 180 L of water. The mixture was added to 500 Kg of crushed rice bran and they were mixed. 180 L of water were added and the moisture content was adjusted to 50%. 1,386 g of the nutrient ingredients was added for the supplement of nitrogen, phosphorous and trace elements. The nutrient ingredients consisted of CH3COONa 600 g, (NH4)2SO460 g, MgSO4 7H2O 40 g, NaCl 20 g, FeCl3 6H2O 1 g, CaCl2 2H2O 10 g, KH2PO4 100 g and yeast extract 10 g. The mixture was cultured in a cylindrical cultivator at the temperature of 25 °C to 40°C for 1 to 2 days. The temperature was periodically adjusted and oxygen was supplied. After cultivation, the number of viable cells was counted. 5 x 1010 to 3 x 10u cells per gram of culture were found to be viable.
C. The Second Mixing and Aging Culture
In the second mixing process, an inorganic carrier, nutrient ingredients and trace elements were added. The inorganic carrier selected should be one that has not effect on a disposal system. Synthetic hydrolysis-silica is a good inorganic carrier, as it results in excellent sedimentation and diffusion. 120 Kg of synthetic hydrolysis-silica was added and well mixed. Then, nutritional ingredients and trace elements were added and the mixture was aged in a cylindrical cultivator at 25°C to
40°C for 1 day, during which the temperature was periodically adjusted and oxygen was supplied. As to nutritional ingredients, 60 Kg of ammonium phosphate was used, supplementing the nitrogen and phosphorous essential for the growth of microorganisms. Trace elements were used in an amount of 10.6 Kg. The moisture content was then adjusted to 35% to 40%. At the end, the viable cells were counted.
3 x 109 to 2 x 1010 per gram of culture were found to be viable.
D. Drying and Screening
The basic microorganism preparation for purifying wastewater was thereby developed. Finally, the basic preparation was dried and screened to create the appropriate moisture content. Masses and large particles were removed. Powders with moisture content of 35% to 38% and with particles of 75 to 100 meshes were obtained. The properties of the powdered microorganism preparation were analyzed. The number of viable cells was at least 3.0 x 109/g, and the apparent density was about 0.45 g/ml. After this preparation was smeared on a plate medium, a kind of microorganism was investigated. The above six strains of the present invention were found to exist in the preparation.
Example 8
Evaluation of the Efficacy of the Microorganism Preparation in the Disposal of Synthetic Composite Wastewater
The synthetic composite wastewater basically contains glucose 20 g/1, yeast extract 10 mg/1, sodium chloride 0.1 g/1, ammonium sulfate 10 g/1, potassium phosphate 0.5 g/1, magnesium sulfate 0.2 g/1, iron chloride 5 mg/1, and calcium chloride 50 ml/1. These basic ingredients are mixed with synthetic wastewater A, consisting of starch 3 g/1, sucrose 3 g/1, lactose 3 g/1 and galactose 3 g/1, synthetic wastewater B, consisting of peptone 3 g/1, tryptone 3 g/1 and beef extract 3 g/1, and synthetic wastewater C, consisting of rice oil 4 g/1, glycerol 4 g/1, stearic acid 4 g/1, oleic acid 4 g/1 and linoleic acid 4 g/1. The synthetic composite wastewater was diluted to an appropriate concentration before it was used.
The BOD of the above synthetic composite wastewater was adjusted to 2,000 ppm and was placed in a 5L reactor. After the microorganism preparation of the present invention was added to 100 ppm, the disposal and sedimentation were measured. The activated sludge collected from a running disposal facility was used as a control. The results are shown in Figure 13. The microorganism preparation of the present invention results in reductions of BOD and COD, an elevation of sedimentation and a decrease in the turbidity of the disposed water.
Example 9
Evaluation of the Efficacy of the Microorganism Preparation in the Disposal of Field Wastewater
As a protein wastewater, wastewaters generated from a factory manufacturing medicament, from a food fermentation factory, from factories processing milk, meat, marine product, leather and livestock, and from factories producing soy and bean pastes were used. As a saccharide wastewater, wastewater generated from factories manufacturing sugar, noodles or beverage and from a fruit processing factory were used. As a lipid wastewater, wastewater generated from an edible oil manufacturing factory, a oil and fat processing factory, a glycerol producing factory, a frozen food factory, and a meat processing factory were used.
Each of the above saccharide wastewaters, protein wastewaters, lipid wastewaters, and a mixture thereof were placed in a 5L reactor. The microorganism preparation of the present invention was added to 100 ppm, and the BOD was measured. The commercially available products, "Bacteria Program" (Product B) and "Polybac" (Product P) were used as controls. These products were applied to the wastewater in the same manner as described above, and the BOD was measured. The results are shown in Figure 14.

Claims

WHAT IS CLAIMED IS:
1. A microorganism preparation for disposing of waterwaste which comprises purple non-sulfur bacteria, Pseudomonas sp. CJ-B25, Lactobacillus sp. CJ-E30, Micrococcus sp. CJ-C14, Pseudomonas sp. CJ-F31, Erwinia sp. CJ-D17, Cellulomonas sp. CJ-G22 and Bacillus sp. in combination with organic and inorganic carriers, nutrients and minerals.
2. A process for producing a microorganism preparation for disposing of wastewater which comprises mixing purple non-sulfur bacteria, Pseudomonas sp. CJ-B25, Lactobacillus sp. CJ-E30, Micrococcus sp. CJ-C14, Pseudomonas sp. CJ-F31,
Erwinia sp. C J-D 17, Cellulomonas sp. C J-G22 and Bacillus sp. with organic carrier, culturing the mixture, mixing the culture with inorganic carrier, nutrients and minerals, and drying and screening the mixture to obtain a powder with appropriate moisture content and with appropriate particle size.
3. A microorganism Pseudomonas sp. CJ-B25 which is well grown under aerobic condition and is able to dispose of organic composite wastewater.
4. A microorganism Lactobacillus sp. CJ-E30 which is well grown under aerobic condition and is able to dispose of organic composite wastewater.
5. A microorganism Micrococcus sp. CJ-C14 which is well grown under aerobic condition and is able to dispose of organic composite wastewater.
6. A microorganism Pseudomonas sp. CJ-F31 which is well grown under aerobic condition and is able to dispose of organic composite wastewater.
7. A microorganism Erwinia sp. C J-D 17 which is well grown under aerobic condition and is able to dispose of organic composite wastewater.
8. A microorganism Cellulomonas sp. CJ-G22 which is well grown under aerobic condition and is able to dispose of organic composite wastewater.
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