WO 00/26341 PCT/KR99/00652 A MIXED MICROBIAL POPULATION DESIGNATED AS JSB 98.0 CAPABLE OF DEGRADING FECAL MATERIAL OF ANIMALS TECHNICAL FIELD 5 The present invention relates to a mixed microbial population, a method intended primarily for the biodegradation of fecal material of animals by using it, particularly a biodegradation purification method, and the use of the treated waste water as a liquid manure for plants. 10 BACKGROUND ART The present invention is directed to the mixture of microorganisms capable of decomposing livestock and poultry waste materials, including human 15 feces into materials which are more environmentally acceptable and thereby also eliminating odors associated with the decay of them by contacting said materials with said microorganisms, preferably microorganisms in aqueous solution. The amounts of waste materials discharged into bodies of water around the world are rising steadily. Increases in population, the dependence of agriculture 20 on massive amounts of fertilizers and pesticides, expansion of the food industry, and growth of other industrial processes all contribute to the volume of sewage and wastewater and to their content of undesirable substances. Most organic waste materials may be degraded by biological treatment. This treatment utilizes the functions of microorganisms to degrade the harmful substances to make them 25 harmless to the environment. The use of microorganisms for the treatment of waste water is an economical alternative to physical treatment systems since biological treatment involves lower capital investment, lower energy requirements, a self-sustaining operation, and finally possibility for product recovery. The biological process may be achieved aerobically or anaerobically in a number of 30 ways. The most widely used aerobic processes are activated sludge processes and their modifications for both domestic and industrial wastewaters. The WO 00/26341 PCT/KR99/00652 2 process consists of aerating and agitating the effluent in the presence of a flocculated suspension of microorganisms on particulated organic water-the activated sludge and thereby gives BOD or COD reduction depending on the nature of the wastewater. In the aerobic phase of the treatment process, a mixed 5 microbial population degrades the complex mixture of organic compounds in liguid waste to carbonate, nitrate, ammonia, phosphate, and sulfate. Major studies on microorganisms which decompose organic sewage and offensive odorous substance are conducted on the sewage treatment by activating sludge method. 10 In recent years, animal industries become specialized and large scale. It is well known that there exists a great abundance of livestock feedlot and poultry excreta which pollutes not only the streams and livers with toxic compounds and pathogenic microorganisms but also the air with offensive odors. Disposal of the excreta is a serious problem which has confronted in various ways but which never 15 has been satisfactorily solved. Countermeasures therefore are strongly needed to prevent the escalation of environmental pollution caused by excreta and recover the polluted environment by them. There is an approach to purify the polluted environment utilizing microorganisms of degradation ability. But, none of the presently known bacteria can satisfy the practical requirements and also high 20 degradation performance when used for microbial excreta degradation. DISCLOSURE OF THE INVENTION In view of the aforementioned prior art, it should be apparent that there 25 exists a need in the art for reliable microorganisms having a excreta degrading power and more advantageous practical characteristics in comparison with the conventionally known strains. In other words, in case of insufficient sewage treatment(particularly in farming places or rural areas), the water is contaminated with fecal mater and pathogenic microorganisms and it will become an extremely 30 serious social problem. At present, in accordance with the present invention, a mixed microbial population isolated from pig houses and designated as JSB 98.0 WO 00/26341 PCT/KR99/00652 3 which consists of 5 active strains having the powerful capacity to decompose fecal waste materials is disclosed. It can be used in the activated sludge process for sewage treatment as powerful agents. Accordingly, it is an object of the present invention to provide a mixed 5 microbial popupation designated as JSB 98.0 having the powerful ability to degrade and deodorize livestock and poultry waste materials, including human feces. It is another object of the invention to provide a method for treating fecal matter of animals in sewage or waste water by utilizing the mixed microbial 10 population of the present invention, JSB 98.0. It is yet another object of the invention to provide a method for deodorize offensive odors from places containing excreta by utilizing the mixed microbial population of the present invention, JSB 98.0. It is further another object of the invention to provide decomposed and 15 deodorized byproduct which is suitable as a liquid manure for plants. The above-mentioned objects can be achieved by the following present invention. The first aspect of the present invention is a mixed microbial population designated as JSB 98.0(accession number in Korean Collection for Type 20 Culture(KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), No. KCTC 0524BP). The second aspect of the present invention is a biological treating method for a medium polluted with excreta, which comprises the degaradation step of excreta by bringing JSM 98.0 into contact with the medium polluted with excreta. 25 The third aspect of the present invention is the use of the treated wastes as a liquid manure for plants. Other objects and advantages will become apparent to those skilled in the art from the following description and appended claims.
WO 00/26341 PCT/KR99/00652 4 BEST MODES FOR CARRYING OUT THE INVENTION The inventors of the present invention screened microorganisms capable of degrading excreta and from the wastewater polluted with excreta from porcine 5 houses a powerful microbial population was obtained which can degrade excreta of high concentration. Thus, a method was found applying this mixed microbial population to the environment containing excreta. As described in the examples given below, the isolates has a feature that it maintains relatively high activity to degrade excreta even at a low temperatures 10 such as about 15 C. In general, the temperature of waste water to be treated is in the range of 10-20 C which is lower than usual optimum growth temperatures of microorganisms. Also to the microorganisms of the present invention is lower than its optimum growth temperature, but it can maintain the activity to degrade excreta sufficient for practical use. In addition, as described hereinafter, the 15 isolated microbial population can also deodorize the offensive odors associated with the decay of excreta when treated to the animal houses, or levatories. The method for treating excreta comprises bringing the above-mentioned microbial mixtures, JSB 98.0 into contact with these excreta in an aqueous medium such as waste water. The contact of microorganism with these excreta in 20 an aqueous medium is accomplished by culturing the microorganism in the aqueous medium containing excreta or by adding the aqueous medium to the culture system of the microorganism, in a batch mode, a semicontinuous mode or a continuous mode. Although no particular restriction is put in the technique for the gas 25 instruction, it is preferable to agitate the culture medium by introduction of the gas to accelerate aeration. The introduction and exhaust of the gas may be continuously carried out, intermittently or batch-wise in compliance with a treatment performance. These conditions are preferably optimized by controlling the system in accordance with the concentration of the remaing waste materials. 30 The fecal material of animals or excreta is treated, in accordance with the present invention, with a mixed microbial population designated as JSB98.0 to WO 00/26341 PCT/KR99/00652 5 form desired liquid manure. The excreta may be either fresh or aged matter obtained from animal, particularly livestock and poultry production waste, including human. For example, fecal matter of animals, such as cows, pigs, horses and the like, can be degraded and deodorized in a liquid by JSB 98.0. 5 The mixed microbial population of the present invention, JSB 98.0, can be applied to a biodegradation purification process and environment remediation method which are useful for purification of waste water pulluted with excreta. It can be also useful for the remediation of soil polluted with them. In one embodiment of the present invention, the microorganisms of the 10 present invention, JSB 98.0 was used as a deodorant. In another embodiment of the present invention, the treated wastewater is used as a liquid manure for plants. Since the treated wastes are substantially odor free, non-air polluting and aesthetically acceptable, they are suitable for use as an inexpensive source of plant nutrients and soil conditioner for the production of 15 flowers, lawns, shrubs, field crops, pastures and the like. In accordance with the aforesaid objects the present invention comprises (a)isolating microbial strains from sources and identifying the potential strains which have the powerful ability to decompose the fecal material of animals (b)treating said fecal materials(or waste water polluted with them) with said 20 isolates (c)Assessing the treated waste water (d)controlling and eliminating the offensive odors from places containing said fecal materials with said isolates and (e)using the said treated waste water as a liguid manure for plants. The processes of isolation, characterization and utilization of the microorganisms of the present invention is illustrated in the examples which 25 follow. Following examples, however are not to be construed as limiting. EXAMPLE 1 Isolation and identification of microorganisms 30 Microorganisms have been isolated from the porcine houses. Among all the isolates, 5 active and predominant strains were identified as Corynebacterium WO 00/26341 PCT/KR99/00652 6 glutamicum H0777, Micrococcus kristinae H0778, Paracoccus denitrificans H0779, Corynebacteriuni glutamicum H0780 and Bacillus mycoides H0781, respectively. On the basis of the results of identification study of the microorganism, the present inventors designated the mixed microbial population 5 of the 5 active strains as JSB 98.0. It was deposited in the Korean Collection for Type Culture(KCTC), placed in Korea Research Institute of Bioscience and Biotechnology(KRIBB), #52, Oun-dong, Yusong-ku, Taejon 305-333, Republic of Korea, on the date of Sep. 28, 1998 and it was accepted under the accession number of KCTC 0524BP. 10 EXPERIMENT 1 Measurement of cell density One ml. of inoculent obtained from samples was added to agar count 15 plates supplied with wastewater from porcine houses for counting bacteria and humic acid plates for actinomycetes, respectively. After inoculation, the plates were placed in room temperature for 48 hours and cell density measurements were taken by serial dilution method. It was found that the total number of bacteria was 1.12 X 10' CFU/mL and that of of actinomycetes was 7.1 X 10' CFU/mL. 20 EXPERIMENT 2 Isolation and pure culture of predominant strains More than 13 strains formed colonies on the agar count plates. The total 25 number of bacterial cells on the plates was measured and 5 bacterial strains which revealed 10% of the total microbial population in number were selected for further study. The selected colonies are as follows. Strain 1 microcolony with cream colony, 20% 30 Strain 2 white and sticky colony, 18% Strain 3 yellow colony, 14% WO 00/26341 PCT/KR99/00652 7 Strain 4 plane brownish colony, 11% Strain 5 rhizoid from colony, 10% EXPERIMENT 3 5 Characterization of bacteria Methods of microorganism identification followed standard procedures as outlined in Bergy's Manual and the manual of Methods for general bacteriology. And the following findings are obtained. 10 Strain I This bacteria was designated as strain H0778 (1) Morphological properties Gram stain : positive 15 Shape and size of cell : spherical, 0.5-1.1 p m in diameter mostly 4 cells in clusters or irregular clusters Colony : round, entire, convex, smooth, pale creamy in color (2) Physiological properties Catalase positive 20 Oxidase positive Asimilation test : L-arabinose, D-ribose, D-xylose, acetic acid, methyl pyruvate, mono-methyl succinate, propionic acid, putrescine (Biolog GP) (3) Fatty acids composition in cell membrane C15:0 anteiso 58.35% 25 C17:0 anteiso 26.04% C16:0 iso 13.08% C16:0 2.53% (4) Identification The above bacteriological properties were compared with those of known 30 strains with reference to Bergy's Manual of Systematic Bacteriology, Volume(2), 1986. As a result, based on the above properties, it was revealed that the WO 00/26341 PCT/KR99/00652 8 microorganism was identified with a microorganism of the species of Micrococcus kristinae (confidence level 98%) Strain 2 5 This bacteria was designated as strain H0780 (1) Morphological properties Gram stain : positive Shape and size of cell : short rod or elipsoid, 0.7-1.0 X 1.0-3.0 , m mostly elipsoidal sphere during late phase of growth, 10 single, pairs or irregular clusters Colony : round, entire, white to pale yellow in color, slightly shiny in apperance Nonsporing, nonmotile, metachromatic granules formation (2) Physiological properties 15 Catalase : positive Urease production : positive Casein decomposition negative Gelatin decomposition negative Nitrate reduction 20 Asimilation test : D-ribose, D-xylose, acetic acid, propionic acid (Biolog GP) (3) Fatty acids composition in cell membrane C18:1 w9c 65.55% C16:0 26.61% 25 C17:0 6.32% C18:0 1.52% major menaquinone MK-9 (H 2 ) (4) Identification The above taxonomical findings are referred to "Bergy's Manual of 30 Systematic Baceriology, 8th Ed., Vol2" and the present strain H0780 has found to have similar taxonomic properties of the strain Corynebacterium glutamicum WO 00/26341 PCT/KR99/00652 9 (confidence level 85%) Strain 3 This bacteria was designated as strain H0777 5 (1) Morphological properties Gram stain : positive Shape and size of cell : rod or elipsoidal sphere, 0.7-1.0 X 1.0-3.0 p m single, pairs or irregular clusters 10 short and mostly rod during late phase of growth Colony : round, entire, smooth, deep yellow in color, slightly shiny in apperance Nonsporing, monmotile, metachromatic granules formation (2) Physiological properties 15 Catalase : positive Urease : positive Nitrate reduction : positive Casein decomposition negative Gelatin decomposition negative 20 Asimilation test : 8 -cyclodextrin, cellobiose, D-fructose, a -D-glucose, maltotriose, D-mannose, 3-methyl glucose, palatinose, D-psicose, D-ribose, sucrose, turanose, D-xylose, acetic acid, L-malic acid, L-asparagine, L-glutamic acid, uridine(Biolog GP) 25 (3) Fatty acids composition in cell membrane C18:1 w9c 57.46% C16:0 38.01% C18:0 1.45% C15:0 1.06% 30 major menaquinone MK-9(H 2 ) (4) Identification WO 00/26341 PCT/KR99/00652 10 The strain was identified as Corynebacteriuni glutamicum (confidence level 99%) Strain 4 5 This bacteria was designated as strain H0779 (1) Morphological properties Gram stain : negative Size and shape of cell : sphere, 0.5-0.9 [p m in diameter or short rod, 0.9-1.2 ,u m in length 10 single, pairs or clusters Colony : mostly round, entire, smooth, shiny, white to pale brown Nonmotile (2) Physiological properties Catalase positive 15 Oxidase positive Nitric acid reduction in anaerobic condition : positive(nitrogen oxides or nitrogen) Gelatin decomposition : negative Starch hydrolysis : negative 20 Asimilation test : L-ribose, D-ribose, D-xylose, 8 -hydroxybutyric acid, D-lactic acid methyl ester, L-lactic acid, methyl pyruvate, mono-methyl succinate (3) Compositions of fatty acids in cell membranes C18:1 w7/w9t/wl2t 82.98% 25 C10:0 30H 5.93% C16:1 iso I/C14:0 30H 3.29% C18:0 2.50% C20:1 w9t 1.65% C17:0 1.30% 30 C19:0 1.27% (4) Identification WO 00/26341 PCT/KR99/00652 1I The strain was identified as Paracoccus denitrificans (confidence level 85%) Strain 5 5 This bacteria was designated as strain H0781 (1) Morphological properties Gram stain : positive Size and shape of cell : rod in chains Colony : rhizoid, frosted glass apperance 10 Spore : elipsoidal , central (2) Physiological properties Catalase : positive Anaerobic growth : positive VP test : negative 15 Acid formation : D-glucose(+), L-arabinose(-), D-xylose, D-mannitol Casein decomposition : positive Starch hydrolysis : positive Propionic acid utilization : negative 20 Citric acid utilization positive Nitric acid reduction positive Indole formation : negative Growth temperature : 301C(+), 37"C(+), 50"C(-), 551C(-) Growth pH : 6.8 in nutrient broth, 5.7 in SDA 25 NaCl growth : 2%(+), 5%(+), 7%(-), 10%(-) Asimilation test : L-arabinose, D-arabitol, D-ribose, D-alanine, adenosine, adenosine-5'-monophosphate(Biolog GP) (3) Compositions of fatty acids in cell membranes C15:0 iso 20.71% 30 C15:0 iso 20H/C16:1 w7c 14.98% C13:0 iso 11.25% WO 00/26341 PCT/KR99/00652 12 C17:0 iso 8.64% C12:0 iso 6.24% C14:0 6.02% Iso C17:1 w5c 5.16% 5 (4) Identification The strain was identified as Bacillus mycoides (confidence level 90%) EXAMPLE 2 10 Treatment of the feedstock waste materials Feedlot waste from a cattle feedlot was collected in an ordinary waste water treating tank and JSB 98.0 was inoculated to determine the presence of microbial activity. The aeration time was maintained 18 hours/day and the 15 dissolved oxygen concentration was maintained as high as about 2ppm. This example was performed three times repeatedly from June 28 to July 6, 1998. The result is shown in table 1. Table 1 20 Assessment of the treated waste water Items BOD SS Test result (mg/L) 24.0 52.5 Assessment Passed Passed It was concluded that JSB 98.0 of the present invention was highly effective to decrease BOD level. 25 EXAMPLE 3 Analysis of the treated wastewater Following the same procedure as in Example 2, the quality of the treated WO 00/26341 PCT/KR99/00652 13 wastewater and the compositions of mineral elements were anaylsed. The results are shown table 2 and 3. Table 2 5 Qualitative and quantitative analysis of the treated waste water Nitrite Nitrate Total Fe pH E.coli Other hardness bacteria Standard (drinking ND >0ppm >300ppm >0.3ppm 5.8- ND/500cc >100/ water) 8.0 lcc Treated waste 0.07 0.12 129.56 0.14 7.87 3,500 370 water * ND = not detectable Table 3 Composition of mineral elements in the treated waste water 10 Mineral elements Contents (ppm) Mineral elements Contents (ppm) Na 314 Ca 29.5 K 710 Fe 0.4 Mg 29.9 Cu 0.5 P 27.1 Zn 0.2 * Mn, Al, Cr, Cd, Pb, As. Ig were not detected in samples It was shown that the treated waster water was acceptable as drinking water in chemical properties, but the number of bacteria was more than that of standard drinking water. Thus, in accordance with the foregoing, the present 15 invention has provided a method for the treatment of livestock and poultry waste for the purpose of assisting in the abatement and management of water pollution caused by those animal excreta materials. By virtue of the hereindescribed JSB 98.0, the chemical properties of the waste water polluted with excreta have been significantly improved.
WO 00/26341 PCT/KR99/00652 14 EXAMPLE 4 Use of of the treated wastewater (1) Deodorization of feces by degradation in the use of the treated waste 5 water has been performed. One liter of the treated wastewater was sprayed to pig houses, cow houses and lavatory three times a day, respectively. Deodorized effect was observed and it was found that odor was barely sensible. That is, JSB 98.0 decomposed odorous substances very strongly from the offensive odor sources. 10 (2) To investigate the use of the treated waste water as a liqud manure, the treated wastewater(diluted with underground water to 50-100 times) was spayed with spring cooler to vegetable gardens(red pepper) and lawns(turfgrass) 3 times a day, respectively. The controls were sprayed with underground water. The 15 results are shown in table 4 and 5. Table 4 Cultivation test of red pepper using the treated waste water Control 50X 100X 150 X dilution dilution dilution Photosynthesis rate 23.7 25.7 24.8 24.2 (Y mol CO 2 -2S-1) Chloroplast content (mg/g) 2.10 2.78 2.92 2.73 Stroma conductance (cm/sec) 3.24 4.54 4.72 4.48 Length growth (cm) 70.9 90.5 88.5 87.7 Total products (number) 34.7 48.4 47.2 45.8 20 Results in table 4 indicated that the treated wastewater according to the present invention improved significantly stroma conductance, length growth and total products of the red pepper compared to the control.
WO 00/26341 PCT/KR99/00652 15 Table 5 Cultivation test of turfgrass using the treated waste water Control 50X 10OX 150 X dilution dilution dilution Photosynthesis rate 5.22 4.30 9.28 8.82 (1 pmol C02,-2S-1) Stroma conductance (cm/s) 0.30 0.82 0.73 0.68 Transpiration rate 4.8 10.8 9.72 8.92 (mg H 2 0/m 2 /sec) 5 It was found that the treated waste water improved photosynthesis rate very highly compared to control. INDUSTRIAL APPLICABILITY 10 As clearly shown in the above, novel mixed microbial population of the present invention designated as JSB 98.0 which can degrade fecal material of animals, including humans enables the biological degradation of these materials utilizing its growth chacteristics different from those of conventional single species, thus efficient biological treatment of waste water containing these 15 materials. While the present invention has been described with reference to particular embodiments, it will be understood that the various modifications may be made by those skilled in the art without actually departing from the scope of the invention therein. According, all modifications and equivalents may be resorted 20 to which fall within the true spirits and scope of the invention, as claimed.
WO 00/26341 PCT/KR99/00652 16 SIMhAPESTY TREAf ONMD Ar10MAM0CNMQX~ OFUDPMr OPIU(RODROj4S POR TPUUM OF PA1XNTPXOcM1J INTERNATIONAL PORM RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule7.1 TO: LEE, Sang Whee Jin-Joo mansion Apt. 2-501, #5-22 Moonrae-dong, Youngdeungpo-ku, Seoul 150-095, Republic of Korea L IDENTIFICATION OF TE MICROORGANISM Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY: JSB-98.0 KOTO 0524BP fl. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The icroorganism identified under I above was accompanied by: [x I a scientific description I I a proposed taxonomic designation (Mark with a cross where applicable) 111 RECEIPT AND ACCEPTANCE This International Depositary Authority accepts the microorganism identified under I above, which was received by it on September 28 1998 IV. RECEIPT OF REQUEST FOR CONVERSION The microorganism identified under I above was received by this International Depositary Authority on and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on V. INTERNATIONAL DEPOSITARY AUTHORITY Name: K orean Collection for Tg pe Culturec Signature(s)ofpcrson(s) having tpowcw to represent the International Depositary Authority or of authorized of ficial(s): Address: Korea Research Institute of Bioscience and Biotechnology (KRIBB) #52, Oun-dong. Yusong-ku. Taejon 305-333. PARK YongHa, Director Republic of Korea Date: October 09 1998 Form BP14 (KCTCForm 17) saepage