TWI377249B - Microbial reagents for scavenging dioxin pollutants present in a contaminated medium and methods of using the same - Google Patents

Description

1377249 Patent Application No. 096,146,052, the entire disclosure of the entire disclosure of the entire disclosure of the entire disclosure of the entire disclosure of the disclosure of the disclosure of Microbial reagents present in dioxins, dioxin-like compounds, and/or polycyclic aromatic hydrocarbons in a contaminated medium It contains a storage number (accessi〇n number) BCRC 910356 deposited at the Food Industry Development Institute's Bioresource Conservation and Research Center (BCRC of FIRDI) Pseudomonas sinensis CPseMi/owowiis mew<Ioci>ia) NSYSU. The microbial agent has the ability to degrading the following environmental pollutants: dioxin, dioxin, and polycyclic aromatic hydrocarbons. The invention also relates to methods and apparatus for using such microbial agents to remove such environmental contaminants present in a contaminated medium. t Prior Art 3 Background of the Invention Due to the excessive development of civilization, many tangible or intangible environmental pollutants, including dioxin and dioxins, polycyclic aromatic carbonitrides (PAHs) and chlorinated phenols, etc. In this way, among the environmental pollutants, dioxins are considered to be the most toxic chemicals and chemicals for humans and animals. In the past two decades of research, it has been found that Dioxin does not affect the body's fertility, causes developmental disorders, damages the immune system, and interferes with the supplement of the patent application of No. 5,377,249, 096, 146, 052. : In December 1999, it was often endocrine and even carcinogenic (DB McGregor βία/. (1998), Pereeci., Apr; 106 Suppl 2: 755-760). However, dioxin is a substance that is not easily decomposed, and it will continue to accumulate and exist in nature and living organisms, forming a serious potential hazard to the entire environment. In particular, because Dioxin is very stable in the general state, even in a hot, acid or alkaline environment, when it is released into the environment and enters the food chain, it will become more and more concentrated as the food chain climbs. high. The chemical structure of Dioxin is similar to that of human hormones (EL 10 Gregoraszczuk (2002), Cad. Saude. Publica., 18(2): 453-462), so when it enters the human body via the food chain, The aspect will form a "false hormone" and produce a hormone-like effect. On the other hand, it will affect the hormone content in the body, both of which will interfere with the body's original endocrine mechanism and seriously affect the human body. Metabolism and interference balance the balance of the hormone, which in turn leads to physical damage. In addition, 'Daysin is also proven to be a powerful tumor promoter' that causes carcinogenicity in animals, including increased incidence of liver cancer, lung tumors, and skin tumors in rats. The US Environmental Protection Agency (US-EPA) and the World Health Organization (WHO) classify Dioxin as a possible human carcinogen, while the International Agency for Research on Cancer (IARC) has been the most toxic in 1997. 3,7,8-tetrachlorodiphenyl-p-dioxin (2,3,7,8-TCDD) is classified as "a human-derived carcinogen" (DB McGregor ei α/. (1998), same as above). 6 1377249 Patent Application No. 096146052 Supplementary, Amendment of Unlined Instructions Replacement Page Revision Date: 99 years π month Dioxin is a collective term for a group of compounds formed by the joining of one or two oxygen atoms to two benzene rings. Such compounds are among the class of halogenated aromatic hydrocarbons (HAHs). Dioxin can be divided into two series of coplanar tricyclic compounds, respectively: 5: (1) polychlorinated dibenzo-p-dioxins (PCDDs) 'The compounds have a chemical formula of C12H8-n02Cln, where n =1~8; and (2) polychlorinated dibenzofurans (PCDFs) having a chemical formula of C^Hs.nOCln 'where n=l~8. When the eight substitution sites of the phenyl ring are attached to different numbers of chlorine atoms, PCDDs have 75 isomers, while PCDFs have 135 isomers. Among the 21 species of Dioxin isomers, the toxicity of 2,3,7,8-TCDD is the strongest, so it is also called "century poison". PCDDs and PCDFs are aromatic compounds with almost planar structure. 15 These two compounds have similar physical properties and chemical properties, and also have a very stable organic molecular structure' and have a south melting point, a south boiling point, lipophilic and not easy. Decomposition characteristics, so the presence of such compounds in various environmental media (such as air 'soil, water, and food) (w· Parzefall (2002), Food 7b; dco/., 20 40 (8) ): 1185-9). In addition, Dioxin has a property of being slightly soluble in most organic solvents and poorly soluble in water. The solubility in water at 25 is about 7.4χ1 (Τ8 to 4.2xl0·4 mg/L, and the solubility will be Decreased as the number of gas atoms on the benzene ring increases. In addition to being exposed to isooctane and ultraviolet light, its chemical properties are changed, and Dioxin is stable for heat, acid, and alkali. Patent application No. 197, 146, 249, 146, 146, 052 Case Supplement, Correction of Unlined Manual Replacement Page Revision Date: December 1999 is highly qualitative, so it is difficult to be decomposed after being produced in nature, and will continue to accumulate in nature. Dioxin compounds (dioxin) -like compounds are a group of compounds with similar chemical and physicochemical properties to 2,3,7,8-TCDD, including: polychlorinated biphenyls (PCBs), Polybrominated diphenyl ethers (PBDEs), polybrominated dibenzo-/?-dioxins (PBDDs), and polybrominated dibeni Ofurans, PBDFs, etc. Dior 10 octyl compounds have good thermal stability, high lipophilicity and non-decomposable properties, so these compounds will continue to accumulate and exist in the natural environment, which will cause serious damage to the organism. Polychlorinated biphenyls (PCBs) are a general term for a group of compounds formed by the joining of two benzene rings by a covalent bond. Such compounds have a chemical formula of 15 Ci2Hi〇-nCln, where n=l ~10. PCBs can be further divided into two types, planar and non-planar. PCBs have a total of 209 isomers depending on the number and position of chlorine atoms attached to the benzene ring. PCBs have been found to be potentially carcinogenic, so the United Nations Environment Programme (UNEP) 20 has classified it as a priority for Persistent Organic Pollutants (POPs). Polybrominated diphenyl ethers (PBDEs) are a collective term for a group of compounds in which only one oxygen atom is bonded between two stupid rings. The formula is C^Hio.nBrnO, where n=l~10. According to the bromine atom attached to the benzene ring 8 1377249 Patent application No. 096146052, the amendments are amended, the instructions are replaced, the correction date is: December, 1999 Depending on the number and location, PBDEs have a total of 209 isomers. Studies have shown that PBDEs interfere with the secretion of thyroid hormone and cause neurotoxicity and carcinogenicity in organisms (T.A. McDonald (2002), Chemosphere, 46: 745-155). 5 PBDDs and PBDFs are collectively referred to as brominated dioxins because of their similar chemical structure to PCDDs/PCDFs. Depending on the number and position of the bromine atoms attached to the phenyl ring, PBDDs have 75 isomers, while PBDFs have 135 isomers. PBDDs/PBDFs have molecular weights higher than 10 PCDDs/PCDFs and have high melting point, low vapor pressure and low water solubility. The physicochemical properties, persistence and toxicity of PBDDs/PBDFs are similar to those of PCDDs/PCDFs, and therefore cause serious harm to the natural environment and humans. Polycyclic aromatic hydrocarbons (PAHs) are a general term for a group of compounds formed by the joining of two or more benzene 15 rings, which can be further classified into low molecular weight (LMW) depending on the number of benzene rings. There are two broad categories of PAHs (having 2 to 3 benzene rings) and high molecular weight (HMW) PAHs (having 4 or more benzene rings). PAHs have a higher n-octanol/water partition coefficient (n-octanol/water partition 20 coefficient, (in particular, the inverse value increases with the number of benzene rings), and is a hydrophobic organic compound (hydrophobic organic compound). Compounds), therefore only soluble in non-polar or low-polar organic solvents. In addition, PAHs have high melting point, high boiling point, and the melting point and boiling point increase with the number of benzene rings, so it will continue Accumulation 9 1377249 No. 0% 146〇52 Patent Application Supplement, Amendment of Unlined Manual Replacement Page Revision Period: December 1999 and exist in nature and living organisms. So far, more than 30 PAHs have been Syndrome is highly carcinogenic' and thus makes it the largest group of known carcinogenic compounds. Chlorinated phenols are usually highly toxic and are one of the most widely distributed 5 garments/losses. Pentachlorophenol (PCP) is one of the most stable and harmful of phenols. Pentachlorophenol (PCP) is a kind of hydroxyl group and five gas atoms attached to it. An aromatic compound (ar〇matic compound) with a molecular weight of 266.35, a melting point of 190 ° C, and a solubility of 14 mg/L in 10 water at 26.7 ° C. Pentaphenol in nature It is difficult to be decomposed and has the risk of carcinogenesis. Its minimum lethal dose (LDl.) for humans is 29 mg/kg. Therefore, the production and use of pentaphenol in the world has been strictly controlled. 0 Because environmental pollutants are serious The land threatens human health and causes damage to the environment. Therefore, how to effectively deal with environmental pollutants has become the focus of attention and research in the world. The current known treatment methods for environmental pollutants include: solidification (solidification) ), rem〇ving method, incineration, activated carbon adsorption, catalytic reduction, photolysis, and biological rejuvenation (bioremediation), etc. Bioremediation is the use of microbial biodegradative activities to remove environmental pollutants and difficult to decompose aliens. Recalcitrant xenobiotics, and in all bioremediation techniques, biological additions 10 1377249 096146052 Patent application supplement, amendments to the unlined instructions Replacement page Revision date: December 1999 (bioaugmentation) ( It is particularly important for adding bacteria to a contaminated environment to enhance the degradation of contaminants, because in the case where indigenous microorganisms cannot degrade the toxins that are difficult to decompose, it may be the only achievable The method of the purpose of biological reproduction. 5 Bioremediation has the advantages of low cost, no secondary to harmful toxic by-products in the process of degrading environmental pollutants, and the ability to operate in situ (with μ). Therefore, the biological re-cultivation method has been widely applied to the remediation of contaminated sites, and the separation and screening of microorganisms suitable for use in biological rehabilitative methods has become an extremely important research and development topic. In H. Habe et al. (2QQ1), Appl. Microbiol. J Biotechnol, 56: 788-795, H. Habe et al. developed a carbazole-utilizing bacterium [pseudomonas] The genus species ίρ·) strain C A10] is a method of biologically rejuvenating 15 soils contaminated by dioxin. Their experimental results show that the Pseudomonas strain CA10 has the potential to degrade Dyrosin with 4 to 7 chlorine atoms (including the most toxic 2,3,7,8-1^00). Afterwards, K. Maeda et al. confirmed that the strain was in the inverse of ;;^^(!&<3/.(2003),··· Mo/. 〇/., 326:21-33 Pseudomonas resin CPsewiiow (10) rehnovorimj·) strain CA10 〇20 in Η·Β. Hong ei 〇/_ (2004), 15: 303-313, Η.Β. Hong et al. by selective proliferation technique (selective enrichment) Techniques) A dioxin-degrading strain (Piewi/omomzs veram) PH-03 was isolated from the contaminated soil. The strain is grown in dibenzo-p-dioxin (DD) and the second is added to the patent application of the patent application No. 096146052. Dibenzofuran (DF) is the only carbon source. It can also metabolize 1-chlorodibenzo-p-dioxin, 2-chlorodibenzo-p-dioxobenzo -p-dioxin) and other dioxin 5 metabolites [eg salicylic acid and catechol] M in M. Bunge ei α/· (2003), iVaiwre, 421: 357-360, Μ.

The results of Bunge et al. showed that a chlorobenzene-dehalorespiring bacterium, 10 strains of CDe/za/ococcozWe®, can produce certain dioxins. [1,2,3-trichlorodibenzo-/?-dioxin, 1,2,3-TrCDD), 1,2,4-TrCDD, l,2,3,4-TeCDD and 1,2,3,7,8-pentachlorodibenzo-p-dioxin (l,2,3,7,8-pentachlorodibenzo-p-dioxin, 15 12,3 , 7,8-PeCDD), etc.] reductively degassing, thus the strain is potentially available for bioremediation of contaminated sites containing artificial PCDD (anthropogenic PCDD).

JP2002300873 discloses a strain of Pseudomonas menedema (FEM P-18187 isolated from an oil field and capable of degrading organochlorine aromatic compounds 2), and using the strain for organic aromatic A method for the degradation treatment of a compound compound, wherein the organic gas aromatic compound may be dioxin [eg, PCDDs, PCDFs, and COpianar polychlorinated biphenyls (Co-PCBs), etc.] 12 1377249 Patent No. 096146052 Case Supplement ' Amendment of the unlined manual replacement page revision period: December 1999 in Cai Xutang, National Institute of Biological Sciences, National Sun Yat-sen University, master's thesis [Name: "Characterization of bacteria Degrading pentachlorophenol)"] revealed a strain isolated from soil contaminated with pentaphenol and capable of using pentachlorophenol as a 5-carbon source. This strain was identified as a Mendoza Pseudomonas mendocina NSYSU. by Zong Zonglin, Department of Marine Environment and Engineering, National Sun Yat-Sen University Master's thesis [name: "Biodegradation of PAHs in Diesel 10 Fuel by Cam/ΰ/α Salty Environment"'] reveals that a strain has been domesticated from diesel for a long time. The Wjwawai/zz./ isolate from the mixed flora in the aerobic biofilter bed is green and degradable. This master's thesis also explores the degradation of polycyclic aromatic hydrocarbons (PAHs). The utility of the strain shows that the strain has the best degradation effect on naphthalene 15 and has poor degradation effect on anthracene, but has no degradation effect on fluoranthene. Therefore, the strain has no degradation effect. It is inferred that there is no ability to degrade PAHs with 4 or more aromatic rings. Although there have been reports of the above-mentioned literature and patents, there is still a need in the art to screen for degradable environmental pollution. Microorganisms (especially Dioxin) for environmental protection. After research, the applicant unexpectedly discovered a Daisin from Sinopec Anshun Plant (Tainan City, Taiwan). Bacterial isolate isolated from contaminated soil (it was later identified as a Pseudomonas cerevisiae NSYS resection 13 1377249 No. 096146052 patent application supplement, revised unlined instructions replacement page revision date: In addition to its ability to degrade pentachlorophenol in December 1999, it also has the ability to degrade environmental pollutants such as dioxin, dioxin and polycyclic aromatic hydrocarbons (pAHs). Therefore, this strain is expected to have great potential in the application of environmental pollution. SUMMARY OF THE INVENTION Accordingly, in a first aspect, the present invention provides a microbial agent for removing dioxin, dioxin, and/or polycyclic aromatic hydrocarbons present in a contaminated medium, It contains a Pseudomonas cerevisiae NS·, a biological resource conservation and research center (BCRC of FIRDI), which is registered with the registration number H). In a second aspect, the present invention provides a method for removing dioxin, dioxin, and/or polycyclic aromatic hydrocarbons present in a contaminated medium, comprising: using - a microorganism as described above Reagents to treat the contaminated medium, such that the dioxin, dioxin, and/or polycyclic aromatic hydrocarbons present in the contaminated medium break the NSYSU of Pseudomonas mendocensis in the microbial agent The above and other objects, features and advantages of the present invention will become apparent after the detailed (4) and the preferred embodiments of the present invention and the accompanying texts of the present invention. For the purpose of this specification, it will be clearly understood that the text "comprising" means "including but not limited to, the application of the patent application of Ref. No. 096146052, the replacement of the unlined specification page. Revision date: December 1999 (comprises) has a corresponding meaning. It is to be understood that if any of the previous publications is quoted here, the reference does not constitute an acknowledgement that in Taiwan or any other country the publication forms part of the common general knowledge of the art. . 5 Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art. In order to effectively deal with environmental pollutants (envir〇nmental pollutants) and to avoid serious damage to the environment and ecology, countries all over the world have invested in human and financial resources to find solutions. Among the current treatment methods for environmental pollution, the biological re-cultivation method has secondary pollution caused by low cost, no toxic by-products in the process of degrading environmental pollutants, and can be carried out in situ. It is widely used in the remediation of contaminated sites. In order to screen out microorganisms suitable for supply to the bioremediation method, 15 people were asked to study a bacterial isolate having the ability to degrade pentaphenol from the soil contaminated by Dioxin from Sinopec Anshun Plant. The bacterial isolate was identified as a Pseudomonas mendoc (sewi/omcmas menc/ociwa) NSYSU by preliminary test and characterization of 16S rDNA sequence analysis, and was deposited on July 6, 2007. The Bioresource Conservation and Research Center (BCRC of FIRDI) of the Food Industry 20 Industry Development Institute in Taiwan, with the registration number BCRC 910356. Applicants have found that Pseudomonas Mendocs NSYSU has the ability to degrade pentoxide, dioxins, and polycyclic aromatic hydrocarbons (PAHs) in addition to its ability to degrade pentoxide. The patent application No. 096146052, the patent application No. 096146052, the replacement page, the replacement page, the replacement page, the correction date: 99 years old, the bacterial isolate also has good mercury resistance, and can resist the presence in a polluted environment. The damage caused by mercury ions. Accordingly, the present invention provides a microbial agent for the removal of dioxin, dioxins and/or polycyclic aromatic hydrocarbons (pAHs) present in a contaminated medium, comprising a storage number BCRC 910356 Pseudomonas mendocina deposited in the Center for Bioresource Conservation and Research (BCRC of FIRDI) of the Food Industry Development Institute NSYSU ° According to the invention, the contaminated medium is a solid, liquid or gaseous 10 Environmental medium, and includes, but is not limited to, soil, sludge, sediment, groundwater, waste water, and exhaust. Preferably, the contaminated medium is selected from the group consisting of agricultural land (eg, field, orchard land, grazing grassland, etc.), drinking water source (eg, well water), fish culture pond, 15 factory Waste water, domestic sewage, and sludge from sewage treatment plants. As used herein, the term "degradation" means the metabolic decomposition of a compound into a less complex molecule. As used herein, the term "dioxin," means a group of compounds comprising polychlorinated dibenzo-p-dioxins (PCDDs) 20 and polychlorinated dibenzofurans (PCDFs). The microbial agent according to the present invention can degrade a dioxin having 4 to 8 gas atoms. In a preferred embodiment of the present invention, the dioxin is selected from the group consisting of 2, 3, 7, 8 - Four gas dibenzo-P-Dioxin, 16 1377249 Patent application No. 096146052, supplementation, amendment, no-line instruction, replacement page, revision date: 99 years, 1, 2, 3, 7, 8-pentachlorodibenzopyrene - Dioxin, 2,3,4,7,8-pentachlorodiphenyl and dioxins, 1,2,3,4,7,8-hexachlorodibenzo-Ocein, 1,2,3,6,7,8 - six gas dibenzo--?-dioxin, 2,3,4,6,.7,8-hexachlorodibenzo-dioxin, 1,2,3,7,8,9-hexachlorodibenzo- -Dioxin, 1,2,3,4,6,7,8-heptachlorodibenzo-5-P-dioxin, 1,2,3,4,7,8,9-heptachlorodibenzo-Ocein, Octachlorodi-p-P-Dioxin, 2,3,7,8-tetra-dibenzofuran, ι, 2,3,7,8-penta-dibenzofuran 1,2,3,4,7,8-hexachlorodibenzofuran, iota, 2,3,6,7,8-hexachlorodibenzofuran, 1,2,3,7,8,9- Hexachlorodibenzofuran, 1,2,3,4,6,7,8-heptachlorodibenzofuran, octachlorodibenzofuran, and combinations thereof. More preferred examples of one of the present inventions 10 The dioxin is octadio-dibenzo-dioxin (OCDD) and/or octachlorodibenzofuran (〇CDF). According to the invention, the dioxin-like compound which can be degraded by the microbial agent is selected from the group consisting of Group: Brominated dioxin, polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs)', and combinations thereof. 15 As used herein, the term "brominated dioxin" means that a group contains more, / Compounds of P〇lybrominated dibenzo-p-dioxins (PBDDs) and p〇iybrominate (j dibenzofurans (PBDFs). The microbial reagent according to the present invention can degrade one containing 4 Brominated dioxin to 8 bromoprotoxins 2. In a preferred embodiment of the invention, the brominated dioxin is selected from the group consisting of 2,3,7,8-tetrabromo Benzo Osin, 1,2,3,7,8-pentabromodibenzo-Doxin, 8_hexabromodibenzo-Ocein, ..., Yiyi Liudi Dibenzoxene Dioxin, 〗 〖,), One benzopyrene, 1,2,3,7,8-five desert dibenzophenone. South, 2,3,4,7,8-five 17 1377249 No. 46052 Patent Application Supplement, Amendment of Unlined Manual Replacement Page Revision Period: December bromodibenzofuran, and combinations thereof . As used herein, the term 'polybromodiphenyl ether," means a group of compounds having from 1 to 10 bromine atoms attached to a diphenyl ether. The microbial reagent according to the present invention can degrade a polybrominated diphenyl ether containing 2 to 10 bromine 5 atoms. In a preferred embodiment of the present invention, the polybrominated diphenyl ether is selected from the group consisting of 2,4-dibromodiphenyl ether, 4,4,_dibromodiphenyl ether, 2 , 2',4-tribromodiphenyl ether, 2,4,4,_tribromodiphenyl ether, 2,2',4,5'-tetrabromodiphenyl ether, 2,3',4',6 -tetrabromodiphenyl ether, 2,2,4,4'-tetrabromodiphenyl ether, 2,3,4,4,-tetrabromodiphenyl ether, 3,3,,4,4,·4 Bromodiphenyl ether, 10 2,2',4,4',6-pentabromodiphenyl ether, 2,3',4,4,6-pentabromodiphenyl ether, 2,2^4,4^ 5-pentaBDE, 2,2',3,4,4'-pentabromodiphenyl ether, 3,3·,4,4',5-pentabromo 2,2,2',4 ,4',5,6'-hexabromodiphenyl ether, 2,2,,4,4',5,5--hexabromodiphenyl ether' 2,2·,3,4,4',6- HexaBDE, 2,2,,3,4,4,6,-hexabromodiphenyl ether 2,2',3,4,4·,5·-hexabromodiphenyl ether, 2, 3,3,,4,4,,5-hexabromodiphenyl ether, 15 2'2',3,4,4',6,6'-heptabromodiphenyl ether, 2,2,3,4 ,4,,5,6-heptabromodiphenyl ether, 2'3,3',4,4',5|,6-heptabromodiphenyl ether, 2,2,3,3,,4, 4,6,6,-octaBDE, 2,2',3,4,4·,5,5' 6-octaBDE, 2,2,3,3,,4,4,5,6,-octabromodiphenyl ether, 2,2,3,3,,4,5,5, ,6,6,-nonabrominated diphenyl ether, 2,2,,3,3,,4,4,5,6,6,-nine/odorous alum, decabromodiphenyl ether, and their combination. >〇' In a more preferred embodiment of the present invention, the polybrominated diphenyl ether is selected from the group consisting of 2, 2, 4, 4, and 6-pentabromodiphenyl ether, 2,3,,4,4',6·pentabromodiphenyl ether, 2,2',4,4,5-pentabromodiphenyl ether, 2,2,3,4,4,-pentabromo Diphenyl ether, 3,3',4,4',5·pentabromodiphenyl ether, 2,2,,4,4·,5,6·-hexabromodiphenyl ether, 2·2', 4,4',5,5'-hexabromodiphenyl ether, 2,2',3,4,4,6-hexabromodiphenyl ether, 18 1377249 No. 46052 patent application for replenishment, correction without Line specification replacement page revision date: 99 years 2,2',3,4,4',6'-hexabromodiphenyl ether, 2,21,3 4,4,5, hexabromo 2 alum 2 3, 3, 4, 4, 5 - six deserts one test 'and their combinations. Month As used herein, the term "polystyrene biphenyl" means a group of compounds having a stack of (7) gas atoms attached to biphenyl.

10 15

20 The microbial agent according to the present invention can degrade a multi-gas biphenyl having 4 to 7 gas. In the preferred embodiment of the present invention, the polychlorination is selected from the group consisting of: 3,3,4,4, tetrachlorobiphenyl, 3,4,4,24 Biphenyl, 2,3,3,,4,4,-pentachlorobiphenyl, 2,3,4,4,,5_penta-biphenyl', · 2,3',4,4',5- Pentachlorobiphenyl, 2',3,4,4,5-pentachlorobiphenyl, 3,31,4,4,5-pentachlorobiphenyl, 2,3,3,,4,4,, 5-hexabenzene, 2,3,3,,4,4,,5,_ hexachlorobiphenyl, 2,3',4,4',5,5'-hexachlorobiphenyl, 3,3 ,, 4, 4i, 5, 5L six gas, stupid, 2,3,3,4,4',5,5'-heptachlorobiphenyl, and combinations thereof. In a more preferred embodiment of the present invention, the polychlorinated biphenyl is selected from the group consisting of 2,3,4,4,5-pentachlorobiphenyl, 231,4,41,5_ Pentachlorobiphenyl, 2',3,4,4',5_ five-gas biphenyl, 2,3,3,,4,4,,5·six gas, stupid, 2,3,3',4,4 ''5'-hexa-biphenyl, 2,3,,4,4,5,5,·hexachlorobiphenyl, 2,3,3,4,4',5,5'-seven-gas biphenyl 'And their combination. As used herein, the term "polycyclic aromatic hydrocarbon," means a group of compounds formed by the joining of two or more benzene rings. The microbial agent according to the present invention can degrade one containing 2 to 6 benzene rings. Polycyclic aromatic hydrocarbons. In a preferred embodiment of the invention, the polycyclic aromatic (tetra) hydrazine compound is selected from the group consisting of: lower naphthalene > ^(acenaphthylene). (aCenaphthene) > 〇 (flU〇rene), phenanthrene, anthracene, and alanidene 19 1377249 December fluoranthene, pyrene, benzo (4) onion [benzo (a) anthracene], chopsticks (chrySene), stupid (b) propadiene [benzo (b) fluoranthene], stupid ( k) benzo(k)fluoranthene, benzo(a)pyrene] 茚 and 5 0,2,3-0(1)芘[indeno(l,2, 3-cd)pyrene], dibenzo(a,h) trial [dibenzo(a,h)anthracene] 'benzo(g,hi) flower [benzo(g,h,i)perylene], and combinations thereof In a more preferred embodiment of the present invention, the polycyclic aromatic hydrocarbon is naphthalene. The microbial reagent according to the present invention may further comprise at least one microorganism capable of removing environmental pollutants. Microorganisms of environmental pollutants include, but are not limited to, microorganisms of the genus Pseudomonas, microorganisms of the genus Dehalobacter, micro-branches of the genus Candida, micro-hosts of the genus Rhodococcus, Microorganisms of the genus Aeromonas, 15 microorganisms of the genus Rhizobium, microorganisms of the genus Sphingomonas, microorganisms of the genus Arthrobacter, microorganisms of the genus Froth's Flavobacterium CP/ανο heart cier/wm Microorganisms and micro-branches of Bacillus'. In a preferred embodiment of the present invention, the microorganism capable of removing environmental pollutants is selected from the group consisting of: Pseudomonas CPjewc/owonas strain CA10, Pseudomonas vonii (corpse jewi/omo such as s verom7) PH-03, Dehalomyces species (Z) e/ia/ococcoiife strain CBDB1, Mendoza Pseudomonas mendocina FERM P-18187 ' Candida 20 1377249

10 15

Patent Application No. 096,146,052, Supplementary, Correction, Replacement of Unlined Instructions Replacement Date: December, December, and their combinations. The microbial agent according to the present invention may optionally contain nutrients which are beneficial for the growth of microorganisms such as glycerol, riboflavin 'casein', polyprotein gland (p〇lypept〇ne), meat Meat extract, soybean cake, yeast extract, cellulose 'glucose, com extract, whey powder 'starch, vitamins [such as thiamine (thiamine), biotin, nicotinic acid amide or calcium panthotenate, or containing enzymes such as amylase, protease or lipase . The microbial agent according to the present invention can be manufactured into a form suitable for use by techniques well known to those skilled in the art, including, but not limited to, culture solutions, suspensions, granules & powders. , recording: tablets, pills, capsules, slurries and the like. In addition, the microbial agent can also be immobilized on an insoluble support. The microbial agent according to the present invention may further comprise a biocompatible carrier. In a preferred embodiment of the invention, the Pseudomonas sp. Pseudomonas NSYSU in the microbial agent is entrapped in the biocompatible carrier. The biocompatible carrier includes, but is not limited to: silica gel, starch, agar, chitin, chitosan 'polyvinyl alcohol, algae Acid (aiginic acid), polyacrylamide, carrageenan 21 1377249 Patent application No. 096146052, amendment, amendment, no-line instructions, replacement page, revision period: December, December (carrageenan), agarose ( Agarose), gelatin, cellulose, cellulose acetate, dextran, and collagen. In another preferred embodiment of the invention, the Pseudomonas donovar NSYSU in the microbial agent is supported on the biocompatible carrier. The biocompatible carrier includes, but is not limited to, glass, ceramic, metal oxide 'activated carbon, kaolinite, bentonite, zeolite ( Zeolite), aluminum, anthracite, glutaraldehyde, 10 polyacrylic acid, p〇lyUrethane, polyvinyl chloride, ion exchange resin (i〇n exchange resin), epoxy resin, photosetting resin, polyester, and polystyrene. The microbial agent according to the present invention can also be manufactured to remove dioxin, dioxin, and/or polycyclic aromatic hydrocarbons in a teaching-contaminated medium by techniques well known to those skilled in the art. Bioreactor or device. For the manufacture of bioreactors, reference is made to, for example, US 5,279,963, US Pat. No. 5,258,303, US Pat. No. 5,552,051, US Pat. No. 5,494, 574, US Pat. No. 6, 030, 533, US PCT PCT PCT PCT PCT And T. Furuichi, (2007), Journal of Hazardous Materials, 148(3): 693-700. The present invention also provides a method for removing dioxin, dioxin, and/or polycyclic ring present in a contaminated medium. A method of aromatic hydrocarbons comprising: the use of a microbial reagent as described above for the treatment of the patent application No. 22 1377249, No. 096146052, the amendment of the unlined specification, the replacement page, the date of correction, the pollution date of December 1999 The medium, such that the dioxin, dioxin, and/or polycyclic aromatic hydrocarbons present in the contaminated medium are degraded and disappeared by Pseudomonas mendocs NSYSU in the microbial agent. In the method according to the invention, the microbial agent can be used in combination with at least one microbe selected from the group consisting of microorganisms capable of scavenging environmental pollutants, microorganisms of the genus Pseudomonas, microorganisms of the genus Dehalobacter , microorganisms of the genus Candida, microorganisms of the genus Rhodococcus, microorganisms of the genus Aeromonas, microorganisms of the genus Rhizobium, microorganisms of the genus Sphingomonas, microorganisms of the genus Arthrobacter, and Fusarium Microorganisms, microbes of the genus Flavobacterium and microorganisms of the genus Bacillus. Preferably, the microorganism capable of removing environmental pollutants is selected from the group consisting of Pseudomonas resin Pseudomonas strain CA10, Pseudomonas vonii ph_〇3, Dehalomyces species Strain CBDB1 'Pseudomonas Mendocs FERM P-18187, CW oil, and combinations thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT OF THE INVENTION The present invention will be further illustrated by the following examples, but it should be understood that these embodiments are for illustrative purposes only and should not be construed as The implementation of the restrictions. 20 Shiguan 1. Degradation of the ability of five (four) degradation _ strain separation and screening experimental materials: h The actual soil samples used in the experiment were taken from Sinopec Anshun Plant (Tainan City, Taiwan) by Dioxin pollution Soil. 2. A defined liquid medium for culturing the isolated microorganisms in the λ test. 23 1377249 Patent Application No. 096146052 Supplementary, Correction of the Unlined Manual Replacement Page Correction Period: December 148 (defined broth medium) The following table! The formula shown. Table 1. Formulations for limiting liquid medium. Ingredients: NH4C1 Na2HP04 * 12H20 KH2PO4 MgS04 · 7H20 CaCl2 trace metal solution* pentachlorophenol deionized water 0.5 g 3_2 g 0.8 g 0.2 g 0.01 g 1 mL Add to 1 L with the stem-seeding experiment. Use 2 1^ out of 3〇4? The 11 value is adjusted to 7.20~7.40. _ The method of preparing the trace metal solution is to mix 5 g FeS04.7H20, 4 g ZnS04 · 7H20, 0.2 g MnCI2-4H2OO.5 g CoCI2·6H2OO.lg NiCl· 6H20> 0.15 g H3B03 and 2.5 g EDTA with 1 L Ionic water. 3. The defined agar medium used to culture the isolated microorganisms in the experiment was prepared by adding the noble agar (Difco) (15 g/L) to the defined liquid medium. Got it. Isolation and Screening of Bacterial Isolates Degrading Pentaphenol: ίο 1. Add 1 gram of real soil samples to a culture flask containing 100 mL of defined liquid medium (containing l〇mg/L of pentaphenol) 'The resulting culture was then cultured in a constant temperature shaking incubator (3 ° C, 160 rpm) for 14 days. 2. Remove 1 mL of the culture obtained in step 1 and add it to a flask containing 15 mL of defined liquid medium (containing 20 mg/L of five gas), followed by a constant temperature shaking incubator. (3 〇. (:, 160 rpm) was cultured for 14 days. 3. Take 1 mL of the culture obtained in step 2 and repeat the operation of step 2, but use 30 mg/L instead. The limited liquid medium is cultured in 2 。. 24 1377249 Patent application No. 096146052 Supplementary, amended, unlined instructions Replacement page Revision date: December, 1999 4. Take 1 mL of the culture obtained in step 3 and weigh The operation of step 2 was repeated, but the culture was carried out by using a defined liquid medium containing 40 mg/L of pentachlorophenol. 5. The culture obtained in step 4 was subjected to a four-zone method (four-quadrant 5 streak method). The method was applied to a defined agar medium (containing 4 mg/L pentachlorophenol), and cultured in an incubator (30 ° C) for 1 day to obtain a bacterial separation having the ability to degrade pentaphenol. Strain 2. Example 2: Bacterial separation with pentaphenol degradation ability . Characterization To confirm the 10 bacterial isolates of species belongs to the above-obtained in Example 1, the following preliminary tests, and 16S rDNA sequence analysis A, preliminary tests: 15

This test was carried out in accordance with J. Cappuccino and N_Sherman (2007) Microbiology: A laboratory manual, 8th ed., Benjamin/ Cummings Science Publishing, California, including the following test items: Gram staining, type observation (morphological observations), motility (m〇tility), temperature growth s test (4 C, 42 C), phenylethyl alcohol agar (PEA) growth test, McConnex agar (McConkeyagar) growth Test, Oxidation-Fermentation test, glucose inoculation test, lactose vinegar test, sugarcane fermentation test, house powder hydrolysis test, citrate utilization test, phenylamine test, h2S Production test, N〇3_reduction test 'indole production test, methyl red-Voges Proskauer (MR-VP) test, urease activity test 'touch enzyme ( Catalase) activity test, oxidase (〇xidase) activity test 25 20 1377249 No. 096146052 Special application to the application, correction of the instructions without a line Replacement page Revision date: December 12, December and gelatin (gelatin) hydrolysis test and the like. Results: The preliminary test results of the isolates having the pentachlorophenol-degrading ability obtained in the above Example 1 are shown in Table 2 below. As can be seen from Table 2, the isolate is a Gram-negative bacillus, has mobility, grows well at 42 ° C, and can grow on the stupid ethyl alcohol agar and MacConkey agar. Both the citrate 'hapase and oxidase activity tests were positive. Based on the results obtained, the applicant initially judged that the bacterial isolate belongs to the Pseudomonas species (10) like W). Table 2. Preliminary test results of the bacterial isolate obtained in Example 1. Test item results Test item results Gram staining - Citrate utilization test + Type observation rod aniline test - Exercise + H2S production test - Growth in 4 ° C — 1 ^ 〇 3 _ reduction test + growth at 42 ° C + 吲哚 production test phenethyl alcohol agar growth test + methyl red test - MacKangkai agar growth test + Berbo two test oxidative - Fermentation test 0/- urease activity test-glucose fermentation test + (acid production) catalase activity test + lactose fermentation test oxidase activity test + sucrose fermentation test starch hydrolysis test a gelatin hydrolysis test - Note: + ' Positive reaction; one, negative reaction; sputum, oxidation. Β, 16S rDNA sequence analysis: For the bacterial isolate obtained in the above Example 1, the genomic DNA was extracted by the following procedure: 1.5 mL of the bacterial liquid [, < More than 〇·3 (〇d6〇〇)] was placed in a microcentrifuge tube (micr〇tube) and centrifuged at 12,000 rpm for 1 minute. After removing the supernatant, 'add 100 pLTE buffer to disperse the cells, then add 1〇〇26 Patent Application No. 096146052 to supplement, correct the instructions without the underline. Replacement page Revision date: December, 1999 μί Lysozyme (lySOZyme) (5 〇 mg/mL) was mixed well and then allowed to act in a 37 ° 〇 water bath for 30 minutes. Thereafter, 350 g of LTE buffer, 30 μM of 10% SDS, and 5 pL of proteinase K (20. mg/mL) were added and mixed well. The resulting mixture was placed at 56. (: 1 hour in a water bath) Then add 1 μμ 5 M NaCl solution and mix well. The resulting mixture was added to 650 cold phenol and subjected to intense shock, then centrifuged at 14,000 rpm. It took 5 minutes to partition the aqueous layer and the organic layer. After that, the upper supernatant was transferred to a new microcentrifuge tube, and the partition separation treatment was repeated several times until the microcentrifuge tube The white matter between the aqueous layer and the organic layer disappeared. Next, 600 pL of ice-cold chloroform/isoamylalcohol (IAA) (v/v = 24:1) was added to the last collected fraction. The supernatant was centrifuged in a microcentrifuge tube, and then centrifuged at 丨4, 〇〇〇 rpm for 5 minutes. After that, 500 μί of the supernatant was transferred.

I Into a new microcentrifuge tube, slowly add 1,000 pL of 95% ice ethanol and mix gently, then centrifuge at 14,000 rpm for 2 minutes. After removing the supernatant, the pellet was washed with 10 〇 70% ice ethanol, and then the pellet was air-dried at room temperature. Finally, 3 〇 to 50 μl of TE buffer and 2 μί RNAase (50 mg/mL) were added to disperse the pellet and placed in a 37 ° C water bath for 30 minutes, thereby obtaining the bacterial isolate. Genomic DNA. · Using the obtained genomic DNA as a template and using a set of 16S rRNA genes directed against bacteria to design a universal primer pair F1 and R1 with the nucleotide sequence shown below, 1377249 Patent Application No. 096146052 Supplementary, Correction of Unlined Instructions Replacement Page Correction Period: December, 1999 Polymerase Chain Reaction (PCR) using the operating conditions shown in Table 3 below: Forward F1 5'-attgaacgctggcggcaggc -3' (SEQ ID NO: 1) Reverse primer R1 5 5'-cccagtcatgaatcactccg-3' (SEQ ID NO: 2) Table 3. Reaction conditions of PCR Content volume (μι) Genomic DNA of five gas degrading strains (5 ng/pL) 10 Forward primer F1 (25 μΜ) 1 Reverse primer R1 (25 μΜ) 1 nucleotide (dNTPs) (50 mM) 2 『Reaction buffer (7bg reaction buffer) (l〇X 10 3"called DNA polymerase (7^DNAp〇lymerase) (2.5 U/uU 0.5 secondary deionized water (dd H20) 75.5 Operating conditions · Denaturing at 94 C for 30 seconds at 52 Primer annealing for 30 seconds at °C, The elongation was carried out at 72 ° C for 90 seconds, and a total of 30 cycles of icvcles were carried out. The amplified product obtained after the completion of the PCR was confirmed by aga-againe gel electrophoresis, and then purified and returned by QIAquick PCR Purification kit (Qiagen), and the consortium was entrusted to the consortium. The Corporate Biotechnology Development Center (Taipei, Taipei) conducted the sequencing of the recovered PCR amplification products, and the resulting sequencing results were analyzed by the Gene Blast software provided on the NCBI website. The results of 16S rDNA sequence analysis of the bacterial isolate 15 having the five-gas phenol degrading ability obtained in Example 1 are shown in Fig. ί. After comparison with the gene database on the NCBI website, the 16SrDNA sequence of the bacterial isolate (SEQ ID NO: 3) was found to be as high as 99〇/〇 between the 16S rDNA sequence of Pseudomonas mendocensis. The sameness. 28 1377249 Patent application No. 096146052, supplementation and correction of the unlined manual replacement page Correction period: December, 1999 Comprehensive characterization results, in the above embodiment! The bacterial isolate with the degradability of pentagas in the medium was identified as “Pseudomonas Mendoc NS-YSU” and was deposited in Hsinchu City, Taiwan on July 6, 2007. Bioresource Conservation and 5 Research Center (BCRC〇fFIRDI) of the Food Industry Development Institute, No. 331, with registration number BCRC 910356. Example 3. Mercury ion tolerance test of Pseudomonas mendocs NSYSU (Hg2+ tolerance test) The bacterial isolate obtained in the above Example 1 was carried out in a nutrient broth (Difco 003-01). (Sub-culture of Pseudomonas Mendoca 10 NSYSU) - After that, the aliquots of the bacterial culture (10 mL) were mixed separately to 10 mL with different concentrations [营养, 1〇, 20, 30, 40, 50, 60, 70, 100, 150, 200, 250, 300, 350 and 400 ppm (w/w) of HgCl2 nutrient broth medium, and oscillated at a constant temperature The culture was carried out in an incubator (30 ° C, 120 rpm) for 15 hours and 2 days. Thereafter, the growth of the bacterial isolate was observed with a naked eye and a microscope. From the observation of the naked eye, it was found that the nutrient broth containing 0~60 ppm (w/w) HgCh showed turbidity or silky floating in the medium, and it was observed under the microscope. The presence of Pseudomonas Mendoca 2〇NSYSU cells. However, the nutrient broth medium containing 70 ppm (w/w) or more of HgCh remained in a clarified state, and the presence of the cells was not observed under the microscope. The results of this experiment show that the bacterial isolate obtained in the above Example 1 can resist the mercury ions present in the contaminated environment because the bacterial isolate has no more than 70 29 1377249 Patent No. 096146052 for the mercury ion. Correction of the unlined manual replacement page correction period: 99 years π month ppm (w / w) of the south tolerance (tolerance). Example 4. Evaluation of Detergent ability of Pseudomonas Mendocs NSYSU to degrade dioxin In order to confirm whether or not the bacterial isolate obtained in the above Example 1 has the ability to degrade dioxin, the following experiment was carried out. 5 Experimental materials: 1. Preparation of dioxin standard: In a suitable trough state, use n-nonane as solvent and prepare 17 kinds of dioxin/furan according to the ingredients listed in Table 4 below. Compounds [they are supplied by the Supermicro Center of Zhengxiu University of Technology] Dai Ou 10 Xin Standard. Table 4. Composition of dioxin standards Compound name concentration (pg/VL) 2,3,7,8-TeCDF 5 l,2,3,7,8-PeCDF 15 2,3,4,7,8-PeCDF 15 1,2,3,4,7,8-HxCDF 20 _____J_,2,3,6,7,8-HxCDF 20 2,3,4,6,7,8-HxCDF 20 1,2,3,7, 8,9-HxCDF 20 ___J^,3,4,6,7,8-HpCDF 20 __^2,3,4,7,8,9-HpCDF 20 _______ OCDF 50 ___2,3,7,8-TeCDD 5 _1,2,3,7,8-PeCDD 20 _____J_,2,3,4,7,8-HxCDD 15 ______J_,2,3,6,7,8-HxCDD 15 _____J_,2,3,7,8, 9-HxCDD 20 __U,3,4,6,7,8-HpCDD 20 OCDD 50 2. Preparation of Mendoza Pseudomonas gNSYSU strain culture solution: Dissolve 3 g of nutrient broth medium powder in 1000 mL Water 30 1377249 Patent Application No. 096146052 Supplementary, Correction of Unlined Instructions Replacement Page Revision Date: December, 1999, and dispensed into a covered glass test tube (6 mL per tube), then at 121 Autoclaving at a temperature of °C (vapor pressure of approximately 1.5 lb/in2) was followed by 15 minutes. Pseudomonas mendocs NSYSU was inoculated into the above sterilized nutrient meat 5 soup medium and cultured in a constant temperature shaking incubator (30. (:, 120 rpm) for 3 days. Experimental method: 50 μί Dioxin The standard samples were separately added to 6 1 mL of covered glass tubes, and then 1 mL of Mendoza Pseudomonas 10 strains of NSYSU strain culture medium was added to each tube. The 6 tubes were placed in a constant temperature. The culture was carried out in a shaking incubator (30 ° C, 120 rpm), and one test tube was taken at the first, first, second, 20, 30, 40, and 50 days. 'High resolution gas Chromatography, HRGC, HP6970)/high resolution mass spectrometry (HRMS, Micromass 15 Autospec Ultimate) was used to analyze the concentration of dioxin/furan compound. The experiment was repeated twice. ·* Results from 2 experiments It was found that 'Pseudomonas sinensis nsysu can degrade the π dioxin/furan compound as listed in Table 4, and the degradation effect of 〇Cdf 2〇 and 〇CDD is most obvious. Therefore, the applicant uses 〇cdf and OCDD The results are further explained. Referring to Figure 2 and Table 5, the concentration of 〇CDf and OCDD will gradually decrease with the increase of culture time, especially when the concentration of OCDF and OCDD is reduced to about 40 days after culture. The original concentration is 50 to 60%. This result shows: Pseudomonas mendocs 31 1377249 Patent application No. 096146052 Supplementary, revised unlined instructions Replacement page Correction period: December 1999 NSYSU has good degradation The ability of 〇CDF and 〇CDD, and the degradation effect will become more and more obvious with the increase of writing time. Table 5. The effect of Pseudomonas Mendocs NSYSU on the degradation of OCDF and OCDD in dioxin standard with the time of application 0 days non-days 0CDF (Pg/PL) OCDF (%) OCDD (pg/"L) OCDD (%\ day 0 51.4583079 100* 66.99054405 100* day 10 39.6764098 77.104 44.61958880 66.605 first experiment day 20 37.0784850 72.056 41.67901485 62.216 Day 30 33.8968179 65.873 36.92067188 55.113 Day 40 26.6726693 51.834 34.91567785 52.12 Day 50 23.2440963 45.19 23.58496628 35.206 Day 0 60.5318687 100* 61.0062094 1 00* Day 10 48.698722 80.451 56.4773418 92.577 2nd experiment 20th day 40.0898431 66.229 55.9026433 91.635 Day 30 40.0434161 66.152 49.31205965 80.831 Day 40 30.7501811 50.8 37.5072404 61.481 * · A flute Heart 50th day 26.9864034 44.582 36.190191 59.322: In the first The 0CDF and OCDD concentrations measured by Haotian were defined as 丨〇〇%, respectively. Example 5 Evaluation of the ability of Pseudomonas mendocs NSYSU to remove dioxin in soil samples. To confirm whether the bacterial isolate obtained in the above Example 1 has the ability to remove dioxin present in contaminated soil. The following actual test is carried out. Experimental materials: 1. Preparation of self-made soil samples: Firstly, 'the soil that was not contaminated by dioxin from the basketball court of Zhenggong University of Science and Technology was screened with a sieve of 1 mesh to make the particles of the soil 15 The size is approximately 〇149 mm. Next, 100 grams of sieved soil was placed in a sample bottle with a foamed bottom, which was then placed in a sample of beta. After that, a cable made of polyvinyl chloride (polyethylene chloride, 32 1377249, 096, 146, 052, pp. 096, 146, 052, pp. The furnace was burned for 3 minutes, and then all the exhaust gas generated after burning the cable was introduced into the sampling device' and the exhaust gas was touched to the soil in the sampling bottle for 80 minutes, thereby using a homemade soil sample. obtain. 5 2. The Pseudomonas Mendocs NSYSU strain culture solution used in this experiment was prepared by the method described in Example 4 above. Experimental methods: A. Removal of dioxin from soil samples: 36 grams of the actual 10 soil samples as described in the "Experimental Materials" of Example 1 above were dispensed equally into 18 10 mL covered glass tubes. So that each tube contains 2 grams of the actual soil sample. After that, 9 tubes were randomly selected and 6 mL of Pseudomonas mendocs NSYSU strain culture solution was added as the experimental group, and the remaining 9 tubes were separately added with 6 mL of sterilized nutrient broth medium. (nutrient br〇th, Difco 003-01) 15 as a control group. Next, the 18 tubes were placed in a constant temperature shaking incubator (30 ° C, 120 rpm) for culture, and were taken out on the first, first, third, fourth, fourth, fourth, fourth, fourth, fourth, third, fourth, fourth, fourth, third, fourth, and fourth, and The experimental group test tube and one control tube were used for the following Dioxin concentration analysis. In addition, the homemade soil samples were also used for the same experiment. 20 B. Analysis of Dioxin concentration: 1. Extraction step: Take 2 g of the soil sample and put it into a cleaned Thimble Filter (ToyoRoshi Kaisha, Ltd.), then The cylinder paper is placed in the Soxhlet extractor return pipe (s〇xhlet), and then added to the patent application of 33 1377249, No. 096146052, and the revised unlined specification replacement page is revised. Period: December 2, 1999 Anhydrous sodium sulfate was added, and 15 mL of toluene was additionally placed in a 250 mL flat-bottomed flask, and the sample was subjected to an extraction treatment with refluxing for 24 hours. Thereafter, the toluene extract was concentrated to about i 5 mL by concentration under reduced pressure for subsequent workup. 2. Acid-wash step: Add the terpene extract obtained in step 1 to a 24 mL vial with a Teflon-lined bottle cap, followed by 7 mL of hexane (n) -hexane) and shake the vial for approximately 5 seconds, then add 4 mL of 10 concentrated sulfuric acid (98%) to pickle and shake the vial vigorously for approximately 20 seconds. Thereafter, centrifugation (2000 ipm '2 minutes) was carried out to produce a layered layer and a sulfuric acid layer was collected, and the n-hexane layer was further pickled with sulfuric acid until the sulfuric acid layer became white (no more than 3 picklings). Collect the positive courtyard layers in a 50 mL sample vial. Each sulfuric acid layer was extracted twice with 7 mL of hexane. A total of 15 positive burned layers were collected in the 50 mL vial and concentrated under reduced pressure to approximately 1 mL for subsequent processing. 3. Purification step using a multi-layered silica gel column: Filled with 20 mm of a pointed bottom of a multi-layered rubber column (0.5 cm in diameter and 20 cm in length) Glass wool), followed by 〇·5 g silica gel, 0.5 g silver nitrate gel (AgN03-silica gel), 0.5 g silicone, 〇·5 g sodium hydroxide* NaOH- Silica gel), 0.5 g of Shiqi gum, 5 g of sulfuric acid-silica gel, 0.5 g of Shiqi gum and 0.5 g of anhydrous sodium sulfate 'and with glass during filling Rod 34 1377249 Patent Application No. 096146052 Supplementary, Amendment, and Unlined Instructions Replacement Page Revision Period: December 1999, compacted. Fill the column with 30 mL of pre-wash and discard the wash solution. The 1 mL of the acid-washed product obtained in the step 2 is transferred to the pre-washed multi-layer rubber hose column, and after the acid-washed product is completely introduced into the column, the acid-washed product is washed with 1 mL of 5 hexane. The vials were totaled 3 times and the positive wash solution was transferred to the column. Next, elute the column twice with 5 mL of n-hexane, then elute the column with 120 mL of n-hexane, and collect the eluate into a 300 mL Erlenmeyer flask. . The collected eluate was blown to near dryness with a nitrogen purge. The purified product obtained in this way contains dioxin, non-planar polychlorinated biphenyl compounds, and planar polychlorinated biphenyl compounds. The total concentration of the above three compounds is 15 degrees, the purified product can be dissolved in DMSO, and high resolution gas chromatograph (HRGC, HP6970) / high resolution mass spectrometer (HRMS, Micromass Autospec Ultimate) is used. A total concentration detection of these compounds is performed. In order to analyze the respective concentrations of the dioxin 'non-planar type 2 bismuth biphenyl compound and the planar poly-biphenyl compound contained in the purified product, the purified product must be further subjected to the following acidic alumina column and activity. The carbon tube column purification step is to separate the dioxin, the non-planar multi-gas biphenyl compound and the planar multi-gas biphenyl compound, and then use a high-resolution gas chromatograph (HRGC, HP6970)/high resolution. Mass spectrometer (HRMS, 35 1377249, Figure 4, No. 4, patent application, supplement, no-line instructions, replacement page, revision date: December, 1999

Micromass Autospec Ultimate) for rich production analysis. 4. Purification step using an acid oxidized column: Fill the glass wool with a pointed bottom at the ae alumhlum Qxide (3)-η (diameter 0.5 cm, length 20 cm), then fill it in sequence. Into 1 g of tannin, 6 g of acid alumina, 1 g of tannin and 丨g anhydrous sodium sulfate. The packed column was pre-washed with 20 mL of hexane and the lotion was discarded. The eluate eluted from the multi-layer rubber hose column in step 3 was purged into 1 mL after being purged with nitrogen, and then transferred to a pre-washed acid alumina column, and then eluted with 5 mL of hexane. Two times in total, the column was eluted with 9 rpm of 1 hexane and the eluate was collected into a 15 〇 mL conical flask. The eluate thus obtained contains a non-planar multi-gas biphenyl compound. If the concentration of the non-planar polychlorinated biphenyl compound contained in the eluate is to be analyzed, the eluate can be purged to near dryness with nitrogen, then dissolved in 15 DMS0, and a high-resolution gas phase layer is used. A concentration analyzer (HRGC, HP6970) / high resolution mass spectrometer (HRMS, Micromass Autospec Ultimate) was used for concentration analysis of the compounds. After completion of the n-hexane elution, the acidic alumina column was further eluted with 20 mL of methylene chloride/n-hexane (20/80, v/v) and washed. The contents were collected in a 5 〇 InL vial and slowly purged to near dryness with nitrogen. The resulting eluate contained a mixture of dioxin and a planar polyglycol compound. The eluate was dissolved in 1 mL of n-hexane and placed in a vial for the following activated carbon camp purification step. 36 1377249 Patent Application No. 096146052 Supplementary, Correction of Unlined Instructions Replacement Page Revision Date: 99 Years of the Month 5. Purification Procedure Using Activated Carbon Pipe Columns: Activated Carbon/celite The pointed bottom of the column (diameter 5·5 cm, length 20 cm) is filled with glass wool, and then sequentially filled with 〇5 g 矽, 0.5 g activated carbon / Shixiazao (18/82, Wv) ) and 〇_5 g Shi Xijiao, 5 is compacted with a glass rod during filling. Thereafter, the filled column is sequentially 5 mL of each of methanol, toluene, dichloropurine/methanol/toluene (75/20/5, v/v/v), The cyclohexane/methylene chloride (50/50, v/v) and the hexane were pre-washed and the lotion was discarded. 10 Transfer the 1 mL of n-hexane solution obtained from the above-mentioned step 4 after elution from dichloromethane/n-hexane to the activated carbon/diatomaceous earth column, after the solution of the positive chamber is completely inside the column. The vials were washed 3 times with 1 mL of hexane and the n-hexane wash was transferred to the column. Next, elute the live 15 carbon/diatomite column 4 times with 2 mL of cyclohexane/diqimethane (50/50, v/v) and then add 1 mL of di-methane/methanol/toluene. (75/20/5, v/v/v) A total of 2 times of the elution column, all the washings were combined in a 50 mL sample σο bottle, and the resulting washings contained a flat type of gas. Biphenyl compound. The right heart is the concentration of the planar poly-biphenyl compound, which can be purged to near dryness with nitrogen and oxygen, then dissolved in DMSO, and using a high-resolution gas chromatograph (HRGC, ΗΡ6970) ) / high resolution mass spectrometer (hrms,

Micromass Autospec Ultimate) for concentration analysis of compounds. After completion of the above elution, the activated carbon/diatomite column is further eluted with 35 toluene, and the eluted material is collected in a 15 〇 11 锥形 conical flask 37 1377249 096146052 Correction of the unlined manual replacement page Revision date: In December 1999, the washout obtained in this step contains Dioxin. To separately analyze dioxin, the collected benzene benzene wash can be purged to near dryness with nitrogen, then dissolved in DMSO, and using a high-resolution gas chromatograph (HRGC, HP6970) / high-resolution mass spectrometer (HRMS, 5 Micromass Autospec Ultimate) for the concentration analysis of dioxin. Results: Tables 6 and 7 below show the results of Deoxin in the soil sample and self-made soil samples of Pseudomonas mendocs NSYSU. The dioxin that was tested was OCDF and OCDD. 10 From Table 6, it can be seen that 'the concentration of OCDF and 〇CDD in the soil samples with the addition of Pseudomonas mendocs NSYSU will gradually decrease with the increase of the writing time, especially when cultivated to the 31st day. When 〇(:1)17 and 〇(::1)1) are reduced to about 47% and 5% of the original concentration, respectively, and on the 59th day, the concentrations of OCDF and OCDD are decreased, respectively. Up to about 12% of the original concentration 15 and 13% (see Figure 3). In contrast, in the actual soil samples without the addition of Pseudomonas mendocs NSYSU, the concentrations of 〇CDF and 〇CDD did not change significantly over time. In addition, 'from Table 7, it can be seen that in the self-made soil samples with the addition of Pseudomonas mendocella, the concentration of 〇CDF and 〇CDD will gradually decrease with the increase of the writing time 2''Specially, #culture At daydays, the concentration of 〇cdd decreased to about the original concentration, while on day 〇, the concentration of 〇cdf decreased to about 44% of the original concentration. If the day is continued, the concentrations of OCDF and 0CDD are reduced to approximately 15% of the original concentration. (See Figure 4). In contrast, in the absence of the addition of Pseudomonas mendocs 38 1377249 Patent Application No. 096146052, the amendments to the unlined instructions page Replacement date: December 1999 NSYSU isolates of homemade soil samples, OCDF and OCDD The concentration does not change significantly over time. From the above results, it can be seen that Pseudomonas mendocs NSYSU has the ability to remove 0CDF and OCDD in the soil contaminated by dioxin, and the scavenging effect of the bacterium 5 isolate becomes more and more obvious with the increase of the writing time.

39 1377249 Patent Application No. 096146052 Supplementary, Amendment of Unlined Instructions Replacement Page Revision Date: December 1999 Table 6. Mendoza Pseudomonas _NSYSU with the time of use to remove OCDF from the actual soil samples OCDD effect days OCDF (pr/r) OCDF (%) OCDD (ρβ/β) OCDD (%) Day 0 382912.172 100.000* 594077.817 100.000* Day 382292.942 99.838 533244.153 89.760 Day 17 373762.042 97.610 491770.787 82.779 Day 210979.667 55.099 523657.619 88.146 Experimental group day 31 181924.845 47.511 301537.269 50.757 Day 38 175315.695 45.785 282277.313 47.515 Day 45 152463.249 39.817 210259.118 35.393 Day 52 128604.841 33.586 154345.444 25.981 Day 59 48174.406 12.581 78360.575 13.190 Day 382912.172 100.000* 594077.817 100.000 * Day 10 384922.532 100.52502 593866.56 99.96443951 Day 7 Day 386523.552 100.94314 594026.8 99.9914124 Day 24 384920.154 100.5244 594080.85 100.0005105 Control Day 31 Day 382802.155 99.971268 594075.87 99.99967227 Day 38 381912 .226 99.738858 594104.76 100.0045353 Day 45 382546.521 99.904508 593067.57 99.82994703 Day 52 382491.526 99.890146 593977.996 99.98319732 Day 59 382886.156 99.993206 594567.852 100.0824867: The OCDF and OCDD concentrations measured on Day 0 were defined as 100%, respectively. Table 7. Effect of Pseudomonas Mendocs NSYSU on the time of removal of OCDF and OCDD in homemade soil samples with OCDF (Ρβ/β) OCDF (%) OCDD (Ρβ/β) OCDD (%) Day 267.121 100.000* 263.988 100.000* Day 10 205.115 76.787 202.954 76.880 Day 17 191.939 71:855 199.889 75.719 Day 24 192.770 72.166 183.470 69.499 Experimental group day 31 194.943 72.979 121.489 46.021 Day 38 180.600 67.610 113.409 42.960 Day 45 118.047 44.192 47.069 17.830 Day 52 91.860 34.389 64.824 24.556 Day 59 61.146 22.891 41.923 15.881 Day 0 267.121 100.000* 263.988 100.000* Day 10 268.203 100.40506 264.866 100.3325909 Day 7 265.621 99.438457 263.886 99.96136188 Day 24 266.665 99.829291 264.125 100.0518963 Control Day 31 265.957 99.564242 264.988 100.3788051 Day 38 268.211 100.40805 262.999 99.62536176 Day 45 267.556 100.16285 264.011 100.0087125 Day 52 266.997 99.953579 264.114 100.0477294 Day 59 266.665 99.829291 264.556 100.2151613: On Day 0 OCDF obtained with OCDD concentration was defined as 100%. 40 Patent Application No. 096146052 Supplementary, Amendment of Unlined Instructions Replacement Page Revision Date: December 1999 Example 6: Mendoza Pseudomonas_NSYSU Evaluation of Degradation of Brominated Dioxin Ability In order to confirm the above embodiment丨Whether the bacterial isolate obtained in the cell has the ability to degrade brominated dioxin' is carried out in the following experiment. Experimental materials: 1. Preparation of brominated dioxins standard: In a suitable container, using n-decane as a solvent and according to the ingredients listed in Table 8 below, a four-polybrominated dibenzo-p_ Dioxin (PBDDs) and three polybrominated dibenzofurans (PBDFs) [they are supplied by the University of Science and Technology's Ultra Micro Center] brominated dioxins standard. Table 8. Composition of brominated dioxin standards. Compound name concentration (pg/μΙΟ 2,3,7,8-TeBDF — 15 1,2,3,7,8-PeBDF 60 2,3,4,7,8- PeBDF 60 2,3,7,8-TeBDD 15 1,2,3,7,8-PeBDD 60 1,2,3,4/6,7,8-HxBDD 100 l,2,3,7,8, 9-HxBDD 50 2. The Pseudomonas mendocs NSYSU strain culture solution used in this experiment was prepared by the method described in the above Example 4. f hair method. · 20 μιη brominated Dioxin standard The product was separately added to 5 1 mL2 covered glass tubes, and then 5 mL of Pseudomonas mendocs NSYSU strain culture solution was added to each tube. The 5 tubes were placed in a constant temperature vibrating culture. The culture was carried out in a box (30 ° C, 120 rpm), and one tester was taken out on the third, third, sixth, ninth and 12th days, and the south resolution gas chromatograph (HRGC, 1377249, No. 096146052) Addition of the application, amendment of the unlined manual replacement page correction period: December 1999 HP697〇) / high resolution mass spectrometer (HRMS, Micromass Autospec Ultimate) for the concentration analysis of PBDDs and PBDFs. The experiment was repeated 2 The results: 5 can be seen from Table 9, brominated wear The concentration of PBDDs and PBDFs contained in the symplectic standard will gradually decrease with the increase of the culture time, especially 'when the culture reaches the 12th day, the concentration of PBDDs and PBDFs is reduced to about 13 to 38 ° of the original concentration. This result shows that Pseudomonas mendocs NSYSU has a good ability to degrade pbdDs and PBDFs, and the effect of degradation 10 will become more and more obvious with the increase of the writing time. 42 1377249 Patent application No. 096146052 Corrected the unlined manual replacement page correction period: December 1999 ^580^3⁄43⁄4^3PBDDs 耘PBDFs 荈谇冷竺淇Μ 驷一驷% 1,-3,7,8,9-HXBDD (S -*L) •--••-HxroDD 2 --3,4/--'hxbd D (Pg-L) -2,3,4/--8-hxbd D _s_ r2,3,7,-peBDD (Pg/ML) ----8-peroDD 90) •--•TeBDD<pg-l- --7,8-TeBDD (%) 2,3>7,s.peBDF (Pg-L) •- --'peroDF (0/0) r--7,8.pe§p (Pg-L> --•-PSDF (%) 2,3,7,8_TemDF (s/ml) ---•TeroDF &lt ;%) -0> Bud v〇loo· 50.4216 1 17.5»6*7 i* -00. 59-06505 1 2.33207loo* 30. 61.6497*· 62,25s -00* 100* l^.o^^l _ 86-4385 43.58366 75.7692 6^.77^9 41.21257 * 70-75°9

S.725S 64.S675 39-9065 40.27S3 7 II 457 1P70323 铒3^ 62.36594 31.44s 5517 6^.-7169^ 67.9^2^2 s-3022 62.U982 7.664356 ^^-200000 34.16S3 ^3.69^3 ^ 33-2602 认7.997°°3 ^.72^2^°0 祺25.6425 12.92833 24.8624 21°.232500 27.79302 16.415s 27-0261 3-42-1 24.29295 14·97654 23.74-s 14.77945

25.7SS 3-67 recognition private *9# 14,9250° • 52582^ 14.25531 16/76236 17-90°°6 10.33098 17.70S9 2-83551 15.10831 9.3M23 13.3354 S.2S4261 15.75S6 2,369576

I S.4216 i* 117,5867

I 59.06505 i* 12-3207 100* 61.6492 10°* 62.252 public 100* l^.OA is 11 Π ii ii'^-^^SYSUIa^^a^s^^^^PBDDS^PBDFSS^^ 82.57131 ±•63378 75.75275 ^^.07^19 ^3-1298 s.32690° 76.24426 9.4S492 71.65418 44.17461 70.25001 Private 3.73232 78·76585 1.S962 45.35597 22-6921 Private 2.S477 Private 9-°°61 έ.94143 28-0728 44.08685 5.436819 Private-°°002 27-21°°1 4'52087 25.8477厶48.79659 7-41013 31.8563 16,06246 30.822= 36.2S71 3°°1厶92 22.53091 34.73662 4.2S742 29.26045 18.03899 32.1U98 19-9235 36- 6S2 ^-3991000 3°°.540792 19.43288 2°°.995362 32.32773 19.09439

28.S67S 3-37671 26.108417 16.0^^77 28.462S95 17.-884 28.30S29 for .2 recognition °691 43 1377249 Patent application No. 096146052 Supplementary, amendments to the instructions without a line Replacement page Revision date: 99 years 12 Month Example 7. Evaluation of the ability of Pseudomonas Mendocs NSYSU isolates to degrade PBDEs Experimental materials: 1. Preparation of PBDE standards: 5 In a suitable container, use N-decane was used as a solvent and a polybrominated diphenyl ether standard containing 29 polybrominated diphenyl ether compounds [provided by the University of Science and Technology Ultramicro Center] was prepared according to the ingredients listed in Table 10 below. Table 10. Composition of polybrominated diphenyl ether standards. Compound name concentration (pg/pL) 2,4-DiBDE 100 4,4'-DiBDE 150 2,2,,4-TrBDE 150 2,4,4'-TrBDE 150 2,2,,4,5,-TeBDE 125 . 2,3',4',6-TeBDE 150 2,2',4,4,-TeBDE 125 2,3,,4,4'-TeBDE 125 3,3',4,4'-TeBDE 150 2,2,,4,4,,6-PeBDE 150 2,3',4,4',6-PeBDE 150 2,2',4,4', 5-PeBDE 150 2,2,,3,4,4,-PeBDE 150 3,3',4,4',5-PeBDE 175 2,2,,4,4,,5,6'-HxBDE 325 2 ,2',4,4,,5,5,-HxBDE 325 2,2',3,4,4',6-HxBDE 325 400 2,2',3,4,4,,5'-HxBDE 325 2,3,3',4,4',5-HxBDE 350 2,2,J3)4,4,)6,6'-HpBDE 325 2,2',3,4,4',5',6 -HpBDE 325 2,3,3',4,4',5',6-HpBDE 350 2,2,,3,3,,4,4',6,6'-OcBDE 450 2,2',3 ,4,4',5,5',6-OcBDE 350 2,2,,3,3,,4,4',5,6'-OcBDE 325 2,2,,3,3,,4,5 ,5,6,6'-NoBDE 950 2,2,,3,3,,4,4',5,6,6,-NoBDE 850 DeBDE 900 44 1377249 Patent application No. 096146052 Instructions for the replacement of the line of the line Revision date: December, 1999. 2. The strain of Pseudomonas mendocs NSYSU used in this experiment. Solution was prepared with reference to the above process in the fourth example embodiment. Experimental Method • Add 20 μL of the BDE standard to 5 mL of 5 covered glass tubes, followed by 5 mL of Pseudomonas mendocs NSYSU. The five tubes were placed in a constant temperature shaking incubator (30. (:, 120 rpm) for culture, and one tube was taken at 0, 3, 6 '9 and 12 days, respectively, to high-resolution gas phase. Concentration analysis of polybrominated diphenyl ether compounds was performed by a chromatograph (HRGC, HP6970) / high resolution mass spectrometer (HRMS, Micromass Autospec 10 Ultimate). The experiment was repeated twice. Results: As can be seen from Table 11, PBDEs The concentration will gradually decrease with the increase of culture time, especially when the culture reaches the 12th day, the concentration of PBDEs drops to 15 to about 12 to 450/〇 of the original concentration, and the degradation effect of PeBDEs and HxBDEs is the most. Both of them can be reduced to about 12 to 23% of the original concentration. This result shows that Pseudomonas mendocs NSYSu has good ability to degrade PBDEs, and the degradation effect will increase with the time of application. 20 45 1377249 Patent Application No. 096146052 Supplementary, Correction of Unlined Instructions Replacement Page Revision Date: December 1999 3.3- >-_TeBDE (Pg-L) 3.3- >-.TiE -/ 0) •3 -3 -4'TewDE (SL) --•TeOTDE (%) 2,2 2,2 4,--TewDm (pg/μ- --TeroDE 9) 2,3 •3 2,2 4-,6_TeBDE (Pg-L) --•TeBDE 2 ---TesE 3) --•- -•TeBDE (Pg-L) -•-•TrsE (Pg-L)••--TrsE i •2-, 'TrBDE (Pg-L) ---•TrBDE 2 irDiBDE (Pg-L) 4,- .DiBS(%) ••DiBDE (SL)-•DiroDE 2 -0> Bud 1^^.6^23loo* 137·01loo* 136.8531 100* 145.5911 100* 127-666 100* 163.0644loo* 161.6554loo* 153- 423loo* 2 2'6957 10* 祺0^ U9.6523 100 137.01 100 136.8531 100 145-5-1 100 127-666 100 163.0644 100 161.6554 100 147.209s S.9503 -0.0315 97.S586 ^3^ 141.3477 94.4532 128.1913 93.56341 133.-65 2-357 133.5269 91.71366 1 16.3575 91.28472 162,3226 S-5S1 | Recognition °°.2906 97.91 855 -7.4781 76.57141 68.68326 60.94574 99.22879 66.30S2 S.817S 6^.20^^3 97-632-71.34132 9S .97599 67.98216 0000.01一私^00 69-4171 I 17.9^300 72.32^61 118.7671 73.46932 99.98157 65-6727

^-5.7782 S.494S 30.36574 20.29S6 35.08828 47.83996 34.95716 32.*70168 22.46131 37,66211 29.54665 Recognition-7768 3^.2672^ 59.86499 37.032^=° 34.82373 22.69786 25·95264 23.S894 $ 12* >11· n^ii^^-KMSYSUSI^^^^^^^lf^pBDESS^^ 149.6523 100* 137.01 100* 136.8531 100* 145.5-1 so* 127.4S6loo*

163.06λλ I 161.6554 100* 153.423 100* two 2.6957 1 125.31s 83.73432 =2.8476 82.36t9 =6.741·° W5.30厶57 two 4.0861 78.3606 two 2.4807 ^==,2^3 200 142.3513 87.2976 U2.8713 88,38019 153 -23 -00 112.S57 100 107.3155 71.70987 101.4342 7λ.03λ1&

Is.8569 79.M2S7 i.6765 69-5017 95-0Ξ17 7Λ.^302^ ls-8012 86.34699 1^-6. Gong 371 90.58596 128.7919 83.94563 87.36034 77.51876 邾2^5rzhong 61.1=51 ^0-356619 57.63272 S .064S3 80.983 ^9.17513100 s-439500 42.8869428 ^1.^^2 7^13 ^3,3730,5 73.01568 44.7772159 69i 43.1439

S.5S53 39,43-M 31.43-27-8-1189 铕9 λ 19.64032 Π-2397

19-32551 14-051S 21--68S 16.0149 21.85434 15.01076

15.231SS 19.41556 28.52875 17.49539 31.20721 19.304*78 22.46164 14.64S3 17.024-15-068°° ¢12^ 46 1377249 The page is replaced by a letter that says there is no repair or replenishment. Please apply for a special case. 2 5 60 4 1A 6 9ο -2 -3,4,4--ixBDE (S,-L) 2,2-3,4,4---HXBDE (y.) -,--4-6.-HXBDE (SL)-2--4 , 4---HXBDE _i 2.2- --4-6-HXBDE (s/-L) 2.2- --4-,6-HXBDE _l90) -2--4--5'HXBDE (Pg-IL)- 2--4-5,-IDE i -2-4,4-5,--HXBDE (s/ml)2,2--4-5,--HXBDE r/0) --.--peroDm { %) 3,->-,5-PeBDE (s-έ月2 11年9 9曰正修. 2,2 2,2 ,3,-4--peBDE (Pg/HL).---- pewD" -1 2,2 ,--,5-peBDE (S-L>>4-5-peBDE (%> -32,3 >4-6-peBDE (SL) ϋΕ (%> • -.-4-,6_peBDE (SL)2,2--4--peBDE i

335.5122 I 423.623 i* 334.337 100* 323.7856-00* 322.9675 loo* 175.5492 100* 159.4585 loo*

-2.2916 I -53.5181 100* 155-7369 100* 335.5122 100 421.3596 99.46572 334.337 100 295.9571 91-40526 253.5S2 7S.5S93

Is.6938 92.67*705 15^.26^1 97.372s 1^0.797^- ^9.01==87 141.3799 92.S329 =3.5910° 72.^3^200 329.8263 s.30532 S3.S3 50 333.3773 99.7129^ 323- 856 100 267.2S6s.76022 17^-^92 100 100 152.2916-00 153.5181 -00 I 1==.3^79 75.998s 231.S97 69.2362 291.23°0 6°°7011 public 229.2°°^^ 6°°. W78 81 219.231° 67.65s 214-755 66.31486 117.0195 66.6^9010 Ι°7·23± 67.2 Ready====7 105.8616 61°.512 Male 6 105-525 68.4952 -04.3654 67.on·0 69.52079 20.72S9 000° .65336 20.92742 72.24^ 2 1.5 Office 7 7^-2507 23.29476 75.2 Office ==^9 23.29924 19.16157 32.22 Office 5 2°20°°7 36-1992 23.7176

29.33706 19.1SS 31.50117 20.227= 335.5122 loo* S3.623 loo* 33 337 loo* S.7S6 — 322.9675 loo* 175.5492 loo* 1^-0^^°0^ 100* 152.2916 100* 153.5181 i* 155.7369 loo* S3 .8S9 90.57S2 385.2138 ^0-3317 3S.8924 92·68°°62 300.S09 92.66962 322.9675 -00 148-998 °°λ-2066 135-746 84.7S99 132-256 ^6.95521° 125.7975 81.S313 155.7369 100 253.3i 75.50236 327:2983 77.2617 261·891 78.33U3 252.0113 77.»3277 2S.S28663-uls 124.4533 70.893600 115--82 72.31862 -~S77 73. Private 365 Office 113.Α9°°1 73.93145 104.3421 66-9894 132.413 39.4659433 174.0982 ± .097431 138.229 Prepared for 1.3 Office 312·? 130.3232 40.24S323 126.9066 39.2939225 69.990000 39.8741711 41.6120736 76.3307 ^0-21 Private 0 Private 3 63.12445 ±-185796 66.6900! 42.8274269 45.04456 13.42561 60.96761 14.39195 ^1-^333 12. Private 70 Office Recognize ^^-7^75 14.16825 45.282G1 14.02061 21.52395 12.26092 2'25172 13.3223 20.^1 Private 6 1 3.73326

20.1225 13-07SS 21.10096 13.5^-^11 47 1377249 Patent Application No. 096146052 Supplementary, Amendment and Replacement of Instructions without Lines Revision Date: December 1999

DeBDm (Pg-L) DeBDE <%) -2-3,->4-5,---NOBDE (Pg/HL) -2-3,3-,4,4---6'2obde l90 )__ 2,2-,3,3-4,5,5-6,6'NOBDE (Pg-L) -2-3,3--5,5-6,6'zOBDE _SI__ 2.2- -3- -4----OCBDE (S-L> 2.2- -3-'4,4-5,--ocroDE 9) 2.2- --4-5,5-6-OCBDE (Pg-L) 2.2- - 4,4-5,5--OCBDE _^__ •2-3,3--4--6-〇CBDE (SL) 2,2-3,3--4----OCBDE _s__

-3,-,·-,5-6-HPBDE (Pg-L) -3,3-4,--5-,6-HPBDE _B 2,2-3,---5-6-HPBDE (Pg -L) 2,2·,-·-,5-6-HPBDE _ (0/0) -2-3,-4----HPBDE (Pg-L)-2-,--4-,6 ,--HPBDE _i -----••HXBDE -gL) --3-,-4-5-HXBDE(./0) 9S.S33 loo* 8S.4709 -00* 9^0.66411^ loo* 3 22-OS loo* 3S.3339 100* 440.6994 loo* 363b412 100* 327.9534 100* 337.3207loo* 375.9084loo* 738.463 --6649 75 厶.W0300 S6.87727 °°31.591» ^^.^0^7 3 286.203⁄4 ==^-586

346.0805 S.046S 3--286 -.94441 339.8286 93-028 312.9102 ^5.413 324-42 96-2278 375.SS 100 791.01s «7.69198 802.5389 92.SS27 ^27.-70° Office 3 98.63075 276.7237 ==^. 91^^7 35CJ.93 --0863 378.3- °°5·8έ 341.2601 94.00038 3S.345 97.68003 334.376 ^^-2705 3S.7S4 95.9562 6S4.5242 75.^^60° 551-765 63-6517 671.253 71.35S5 188.3621° 241.391 62.S764 267.2743 s.64775 237.£42 65-01 Fen 6 223.8652 68.26128 221.63U 65.70349 254.3523 67-6337 S8.2536 Office 5.25026 3S.9872 40.29925 350.5729 37-°'000'l"

16s 33.S996S 141.2612 36.75482 173.3933 39.345s 1 14-636 31.63926 99-041 30.21S95 103.2131° 30.59816 78.25880° 20.8186 S*°S33 100* 868.4709 loo* 9 Office 0.66λλ 100* 322.0s00 loo* 384.3331° 100* 440.699^ Loo* 363.0412 so* 327.9534 loo* 337.3207loo* 375.9084loo* 902.0333 100 8S.4S9 100 9λ0.66λλ 100 322.08s 100 3S,3339 100 έο.294 100 363.0412 100 327-9534 100 337.3207 i 346.9821 92-298 S36.6472 ^2-75125 -8.0904 94.1S95 ==--86 91.58272 287.6541 S.3S16 371.5531 96.3^51 393.S35 ^^-^538 S6-3-6 84-3793 283.7866 86.5326 295.7542 S7.67S6 281.36M 74.84947 706.soos 78-3652787 237.3596 26,31384 360.8478 41.5497883 212-ss 24.50742 363.72» 38.67-72 206.3327 21.93478 =8.341°1 36.7S0055 60-9464 18.68000000 -44.6168 37.6279009 147.SIS 33.5417754

14~0345 3S.848067S 129.8206 39.5850797 -32-8057 39.3707453 -0.0441 39.9150723 74-6S501 19-43233 85-03 19-37897 S.5S4 15.59008 52.58872 16-S542 56.2-74 16.67-2 47-2837 12.5372 48 1377249 096146052 No. Patent Application Supplement, Amendment of Unlined Manual Replacement Page Revision Date: December 1999 Example 8. Evaluation of the ability of Pseudomonas Mendocs NSYSU to degrade multi-gas biphenyls (PCBs) Experimental materials: 1. Multi Preparation of chlorobiphenyl standards: In a suitable container, n-decane was used as a solvent and 12 polychlorinated biphenyl compounds were formulated according to the ingredients listed in Table 12 below [they are from the University of Science and Technology) Polychlorinated biphenyl standards provided by the ultra-micro center. Table 12. Composition of Multi-Gas Stupid Standards Compound Name Concentration (pg/μί) 3,3,,4,4,-Te Qiao 100 3,4,4',5-TeCB 100 2,353',4,4 '-Ρ^〇Β 100 2,3,4,4',5-PeCB 100 2,3',4,4',5-P^i — 100 2,3,4,4,5'Pe(j_3 100 卜100 2,3,3,4,4,b-HxCR 100 2,3,3-,4,4-,57^3⁄4^ 100 2,3,,4,4-,5,5^ϋ^ 100 3,3 ,4,4 ,5,i'-HxCR 100 2,3,3 ,4,4 ,5,5'-Ht)CR 100 10 2. The Η多萨假单单 used in this experiment_ 8 _ culture solution was prepared by the method described in Example 4 above. Experimental method: Add 20% of the gas-stirred standard products to 5 sets of 1 covered glass test f, and then add 5 mL of Pseudomonas mendocensis NSYS (10) culture solution to each test tube. . The five test tubes were placed in a -wet temperature vibrating incubator (3 ° ° C, 12 〇 rPm) for cultivation, and the third, third, sixth, ninth, and twelveth day 49 15 1377249 No. 096146052 patent application was supplemented and amended. Unlined instructions Replacement page Correction date: 1 test officer was taken out in December 1999's high resolution gas chromatograph (HRGC, HP6970) / High resolution mass spectrometer (HRMS, Micromass Autospec Ultimate) Concentration analysis of poly-biphenyl compounds was performed. The experiment was repeated twice. 5 Results: It can be seen from Table 13 that the concentration of PCBs will gradually decrease with the increase of culture time. In particular, when cultured to the 12th day, the concentration of certain PeCBs, hxcBs and HpCBs can be reduced to about the original concentration. 〇6 to 4%, of which 2,3',4,4',5,5,-1^€6 has the best degradation effect, and its concentration is reduced to about 1〇 as the original concentration of 〇·6 to 1.6%. . This result shows that Pseudomonas mendocs NSYSU has a good ability to degrade PCBs, and the degradation effect will become more and more obvious as the action time increases. 50 Patent Application No. 096146052 Supplementary, Correction of Unlined Instructions Replacement Page Revision Date: December 1999 Table 13. Effect of Pseudomonas Mendocs NSYSU of the Invention on Degradation of PCBs with Working Time 1st Experiment 2nd day of the experiment Day 0 Day 3 Day 6 Day 9 Day 12 Day 0 Day 3 Day 6 Day 9 Day 12 3.3. 4,4*-TeCB (pg/μί 3.3. , 4,4,-TeCB (%) 99.971 76.086 60.947 62.071 62.008 99.971 75.617 66.547 60.829 60.148 100* 76.108 60.965 62.089 62.026 !00* 75.638 66.566 60.847 60.165 3,4,4.,5-TeCB (pg/μί 98.580 67.794 60.203 20.539 45.828 98.580 76.418 58.397 61.617 25.534 3,4,4',5-TeCB (%) 100* 68.771 61.070 20.835 46.488 100* 77.5Ϊ9 59.238 62.505 25.902 2,3,3,,4,4'-PeCB (pg/μΐθ 2,3,3_,4,4.-PeCB (%) 100.679 73.686 67.565 16.225 24.587 100.679 73.953 65.538 15.901 9.840 100* 73.189 67.110 16.116 24.421 100* 73.455 65.096 15.794 9.774 2,3,4,4., 5-PeCB (pg/μΐ) 2,3,4,4,,5-PeCB (%) 99.655 66.879 66.475 6.766 3,245 99.655 81.225 64.480 6.630 0.902 1 00* 67.111 66.705 6.789 3.257 100* 81.507 64.704 6.653 0.905 2,3',4,4',5-PeCB (pg/μί) 2,3',4,4',5-PeCB (%) 99.679 80.260 73.750 5.287 3.05] 99.679 75.063 71.537 5.181 0.871 100* 80.519 73.987 5.304 3.061 100* 75.305 71.768 5.198 0.874 2-,3,4,4.,5-PeCB (pg/μί) 2',3,4,4',5-PeCB (%) 99.981 67.854 67.046 4.366 2.394 99.981 86.708 65.034 4.279 0.717 100* 67.867 67.058 4.367 2.394 100* 86.725 65.047 4.279 0.717 3,3',4,4',5-PeCB (pg/μΐ) 3,3_,4,4 ,,5-PeCB (%) 99.417 78.583 66.443 22.098 45.483 99.417 74.515 64.450 55.245 25.861 100* 79.044 66.833 22.227 45.750 100* 74.952 64.828 55.569 26.013 2t3,3,,4,4_,5-HxCB (pg/μ^) 2, 3,3',4,4·,5-ΗχΟΒ (%) 100.997 76.760 75.248 7.529 3.413 100.997 73.215 72.991 7.379 0.979 100* 76.003 74.506 7.455 3.379 100* 72.492 72.270 7.306 0.969 2,3,3i,4,4_,5_- HxCB (Pg/pL) 2,3>3,,4,4,(5,-HxCB (%) 100.993 68.022 74.678 9.823 4.625 100.993 78.143 72.437 9.626 1.169 100* 67.353 73.943 9.726 4.580 100* 77.375 71.725 9.532 1.157 2,3 4,4,,5,5,-HxCB (Pg~L) 2,3,,4,4',5,5,-HxCB (%) 99.978 71.159 73.375 2.196 1.651 99.978 78.824 71.174 2.152 0.627 100* 71.174 73.391 2.196 1.651 100* 78.841 71.189 2.152 0.627 (pg/μΐ) (%) 99.316 84.620 75.106 15.194 13.553 99.316 79.251 72.853 14.890 3.851 100* 85.203 75.624 15.298 13.647 100* 79.797 73.355 14.992 3.877 2,3,3.,M,,5,5 ,-HpCB (pg/μί) 2,3,3.,4,4_,5,5--HpCB (%) 100.007 74.593 80.736 3.436 2.011 100.007 89.670 78.314 3.368 0.682 100* 74.588 80.731 3.436 2.010 100* 89.664 78.309 3.367 0.682 * : The PCBs concentration measured on day 0 was defined as 100%. Example 9. Evaluation of the ability of Pseudomonas Mendocs NSYSU isolates to degrade polycyclic aromatic hydrocarbons (PAHs) Experimental materials: 1. Preparation of polycyclic aromatic carbon oxime compound standards: in a suitable container Using n-decane as a solvent and formulating a compound containing 16 kinds of polycyclic aromatic hydrocarbons according to the components listed in Table 14 below, supplementing the patent application No. 096146052 The flood season: the polycyclic aromatic hydrocarbon standard of December 1999 [they are provided by the ultra-micro center of Zhengxiu University of Science and Technology]. Table 14. Composition of Polycyclic Aromatic Hydrocarbon Standards Compound Name Concentration (ng/pL) naphthalene 1 %, (acenaphthylene) 1 acenaphthene 1 % (fluorene) 1 l^(phenanthrene) 1 onionracene 1 fluoranthene 1 芘 (pyrene) 1 benzo (a) hui [benzo (a) anthracene] 1 qi (chrysene) 1 stupid (b) propylene two women 爇 [benzo (b)fluoranthene] 1 benzo(k)propanthene 1 benzo(a)芘[benzo(a)pyrene] 1 茚(), 2,3-cd)芘[indeno(l,2,3-cd)pyrene] 1 dibenzo(a,h) onion [dibenzo(a,h)anthracene] 1 benzo(g,h,i)茈[benzo(g,h, i) Perylene] 1 2. The Pseudomonas Mendocs NSYSU strain culture solution used in this experiment was prepared by following the method described in Example 4 above. Experimental method: 50 μί polycyclic aromatic hydrocarbon standard was added to 5 10 mL covered glass tubes, and then 5 mL of Pseudomonas mendocs NSYSU strains were added to each tube. liquid. The five tubes were cultured in a 10 constant temperature shaking incubator (30 ° C, 120 rpm), and one tube was taken out on the third, third, sixth, ninth and 12th days to form a high-resolution gas phase layer. The analyzer (HRGC, HP6970) / Southern Resolution (JJRMS, Micromass Autospec Ultimate) was used to analyze the concentration of polycyclic aromatic hydrocarbons. The experiment was repeated twice. 52 1377249 Supplementary result of patent application No. 096146052: Correction of the unlined specification replacement page Revision date: From December 15, 1999, it can be seen from Table 15 that the concentration of pAHs will gradually decrease with the increase of culture time, _ ground, when On the 12th day of cultivation, some fine concentrations can be reduced to about 5 to 2 原始 of the original concentration. /. Among them, the degradation effect of naphthalene is the best', and its concentration is lowered to about 5 to 16% of the original concentration. This result shows that: Pseudomonas mendocs NSYSU has a good ability to degrade PAHs, and the degradation effect will become more and more obvious with the increase of the writing time.

53 1377249 Patent Application No. 096146052 Supplementary, Amendment of Unlined Instructions Replacement Page Revision Date: December 1999 Table 15. Effect of Pseudomonas Mendocs NSYSU on the Degradation of PAHs with the Time of Application The First Experiment 2 days of experimentation Day 0 Day 3 Day 6 Day 9 Day 12 Day 0 Day 3 Day 6 Day 9 Day 12 Day naphthalene (ng/μΙΟ 1.001 0.890 0.640 0.551 0.052 1.001 0.810 0.570 0.134 0.170 _ naphthalene (%) 100* 88.879 63.913 55.007 5.219 100* 80.890 56.923 13.399 16.976 卮(ng/μΐ) 0.991 0.660 0,520 0.536 0.089 0.991 0.630 0.440 0.390 0.293 苊(〇/〇) 100* 66.573 52.451 54.096 8.978 100* 63.546 44.382 39.357 29.525 · Dihydroanthracene (ng/pL) 0.999 0.790 0.640 0.646 0.108 0.999 0.780 0.610 0.430 0.334 Dihydroanthracene (%) 100* 79.066 64.053 64.677 10.818 100* 78.065 61.051 43.052 33.444 ' 格 (ng/μί) 0.987 0.770 0.620 0.626 0.190 0.987 0.750 0.630 0.525 0.401 M%) 100* 78.004 62.808 63.367 19.295 100* 75.978 63.821 53.234 40.586 Philippine (ng>L> 0.971 0.780 0.710 0.619 0.335 0.971 0.75 0 0.720 0.617 0.428 Philippine (%) 100* 80.300 73.093 63.755 34.485 100* 77.21 1 74.123 63.557 44.064 £(ng/ML) 0.993 0.830 0.716 0.610 0.453 0.993 0.840 0.669 0.620 0.455 蒽(%) 100* 83.573 72.102 61.421 45.644 !00♦ 84.580 67.365 62.428 Propadiene oxime (ng/μΐ) 0.997 0.890 0.784 0.760 0.498 0.997 0.900 0.814 0.760 0.480 * Propadiene (%) 100* 89.226 78.607 76.193 49.905 100* 90.228 81.561 76.193 48.143 芘(ng/pL) 0.995 0.880 0.800 0.773 0.482 0.995 0.900 0.810 0.795 0.462 芘 (%) 100* 88.429 80.390 77.628 48.416 100* 90.439 81.395 79.876 46.436 Stupid and 蒽 (ng/pL) 0.990 0.820 0.670 0.634 0.394 0.990 0.790 0.710 0.578 0.343 Stupid and U) onion ( %) 100* 82.836 67.683 64.091 39.795 100* 79.805 71.724 58.372 34.607 Chopsticks (ng/pL) 1.061 1.100 0.860 0.898 0.662 1.061 1.090 0.880 0.825 0.553 «(%) 100* 103.630 81.020 84.601 62.367 100* 102.688 82.904 77.756 52.118 Stupid (b ) Propadiene oxime (ng/μί) Stupid (b) Propadiene oxime (%) 0.986 0.810 0.620 0.561 0.375 0.986 0.810 0. 660 0.503 0.302 100* 82.178 62.902 56.898 38.078 100* 82.178 66.960 51.046 30.596 Benzo(k)propadiene (ng/pL) 1.165 1.120 0.960 0.844 0.597 1,165 1.090 0.990 0.803 0.512 and (k) and body chess (%) 100* 96.162 82.425 72.423 51.262 100* 93.586 85.000 68.939 43.926 Stupid (a) 芘 (ng/pL) 1.037 1.010 0.710 0.621 0.420 1.037 0.980 0.740 0.499 0.322 Stupid (a) 芘 (%) 100* 97.387 68.460 59.870 40.520 100* 94.494 71.353 48.155 茚(l,2,3-cd)芘(ng/μυ 0.993 0.850 0.650 0.508 0.332 0.993 0.840 0.710 0.368 0.259 茚 and (1,2,3-cd)芘(%) 100* 85.559 65.427 51.170 33.412 100* 84.552 71.467 37.076 26.067 Two stupid (a, h) Jfe (ng~L) Two stupid (a, h) onion (%) 1.067 0.990 0.810 0.659 0.474 1.067 1.000 0.850 0.495 0.422 100* 92.748 75.885 61.763 44.390 100* 93.685 79.632 46.406 39.507 stupid (g,h,i)茈(ng/pL) 1.099 1.130 0.860 0.778 0.544 1.099 1.130 0.890 0.626 0.464 stupid (g,h,i)茈(%) 100* 102.793 78.232 70.753 49,481 100* 102.793 80.961 56.952 42.239 * : The measured concentration of PAHs on day 0 was defined as 100% of all patents and publications in this specification * is quoted and is in its entirety into the case as a reference. In case of conflict, the detailed description of the case (including 5) will prevail. 54 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 Many modifications and changes can be made. It is therefore intended that the present invention be limited only by the scope of the appended claims. 5 [Simple description of the schema] Figure 1 shows the nucleotides of 16SrDNA of Pseudomonas mendocs NSYSU (which is a bacterial isolate isolated in Example J and characterized by Example 2) Sequence; Figure 2 shows the effect of Pseudomonas mendocs NSYSU on the OCDF and OCDD in the Dyros standard for the duration of the study. The upper part is the result of the first experiment, and the lower part is the second experiment. Results; Figure 3 shows the effect of Pseudomonas mendocs NSYSU on the removal of 0CDF and 〇CDE in the soil samples of the plant; and Figure 4 shows the hypothesis of Pseudomonas mendocs NSYSU Qing 15 In addition to the effect of OCDF and OCDD in homemade soil samples. [Explanation of main component symbols] (None) 55 1377249 Patent application No. 096146052 Supplementary, amendment of unlined manual Replacement page Revision date: December 1999 Sequence Listing <110> National Sun Yat-Sen University University of Science and Technology<120> Microbial test 5 for removing dioxin pollutants present in a contaminated medium and method of using the same <130> NSYSU <160> 3 <170> Patentln version 3.4 10 <210> 1 <211 〉 20 <212> DNA <213> Artificial <220> 15 <223> General forward primer for PCR amplification of 16S rRNA gene of bacterial strain F1 <400> 1 20 attgaacgct ggcggcaggc <210 〉 2 20 <211> 20 <212> DNA <213> Artificial <220><223><223> General purpose reverse primer for PCR amplification of i6S rRNA gene of bacterial strain R1 1377249 Patent Application No. 096146052 Case Supplement, Correction of Unlined Manual Replacement Page Revision Date: December 1999 <400> 2 20 cccagtcatg aatcactccg

<210> 3 5 <211> 1453 <212> DNA <213> Pseudomonas Mendocs NSYSU <400> 3 at tgaacgct ggcggcaggc ttaacacatg caagtegage ggatgaaggg aget tgctcc 60 10 ctgatt tagc ggcggacggg tgagtaatgc etaggaatet gcctggtagt gggggataac 120 gt tccgaaag gaacgctaat accgcatacg tcctacggga gaaagcaggg gacct teggg 180 cct tgcgcta tcagatgagc ctaggtcgga t tagetagt t ggtgaggtaa tggctcacca 240 aggcgacgat ccgtaactgg tctgagagga tgatcagtca cactggaact gagacacggt 300 ccagactcct acgggaggca gcagtgggga atat tggaca atgggcgaaa gcctgatcca 360 15 gccatgccgc gtgtgtgaag aaggtct teg gat tgtaaag cact t taagt tgggaggaag 420 ggcat taacc taatacgt ta gtgt 11 tgac gt taccaaca gaataageae cggcaact tc 480 gtgccagcag ccgcggtaat acgaagggtg caagcgt taa teggaat tac tgggcgtaaa 540 gcgcgcgtag gtggt tcgt t aagt tggatg tgaaagcccc gggctcaacc tgggaactgc 600 atccaaaact ggcgagctag agtacggtag agggtggtgg aat t tcctgt gtagcggtga 660 20 aatgcgtaga tataggaagg aacaccagtg gegaaggega c cacctggac tgatactgac 720 actgaggtgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgccgta 780 aacgatgtca actagccgt t ggaatcct tg agat 11 tagt ggcgcagcta acgcat taag 840 t tgaccgcct ggggagtacg gccgcaaggt taaaactcaa atgaat tgac gggggcccgc 900 acaagcggtg gagcatgtgg 11 taat tega agcaaegega agaacct tac ctggccttga 960 2 1377249 No. 096H6052 Patent Application supplement 'no ruled line correction of corrected page description Alternatively date: December 99 catgctgaga act t tccaga gatggattgg atggctgtcg tcagctcgtg tcgtgagatg tgtcct tagt taccagcacc tcgggtgggc aggaaggtgg ggatgacgtc aagtcatcat acaatggtcg gtacaaaggg t tgccaagcc cgtagtccgg atcgcagt ct gcaactcgac aatcagaatg tcacggtgaa tacgt tcccg ggagtgggt t gctccagaag tagctagtct at tcatgact Ggg tgcct tcggg aactcagaca caggtgctgc 1020 tgggt taagt cccgiaacga gcgcaaccct 1080 actctaagga gactgccggt gacaaaccgg 1140 ggccct tacg gccagggcta cacacgtgct 1200 gcgaggtgga gctaatccca t aaaaccga t 1260 tgcgtgaagt cggaa t cgc t agtaatcgtg 1320 ggcct tgtac acaccgcccg tcacac Catg 1380 aacct tcggg aggacggt ta ccacggagtg 1440 1453 10 3

Claims (1)

1377249 Patent application No. 096146052, patent application scope: January 1st - Replacement - page - a microbial reagent used to remove dioxin, dioxins and/or polycyclic aromatic hydrocarbons present in a contaminated medium. It contains a registration number BCRC 910356 is deposited in the bioresource conservation and research center (bcrc of FIRDI) of the 5th Industrial Development Institute, Pseudosm〇nas men (jocina) 10 NSYSU' and a biocompatible carrier (bi〇c〇mpatibie carrier). 2. The microbial agent of claim 1, wherein the microbial agent degrades a dioxin having 4 to 8 chlorine atoms. 3. The microbial reagent of claim 2, wherein the dioxin is selected from the group consisting of 2, 3, 7, 8 • tetrachlorodibenzo-p_Dauol 15 sin, 1, 2,3,7,8-penta-dibenzo- _ dioxin, 2,3,4,7,8-pentachlorodibenzo-Ocein, 1,2,3,4,7,8-hexachlorodi Stupid and / / 7_ Dioxin, to add - six gas dibenzophenone Dioxin ^ hexachlorodiphenyl And ρ-Dioxin, 123^9 six gas two stupid and 卞 _ Dai Osin, If, 3,4,6,7,8. Heptachlorodibenzo- Ρ_Dioxin, ..., ^... seven gas two = and; Dioxin, popular dibenzo-Dioxin, four-gas two-dimensional from 1,2,3,7,8-pentachlorodibenzofuran, 1,2,3,4,7,8-hexachlorobenzin 1,2,3,6,7,8-hexachlorodibenzofuran, 1,2,3,7,8,9·^dibenzo. Furu, like the cut-off domain diphenyl ^ octachloro-benzofuran, and combinations thereof. ^Please request the microbial reagent of the first item of the patent range, wherein the microorganism test = degradation - selected from the group consisting of - dioxins in the group - odorized dioxin, polybrominated bismuth, polychlorinated biphenyl, and 20 thereof 1377249 Patent No. 096146052, Replenishment of Requests, Correction, and Replacement of Instructions without Lines Replacement Date: Combination of 100 Years of U Months 5. For microbiological reagents of claim 4, wherein the brominated Dioxin contains 4 Up to 8 bromine atoms. 6. If the microbial reagent of claim 5 is applied, the brominated dioxane 5 sen is selected from the group consisting of 2,3,7,8-tetrabromodibenzo- household_Dioxin , 1,2,3,7,8-five> odorous dibenzo-households - dioxin, 1,2,3,4/6,7,8-hexabromodibenzo-P-dioxin, 1,2 , 3,7 8 9_hexabromodibenzo- _ dioxin, 2,3,7,8-tetrabromodibenzofuran, 12,3,7,8-pentabromodibenzofuran, 2,3, 4,7,8-pentabromodibenzofuran, and combinations thereof. 10. 7. The microbial agent of claim 4, wherein the polybrominated dip has 2 to 1 bromine atom. 8. The microbial reagent of claim 7, wherein the polybrominated diphenyl ether is selected from the group consisting of 2,4-dibromodiphenyl ether, 4,4, dibromodi Phenyl ether, 2,2,4-tribromodiphenyl ether, 2,4,4,-tribromodiphenyl ether, 15 2,2',4,5'-tetrabromodiphenyl ether, 2,3, ,4,6-tetrabromodiphenyl ether, 2,2',4,4,-tetrabromodiphenyl scale, 2,3',4,4,·tetrabromodiphenyl ether, 3,3',4 , 4'-tetrabromodiphenyl ether, 2,2',4,4,6-pentabromodiphenyl ether, 2,3,,4,4,6-pentaBDE, 2,2'' 4,4',5-pentabromodiphenyl ether, 2,2,3,4,4,-pentabromodiphenyl ether, 3,3',4,4',5-five desert diphenyl ether, 2 ,2,,4,4',5,6,_ Liu desert diphenyl ether, 20 2,2',4,4',5,5'_ hexabromodiphenyl ether, 2,2',3,4 ,4,6-hexabromodiphenyl ether, 2,2·,3,4,4·,6·-hexabromodiphenyl ether, 2,2,3,4,4,,5,-hexabromo Diphenyl ether, 2,3,3',4,4',5-hexabromodiphenyl ether, 2,2,3,4,4,6,6,-heptabromodiphenyl ether, 2,2 ',3,4,4',5',6-heptabromodiphenyl ether, 2,3,3,,4,4|,5,6-heptabromodiphenyl ether, 2,2',3, 3',4,4',6,6·- Bromodiphenyl ether, 2,2',3,4,4,5,5,6-octabromobiphenyl 2 No. 0061 Na 2_Additional case, no correction after the correction of the book Period: 100 years η month key, 2, 2', 3, 3', 4, 4', 5, 6'. octabromodiphenyl _, 2, 2, 3, 3, 4, 5, 5 | ,6,6,- decabromodiphenyl ether, 2,2',3,3,4,4,5,6,6,-nonabromodiphenyl ether decabromodiphenyl ether, and combinations thereof . 9. The microbial reagent of claim 4, wherein the polychlorinated biphenyl contains 4 to 7 gas atoms. 1. The microbial reagent of claim 9, wherein the polyglycol is selected from the group consisting of 3, 3, 4, 41_tetrahydrobiphenyl, 3, 4, 4 , 5-tetrachlorobiphenyl, 2,3,3,,4,4,·pentachlorobiphenyl, 2,m,5·pentachlorobiphenyl, 2,3,,4,4,,5-five Biphenyl, 2, 3, 4, 4i, 5_ five gas biphenyl, 3, 3', 4, 4', 5 - five gas biphenyl, 2, 3, 3,, 4, 4', 5_ six Chlorinated biphenyl, 2,3,3',4,4',5,·hexachlorobiphenyl, 2,3,,4,4,,5,5,_ hexachlorobiphenyl, 3,3',4 , 4, 5, 5'. hexa-biphenyl, 2,3,31,4,4,5,5,_heptachlorobiphenyl, and combinations thereof. 11- The first dose of the patent application can degrade one containing two substances. The microbial reagent of the present invention, wherein the microorganism is tested to a polycyclic aromatic aromatic carbon gas of 6 benzene rings, and the microbial reagent of the U (N) U, wherein the polycyclic aromatic hydrocarbon is selected from the group consisting of Philippine propylene carbonate..., and (4) dithiline bismuth, benzo(8) propylene diene. (2,2,2,3-cd), dibenzo ((4) onion, benzo and their combination. De... (4)' and the microbial reagent of claim 1 of the patent scope, further enveloping at least - A variety of patents selected from the following groups: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Micro-mains of (Pseudomonas), micro-substances of Dehabcoccoides, microorganisms of Candida, microorganisms of the genus Escherichia (Λ/ιθί/ococci^), Aeromonas (Aro qing / uw) microorganisms, Rhizobium (and / ^ ~ ~) microorganisms, sphingomonas (5 > / ^ «gowo « 〇 s) microorganisms, Arthrobacter microorganisms, Microorganisms of the genus Flavobacterium, microorganisms of the genus Flavobacterium, and trees of the genus Bacillus. 14 · The microbial reagent of the 13th item of the patent application, the microorganism capable of removing environmental pollutants Is selected from the group consisting of Pseudomonas resin (corpse·sewi/owowfls mszT) Zovora/w) strain CA10, Pseudomonas vonii (corpse πΜί/ίϊΑΜΟΛα? verom.O PH-03, De/A/ococco Wes #.) strain CBDB1, Mendoza Pseudomonas mendocina FERM, Candida ν^>να«αί/ϊ//, and combinations thereof 15. The microbial agent of claim 1 is manufactured to be selected from the following Group dosage forms: culture solutions, suspensions, granulis, powders, tablets, pills, capsules, and siurry. The microbial agent of the first aspect, wherein the Pseudomonas parasiticus NSYSU is entrapped in the biocompatible carrier. 17. The microbial agent of claim 16 wherein the organism Pp. 1377249, the patent application No. 096, 146, 052, the disclosure of which is incorporated herein by reference. Cyclolipid, chitin Chitin (chit〇san), polyvinyl alcohol, aginic acid, polyacrylamide, carrageenan, agarose (agarose), Mingsheng ( Gelatin), cellulose, cellulose acetate, dextran, and collagen. 18. The microbial agent of claim 1, wherein the Pseudomonas puteria NSYSU is supported on the biocompatible carrier. 10. The microbial agent of claim 18, wherein the biocompatible carrier is selected from the group consisting of: glass, ceramic, metal oxide, activated carbon Activated carbon, kaolinite, bentonite, zeolite, aluminum, anthracite, glutaraldehyde, polyacrylic acid, polyamine曱酸6, (polyvinyl chloride), ion exchange resin, epoxy resin, photosetting resin, polyester, and polyphenylene Polystyrene. 2. A microbial agent as claimed in claim 1, wherein the microbial agent is also resistant to mercury. 21. The microbial agent of claim 1, wherein the contaminated medium is selected from the group consisting of: soil, sludge, sediment, groundwater (groundwater) ), Waste 5 No. 096146052 Patent Application Supplement, Corrected No-Lined Instruction Replacement Page Revision Date: November water (waste water) and exhaust gas (exhaust). 22. The microbial reagent of claim 21, wherein the contaminated medium is selected from the group consisting of: land, orchard land, grazing grassland 'well water, fish culture pond, factory wastewater, Domestic sewage and sludge from sewage treatment plants. 23. A method for removing dioxin, dioxin-like compounds and/or polycyclic aromatic hydrocarbons present in a contaminated medium, comprising: using a microbial agent as in claim 1 Treating the contaminated medium, such that the dioxin, dioxin, and/or polycyclic aromatic hydrocarbons present in the contaminated medium are treated by Pseudomonas mendosus nsysu in the microbial agent Degradation and disappearance 0 2 = method of claim _ 23, wherein the contaminated medium 自 k from the following group: soil sludge, sediment, groundwater, wastewater and waste gas. 25·^μμ Patent (4) Method 24, wherein the contaminated medium: the following group: field, orchard land, grazing grass =, well water, Μ养统, Wei factory wastewater, domestic sewage And the sludge of the sewage treatment plant. 26·ΙΙ^· The method of the 23rd item, the ## lies in the contaminated "quality of the dioxin containing 4 to 8 chlorine atoms. The method of the 26th item, the t exists in the contaminated four: + Dioxin is selected from the group consisting of: 2,3,7,8-^本和··················································· Supplementary, revised, no-line instructions to replace the page revision period: 100 years November, Xin, 2,3,4,7,8-five gas dibenzo-household-Dioxin, ι,2,3,4,7 , 8-hexachlorodibenzo-/?-dioxin, 1,2,3,6,7,8-hexachlorodibenzo-/7-dioxine, 2.3.4.6.7.8-hexachlorodiphenyl and dioxin, 1,2,3,7,8,9-hexachlorodibenzo-/?-dioxin, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin, 5 1, 2,3,4,7,8,9-seven gas dibenzo-household-Dioxin, octachlorodibenzo-;?-Dioxin, 2,3,7,8-tetra-dibenzofuran, ι, 2,3,7,8-penta-dibenzofuran, 1,2,3,4,7,8-hexa-benzo-benzofuran, 1,2,3,6,7,8-hexahydrate Benzofuran, ^ on the child ^ six gas dibenzofuran ~ ^ one / nine Yiyi seven gas dibenzofuran, eight gas diphenyl The method of claim 23, wherein the dioxin compound present in the contaminated medium is selected from the group consisting of brominated dioxin and polybromine. Diphenyl ether, polychlorinated biphenyl, and combinations thereof. 29. The method of claim 28, wherein the dioxin compound 15 is brominated dioxin and contains 4 to 8 bromine atoms. The method of claim 29, wherein the brominated dioxin is selected from the group consisting of 2,3,7,8-tetrabromodiphenyl and dioxin, 1,2,3,7 , 8--five dibenzo-dioxin, nwns hexabromo-di- and dioxins, ^'^^hexabromo-di-bromo-dioxin, 2〇2,3,7,8-tetrabromodi-p-furan, mis· Pentabromodibenzofuran, 2.3.4.7.8-pentabromodibenzofuran, and combinations thereof. 31. The method of claim 28, wherein the dioxin compound 疋 漠 _ 苯 喊 and contains 2 to 1 演 an atom. A as claimed in the scope of the 31st method, wherein the polybromine is 7 1377249 096146 Supplementary to the 052 patent application, revised or unlined instructions Replacement page Revision date: November 1st is selected from the group consisting of 2,4-dibromodiphenyl hydrazine, 4,4'- Erqin diphenyl mystery, 2,2',4-tribromodiphenyl mystery, 2,4,4'-three desert diphenyl mystery, 2/,4,5.-tetrabromodiphenyl ether, 2,3' , 4',6-tetrabromodiphenyl ether, 2,2,,4,4,-tetra-diphenyl ether, 2,3,,4,4,-tetra-diphenyl ether, 3,3,,4 , 4,-tetrabromobiphenyl mystery, 2,2·,4,4',6-pentabromodiphenyl ether, 2,3,4,4,6-pentabromodiphenyl ether, 2,2' , 4,4,,5-pentabromodiphenyl ether, 2,2',3,4,4,-pentabromodiphenyl ether, 3,3·,4,4|,5-pentabromodiphenyl ether, 2,2,,4,4,5,6,·HBromodiphenyl ether, 2,2',4,4',5,5'-six> Odor 2, 2', 3 , 4,4',6-six desert diphenyl mystery, 2,2',3,4,4,6,-hexabromodiphenyl ether, 2,2',3,4,4',5,- Hexabromodiphenyl ether, 2,3,3',4,4',5-hexabromodiphenyl ether, 2,2,3,4,4|,6,6,-heptabromodiphenyl ether, 2 , 2,3,4,4,5,6-seven desert one key, 2,3,3',4,4',5',6-heptbromobiphenyl mystery, 2,2,3,3, 4,4,6,6-octabromo This shout, 2,2',3,4,4',5,5',6-octabromobiphenyl, 2,2',3,3',4,4',5,6'-eight&gt Odorous benzene test '2,2|,3,3',4,5,5,6 6,· nonabrominated diphenyl ether, 2,2·,3,3',4,4',5,6 , 6'-nonabrominated diphenyl ether, decyl diphenyl ether, and combinations thereof. 33. The method of claim 28, wherein the dioxin compound is multi-gas and has 4 to 7 gas atoms. 34. The method of claim 33, wherein the polyglycol is selected from the group consisting of 3, 3', 4, 4, - tetrahydrobiphenyl, 3, 4, 4, , 5-tetrahydrobiphenyl, 2,3,3',4,4·-five gas stupid, 2,3,4,4,5_pentachlorobiphenyl, 2,3',4,4' , 5-pentachlorobiphenyl, 2,3,4,4,5-pentachlorobiphenyl, 3,3,,4,4,,5_ five gas, stupid, 2,3,3',4, 4',5-six gas, stupid, 2,3,3',4,4,,5'-six gas, stupid, 2,3',4,4',5,5'-hexabenzene, 3,3',4,4',5,5,-six gas, stupid, 2,3,3,4,4,5,5 - seven gas joints' and combinations thereof. 8 1377249 No. 2 Patent Application No. 2 of the No. 96 丨 _ 充 、 、 、 、 替换 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正 修正" Polycyclic aromatic hydrocarbons in shellfish contain 2 to 6 benzene rings. 36. The method of claim 35, wherein the polycyclic aromatic hydrocarbon present in the contaminated medium is selected from the group consisting of f, and naphthalene, Er' dihydrogen , the first, the Philippine 'onion', the second is the second, and the other is (a) onion, Qi's stupid and (b) the second association of the second and the second, stupid (8) the second generation, stupid and (four), material (123_ Cis, dibenzo (10) onion, sputum (g, h, sputum, and combinations thereof. 10 37.2 Please add (4) 23 methods, the cut property reagent can be = two _ from the following groups can be cleared Environmental pollutants =: = microorganisms of the genus Pseudomonas, microorganisms of the genus Desmoococcus, microorganisms of the genus Narcissus, microorganisms of the genus Aeromonas, Rhizobium, microorganisms of the genus Cyanobacteria, and椁 属 、 勒 勒 勒 勒 勒 勒 勒 勒 勒 勒 勒 勒 勒 勒 勒 勒 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 From: into the clearing of the environmental bacteria strain CA10, Wei Wei. Group: resin pseudo-single year Pseudomonas m. ΡΗ 4 species _, Pseudomonas Mendocs FER: p: cocci-wi and combinations thereof. - (10), 20 1377249 Patent application No. 096146052, amendments to the instructions without replacement of the line Replacement date: December 1999 VII. Designation of representative drawings: (1) The representative representative of the case is: (3). (2) The symbol of the symbol of the representative figure is simple: (none) 8. If there is a chemical formula in this case, please reveal the best display invention. Characteristic chemical formula: