JP4172992B2 - NOVEL MICROORGANISM, FAT AND FAT CONTAINING AGENT CONTAINING THE SAME, AND METHOD FOR TREATING OIL AND FAT CONTAINING SUBSTANCE USING THE SAME - Google Patents
NOVEL MICROORGANISM, FAT AND FAT CONTAINING AGENT CONTAINING THE SAME, AND METHOD FOR TREATING OIL AND FAT CONTAINING SUBSTANCE USING THE SAME Download PDFInfo
- Publication number
- JP4172992B2 JP4172992B2 JP2002334047A JP2002334047A JP4172992B2 JP 4172992 B2 JP4172992 B2 JP 4172992B2 JP 2002334047 A JP2002334047 A JP 2002334047A JP 2002334047 A JP2002334047 A JP 2002334047A JP 4172992 B2 JP4172992 B2 JP 4172992B2
- Authority
- JP
- Japan
- Prior art keywords
- fat
- oil
- acinetobacter
- medium
- microorganism
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 244000005700 microbiome Species 0.000 title claims description 31
- 238000000034 method Methods 0.000 title claims description 21
- 239000000126 substance Substances 0.000 title claims description 4
- 238000012360 testing method Methods 0.000 claims description 16
- 241000589291 Acinetobacter Species 0.000 claims description 14
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 241000588625 Acinetobacter sp. Species 0.000 claims description 10
- 239000002351 wastewater Substances 0.000 claims description 10
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 238000000855 fermentation Methods 0.000 claims description 7
- 230000004151 fermentation Effects 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 7
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 6
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 6
- 230000003647 oxidation Effects 0.000 claims description 6
- 238000007254 oxidation reaction Methods 0.000 claims description 6
- 102000004316 Oxidoreductases Human genes 0.000 claims description 5
- 108090000854 Oxidoreductases Proteins 0.000 claims description 5
- 230000001766 physiological effect Effects 0.000 claims description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- XUCIJNAGGSZNQT-JHSLDZJXSA-N (R)-amygdalin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O[C@@H](C#N)C=2C=CC=CC=2)O1 XUCIJNAGGSZNQT-JHSLDZJXSA-N 0.000 claims description 3
- 108010082340 Arginine deiminase Proteins 0.000 claims description 3
- 102100026189 Beta-galactosidase Human genes 0.000 claims description 3
- 108010053835 Catalase Proteins 0.000 claims description 3
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 3
- 102000013382 Gelatinases Human genes 0.000 claims description 3
- 108010026132 Gelatinases Proteins 0.000 claims description 3
- 238000003794 Gram staining Methods 0.000 claims description 3
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 3
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 claims description 3
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 3
- 108010048581 Lysine decarboxylase Proteins 0.000 claims description 3
- 101100447665 Mus musculus Gas2 gene Proteins 0.000 claims description 3
- 241000588653 Neisseria Species 0.000 claims description 3
- 108700005126 Ornithine decarboxylases Proteins 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 3
- 108010046334 Urease Proteins 0.000 claims description 3
- ROWKJAVDOGWPAT-UHFFFAOYSA-N acetyl methyl carbinol Natural products CC(O)C(C)=O ROWKJAVDOGWPAT-UHFFFAOYSA-N 0.000 claims description 3
- YZLOSXFCSIDECK-UHFFFAOYSA-N amygdalin Natural products OCC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC(C#N)c3ccccc3 YZLOSXFCSIDECK-UHFFFAOYSA-N 0.000 claims description 3
- SIKJAQJRHWYJAI-UHFFFAOYSA-N benzopyrrole Natural products C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 claims description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 3
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 3
- 238000010876 biochemical test Methods 0.000 claims description 3
- GFAZHVHNLUBROE-UHFFFAOYSA-N hydroxymethyl propionaldehyde Natural products CCC(=O)CO GFAZHVHNLUBROE-UHFFFAOYSA-N 0.000 claims description 3
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 claims description 3
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 claims description 3
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 3
- 229960000367 inositol Drugs 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 238000003672 processing method Methods 0.000 claims description 3
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 238000005516 engineering process Methods 0.000 claims description 2
- 102100035882 Catalase Human genes 0.000 claims 1
- 102100021079 Ornithine decarboxylase Human genes 0.000 claims 1
- 239000003921 oil Substances 0.000 description 26
- 239000002609 medium Substances 0.000 description 25
- 235000019198 oils Nutrition 0.000 description 23
- 239000003925 fat Substances 0.000 description 19
- 235000019197 fats Nutrition 0.000 description 14
- 238000002360 preparation method Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 239000003002 pH adjusting agent Substances 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 5
- 239000004519 grease Substances 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 239000004006 olive oil Substances 0.000 description 5
- 235000008390 olive oil Nutrition 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 239000008187 granular material Substances 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 239000006916 nutrient agar Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000006150 trypticase soy agar Substances 0.000 description 3
- 241001135518 Acinetobacter lwoffii Species 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000003262 industrial enzyme Substances 0.000 description 2
- 238000005339 levitation Methods 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 235000019645 odor Nutrition 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 241000588626 Acinetobacter baumannii Species 0.000 description 1
- 241000588624 Acinetobacter calcoaceticus Species 0.000 description 1
- 241001250710 Acinetobacter genomosp. 13 Species 0.000 description 1
- 241000759681 Acinetobacter genomosp. 13BJ Species 0.000 description 1
- 241001148231 Acinetobacter haemolyticus Species 0.000 description 1
- 241000122229 Acinetobacter johnsonii Species 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 241000224489 Amoeba Species 0.000 description 1
- 235000019737 Animal fat Nutrition 0.000 description 1
- 241000206761 Bacillariophyta Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 229920003043 Cellulose fiber Polymers 0.000 description 1
- 241000195628 Chlorophyta Species 0.000 description 1
- 241000223782 Ciliophora Species 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- 241000813862 Streptomyces saprophyticus Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- WHWDWIHXSPCOKZ-UHFFFAOYSA-N hexahydrofarnesyl acetone Natural products CC(C)CCCC(C)CCCC(C)CCCC(C)=O WHWDWIHXSPCOKZ-UHFFFAOYSA-N 0.000 description 1
- 239000002035 hexane extract Substances 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- GVALZJMUIHGIMD-UHFFFAOYSA-H magnesium phosphate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GVALZJMUIHGIMD-UHFFFAOYSA-H 0.000 description 1
- 239000004137 magnesium phosphate Substances 0.000 description 1
- 229960002261 magnesium phosphate Drugs 0.000 description 1
- 229910000157 magnesium phosphate Inorganic materials 0.000 description 1
- 235000010994 magnesium phosphates Nutrition 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 108010050327 trypticase-soy broth Proteins 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Description
【０００１】
【発明の属する技術分野】
本発明は、新規な微生物、特に油脂分解性を有する微生物、それを含む油脂分解剤およびそれを用いた油脂含有物質の処理方法に関する。
【０００２】
【従来の技術】
油脂分を含む排水の処理は、工業的には加圧浮上装置、自然浮上槽（オイルピット）あるいは油分分離槽（グリーストラップ）等の処理施設が用いられている。
しかし、従来用いられているグリーストラップなどの油脂分除外設備の設置では、固形化油脂の蓄積、下水管の閉塞、悪臭の発生、排水基準値の超過などの問題が煩雑に発生する。
この問題を解決する排水の処理技術の一つに自然界に存在する種々の生物、特に微生物を利用して排水中の油脂分を含む汚濁物質を除去したり分解する生物学的処理法がある。このための微生物としてはＢａｃｉｌｌｕｓ ｓｐ．，Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．ＳＫ０４０１株（微工研菌１３２３１），Ａｃｉｎｅｔｏｂａｃｔｅｒ ｓｐ．ＳＫ０４０２Ａ株（微工研菌１３２３２），Ｓ．ｓａｐｒｏｐｈｙｔｉｃｕｓ ＯＤ−１（微工研菌１７２０１）などが、原生動物としてはアメーバー類、繊毛虫類、鞭毛虫類などが、また藻類としては藍藻類、緑藻類、ケイ藻類などがある。本発明と同じＡｃｉｎｅｔｏｂａｃｔｅｒ属に属する菌については、特許文献１において、Ａｃｉｎｅｔｏｂａｃｔｅｒ ｓｐ．ＳＫ０４０２Ａ（微工研菌１３２３２）が開示されているが、これは、運動性短桿菌であって、本発明のものが非運動性球状桿菌である点で異なっている。
【０００３】
【特許文献１】
特開平６−１５３９２２号公報
【０００４】
【発明が解決しようとする課題】
本発明の目的は、比較的簡単な装置により、工場排水は勿論、ラーメン屋、洋食屋、てんぷら屋などの排水を手軽に処理するために有用な新規な微生物、それを含有する油脂分解剤、および該分解剤を用いた油脂含有物質の処理方法を提供する点にある。
【０００５】
【課題を解決するための手段】
本発明の第1は、Ａｃｉｎｅｔｏｂａｃｔｅｒ属に属し、非運動性、グラム染色陰性でカタラーゼ陽性、オキシダーゼ陰性を示す球状桿菌であって、コロニー形態として、円形、周縁波状、低凸状であり、光沢のある淡黄色を呈し、かつ、油脂分解性を有するとともに、配列番号１で示される１６ＳｒＤＮＡの塩基配列を有することを特徴とする微生物に関する。
本発明の第２は、生理性状試験結果が下記のものである請求項１記載の微生物に関する。
生理性状試験結果
１．生化学試験
β−ガラクトシダーゼ −
アルギニンジヒドロラーゼ −
リシンデカルボキシラーゼ −
オルニチンデカルボキシラーゼ −
クエン酸の利用性 ＋
Ｈ２Ｓ産生 −
ウレアーゼ −
トリプトファンデアミナーゼ −
インドール産生 −
アセトイン産生 −
ゼラチナーゼ −
オキシダーゼ −
ＮＯ２産生 −
Ｎ2ガスへの還元 −
２．発酵／酸化試験
ブドウ糖 ＋
Ｄ−マンニトール ＋
イノシトール −
Ｄ−ソルビトール −
Ｌ−ラムノース ＋
白糖 −
Ｄ−メリビオース ＋
Ｄ−アミグダリン ＋
Ｌ−アラビノース ＋
ＯＦ培地での酸化 ＋
ＯＦ培地での発酵 ＋
なお、上記試験において「＋」は陽性、「−」は陰性を示す。
本発明の第３は、独立行政法人産業技術総合研究所 特許生物寄託センター 受託番号ＦＥＲＭ Ｐ−１８８０５で示されるアシネトバクター（Ａｃｉｎｅｔｏｂａｃｔｅｒ） ｓｐ．ＧＫＮ−４株に関する。
本発明の第４は、請求項１〜３のいずれかに記載の微生物を含有することを特徴とする油脂分解剤に関する。
本発明の第５は、請求項４記載の油脂分解剤を用いることを特徴とする油脂含有物質の処理方法に関する。
本発明の第６は、前記油脂含有物質が排水である請求項５記載の油脂含有物質の処理方法に関する。
【０００６】
本発明者等は、油脂を効率的に分解する微生物を分離することに成功し、本発明を完成した。
本発明における油脂とは、主として動植物由来のものであるが、必ずしもそれに限定するものではなく、合成法により得られた油脂類やその誘導体およびその他の種々の炭化水素等を含むものである。
【０００７】
本発明において用いた培地の組成
＜無機培地の組成＞
（ＮＨ４）２ＰＯ４ ２．０ｇ／ｌ
Ｋ２ＨＰＯ４ ２．０ｇ／ｌ
ＮａＨ２ＰＯ４ １．０ｇ／ｌ
ＭｇＳＯ４ ０．２ｇ／ｌ
蒸留水を加えて、全体で１リットルとした。
ｐＨ：７．０
＜栄養寒天培地の組成＞
ポリペプトン １０．０ｇ／ｌ
酵母エキス ５．０ｇ／ｌ
ＮａＣｌ ５．０ｇ／ｌ
寒天 １０．０ｇ／ｌ
蒸留水を加えて、全体で１リットルとした。
ｐＨ：７．３
＜栄養培地の組成＞
ポリペプトン １．８ｇ／ｌ
肉エキス １．２ｇ／ｌ
尿素 ０．３ｇ／ｌ
ＮａＣｌ ０．０９ｇ／ｌ
Ｋ２ＨＰＯ４ ０．３ｇ／ｌ
ＫＣｌ ０．０４２ｇ／ｌ
ＣａＣｌ２・２Ｈ２Ｏ ０．０４２ｇ／ｌ
ＭｇＳＯ４・７Ｈ２Ｏ ０．０３ｇ／ｌ
蒸留水を加えて、全体で１リットルとした。
＜トリプチカーゼ 大豆 寒天培地 （Ｔｒｙｐｔｉｃａｓｅ Ｓｏｙ Ａｇａｒ）＞
トリプチカーゼ ペプトン（Ｔｒｙｐｔｉｃａｓｅ Ｐｅｐｔｏｎｅ） １５ｇ
フィトン ペプトン（Ｐｈｙｔｏｎｅ Ｐｅｐｔｏｎｅ） ５ｇ
ＮａＣｌ ５ｇ
寒天（Ａｇａｒ） １５ｇ
＜培地Ａ＞
ペプトン １０ｇ／ｌ
肉エキス １０ｇ／ｌ
ＮａＣｌ ２ｇ／ｌ
蒸留水を加えて、全体で１リットルとした。
ｐＨ：７．０
＜培地Ｂ＞
リン酸２アンモニウム ２ｇ／ｌ
リン酸２カリウム ２ｇ／ｌ
リン酸１ナトリウム １ｇ／ｌ
リン酸マグネシウム ０．２ｇ／ｌ
酵母エキス ０．１ｇ／ｌ
蒸留水を加えて、全体で１リットルとした。
ｐＨ：７．０
【０００８】
（イ）新潟県黒川村油田近郊から採取した土壌を蒸留水に加え振とうした後、その上澄み液を分取した。
（ロ）水道水１リットルを入れたビーカーに（イ）で分取された上澄み液とオリーブオイルを加え水中ポンプで３日間撹拌した。
（ハ）上記無機培地１０ｍｌを加えたＬ−字管に（ロ）によって得られた撹拌液１００μｌ及びオリーブオイル１００μｌを加え、３０℃で培養液が懸濁するまで振とう培養した。
（ニ）培養液の懸濁が認められた後、上記無機培地１０ｍｌを入れたＬ−字管に（ハ）の懸濁液１００μｌ及びオリーブオイル１００μｌを加え、３０℃で再度培養液が懸濁するまで振とう培養した。
（ホ）培養液の懸濁が認められた後、（ニ）の操作を５回繰り返した。
（ヘ）（ホ）の操作が終了した後、培養液１００μｌを１０００倍に希釈し、上記栄養寒天培地上に滴下し、コラージ棒で均一にのばし一晩３０℃で培養する。
（ト）栄養寒天培地上に発現したコロニーを単離し、オリーブオイルの分解実験を行い、分解率の高い菌株を選択し、ＧＫＮ−４とした。
【０００９】
本発明微生物であるＡｃｉｎｅｔｏｂａｃｔｅｒ ｓｐ．ＧＫＮ−４と前記Ａｃｉｎｅｔｏｂａｃｔｅｒ ｓｐ．ＳＫ０４０２Ａとの相違点を下記表に示す。
【００１０】
【表１】
本発明の微生物を油脂分解剤として提供するためには、本発明の微生物を、種々の担体により固定化し、製剤化すればよい。具体的には、例えば１９９３年培風館発行、村尾澤夫他１名編「応用微生物学改訂版」や１９９５年丸善発行、上島孝之著、「産業用酵素」などに記載の製剤法を採用することができる。
【００１２】
液状製剤とするには、下記の方法を使用することができる。
（１）栄養培地で本発明の微生物を１２〜３６時間程度培養し、必要に応じてこれにｐＨ調整剤などを加えて製剤とする。
（２）前記（１）の培養物から菌体を遠心分離などで回収し、生理食塩水などの媒体に適当な濃度となるように懸濁し、必要に応じてこれにｐＨ調整剤などを加えて製剤とする。
（３）前記（１）の培養物を凍結乾燥などにより適度に濃縮し、必要に応じてこれにｐＨ調整剤などを加えて製剤とする。
（４）前記（１）の培養物から菌体を遠心分離などで回収し、栄養培地に懸濁し、必要に応じてこれにｐＨ調整剤などを加えて製剤とする。
（５）前記（４）をさらに凍結乾燥などにより適度に濃縮して製剤とする。
【００１３】
粉末製剤とするには、下記の方法を採用することができる。
（ａ）栄養培地で本発明の微生物を１２〜３６時間程度培養し、必要に応じてｐＨ調整剤などを加え、凍結乾燥などにより乾燥したものを製剤とする。
（ｂ）前記（ａ）の培養物から菌体を遠心分離などで回収し、生理食塩水あるいはスキムミルクとグルタミン酸ナトリウムなどからなる溶液などの媒体に適当な濃度となるように懸濁し、必要に応じてｐＨ調整剤を加え、凍結乾燥などにより乾燥したものを製剤とする。
（ｃ）前記（ａ）の培養物から菌体を遠心分離などで回収し、栄養培地に懸濁し、必要に応じてｐＨ調整剤などを加え、凍結乾燥などにより乾燥し製剤とする。
（ｄ）前記（ａ）〜（ｃ）のものに、繊維くず、おがくずなどの微粉体を適度に加えたものを製剤とする。
（ｅ）例えば１９９５年丸善発行、上島孝之著、「産業用酵素」に記載されている方法により、本発明の微生物を顆粒化することができる。具体的には（ａ）〜（ｄ）と同様にして調製した菌体懸濁液をプリム顆粒、マルム顆粒、フィルムコーティングしたマルム顆粒、セルロース繊維配合のＴ顆粒などとする。
【００１４】
また、前記方法以外にも、担体結合法、架橋法、包括法、複合法などの公知の技術により、本発明の微生物を種々の固定化用材料を用いて、固定化することができ、さらに、公知の錠剤化技術により、本発明微生物を錠剤化することもできる。
【００１５】
本発明の微生物を使用するにあたっては、微生物の高い活性を維持するために空気を吹き込むための散気手段、すなわちエアレーション手段を採用することが好ましい。また本発明の微生物を用いた処理装置においては本発明の微生物もしくは本発明の油脂分解剤の流出を防止し、かつ油脂分と分離するためのトラップを付設することが好ましい。トラップとしては、所定のポアサイズ（微生物は透過せず、水や低分子化合物などのみを通過させる）を有する膜フィルターをもつものが好ましい。また低温で固形化するような動物油脂を多く含む排水の場合には、ヒーターなどによるトラップ内の排水温度をコントロールすることにより一層高い効果を挙げることができる。
【００１６】
【実施例】
以下に実施例を挙げて本発明を説明するが、本発明はこれにより何ら限定され得るものではない。
【００１７】
実施例１
本発明のＧＫＮ−４株について、１２１℃で１５分間滅菌したトリプチカーゼ大豆 寒天培地 （Ｔｒｙｐｔｉｃａｓｅ Ｓｏｙ Ａｇａｒ）（ＢＢＬ、米国）上でのコロニー形態、顕微鏡観察による細胞形態、グラム染色、胞子の有無、鞭毛による運動性の有無およびカタラーゼ、オキシダーゼ、ブドウ糖の酸化発酵（ＯＦ）などの観察・試験を行った結果を表２に示す。
【表２】
実施例２
本発明のＧＫＮ−４株の生化学的同定を行った。各種試験は、ＫＲＩＥＧ，（Ｎ．Ｒ．） ａｎｄ ＨＯＬＴ，（Ｊ．Ｇ．）１９８４：Ｂｅｒｇｅｙ’ｓ ｍａｎｕａｌ ｏｆ Ｓｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ．Ｖｏｌ．１． Ｗｉｌｌｉａｍｓ ａｎｄ Ｗｉｌｋｉｎｓ， Ｂａｌｔｉｍｏｒｅ．を参考に、ＡＰＩ ２０Ｅの測定方法にしたがって測定した測定結果を下記に示す。
１．生化学試験
β−ガラクトシダーゼ −
アルギニンジヒドロラーゼ −
リシンデカルボキシラーゼ −
オルニチンデカルボキシラーゼ −
クエン酸の利用性 ＋
Ｈ２Ｓ産生 −
ウレアーゼ −
トリプトファンデアミナーゼ −
インドール産生 −
アセトイン産生 −
ゼラチナーゼ −
オキシダーゼ −
ＮＯ２産生 −
Ｎ2ガスへの還元 −
２．発酵／酸化試験
ブドウ糖 ＋
Ｄ−マンニトール ＋
イノシトール −
Ｄ−ソルビトール −
Ｌ−ラムノース ＋
白糖 −
Ｄ−メリビオース ＋
Ｄ−アミグダリン ＋
Ｌ−アラビノース ＋
ＯＦ培地での酸化 ＋
ＯＦ培地での発酵 ＋
なお、上記試験において「＋」は陽性、「−」は陰性を示す。
【００１９】
実施例３
ＧＫＮ−４株を１２１℃で１５分間滅菌したトリプチカーゼ 大豆 寒天培地（Ｔｒｙｐｔｉｃａｓｅ Ｓｏｙ Ａｇａｒ）（ＢＢＬ、米国）に植菌し、３０℃での培養物を供試菌体とし、菌体からゲノムＤＮＡを抽出した。抽出したゲノムＤＮＡを鋳型としてＰＣＲにより１６ＳｒＤＮＡの塩基配列約１６００ｂｐを増幅し、塩基配列をシーケンスして解析に使用した。ＰＣＲ産物の精製、サイクルシークエンスにはＭｉｃｒｏＳｅｑ（商標名）５００ １６ＳｒＤＮＡ バクテリアル シークエンシング キット〔Ｂａｃｔｅｒｉａｌ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ，米国）〕を使用した。ゲノムＤＮＡ抽出からサイクルシークエンスまでの操作に関してはＡｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社のプロトコール（Ｐ／Ｎ４３０８１３２ Ｒｅｖ．Ａ）に従った。サーマルサイクラーにはＧｅｎｅＡｍｐ（商標名）ＰＣＲＳｙｓｔｅｍ９６００（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社 米国）、ＤＮＡシークエンサーにはＡＢＩ ＰＲＩＳＭ（商標名）３７７ ＤＮＡ Ｓｅｑｕｅｎｃｅｒ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社 米国）を使用した。
ＤＮＡシークエンサーによって解析した結果、本発明菌株ＧＫＮ−４の１６ＳｒＤＮＡをコードする遺伝子は、配列番号１に示す１４９４ｂａｓｅの塩基対からなることが確認された。
【００２０】
得られた１６ＳｒＤＮＡの塩基配列を使って相同性検索、系統樹の作製をし、検体の近縁種および帰属分類群の検討を行った。相同性検索および系統樹の作製にはＭｉｃｒｏＳｅｑ（商標名）Ｍｉｃｒｏｂｉａｌ Ｉｄｅｎｔｉｆｉｃａｔｉｏｎ Ｓｙｓｔｅｍ Ｓｏｆｔｗａｒｅ Ｖ．１．４．１およびＭｉｃｒｏＳｅｑ（商標名）Ｂａｃｔｅｒｉａｌ ５００ Ｌｉｂｒａｒｙ ｖ．００２３（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社 米国）を使用した。
【００２１】
本発明の菌株と近縁とされる菌株とその相違性および近縁上位株との相違点を以下に示す。
ＧＫＮ−４株と近縁とされる菌株とその相違性
【表３】
Library: FG0001 0.9 1209/1209
BLAST: 1494 GKN-4
1.00% 1533 Acinetobacter genomospecies 3
1.34% 1533 Acinetobacter genomospecies 13
2.95% 1533 Acinetobacter calcoaceticus
3.35% 1532 Acinetobacter baumannii
3.35% 1533 Acinetobacter genomospecies 6
4.08% 1533 Acinetobacter haemolyticus
4.35% 1533 Acinetobacter genomospecies 14
4.89% 1533 Acinetobacter lwoffii
4.89% 1533 Acinetobacter johnsonii
4.89% 1533 Acinetobacter genomospecies 9
近縁上位株との相違点
【表４】
ＧＫＮ−４と近縁株との近隣結合法による系統樹を表５に示す。
【表５】
本発明のＧＫＮ−４株の１６ＳｒＤＮＡ塩基配列は相同率９９．００％でＡｃｉｎｅｔｏｂａｃｔｅｒ ｇｅｎｏｍｏｓｐｅｃｉｅｓ ３に対し最も高い相同性を示した。分子系統樹上でもＧＫＮ−４株は、Ａｃｉｎｅｔｏｂａｃｔｅｒ ｇｅｎｏｍｏｓｐｅｃｉｅｓ ３、Ａｃｉｎｅｔｏｂａｃｔｅｒ ｃａｌｃｏａｃｅｔｉｃｕｓとクラスターを形成し近縁であることが示された。
Ａｃｉｎｅｔｏｂａｃｔｅｒ ｇｅｎｏｍｏｓｐｅｃｉｅｓ ３（ＡＴＣＣ１７９２２株）は、Ａｃｉｎｅｔｏｂａｃｔｅｒ ｃａｌｃｏａｃｅｔｉｃｕｓの一系統株で、ｇｅｎｏｍｏｓｐｅｃｉｅｓとはＡｃｉｎｅｔｏｂａｃｔｅｒにおいてＤＮＡ−ＤＮＡ相同性の比較から認められた集団に適用されたグループ名である。本発明のＧＫＮ−４株は、別に行った形態学的・生理性状学的試験から腸内細菌群の性状に類似している。本発明のＧＫＮ−４株の生理性状は多くの性状においてＡｃｉｎｅｔｏｂａｃｔｅｒの典型性状にも一致したが、ブドウ糖の発酵性を示す点で絶対好気性菌として記載されているＡｃｉｎｅｔｏｂａｃｔｅｒの典型性状とは異なるが、１６ＳｒＤＮＡを用いた解析から本発明のＧＫＮ−４株は、Ａｃｉｎｅｔｏｂａｃｔｅｒへの帰属が示唆されており、本発明のＧＫＮ−４株は、従来公知のＡｃｉｎｅｔｏｂａｃｔｅｒ ｓｐ．に比較して極めて優れた油脂分解性能を有することから、新規微生物と判断し、Ａｃｉｎｅｔｏｂａｃｔｅｒ ｓｐ．ＧＫＮ−４株とした。
【００２４】
実施例４
培地Ａ、培地Ｂはともに調整後、オートクレーブ滅菌し、培地Ｂについては、さらにフィルターを用いて濾過をした。
Ｌ字管に培地Ａを１０ｍｌに微生物（ＧＫＮ−４）の栄養寒天培地に発現した単コロニーを植菌し、２５℃、４０ｒｅｐ／ｍｉｎで振とう培養を行い、前培養液とした。
次いで、５００ｍｌ坂口フラスコに培地Ｂを１４０ｍｌ、上記前培養液１０ｍｌおよび基質としてオリーブオイルを添加し、４８時間１２０ｒｅｐ／ｍｉｎで振とう培養を行った。また、前培養液を加えず、同様の実験を行い、対照例とした。
【００２５】
オートクレーブ滅菌後、ＪＩＳ法に基づきｎ−ヘキサン抽出を行った。
１．ｐＨ７での温度変化による分解性能を表６に示す。
【表６】
２．表６で判明した最適温度（２８℃）にてｐＨ変化による分解性能を表７に示す。
【表７】
３．最適温度（２８℃）、最適ｐＨ（ｐＨ８）を用いての油脂添加量別分解性能を表８に示す。
【表８】
【００２８】
Ａホテル厨房内グリーストラップにおいて、平成１３年１０月６日から１０月２４日の１９日間にわたり、実際の排水について、本発明のＡｃｉｎｅｔｏｂａｃｔｅｒ ｓｐ．ＧＫＮ−４株からなる製剤を添加した場合の、水温、ｐＨ、溶存酸素（ＤＯ）、アンモニア性窒素、ノルマルヘキサン抽出物質（ｎ−Ｈｅｘ値）（ＪＩＳ Ｋ ０１０２による）の５項目について測定を行った。
このＡホテル厨房内グリーストラップは、４００ｍｍ×５００ｍｍ×１０００ｍｍ＝２００リットルで、油分量は、約５〜６リットル／日、使用水量４０ｔ／日、油分濃度は約１５０００〜約３０００ｐｐｍであり、通常は、１日１回、凝集剤により凝集させ、手作業による汲み取りを実施することによりグリーストラップの清掃を実施していた。
実験スケジュールは以下の表９に示す通りである。
【表９】
油分解微生物製剤ＧＫＮ−４によるフィールド試験結果を表１０に示す。
【表１０】
【００３０】
通常通り、手作業による油脂分を完全に回収することは難しく、実際に清掃後、採取した排水の測定結果を見ても１９６１ｐｐｍという高い数値を示していた。微生物製剤投入後、２日目（１０月８日）は７２１ｐｐｍという数値を示しているが、投入前と比較すると約１／３の値に減少していることが分かる。（これは微生物増殖段階と考えられる。）４日目以降は、多少の増減は見られるが、平均６３．９ｐｐｍという数値が示され、投入前と比較すると明らかな差があり、微生物製剤による投入効果が充分に発揮することができた。
また、試験期間中は槽内からの悪臭の発生も感じられなかった。
【００３１】
【発明の効果】
本発明により、油脂を分解する能力を有する新規な微生物、それを含有する油脂分解剤、それを用いた油脂含有物質の処理方法を提供することができた。
【００３２】
【配列表】
BACKGROUND OF THE INVENTION
The present invention relates to a novel microorganism, particularly a microorganism capable of decomposing oil and fat, an oil-and-oil decomposing agent containing the same, and a method for treating an oil-containing material using the same.
[0002]
[Prior art]
For the treatment of wastewater containing oil and fat, industrial treatment facilities such as a pressurized levitation device, a natural levitation tank (oil pit) or an oil separation tank (grease trap) are used.
However, installation of oil and fat content exclusion equipment such as grease traps that have been used in the past causes complicated problems such as accumulation of solid oil and fat, clogging of sewage pipes, generation of bad odors, and exceeding drainage standard values.
One of the wastewater treatment techniques for solving this problem is a biological treatment method that removes or decomposes pollutants including fats and oils in wastewater using various organisms existing in nature, particularly microorganisms. As a microorganism for this purpose, Bacillus sp. , Pseudomonas sp. SK0401 strain (MICROKEN 13132), Acinetobacter sp. SK0402A strain (MICROKEN 13132), S. saprophyticus OD-1 (Kojiken 17201) and the like, protozoa such as amoeba, ciliate and flagellate, and algae include cyanobacteria, green algae and diatoms. Regarding the bacteria belonging to the same Acinetobacter genus as in the present invention, Patent Document 1 discloses Acinetobacter sp. SK0402A (Mikken Ken 13232) is disclosed, which is a motile short bacillus and differs in that the present invention is a non-motile gonococcus.
[0003]
[Patent Document 1]
Japanese Patent Application Laid-Open No. 6-153922
[Problems to be solved by the invention]
An object of the present invention is to provide a novel microorganism useful for easily treating wastewater from a ramen shop, a Western restaurant, a tempura shop, etc., as well as factory wastewater, by a relatively simple device, an oil-and-fat decomposer containing the same, Another object of the present invention is to provide a method for treating an oil-containing material using the decomposition agent.
[0005]
[Means for Solving the Problems]
The first of the present invention is a gonococcus belonging to the genus Acinetobacter, non-motile, gram staining negative, catalase positive, oxidase negative, colony form, round, fringe, low convex, glossy The present invention relates to a microorganism that exhibits a certain light yellow color, has oil-degradability, and has the base sequence of 16SrDNA represented by SEQ ID NO: 1 .
A second aspect of the present invention relates to the microorganism according to claim 1, wherein the physiological property test result is as follows.
Results of physiological property test Biochemical test β-galactosidase −
Arginine dihydrolase −
Lysine decarboxylase −
Ornithine decarboxylase −
Availability of citric acid +
H 2 S production −
Urease −
Tryptophan deaminase −
Indole production −
Acetoin production −
Gelatinase −
Oxidase −
NO 2 production −
Reduction to N 2 gas −
2. Fermentation / Oxidation Test Glucose +
D-mannitol +
Inositol −
D-sorbitol-
L-rhamnose +
Sucrose −
D-melibiose +
D-Amygdalin +
L-arabinose +
Oxidation in OF medium +
Fermentation in OF medium +
In the above test, “+” indicates positive and “−” indicates negative.
A third aspect of the present invention is an Acinetobacter sp. Represented by the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Accession No. FERM P-18805. It relates to GKN-4 strain.
4th of this invention is related with the fats and oils decomposition agent characterized by containing the microorganisms in any one of Claims 1-3.
5th of this invention is related with the processing method of the fat-and-oil containing substance characterized by using the fat-and-oil decomposer of Claim 4.
6th of this invention is related with the processing method of the oil-and-fat containing material of Claim 5 whose said oil-and-fat containing material is waste_water | drain.
[0006]
The present inventors have succeeded in separating microorganisms that efficiently decompose fats and oils, thereby completing the present invention.
The fats and oils in the present invention are mainly derived from animals and plants, but are not necessarily limited thereto, and include fats and oils obtained by a synthesis method, derivatives thereof, and other various hydrocarbons.
[0007]
Composition of medium used in the present invention <Composition of inorganic medium>
(NH 4 ) 2 PO 4 2.0 g / l
K 2 HPO 4 2.0 g / l
NaH 2 PO 4 1.0 g / l
MgSO 4 0.2 g / l
Distilled water was added to make a total of 1 liter.
pH: 7.0
<Composition of nutrient agar>
Polypeptone 10.0g / l
Yeast extract 5.0 g / l
NaCl 5.0 g / l
Agar 10.0 g / l
Distilled water was added to make a total of 1 liter.
pH: 7.3
<Composition of nutrient medium>
Polypeptone 1.8g / l
Meat extract 1.2g / l
Urea 0.3g / l
NaCl 0.09 g / l
K 2 HPO 4 0.3 g / l
KCl 0.042g / l
CaCl 2 · 2H 2 O 0.042 g / l
MgSO 4 · 7H 2 O 0.03 g / l
Distilled water was added to make a total of 1 liter.
<Trypticase Soy Agar>
Trypticase Peptone 15g
Phytone Peptone 5g
NaCl 5g
Agar 15g
<Medium A>
Peptone 10g / l
Meat extract 10g / l
NaCl 2g / l
Distilled water was added to make a total of 1 liter.
pH: 7.0
<Medium B>
Ammonium phosphate 2g / l
Dipotassium phosphate 2g / l
Monosodium phosphate 1g / l
Magnesium phosphate 0.2g / l
Yeast extract 0.1g / l
Distilled water was added to make a total of 1 liter.
pH: 7.0
[0008]
(B) The soil collected from the oil field near Kurokawa Village in Niigata Prefecture was added to distilled water and shaken, and then the supernatant was collected.
(B) The supernatant and olive oil separated in (a) were added to a beaker containing 1 liter of tap water, and the mixture was stirred with a submersible pump for 3 days.
(C) To the L-shaped tube to which 10 ml of the inorganic medium was added, 100 μl of the stirring solution obtained in (b) and 100 μl of olive oil were added, and cultured with shaking at 30 ° C. until the culture solution was suspended.
(D) After the suspension of the culture solution was observed, 100 μl of the suspension of (c) and 100 μl of olive oil were added to the L-shaped tube containing 10 ml of the inorganic medium, and the culture solution was suspended again at 30 ° C. Shake culture until complete.
(E) After the suspension of the culture solution was observed, the operation (d) was repeated 5 times.
(F) After the operations of (e) are completed, 100 μl of the culture solution is diluted 1000 times, dropped onto the nutrient agar medium, spread uniformly with a collagen rod, and cultured at 30 ° C. overnight.
(G) A colony expressed on a nutrient agar medium was isolated, and an olive oil degradation experiment was performed. A strain with a high degradation rate was selected and designated GKN-4.
[0009]
Acinetobacter sp. Which is the microorganism of the present invention. GKN-4 and the Acinetobacter sp. Differences from SK0402A are shown in the table below.
[0010]
[Table 1]
In order to provide the microorganism of the present invention as an oil and fat decomposing agent, the microorganism of the present invention may be fixed by various carriers and formulated. Specifically, the formulation method described in, for example, 1993 Baifukan, Murao Sawao et al. “Applied Microbiology Revised Edition”, 1995 Maruzen, Ueshima Takayuki, “Industrial Enzymes” may be adopted. it can.
[0012]
In order to obtain a liquid preparation, the following method can be used.
(1) The microorganism of the present invention is cultured in a nutrient medium for about 12 to 36 hours, and a pH adjuster or the like is added thereto as necessary to prepare a preparation.
(2) Bacteria are collected from the culture of (1) by centrifugation, suspended in a medium such as physiological saline, etc., and added with a pH adjuster or the like as necessary. To make a preparation.
(3) The culture of (1) above is moderately concentrated by freeze-drying or the like, and if necessary, a pH adjuster or the like is added to prepare a preparation.
(4) Bacteria are collected from the culture of (1) by centrifugation, suspended in a nutrient medium, and added with a pH adjuster or the like as necessary to prepare a preparation.
(5) The above (4) is further appropriately concentrated by freeze-drying or the like to prepare a preparation.
[0013]
In order to obtain a powder formulation, the following method can be employed.
(A) The microorganism of the present invention is cultured for about 12 to 36 hours in a nutrient medium, a pH adjuster or the like is added as necessary, and the product dried by lyophilization or the like is used as a preparation.
(B) The bacterial cells are collected from the culture of (a) by centrifugation and suspended in a medium such as physiological saline or a solution comprising skim milk and sodium glutamate to obtain an appropriate concentration. Add a pH adjuster and dry the product by freeze-drying.
(C) Bacteria are collected from the culture of (a) by centrifugation, suspended in a nutrient medium, added with a pH adjuster or the like as necessary, and dried by lyophilization to obtain a preparation.
(D) A preparation obtained by appropriately adding fine powders such as fiber waste and sawdust to the above (a) to (c) is used.
(E) The microorganism of the present invention can be granulated by the method described in, for example, 1995 published by Maruzen, written by Takayuki Kamijima, “industrial enzyme”. Specifically, the cell suspension prepared in the same manner as in (a) to (d) is used as prim granules, malm granules, film-coated malm granules, cellulose fiber-containing T granules, and the like.
[0014]
In addition to the above methods, the microorganism of the present invention can be immobilized using various immobilization materials by known techniques such as a carrier binding method, a crosslinking method, a comprehensive method, and a composite method, The microorganisms of the present invention can also be tableted by a known tableting technique.
[0015]
In using the microorganism of the present invention, it is preferable to employ an aeration means for blowing air, that is, an aeration means, in order to maintain a high activity of the microorganism. Moreover, in the processing apparatus using the microorganisms of the present invention, it is preferable to provide a trap for preventing the microorganisms of the present invention or the oil decomposing agent of the present invention from flowing out and separating them from the oils and fats. The trap preferably has a membrane filter having a predetermined pore size (which does not allow microorganisms to permeate and allows only water, low molecular weight compounds, etc. to pass through). Further, in the case of wastewater containing a large amount of animal fat that solidifies at a low temperature, a higher effect can be obtained by controlling the temperature of the wastewater in the trap with a heater or the like.
[0016]
【Example】
Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.
[0017]
Example 1
About the GKN-4 strain of the present invention, colony morphology on Trypticase Soy Agar (BBL, USA) sterilized at 121 ° C. for 15 minutes, cell morphology by microscopic observation, Gram staining, presence of spores, flagella Table 2 shows the results of observation and testing of the presence or absence of motility and catalase, oxidase, glucose oxidative fermentation (OF), and the like.
[Table 2]
Example 2
Biochemical identification of the GKN-4 strain of the present invention was performed. Various tests were performed according to KRIEG, (N.R.) and HOLT, (J.G.) 1984: Bergey's manual of Systematic Bacteriology. Vol. 1. Williams and Wilkins, Baltimore. The measurement results measured in accordance with API 20E measurement method are shown below.
1. Biochemical test β-galactosidase −
Arginine dihydrolase −
Lysine decarboxylase −
Ornithine decarboxylase −
Availability of citric acid +
H 2 S production −
Urease −
Tryptophan deaminase −
Indole production −
Acetoin production −
Gelatinase −
Oxidase −
NO 2 production −
Reduction to N 2 gas −
2. Fermentation / Oxidation test glucose +
D-mannitol +
Inositol −
D-sorbitol-
L-rhamnose +
Sucrose −
D-melibiose +
D-Amygdalin +
L-arabinose +
Oxidation in OF medium +
Fermentation in OF medium +
In the above test, “+” indicates positive and “−” indicates negative.
[0019]
Example 3
GKN-4 strain was inoculated into Trypticase Soy Agar (BBL, USA) sterilized at 121 ° C. for 15 minutes, and a culture at 30 ° C. was used as a test cell. Extracted. Using the extracted genomic DNA as a template, a base sequence of about 1600 bp of 16S rDNA was amplified by PCR, and the base sequence was sequenced and used for analysis. For the purification and cycle sequencing of the PCR product, MicroSeq (trade name) 500 16SrDNA bacterial sequencing kit (Bacterial Sequencing Kit (Applied Biosystems, USA)) was used. The procedure from genomic DNA extraction to cycle sequence was in accordance with Applied Biosystems protocol (P / N4308132 Rev. A). GeneAmp ™ PCR System 9600 (Applied Biosystems USA) was used for the thermal cycler, and ABI PRISM ™ 377 DNA Sequencer (Applied Biosystems USA) was used for the DNA sequencer.
As a result of analysis by a DNA sequencer, it was confirmed that the gene encoding 16S rDNA of the strain GKN-4 of the present invention consists of the 1494 base base pair shown in SEQ ID NO: 1.
[0020]
Using the obtained 16S rDNA base sequence, homology search and phylogenetic tree construction were carried out, and related species of species and belonging taxonomic groups were examined. For the homology search and generation of phylogenetic trees, MicroSeq ™ Microbib Identification System Software V. 1.4.1 and MicroSeq ™ Bacterial 500 Library v. 0023 (Applied Biosystems USA) was used.
[0021]
The difference between the strain of the present invention and the related strain and the difference between the related strain and the related upper strain is shown below.
Strains related to GKN-4 and their differences [Table 3]
Library: FG0001 0.9 1209/1209
BLAST: 1494 GKN-4
1.00% 1533 Acinetobacter genomospecies 3
1.34% 1533 Acinetobacter genomospecies 13
2.95% 1533 Acinetobacter calcoaceticus
3.35% 1532 Acinetobacter baumannii
3.35% 1533 Acinetobacter genomospecies 6
4.08% 1533 Acinetobacter haemolyticus
4.35% 1533 Acinetobacter genomospecies 14
4.89% 1533 Acinetobacter lwoffii
4.89% 1533 Acinetobacter johnsonii
4.89% 1533 Acinetobacter genomospecies 9
Differences from close relatives [Table 4]
Table 5 shows the phylogenetic tree of GKN-4 and related strains based on the neighborhood binding method.
[Table 5]
The 16S rDNA base sequence of the GKN-4 strain of the present invention showed the highest homology to Acinetobacter genomospecies 3 with a homology rate of 99.00%. It was shown that the GKN-4 strain formed a cluster with Acinetobacter genomospecies 3 and Acinetobacter calcaceticus on the molecular phylogenetic tree.
Acinetobacter genomospecies 3 (ATCC 17922 strain) is a strain of Acinetobacter calcaceticus, and genomospecies is a group name applied to a group recognized from DNA-DNA homology in Acinetobacter. The GKN-4 strain of the present invention is similar to the properties of the intestinal bacteria group from morphological and physiological characterization tests conducted separately. Although the physiological properties of the GKN-4 strain of the present invention coincided with the typical properties of Acinetobacter in many properties, they differ from the typical properties of Acinetobacter described as absolute aerobic bacteria in that they exhibit glucose fermentability. From the analysis using 16S rDNA, it is suggested that the GKN-4 strain of the present invention is assigned to Acinetobacter, and that the GKN-4 strain of the present invention is a known Acinetobacter sp. Therefore, it is judged as a novel microorganism, and Acinetobacter sp. GKN-4 strain was used.
[0024]
Example 4
Both medium A and medium B were prepared and then autoclaved, and medium B was further filtered using a filter.
A single colony expressed on a nutrient agar medium of microorganisms (GKN-4) was inoculated into 10 ml of medium A in an L-shaped tube, and cultured with shaking at 25 ° C. and 40 rep / min to obtain a preculture solution.
Next, 140 ml of medium B, 10 ml of the above preculture solution, and olive oil as a substrate were added to a 500 ml Sakaguchi flask, and cultured with shaking at 120 rep / min for 48 hours. In addition, a similar experiment was performed without adding the preculture solution, and used as a control example.
[0025]
After autoclave sterilization, n-hexane extraction was performed based on the JIS method.
1. Table 6 shows the degradation performance due to temperature change at pH 7.
[Table 6]
2. Table 7 shows the degradation performance due to pH change at the optimum temperature (28 ° C.) found in Table 6.
[Table 7]
3. Table 8 shows the decomposition performance according to the addition amount of fats and oils using the optimum temperature (28 ° C.) and the optimum pH (pH 8).
[Table 8]
[0028]
In the A Hotel Kitchen Grease Trap, the actual drainage of the Acinetobacter sp. Of the present invention was carried out for 19 days from October 6 to October 24, 2001. Measurements were made on five items of water temperature, pH, dissolved oxygen (DO), ammoniacal nitrogen, normal hexane extract (n-Hex value) (according to JIS K 0102) when a formulation comprising GKN-4 strain was added. It was.
The grease trap in this A hotel kitchen is 400 mm × 500 mm × 1000 mm = 200 liters, the oil amount is about 5 to 6 liters / day, the amount of water used is 40 t / day, and the oil concentration is about 15,000 to about 3000 ppm. The grease trap was cleaned once a day by agglomerating with a flocculant and manually pumping.
The experimental schedule is as shown in Table 9 below.
[Table 9]
Table 10 shows the results of field tests using the oil-degrading microbial preparation GKN-4.
[Table 10]
[0030]
As usual, it was difficult to completely recover the fats and oils by hand, and even after the actual cleaning, the measured result of the collected wastewater showed a high value of 1961 ppm. On the second day (October 8) after the introduction of the microbial preparation, a value of 721 ppm is shown, but it can be seen that the value is reduced to about 1/3 compared to before the introduction. (This is considered to be a microbial growth stage.) After 4th day, there is a slight increase and decrease, but the average value is 63.9ppm, there is a clear difference compared to before the input, the input by the microbial preparation The effect was fully demonstrated.
Further, no bad odor was generated from the tank during the test period.
[0031]
【The invention's effect】
INDUSTRIAL APPLICABILITY According to the present invention, it was possible to provide a novel microorganism having an ability to decompose fats and oils, an oil-and-fat decomposer containing the microorganisms, and a method for treating fat-and-fat-containing substances using the microorganisms.
[0032]
[Sequence Listing]
Claims (6)
生理性状試験結果
１．生化学試験
β−ガラクトシダーゼ −
アルギニンジヒドロラーゼ −
リシンデカルボキシラーゼ −
オルニチンデカルボキシラーゼ −
クエン酸の利用性 ＋
Ｈ２Ｓ産生 −
ウレアーゼ −
トリプトファンデアミナーゼ −
インドール産生 −
アセトイン産生 −
ゼラチナーゼ −
オキシダーゼ −
ＮＯ２産生 −
Ｎ2ガスへの還元 −
２．発酵／酸化試験
ブドウ糖 ＋
Ｄ−マンニトール ＋
イノシトール −
Ｄ−ソルビトール −
Ｌ−ラムノース ＋
白糖 −
Ｄ−メリビオース ＋
Ｄ−アミグダリン ＋
Ｌ−アラビノース ＋
ＯＦ培地での酸化 ＋
ＯＦ培地での発酵 ＋
なお、上記試験において「＋」は陽性、「−」は陰性を示す。The microorganism according to claim 1, wherein the physiological property test result is as follows.
Results of physiological property test Biochemical test β-galactosidase −
Arginine dihydrolase −
Lysine decarboxylase −
Ornithine decarboxylase −
Availability of citric acid +
H 2 S production −
Urease −
Tryptophan deaminase −
Indole production −
Acetoin production −
Gelatinase −
Oxidase −
NO 2 production −
Reduction to N 2 gas −
2. Fermentation / Oxidation Test Glucose +
D-mannitol +
Inositol −
D-sorbitol-
L-rhamnose +
Sucrose −
D-melibiose +
D-Amygdalin +
L-arabinose +
Oxidation in OF medium +
Fermentation in OF medium +
In the above test, “+” indicates positive and “−” indicates negative.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002334047A JP4172992B2 (en) | 2002-11-18 | 2002-11-18 | NOVEL MICROORGANISM, FAT AND FAT CONTAINING AGENT CONTAINING THE SAME, AND METHOD FOR TREATING OIL AND FAT CONTAINING SUBSTANCE USING THE SAME |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002334047A JP4172992B2 (en) | 2002-11-18 | 2002-11-18 | NOVEL MICROORGANISM, FAT AND FAT CONTAINING AGENT CONTAINING THE SAME, AND METHOD FOR TREATING OIL AND FAT CONTAINING SUBSTANCE USING THE SAME |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2004166533A JP2004166533A (en) | 2004-06-17 |
| JP4172992B2 true JP4172992B2 (en) | 2008-10-29 |
Family
ID=32698596
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2002334047A Expired - Fee Related JP4172992B2 (en) | 2002-11-18 | 2002-11-18 | NOVEL MICROORGANISM, FAT AND FAT CONTAINING AGENT CONTAINING THE SAME, AND METHOD FOR TREATING OIL AND FAT CONTAINING SUBSTANCE USING THE SAME |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4172992B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5715477B2 (en) * | 2011-04-25 | 2015-05-07 | 石川県 | NOVEL MICROORGANISM HAVING FUEL OIL RESOLUTION, ENVIRONMENT PURIFYING AGENT, AND ENVIRONMENT PURIFYING METHOD USING THE SAME |
| KR102096662B1 (en) * | 2019-01-23 | 2020-04-02 | 지병주 | Reagent for the selection assay of acinetobacter baumannii like gram negative bacteria and selection assay of acinetobacter baumannii like gram negative bacteria using the same |
-
2002
- 2002-11-18 JP JP2002334047A patent/JP4172992B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2004166533A (en) | 2004-06-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Arahal et al. | Moderately halophilic and halotolerant species of Bacillus and related genera | |
| KR20190061210A (en) | Bacillus siamensis 1HC-NA strain having nitrogen removal activation, and microbial agent comprising the same for removing nitrogen | |
| Tarhriz et al. | Isolation and characterization of naphthalene-degradation bacteria from Qurugol Lake located at Azerbaijan | |
| KR102051868B1 (en) | Bacillus tequilensis 2F-NA strain having nitrogen removal activation, and microbial agent comprising the same for removing nitrogen | |
| Zhang et al. | Isolation and characterization of a new carbendazim-degrading Ralstonia sp. strain | |
| CN110283741B (en) | A rose chromogen with efficient polycyclic aromatic hydrocarbon degradation and its application | |
| Sarkar et al. | Gordonia sp. BSTG01 isolated from Hevea brasiliensis plantation efficiently degrades polyisoprene (rubber) | |
| JP4088690B2 (en) | New microorganisms and methods for removing arsenic by microorganisms | |
| JP5953078B2 (en) | NOVEL MICROORGANISM AND ITS VARIANTS AND METHOD FOR PRODUCING POLYsaccharideS | |
| JP4172992B2 (en) | NOVEL MICROORGANISM, FAT AND FAT CONTAINING AGENT CONTAINING THE SAME, AND METHOD FOR TREATING OIL AND FAT CONTAINING SUBSTANCE USING THE SAME | |
| JP4540211B2 (en) | New microorganisms belonging to Bacillus subtilis | |
| JP6675714B2 (en) | Sludge volume reduction method for microbial material and excess sludge | |
| CN105829522A (en) | Novel microorganism | |
| KR100325252B1 (en) | A novel microorganism Rhodococcus pyridinovorans PDB9 degrading aromatic compounds | |
| Sivakumar et al. | SCREENING OF KERA SCREENING OF KERATINOLYTIC BACTERIA BACILLUS CEREUS US CEREUS US CEREUS FROM THE FEATHER DUMPING SOIL OF SIV THER DUMPING SOIL OF SIV THER DUMPING SOIL OF SIVAKASI | |
| JP2002301494A (en) | Activated sludge and wastewater treatment method | |
| Navrátilová et al. | Characterization of Rhodococcus wratislaviensis strain J3 that degrades 4-nitrocatechol and other nitroaromatic compounds | |
| El-Tarrs et al. | In vitro screening for oil degrading bacteria and evaluation of their biodegradation potential for hydrocarbon | |
| JP4654437B2 (en) | Novel microorganism and method for treating organic sludge using the same | |
| JP4416470B2 (en) | High-performance denitrifying oil-degrading bacteria, wastewater purification method using the high-performance denitrifying oil-degrading bacteria | |
| JP4540210B2 (en) | A new microorganism belonging to Bacillus schurigensis | |
| JP5790983B2 (en) | Novel oil-degrading bacteria, oil-degrading bacteria preparation, and method for treating oil-containing wastewater | |
| JP3091758B1 (en) | Oil-decomposing bacteria and method for treating oil-containing wastewater using the same | |
| Shahbaz et al. | Growth promoting attributes of chromium (VI) resistant bacillus strains for Triticum aestivum L | |
| JP2003235547A (en) | Pseudomonas sp. FK916 strain producing organic matter-degrading enzyme and method for treating organic waste using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A711 | Notification of change in applicant |
Free format text: JAPANESE INTERMEDIATE CODE: A711 Effective date: 20050124 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20050124 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20050323 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20051110 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20051110 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20060203 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20080325 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20080523 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20080715 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20080812 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20110822 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20110822 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20140822 Year of fee payment: 6 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |