LT6625B - Bacterial preparation for oxydation of oil products and their use - Google Patents
Bacterial preparation for oxydation of oil products and their use Download PDFInfo
- Publication number
- LT6625B LT6625B LT2017079A LT2017079A LT6625B LT 6625 B LT6625 B LT 6625B LT 2017079 A LT2017079 A LT 2017079A LT 2017079 A LT2017079 A LT 2017079A LT 6625 B LT6625 B LT 6625B
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- Prior art keywords
- soil
- bacterial preparation
- oil
- preparation according
- nitrogen
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- 238000002360 preparation method Methods 0.000 title claims description 31
- 230000001580 bacterial effect Effects 0.000 title claims description 24
- 238000007254 oxidation reaction Methods 0.000 title claims 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000002689 soil Substances 0.000 claims abstract description 37
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 22
- 241000588624 Acinetobacter calcoaceticus Species 0.000 claims abstract description 15
- 230000015556 catabolic process Effects 0.000 claims abstract description 5
- 238000006731 degradation reaction Methods 0.000 claims abstract description 5
- 239000003344 environmental pollutant Substances 0.000 claims abstract description 5
- 231100000719 pollutant Toxicity 0.000 claims abstract description 5
- 244000005700 microbiome Species 0.000 claims description 22
- 229910052698 phosphorus Inorganic materials 0.000 claims description 7
- 229910052700 potassium Inorganic materials 0.000 claims description 7
- 239000000356 contaminant Substances 0.000 claims description 5
- 238000000354 decomposition reaction Methods 0.000 claims description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 4
- 239000003209 petroleum derivative Substances 0.000 claims description 4
- 239000011574 phosphorus Substances 0.000 claims description 4
- 239000011591 potassium Substances 0.000 claims description 4
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 3
- 238000005273 aeration Methods 0.000 claims description 2
- 239000011785 micronutrient Substances 0.000 claims description 2
- 235000013369 micronutrients Nutrition 0.000 claims description 2
- 239000011573 trace mineral Substances 0.000 claims description 2
- 235000013619 trace mineral Nutrition 0.000 claims description 2
- 230000002262 irrigation Effects 0.000 claims 1
- 238000003973 irrigation Methods 0.000 claims 1
- 230000003647 oxidation Effects 0.000 claims 1
- 229930195733 hydrocarbon Natural products 0.000 abstract description 18
- 150000002430 hydrocarbons Chemical class 0.000 abstract description 18
- 229910052500 inorganic mineral Inorganic materials 0.000 abstract description 14
- 239000011707 mineral Substances 0.000 abstract description 14
- 239000000203 mixture Substances 0.000 abstract description 9
- 241000894006 Bacteria Species 0.000 abstract description 8
- 230000001717 pathogenic effect Effects 0.000 abstract description 2
- 239000003921 oil Substances 0.000 description 20
- 235000010755 mineral Nutrition 0.000 description 12
- 239000003337 fertilizer Substances 0.000 description 10
- 239000003208 petroleum Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 229910001868 water Inorganic materials 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 7
- 241000589291 Acinetobacter Species 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000006065 biodegradation reaction Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 239000004215 Carbon black (E152) Substances 0.000 description 3
- 108010079246 OMPA outer membrane proteins Proteins 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000011109 contamination Methods 0.000 description 3
- 239000003995 emulsifying agent Substances 0.000 description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 3
- 235000013379 molasses Nutrition 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 230000003442 weekly effect Effects 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- PBAYDYUZOSNJGU-UHFFFAOYSA-N chelidonic acid Natural products OC(=O)C1=CC(=O)C=C(C(O)=O)O1 PBAYDYUZOSNJGU-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 238000009533 lab test Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 235000011007 phosphoric acid Nutrition 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 241000588625 Acinetobacter sp. Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 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 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000001183 RAG-1 Human genes 0.000 description 1
- 108060006897 RAG1 Proteins 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004939 coking Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000010779 crude oil Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003795 desorption Methods 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
- 238000005516 engineering process Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000010169 landfilling Methods 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 238000011197 physicochemical method Methods 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 235000020071 rectified spirit Nutrition 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000006150 trypticase soy agar Substances 0.000 description 1
- 235000013522 vodka Nutrition 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C2101/00—In situ
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Soil Sciences (AREA)
- Microbiology (AREA)
- Environmental & Geological Engineering (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Processing Of Solid Wastes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Išradimo sritisField of the Invention
Šis išradimas yra susijęs su naftą skaidančio mikroorganizmo kompozicija ir jos panaudojimu naftos, ir jos darinių utilizavimui iš užteršto grunto.The present invention relates to a composition of an oil-decomposing microorganism and its use for the recovery of oil and its derivatives from contaminated soil.
Technikos lygisState of the art
Nafta yra sudėtingas angliavandenilių ir kitų organinių junginių mišinys. Jame yra šimtai ar tūkstančiai alifatinių, šakotų ir aromatinių angliavandenilių (Wang et ai. 1998), kurių dauguma yra toksiški gyviems organizmams.Oil is a complex mixture of hydrocarbons and other organic compounds. It contains hundreds or thousands of aliphatic, branched, and aromatic hydrocarbons (Wang et al. 1998), most of which are toxic to living organisms.
Užteršto dirvožemio valymui dažniausiai naudojami fiziko-cheminiai ir biologiniai būdai. Fiziko-cheminiai apdorojimo būdai yra deginimas, terminė desorbcija, koksavimas, tirpiklių ekstrahavimas, talpinimas į sąvartynus irt.t. Tačiau šie būdai arba paverčia gruntą nebenaudotinu (deginimas), arba nepašalina esminės problemos (sąvartynai). Be to, Europoje, įstatymai reikalauja, kad būtų mažinamas visų sąvartynų skaičius ir kuo daugiau atliekų būtų perdirbama.Physico-chemical and biological methods are commonly used for cleaning contaminated soil. Physico-chemical treatment methods include incineration, thermal desorption, coking, solvent extraction, landfilling and so on. However, these techniques either render the soil unusable (incineration) or do not eliminate the underlying problem (landfill). In addition, in Europe, legislation requires that all landfills be reduced and that as much waste as possible be recycled.
Dar 1946 m. Claude E. ZoBelI atrado, kad daugelis mikroorganizmų gali naudoti angliavandenilius kaip vienintelį anglies ir energijos šaltinį. Jis taip pat nustatė, kad angliavandenilių panaudojimas labai priklausė nuo naftos mišinių sudedamųjų dalių cheminio pobūdžio ir aplinkos veiksnių (Jain et ai. 2011). Mikroorganizmų panaudojimas angliavandeniliais užterštos teritorijos atkūrimui (remediacijai) yra vykdomas panaudojant naftą oksiduojančius mikroorganizmus, ypač dirvožemyje esančias vietines bakterijas ir grybus. Šie mikroorganizmai gali skaidyti daugybę naftos atliekose esančių sudedamųjų dalių (Eriksson, Dalhammar, ir Borg-Karlson 1999; Barathi ir Vasudevan 2001; Mishra et ai. 2001). Nors grybai taipogi pasižymi naftą oksiduojančiomis savybėmis, tačiau jų augimas yra sąlyginai lėtas, todėl jie sunkiai pritaikomi lauko sąlygomis ir esant dideliam užterštumui (Mohsenzadeh, Chehregani Rad, ir Akbari 2012). Siekiant paspartinti biologinį skaidymą naftos produktais užterštas gruntas apdorojamas dirbtinai užaugintais naftą oksiduojančiais mikroorganizmais, kurie naftos angliavandenilius naudoja kaip maisto šaltinį ir skaido juos iki nekenksmingų medžiagų: CO2 ir H2O.Back in 1946 Claude E. ZoBelI discovered that many microorganisms can use hydrocarbons as their sole source of carbon and energy. He also found that the use of hydrocarbons was highly dependent on the chemical nature and environmental factors of the components of petroleum blends (Jain et al. 2011). The use of microorganisms for the rehabilitation of hydrocarbon contaminated sites is carried out by utilizing oil oxidizing microorganisms, in particular native bacteria and fungi in the soil. These microorganisms can degrade many of the constituents present in petroleum waste (Eriksson, Dalhammar, and Borg-Karlson 1999; Barathi and Vasudevan 2001; Mishra et al. 2001). Although fungi also have oil-oxidizing properties, their growth is relatively slow, making them difficult to adapt to field conditions and high levels of contamination (Mohsenzadeh, Chehregani Rad, & Akbari 2012). To accelerate the biodegradation of oil-contaminated soils, the soil is treated with artificially cultivated oil-oxidizing microorganisms that use petroleum hydrocarbons as a food source and decompose them into harmless substances: CO2 and H2O.
Acinetobacter rūšys yra plačiai paplitę ir gali būti išgaunamos iš vandens, dirvožemio, gyvų organizmų ir net iš žmogaus odos. Tai nejudrios, neigiamos oksidazės, griežtai aerobinės, gram neigiamos kokobacilos. Jos gali naudoti įvairius anglies šaltinius augimui ir gali būti auginamos palyginti paprastose terpėse, įskaitant maistinį agarą arba triptikazės sojų agarą (Abdelhaleem 2003). Acinetobacter genties bakterijos dėl savo įvairovės gamina daugybę medžiagų naudojamų biotechnologijose. Kai kurios Acinetobacter rūšys gamina didelius kiekius polisacharidų, poliesterių ir lipazių. Tačiau industriniu požiūriu svarbiausi yra emulsikliai, tokie kaip, pvz., emulsinas (Gutnick et ai. 1980), OmpA (VValzer, Rosenberg, ir Ron 2006) ar alasanas (Toren et ai. 2002). Emulsikliai ypač naudingi angliavandenilių skaidyme, nes padeda emulguoti naftą ir jos darinius, taip paspartindami degradavimo procesus. Jau prieš porą dešimtmečių buvo pastebėta, kad kai kurios Acinetobacter rūšys geba skaidyti naftą ir jos darinius, tačiau geri rezultatai buvo pasiekiami tik laboratorinėmis sąlygomis, dėl naftos inhibicinių savybių bei aeracijos trūkumo dirvoje bei skystoje terpėje (Hanson et ai. 1997). Visų mikroorganizmų augimui ir dauginimuisi yra būtinos ne tik maisto medžiagos, bet ir mikroelementai, tokie kaip azotas, kalis, fosforas, siera, magnis, kalcis ir tt. Kai kurie iš jų - gyvybiškai svarbūs, nes yra DNR sudėtyje (azotas, fosforas), amino rūgščių sudėtyje (azotas, siera) ir ląstelių sienelėse (fosforas, kalcis).Acinetobacter species are widespread and can be extracted from water, soil, living organisms and even from human skin. These are immobile, negative oxidases, strictly aerobic, gram negative cocobacilli. They can use a variety of carbon sources for growth and can be grown in relatively simple media, including nutrient agar or trypticase soy agar (Abdelhaleem 2003). The bacteria of the genus Acinetobacter, due to their diversity, produce many substances used in biotechnology. Some species of Acinetobacter produce large amounts of polysaccharides, polyesters and lipases. However, industrially important emulsifiers such as emulsin (Gutnick et al. 1980), OmpA (Walzer, Rosenberg, and Ron 2006) or Alasan (Toren et al. 2002) are important. Emulsifiers are particularly useful in the decomposition of hydrocarbons because they help to emulsify petroleum and its derivatives, thereby accelerating the degradation process. Some Acinetobacter species have been observed for a couple of decades ago to decompose oil and its derivatives, but good results have been achieved only under laboratory conditions due to the inhibitory properties of oil and the lack of aeration in soil and liquid media (Hanson et al. 1997). Not only nutrients, but also micronutrients such as nitrogen, potassium, phosphorus, sulfur, magnesium, calcium, etc., are essential for the growth and growth of all microorganisms. Some of them are vital because they contain DNA (nitrogen, phosphorus), amino acids (nitrogen, sulfur) and cell walls (phosphorus, calcium).
Tačiau mikroelementai taip pat gali įtakoti įvairių naftos komponentų bioremediaciją. Aromatinių angliavandenilių biologinis skaidymas yra ypač jautrus pH pokyčiams. Foght ir kt. 1999 m. ištyrė azoto šaltinio vaidmenį naftos komponentų biologiniam skaidymui esant šaltoms jūrinėms sąlygoms (10 °C). Nitratai neturėjo įtakos pH, tačiau amonio pokyčiai lėmė rūgštėjimą, dėl kurio susilpnėjo aromatinių junginių skaidymas (Margesin irSchinner2001). Tokie rezultatai rodo, kad labai svarbu tinkamai adaptuoti sąlygas, kuriose mikroorganizmai skaido teršalus ir nuolat stebėti tų sąlygų pokyčius. Dėl nepastovių oro sąlygų ir nevykdant stebėjimų, daugelis bakterinių preparatų neveikia taip kaip yra tikimąsi atlikus tik laboratorinius eksperimentus. Taigi daug su naftos biologiniu skaidymu susijusių išradimų yra nepritaikomi praktiškai arba veikia tik uždaromis ir kontroliuojamomis sąlygomis ir negali būti pritaikyti dideliems užteršto grunto plotams.However, trace elements can also influence the bioremediation of various petroleum components. Biodegradation of aromatic hydrocarbons is particularly sensitive to changes in pH. Foght et al. 1999 investigated the role of the nitrogen source in the biodegradation of petroleum components under cold marine conditions (10 ° C). Nitrates had no effect on pH, but changes in ammonium led to acidification, which reduced the degradation of aromatic compounds (Margesin and Schinner2001). Such results indicate the importance of proper adaptation of the conditions in which the microorganisms decompose contaminants and the constant monitoring of changes in those conditions. Due to the volatile weather conditions and the absence of observations, many bacterial agents do not perform as expected in laboratory experiments alone. Thus, many inventions related to the biodegradation of petroleum are not practicable or operate only under closed and controlled conditions and cannot be applied to large areas of contaminated soil.
Išradimo esmėThe essence of the invention
Bakterinio preparato skaidančio naftos teršalus, apimančio Acinetobacter calcoaceticus BT 8 mikroorganizmus, panaudojimo technologija yra pagrįsta bakterijų, atrinktų iš dirvožemio, kultivavimu ir jų panaudojimu naftos degradacijai. Šis išradimas paspartina dirvožemio valymo procesą ir yra pranašesnis už kitus pramoniniu būdu gaminamus preparatus kadangi panaudojamas mikroorganizmo gebėjimas fiksuoti ore esantį azotą, taip sumažinant pridėtinių trąšų poreikį.The technology for utilizing a bacterial agent to decompose oil contaminants comprising microorganisms of Acinetobacter calcoaceticus BT 8 is based on the cultivation of bacteria selected from the soil and their use for oil degradation. The present invention accelerates the soil remediation process and is superior to other industrially prepared formulations because it utilizes the ability of the microorganism to fix nitrogen in the air, thereby reducing the need for added fertilizers.
Detalus išradimo aprašymasDetailed Description of the Invention
Šiame išradime aprašomas naujas bakterinis preparatas gebantis efektyviai, saugiai ir pigiai oksiduoti naftos teršalus dirvožemyje ir paversti juos nekenksmingomis cheminėmis medžiagomis. Sukurtas bakterinis preparatas pasižymi mažesniu mineralinio azoto poreikiu, nes jame naudojama bakterija A. calcoaceticus BT 8 geba fiksuoti ore esantį dujinį azotą ir versti jį mineraline medžiaga reikalinga įvairių gyvybinių funkcijų palaikymui.The present invention describes a novel bacterial preparation capable of efficiently, safely and cheaply oxidizing oil pollutants in soil and converting them into harmless chemicals. The developed bacterial preparation has a lower mineral nitrogen requirement because it uses the A. calcoaceticus BT 8 bacterium to capture gaseous nitrogen in the air and convert it into a mineral to support a variety of vital functions.
Viename išradimo įgyvendinimo pavyzdyje naudojamas bakterinis preparatas susidedantis iš atrinktų A. calcoaceticus BT 8 bakterijų ir mineralinių trąšų, kurios neapima azoto. Šis preparatas buvo išbandytas laboratorinėmis sąlygomis, kur naftos teršalų koncentracija buvo 181 g/kg.In one embodiment of the invention, a bacterial preparation consisting of selected A. calcoaceticus BT 8 bacteria and a mineral fertilizer that is nitrogen-free is used. This preparation was tested under laboratory conditions at a petroleum contaminant concentration of 181 g / kg.
Dar viename išradimo įgyvendinimo pavyzdyje naudojamas bakterinis preparatas susidedantis iš atrinktų A. calcoaceticus BT 8 bakterijų ir mineralinių trąšų apimančių azotą. Preparatas buvo išbandytas laboratorinėmis sąlygomis, kur naftos teršalų koncentracija buvo 181 g/kg.In another embodiment of the invention, a bacterial preparation comprising selected A. calcoaceticus BT 8 bacteria and a mineral fertilizer comprising nitrogen is used. The preparation was tested under laboratory conditions at a concentration of 181 g / kg of oil pollutants.
Dar viename išradimo įgyvendinimo pavyzdyje bakterinis preparatas yra naudojamas specializuotu būdu, po auginimo užsaldant bakterinį preparatą ir jį atšildant tik prieš purškimą ant nafta užterštos dirvos, kai preparato bei mineralinių trąšų kiekiai yra parenkami pagal dirvos užterštumo laipsnį bei numatomas papildomo purškimo grafikas.In another embodiment of the invention, the bacterial preparation is used in a specialized manner by freezing and thawing the bacterial preparation after cultivation only prior to spraying on oil-contaminated soil, wherein the amounts of formulation and mineral fertilizers are selected based on soil contamination and expected additional spraying schedules.
Brėžinių paveikslų aprašymas pav. Parodytas naftos angliavandenilių kiekis po mėnesį trukusio laboratorinio eksperimento, kai pradinis teršalų kiekis buvo 181 g/kg. Palyginamas biopreparato poveikis su mineralinio azoto šaltiniu ir be jo.Description of Drawings Fig. Shown are the petroleum hydrocarbons content after a month-long laboratory experiment with an initial contaminant of 181 g / kg. The effect of the biopreparation with and without mineral nitrogen source is compared.
pav. Parodytas naftos angliavandenilių skaidymas lauko sąlygomis, kai pradinė naftos koncentracija -189 g/kg. Eksperimento trukmė - 8 sav.Fig. The breakdown of petroleum hydrocarbons under field conditions with an initial oil concentration of -189 g / kg is shown. Experiment duration - 8 weeks.
Priemonės ir metodaiTools and methods
Prietaisai: techninės svarstyklės KERN PCB 2500, analitinės svarstyklės KERN ABJ 220, laminarinė oro srauto spinta „Thermo Scientific“, termostatuojama purtyklė „Thermo Scientific“ MaxQ 4450, termostatuojamas mikroorganizmų inkubatorius „Thermo Scientific“ Heratherm IGS60, fermentatorius EDF-5.4_1 „Biotehniskais centrs“, autoklavas AL02-10 „Advantage-Lab“, spektrofotometras GENESYS 10S UV-Vis „Thermo Scientific“.Appliances: KERN PCB 2500 technical scales, KERN ABJ 220 analytical balance, Thermo Scientific laminar flow cabinet, Thermo Scientific MaxQ 4450 shaker, Thermo Scientific Heratherm IGS60 thermo-incubator, EDF-5.4_1 Biotechnical Center , Autoclave AL02-10 Advantage-Lab, GENESYS 10S UV-Vis Spectrophotometer Thermo Scientific.
Reagentai:Reagents:
Lentelė. Bakterinio preparato gamyboje naudojami reagentai.Table. Reagents used in the preparation of the bacterial preparation.
Metodai:Methods:
Mikroorganizmų atrankaSelection of microorganisms
Siekiant sukurti bakterinį preparatą naftos teršalų valymui dirvožemyje buvo naudojamas selektyvios atrankos metodas išgaunant mikroorganizmus iš angliavandeniliais užteršto dirvožemio mėginių. Mėginiai buvo imti iš seno garažo automobilių remonto dirbtuvių, paėmus naftos produktais užteršto šlamo pavyzdžių. Norimų izoliatų išskyrimas buvo atliekamas ant agarizuotos terpės auginant iš mėginių išskirtus mikroorganizmus, kai vienintelis anglies šaltinis terpėje buvo angliavandeniliai. Sėkmingiausiai augę mikroorganizmai buvo naudojami kituose eksperimentuose.In order to develop a bacterial preparation for the treatment of oil pollutants in soil, a selective selection method was used to extract microorganisms from soil samples contaminated with hydrocarbons. Samples were taken from an old garage car repair shop, taking samples of oil-contaminated sludge. Isolation of the desired isolates was performed on the agarized medium by culturing isolated microorganisms where the only source of carbon in the medium was hydrocarbons. The most successful microorganisms were used in other experiments.
Azotą fiksuojančių mikroorganizmų atrankaSelection of nitrogen fixing microorganisms
Išskirti naftą oksiduojantys izoliatai buvo auginami ant agarizuotos terpės, be azoto šaltinių. Eksperimento metu išaugo tik tie naftą oksiduojantys izoliatai, kurie gebėjo fiksuoti ore esantį dujinį azotą ir versti jį mineraline medžiaga reikalinga įvairių gyvybinių funkcijų palaikymui.Isolated oil-oxidizing isolates were grown on agarized medium without nitrogen sources. During the experiment, only those oil-oxidizing isolates that were capable of trapping gaseous nitrogen in the air and converting it into mineral matter were required to support various vital functions.
Mikroorganizmų rūšies nustatymasSpecification of the micro-organism
Siekiant patvirtinti išskirto mikroorganizmo rūšį jis buvo identifikuojamas BASECLEAR laboratorijoje Olandijoje. Mikroorganizmas buvo identifikuotas panaudojant validuotą MicroSeų sistemą iš „Applied Biosystems“. Ši automatinė sistema yra paremta bakterinio 16S rRNR geno PGR amplifikacija ir DNR sekoskaita. Buvo nustatyta A. calcoaceticus BT8 16S rRNR geno seka - SEQ ID Nr. 1:In order to confirm the species isolated, it was identified in the BASECLEAR laboratory in the Netherlands. The microorganism was identified using a validated MicroSeai system from Applied Biosystems. This automated system is based on PCR amplification and DNA sequencing of the bacterial 16S rRNA gene. The A. calcoaceticus BT8 16S rRNA gene sequence - SEQ ID NO. 1:
GATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGAGTGA TGGTGyTTGCACTATCACTTAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTG CCTATTAGTGGGGGACAACATTTCGAAAGGAATGCTAATACCGCATACGTCCTA CGGGAGAAAGCAGGGGATCTTCGGACCTTGCGCTAATAGATGAGCCTAAGTCG GATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGT CTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGG GAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGC CGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGG CTACTGAAGTTAATACCTTCAGATAGTGGACGTTACTCGCAGAATAAGCACCGGGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGAGTGA TGGTGyTTGCACTATCACTTAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTG CCTATTAGTGGGGGACAACATTTCGAAAGGAATGCTAATACCGCATACGTCCTA CGGGAGAAAGCAGGGGATCTTCGGACCTTGCGCTAATAGATGAGCCTAAGTCG GATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGT CTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGG GAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGC CGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCGAGGAGGAGG CTACTGAAGTTAATACCTTCAGATAGTGGACGTTACTCGCAGAATAAGCACCGG
CTAACTCTGT.CTAACTCTGT.
Nustatyta, jog geno seka 99,94 % sutampa su laboratorijoje naudojamoje duomenų bazėje esančia A. calcoaceticus 16S rRNR geno seka. Rūšies nustatymas patvirtino, jog mūsų išskirta bakterija yra nepatogeninė ir tinkama naudojimui dirvožemio bioremediacijoje.The gene sequence was found to be 99.94% identical to the A. calcoaceticus 16S rRNA gene sequence found in the laboratory database. Species identification confirmed that the bacterium we isolated was non-pathogenic and suitable for use in soil bioremediation.
A. calcoaceticus BT 8 biopreparato paruošimasPreparation of A. calcoaceticus BT 8 biopreparation
Norint užtikrinti, kad A. calcoaceticus BT8 bus veiksmingas lauko sąlygomis, mikroorganizmas kultivuojamas paprastoje mitybinėje terpėje su sumažintu mineralinio azoto kiekiu, aerobinėmis sąlygomis. Auginimo pabaigoje biopreparatas yra paruošiamas užšaldymui -18 °C temperatūroje, pridedant 10 % (pagal tūrį) melasos. Kultūrinis skystis su melasa gerai išmaišomas, išpilstomas ir užšaldomas.To ensure that A. calcoaceticus BT8 will be effective under field conditions, the microorganism is cultured in a simple nutrient medium with reduced mineral nitrogen under aerobic conditions. At the end of cultivation, the biopreparation is prepared for freezing at -18 ° C by adding 10% (v / v) molasses. Mix the culture liquid with molasses well, dispense and freeze.
Biopreparato panaudojimo pavyzdžiaiExamples of use of a biopreparation
Čia nurodomi bakterinio preparato panaudojimo pavyzdžiai. Išradimas nėra ribojamas šiais pavyzdžiais.Examples of the use of the bacterial preparation are given here. The invention is not limited to the following examples.
pavyzdysexample
Išaugintas ir užšaldytas A. calcoaceticus BT8 preparatas laboratorinėmis sąlygomis buvo atšildytas, atskiestas su vandeniu papildytu mineralinėmis trąšomis (P, K) ir po 50 ml išpilstytas į 1 L tūrio indus su nafta užterštu gruntu. Indai buvo laikomi kambario temperatūroje (~22 °C) mėnesį laiko. Kiekvieną savaitę gruntas buvo permaišomas ir papildomai užpilami skirtingi kiekiai biopreparato bei vandens. Kas savaitę buvo matuojamas angliavandenilių kiekis grunte. Eksperimento sąlygos pateikiamos 2 lentelėje.The cultivated and frozen preparation of A. calcoaceticus BT8 was thawed under laboratory conditions, diluted with water-supplemented mineral fertilizer (P, K) and poured into 50 ml 1 L containers containing oil-contaminated soil. The dishes were stored at room temperature (~ 22 ° C) for one month. Each week, the soil was mixed and additional amounts of biopreparation and water were added. Weekly soil hydrocarbons were measured. The experimental conditions are shown in Table 2.
lentelėtable
pavyzdysexample
Išaugintas ir užšaldytas A. calcoaceticus BT8 preparatas laboratorinėmis sąlygomis buvo atšildytas, atskiestas su vandeniu papildytu mineralinėmis trąšomis (N, P, K) ir po 50 ml buvo išpilstytas į 1 L tūrio indus su nafta užterštu gruntu. Indai buvo laikomi kambario temperatūroje (~22 °C) mėnesį laiko. Kiekvieną savaitę gruntas buvo permaišomas ir papildomai užpilami skirtingi kiekiai biopreparato bei vandens. Kas savaitę buvo matuojamas angliavandenilių kiekis grunte. Eksperimento sąlygos pateikiamos 3 lentelėje.The cultivated and frozen preparation of A. calcoaceticus BT8 was thawed under laboratory conditions, diluted with water-supplemented mineral fertilizer (N, P, K) and placed in 50 ml 1 L containers containing oil-contaminated soil. The dishes were stored at room temperature (~ 22 ° C) for one month. Each week, the soil was mixed and additional amounts of biopreparation and water were added. Weekly soil hydrocarbons were measured. The experimental conditions are shown in Table 3.
Šio eksperimento metu nustatyta, kad į terpę pridėjus papildomą azoto šaltinį angliavandenilių skaidymas pasikeitė nežymiai. Pirmo ir antro pavyzdžių rezultatų palyginimas pateikiamas 1 pav.In this experiment, it was found that the addition of nitrogen to the medium resulted in a slight change in hydrocarbon decomposition. A comparison of the results of the first and second examples is presented in Fig. 1.
lentelėtable
pavyzdysexample
Išaugintas ir užšaldytas A. calcoaceticus BT8 preparatas prieš naudojimą buvo atšildytas ir po 1 L buvo išpilstytas ant 4 m2 ploto dirvos, kuri buvo užteršta naftos produktais (189 g/kg). Preparatas buvo skiedžiamas su mineralinėmis trąšomis (P, K, N) papildytu vandeniu santykiu 1:20. Azoto kiekis mineralinių trąšų mišinyje buvo sumažintas iki 50 mg/kg užteršto grunto. Pusė praskiesto biopreparato buvo išpurškiama ant ~30 cm sluoksnio storio sukasto grunto, po išpurškimo gruntas vėl sukasamas ir išpurškiama antra biopreparato dalis. Kiekvieną savaitę gruntas buvo permaišomas ir papildomai užpilama biopreparato bei vandens. Kas savaitę buvo matuojamas angliavandenilių kiekis grunte. Lauko eksperimentas truko 2 mėn. Eksperimento sąlygos pateikiamos 4 lentelėje.The cultivated and frozen preparation of A. calcoaceticus BT8 was thawed before use and after 1 L was applied to 4 m 2 soil contaminated with oil (189 g / kg). The preparation was diluted 1:20 with mineral fertilizers (P, K, N) and water. The nitrogen content of the mineral fertilizer mixture was reduced to 50 mg / kg of contaminated soil. Half of the diluted biopreparation was sprayed on the rotated soil with a layer thickness of ~ 30 cm, after the application the soil was rotated again and the second part of the biopreparation was sprayed. Each week the soil was remixed and biopreparation and water were added. Weekly soil hydrocarbons were measured. The field experiment lasted 2 months. The experimental conditions are shown in Table 4.
lentelėtable
Šis eksperimentas parodė jog, naudojant mūsų išradimą, efektyviam angliavandenilių skaidymui lauko sąlygomis reikalingi mažesni azoto trąšų kiekiai.This experiment has shown that, according to our invention, smaller amounts of nitrogen fertilizers are required for efficient hydrocarbon decomposition under field conditions.
Optimalios mūsų sukurto bakterinio preparato veikimo sąlygos bei paaiškinimai pateikti 5 lentelėje.The optimal operating conditions and explanations of the bacterial preparation we have developed are presented in Table 5.
Lentelė. Optimalūs bakterinio preparato parametrai.Table. Optimal parameters of bacterial preparation.
LiteratūraLiterature
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| LT7087B (en) | 2024-02-07 | 2024-09-10 | Gamtos Tyrimų Centras | BIO PREPARATION FOR CLEANING SOIL CONTAMINATED BY PETROLEUM PRODUCTS AND CLEANING METHOD |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2019073308A1 (en) | 2019-04-18 |
| LT2017079A (en) | 2019-04-25 |
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