RU2759807C1 - Strain of serratia plymuthica bacteria rncim b-12668 extracted from biomaterial of paleontological sample (calf of fossil woolly rhinoceros) - destructor of petroleum and petroleum products - Google Patents

Abstract

FIELD: microbiology.SUBSTANCE: strain of Serratia plymuthica bacteria "BMNs-L" capable of degrading petroleum and petroleum products is deposited in the RNCIM under the registration number B-12668. The strain of Serratia plymuthica bacteria RNCIM B-12668 can be used to purify environmental objects from contamination with petroleum and petroleum products.EFFECT: invention provides a possibility of increasing the degree of purification of environmental objects from petroleum and petroleum products.1 cl, 1 dwg, 2 tbl, 4 ex

Description

The invention relates to the field of microbiology and is a new bacterial strain Serratia plymuthica VKPM B-12668, isolated from the biomaterial of a paleontological sample (baby woolly rhinoceros), which has the ability to degrade oil and oil products (diesel fuel, motor oil, gas condensate, hydraulic oil, aviation kerosene , gasoline).

The invention improves the efficiency of cleaning substrates from oil and oil products (diesel fuel, motor oil, gas condensate, hydraulic oil, aviation kerosene, gasoline), in a wide temperature range from +2 to + 37 ± 1 ° C.

State of the art.

Known bacterial strain Serratia species - producer of lipase used to catalyze hydrolysis of triacylglycerides with the formation of fatty acids and glycerol (1. Patent RU No. 2308485. Bacterial strain Serratia species - producer of lipase. C1, C12N 9/20, C12N 1/20. Application: 2006107803/13. Declared: 03/13/2006. Published: 10/20/2007. Authors: Loginov O.N., Pakaneshchikova N.V., Silischev N.N., Galimzyanova N.F., Boyko T.F. NPP "Biomedkhim" (RU)), which can be used in the food industry, household chemicals, cosmetics.

The disadvantage of the known strain is that it is not intended for use as a destructor of oil and oil products.

Known strain of bacteria Serratia species, which has antiviral activity (2. Patent RU No. 2650762. The strain of bacteria Serratia species, which has antiviral activity. C1, C12N 1/20, C12N 9/14, C12N 9/22, A61P 31/16. Application: 2017110331. Declared: 28.03.2017. Published: 17.04.2018 Authors: Puchkova L.A., Andreeva I.S., Mazurkova N.A.

The disadvantage of the known strain is that it is also not intended for use as a destructor for oil and oil products.

Known bacterial strain Serratia marcescens m-10 - producer of extracellular nonspecific endonuclease with a pronounced antiviral effect (3. Patent RU No. 2604183. Bacterial strain Serratia marcescens m-10 - producer of extracellular nonspecific endonuclease, which has a pronounced antiviral effect. C202, C12N 1/202, C12 , C12N 9/16, C12R 1/01. Application: 2015107292/10. Declared: 03.03.2015. Published: 10.12.2016. Bulletin No. 27. Authors: Kelin L.V. Patentee: Limited Liability Company "Northern Style "(RU)).

The disadvantage of the known strain is that it is not intended for cleaning substrates from oil and oil products.

Known strain Serratia plymuthica, which has antiviral activity against influenza viruses a (its variants) (4. Title of protection No. 0002551316. Date of title of title 20.05.2015. Authors: FGBUN SSC VB "Vector", Afonina BC, Puchkova LA, Makarevich E.V., Mazurkova N.A., Solovyanova N.A., Selivanova M.A., Andreeva I.S.Patent holders: Solovyanova N.A., Makarevich E.V., Mazurkova N.A., Selivanova M.A., Andreeva I.S., Afonina V.S., Puchkova L.A., FGBUN SSC VB "Vector" (RU)).

However, the known strain Serratia plymuthica was not found to have the ability to utilize oil and oil products, which excludes the possibility of its use for cleaning environmental objects (soil and water) from oil and oil products pollution.

Known strains: Pseudomonas putida 36 (5. USSR author's certificate N1076446, Strain Pseudomonas putida 36, used to purify water and soil from oil and oil products. A1, C02F 3/34, B09C 1/10, C12N 1/20 C02F 3/34 , C02F 101: 32, C12N 1/20, C12R 1:40. Application: 3474745, 22.07.1982. Published: 28.02.1984. Applicant: West Siberian Research Oil Geological Institute. Authors: Dyadechko V.N., Tolstokorova L.E., Morozova T.N.); Acinetronacter sp.НВ-1 (6. Patent RU N2077579. The strain of bacteria Acinetobacter species used to purify water and soil from oil and oil products. C1, C12N 1/20, C02F 3/34, В09С 1/10, C12R 1: 01 , C12N 1/20, В09С 101: 00. Application: 94043331/13, 06.12.1994. Published: 20.04.1997. Applicant: State Research Institute of Applied Microbiology. Authors: Zhirkova N.A., Kobelev BC, Holodenko VP Patentee: State Research Institute of Applied Microbiology); Pseudomonas alcaligenes E7 (7. Patent RU N2134723. The bacterial strain Pseudomonas alcaligenes E7, used to purify water and soil from oil and oil products. C1, C12N 1/20, C02F 3/34, B09C 1/10, C12N 1/20, C12R 1:38. Application: 98105707/13, 25.03.1998. Published: 20.08.1999. Applicant: State Scientific Center for Applied Microbiology. Authors: Ermolenko Z.M., Holodenko V.P., Chugunov V.A. Scientific Center for Applied Microbiology).

The disadvantage of the above known strains is that they utilize oil at relatively high temperatures and cannot be used to clean substrates from oil pollution at a low positive temperature (+ 2 ° C), which is not promising for the use of the strain in the climatic conditions of the Far North.

Known consortium of oil-oxidizing microorganisms consisting of Pseudomonas putida PI Ko-1, Pseudomonas fluorescens PI-896, Micrococcus sp. PI Ku-1, Burkholderia caryophylli Jap-3, Serratia odorifera Jap-1 with a weight ratio of 3-12 wt. % of each microorganism, while together with the suspension of the biological product, rhizotorfin is introduced into the contaminated environment in an amount of 30-120 g / m3 (8. Patent RU 2191643. Method for cleaning soil from oil and oil product contamination. C1, B09C 1/10, C12N 1/20 , C12N 1/20, C12R 1:01. Application: 2001119562/13, 09.07.2001. Published: 27.10.2002. Applicant: Closed Joint Stock Company "Polyinform" Authors: Saxon V.M., Kuznetsov S.A., Boykova I.V., Novikova I.I.Patentee: Closed Joint Stock Company "Polyinform").

The disadvantage of the known consortium is that when preparing a working suspension of microorganisms, the temperature is maintained at + 18 + 22 ° C, i.e. microorganisms that are part of the consortium are not capable of degradation of oil products at a low positive temperature (+ 2 ° С), which also excludes their use for cleaning permafrost soils from oil pollution in the climatic conditions of the Far North.

Known bacterial strain "Bacillus subtilis Kolyma 7/2" (9. Patent RU 2446900. Method for cleaning permafrost soils from oil spore-forming bacteria Bacillus subtilis. C2. B09C 1/10 (2006.01). Application: 2010129158/13, 13.07.2010. Published. Published : 10.04.2012 Authors: Neustroev M.P., Tarabukina N.P., Neustroev M.M., Sazonov N.N., Parnikova S.I., Stepanova AM farms of the Russian Agricultural Academy), which has the ability to reduce the oil concentration to 0.48%, that is, the oil level from 135143 mg / kg decreases to 645 mg / kg within 3 months of the summer period.

However, the strain degrades only oil and is not able to utilize oil products (diesel fuel, motor oil, gas condensate, hydraulic oil, aviation kerosene, gasoline), including under conditions of a low positive temperature (+ 2 ° C), which would be a promising method for use in natural and climatic conditions of the Far North.

Known bacterial strain Serratia marcescens H ₃ K, used to purify wastewater from oil and oil products (10. Patent KZ No. 1732. Bacterial strain Serratia marcescens H ₃ K, used to purify waste water from oil and oil products. U, C12N 1/20 , C02F 3/34, B09C 1/10. Application: 2015 / 0326.2. Declared: 05.10.2015. Published: 30.09.2016. Bulletin No. 12. Authors: Ramankulov E.M., Moldagulova N.B., Khasenova E .Zh., Makhatova A.S., Shevtsov A.B.

The disadvantage of the known strain Serratia marcescens H ₃ K is that it is not capable of degradation of oil and oil products at a temperature of + 2 ° C, which excludes its use in the Far North.

To date, not a single strain of microorganisms isolated from paleontological finds of prehistoric animals that are used as destructors of oil and oil products is known, including none of the above known strains isolated from paleontological finds of prehistoric animals.

The closest analogue is the bacterial strain Serratia plymuthica VKM B-2819D - a destructor of oil and oil products (11. Patent RU 2560272. The bacterial strain Serratia plymuthica is a destructor of oil and oil products. IPC C1, C12N 1/20, C02F 3/34, В09С 1 / 10, C12R 1/01. Application: 2014138294/10. Declared: 22.04.2014. Published: 20.08.2015. Author: Erofeevskaya L.A. Patentee: Federal State Budgetary Institution of Science Institute of Oil and Gas Problems of the Siberian Branch of the Russian Academy of Sciences (RU)).

The disadvantage of the known solution is that experiments on testing the bacterial strain Serratia plymuthica VKM B-2819D were performed only at temperatures of +4, +20, + 30 ° C and the ability of the strain to destroy oil and oil products at a low positive temperature of + 2 ° C is not known. and at a higher temperature (+ 37 ° С), which would make the known strain more promising for use in various natural and climatic conditions, including in the Far North, in addition, the ability of the Serratia plymuthica VKM B-2819D bacterial strain is not known to the degradation of petroleum products such as aviation kerosene and gasoline.

The objective of the invention is to expand the range of strains that have the ability to degrade both oil and oil products (diesel fuel, engine oil, gas condensate, hydraulic oil, aviation kerosene, gasoline), in a wide temperature range (+ 2 ... + 37 ° C), in relatively short periods (10-60 days), which will be promising for the use of the strain in various natural and climatic conditions, including in the Far North during the restoration of technogenically disturbed ecosystems of the cryolithozone.

The problem was solved by the fact that the obtained bacterial strain Serratia plymuthica VKPM V-12668 is a destructor of oil and oil products, which has a utilizing ability in relation to oil and oil products (diesel fuel, motor oil, gas condensate, hydraulic oil, aviation kerosene, gasoline), promising for cleaning environmental objects from oil and oil products pollution (diesel fuel, motor oil, gas condensate, hydraulic oil, aviation kerosene, gasoline), in a wide temperature range (from +2 to + 37 ° С).

The origin of the strain.

верхняя челюсть до 7 см), найденного осенью 2014 г. охотниками на берегу реки Семюэлях в Абыйском районе Республики Саха (Якутия).The bacterial strain Serratia plymuthica VKPM B-12668 (author number BMNs-L) was isolated by the method of targeted research from the biomaterial of a frozen prehistoric baby woolly rhinoceros (Coelodonta antiquitatis Blumenbach, 1799) (sample head

upper jaw up to 7 cm), found in the fall of 2014 by hunters on the banks of the Semyuelakh River in the Abyisky District of the Sakha Republic (Yakutia).

Sampling was carried out on 03.04.2015 at the Academy of Sciences of the Republic of Sakha (Yakutia).

A frozen mummy of a fossil woolly rhino cub was found in a thawed outcrop of an unnamed tributary of the river. Semyuelakh in the vicinity of the Belaya Gora village, where the ice deposits of the Upper Pleistocene were exposed. Judging by the size of the body (172 cm from the nostrils to the base of the tail) and the state of the dental system, the baby sucker was from one to one and a half years old.

Radiocarbon dating of the bones of the animal showed its age at 42 thousand years ago, and the dating of the skin sample - 34.7 thousand years ago (12. Protopopov A., Potapova O., Plotnikov V., Maschenko E., Boeskorov G., Klimovskii A., Banderov A., Ivanov S., Kolesov S., Pavlov I. Abstrs 75th Annual SVP Meeting. October 14-17th, 2015, Dallas (TX), 2015. P. 199).

The resulting strain was identified by analysis of genes encoding 16S RNA and deposited at the National Bioresource Center of the All-Russian Collection of Industrial Microorganisms (VKPM) of the Federal State Budgetary Institution "GosNIIGENETIKA" of the Ministry of Education and Science of Russia under the registration number VKPM: B-12668.

The result of studies of the carcass of a woolly rhinoceros according to examination No. 373 dated 04/09/2015 for anthrax antibodies is negative. Place of testing GBU PC (Y) "Yakutsk Republican Veterinary Testing Laboratory".

The resulting strain is characterized by the following features.

Morphology: small rods arranged singly and in irregular clusters.

Cultural characteristics: on mesopatamia agar (MPA), mass. %: enzymatic peptone - 1.0; sodium chloride - 0.5; agar - 1.0; meat water - the rest, pH 7.0-7.2) and timing agar (based on fish meal hydrolyzate), wt. %: fish meal hydrolyzate - 1.2; enzymatic peptone - 1.2; sodium chloride - 0.6; agar - 1.0; distilled water the rest; pH 7.1-7.5, forms raspberry or red-orange pasty, round-shaped colonies, 1-3 mm in diameter. The consistency is soft, easily removed from the surface of the agar, easily smeared.

On Sabouraud's medium (composition, g / l: peptone - 10.0 maltose - 40.0; agar - 12.0 ± 2.0; purified water up to 1 l), forms raspberry or red-orange pasty rounded colonies with a diameter 1-3 mm. The consistency is soft, easily removed from the surface of the agar, easily smeared.

On agar Münz medium (13. Kersten D.K. Morphological and cultural properties of indicator microorganisms of oil and gas survey - Microbiology, No. 5. - 1963. - P. 1024-1030) with oil and oil products (diesel fuel, motor oil, gas condensate, gasoline, aviation kerosene) of the following composition: (wt.%) KNO ₃ - 0.4; MgSO ₄ ⋅7H ₂ O 0.08; NaCl - 0.1; K ₂ HPO ₄ - 0.14; KH ₂ PO ₄ - 0.06; agar - 2.0; oil or oil products - 1.0; distilled water - the rest, pH - 7.2, forms raspberry or red-orange pasty rounded colonies, 1-2 mm in diameter. The consistency is soft, easily removed from the surface of the agar, easily smeared.

In mesopatamia broth it forms a haze without a film.

The studied characteristics of the strain: grows on a medium with 2% NaCl at pH 5.5-9.0 (optimum at pH 6.5-7.4) conv. units, at a temperature of + 2 ... + 37 ° С, (+ 4 ... + 20 ° С).

Attitude to oxygen: aerobic, but it can develop in the absence of oxygen, but more slowly.

Used carbon sources: sorbitol, lactose, glucose.

Other tests: Indole positive. The Voges-Proskauer reaction is negative. Phenylalanine deoxaminase is negative. Does not ferment beta galactosidase. Does not produce hydrogen sulfide. Not active against ornithine and lysine. Does not use sodium citrate and sodium malonate. Utilizes inositol Utilizes petroleum hydrocarbons. Does not produce hemolysins.

Method, conditions and composition of solid media for culture growth: sterile nutrient agar based on fishmeal hydrolyzate or industrial meat-peptone agar, poured into Petri dishes, after solidification, dried in a thermostat, inoculated and cultivated at a temperature of 20-30 ° C, within 72 hours.

The strain is non-virulent, non-toxic, non-toxigenic (tested on white mice).

The strain is not pathogenic for plants (the test for phytotoxicity was carried out on the seeds of sowing oats (Avéna sativa), variety Skakun, wheat (Triticum), variety Prilenskaya-19, and meadow clover (Trifolium pretense).

The strain can be maintained by regular subcultures (1 time in 10-14 days) on a canted MPA or stored in a lyophilized state in ampoules at a temperature of + 4 ° C.

The strain can be obtained in the laboratory of geochemistry of caustobiolites, 677007, Republic of Sakha (Yakutia), Yakutsk, st. Avtodorozhnaya, house 20, IPNG SB RAS, office. 401.

The invention is illustrated by the following examples.

Example 1. Isolation and cultivation of the bacterial strain Serratia plymuthica VKPM B-12668.

верхняя челюсть до 7 см), найденного осенью 2014 г. охотниками на берегу реки Семюэлях в Абыйском районе Республики Саха (Якутия).The bacterial strain Serratia plymuthica VKPM B-12668 was isolated by targeted research from the biomaterial of a frozen prehistoric baby woolly rhinoceros (sample head

upper jaw up to 7 cm), found in the fall of 2014 by hunters on the banks of the Semyuelakh River in the Abyisky District of the Sakha Republic (Yakutia).

For this, 1.0 g of biological material taken from the frozen carcass of the above object was ground in a mortar under aseptic conditions, introduced into a 250 ml flask containing a pre-prepared 100 ml of a mineral medium of the following composition: (wt%) KNO ₃ - 0 ,4; MgSO ₄ 7H ₂ O - 0.08; NaCl - 0.1; K ₂ HPO ₄ 0.14; KH ₂ PO ₄ 0.06; distilled water - the rest, pH 7.2.

As a source of carbon and energy, oil was used, which was introduced into the mineral medium in an amount of 1000 mg per 1.0 dm3 of the medium.

The incubation was carried out in thermostatically controlled rocking conditions on a UVMT-12-250 installation at 200 rpm and a temperature of + 20 ± 1 ° C.

The growth of bacteria was observed already after 24 hours of incubation by the formation of a turbid emulsion and disintegration of the oil layer.

A pure culture of bacteria was obtained by cultivation under the above conditions and repeated subcultures (more than 10) of the enrichment culture on Petri dishes with MPA.

Then the inoculations were incubated under stationary conditions at various temperatures (+2, + 4, + 20, +30, +37, + 40 ° C).

After 72 hours, during cultivation of the strain at temperatures from +20 and + 30 ° С and after 10 days, during cultivation at temperatures of +2, +4 and + 37 ° С, the appearance of convex, raspberry or red-orange colonies was observed on the surface of the MPA. colors that are cultural-morphological and biochemical descriptions given in Bergey's Guide (14. Bergey's Manual of Systematic Bacteriology Book Review Int. J. of Syst. Bact; July 1985, p. 408.). identified as the genus Serratia bacteria.

At a temperature of + 40 ° C, the growth of colonies was not observed.

Initial screening in the GenBank database showed that the studied strain belongs to the following systematic group: Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Serratia.

The criterion for assigning a microorganism to a particular species is considered to be a homology of at least 97% (16. Biochemistry. - No. 12. - Volume 72. - 2007. - S. 1659-1667).

The analysis of phylogenetic relationship, constructed using strains of closely related microorganisms, showed that the species Serratia plymuthica is the closest to the studied strain (99%).

Example 2. Preparation of a bacterial preparation based on cells of the bacterial strain Serratia plymuthica VKPM B-12668

Isolated colonies of the bacterial strain Serratia plymuthica VKPM B-12668 (Fig. 1), obtained according to example 1, are subcultured on a slant MPA and incubated for 48 hours at a temperature of + 20 ± 1 ° C.

After incubation, cells of the bacterial strain Serratia plymuthica VKPM B-12668 are washed off with a 0.9% NaCl solution and an initial microbial suspension of a bacterial isolate is prepared with a titer of 1 × 10 ⁹ microbial cells / cm ³ according to the optical standard of GISK im. AM Tarasevich.

To obtain a bacterial isolate, a liquid mineral Muntz medium of the above described composition with oil (oil products) is prepared and cultivated in flasks under shaking conditions for 48 hours at a temperature of plus 20 ± 1 ° C at 200 rpm.

A liquid turbid mixture of cells of the bacterial strain Serratia plymuthica VKPM B-12668 with a titer of 1-5 × 10 ⁹ cells / cm ^{3 is} obtained, which can be used as a biological product for processing objects contaminated with oil or oil products.

Example 3. Study of the oil-oxidizing activity of the bacterial strain Serratia plymuthica VKPM B-12668

The cells of the bacterial strain Serratia plymuthica VKPM B-12668 are washed off with a 0.9% NaCl solution from the slanted MPA (according to example 2), an initial microbial suspension of a bacterial isolate with a volume of 25 cm is prepared, with a concentration of 1 × 10 microbial cells / cm ³ according to the GISK optical standard them. AM Tarasevich.

The resulting bacterial isolate of Serratia plymuthica bacteria VKPM B-12668 is cultivated in an aqueous medium with mineral components (wt%): KNO ₃ - 0.4; MgSO ₄ ⋅7H ₂ 0 - 0.08; NaCl - 0.1; K ₂ HPO ₄ 0.14; KH ₂ PO ₄ 0.06; distilled water - the rest; pH of the medium - 7.2 services. units

In cones with a volume of 200.0 cm ³ add 100.0 cm ^{3 of the} finished mineral medium and 1000 mg of oil.

Flasks are inoculated with cells of the bacterial strain Serratia plymuthica VKPM B-12668 to a concentration of 1 × 10 ⁹ cells / cm ³ .

As a control, put the same flask with medium, oil and without bacteria to determine the total (natural) losses.

The natural evaporation of oil and oil products is calculated after 48 hours and after the end of the experiment.

The flasks are cultivated at temperatures of +2, +10, +20 and + 37 ° C for 10 days.

The results of the experiment are presented in table 1.

Biodegradation of oil and oil products is determined by the spectrometric method using the TSC-025 concentrometer (16. FR. 1.31.2007.03234 MVI 01.02.117. Methods for measuring the mass concentration of oil products in drinking, natural and waste waters by the IR spectrometric method using the concentrometer " TSC-0.25 ").

The experimental data show that the natural loss of oil and oil products in the medium without the addition of bacterial isolate after 48 hours averaged at temperatures: + 2 ° С - 0.2%; + 10 ° С - 0.3%; + 20 ° С - 0.6%; + 37 ° С - 1.0%.

On the 10th day, the degradation of oil and oil products in the control variants was insignificant, while under the influence of the proposed bacterial strain Serratia plymuthica VKPM B-12668, the degradation of oil and oil products was at a temperature of + 2 ° C - 2.98-3.64 %; at a temperature of + 10 ° C - 5.84-17.21%; at a temperature of + 20 ° C - 63.04-67.16%; at a temperature of + 37 ° С - 46.88-64.47%) (table 1).

Example 4. Purification of fresh water from oil and oil products using cells of the bacterial strain Serratia plymuthica VKPM B-12668.

In a desiccator with a volume of 5.0 dm ^3, 3.0 dm ^{3 of} tap water, which has settled for a day at room temperature, is introduced.

30,000 mg of crude crude oil (petroleum products) and 0.03 dm ^{3 of the} bacterial isolate of the strain are added to the water. bacterial strain Serratia plymuthica VKPM B-12668, prepared according to example 2, containing 1.0-10 ⁹ cells / cm ³ suspension.

As a control, water is used in the amount of 3.0 dm ³ with the addition of 30,000 mg of oil (petroleum products) and without the introduction of a microbial suspension of the bacterial isolate.

The experiment is carried out for 60 days at a temperature of + 20 ° C.

It was shown that under the influence of the bacterial strain Serratia plymuthica VKPM B-12668 for 60 days, destruction in fresh water for oil was 57.67% and for diesel fuel and motor oil - 57.07%; for hydraulic oil - 56.87%; for gas condensate - 56.69%; for aviation kerosene - 59.99%; for gasoline - 59.29% (table 2).

Thus, the advantage of the proposed bacterial strain Serratia plymuthica VKPM B-12668 is that it has a high destructive ability in relation to both oil and oil products in a relatively short time (from 10 to 60 days), in a wide temperature range from + 2 to + 37 ° С, which can be used to clean environmental objects from oil and oil product pollution (diesel fuel, motor oil, gas condensate, hydraulic oil, aviation kerosene, gasoline).

Claims (1)

The bacterial strain Serratia plymuthica VKPM B-12668, isolated from the biomaterial of a paleontological sample (fossil fossil woolly rhinoceros calf), is a destructor of oil and oil products.

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