RU2487933C1 - STRAIN Penicillium sp, HAVING POLYFUNCTIONAL PROPERTIES AND REALISING TRANSFORMATIONS OF ORGANIC REMAINS OF NATURAL ORIGIN - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: strain Penicillium sp. PBT-2 extracted from samples of humic pod mixed with cereal straw and having polyfunctional properties and promoting increased productivity of agroecosystem, participating in processes of transformation of organic remains of natural origin, is deposited in the Departmental Collection of Beneficial Microorganisms of Agricultural Purpose of the Russian Academy of Agricultural Sciences (RCAM) (GNU VNIISHM) under the registration number RCAM 01017 and may be used in transformation of organic remains of natural origin.

EFFECT: improved productivity of agroecosystem.

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Description

The invention relates to the microbiological industry and the use of a culture of the genus Penicillium of the species Penicillium sp. To obtain a new strain of Penicillium sp., Which has multifunctional properties and helps to increase the productivity of the agro-ecosystem, participating in the processes of transformation of organic residues of natural origin.

Known methods for preparative chromatographic separation of complex enzyme preparations of producer strains Trichoderma sp., Penicillium sp., Chrysosporium sp., Aspergillus sp. And isolation of individual enzymes, inbi.ras.ru/labs/sinitsyn/index ...

A known method of processing sludge in a sewage treatment plant, including the stage of treating sludge with micro fungi of the genus Penicillium, Trichoderma, Fusarium, Phoma, Mucor, Galactomyces, Aspergillus, Botrytis, Geomyces and mixtures thereof, and culturing in a parallel tank, RU No. 2303572 C2, C02F 3 / 34, C02F 11/02, C02F 1/72, 07.27.2007.

A known method of cleaning the soil from oil pollution, involving the periodic introduction into the soil of a suspension of microorganisms in the form of strains of micromycetes selected from the genera Penicillium, Mucor, Aspergillus, Trichoderma, RU No. 2049739 C1, C02F 3/34, 12/10/1995.

A known method of saccharification of lignocellulosic raw materials, including the preparation of raw materials and processing the prepared raw materials with an enzyme preparation, which is used as a culture fluid obtained by culturing a strain of Penicillium verruculosum BKMF-3972D, RU No. 2405838 C1, C13K 1/02, 10.12.2010.

A known method of obtaining a strain of fungus. Penicillium verruculosum is a producer of the enzyme encoded by the nucleotide sequence, which provides for the transformation of Penicillium verruculosum fungal cells by genetic construction, RU No. 2378372 C2, C12N 15/00, C12N 9/24, C12N 1/15, 01/10/2010.

Known strain of the fungus Penicillium verrucosum VKPM F-984, used to obtain funds with immunomodulating properties, RU No. 2373276 C1, C12N 1/14, A61K 36/06, 11/20/2009.

Known strain of mycelial fungus Penicillium verruculosum VKM F-3972D is a producer of a complex of cellulases containing cellobiohydrolase, endoglucanase, β-glucosidase, cellobiasis, xylanase and xyloglucanase, RU No. 2361918 C1, C12N 9/00, 20.07.2009 19.07.2009.

Known strain of the fungus Penicillium vermiculatum Dang.BH3P-24 for obtaining a drug against phytopathogenic fungi, RU No. 2322490 C1, C12N 1/14, 04/20/2008.

Known DNA fragment of the mycelial fungus Penicillium verruculosum, RU No. 2238974 C2, C12N 1/21, C12N 15/00, 10.27.2004.

Famous enzyme preparations obtained on the basis of new recombinant strains of Penicillium verruculosum that efficiently hydrolyze various types of pre-treated coniferous and deciduous wood, inbi.ras.ru/dissertation/disser ...

Known for the novel endo- (1-4) -β-D-xylanase XylB (SEQ ID NO: 1) of the mycelial fungus Penicillium canescens, RU No. 2412246 C2, C12N 9/42, C12N 15/55, C12N 15/11, C12N 1/14, 02.20.2011.

Known strain of the fungus Penicillium canescens - producer of secreted beta-galactosidase, RU No. 2197526 C1, C12N 15/56, C12N 1/15, 01/27/2003; RU No. 2126049 C1, C12N 15/56, C12N 15/80, C12N 1/15, 02/10/1999; RU No. 2080387 C1, C12N 9/38, C12N 15/56, C12N 15/80, C12N 1/14, 05/27/1997.

A known technology for the production of technical enzymes β-glucanases (cellulases) and xylanases based on highly active mutant producer strains Penicillium funiculosum and Trichoderma longibrachiautum for use as feed additives in diets of farm animals and poultry, inbi.ras.ru/labs/simtsyn/index ...

A known method of biotechnological production of fermentable sugars from lignocellulosic materials, including processing the raw material with an enzyme preparation obtained by culturing the Penicillium funiculosum S-2006 strain on medium RU No. 2316584 C1, C12N 1/14, C12P 19/02, C12P 19/04, C12N 9/04 42, 02/10/2008.

Known strain of mycelial fungus Penicillium funiculosum BKM F-3668D (All-Russian collection of microorganisms at the IBPM named after G.K.Skryabin RAS) is a producer of a complex of carbohydrases containing cellulases, β-glucanase, xylanase, pectinase and mannanase, RU No. 2287070, C 128755 / 14, C12N 9/42, 11.20.2006.

Known strain Penicillium funiculosum IMI378536, producing a complex of enzymes - cellulase, endo-1,4-β-xylanase, cellobiohydrolase, β-glucosidase, endo-1,3 (4) -β-glucanase, feruloyl esterase, RU No. 2261910 C2, C12N 9/16, C12N 9/24, C12N 9/42, C12N 1/16, 10/10/2005.

Known strain Penicillium funiculosum KM MSU 433 - a product of glucose oxidase, RU No. 2170762 C2, C12N 1/14, C12N 904, 07.20.2001.

Known strain of micromycete Penicillium sizovae P11 (stored in the collection of bacteria of Vyatka State University under registration number VGUCM-P11) is a wood lignin destructor, RU No. 2355753 C2, C12N 1/14, 05.20.2009.

Known strain of the fungus Penicillium chrysogenum - producer of benzyl penicillin and phenoxymethylpenicillin, RU No. 2035514 C1, C12P 37/00, 05/20/1995; RU No. 1594991 C, C12P 37/00, C12N 1/14, July 9, 1995; RU No. 1549075 A1, C12P 37/00, 12/15/1994.

Known strain of the fungus Penicillium chrysogenum BKMF-3330D, used to obtain biomass extracting radionuclides and heavy metals from liquids, RU No. 2024078 C1, G21F 9/18, C02F 3/34, 11/30/1994.

Known strain of the fungus Penicillium species CCM Fn 37B used to clean the surface of water from floating oil, RU No. 2063435 C1, C12N 1/14, C02F 3/34, 07/10/1996.

A known method for the biological treatment of sulfur-containing wastewater, including biodegradation of organosulfur compounds using destructive microorganisms, which use the Penicillium sp. Micromycete association. No. 15 and bacteria Thiobacillus sp. No. 3 in the ratio 4: 1, RU No. 2314267 C2, C02F 3/34, C02F 103/36, C12N 1/20, C12N 1/14, 01/10/2008.

A known microbiological method for removing a nitroaromatic compound present in a solution or in soil, comprising contacting the solution or soil with the biomass of Penicillium sp. (deposit number 1-2081, CNCM, Pasteur Institute, France) under conditions suitable for the mineralization of the nitroaromatic compound by the indicated strain of Penicillium sp. in the presence of a carbon source for the specified strain, RU No. 2249564 C2, C02F 3/34, B09C 1/10, C12N 1/14, 04/10/2005.

In known microorganisms of the genus Penicillium strain Penicillium sp., With the claimed properties, was not found.

The basis of the present invention is the solution of the problem, allowing to identify from the genus Penicillium a strain having multifunctional properties and contributing to increased productivity of the agroecosystem.

The technical result of the present invention is the isolation of a strain of Penicillium sp., Participating in the processes of transformation of organic residues of natural origin.

According to the invention, this problem is solved due to the fact that a new strain of Penicillium sp.PBT-2 has been identified, which has multifunctional properties, transforms organic residues of natural origin and increases the productivity of the agroecosystem.

The applicant has not identified sources containing information about technical solutions identical to the present invention, which allows us to conclude that it meets the criterion of "novelty."

The applicant is not aware of any publications that would contain information on the influence of the distinguishing features of the invention on the achieved technical result. In this regard, according to the applicant, it can be concluded that the claimed technical solution meets the criterion of "inventive step".

A new strain of Penicillium sp. PBT-2, which has multifunctional properties and helps to increase the productivity of the agroecosystem, participating in the processes of transformation of organic residues of natural origin.

The specific name is Penicillium sp. Thom.

Strain Penicillium sp. PBT-2 was isolated in July 2009 from samples of sod-podzolic soil mixed with grain straw in the Leningrad Region.

Cultural and morphological, physiological and biochemical characteristics of culture

Slow-growing strain. Microscopy (phase-contrast and light) of a 5-day-old culture on the Chapek medium showed the presence of multicore mycelium, erect conidiophores with branches at the top, the upper row of whorls consists of phialids, in which there are round spores. Aerobe. Catalase positive. On Wednesday, Chapek appears in two days, the colonies are white, on the 3-5th day in the middle of green, on the 7th day the colonies darken to gray-green, with a white rim, a light brown pigment appears.

Relation to carbon sources.

The strain uses carbohydrates on the 5-7th day with acidification of the medium: good - arabinose, galactose, glucose, weaker - maltose, rhamnose, sorbitol, sucrose, raffinose. Does not use - dulcite.

It grows on the breast cancer, actively dilutes the gelatin layer by layer from above, growth from above. On BCH, ammonia forms, nitrates restores. Starch weakly hydrolyzes. On Ashby medium, moderate growth, capable of nitrogen fixation. Grows on MPA, good growth on MPA with glycerin. Slowly grows on liquid and agarized Getchinson’s medium with cellulose (filter paper). Salt tolerant, grows well on BCH with 2.5% and 6.5% NaCl.

Identification of the strain Penicillium sp.

The culture is investigated by the numerous properties used in modern taxonomy.

To deposit the isolated strains of microorganisms in the Departmental collection of useful agricultural microorganisms of the Russian Agricultural Academy (RCAM), their genetic identification was additionally carried out.

To identify the genus of the micromycete strain, the ITS region (ITS + 5S + ITS2) was sequenced. The greatest similarity was revealed with the genus Penicillium, 100% similarity (the closest relative is Penicillium crustosum).

Identification data characterizing the Penicillium sp. Strain are presented in the figure at the end of the description.

The culture is investigated by the numerous properties used in modern taxonomy. The strain is identified using publications: "Soil micromycetes", ed. Bilay V.I. (Kiev, 1982), Raper K.B., Thom Ch. A manual of the Penicillia. Baltimore (1949), “Soil Mycology” Mirchink T.G. (Moscow State University, 1988), “Workshop on Microbiology” edited by N. Egorov. (1976), Internet resources, etc., and assigned to the genus Penicillium, species sp.

The strain Penicillium sp.PBT-2 was deposited November 29, 2011 under registration number RCAM 01017 in the Departmental collection of beneficial microorganisms for agricultural purposes “State Scientific Institution. All-Russian Scientific Research Institute of Agricultural Microbiology (GNU VNIISKhM of the Russian Agricultural Academy). ”

Example 1

Isolation of Penicillium sp. PBT-2

The Penicillium sp.PBT-2 strain was isolated from samples of sod-podzolic soil mixed with grain straw by sowing on Chapek's agar medium (limiting dilution method, 3rd dilution).

The number of CFU (colony forming units) of the strain Penicillium sp.PBT-2 in the sample reaches 20-30 thousand / g of soil.

Example 2

Cellulolytic activity

When grown on a standard agar or liquid Hetchinson medium, a strain of Penicillium sp. PBT-2 has the ability to decompose cellulose (filter paper).

On Wednesday, 1 ml of culture fluid is added to Gatchinson’s filter paper.

After 3 weeks of cultivating a micromycete culture grown on Chapek’s beveled agar medium, a change in substrate is observed.

Decomposition zones of the filter paper are formed, which darken.

The determination of cellulase activity is carried out on the basis of the residual amount of undigested cellulose.

The residual amount of unsplit pulp is 20%.

Example 3

Transformation processes of organic residues of natural origin

Strain Penicillium sp. PBT-2 is actively involved in the oxidation of organic compounds of natural origin.

Strain Penicillium sp. PBT-2 produces enzymes of the oxidoreductase class - catalase, peroxidase (participating in the decomposition of organic residues, including humic substances) and polyphenol oxidase (contributing to the synthesis of humic compounds) (A.Sh. Galstyan. Enzymatic activity of soils in Armenia. 1974. - 275 s .).

Strain Penicillium sp. PBT-2, grown on a special nutrient medium with organic additives, decomposes plant debris (cereal straw, wood waste).

After 1 month, a discoloration of the substrate (browning) is observed, the substrate becomes loose, easily breaks. After 2-3 months, the appearance of humic substances (fulvic acids and humic acids - in trace amounts) is observed.

Strain Penicillium sp. PBT-2 is stored in the collection of Petersburg Biotechnologies LLC, 196603, St. Petersburg - Pushkin, Krasnoselskoye Shosse, 14/28, building AB, room. 1-H.

The way to maintain and store:

The culture is grown on Chapek medium at 25 ° -30 ° C.

It is stored at a temperature of + 4 ° C in the refrigerator.

Reseeding I every 3 months.

Long-term storage (3 years or more) is carried out in a cryopreserved state at -80 ° C.

The proposed strain of Penicillium sp. PBT-2 refers to the microbiological industry and includes well-known isolation methods, confirmed by studies of Petersburg Biotechnologies LLC, which determines, according to the applicant, that his criterion is “industrial applicability”.

New strain of Penicillium sp. PBT-2 has semi-functional properties and helps to increase the productivity of the agroecosystem, participating in the processes of transformation of organic residues of natural origin.

Identification data of the strain Penicillllium sp.

Claims (1)

The Penicillium sp. Strain, possessing multifunctional properties, transforming organic residues of natural origin and increasing the productivity of agroecosystems, is deposited in the Departmental Collection of Useful Microorganisms of Agricultural Purpose of the Russian Agricultural Academy (GNAM VNIISCHM) under registration number RCAM 01017.