RU2565561C1 - Method of obtaining of biomass of activated autochthonic microorganisms-biodestructors of n-phosphon methyl glycine (glyphosate) - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method of obtaining of biomass of activated autochthonic microorganisms-biodestructors of N-phosphon methyl glycine (glyphosate) is offered. Before cultivation the composition of the liquid medium is added by 0.05-0.4 mcg·cm^-3 of glyphosate and perfluordecalin emulsion stabilized by the emulsifier monostearate sorbitan. The final content of perfluordecalin in the liquid nutrient medium is 1.0% by weight, a monostearate sorbitan - 0.0005% by weight. Throughout all the cycle of cultivation fractionally, with the interval 2-4 hours, glyphosate is added with the increment 1.25-2.5 after each addition, starting from the amounts of glyphosate equal to its initial contents in the cultural medium.

EFFECT: possibility to obtain in one cycle of deep cultivation the cultures of bacteria and micromycetes with the glyphosate resistance increased 150-200 times, and ability to destruction of glyphosate, 7-11 times exceeding the biodestructive activity of initial autochthonic isolates.

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Description

I. The technical field to which the invention relates.

The invention relates to microbiology, biotechnology, ecology, and also to agriculture and can be used to produce activated biomass of soil autochthonous microorganisms-biodestructors of N-phosphonomethylglycine (glyphosate), the systemic herbicide most used in the world practice, registered in more than 120 countries under various trademarks (Roundup, Ground Bio, Hurricane, Glysol, etc.), intended for processing crops of a wide range of crops, in gardens and parks x, in the water and forestry sectors, the ability to accumulate in the soil, plants and aquatic environment, thereby creating conditions for intensive pollution and negative impact on humans and animals.

II. State of the art

Known methods for producing biomass of microorganisms-biodestructors of xenobiotics phosphonates by isolating accumulative and pure cultures from soils contaminated with them, including glyphosate, followed by selection of strains with increased biodestructive activity against glyphosate in phosphorus-limited "hungry" environments using in as a source of glyphosate phosphorus or AMPA, selection and cultivation on media containing certain concentrations of these xenobiotics (Kharechko A.T. et al. of organisms for the destruction of hazardous substances polluting the environment / J. Russian Chemical Chemical Society named after D.I. Mendeleev. 1993, v. XXXVII, No. 3. - P. 40-43; Kononova S.V., Nesmeyanova M.A. Phosphonates and their degradation by microorganisms / Biochemistry, 2002. - V. 67. - No. 2. - P. 220-233; Kuznetsova E.M., Chmip V.D. Glyphosate: environmental behavior and levels of residues / Current problems of toxicology, 2010. - No. 1. - P. 87-95; Kravtsov KS et al. Isolation of microorganisms capable of decomposing phosphonates from the environment / Chemical and Biological Safety, 2006, No. 6. - S. 3-9; Shushkova T.V. Biodegradation of glyphosate by soil bacteria / Dis. Cand. biol. Sciences / Pushchino. - 2010 .-- 132 p.).

The disadvantage of the methods used is that none of them allows you to get the biomass of autochthonous activated microorganisms, glyphosate biodestructors in a single cultivation cycle. To obtain it, a long preliminary work is required to prepare microbial cultures of biodestructors and optimize media, cultivate in media “hungry” for phosphorus and other components, as well as nutritious media, which leads to a decrease in biodestructive activity and / or yield of biomass suitable for introduction into the soil contaminated with xenobiotic, loss of natural adaptability of the resulting crops with low resistance to stressful chemical, physical and biological environmental factors.

The closest analogue to the claimed method is a method based on the use of soil glyphosate-resistant isolates of bacteria, selected sequentially on media with increasing concentrations of xenobiotics, and their deep cultivation in a liquid medium with a certain concentration of xenobiotics to obtain biomass biodestructor with increased resistance to glyphosate (see. article Bakulin VM The use of soil glyphosate-resistant isolates of bacteria of the genus Pseudomonas in biotechnology of phosphonome degradation tilglycine / V.M. Bakulin and others // Veterinary medicine. - 2012, No. 3-4. - S. 17-19).

A common essential feature of the proposed method with an analogue is the use of microorganisms contaminated with glyphosate and its decomposition products isolated from soils and their use for biomass biodestructors by deep cultivation in liquid nutrient media with glyphosate (see Semenov S.M. Laboratory media for actinomycetes and fungi / SM. Semenov // Moscow. - 1990. - 240 p .; Ravilov, A.Z. Microbiological media / A.Z. Ravilov et al. // Kazan. - 1999. - 398 p.).

The disadvantages of this analogue include:

- the need for lengthy breeding work to obtain a high level of resistance to glyphosate;

- the use of only one concentration of glyphosate in a liquid medium when producing biomass, and if it is large, then the growth of the culture will be suppressed by an excess of glyphosate, if it is small, then the biodestructive activity of the culture will be low;

- the absence of a correlation between the achieved level of stability of the isolated strain and the biodestructive activity obtained on its basis during deep biomass cultivation (a high level of stability does not guarantee the activity of glyphosate biodegradation);

- the biomass of the biodestructor obtained in this way loses its speed of adaptation to changing environmental conditions and is not able to occupy an ecological niche in comparison with the native (of the same species) microflora native to a given locality (soil) having specific mechanisms of natural antagonism (competitive displacement, bacteriocins, etc. .).

III. The invention consists in the following.

The task was to develop a method for producing biomass of activated microorganisms-glyphosate biodestructors in a single cultivation cycle.

The problem is solved by using the developed method for the deep cultivation of crops during one cycle of cultivation of indigenous biodestructive microorganisms in liquid media on a conventional nutrient basis, into which submicrobostatic glyphosate concentrations and perfluorodecalin emulsified with sorbitan are added before cultivation, followed by fractional with an interval of 2-4 hours the introduction of submicrobostatic, gradually increasing 1.25-2.5 times the amount of glyphosate.

The proposed method for producing biomass of activated autochthonous microorganisms-glyphosate biodestructors makes it possible to obtain cultures of bacteria and micromycetes that are 150-200 times more resistant to glyphosate in the culture medium and capable of glyphosate degradation by the obtained biomass, 7-11 times higher than biodestructive for one deep-cycle cultivation. activity of the original autochthonous isolates. Moreover, the yield of biomass this method is not inferior to the accepted method of culturing glyphosate biodestructors.

The essence of the technical solution - a method for producing biomass of activated microorganisms-glyphosate biodestructors is explained as follows. The introduction of submicrobostatic concentrations of glyphosate into the culture medium leads to selective pressure in the culture medium and the selection of biodestructive microorganisms combining the ability to intensive growth and decomposition of xenobiotics during growth. In this case, the subsequent fractional introduction of increasing submicrobostatic concentrations of glyphosate under the control of the growth of cultures of biodestructors leads to a constant increase in selective pressure and the selection of the most stable and active in terms of biodegradation of the cell population and a simultaneous increase in the biomass of biodestructors due to the usefulness of the natural environment and the presence of emulsified perfluorodecalin in the medium.

The presence of glyphosate in the culture medium is a stressful chemical factor for microorganisms; therefore, emulsified perfluorodecalin is introduced into the culture medium as a powerful anti-stress component, providing improved nutrition and growth intensification of biodestructive microorganisms under the negative influence of glyphosate (Organofluorine compounds, stresses and survival of microorganisms / C. V. Darmova, MK Bakulin and others. Collection "Science-Production-Technology-Ecology. - 2009. - S. 137-139).

Perfluorodecalin (PFD, chemical formula - C ₁₀ F ₁₈ ), TU 95-1233 - a colorless, transparent, non-combustible, odorless liquid, with a viscosity of 2.74 mm ² × cSt ^-1 at 25 ° C, a density of 1.945 g / cm ³ and boiling point 155 ° C. PFD does not serve as a nutrient, is not utilized by microorganisms, and is the best organofluorine compound for use in biotechnological processes as a powerful anti-stress factor and at the same time an activator of the growth of the microbial culture of biodestructors (Ivanitsky G.R. Biophysics on the threshold of the new millennium: perfluorocarbon media and gas-transporting blood vessels G.R. Ivanitsky // Biophysics. - 2001. - No. 1. - S. 5-33; Ivanitsky G.R. Nanocontainers based on perfluorocarbons with the function of transfer of nitric oxide / G.R. Ivanitsky // Biophysics. - 2008. - No. 2. - P. 367-377; Bakulin MK The theory and practice of using perfluorocarbons of “blue blood” in deep cultivation of biodestructors / MK Bakulin and others. Theoretical and applied ecology . 2010, No. 4, p. 4-8).

Biodestructors were microbial isolates isolated from soil subjected to eight-fold glyphosate treatment for four years: gram-negative aerobic bacterium Pseudomonas fluorescens 047, capable of growth in a liquid medium containing 0.4 μg cm- ³ glyphosate, gram-positive facultative anaerobic bacterium Bacillus cereus 41 and mitosporic mikromitcetami Aspergillus niger 17 capable of growing containing 0.05 ug in liquid glyphosate · cm ^-3 (see. article Bakulin VM Isolation of bacteria of the genus Pseudomonas from soils contaminated phosphono xenobiotic etilglitsinom / VM Bakulin, etc. // Veterinary Medicine -.. 2012. - №1 -. pp 9-11).

The cultivation was carried out at a temperature of 30 ° C in biological reactors BIOR (Yoshkar-Ola) or BIOSTAT B Plus (Sartorius) under continuous mixing and forced aeration (air flow: 0.5 air volume per volume of medium per minute), ceteris paribus.

In common with the claimed method is that for cultivation used the environment, usually used for growing these microorganisms.

For the growth of biodegradable microorganisms based on bacterial cultures of P. fluorescens 047 to Bacillus cereus 41, a liquid nutrient medium based on enzymatic casein hydrolyzate was used, having the following ratio of components, g / l:

KH ₂ PO ₄ 2.2 K ₂ HPO ₄ 1.7 MgSO ₄ 0.6 Na ₂ S ₂ O ₃ 0.6 amine nitrogen 130 mg% hydrogen ions 7.1 units pH

To grow biodestructors based on the Aspergillus niger 17 mitospore micromycete culture, Steinberg’s liquid synthetic medium with the addition of soy flour was used, which has the following ratio of components, g / l:

soy flour 20,0 sucrose 50,0 NH ₄ NO ₃ 1.9 KH ₂ PO ₄ 0.35 MgSO ₄ × 7H ₂ O 0.25 FeCl ₂ 0,0004 ZnCl ₂ 0,0004 CuCl ₂ 0.0001 MnCl ₂ 0.0001 MoCl ₂ 0.00002 CaCl ₂ 0.00002; hydrogen ions 6.3 units pH

The finished emulsion of perfluorodecalin stabilized using sorbitan monostearate is aseptically introduced into sterile liquid culture media (the final content of perfluorodecalin in liquid culture media is 1.0% by weight; the final content of sorbitan monostearate in liquid culture media is 0.0005% by weight of the total amount used nutrient media), which is then used to grow biodestructors.

Glyphosate is added to the media before cultivation in amounts that provide an initial concentration of xenobiotic, to which microorganisms are resistant.

Sowing cultures were introduced into the medium in quantities sufficient to create a concentration of bacteria in the medium when sowing 1.0 · 10 ⁸ bacteria per cm, aspergillus culture - 1.0 · 10 ⁷ conidia in cm ³ . For a culture of P. fluorescens 047, the initial concentration of glyphosate in a liquid medium is 0.4 μg cm ^–3 ; for Bacillus cereus 41 and Aspergillus niger 17-0.05 μg · cm ^-3 . 4 hours after the start of cultivation, glyphosate for the culture of P. fluorescens 047 glyphosate is additionally added to the medium at the rate of 0.4 μg cm ^–3 and then according to the scheme (Scheme 1) every two hours in increasing concentrations: 0.8-1.5 -4.0-8.0-10.0-20.0-50.0-80.0 mcg · cm ^-3 ; for the culture of Bacillus cereus 41, 4 hours after the start of cultivation, an additional 0.05 μg · cm ^-3 glyphosate is added and then every two hours according to the scheme (Scheme 2): 0.1-0.2-0.5-1.0- 2.5-6.0-15.0-20.0 mcg · cm ^-3 ; for the culture Aspergillus niger 17, 4 hours after the start of cultivation, an additional 0.05 μg · cm ^-3 glyphosate is added and then every 4 hours according to the scheme (Scheme 3): 0.1-0.5-1.0-2.0 -5.0-10.0-15.0-15.0 mcg · cm ^-3 . The cultivation of bacteria lasts 28 hours, aspergillus - 56 hours.

Example 1. Obtaining the biomass of microorganisms-glyphosate biodestructors based on the culture of bacteria P. fluorescens 047 and B. cereus 41 (tables 1 and 2) is carried out in a liquid nutrient medium based on an enzymatic hydrolyzate of casein of the following composition, g / l:

KH ₂ PO ₄ 2.2 K ₂ HPO ₄ 1.7 MgSO ₄ 0.6 Na ₂ S ₂ O ₃ 0.6 amine nitrogen 130 mg% hydrogen ions 7.1 units pH initial concentration of hydrogen ions 7.2 units pH

Before cultivation under sterile conditions, a prepared perfluorodecalin emulsion stabilized using a sorbitan monostearate emulsifier is added to the medium (the final content of perfluorodecalin in the liquid nutrient medium is 1.0% by weight; the final content of sorbitan monostearate in the liquid nutrient medium is 0.0005% by weight of the total the amount of nutrient medium used).

The initial inoculum concentration of bacteria in the medium is 1.0 · 10 ⁸ live microbial cells / cm ³ . Glyphosate is introduced into the culture medium with P. fluorescens 047 according to Scheme 1. Its initial concentration in the liquid medium is 0.4 μg cm ^–3 . 4 hours after the start of cultivation, 0.4 μg · cm ^-3 glyphosate is additionally added to the medium and then every two hours in increasing concentrations: 0.5-1.5-4.0-8.0-10.0-20, 0-50.0-80.0 μg glyphosate / cm ^-3 culture medium.

The cultivation of bacteria lasts 28 hours (table 1).

For the Bacillus cereus 41 culture, the initial concentration of glyphosate in the liquid medium is 0.05 μg · cm ^-3 , 4 hours after the start of cultivation, 0.05 μg · cm ^-3 glyphosate is additionally added and then every two hours according to the scheme (Scheme 2): 0 , 1-0.2-0.5-1.0-2.5-6.0-15.0-20.0 μg · cm ^-3 .

The cultivation of bacteria lasts 28 hours (table 2).

Example 2. Obtaining the biomass of microorganisms-biodestructors of glyphosate based on the culture of Aspergillus niger 17 micromycetes is carried out in a nutrient medium based on Steinberg liquid synthetic medium with the addition of soy flour, having in its composition the following ratio of components, g / l:

soy flour 20,0 sucrose 50,0 NH ₄ NO ₃ 1.9 KH ₂ PO ₄ 0.35 MgSO ₄ × 7H ₂ O 0.25 FeCl ₂ 0,0004 ZnCl ₂ 0,0004 CuCl ₂ 0.0001 MnCl ₂ 0.0001 MoCl ₂ 0.00002 CaCl ₂ 0.00002 hydrogen ions 6.3 units pH

Before cultivation under sterile conditions, a prepared perfluorodecalin emulsion stabilized using a sorbitan monostearate emulsifier is added to the medium (the final content of perfluorodecalin in the liquid nutrient medium is 1.0% by weight; the final content of sorbitan monostearate in the liquid nutrient medium is 0.0005% by weight of the total the amount of nutrient medium used).

Glyphosate is added to the culture medium with Aspergillus niger 17 before cultivation at a concentration of 0.05 μg · cm ^-3 , 4 hours after the start of cultivation, an additional 0.05 μg · cm ^{-3 of} glyphosate is added and then every 4 hours according to the scheme (Scheme 3 ): 0.1-0.5-1.0-2.0-5.0-10.0-15.0-15.0 μg · cm ^-3 Cultivation of aspergillus continues for 56 hours (table 3).

Claims (1)

A method for producing biomass of activated autochthonous microorganisms-biodestructors of N-phosphonomethylglycine (glyphosate) as a result of one cycle of deep cultivation of cultures of native microorganisms-biodestructors in liquid media on conventional nutrient bases used for their cultivation, characterized in that before the start of cultivation, the composition of the liquid medium submikrobostaticheskie making glyphosate concentration - 0.05-0.4 g · cm ^-3 and stabilized using emulsifier sorbitan monostearate emulsion per tordecalin, and the final content of perfluorodecalin in the liquid nutrient medium is 1.0% by mass, the final content of sorbitan monostearate is 0.0005% by mass of the total amount of used liquid nutrient medium, then it is fractional throughout the cultivation cycle repeatedly, with an interval of 2 4 hours, submicrobostatic is administered, increasing at each injection 1.25-2.5 times the amount of glyphosate, starting with the amounts of glyphosate equal to its initial content in the culture medium.