Nutrient medium for cultivating soil microorganisms

Abstract

FIELD: biotechnology.SUBSTANCE: creation of dense nutrient media intended for cultivating soil-derived microbiota, used to study the morphological and physiological characteristics of soil microorganisms. The nutrient medium includes soy milk, sodium pyruvate, pectin, calcium chloride, vitamins, gellan gum, and water in the following ratio of components, g / l: soy milk 30–75, sodium pyruvate 0.1–0.3, pectin 0.05–0.1, CaCl2 0,3–0.5, vitamins 1–2, gellan gum 10–15, water – the rest.EFFECT: declared nutrient medium allows cultivating, in addition to representatives of the phyla Acidobacteriota and Verrucomicrobiota, also slow-growing representatives of other groups of bacteria.3 cl, 1 dwg

Description

[0001] The invention relates to biotechnology and microbiology, namely to the creation of dense nutrient media intended for cultivating soil-origin microbiota, and can be used to study the morphological and physiological characteristics of soil microorganisms.

[0002] There are a wide variety of culture media used for isolating microorganisms from soil. To increase diversity and the number of colony-forming units (CFU), various modifications are made to the composition of these media.

[0003] Soy milk is widely used as a nutrient substrate for cultivating various microorganisms [1]. Due to its content of plant proteins, peptides, and polyphenols, soy milk provides favorable conditions for the vital activity of microorganisms, as confirmed by studies involving Lactobacillus, Bifidobacterium, and Propionibacterium. Cultivation on soy milk promotes high biomass growth and active accumulation of lactic acid, which is important for the production of fermented products and the study of the physiological characteristics of bacteria [2].

[0004] Sodium pyruvate is used in nutrient media for cultivating soil bacteria as an energy substrate and carbon source. It is particularly useful in nutrient media formulations with low levels of primary carbon sources, which allows simulating oligotrophic soil conditions [3].

[0005] Agar, a polysaccharide extracted from seaweed, is the most commonly used gelling agent in bacterial culture media. It provides a strong and transparent structure to the medium. Agar is heat-stable and resistant to most bacteria, making it a versatile and reliable solidifier for solid and semi-solid culture media. In addition to agar, exotic gelling agents such as carrageenan, alginate, and other polysaccharides can be used to vary the physical properties of the medium and create optimal conditions for the growth of specific microorganisms [4].

[0006] Gellan gum is a water-soluble polysaccharide produced by the bacterium Sphingomonas elodea. In microbiology and biotechnology, it is used as an effective gelling agent and an alternative to agar. Gellan gum forms a highly transparent and heat-stable gel, capable of withstanding temperatures up to 120°C, making it particularly useful for culturing thermophilic microorganisms. Approximately half as much gellan gum is required to achieve equivalent gel strength compared to agar. This substance is also widely used to create transparent culture media that facilitate the microscopic analysis of cells and tissues. Due to its resistance to heat and acids, gellan gum is widely used not only in microbiology but also in the food, pharmaceutical, and cosmetic industries as a thickener, stabilizer, and texturizing agent.Its properties allow the creation of rigid or elastic gels depending on the degree of acetylization, which expands the range of its application in various biotechnological processes and products [5]. Other advantages of gellan gum over agar include the absence of agar-associated organic and inorganic contaminants, such as 5-hydroxymethylfuran-2-carboxylic acid and furan-2-carboxylic acid, sugars, peptides, and, in some brands of agar, heavy metals.

[0007] A nutrient medium (US Patent No. 4,329,431A, USA) and a method for preparing a sterile culture medium, known as "instant culture media," are known. The medium contains a gelling agent, a halogen-based disinfectant bleach (e.g., chlorine, iodine, bromine), an agent for inactivating residual halogen, and standard nutrients for microbial growth. A method for sterilizing the medium without autoclaving is proposed: the gelling agent is dissolved in a buffered water solution, then a disinfectant (halogen-containing bleach) is added, forming a suspension that is heated to boiling. After heating, an excess of the agent is added to the suspension, which neutralizes the residual halogen. This method allows obtaining a sterile culture without the need for high-temperature autoclaving, which simplifies and speeds up the process of medium preparation [6].

[0008] Another invention (US Patent No. 11820974B1, USA) describes a cultivation medium based on pomegranate molasses with soil extract and water. The medium is intended for growing beneficial microorganisms such as Cyanotheca (cyanobacteria) and beneficial fungi.

[0009] Main characteristics of the environment:

[0010] - contains approximately 20 ml of pomegranate molasses diluted with water in a ratio of 1:100 by volume,

[0011] - about 200 ml of soil extract obtained by filtering a mixture of soil (without herbicides) with water in the proportion of 1 g of soil to 10 ml of water,

[0012] - the remaining volume (about 780 ml) is distilled water,

[0013] - may additionally contain potassium dihydrogen phosphate and iron ammonium EDTA to enrich nutrients.

[0014] The medium is characterized by the use of natural components and is suitable for growing beneficial microorganisms that help improve the quality of soil and plants [7].

[0015] A culture medium (US Patent No. 11261422B2, USA) is also known for culturing cells in suspension with the possibility of their easy extraction. The basic idea is to use a mixture of gellan gum and an acidic polysaccharide (e.g., alginate), which, in the presence of divalent metal ions, form a cross-linked three-dimensional network. The concentrations of the components are selected so that the cells are cultivated efficiently and easily separated after cultivation. The medium is prepared in concentrations of gellan 0.002-0.01% and acidic polysaccharide 0.004-0.02%, optimized for maximum efficiency of cell cultivation and collection [8].

[0016] The closest in execution to the proposed nutrient medium is the medium described in the article [9]. The medium with diluted soy milk as a carbon source and gellan gum as a gelling agent (tryptone, 0.17 g / l; soy milk protein, 0.03 g / l; dextrose, 0.025 g / l; sodium chloride 0.05 g / l; potassium phosphate, 0.025 g / l; gellan gum, 15 g / l) gives the highest indicators of the diversity of cultured microorganisms. With its help, representatives of the phyla Acidobacterota and Verrucomicrobiota are isolated.

[0017] However, slow-growing representatives of other bacterial groups are not cultivated on the medium.

[0018] The technical result is the possibility of cultivating on a nutrient medium, in addition to representatives of the phyla Acidobacterota and Verrucomicrobiota, also slow-growing representatives of other groups of bacteria.

[0019] The technical result is achieved in that the nutrient medium includes soy milk, sodium pyruvate, pectin, calcium chloride, vitamins, gellan gum, and water in the following ratio of components, g / l:

[0020] soy milk - 30-75

[0021] sodium pyruvate - 0.1-0.3

[0022] pectin - 0.05-0.1

[0023] CaCl2- 0.3-0.5

[0024] Vitamins - 1-2

[0025] Gellan gum - 10-15

[0026] Water - the rest

[0027] It is preferable to use tap water as a water source.

[0028] As vitamins, pharmaceutical vitamin stocks are preferred, usually containing ascorbic acid - 0.075 mg; vitamin B1 - 0.015 mg; vitamin B2 -0.015 mg; vitamin B3 - 0.015 mg; vitamin B6 - 0.037 mg; folic acid - 0.015 mg; nicotinic acid - 0.037 mg; nicotinamide - 0.037 mg; biotin - 0.015 mg; para-aminobenzoic acid - 0.074 mg; choline chloride - 60 mg; vitamin D3 - 0.015 mg. To maintain activity, vitamins are introduced after autoclaving.

[0029] Natural soy milk provides a set of proteins and sugars that closely imitates those found in natural environments (upper layers of soil in contact with leaf litter).

[0030] Sodium pyruvate acts as an additional energy substrate and antioxidant.

[0031] Gellan gum as a gelling agent improves the physicochemical properties of the medium, giving it transparency and stability, and is also non-toxic to soil bacteria.

[0032] Pectin acts as a carbon source.

[0033] This combination of components and factors is aimed at the effective cultivation of oligotrophic and indigenous soil microorganisms in natural conditions.

[0034] The method for obtaining a nutrient medium consists of mixing the components in an aqueous medium.

[0035] The distinctive feature of the proposed nutrient medium is the use of soy protein and pectin as a source of carbon, sodium pyruvate, which helps, among other things, reduce oxidative stress and remove bacterial cells from a state of energy stress, vitamins and calcium chloride.

[0036] Below is an example of the invention.

[0037] Example

[0038] The medium was prepared by mixing the components in an aqueous medium, with the following ratio of components: soy milk, 50 ml / 75 g; sodium pyruvate, 100 mg; citrus pectin, 100 mg; CaCl2, 300 mg; vitamins (after autoclaving), 1 g; gellan gum, 15 g; water - the rest up to 1000 ml.

[0039] The following medium is also prepared: soy milk, 30 g; sodium pyruvate, 300 mg; citrus pectin, 50 mg; CaCl2, 500 mg; vitamins (after autoclaving), 2 g; gellan gum, 10 g; water, the rest, up to 1000 ml.

[0040] A microbiological sowing of an aqueous suspension of soil (Ordinary carbonate chernozem, Botanical Garden of SFedU, 47.2373, 39.6584), diluted in a ratio of 1:10000, was carried out on solid nutrient media with soy milk of the above compositions.

[0041] Twenty Petri dishes were seeded. During the first and third weeks of incubation, 10 dishes were selected from each treatment, and all biomass that had grown since the experiment was set up was washed off with distilled water into sterile plastic tubes and centrifuged (10 minutes, 7000g). The supernatant was then removed, and the cell pellet was frozen. After thawing, the biomass was subjected to DNA extraction. DNA was isolated using the DNA-Extran kit (Synthol, Russia) according to the instructions. Sequencing was performed on a MinION instrument using the Rapid Sequencing Kit V14 reagents according to the instructions. For in-depth taxonomic and functional analysis, the sequenced DNA reads were assembled into contigs using the MEGAHIT program. Contigs were assigned taxonomic names using the Cat_pack program and the GTDB database.

[0042] Analysis of the obtained culturomes revealed that a significant increase in the rare taxonomic group, Verrucomicrobiota, was observed during the first week of incubation on the soy milk medium. Figure 1 shows dot plots of the representation of DNA fragments of different bacteria in washes from the surface of Petri dishes with the proposed nutrient medium. The size and color of the dots encode the number of DNA fragments. Fig. 1(a) is a dot plot of the representation of four difficult-to-cultivate phyla in washes from weeks 1 and 3. Fig. 1(b) is a dot plot as in Fig. 1a, but divided by generic categories. Fig. 1(c) is a dot plot of the length and taxonomy of DNA fragments (each dot corresponds to a fragment, only fragments longer than 1000 nucleotides are shown).

[0043] A smaller increase in some other groups was also observed. For example, the number of contigs affiliated with this taxon detected in the first week was 14, while 97 contigs (with a maximum length of 56,023 bp and an average length of 14,253 bp) were detected in washes from the third week of incubation. Most of the Verrucomicrobiota contigs were affiliated with the genus Luteolibacter (in particular, L.yonseiensis), the genus Haloferula, and the genus Roseimicrobium.

[0044] Furthermore, by the third week, the number of DNA fragments of various difficult-to-cultivate actinobacteria increases on this nutrient medium. These include Micromonospora, Saccharopolyspora, Chryseoglobus, Isoptericola, Microterricola, Promicromonospora, as well as representatives of the order Solirubrobacterales, the genera Solirubrobacter and Patulibacter, which are characterized by slow growth and are outcompeted by other bacteria when grown on standard nutrient media. At the same time, common representatives of soil actinobacteria maintain a constant number during three weeks of incubation (Microbacterium, Arthrobacter, Rhodococcus spp.), or decrease in relative proportion (Paenarthrobacter spp.).

[0045] Thus, the soy milk medium effectively supports the development of various bacterial groups and can be used to study the dynamics of microbiota and its morphophysiological characteristics.

[0046] Sources of information

[0047] 1. Hati S., Patel N., Mandal S. Comparative growth behaviour and biofunctionality of lactic acid bacteria during fermentation of soy milk and bovine milk / / Probiotics and antimicrobial proteins. - 2018. - Т. 10. - №. 2. - С. 277-283.

[0048] 2. do Amaral Santos C. C. A., da Silva Libeck B., Schwan R. F. Co-culture fermentation of peanut-soy milk for the development of a novel functional beverage / / International Journal of Food Microbiology. - 2014. - Т. 186. - С. 32-41.

[0049] 3. Imazaki I., Kobori Y. Improving the culturability of freshwater bacteria using FW70, a low-nutrient solid medium amended with sodium pyruvate / / Canadian journal of microbiology. - 2010. - Т. 56. - №. 4. - С. 333-341.

[0050] 4. Das N. et al. Progress in the development of gelling agents for improved culturability of microorganisms / / Frontiers in microbiology. - 2015. - V. 6. - P. 698; p. 2.

[0051] 5. Klein T. et al. Cultivation of ammonia-oxidizing archaea on solid medium / / FEMS Microbiology Letters. - 2022. - V. 369. - №. 1. - P. fnac029.

[0052] 6. US4329431A, USA, 1980.

[0053] 7. US11820974B1, USA, 2022.

[0054] 8. US11261422B2, USA, 2017.

[0055] 9. Nishioka T., Tamaki H. Improved cultivation and isolation of diverse endophytic bacteria inhabiting Dendrobium roots by using simply modified agar media / / Microbiology Spectrum. - 2022. - Т. 10. - №. 6. - С. e02238-22.

Claims

1. A nutrient medium for cultivating soil microorganisms, characterized by the fact that it includes soy milk, sodium pyruvate, pectin, calcium chloride, vitamins, gellan gum, and water in the following ratio of components, g / l: soy milk 30-75 sodium pyruvate 0,1-0,3 pectin 0,05-0,1 CaCl2 0,3-0.5 vitamins 1-2 gellan gum 10-15 water rest 2. A nutrient medium according to paragraph 1, characterized in that tap or distilled water is used as water.

3. The nutrient medium according to paragraph 1, characterized in that the vitamins used are pharmacy vitamin stocks containing ascorbic acid, vitamin B6, folic acid, nicotinic acid, nicotinamide, biotin, para-aminobenzoic acid, choline chloride, and vitamin D3.

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