RU2687023C1 - Pediococcus pentosaceus strain for processing organic wastes and a preparation based thereon - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology, microbiology, agriculture and environmental protection. Strain of bacteria Pediococcus pentosaceus 3L for processing of biogenic organic wastes is deposited in the Departmental collection of useful microorganisms for agricultural purposes of All-Russian Research Institute of Agricultural Microbiology under number RCAM04480. Invention also relates to a strain-based preparation bacteria Pediococcus pentosaceus 3L RCAM04480 in the form of a liquid cell culture or in the form of biomass of cells of said strain in combination with neutral components subjected to drying and / or grinding if necessary.

EFFECT: group of inventions provides faster processing of biogenic organic wastes and enables to obtain a fertilizer safe for sanitary and hygienic indices.

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Description

Technical field

The invention relates to the field of biotechnology, microbiology, agriculture, food and processing industry, veterinary microbiology and environmental protection and can be used in the processing of organic waste, as well as in obtaining on their basis of fertilizers.

The level of technology

Recycling of organic waste is a pressing problem for the environment. For the disposal of organic waste used chemical, physical and biological methods of processing. One of the biological methods of processing organic waste involves the use of cultures of microorganisms. The use of cultures of microorganisms contributes to the destruction of organic waste, as well as the accumulation of useful substances (products of metabolism of microorganisms), for example, stimulating the growth of plants.

The preparations “Baikal-EM1” and Vostok-EM1 ”are known on the basis of a complex of microorganisms for the processing of organic waste (Sukhamera S.A. EM-technology is a biotechnology of the 21st century. A collection of materials on the practical use of the preparation Baikal EM-1 / Almaty. 2006 P.11; Severina V.Ya. Biological preparation “Vostok-EM1". Theory and Practice. / Vladivostok. 2009. P.9-11.). They include a complex of various types of microorganisms that can accelerate the processing of carbon-containing organic waste. Preparations "Baikal-EM1" and "Vostok-EM1" are a liquid suspension of microbial cells.

There are well-known dry preparations of a similar purpose, for example, “Microzim ^(tm) TRIT COMPOST”, “VODOGRAY”, which are described on the websites of supplier companies or manufacturers.

One way to recycle organic waste is composting. The classic composting process takes 40-60 days, and one of the important issues is its acceleration. The most common composting preparations contain the bacteria Bacillus sp., Pseudomonas sp. and others, fungi that are classified as conditionally pathogenic (third class of pathogenicity) (SP 1.2.731-99 Safety of work with microorganisms of groups III-IV pathogenicity and helminths).

Preparations "Baikal-EM1", "Vostok-EM1", "Microzim ^(tm) COMPOST TRIT", "VODOGRAY", which are also used as activators of composting, are multicomponent, containing various types of microorganisms. Obtaining multicomponent preparations is laborious: for their production, it is first necessary to develop several different types of microorganisms separately, then mix them with strict observance of the quantitative ratio of each of them to eliminate the loss of drug properties - as a mixture of species and strains of microorganisms.

Lactic acid bacteria can be used to process organic waste as well as speed up composting, since these types of microorganisms are not pathogenic for humans and animals. Lactic acid bacteria are able to inhibit the growth of pathogenic organisms, and some species also have the ability to destroy organic waste, which allows the use of cultures of microorganisms to produce effective fertilizers based on organic waste. The ability of lactic acid microorganisms to suppress the growth of pathogenic organisms allows them to be used for processing biogenic wastes that pollute the environment and pose a danger to humans.

On the basis of lactic acid microorganisms, preparations can be obtained, including one type of bacteria, which ensure the processing of organic waste, and also have a simplified process for the preparation of the preparation and make it possible to obtain a preparation in liquid or dry form, which has long shelf life and accelerates the processing of organic waste, including composting.

To process large volumes of organic waste, a large amount of the drug is needed. Therefore, it is more convenient to use preparations of microorganisms, the volumes of which can be easily scaled at the place of processing waste (application), that is, for example, having 1 liter of liquid, or 1 kg of dry preparation to get more of it, at least 10 times, according to the known the principle of reproduction of microorganisms in liquid media in the ratio of sowing culture: medium - 1:10 in volume. In addition, it is known that the best uniformity of waste treatment with preparations for composting is achieved by spraying cell suspensions, i.e. liquid, not dry preparations. Therefore, in the presence of a dry form of the drug, it is more convenient to get liquid on the spot.

DISCLOSURE OF INVENTION

The present invention is to obtain a strain of lactic acid microorganism that allows the processing of biogenic organic waste, as well as a drug based on it, quickly and easily scalable at the site of application, which can be used in both liquid and dry form, as well as developing a method of obtaining the drug on the basis of the bacterial strain, providing the drug in a short time.

The problem is solved by obtaining a new strain of bacteria Pediococcus pentosaceus 3L for the processing of biogenic organic waste.

The strain Pediococcus pentosaceus 3L is deposited in the Departmental collection of useful microorganisms for agricultural purposes VNIISM under the number RCAM04480.

In some particular embodiments of the invention, nutrient organic wastes are: animal waste, poultry waste, sewage sludge, municipal solid waste, crop waste and / or waste from the timber industry.

In some embodiments of the invention, the problem is solved by developing a preparation for processing biogenic organic waste, including the bacterial strain Pediococcus pentosaceus 3L RCAM04480.

In some particular embodiments of the invention, the content of the bacterium strain Pediococcus pentosaceus 3L RCAM04480 is at least 10 ³ cells / ml.

In some particular embodiments of the invention, the preparation is made in dry or liquid form.

In some particular embodiments of the invention, the formulation further includes wheat bran, barley bran, rye bran, rice bran, sunflower husk, rice husk and / or oatmeal.

In some embodiments of the invention, the problem is solved by developing a method for producing a preparation for processing organic waste, the method including cultivating the strain Pediococcus pentosaceus 3L RCAM04480 in an environment and conditions favorable for its growth, until a concentration of at least 10 ³ cells / ml is reached.

In some particular embodiments of the invention, the method further comprises drying and pulverizing the preparation.

In some particular embodiments of the invention, the medium includes wheat bran, barley bran, rye bran, rice bran, sunflower husk, rice husks, and / or oatmeal husks.

In some particular embodiments of the invention, the medium further comprises molasses.

In some particular embodiments of the invention, the bacteria Pediococcus pentosaceus 3L RCAM04480 are cultivated in a liquid medium including beet molasses.

In some particular embodiments of the invention, cultivation includes: seeding a liquid culture of the bacterium strain Pediococcus pentosaceus 3L RCAM04480 medium in a ratio medium: 10: 1 culture - 100: 1 (mass: volume), aging at 50-55 ° C - 6-24 hours, then cooling the mass to 37-50ºС and keeping not less than 24 hours.

In some particular embodiments of the invention, the environment has a moisture content of at least 50%.

In some particular embodiments of the invention, the method further comprises stirring intermittently.

In some particular embodiments of the invention, the method further comprises drying the obtained bacterial strain Pediococcus pentosaceus 3L RCAM04480 on bran to a moisture content of not more than 15% and grinding to powder.

As a result of carrying out the invention, the following technical results are achieved:

- the bacterial strain Pediococcus pentosaceus 3L RCAM04480 was obtained, which allows for the efficient processing of biogenic organic waste;

- a bacterial strain and a drug have been obtained that can be used for processing as well as neutralizing a wide range of biogenic organic waste;

- a bacterial strain and a drug that accelerates the processing of biogenic organic waste;

- a preparation based on the bacterial strain was obtained, which can be used both in dry and in liquid form with the possibility of increasing the volume of the preparation several times at the place of use, as well as having a long shelf life;

- developed a method of obtaining the drug on the basis of the bacterium strain Pediococcus pentosaceus 3L RCAM04480, which allows to obtain the drug in a short time, as well as being easy and time-consuming;

- the possibility of obtaining in the processing of biogenic organic waste using a strain of bacteria and a drug based on them highly effective fertilizers (including those with a high nitrogen content);

- the possibility of using the strain and the preparation on the basis of the strain for the processing of biogenic organic waste by composting.

Detailed Description of the Invention

The present invention is directed to obtaining an effective bacterial strain for processing organic waste and a preparation based on the bacterial strain, as well as developing a method for producing a preparation based on the bacterial strain.

The bacterial strain Pediococcus pentosaceus 3L RCAM04480 was isolated from animal feed using selective medium.

The bacterial strain Pediococcus pentosaceus 3L was identified to a species by cultural and morphological characteristics, as well as using the method of sequencing part of the nucleotide sequence of the 16S rRNA gene, deposited in the Departmental collection of useful microorganisms for agricultural purposes VNIISM under the number RCAM04480.

Description of the strain. The thermophilic strain of lactic acid bacteria P. pentosaceus 3L RCAM04480 has physiological and morphological characteristics typical of this species. Gram-positive spherical cells (cocci), 1-2 µm in size, are arranged in pairs or separately. In smears can be combined into groups of various shapes. Motionless. On solid nutrient media forms a small convex colonies of a round shape with smooth edges, whitish cream or beige, with a diameter of 1-2 mm. The surface of the colonies is smooth. It grows well on rich carbohydrate-containing media. Resists culturing in liquid and on solid media at a temperature up to 55 ^C.

The strain is not pathogenic and does not carry artificial genetic structures.

The strain P. pentosaceus 3L RCAM04480 is used in the processing of organic waste, ensuring their destruction, as well as the neutralization of pathogenic organisms by suppressing their reproduction and growth. The strain P. pentosaceus 3L RCAM04480 is able to act as an active destructor of organic waste due to the presence of enzyme systems affecting organic substances (for example, cellulose, proteins, lipids and their derivatives). Also, the strain P. pentosaceus 3L RCAM04480 contributes to the suppression of pathogenic organisms, for example, by forming lactic acid and antimicrobial substances (bacteriocins). In this case, pathogenic organisms are understood as E. coli, Salmonella and other microorganisms.

Using a bacterial strain and a drug based on it, various types of organic waste can be recycled, for example, biogenic waste. As biogenic waste, but not limited to, there may be: waste from agriculture and processing industry (animal dung, poultry dung, whole carcasses and parts of them from dead animals and poultry, oilcake fruits, berries, vegetables, canyon, etc.) plant waste, waste from the timber industry, sewage sludge, municipal solid waste. Using the strain and the preparation based on it, the listed wastes can be processed in solid and liquid form, either individually or in combination with each other, since it is known that proteins, carbohydrates and lipids form the basis of all biogenic wastes in aggregate or separately. that can be used as sources of carbon and / or nitrogen - a nutrient medium for the cultivation of this and other types of lactic acid bacteria.

The resulting strain and the preparation based on it can be used for processing waste using various technologies, in a particular case, the strain and preparation can be used for processing waste using the composting method.

The resulting strain and the drug can significantly speed up the processing of organic waste. In the particular case, in the processing of biogenic organic waste by composting, the processing of waste can be carried out for no more than 10 days.

During the processing of biogenic waste, in the particular case of animal waste (animal manure) and / or poultry waste (bird droppings), an effective organic fertilizer complies with GOST R 53117-2008, which is safe for sanitary and hygienic indicators, is obtained.

During the processing of organic waste to obtain organic fertilizer using a bacterial strain and a preparation based on it, significant nitrogen losses are excluded.

Organic waste can be recycled using a pure culture of the strain of bacteria P. pentosaceus 3L RCAM04480, or using a preparation based on the strain of bacteria.

Depending on the tasks set during the processing of waste and types of waste, the preparation may contain different numbers of bacterial strain cells, and the preparation based on the strain may be used and be performed both in dry and in liquid form.

The drug based on the strain of bacteria P. pentosaceus 3L RCAM04480 may include one or more additional components that positively affect the properties of the drug. Additional components can be selected (but not limited to) from plant waste, for example, such as: wheat bran, barley bran, rye bran, rice bran, sunflower husk, rice husk, oatmeal. Also, the drug may include an additional molasses, for example, beet molasses.

The claimed method of obtaining the drug includes, but is not limited to these steps: the cultivation of the strain of bacteria P. pentosaceus 3L RCAM04480 in an environment and conditions favorable for its growth. In particular cases, the method of preparation may additionally include a step of drying and / or grinding to a powder. In the course of preparation, the culture medium may also vary, and the medium may include carbon sources as components that are favorable for growth. At the same time, the medium can be both liquid and solid. Cultivation of bacteria to obtain the drug can be carried out both on standard media and on media, including, for example, plant waste. In particular cases of the invention, the medium may include: wheat bran, barley bran, rye bran, rice bran, sunflower husk, rice husk and / or oatmeal husks (or a combination of these). Also, the medium may additionally include molasses, for example, beet molasses.

In particular cases, the cultivation of the P. pentosaceus 3L RCAM04480 bacterial strain is carried out in a liquid medium containing wheat bran, barley bran, rye bran, rice bran, sunflower husk, rice husk and / or oatmeal, as well as beet syrup. This method can be used as one of the methods for obtaining a liquid preparation.

Also in the particular case, the cultivation of bacteria includes the following stages: seeding a culture of the P. pentosaceus 3L RCAM04480 bacterial strain medium and aging. Cultivation medium for bacteria, in particular cases, may include wheat bran, barley bran, rye bran, rice bran, sunflower husk, rice husk and / or oatmeal. Also in the particular case of the cultivation additionally includes periodic mixing.

The invention is as follows.

In one of the embodiments of the invention, the strain Pediococcus pentosaceus 3L RCAM04480 and / or a preparation based on it can be used for processing agricultural waste (animal dung, poultry dung, whole carcasses and parts of their dead animals and poultry), processing and food industry (cake and fruit , berries, vegetables, fat-containing wastes, canyga), plant wastes (husks, husks, bran), wastes of the wood processing industry, sewage sludge, municipal solid waste using the composting method.

In the particular case of the invention, the preparation based on the bacterial strain contains at least 10 ³ cells / ml or at least 1% by weight of the dry preparation.

The method of obtaining the drug was carried out as follows. The cultivation of the P. pentosaceus 3L RCAM04480 bacterial strain was carried out in a liquid medium, including bran, molasses (for example, beet molasses), water, optimal for cell proliferation, and a culture of the bacterial strain was obtained during cultivation. Liquid culture of the strain obtained by this method, in the particular case was used as a finished product, made in liquid form.

In another embodiment, the preparation of the drug is as follows. The cultivation of the P. pentosaceus 3L RCAM04480 bacteria strain is carried out under aseptic conditions in an environment including: wheat bran, barley bran, rye bran, rice bran, sunflower husk, rice husk and / or oatmeal (or a combination of them) with a moisture content of at least 50% ; the culture medium is seeded with a liquid culture of the strain of bacteria P. pentosaceus 3L RCAM04480 in a ratio of 10: 1 - 100: 1 (weight: volume); incubated at a temperature of 50-55 ^o C - 6-24 hours, then the mass is cooled to 37-50 ^o C and kept culture for at least 24 hours, stirring occasionally. Also in the particular case, the cultivation additionally includes periodic mixing, while mixing is carried out both during sowing and during aging. After the cultivation process is complete, the mass of P. pentosaceus 3L RCAM04480 bacteria strain is dried to a moisture content of not more than 15% and ground to a homogeneous powder. This method of obtaining in the particular case is used to obtain the drug, made in a dry form.

The preparation obtained by the method described above can be scaled at the place of use as follows: to obtain a working solution, an environment is prepared, including bran, molasses and water, and mixed with a dry preparation in a container (with the mass of the preparation being 1-2% by weight of the medium) , mix and incubate with occasional stirring. The resulting working solution is used for composting nutrient organic waste.

It should be understood that these and all the examples given in the application materials are not limiting and are given only to illustrate the present invention.

Example 1. The method of obtaining the drug on the basis of the strain

A stock culture Pediococcus pentosaceus 3L RCAM04480 aseptically inoculated in sterilized medium, autoclaved at ¹²⁰ ° C, a pressure of 1 atm. 20 minutes, containing 10 g of wheat bran, 20 g of beet molasses in 1 liter of distilled water. The ratio of the initial liquid culture to the nutrient medium is 1:10 by volume. The culture of bacteria is grown under aseptic conditions at 40-45 ^o C, mixing 2 times a day. A liquid culture is obtained with a cell titer from 100 million to 1 billion in 1 ml.

To dry wheat bran was added distilled water to a weight of 50-55% humidity, autoclaved at ¹²⁰ ° C and 1 atm. 20 minutes. Then bran cooled to 55 ^{° C} and aseptically added to a liquid culture of bacteria Pediococcus pentosaceus 3L RCAM04480 bran in a ratio of: culture of 100: 1 (dry weight bran, r: Culture mL). Was stirred and heated at 55 ° ^C for 24 hours. Then the mass was cooled to 50-45 ^{° C} and grown bacterial culture for 2-3 days with stirring periodically until sour odor. After that, the resulting mass of bacteria is dried under aseptic conditions to a moisture content of not more than 15% and ground to a homogeneous powder.

Example 2. Preparation of the working solution of the drug (an increase in the volume of the drug in place and the activation of bacteria) for the processing of organic waste

Nutrient medium containing wheat bran, beet syrup and water in the ratio by weight 1: 2: 100, sterilized by boiling for 20 minutes. The dry preparation is poured into a clean container with a tight-fitting lid and a volume of warm (40 ± 3 ^° C) nutrient medium is added so that the mass of the preparation is 1-2% of the mass of the medium. The suspension is well mixed, the container is tightly closed with a lid and the preparation is kept for at least three days at the specified temperature, mixing 2 times a day. The degree of readiness is judged by the achievement of a pH of 3.5 ± 0.5 and / or an acidic smell. After that, the drug is mixed, defend for at least one hour, drain the liquid with the culture of cells above the precipitate in a clean container. Analysis of the number of bacteria in the preparation showed their content of at least 1x10 ⁸ cells per 1 ml, which corresponds in concentration, for example, to Vostok-EM1 (Severina V.Ya., 2009).

For the treatment of manure, use 1-2 liters of the preparation per 1 ton of waste, for example, as follows: 350 kg of litter chicken manure with 49% moisture (using sawdust as a litter) are sprayed with 0.7 l of a liquid preparation obtained from the dry form as described above diluted in 10 liters of drinking water. Then they fall asleep in a box measuring 1.2 m by 1.2 m, with a wall height of 0.3 m, and put into a collar in the form of a pyramid. After 2 days, the temperature of the mass of waste in the collar rises to 55 ^o C and more. Litter quickly ted, again forming a collar out of it. The results of the product (compost) obtained after 10 days are shown in Table 1. As can be seen, for most agrochemical characteristics, the compost obtained by treating the waste with the preparation obtained by the claimed method is not only not inferior to the compost obtained using the Vostok-EM1 multicomponent liquid preparation on the basis of lactic acid bacteria (Severin V.Ya., 2009.), but also surpasses it.

The resulting compost can be used as an organic fertilizer, as it contains nutrients in an amount not less than those specified in GOST R 53117-2008, and is safe in terms of sanitary and hygienic indicators (Table 2).

* Note - not detected

Although the invention has been described with reference to the disclosed embodiments, it should be obvious to those skilled in the art that the specific experiments described in detail are given only for the purpose of illustrating the present invention and should not be construed as limiting the scope of the invention in any way. It should be clear that it is possible to implement various modifications without departing from the gist of the present invention.

Claims (4)

1. The bacterial strain Pediococcus pentosaceus 3L RCAM04480, used for the processing of biogenic organic waste. 2. The preparation on the basis of the strain according to claim 1 for the processing of biogenic organic waste containing cells of the specified strain in the form of liquid cell culture of the specified strain or in the form of the biomass of cells of the specified strain in combination with one or more additional neutral components, subjected, if necessary, drying and / or grinding. 3. The preparation according to claim 2, in which the content of the strain of bacteria Pediococcus pentosaceus 3L RCAM04480 is at least 10 ³ cells / ml. 4. The preparation according to claim 2, in which the additional neutral components are wheat bran, barley bran, rye bran, rice bran, sunflower husk, rice husk and / or oatmeal.