Method and plant for the production of biological products based on entomopathogenic nematodes
The invention relates to the microbiological industry, and in particular to methods and lines for the production of biological products based on entomopathogenic nematodes used as biological agents in the control of insect pests. The method consists in the fact that autoclaving of the nutrient medium is carried out in a separate container, which is then inoculant symbiotic bacteria, and after their reproduction medium is transferred into another container with simultaneous inoculation in the same capacity monoxenic culture of nematodes. Line for production of biological products based on entomopathogenic nematodes, characterized in that the bioreactor consists of two blocks: a bioreactor for pre-breeding symbiotic bacteria, made in polypropylene bag, and bioreactor for growing nematode-bacterial complex, designed in the form of a plastic trough with cover foil with holes; additionally introduced sterilization of plastic troughs, made using an open flame, and the unit of nematodes from inert carrier made using a standard washing machine. This embodiment bioreaktoren, that provides a stable increase of biomass yield of nematodes per unit mass of the medium. 2 N. p. F.-ly, 3 ill., table 2.
The invention relates to the microbiological industry, and in particular to methods and lines for the production of biological products based on entomopathogenic nematodes used as biological agents in the control of insect pests.
A method of obtaining biomass of the entomopathogenic nematodes, which lies in the fact that the components of the nutrient medium - chicken giblets, pork fat and water are homogenized, impregnated with a homogenate of inert media, fall asleep nutrient medium in polypropylene bags and sterilized. In chilled medium inoculant liquid culture of symbiotic bacteria. After the 48-hour cultivation of bacteria in a nutrient medium inoculant invasive larvae of nematodes. In the process of development of nematodes and bacteria in bags with a nutrient medium is supplied with sterile air. Office Mature culture of nematodes from the inert carrier is carried out by distributing the foam crumbs on the sieve, placed in a trough of water. The precipitate is not what nedostatkov this method of cultivation of nematodes should be noted the following: low yield of Mature culture of nematodes with 1 g of the nutrient medium, the complexity of the processes inoculation of microorganisms in polypropylene bags with a nutrient medium and low-tech process that is associated with forced aeration environment to ensure the development of bacteria and nematodes.
The closest known adopted for the prototype is a method that includes obtaining biomass of nematodes using semisolid nutrient medium consisting of the following composition: soy flour, corn oil, egg, brewers yeast, foam crumb and water. The foam crumb soaked homogenate medium, placed in metal containers (boxes) are made of stainless steel, which is sterilized and after cooling in a nutrient mixture inoculant liquid culture of symbiotic bacteria. After the 48-hour cultivation of bacteria in a nutrient medium inoculant invasive larvae of nematodes .
The disadvantages of the prototype are determined by the unstable output of a Mature culture of nematodes due to imperfect aeration system the internal cavity of the mailbox with a nutrient medium, which develop bacteria and nematodes, and use in the production process expensive materials and equipment.
Task d what about the product.
The proposed method for the cultivation of entomopathogenic nematodes is that in the production process after impregnation of the foam crumb the homogenate components of the nutrient medium, it is transferred into a container and autoclave. After cooling in a nutrient medium inoculant symbiotic bacteria. After 48 hours of exposure the contents of the bags under sterile conditions pour into another container.
The cultivation process starts with preparation of a pure culture of the primary forms of symbiotic bacteria and monoxenic culture (i.e. the culture of the invasive larvae of nematodes with one type of bacteria symbiotic) nematodes. Pure cultures of symbiotic bacteria get method, Poinar  by infecting nematodes larvae Galeria mellonella with subsequent selection of bacterial cells from the hemolymph of insects and planting them on nutrient YS agar (NH4H2PO4- 0.5 g; K2HPO4- 0.5 g; MgSO4·7H2O - 0.2 g; NaCl, 5 g; yeast extract 5 g; agar 12 g; water - 1 l) , containing triphenyltetrazolium chloride (0,004%) and bronchiology blue (0,025%), followed by identification of the primary forms of symbiotic bacteria .
Monoxenic nematodes carried by the female sterilization in the respective placing them on nutrient agar with a pure culture of symbiotic bacteria. After five days on agar develop Mature females who undergo sterilization by posting through merthiolate and a set of antibiotics . Obtained from females axenic larval nematodes of the first age carry on lipid agar  with symbiotic bacteria. After completion of the development cycle of nematodes collect monoxenic culture infective larvae of nematodes and inoculant them in a conical flask (500 cm3with a foam rubber sponge, soaked in a nutrient medium with pre-multiplied her symbiotic bacteria Xenorhabdus nematophilus .
The flask is kept at a temperature of 25°C and after completion of the development cycle of nematodes obtained monoxenic larvae used for further breeding in artificial nutrient medium.
Pure cultures of symbiotic bacteria stored on nutrient agar at a temperature of 12°C and checked for purity on a monthly basis.
Obtaining biomass of nematode species Steinernema carpocapsae strain "agriotos conducted in polypropylene bags and plastic troughs (bioreactors) (experience) and in metal boxes (bioreactors) (control), filled with a nutrient medium containing: soy flour - 16%, egg powder - 20%, corn oil and 5%, PI;10-9g/ml of culture medium ).
In the experimental embodiment, the culture medium was first filled in polypropylene bags of calculation 1.4 kg environment on one bag. Bags with a nutrient medium autoclaved (121°C for 30 min) and after cooling in each bag was inoculable symbiotic bacteria (Xenorhabdus nematophilus) in an amount of 100 ml of bacterial suspension with a titer of 3·109bacterial cells. Bacteria in a nutrient medium was inoculable in the form of an aqueous suspension of bacterial cells.
The contents of the bags after inoculation of bacteria were thoroughly stirred and kept at a temperature of 25-28°C. After 48 hours of exposure the contents of the bags under sterile conditions poured into a plastic trough, pre-sterilized over a flame of a gas burner for 10 seconds. At the same time in a nutrient medium with grown culture of bacteria in sterile conditions were inoculable nonoxinol culture infective larvae of nematodes from 60 per million individuals per one trough. Then the trough was covered with a lid of foil and transferred them in a temperature-controlled room with a temperature of 23-25°C for development nematode-bacterial complex.
In the control variant nutrient medium for the after sterilization and cooling medium under sterile conditions in a nutrient medium was inoculable liquid symbiotic culture of bacteria and 48 hours later an additional inoculable invasive larvae of nematodes, as in the advanced options. All variants of the experience and control had a five-fold repetition. Productive output of the biomass of nematodes in the experimental and control variants was determined after completion of the development cycle of nematodes in nutrient medium.
Mature culture of nematodes were isolated from the foam crumbs by washing using a washing machine for 10 minutes in the wash cycle, followed by counting the number of nematodes in the resulting aqueous suspension in the test and control versions. At the same time identified invasive (entomopathogenic) activity of nematodes in figure LD50for the test insects .
The results of the assessment of the productive output of nematodes per 1 g of the nutrient medium are shown in table 1, and their invasive activity are shown in table 2.
The example 1 shows the high efficiency of biomass production of entomopathogenic nematodes with prior cultivation of symbiotic bacteria in polypropylene bags and followed by cultivation nematode-bacterial complex in plastic troughs. The yield of biomass of nematodes in a test version of policiesare in polypropylene bags with a nutrient medium promotes the best and stable development nematode-bacterial complex in plastic troughs, that is accompanied by a significant increase in the yield of nematodes per unit mass of the medium and reducing the cost of production nematode drugs.
The prototype line for the cultivation of nematodes is a Line for the production of biological substances of entomopathogenic nematodes", with each bioreactor for growing nematode-bacterial complex represents a tank having in cross section a rectangular shape and features a removable lid and a hole for the flow of air inside and underneath the hole biofilter .
The disadvantages of the prototype are defined: 1) the presence of bioreactor device structure that does not provide optimal aeration conditions for the development of bacteria and nematodes; 2) expensive material - stainless steel, which is used in bioreactors for the cultivation of symbiotic bacteria and nematodes; 3) the need for autoclaves with a large amount of cameras for sterilization of bioreactors.
The scheme proposed line (Fig.3) includes two new devices that perform the functions of the bioreactor. For pre-cultivation of symbiotic bacteria are polypropylene and hole size of 4 μm. For growing nematode-bacterial complex uses plastic trough rectangular size HH mm with flanges on the outer side of the width of 10 mm (Fig.2, POS.2), provided with a removable cover foil, the edges soften the outer side of the trough. In the center of the cover on the area of 40000 mm2there are openings with a diameter of 0.5 mm, in the amount of five holes on each square centimeter (Fig.2, POS.1).
Line for production of biological products on the basis of entomo-pathogenic nematodes (Fig.3) operates as follows.
Production of nematodes begin with cooking homogenate nutrient medium and impregnating her foam crumb (1), which is then filled polypropylene bags (2). The opening of the bag closed by the sealing tape and bags with a nutrient medium is sterilized by autoclaving (3). After cooling the nutrient medium bags inoculant symbiotic bacteria in sterile box (4), which pre-cultivated for two days on a liquid nutrient medium in a rocking chair or in the fermenter (5). The contents of the bags after inoculation of bacteria are thoroughly mixed and after 48 hours of exposure under sterile conditions (4) pour in plantacii (7). At the same time in a nutrient medium with grown culture of bacteria in sterile conditions (4) inoculant nonoxinol culture infective larvae of nematodes from troughs, taken from the block growing (8), sterilized outside the unit sterilization (7). Then trough cover and put in the block growing nematode-bacterial complex (8). After 20-25 days a Mature culture of nematodes secrete leaching in water using standard washing machines in the separation unit of the invasive larvae from inert carrier (9) with the subsequent concentration in the block thickening biomass of nematodes to obtain concentrate (10), on the basis of which receive preparative form nematode preparations (11). Released in the production process bags and trough after washing (12) and sterilization (3, 7) are included in the production process. The water used in the production process, closed cycle, including cleaning of residue from the nutrient medium and waste products nematodes and bacteria (13).
Thus, the proposed line for the production of biological products based on entomopathogenic nematodes eliminates the use in the production process expensive materials (stainless steel the environment. The introduction of new units significantly increases the capacity of the line by reducing operations, which requires large amounts of manual labor, and Miniset square, employed for the production, which generally reduces the cost of the final product.
Sources of information
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1. Method for the production of biological products based on entomopathogenic nematodes, including the impregnation of the inert carrier homogenate nutrient medium components, autoclaving, cooling and inoculation with symbiotic bacteria and monoxenic culture of nematodes in nutrient medium, wherein the autoclaving of the nutrient medium is carried out in a separate container, which is then inoculant symbiotic bacteria, and after completing their breeding nutrient medium with bacteria pour into another container, where inoculant and nonoxinol culture of nematodes.
2. Line for production of biological products based on entomopathogenic nematodes, including bioreactor, washer, dryer, installation, preparation of artificial nutrient medium on a solid medium loading device nutrient medium bioreactors, the device sterilization past, sterile unit for insulinopenia symbiotic bacteria and nematodes in nutrient medium, block them versiani the thickening of the suspension of the biomass production of concentrate biological drug and device packing it in a container, characterized in that the bioreactor is made of two blocks: one for pre-breeding symbiotic bacteria, made in polypropylene bag, provided with an air filter, and the other for growing nematode-bacterial complex, designed in the form of a rectangular plastic trough and provided with a removable cover foil with holes for aeration of the nutrient medium for sterilization of plastic troughs has a unit using an open flame; as a unit for separating nematodes from the inert carrier used in a standard washing machine.