RU2162833C2 - Biopreparation as activating agent of plant material composting and stubble decomposition, method of its preparing and bacterium consortium for preparing biopreparation as activating agent of plant material composting and stubble decomposition - Google Patents

Abstract

FIELD: biotechnology, agriculture microbiology. SUBSTANCE: invention relates to bioconversion of plant waste. Consortium of bacteria Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus 384-5KS is stored in collection of microorganisms of enclosed joint-stock "Bioflora" at number B-04. Preparation formulation based on consortium is developed and can be used for composting plant material and stubble decomposition. Preparation has, %: consortium of the above indicated bacteria grown in liquid nutrient medium and taken in the amount 25-30; carbamide, 0.7-7.0; dry algae or their alginates, 0.5-3.5; potassium hydrogen phosphate, 0.0005-0.03; trace elements B, Zn, Mg, Mn, Mo, Cu, Co as chelates taken in the equal amounts and in the total concentration 0.0001-0.003; surface-active substance, 0.001-0.05; sodium benzoate, calcium propionate, ascorbic acid as stabilizing agents taken in the equal amounts, 0.001-0.01. Preparation improves the composition of soil microflora, it shows fungicide properties, its composition provides ecological purity and safety. Preparation is useful as component in making soil-improving compositions. EFFECT: improved method of preparing, enhanced effectiveness of biopreparation. 11 cl, 1 tbl, 5 ex

Description

 The present invention relates to the biotechnology industry and agricultural microbiology, and relates to the production of preparations for increasing soil fertility and processing of plant material, and in particular, to obtaining a consortium of bacteria and a preparation based on it.

 The microorganisms used to process plant materials are widely known. Thus, the culture of lactic acid bacteria Lactobas isolated by E.I.Kvasnikov. plantarum AN-11/16 and used in silage of the green mass of corn, accelerated the maturation of silage (1). The use of marsupials mushrooms Lophodermium pinasti and Lophodermium macrosporum accelerated the composting process of spent needles (2). When compost was prepared on the basis of bird droppings, peat, sawdust, and cattle manure, bacterial suspensions of Klebsiella, Pseudomonas putida, and Bacillus were used as inoculum (3).

Known methods of composting and composting preparations, which include the preparation of compost mixtures (preparations) with the introduction of various kinds of starter cultures, consisting of a mixture of microorganisms of uncertain composition (4): for example, a compostable mixture consisting of organic waste, bird droppings, peat, was heated to plus 40 ^o C, while creating the conditions for the development of microflora introduced from a neighboring, exploited (apparently actively "wandering") fermenter; in another case, when composting fiber separation waste during the processing of cotton, in order to turn it into a humus-like material, the temperature of the composted mass is increased to plus 56 - 65 ^o C, which occurs due to the growth of the population of spontaneous microflora, for example, thermophilic fungi, actinomycetes, bacteria, at the same time, the death of pathogenic microorganisms is noted (5).

 The closest is the use of Lactobac culture. plantarum AN-11/16, but the disadvantage is the limited range of applications, namely, its use only in the silage of plant material.

 The aim of the present invention is to eliminate this drawback and to obtain a microorganism suitable for use not only for composting plant material, but also for obtaining a soil preparation that is used, for example, to decompose plant crop residues - stubble in the field after harvesting cereals, cotton, and others agricultural crops.

 A consortium of lactic acid bacteria has been obtained, which makes it possible to eliminate the aforementioned disadvantage and to obtain a drug with a wider spectrum of action, namely: - to compost plant material in order to obtain compost mass for subsequent use as a soil-improving substrate (weed grass, plant residues, household carbon-containing waste etc. ); - decompose crop residues, stubble left on the field after harvesting cotton, corn, grain and other crops directly in the field to accelerate the decomposition of plant residues, enrich the soil with stubble bioconversion products, increase soil fertility, destroy weed seeds after harvesting.

 The consortium was isolated from soil samples (sierozem) taken in the territory adjacent to the milk processing enterprise (suburb of Andijan, Uzbekistan).

 To isolate lactic acid bacteria, direct seeding from selected soil samples was used, followed by obtaining an accumulative culture, first on liquid media (hydrolyzed milk medium with alcohol), and then an agar medium with chalk.

 Stepwise analytical selection was carried out according to the principle of selection of thermophilic, highly productive and active producers of lactic acid in combination with a high level of cytokinin synthesis.

The morphology of the culture colonies — the components of the consortium — was described by microscopy of the deep colonies: Petri dishes with a 2-day-old culture of the consortium (no more than 100 colonies per 1 cup) were filled with nutrient medium (layer height 3–5 mm), melted and cooled to plus 45 - 43 ^o C, cultivation was another 48 hours.

 Colonies of lactic acid bacteria according to morphology are curl-shaped and star-shaped: with outgrowths on the periphery, with a dense center.

 The consortium is a natural indivisible community.

 Sowing of monocolonial isolates on a dairy medium, the study of their acid-forming activity, their attitude to carbohydrates by their ability to form them, substantiated the determination of the species composition of the consortium.

 The main physiological feature of the consortium, which is of practical importance, is the production of biologically active substances.

All cultures of the consortium are thermophiles, with boundaries of good growth and high activity in the range of 35 - 45 ^o C at optimum plus 39 ± 1 ^o C. Lactobacillus acidophillus. Grow at plus 50 ^o C; do not grow at plus 20 ^o C, never grow at plus 15 ^o C. Fermented: cellobiose, lactose, maltose, galactose, melibiosis, salicin, sucrose, trehalose; do not ferment: rhamnose, mannitol, sorbitol; weakly fermented: raffinose. Optimum growth temperature plus 39 ± 1 ^o C.

 Lactobacillus plantarum - small sticks located individually, in pairs, very rarely - chains of 3-4 cells. Gram-positive, spores, capsules do not form. In meat-peptone broth gives a slight turbidity, disappearing by 48 hours of growth; form a dense, white, sparingly soluble precipitate. On solid nutrient media, small, round, drop-shaped colonies with smooth edges, whitish-cream color.

 The surface of the colonies is smooth, shiny. They grow on cabbage-chalk agar with the formation of zones of enlightenment around the colonies. Ferments: sucrose, glucose, galactose, cellobiose, weakly ferments lactose, mannitol.

Streptococcus thermophilus - grows at plus 50 ^o C, does not grow at plus 10, 13, 16 ^o C; ferments maltose, raffinose, sucrose, lactose; weakly ferments glucose; does not ferment mannitol, sorbitol. Optimum growth temperature plus 39 ± 1 ^o C.

 Such a consortium is not described in the literature and is distinguished as natural, inseparable, for the first time.

 The dominant component of the consortium are sticks; their number is 60 - 70%. Varying the ratio of bacteria - components of the consortium depends mainly on the composition of the nutrient medium. With an increase in the concentration of nitrogen compounds and a decrease in the concentration of carbohydrates, lactic streptococci can become the dominant component, up to 70% and higher.

 The main properties of the consortium are high hydrolytic activity, antibiotic activity against phytopathogenic fungi Helminthosporium sativum, Verticillum dahliae, Fusarium lini, Fusarium oxysporum, high cytokinin activity.

 Cellulose activity was determined according to the method adopted in the enzyme industry (6); cytokinin activity - according to the biotest, expressing it in %% to the control; antibiotic activity - according to the minimum inhibitory concentration, comparable with the diameter of the zone of growth inhibition of the tested fungus - pathogen.

Cellulose activity was 1.3 - 4.2 mg
The cytokinin activity was - 120 - 142 %%
Antibiotic activity was 4 - 11 mm in diameter (zone of inhibition of growth of the pathogen) when diluted 1000 times.

 Phosphatase activity was determined by the transformation of insoluble phosphorus compounds (the clarification zone of the medium on Petri dishes when tricalcium phosphate, sodium nucleonate, fluoroapatite was added to the medium), the clarification zone was 4–7 mm when sodium nucleonate was added to the medium, and 3–8 mm when tricalcium was added to the medium phosphate.

The consortium is assigned the number 384-5KS; the consortium is stored in the Museum of Microorganism Cultures at AOZT Bioflora under collector number B-04. The consortium retains its properties when stored in a lyophilized state (drying using a sucrose-gelatin protective environment), storage temperature - not higher than minus 4 ^o C.

 The resulting consortium eliminates the disadvantages of the drug, obtained on the basis of the Lactobac strain. plantarum AN-11-16 and receive a preparation with a wider spectrum of action, namely, compost the plant material in order to obtain compost mass for subsequent use as a soil improving preparation and, in addition, decompose the crop residues, stubble remaining after the crop, such for example, like corn, cotton, sunflower, directly in the field.

 The resulting culture can be used independently, as a preparation for composting or decomposition of stubble.

 Example 1

Vitamin supplements in the form of an aqueous sterile solution of vitamins PP, B ₁₂ , para-aminobenzoic acid, riboflavin, B ₆ with a total concentration of 60 mg / 1000 ml of skim milk are added to 1 liter of sterile skim. Inoculate a lyophilized culture of a consortium of lactic acid bacteria 384-5KS, consisting of Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus, incubate under anaerobic conditions at a temperature of plus 39 ± 2 ^o C for 18 hours until a pH of 3.6 is reached; the microbial population density reached 4.5 · 10 ¹¹ cells / ml.

The resulting bacterial culture was diluted 100 times with tap water and the resulting suspension was sprayed with stubble after harvesting spring wheat at the rate of 1000 ml of suspension per 100 m ² field; the stubble was smelled, left for the winter. In the spring, soil analysis was carried out and compared with the analysis of samples taken on an untreated field. The results showed that in the treated area the soil contains 1.4 times higher carbon, 2.1 times higher abundance of saprophytic microflora, 3.3 times lower abundance of fungal microflora, and phosphorus content (in terms of P ₂ O ₅ ) more than 6 times its content in the soil on the untreated part of the field, which allowed us not to use phosphorus fertilizers on the treated part of the field. The stubble remains were thin macerated particles of a fibrous structure, while the stubble remained almost unchanged in the untreated part of the field.

 Based on this culture - a consortium of bacteria consisting of Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus, which is assigned the number 384-5KS (collection No. B-04), a biological product formulation has been developed that is proposed to be used both for composting plant material and for stubble decomposition, i.e. directly in the field.

To obtain the drug in the liquid culture of the consortium 384-5KS expressed in a 10% sterile way with vitamin supplements at a temperature of plus 40 ^o C for 18-24 hours under anaerobic conditions until a population density of 10 ¹¹ -10 ¹² cells / ml, and having a cellulosic activity of at least 1.3-4.0 mg, add 3-5 volumes of tap water, and then dry algae (or their alginates) in an amount of 0.3-3.5%, a surfactant (laprol, propyl B-400) in an amount of 0.001%, urea in an amount of 0.7 - 7.0%, potassium phosphate in an amount of 0.0005-0.030%, phosphoric acid, trace elements Zn, Mg, Mn, Mo, Co, Cu, B, in the form of glycine complexonates or, for example, HEDP in equal amounts at a total concentration of 0.0001-0.003%, stabilizers (preservatives) Na benzoate, Ca propionate , ascorbic acid - with a total concentration of 0.001 - 0.01%, the volume of the drug is adjusted to the calculated tap water, mixed thoroughly.

 The resulting preparation is used for treatments.

 Example 2

To 20 ml of a liquid culture of the consortium, bacteria 384-5KS, grown on a 10% sterile sample at plus 40 ^o C under anaerobic conditions for 24 hours, 80 ml of tap water, 0.3 g of algae algae, 0.7 were added % urea, 10 mg of disubstituted phosphate K, 5 ml of a solution of a mixture of trace elements containing 54 mg of trace elements Zn, Mg, Mn, Mo, Cu, Co, B, in the form of HEDP complexonates, a mixture of Ca propionate and Na benzoate (in equal amounts) - only 4 mg, ascorbic acid 20 mg, propinol B-400 in an amount of 0.3 ml.

The preparation obtained in a dilution of 1: 100 was treated with 500 mg of weed grass accumulated after weeding the crops of table beets and carrots. The treatment was carried out by spraying with a sprayer "Quasar", followed by mixing the treated mass, compacting it; the surface of the treated mass was covered with black plastic wrap. After 5 days, the treated mass settled, and the temperature at a depth of 12 - 15 cm increased to 52 - 57 ^o C. After 15 days, a sample of the fermented mass was taken. It was a macerated fiber of grass, had a greenish-brown paint, a pleasant specific smell. The resulting compost was analyzed for the content of some groups of microorganisms, the same analysis was carried out with grass samples not treated with the preparation, but also covered with a black film. Only single fungal colonies were found in composted (treated) grass when plated on Chapek’s medium, and 7 · 10 ² - 1 · 10 ³ fungal cells / g were found in samples of untreated grass; samples of untreated grass looked dark, with an unpleasant odor of the mass, which confirms the presence of ammonifiers.

Example 3
The composted grass mass obtained in Example 2 was added as a top dressing when growing strawberries. Top dressing was carried out when the first ovary appeared at the rate of 300-500 g of compost mass around each “outlet” in the experimental plot. Plants were used as control; they were not fed with compost. A sharp decrease in the number of berries affected by gray rot was noted (in the control, i.e., without top dressing with compost mass, the number of affected berries was 85%, while in the bushes treated, i.e. with top dressing, the number of affected berries was only 11%); a berry yield increase of 19 kg / ha was noted, while a berry crop without top dressing with compost weight was 15.5 kg / ha, thus, a yield increase of 3.5 kg / ha or 22%.

 Example 4

 20 ml of the preparation obtained in example 2 was diluted with tap water in a ratio of 1:50. The resulting solution was tested for relation to the causative agents of some of the most common plant diseases. The test was carried out on Petri dishes using the well method.

 The following results were obtained (see table. 1).

 Thus, it was found that the drug has the ability to inhibit the growth of certain pathogens of plants, i.e. It has a healing effect on the soil and on plants, preventing their infection.

 Example No. 5.

10 ml of the preparation obtained in example 2 was diluted with tap water to 10 liters. The resulting solution treated the area (100 m ² ) of the field after harvesting the corn, for which the solution was sprayed, and the treated area was scented, the mass of crop residues per 1 m ² reached up to 3.7 kg. In the soil sample taken before treatment with the preparation, the number of saprophytic microflora, as well as actinomycetes, fungi, anaerobic bacteria that decompose fiber, was determined. A second sample was taken in the spring, after the snow melted, and the same determinations were made. The same analysis was performed with a soil sample taken from an untreated field. The results are shown in table 2.

 Thus, a new consortium of lactic acid bacteria with original properties was obtained, which made it possible to develop a drug — an activator of composting plant material and stubble decomposition, which additionally has the ability to improve the composition of soil microflora, has fungicidal properties; the composition of the drug ensures its environmental cleanliness and safety, as all the components that make up its composition are completely consumed during the action of the drug, participating in the processes that occur during composting of plant material, or participating in the decomposition of stubble, as components of the nutrition of soil microorganisms involved in this process.

References
1. E.I. Kvasnikov, O.A. Nesterenko "Lactic acid bacteria and ways of their use. M.," Science ", 1977, p. 322.

 2. Copyright certificate, USSR, N 962279, publ. 09/30/82

 3. RF patent N 2057103. (Bulletin of inventions N 9, 1996, p. 214).

 4. RF patent N 2028998. (Bulletin of inventions N 5, 20.02, 1995).

 5. RF patent N 2025470. (Bulletin of inventions N 24, 12/30/1994).

 6. Sagi J. "Methods of soil microbiology". M., 1983, with. 239 - 242.

Claims (11)

 1. A consortium of bacteria Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus 384-5КС (CJSC "Bioflora" V-04) to obtain a biological product - an activator of composting of plant material and decomposition of stubble.  2. A method of obtaining a biological product - an activator of composting plant material and decomposition of stubble, characterized in that a consortium of bacteria Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus 384-5KS (CJSC "Bioflora" B-04) is cultivated on a sample containing starting vitamin supplements in the form an aqueous solution of a mixture of vitamins: paraaminobenzoic acid, thiamine, ascorbic acid, riboflavin in a total concentration of 0.005 - 0.06%.  3. The method according to claim 2, characterized in that the resulting culture fluid is diluted with water and add algae or alginates, a surfactant, carbamide, phosphoric acid, trace elements Zn, Mg, Mn, Mo, Cu, Co, B in the form of complexonates and stabilizers.  4. The method according to claim 2 or 3, characterized in that algae or alginates are added to the culture fluid in an amount of 0.5 - 3.5%, carbamide 0.7 - 7%, potassium phosphate 0.0005 - 0.03% , trace elements in the total amount of 0.0001 - 0.003% in the form of complexonates.  5. The method according to claim 2 or 3, characterized in that as stabilizers use calcium propionate, sodium benzoate and ascorbic acid.  6. Biological product-activator of composting of plant material and stubble decomposition containing lactic acid bacteria, characterized in that it contains lactic acid bacteria in the form of a consortium of bacteria Lactobacillus plantarum, Lactobacillus acidophilus, Streptococcus thermophilus 384-5KS (AOZT "Bioflora", B-04, B-04) additionally contains dry algae or alginates, urea, potassium phosphate, phosphoric acid, trace elements Zn, Mg, Mn, Mo, Cu, Co, B, stabilizers: calcium propionate, ascorbic acid and sodium benzoate, surfactant and water.  7. The biological product according to claim 6, characterized in that it contains algae in the form of dry seaweed or alginates in an amount of 0.5 - 3.5%.  8. The biological product according to claim 6, characterized in that the carbamide it contains in an amount of 0.7 - 7%, potassium phosphate 0.0005 - 0.03%.  9. The biological product according to claim 6, characterized in that it contains trace elements in a total amount of 0.0001 - 0.003% in the form of complexonates Zn, Mg, Mn, Mo, Cu, Co, B, obtained, for example, on the basis of glycine or hydroxyethylenediamine phosphate .  10. The biological product according to claim 6, characterized in that the stabilizer-calcium propionate and sodium benzoate, ascorbic acid, it contains in amounts in total equal to 0.001 - 0.01%, and as surface-active substances contains laprol or propinol B-400 in an amount of 0.001 to 0.05%.  11. The biological product according to claim 3, characterized in that it contains water in an amount equal to 2.5 to 5 volumes to the volume of a consortium of lactic acid bacteria used as the basis of the biological product.

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