RU2081867C1 - Strain of bacterium bacillus mucilaginosus for fertilizer and exopolymer producing - Google Patents

Abstract

FIELD: agriculture microbiology, biotechnology. SUBSTANCE: strain of bacterium Bacillus mucilaginosus is isolated from the cultured soil and deposited in All-Union Collection of industrial microorganisms at N B-5987. Strain is cultured on liquid nutrient medium containing carbon source and mineral salts at 28 ± 1 C for 48 h under aeration condition. The grown culture containing 300 mln spores/ml and exopolysaccharides (0.2 g/l) is mixed with bentonite and dried. The obtained preparation is used for presowing plant seeds, vegetating plants, fruit and decorative culture cuttings treatment and added to the soil also. EFFECT: bacterial strain indicated above, enhanced effectiveness of the strain. 4 tbl

Description

 The invention relates to agricultural microbiology and biotechnology, in particular to the production of bacterial preparations used in crop production, and relates to a new strain for one of the bacterial fertilizers.

 Bac.mucilaginosus strains are known that differ in their properties, for example, a strain having the ability to transform siliceous rocks [1] or strain 4 [2] with phosphate mobilizing activity from the collection of the All-Russian Research Institute of Agricultural Microbiology.

Strain 4 turned out to be closest in its properties. The disadvantage of this strain is its very low productivity and unstable phosphate mobilizing activity when grown on an industrial nutrient medium of the following composition, molasses 0.6; K ₂ HPO ₄ 0.03; MgSO ₄ • 7H ₂ O 0.1; NaCl 0.05; KNO ₃ 0.1; chalk 0.1.

 The aim of the present invention is to obtain a strain of Bac.mucilaginosus with high activity to transform insoluble and sparingly soluble phosphates into a form accessible to plants and synthesize exopolysaccharides.

 This goal was achieved by the method of selection of natural isolates isolated from cultivated soils of the Kuibyshev region, a new strain was obtained that has stable ability to transform insoluble and poorly soluble (organic and mineral) phosphorus compounds and stable ability to synthesize exopolysaccharides.

 Collection number Bac.mucilaginosus, pcs Z-B2-5987 in the All-Russian collection of industrial microorganisms.

 Cultural and morphological characters.

1. When grown on an Ashby agar medium containing the composition,
sucrose 2
K ₂ HPO ₄ 0,02
MgSO ₄ • 7H ₂ O 0.02
NaCI 0.02
NH ₄ NO ₃ 0,02
CaCO ₃ 0.5
K ₂ SO ₄ 0,0
Agar-agar 20 g / l
at a temperature of 28 ± 1 ^o C for 72 hours: round, regular forms, convex, colorless, transparent, mucous colonies. During microscopy ("crushed drop") large sticks, sticks with spores; when grown for more than 72 hours, single sticks, mainly sticks with spores, spores.

2. When grown on a nitrogen-free liquid nutrient medium Aleksandrova N 27, having the compositions
Sucrose 0.5
Crushed glass with 1% chalk 0.1
Na ₂ HPO ₄ 0.2
MgSo ₄ • 7H ₂ O 0.05
FeCI ₃ 1-2 drops of 1% solution per 100 ml of medium,
at a fermentation temperature of 28 ± 1 ^o C and an azration regime of 180-200 rpm, provided that the pH is naturally changed and grown for 48 hours, the microbial number at 48 hours of fermentation is 8-12 million ml ^-1 ; upon microscopy, large sticks with capsules, single cells with spores; the dissolution activity of tricalcium phosphate (in the clarification zones in mm) is 4-10 mm. Synthesis of exopolysaccharides 1.0 1.4 g / L.

3. When grown on medium N 1 with molasses having the composition
Molasses 0.6
K ₂ HPO ₄ 0,03
MgSO ₄ • 7H ₂ O 0.1
NaCI 0.05
KNO ₃ 0.1
Chalk 0.1
at a fermentation temperature of 28 ± 111 ^o C and a fermentation time of 48 hours (aeration-mixing mode V _air. V _medium = 1 1, at 18-24 hours of fermentation, the culture is a viscous liquid; when microscopic, bacterial cells are large sticks, single sticks, spores up to 200-250 million / ml; the activity of dissolution of tricalcium phosphate (in the clarification zones on Petri dishes) is 3-8 mm; the content of exopolysaccharide is 1.2 1.6 g / l.

 Physiological and biochemical characteristics.

Aerob, optimal growth temperature plus 28 ± 1 ^o C, optimal pH value of 6.8-7.0; with deep fermentation in a liquid nutrient medium, the optimal mixing aeration regime is 1: 1, which corresponds to a sulfite number of 1.

 Attitude towards carbohydrates and organic acids.

 Absorbs glucose, sucrose, lactose, starch; poorly assimilates cellulose; does not absorb succinic, lactic, citric, acetic acids.

 Attitude to nitrogen sources.

 Ammonium, nitrates, amino acids, urea can serve as a source of nitrogen. The maximum productivity of the strain is found when using sucrose and ammonium, the minimum when using glucose and sodium nitrite.

 Relation to insoluble phosphorus compounds.

 The strain has the ability to dissolve tricalcium phosphate, fluorapatite, sodium nucleonate.

To maintain strain "Z" it is grown on a beveled agar medium composition,
Starch 1.0
K ₂ HPO ₄ 0,03
NaCI 0.1
KNO ₃ 0.1
Agar Agar 2.0
The temperature of cultivation is 28 ± 1 ^o C, the duration of cultivation is 72-78 hours. Storage in test tubes on a mown agar medium at a temperature of plus 6 ± 2 ^o C for up to 2 years (Table 1).

Characteristic features of strain "Z" are:
high productivity
phosphate mobilizing activity,
high level of synthesis of exopolysaccharides.

 Examples of the use of the present invention.

 Example 1. Obtaining a bacterial preparation using pcs. "Z".

Pieces "Z" were grown on a liquid nutrient medium, including molasses 0.6, K ₂ HPO ₄ 0.03, MgSO ₄ • 7H ₂ O 0.1, KNO ₃ 0.1, chalk 0.1. The fermentation temperature was 28 ± 1 ^o C, the fermentation time was 48 hours, and under aeration-mixing mode V _air : V _{nutrient medium} = 1: 1 obtained a liquid culture containing 300 million / ml spores, the content of exopolysaccharides 0.2 g / L. The grown culture was mixed with a filler, which was used bentonite. The resulting mass was dried on a spray dryer under mild conditions: t ^o _in. = 120 ^o C, t ^o out. 65 ^o C. The output of the drug on the balance of viable spores 95%
The drug was given for pre-sowing treatment of carrot seeds (I var.) And in experiments on the reclamation of dumps of Poltava GOK (II var.).

In the first application, the yield increase was 23.8% compared with the control
In the II embodiment, the drug was introduced into a vessel with a sample blade at the rate of 90 mg of the drug per 1 kg of blade control the sample blade without making the drug. White clover seeds were sown in each vessel. The change in the phosphorus content (in terms of P ₂ O ₅ ) is shown in Table 2.

 The phosphorus content in biota increased after 2 months, and after 3 months during the flowering period of clover reached 3.68 mg / kg, i.e. almost 6 times per month, or more than 50 times compared with the control, which indicates the active dissolution of phosphorus under the influence of the introduced drug.

 Example 2. The preparation obtained as in example 1 was used to treat tomato plants in a film greenhouse when growing them on ordinary greenhouse soil; the nutrient content was maintained at an optimal level. The drug was added to the soil before transplanting. The effect of the drug on the tomato crop (hybrid, variety "Swallow") is given in table.3.

 Example 3. Liquid culture of Bac. mucilaginosus pcs. "Z", obtained at 48 hours, grown on medium No. 1, was used to process cuttings of fruit and ornamental crops when planting them in boxes and the rooting of cuttings in the variant with the use of the drug and in the control was compared (i.e. without drug). The effect of treatment (application to the soil) with the culture fluid of strain "Z" is given in table 4.

 The use of a liquid culture of a piece of “Z” increases the number of rooted cuttings as compared to a control, namely: cherry cuttings by 14% of mock up of cuttings of a mock bilberry by 40%.

Example 4. Liquid culture of Bac.mucilaginosus, pcs. "Z" grown on a medium with sucrose was precipitated with ethyl alcohol in a ratio of 1: 1; the resulting precipitate was dried at 60 ^o C to constant weight. With 150.0 ml of liquid culture, 1.2 g of exopolysaccharide, i.e. the yield was 0.8%. Analytical studies of the polysaccharide revealed the content of galactose and mannose in it.

 Thus, it was found that pcs. “Z” is promising not only for obtaining a bacterial “soil-fertilizing” biological product, but also for producing an exopolysaccharide.

References:
1. Microbiological journal, 1989, t.51, N 5, p. 13-19.

 2. Passport of the bacterial strain Bac.mucilaginosus, pc. 4 (Museum of Microorganism Culture, All-Russian Research Institute of Agricultural Microbiology), 1983.

Claims (1)

 The bacterial strain Bacillus mucilaginosus VKPM B-5987 to obtain bacterial fertilizer and exopolymer.

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