RO120556B1 - Phosphatase-producing bacillus megaterium strain with biofertilizer function - Google Patents

Abstract

The invention relates to a Bacillus megaterium strain CA1ICCF280 obtained in stages, by laboratory selection, without mutagenic agents, from a population of wild cells which, on solid culture media is presented as round colonies having the diameter of 3-5 mm, smooth surface, viscous consistency and whitish colour and having the capacity of solubilizing the phosphorus immobilized in the soil resulting in the 16-times increase of the soluble phosphorus form and which can ensure the mobilization of the phosphorus in the rocks, such as apatite and phosphorite, of the iron, molybdenum and other microelements in sulphurous rocks, such as pyrite, chalcopyrite and blende.

Description

The invention relates to a new strain of Bacillus megaterium, obtained by laboratory selection, in stages, without mutagenic agents, on a wild variant isolated from the wheat rhizosphere.

Microorganisms are known to possess the ability to solubilize immobilized phosphorus in insoluble forms, due to the presence of phosphatase activity. These microorganisms belong to the genera Bacillus, Pseudomonas and Chromobacterium. The total phosphorus, contained in the arable soil layer, is composed of organic and inorganic phosphates, which can be soluble or immobilized in various insoluble forms. The provision of phosphorus crops from soil reserves depends solely on the soluble phosphate content of the arable layer. (Daughton CG, CookA. M., Alexander M., 1979, J. Agric. FoodChem., 27,1375-1382; Hans Jurgen Rehm, 1983, Biotechnology, vol. 3, H. Delweg (ed), Verlag Chemistry, Weinheim, Deerfield Beach, Florida Basel, 204-208; 3, Quinn PH, Joseph M. Peden, R. Elaine Dick, 1989, Microbiol Appl. Biotechnol., 31, 283-287; ACA Coast, Medronho AR, Pecanha PR, 1992, Biotech. Letters, 14,233-238). Bacillus megaterium is one of the most popular bacteria that solubilizes phosphorus. Barea (Barea JM, Navarro E., Montoya E., 1976, J. Appl. Bacteriol. 40,129-134) isolated 108 strains of Bacillus megaterium from the rhizosphere of different cereals, some having phytase activity, and others being able to dissolve phosphate rocks. what the.

The present invention extends the range of bacterial biofertilizers by the fact that the Bacillus megaterium strain obtained by laboratory selection, in steps, called CA! ICCF 280 and deposited in the culture collection of the National Institute of Chemical-Pharmaceutical Development, has the capacity to solubilize the immobilized phosphorus in the soil, leading to an increase of the concentration of the soluble form of phosphorus by about 16 times. Due to the presence of phosphatase activity, the Bacillus megaterium strain of ICCF 280 can ensure the mobilization of phosphorus from rocks such as apatite and phosphorite, iron, molybdenum and other microelements, from sulfurous rocks such as pyrite, chalcopyrite and blende.

Bacillus megaterium CA strain, ICCF 280, obtained by laboratory selection, in stages, without mutagenic agents, from a population of wild cells, which, on solid culture media, are in the form of round colonies, with a diameter of 3- 5 mm, with a smooth surface, whitish color and viscous consistency, and which has the ability to solubilize the phosphorus immobilized in the soil, leading to an increase of the soluble phosphorus form concentration about 16 times and which can ensure the mobilization of phosphorus from rocks such as apatite and phosphorite. , of iron, molybdenum and other microelements, from sulphurous rocks such as pyrite, chalcopyrite and blende.

The invention has the following advantages:

- realizes a new spontaneous mutant of Bacillus megaterium, which has the ability to solubilize the immobilized phosphorus in the soil, leading to the increase of the concentration of the soluble form of phosphorus approximately 16 times and can ensure the mobilization of phosphorus from rocks such as apatite and phosphorite, iron, molybdenum and of other microelements, from sulphurous rocks such as pyrite, chalcopyrite and blende, this mutant could be a very useful biofertilizer in agriculture.

On solid cultivation media, the Bacillus megaterium strain CAi ICCF 280 develops after 72 h of incubation at 28 ° C, in the form of round colonies, with a diameter of 3-5 mm, with a smooth surface, whitish color and viscous consistency.

Here is an example of getting the strain. Table 1 describes the strain obtained by the laboratory selection, in steps, carried out on a wild strain isolated from the wheat rhizosphere, which fall into the genus Bacillus specia megaterium.

RO 120556 Β1

Table 1 1

Cultural and morphological features of Bacillus megaterium strain CAJCCF 280

test Specific response to Bacillus megaterium strain CAJCCF 280 Colony shape round Colony diameter (mm) 3-5 Colony relief Umbonat T ransparency matte The appearance of the surface Smooth Consistency viscous color whitish The shape of the cell bacillary Cell length (pm) 2-5 Cell diameter (pm) 1.2-1.5 scourge present sporulation Present The shape of the spores oval Arrangement of spores in the cell plant Gram coloration positive Cellular mobility Present Polibetahidroxibutirat Present test Specific response to Bacillus megaterium strain CAJCCF 280 Minimum rise temperature (° C) 3-20 Maximum rise temperature ("C) 35-45 Optimum rise temperature (° C) 28 Anaerobic growth Absence Phosphatase synthesis Present Environmental browning at prolonged incubation Present Catalase synthesis Present Increase in 0.001% lysozyme Present Increase in 7% NaCl Present Use of citrate Present

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Table 1 (continued)

test Specific response to Bacillus megaterium strain CAJCCF 280 Reduction of NO ₃ to NO ₂ Present Gelatine liquefaction Present Decomposition of casein Present Examination of phenylalanine Present Multiplication without growth factors Present Acid formation from: Glucose Present arabinose Present xylose Present mannitol Present Carbohydrate metabolism Maltese (D) ++ Ribose (D) + Xylose (D) + Mannose (D) + Raffinosis (D) +++ Galactose (D) + Fructose (D) ++ Melibiosis (D) ++ test Specific response to Bacillus megaterium strain CAJCCF 280 Trehalose (D) +++ Arabinose (D) + sucrose +++ Glucose (D) +++ Dextrose +++ sorbose + Fucose (D) + cellobiose +++ Lactose ++

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Table 1 (continued)

test Specific response to Bacillus megaterium strain CAJCCF 280 Lyxoză + mannitol ++ sorbitol + myo + inulin +++ dulcitol - glycerol ++ Adonitol + xylitol - Starch +++ Dextrin +++ Sugar beet molasses +++ Metabolism of some organic acids Malic acid ++ Succinic acid + 2-Ketoglutaric acid + Cisaconitic acid + Pyruvic acid ++ Citric acid + Lactic acid + Acetic acid + Fumaric acid + Metabolism of some amino acids L-Tyrosine + L-Methionine + Ac.L-Glutamic ++ L-Lysine + L-Alanine +++ L-Histidine + L-Threonine +++

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Table 1 (continued)

test Specific response to Bacillus megaterium strain CAJCCF 280 L-Tryptophan + L-Phenylalanine ++ L-homoserine + L-Leucine ++ L-Valine ++ L-Cystine + L-Seri nă +++ L-Arginine +++ L-Cysteine ++ L-Glycine + L-Proline + L-Hydroxyproline + Ac.DL aspartic +++ Metabolization of inorganic nitrogen sources NH ₄ CI +++ nh ₂ conh ₂ ++ kno ₃ +++ NaNO ₃ ++ NaNO ₂ + (NH ₄ ) ₂ HPO ₄ + Metabolization of complex sources of organic nitrogen Soybean flour +++ Proflo cotton flour +++ Pharmamedia cotton flour +++ Corn extract +++

: Culture does not develop;

: Questionable use;

+: Poor use;

++: Good use;

+++: Very good use.

As shown in Table 2, the Bacillus megaterium strain CAJCCF 280 can use several energy sources, but for reasons of economy and in order to increase the concentration of soluble phosphorus form in the soil by approximately 16 times, the media corresponding to the multiplication

RO 120556 Β1 at pilot and industrial level, for Bacillus megaterium strain CAi ICCF 280, contains as 1 carbon sources beet molasses, sucrose, corn extract, pyruvic acid, malic acid and others. The culture media do not require the use of inorganic nitrogen sources, and as far as mineral salts are concerned, they must contain phosphorus, magnesium, sodium, calcium, iron, molybdenum and others. 5

As a source of growth factors, yeast extracts, feed yeast, mixtures of amino acids and others can be introduced into the environment. 7 The pH of the culture medium may vary between 5.5 and 8, but preferably it is 7.0-7.5. During multiplication, the pH of the culture liquid can increase to 8.5. The temperature of cultivation under submerged conditions varies between 26 and 34 ° C, with the optimum in the range 28-32 ° C. The air requirement varies within the range 0.5-1.5 v / v / m and changes depending on the stage 11 of culture development.

The cultivation cycle is 36-48 h, at 28-32 ° C, under diarrhea and agitation conditions. Tulpina 13 obtained by laboratory selection is kept at + 4 ° C in lyophilized form and is maintained by the usual cultivation techniques. agarized media, making periodic laboratory selections based on the clonelortipic retention in morphological aspect and preliminary testing of the solubilization capacity of insoluble mineral phosphates. The colonies that have the highest solubilization capacity of insoluble phosphates, 17 serve to obtain pure cultures and then to prepare the vegetative inoculum by deep cultivation. 19

Claims (2)

Claims 21 1. Bacillus megaterium strain CA ₁ ICCF 280, obtained by laboratory selection, 23 in stages, without mutagenic agents, from a population of wild cells, which on the solid culture media are in the form of round colonies, with the diameter of 3-5 mm, with a smooth, whitish upper surface and a viscous consistency, which has the ability to solubilize the immobilized phosphorus in the soil, leading to an increase in the soluble form of phosphorus 27 approximately 16 times and which can ensure phosphorus mobilization. from rocks such as apatite and phosphorite, iron, molybdenum and other microelements, from sulfurous rocks such as pyrite, calcite-pyrite and blende. 2. Use of Bacillus megaterium CA strain, ICCF 280, to obtain a 31 phosphorus solubilizing biofertilizer preparation.