FR2806420A1 - Microbial mixture for fixing nitrogen and increasing solubility of phosphorus compounds to improve fertility of soil, comprises the co-fermentation of Azotobacter croococcum, Azospirillum brasiliense and Bacillus megaterium - Google Patents

Abstract

Preparing a mixture (I) of micro-organisms by co-fermentation of the micro-organisms in nutrient soil, which is nitrogen free but contains phosphorus, is new. Preparation of a mixture (I) of microorganisms, suitable for fixing nitrogen in the air and increasing the solubility of phosphorus compounds, by co-fermentation of microorganisms in nutrient soil free of nitrogen but containing phosphorus compounds, involves multiplying the bacterial strains Azotobacter croococcum CCM 4642 and Azospirillum brasiliense CCM 4644 for 22 hours at 28 deg C, adding Bacillus megaterium CCM 4643 and fermenting the microorganisms together for 44-46 hours at 28 deg C, under aeration at 0.6 liters of air per liter of nutrient soil. An Independent claim is also included for a new mixture (I') of microorganisms, useful as for (I), comprising CCM 4642 and CCM 4644, each at 2-4 x 10<9> cells per ml in nutrient soil free of nitrogen but containing phosphorus compounds, together with CCM 4643 at 3-4 x 10<8> cells per ml, where the grown biomass contains a total of 4.25-8.4 x 10<9> cells per ml and has pH 7.5-8.5.

Description

<B> Mixture of microorganisms, intended for the </B> fixation <B> of nitrogen of </B> the air <B> and </B> <B> for the increase of </B> <B> solubility of phosphorus compounds in </B> <B> water, and method of preparation. </B> Technical field </U> The invention relates to a method of preparing a mixture of microorganisms for fixing nitrogen in the air and increasing the solubility of insoluble phosphorus compounds in soil. The invention also relates to the mixture in question.

The invention is important from the point of view of environmental protection, thanks to a metabiosis action of microorganisms.

State of the art In recent decades agriculture has mainly used artificial nitrogen fertilizers. Significant yields have thus been achieved. If these fertilizers were not combined with farm manure, their overuse made the soil acidic and microbial life was suppressed; the soil became more compact and the water and soil conditions worsened.

In addition, harmful nitrates have entered groundwater. The current of water accompanying the heavy rains carried the artificial granulated fertilizer to streams and the nitrates therefore entered the rivers.

Due to the high doses of artificial nitrogen fertilizers applied at once the plants - especially garden plants - received an excessive amount of nitrates, which negatively affect the biological organism and cause harmful effects, especially in infants and children. small children because they cause methemoglobinemia in the blood, which can be fatal.

A control of the nitrate content, especially in vegetables, was introduced their upper limit was determined. But it is very difficult to make a permanent control of the nitrate content of all vegetables. For this reason, the practical possibility of bacterial fixation of nitrogen in the air is becoming more and more topical. The increase in the prices of artificial nitrogen fertilizers, following the increase in the cost of energy, of which the production of artificial nitrogen fertilizers is a major consumer, decreases this need. After discovering the bacterial fixation of nitrogen in the air, efforts directed towards the use of nitrogen in the air in agriculture have increased, especially in recent years.

Nitrogen fixation is known using bacteria of the Rhizobium family which, on the roots of plants such as beans, soybeans, clover, form tubers which fix nitrogen from the air. But this nitrogen fixation is limited to plants in the papilionaceous domain.

Methods of preparing mixtures are also known which use microorganisms for the treatment of soils. Russian patent application No. 2072756 mentions, for example, how to put soil damaged by coal mining back into cultivation. The essence of this process consists in a mechanical treatment of the soil and in the sowing of grains which have been inoculated by microorganisms, to which, optionally, if necessary, in the case of a very acidic soil, is added. another microorganism. This process solves neither the problem of preparing the mixture by common fermentation, nor that of the mixture thus formed.

The articles mentioned below, which relate to the problem in question, describe certain methods of applying microorganisms. Mention will be made, for example, of Chemical Abstracts, Vol. 124, No. 2, Columbus Ohio, US, No. 147,114, which discloses a process for the biological removal of hydrocarbons from petroleum in the form of a mixture of gasoline, petroleum and lubricating oils contained in the ground. In principle, it is a regeneration of groundwater and wastewater. Another article, published in Compendex Engineering Information, US, Biol. Wastes 1987, describes information on the influence of non-symbiotic nitrogen fixation by organic waste, with the use of microorganisms. The articles mentioned do not answer either the question related to the way of preparing the mixture of microorganisms, nor to the mixture produced.

Another concept known from HU Patent No. 207,751 partially eliminates the application of inorganic nitrogenous and phosphorous fertilizers, but does not answer the question of potassium return. Another disadvantage of this mixture is the fact that its effects are thermally limited up to a temperature of 28 C. This mixture is therefore only effective during a short period of spring.

International patent application WO 96/34840 describes the use of new bacterial strains, which fix nitrogen with a higher resistance against alkalis, and the transformation of two bacteria in the medium containing additives and microelements. This mixture has a disadvantage, namely that its use is very limited, especially with soils with a high pH.

US Patent No. 4,952,229 describes the use of certain soil bacteria and nitrogen fixing bacteria with additions of microelements, for example in the form of chelates and various minerals. The subject of this patent is an additive and a method aimed at improving the fertility and quality of plants, applied to the soil or to the leaves of plants. The additive contains specific microorganisms and an organic acid, such as humic acid, fulminic acid and ulvinic acid. It is possible to add mineral trace elements, moisture stabilizers. In principle, it is a determination of the choice of additives and how to apply the additives to different species of plants. The mentioned patent does not understand how to prepare (produce) the mixture of microorganisms using common fermentation of microorganisms, without minerals or nitrogen containing microelements, and does not mention the important bacterial strain Bacillus megatherium. A drawback is that the invention requires the use of a large number of minerals and microelements.

<U> Summary of the invention </U> The drawbacks mentioned above are substantially eliminated by the invention described below, which aims to prepare a mixture which fertilizes the soil, enriches it with nitrogen from air and, at the same time, transforms insoluble phosphorus in the soil into soluble phosphorus.

The invention is based on the unexpected discovery of the common fermentation of microorganisms, without the addition of minerals and microelements containing nitrogen, this mixture being formed under certain determined conditions and, after application to the soil, ensuring for the plants the fixing nitrogen from the air and at the same time converting insoluble phosphorus into soluble phosphorus, this in a single application. The mixture is active up to 36-38 C, and thus produces its effects throughout the period of plant growth.

The process for preparing the mixture intended for fixing nitrogen according to the invention consists essentially in the use of certain species of bacteria of the <I> Azotobacter family, </I> which are capable of fixing the nitrogen of the air during their multiplication in the soil, taking up during their lifetime the synthesized forms of nitrogen in the soil, such as nitrates, nitrites, ammonium, amino acids, albumins with small molecules, which fix organic matter soil, from where the roots absorb them. The nitrogenous matter thus created is not eliminated by washing the soil and does not pollute the environment. This results in the advantage that, during the period of plant growth, nitrogen supplies the plants in a continuous and harmonious manner.

The bacterial strains concerned are <I> Azotobacter </I> croococcum 4642 and Azospirillium brasiliense CCM 4644. These bacterial strains have been deposited, in application of the Budapest Treaty, under the references mentioned above, with from the Czech Collection of Microorganisms at Masaryk University, Tvrdého 14, Brno, Czech Republic.

The most favorable conditions for the multiplication of the bacterial strain <I> Azotobacter </I> croococcum are between 16 C and 30 C, while the optimum temperature for the bacterial strain Azospirillium brasiliense is 24 C to 38 C. Soil is inoculated by these two species of bacterial strains and they are cultivated in the soil brought to 16 C in spring and to 38 C in summer. This is how plants are harmoniously supplied with nitrogen.

Also, due to the excessive use of artificial fertilizers containing phosphorus, a large amount of phosphorus compounds has accumulated in the soil. This phosphorus is insoluble in water and, therefore, it is not usable by plants which need phosphorus. The microorganism of the species Bacillus megatherium CCM 4643, which is the subject of a deposit, under this reference, in the Collection mentioned above, is capable, within the framework of its energy metabolism, of transforming phosphorus compounds having large molecules, which are insoluble in water, in water soluble phosphorus compounds, and therefore to supply the soil with soluble phosphorus compounds usable by plants.

The result of inoculation of the soil with nitrogen-retaining microorganisms and, at the same time, strains of microorganisms breaking down insoluble phosphorus compounds, is metabiosis, in which nitrogen fixers provide the nitrogen required and the microorganism Bacillus megatherium converts insoluble phosphorus compounds to water soluble phosphorus compounds. Both species of bacteria use energy for their own growth and multiplication and at the same time supply nitrogen and phosphorus to plants.

The invention can be implemented as follows: the compact biomass of the microorganisms Azospirillium brasiliense, <I> Azotobacter </I> croococcum and Bacillus megatherium, after having been diluted to a suitable concentration of 10 liters per hectare, is pulverized on the ground, then the ground is plowed to the depth of sowing, at the same time as it is inoculated in such a way that it is possible to abandon the artificial fertilizers containing nitrogen and phosphorus.

This three-component preparation can be used instead of artificial fertilizers.

The essence of the invention lies in the way of producing the mixture of microorganisms intended for fixing nitrogen in the air, and for increasing the solubility of insoluble phosphorus in the soil.

According to the invention, the bacterial strain <I> Azotobacter </I> croococcum CCM 4642 multiplies on the nutrient soil, which contains neither organic nitrogen nor inorganic nitrogen, by fermentation at a temperature of approximately 28 C, for 22 hours , then the Azotobacter ferment is inoculated with 10% of the inoculum of the bacterial strain Bacillus megatherium CCM 4643 at 22 hours. For 22 hours, the microorganism of the Azotobacter family produces in the fermentation medium the quantity of nitrogen necessary for the growth of the microorganism of the Bacillus megatherium family. The double fermentation continues for another 22 - 24 hours, aerating with 0.6 liters of air per liter of nutrient soil, with rotary agitation at 120 revolutions / minute and at a temperature of 28 C. After the end of the culture, after 44-46 hours, the pH of the culture soil is between 6.8 and 8.5, with an increase of 4-6 billion cells / ml of the medium of the <I> Azotobacter </I> strain croococcum and of 150-200 million cells / ml of the strain Bacillus megatherium CCM.

In addition, according to the invention, the bacterial strain Azospirillium brasiliense CCM 4644 multiplies on the nutrient soil which contains neither organic source nor inorganic source of nitrogen, thanks to fermentation at a temperature of 28 C for 22 hours, then the multiplied culture of the microorganism Azospirillium brasiliense and inoculated with 10% of the inoculum of the bacterial strain Bacillus megatherium CCM 4643 by 22 hours and, until this period, the bacterial strain Azospirillium brasiliense produces an amount of nitrogen necessary for growth of the microorganism Bacillus megatherium. The double fermentation continues for another 22-24 hours, aerating with 1 liter of air per liter of the fermentation medium, at 120 rpm and at a temperature of 28 C. After the end of the fermentation, the pH of the fermentation medium fermentation is 6.8 - 8.5 and the growth of the Azospirillium brasiliense strain is 4 - 6 billion cells / ml of the medium and that of the Bacillus megatherium strain is 150 - 200 million cells / ml of the medium.

During the practice of the invention, no side product occurs. The biomass of microorganisms can be used as a starter, which does not undergo further processing. Water from washing ferments can be poured into the drains, as it enriches the water in rivers with different species of rare bacteria.

<U> Examples of implementation </U> <U> of the invention </U> <U> Example 1 </U> In a flask, a mixture of 200 ml of potassium dihydrogen phosphate 0, 3 g, calcium chloride 0.2 g, magnesium sulfate 0.3 g, potassium sulfate 0.2 g, sodium chloride 0.4 g, ferric chloride 0.05 g, sodium carbonate calcium 5.0 g, molasses 30.0 g, fermentable sugar 10.0 g, per 1000 ml of nutrient soil, and for 40 minutes the soil is sterilized at a temperature of 125 C, then 1 ml is added of trace element solution: boric acid 5.0 g, ammonium molybdate 6.0 g, potassium iodide 0.5 g, sodium bromide 0.5 g, zinc sulphate 0.3 g, d sulphate aluminum 0.3 g, per 1000 ml of nutrient soil.

To the contents of the lyophilized ampoule of two bacterial strains <I> Azotobacter </I> croococcum and Azospirillium Brasiliense, add 5 ml of sterilized water and a suspension is obtained. The two flasks are inoculated with 1.0 ml of suspension. The inoculated flasks are placed on a horizontal stirring installation and are cultured at a temperature of 28 ° C, 160 rpm, for 48-72 hours, until sufficient growth of the biomass. This is how the basic inoculum is prepared.

Then, 400 ml of nutrient soil of the above-mentioned composition are placed in the flask and 3-4 ml of base inoculum are inoculated and cultivated on the horizontal stirring installation, the temperature of 28 C, for 48 hours. This is how the preparation inoculum is obtained.

Then 1000 liters of nutrient soil are sterilized at a temperature of 125 C for 40 minutes. After cooling, the inoculation is made with 1.6 l of the preparation inoculum. The inoculated soil ferments for 22 hours at a temperature of 28 ° C., aerating with 0.6 liters of air per liter of fermentation medium, at 120 rpm. Thus, the working inoculum of suitable consistency is obtained from two species of microorganisms. To limit the production of foam, add 0.2 - 1.0 1 / m3 of skimming product. The pH value of nutrient soil is 6.8 - 7.0.

<U> Example 2 </U> In a flask, we put a mixture of 200 ml of potassium dihydrogen phosphate 0.3 g, molasses 30.0 g, coorn-steep 0.5 g, peptone 0, 3 g of calcium carbonate 5.0 and sterilized at a temperature of 125 C for 40 minutes.

In the contents of the lyophilized vial of the bacterial strain Bacillus megatherium, 5 ml of sterile water is added for each vial and, thus, a suspension is formed, then the two flasks are inoculated with 1.0 ml of the suspension. The inoculated flasks are placed on the horizontal stirring installation and are cultured at a temperature of 28 ° C., for 48 hours, at 160 revolutions / minute. Then 400 ml of nutrient soil are put into the flask and 1-2 ml of base inoculum are inoculated and mixed on the horizontal stirring device, at the temperature of 28 C, for 24-48 hours and, at With the help of a microscope, the vegetation condition of the crop and the formation of spores are checked. This is how the preparation inoculum is obtained.

Then, 1000 liters of nutrient soil are sterilized at a temperature of 125 C for 40 minutes. After cooling, the inoculation is made with 3 pieces of preparation inoculum with a volume of 400 ml. The inoculated soil ferments for 22 hours at a temperature of 28 C, aerating with 0.6 liters of air per liter of nutrient soil, at 120 rpm. To limit the production of foam it is necessary to add 0.2 - 1.0 1 / m3 of skimming product. The growth of the culture is followed with the aid of a microscope, so that the growth develops to the state of vegetation, without formation of spores. The pH value of nutrient soil is 6.8 - 7.0. <U> Example 3 </U> In two fermenters with a total content of 10-12 m3, useful volume of 5.0 - 6.0 m3, a mass of 4.5 and 4.5 m3 of nutrient soil without nitrogen content described in Example 1 is sterilized and is inoculated with 10% of working inoculum containing the bacterial strain Azotobacter croococcum according to Example 2. Then it is cultured at the temperature of 28 C for 22 hours, in providing simultaneous aeration with 0.6 liters of air per liter of nutrient soil, at 120 rpm. Then, the fermented soil is inoculated with 10% of the culture of the microorganism Bacillus megatherium according to Example 2 and the double fermentation continues for another 22 hours. After fermentation for 44-46 hours, the pH of the nutrient soil is 7.7 - 8.5 and the bacterial strain <I> Azotobacter </I> croococcum achieves a growth of 4-8 billion cells / ml of the medium.

In two fermenters with a useful volume of 5.5 - 6.0 m3, 4.5 and 4.5 m3 of the nitrogen-free nutrient soil described in Example 1 are placed, the mass is inoculated with 10% of working inoculum having the content of the bacterial strain Azospirillium brasiliense according to Example 1. The culture lasts 22 hours, at a temperature of 28 C, ensuring aeration with 0.6 liter of air per liter of nutrient soil, at 120 revolutions /minute. After the end of the fermentation, the pH of the fermentation medium is 7.5 8.5 and the Azospirillium brasiliense strain <I> a </I> reaches a growth of 6 billion cells / ml of medium and the Bacillus strain megatherium reaches -200 million cells / ml of medium.

Then, by mixing the two types of ferments and homogenizing them, we obtain the defined product composed of three elements Azotobacter croococcum at 2-4 billion cells / ml, Azospirillium brasiliense at 2-4 billion cells / ml, Bacillus megatherium at - 400 million cells / ml of medium, and the multiplied biomass comprises a total of 4.1 billion cells / ml up to 8.2 billion cells / ml of nutrient medium, with a pH of 7.5 - 8 , 5.

<U> Use in industry </U> A mixture of microorganisms intended for fixing nitrogen and increasing the solubility of phosphorus compounds in water is prepared according to the process described below above and this mixture can be used in agriculture.

Claims (2)

1. Process for the preparation of a mixture of microorganisms intended for fixing nitrogen in the air, and for increasing the solubility of phosphorus compounds, characterized in that, by the joint fermentation of two strains of microorganisms in the less in nutritious soil without nitrogen and containing phosphorus compounds, the bacterial strains <I> Azotobacter </I> croococcum CCM 4642 and Azospirillium brasiliense CCM 4644 are multiplied for about 22 hours, a temperature of about 28 C, then the bacterial strain Bacillus megatherium CCM 4643 is added and they ferment together for about 44-46 hours at a temperature of about 28 ° C, with aeration of about 0.6 liters of air per liter of nutrient soil. 2. Mixture of microorganisms intended for fixing nitrogen in the air and increasing the solubility of phosphorus compounds, characterized in that it consists of two strains of microorganisms, <I> Azotobacter </I> croococcum CCM 4642, at 2 - 4 billion cells / ml and Azospirillium brasiliense CCM 4644, at 2 - 4 billion cells / ml in nitrogen-free nutrient soil, containing phosphorus compounds, and Bacillus megatherium strains CCM 4643, at 300 - 400 million cells / ml, the multiplied biomass comprising a total of 4.25 8.4 billion cells / ml of the nutrient medium, with a pH of?, 5 to 8.5