SK3862000A3 - Method for the preparation of microorganism mixture for binding air nitrogen and for increasing the phosphorous compound solubility in soil and mixture listed above - 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

Process for preparing a mixture of microorganisms for binding nitrogen from air and for increasing the soil solubility of phosphorus compounds and said mixture.

Technical field

The invention relates to a process for the preparation of a mixture of microorganisms for the bacterial binding of nitrogen from air, for increasing the solubility of the water-insoluble phosphorus contained in the soil and said mixture. The invention is important in terms of environmental protection by the action of metabiosis of microorganisms.

BACKGROUND OF THE INVENTION

In the last few decades, mainly nitrogen fertilizers have been used in agricultural production. This resulted in large crop yields. The continued use of artificial fertilizers, if not combined with the use of granulated fertilizers, caused the soil to become acidic due to the large amount of chemicals, and the microbial life in the soil was suppressed, the soil compacted, soil and water conditions deteriorated. In addition, harmful nitrate has penetrated the groundwater. During heavy rains, the water stream washed away the granulated fertilizer into the streams, and thus nitrate also entered the watercourses. By a single dose of large amounts of nitrogen fertilizer, plants, especially garden plants, have received excessive amounts of nitrate, which have a negative effect on the biological organism, * particularly harming nitrate to infants and young children because it causes methemoglobinemia in the blood, which can be fatal. Monitoring of nitrate levels has therefore been introduced, particularly in vegetables, and their upper acceptable limit has been established. However, in practice, it is very difficult to carry out a constant control of the nitrate content in the desired wide range.

Therefore, the practical possibility of bacterial binding of nitrogen from the air is becoming increasingly important. The problem of the increase in the price of nitrogenous fertilizers in connection with the increase in energy prices is also supportive, because the production of nitrogenous fertilizers requires a large amount of energy. Upon the discovery of bacterial binding of air nitrogen, efforts are being made to utilize nitrogen from the air at large

- the extent of land management in agricultural practice, especially in the recent period.

Known is the binding of nitrogen by bacteria of the genus Rhizobium, which on the roots of flowering plants e.g. beans, soybeans, clover create tubers that bind nitrogen from the air. However, this nitrogen bonding is limited only to the flowering plant region.

Further, methods of preparing mixtures that utilize microorganisms to treat soils, e.g. in Russian patent no. 2072756 discloses a method of reclaiming land damaged by coal mining. Its essence lies in the mechanical treatment of the soil with subsequent sowing of seeds which are inoculated with the microorganism, or if necessary in the case of very acidic soil, additional microorganism is additionally added. The present invention does not solve the process of preparing the mixture by co-fermentation or the mixture thus formed.

Other published articles on the subject of investigation describe some methods of application of microorganisms such as e.g. Chemical Abstract, vol. 124, no. 2, Columbus Ohio, US, no. No. 147114 discloses a process for the biological removal of petroleum hydrocarbon in the form of a mixture of gasoline, diesel and lubricating oils contained in water. Basically, it is the regeneration of groundwater and wastewater. Another article in Compendex Engineering Information, US, Biol Wastes 1987, publishes information on the effect of non-symbiotic nitrogen bonding on organic waste using a microorganism. The cited strands do not solve the method of preparation of the mixture of microorganisms nor the mixture thus prepared according to the invention.

Another known solution according to HU patent no. 207751 partially removes the application of inorganic nitrogen and phosphorus fertilizers to the soil, but does not address the return of potassium. Another disadvantage of this mixture is that its effects are thermally limited to a temperature of 28 ° C, therefore this mixture only acts in a short spring period.

The published patent application WO 96/34840 describes and claims the use of novel strains of nitrogen-binding bacteria with increased resistance to alkali and a mixture of two bacteria in an aqueous medium containing additives and microelements. The disadvantage of this mixture is that its use is very limited especially on soils with a high pH content.

U.S. Pat. No. 4,952,229 discloses the use of certain soil and nitrogen-binding bacteria with the addition of microelements e.g. in the form of chelates and various minerals The subject matter of the patent is an additive and method for improving the fertility and quality of plants for use in soil or plant leaves. The additive contains specific microorganisms, and an organic acid such as humic acid, fulminic acid and ulvic acid, trace minerals, a moisture stabilizer may be added. Essentially, it is a matter of determining the choice of ingredients and the method of applying the ingredients to different plant species. This patent does not include a process for preparing (producing) a mixture of microorganisms by co-fermenting microorganisms without minerals or microelements containing nitrogen, and does not contain in any application an important bacterial strain Bacillus megatherium. A disadvantage is that the invention requires the use of a large number of minerals and microelements.

SUMMARY OF THE INVENTION

The above-mentioned disadvantages are largely overcome by the invention described below which aims to prepare such a mixture which fertilizes the soil, enriches it with nitrogen from the air, and at the same time converts the insoluble phosphorus in the soil into a soluble form of phosphorus. The present invention is based on the unexpected knowledge of the co-fermentation of microorganisms, free of minerals and nitrogen-containing microelements, and under certain specific conditions, a mixture is formed which, when applied in the soil system, simultaneously provides nitrogen to the plants insoluble phosphorus to soluble, in one application, while the mixture is active over a wide range of ambient temperatures up to 36 to

38 ° C, which means that it acts throughout the growing season.

The present invention relates to a method for the preparation of a nitrogen-binding composition according to the invention which utilizes certain species of the genus Azotobacter which are capable of binding nitrogen from the air by diffusing the synthesized forms of nitrogen into the soil during their propagation in soil. nitrates, nitrites, ammonium, amino acids, small molecule proteins that bind to the organic matter of the soil from where their plant roots absorb. thus

- Continuously produced nitrogenous substances are not washed out of the soil and do not pollute the environment. The big advantage is that during the vegetative period nitrogen is supplied to plants continuously and harmoniously. Suitable species are the bacterial strain Azotobacter croococcum CCM 4642 and the bacterial strain Azospirillium brasiliense CCM 4644. These bacterial strains were deposited in the authenticated Czech Collection of Microorganisms, Brno, Tvrdého no. 14 under the above numbers.

The bacterial strain Azotobacter croococcum is best propagated at a temperature of 16 ° C to 30 ° C, while the bacterial strain Azospirillium brasiliense has a temperature optimum of 24 ° C to 38 ° C. The two strains of bacteria are inoculated and then cultivated in soil heated to 16 ° C in the spring and 38 ° C in the summer. In this way, a harmonic nitrogen supply is provided to the plants.

In addition, very many phosphorus compounds, which are insoluble in water, have accumulated in the soil due to the excessive use of phosphorus-containing fertilizers and are therefore unusable for plants requiring phosphorus. A microorganism of the species Bacillus megatherium CCM 4643, deposited in the authenticated Czech Collection of Microorganisms, Brno, Tvrdého no. 14 under the above-mentioned heading, it is able, within its energy metabolism, to convert phosphorus compounds with large water-insoluble molecules to water-soluble phosphorus compounds and thereby supply the soil with plant-soluble, soluble phosphorus compounds.

Inoculation of the soil with nitrogen-binding microorganisms and simultaneous inoculation of the soil with micro-organisms decomposing insoluble phosphorus compounds results in so-called metabiosis, where the nitrogen binders supply the necessary nitrogen and the Bacillus megatherium converts the insoluble phosphorus compounds into water-soluble phosphorus species. their own growth and multiplication, while supplying nitrogen and phosphorus to plants.

The invention can be utilized by spraying the dense biomass of the microorganisms Azospirillium brasiliense, Azotobacter croococcum and Bacillus megatherium after dilution to a suitable concentration of 10 liters per hectare onto the soil, then turning the soil to the depth of sowing, thereby inoculating the soil. the use of nitrogen and phosphorus fertilizers may be waived in part or in whole.

This three component preparation can be used as a substitute for fertilizer.

The present invention provides a process for the preparation of a mixture of microorganisms for binding nitrogen from air, increasing the solubility of non-permeable phosphorus in the soil, and preparing the mixture. According to the invention, the bacterial strain Azotobacter croococcum CCM 4642 is propagated on a culture medium containing no organic or inorganic nitrogen by submerged fermentation at about 28 ° C for 22 hours, after which the Azobacter ferment is inoculated with a 10% 22 hour inoculum of Bacillus megatherium CCM. 4643. The microorganism of the genus Azobacter produces within 22 hours as much nitrogen into the fermentation medium as the microorganism of the genus Bacillus megatherium needs to grow. The double fermentation is continued for 22-24 hours, with aeration of 0.6 liters of air per liter of nutrient medium, at a speed of 120 rpm. and at 28 ° C. At the end of the culture, after 44-46 hours, the pH of the culture broth is in the range of 6.8-8.5 and an increase of 4-6 billion cells / ml. media of the strain Azotobacter croococcum and 150-200 million cells / ml Bacillus megatherium CCM strain.

Further, according to the invention, the bacterial strain Azospirillium brasiliense CCM 4644 is propagated on a culture medium containing no organic or inorganic nitrogen source by fermentation at 28 ° for 22 hours, after which the propagated culture of the microorganism Azospirillium brasiliense is inoculated with 10% of a 22 hour inoculum of Bacillus megatherium. 4643, while the bacterial strain Azospirillium brasiliense has been producing as much nitrogen into the nutrient medium as the Bacillus megatherium needs to grow. Double submerged fermentation is continued for 22-24 hours while aerating 1 liter of air per liter of fermentation medium at 120 rpm and at 28 ° C. At the end of the fermentation, the pH of the fermentation medium is 6.8-8.5 and the growth of the Azospirillium brasiliense strain is 4-6 billion cells / ml medium and the Bacillus megatherium strain 150-200 million cells / ml medium.

In the technical implementation of the invention, no by-product is formed. Biomass of microorganisms can be used as a ferment which is no longer used

-6nespracuváva. The water from the washing of the fermenters can reach the sewage system as it advantageously enriches the water of the rivers with rare species of bacteria.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

200 ml of a mixture 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, calcium carbonate 5.0 g, molasses 30.0 g, fermentable sugar 10.0 g per 1000 ml of nutrient broth and sterilized at 125 ° C for 40 minutes, then 1 ml of the trace element composition boric acid 5 is added, 0 g, ammonium molybdate 6.0 g, potassium iodide 0.5 g, sodium bromide 0.5 g, zinc sulfate 0.3 g, aluminum sulfate 0.3 g per 1000 ml of culture medium. To the contents of the lyophilized ampoule of both the bacterial strains of Azotobacter croococcum and Azospirillium brasiliense, 5 ml of sterilized water is added and a suspension is formed, 1.0 ml of the suspension is inoculated with both flasks. The inoculated flasks are placed on a horizontal shaker and cultured at 28 ° C and 160 rpm for 48-72 hours, until sufficient biomass growth occurs. A basic inoculum is thus prepared.

Next, 400 ml of the broth of the above composition is placed in a flask and inoculated with 3-4 ml of the basic inoculum and cultured on a horizontal shaker at 28 ° C for 48 hours. A preparative inoculum is thus obtained.

Next, 1000 liters of nutrient broth are sterilized in a fermenter at 125 ° C for 40 minutes, and after cooling, they are inoculated with 1.6 l of preparative inoculum. The inoculated soil is fermented for 22 hours at 28 ° C with aeration of 0.6 liters of air per liter of fermentation medium at 120 rpm. In this way, an inoculum of suitable consistency is obtained from both microorganisms. To prevent the formation of foam, an antifoam is added in an amount of 0.2 - 1.0 liters / m ³ . The pH of the broth is in the range 6.8 - 7.0.

Example 2

200 ml of potassium dihydrogen phosphate 0.3 g, calcium chloride 0.2 g, magnesium sulphate 0.3 g, molasses 30.0 g, coorn-steep 0.5 g, peptone 0.3 g , calcium carbonate 5.0 g and sterilized at 125 ° C for 40 minutes.

To the contents of two lyophilized ampoules of Bacillus megatherium strain, 5 ml of sterilized water is added to each ampoule and a suspension is formed, then 1.0 ml. suspensions were inoculated with both flasks. The inoculated flasks are placed on a horizontal shaker and cultured at 28 ° C for 48 hours at 160 rpm.

Next, 400 ml of the above nutrient broth are placed in a flask and inoculated with 1-2 ml of the basic inoculum and mixed on a horizontal shaker at 28 ° C for 24-48 hours, the microscopic examination to determine the vegetative state of the culture and the formation spores. This procedure yields a preparative inoculum.

Next, 1000 liters of nutrient broth are sterilized in a fermenter at 125 ° C for 40 minutes, inoculated with 3 pieces of 400 ml preparative inoculum after cooling. The inoculated soil is fermented for 22 hours at 28 ° C with aeration of 0.6 liters of air per liter of nutrient medium at 120 rpm. To prevent the formation of foam, an antifoam is added in an amount of 0.2 - 1.0 liters / m ³ . The growth of the culture is monitored microscopically so that it proceeds only to the vegetative state without the formation of spores. The pH of the broth is in the range 6.8 - 7.0.

Example 3

In two fermenters with a total content of 10-12 m ³ , a single payload volume of 5.0 - 6.0 m ³ , 4.5 and 4.5 m ³ of nitrogen-free broth described in Example 1 are sterilized and inoculated with 10 % of the inoculum containing the bacterial strain Azotobacter croococcum according to Example 2. It is then cultured at 28 ° C for 22 hours while aerating 0.6 liters of air per liter of culture medium, at a speed of 120 rpm. The fermented broth is then inoculated with a 10% culture of Bacillus megatherium according to Example 2, and the double fermentation is continued for 22-24 hours. After 44 46 hours of fermentation, the pH of the broth is 7.7-8.5 and the bacterial strain Azotobacter

-8roococcum achieved an increase of 4-8 billion cells / ml medium.

4.5 and 4.5 m ^{3 of} nitrogen-free broth described in Example 1 are introduced into two fermenters with a useful volume of 5.5-6.0 m ³ and inoculated with 10% operational inoculum containing the bacterial strain Azospirillium brasiliense according to Example 1, cultivation takes 22 hours at 28 ° C with aeration of 0.6 liters of air per liter of culture medium at 120 rpm. At the end of the fermentation, the pH of the fermentation medium is 7.5-8.5 and the Azospirillium brasiliense strain achieved an increase of 4-6 billion cells / ml medium and the Bacillus megatherium strain 150-200 million cells / ml medium.

Further mixing and homogenizing the two types of ferments yields a three-component final product of the composition: Azotobacter croococcum 2-4 billion cells / ml, Azospirillium brasiliense 2-4 billion cells / ml, Bacillus megatherium 300-400 million cells / ml medium, in expanded form. biomass is a total of 4.1 billion cells / ml to 8.2 billion cells / ml of nutrient medium and the pH is in the range

7.5 - 8.5.

Industrial Applicability

A mixture of microorganisms for binding nitrogen from air to increase the water solubility of phosphorus compounds for use in agriculture is prepared according to the process of the invention.

Claims (2)

PATENTOVÉNÁROKY Process for preparing a mixture of microorganisms for binding nitrogen from air, for increasing the solubility of phosphorus compounds, characterized in that the bacterial strains Azotobacter croococcum CCM 4642 and Azospirillium brasiliense CCM 4642 are multiplied by co-fermentation of at least two strains of microorganisms in a nitrogen-free culture medium. 4644 for 22 hours at 28 [deg.] C., then the bacterial strain Bacillus megatherium CCM 4643 is added thereto and then co-fermented for 44 to 46 hours at 28 [deg.] C. by aerating 0.6 liters of air per liter of culture medium. A composition comprising a mixture of microorganisms for binding nitrogen from air to enhance the solubility of phosphorus compounds, comprising two strains of microorganisms Azotobacter croococcum CCM 4642 at 2-4 billion cells / ml and Azospirillium brasiliense CCM 4644 at 2-4 billion cells / ml in nitrogen-free broth containing phosphorus compounds and Bacillus megatherium CCM 4643 strains at 300-400 million cells / ml, with a total biomass of 4.25 billion cells / ml to 8.4 billion cells / ml of the nutrient medium and the pH is in the range of 7.5-8.5.