SK25097A3 - Method for preparing a mixture of microorganisms for binding nitrogen from air, for improving the solubility of phosphorus compounds and for decomposition of food oil sediment - Google Patents

Abstract

The process consists in cultivating together at least two micro-organism strains in a nitrogen-free culture medium containing phosphorus compounds. The Azotobacter croococcum CCM 4642 and Azospirillum brasiliense CCM 4644 strains are cultivated for 22 hours at a temperature of 28 DEG C, then the Bacillus megatherium CCM 4643 and Pseudomonas putida CCM 4641 strains are added and cultivated for 44 to 46 hours at a temperature of 28 DEG C, while airing the culture medium with 0.6 litre air per litre of culture medium.

Description

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 for the bacterial decomposition of unprocessed sludge in the oil resulting from edible oil production. 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, especially nitrate, especially on infants and young children, since 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. Efforts to utilize nitrogen from air to a large extent in soil

-Agriculture in agricultural practice, especially in recent times.

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.

SUMMARY OF THE INVENTION

The aforementioned disadvantages are largely eliminated by the process for preparing the nitrogen-binding composition according to the invention, which is based on the use of certain species of bacteria of the genus Azotobacter which are capable of binding nitrogen from the air by diffusing them during their propagation in soil. synthesized forms of nitrogen into the soil such as nitrates, nitrites, ammonium, amino acids, small molecule proteins that bind to the organic matter of the soil from where their plant roots absorb. The 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.

Furthermore, 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.

-3 Inoculating the soil with nitrogen-binding microorganisms and simultaneously inoculating the soil with insoluble phosphorus decomposing microorganisms produces so-called metabiosis, where the nitrogen binders supply the necessary nitrogen and the Bacillus megatherium converts the insoluble phosphorus compounds into the water-soluble phosphorus species, for their own growth and multiplication, while supplying nitrogen and phosphorus to plants.

In the production of edible oils, especially at the end of production, oil sludge is formed which can no longer be cleaned; it is not suitable for use in the food industry or for other uses, e.g. in the automotive industry. This oil sludge cannot be disposed of freely in the environment, therefore neutralization and cleaning equipment is required for its disposal. Therefore, it is advantageous to use the described property of microorganisms of the strain Pseudomonas putida CCM 4641, which has been deposited in the authenticated Czech Collection of Microorganisms, Brno, Tvrdý no. 14 under the above-mentioned number. These microorganisms, by their action, decompose open-chain oil compounds into glycerin and organic acids.

The oil sludge is mixed with the fertile soil and inoculated with cultures of Azotobacter croococcum, Azospirillium brasiliense, Bacillus megatherium and then with Pseudomonas putida, mixed several times, and the oil sludge content is mineralized and decomposed in two to three months. When applied to arable land together with a large number of microbes, it is a nutrient with the characteristic properties of maize manure.

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, then turning the soil to the sowing depth, thereby inoculating the soil. the use of nitrogen and phosphorus fertilizers may be waived in part or in whole.

In addition, another Pseudomonas putida culture can be added to the above three microorganism cultures, and the mixture is used by adding 10 liters of a suitably diluted mixture of microorganisms per 10 tonnes of soil and mixing the soil several times. 500 liters of oil sludge can be decomposed by this method. The resulting product of the process of the invention is a nutritional composition rich in microorganisms binding

-4 nitrogen, decomposing phosphorus and oil sediment. This four-component preparation can be used as a substitute for fertilizer.

The present invention provides a process for producing a mixture of microorganisms for binding nitrogen from air, increasing the solubility of non-permeable phosphorus in soil, and mineralizing sludge from edible oil.

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 Pseudomonas putida CCM 4641. The microorganism of the genus Azobacter produces within 22 hours as much nitrogen into the fermentation medium as the microorganism of the genus Pseudomas 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 cultivation, after 44-46 hours, the pH of the culture medium is in the range of 7.5 8.5 and an increase of 4-6 billion cells / ml. medium of Azotobacter croococcum and 150-200 million cells / ml of Pseudomonas putida.

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

I 1

- 24 hours with aeration of 1 liter of air per liter of fermentation medium at 120 rpm and at 28 ° C. At the end of 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 200-300 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 that is no longer processed. The water from the washing of the fermenters can reach the sewage system as it enriches advantageously

-5water rivers by 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 medium 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, co-steep 0.5 g, peptone are placed in a flask.

-60.3 g, calcium carbonate 5.0 g and sterilized at 125 ° C for 40 minutes.

To the contents of the lyophilized ampoule of both bacterial strains of Bacillus megatherium and Pseudomonas putida is added 5 ml of sterilized water 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 the 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 Pseudomonas putida according to Example 2, and the double fermentation is continued for 22-24 hours. At the end of the 44-46 hour fermentation, the pH of the broth is 7.7-8.5 and the bacterial strain Azotobacter croococcum achieved an increase of 48 billion cells / ml medium and the Pseudomonas putida strain 200-300 million cells / ml medium.

-7 Into two fermenters with a useful volume of 5.5-6.0 m ³ , 4.5 and 4.5 m ^{3 of} nitrogen-free broth described in Example 1 are introduced 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 nutrient medium at 120 rpm. The fermentation medium is then inoculated with a 10% operational inoculum of a culture of Bacillus megatherium according to Example 1, and the double fermentation is continued for 22-24 hours. 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 200-300 million cells / ml medium.

Further mixing and homogenizing the two types of ferments yields a four-component final product of the composition: Azotobacter croococcum 2-4 billion cells / ml, Azospirillium brasiliense 2-4 billion cells / ml, Pseudomonas putida 150-200 million cells / ml and Bacillus megatherium 150-200 million cells / ml, with a total of 4.3 billion cells / ml to 8.4 billion cells / ml of nutrient medium in the propagated biomass and a pH in the range of 7.5-8.5.

Industrial usability

A mixture of microorganisms for binding nitrogen from air, for increasing the water solubility of phosphorus compounds, and for decomposing a food oil sediment for use in agriculture is prepared according to the method of the invention.

Claims (1)

PATENTOVÉNÁROKY Process for preparing a mixture of microorganisms for binding nitrogen from air, for increasing the solubility of phosphorus compounds and for decomposing food oil sediments, characterized in that bacterial Azotobacter strains are propagated by co-cultivation of at least two strains of microorganisms in a nitrogen-free culture medium. croococcum CCM 4642 and Azospirillium brasiliense CCM 4644 for 22 hours at 28 ° C, then bacterial, Bacillus megatherium (CCM 4643) and Pseudomonas putida (CCM 4641) strain are added thereto, and then cultured together for 44 to 46 hours at room temperature. 28 ° C with aeration of 0.6 liters of air per liter of broth.