HU188434B - Process for preparing a bacterial substance for the inoculation of soil - Google Patents

Abstract

Treating prepns. contg. microorganisms are produced by propagating the Azotobacter chroococcum strain registered at the National Institute for Public Health under number OKI 00239 in a nitrogen-free liq. nutrient in submerged culture at around 28 deg.C for 24-80 hrs., concentrating the fermentation liquor or drying it into a powder, and adding to the concentrate or powder. (a) opt. vitamins, plant growth regulators and trace elements, and (b) opt. the fermentation liquor or dried powder from the fermentation under the above conditions of the Rhizobium strain OKI 00238.

Description

The present invention relates to a process for the preparation of a bacterial inoculum. The soil inoculum produced according to the invention contains a large amount of nitrogen bacteria, and its application improves the microbiological soil life, which has been sufficiently degraded over the last two to three decades, resulting in surprisingly high yields.

According to earlier findings, the spread of microorganisms in the soil was dependent on the temperature and the water content of the soil. This is no longer the case today. Due to the often excessive application of fertilizers and pesticides, the soil became nitrated and acidified. The chemicals and their metabolites accumulate in the soil and as a result the number of microorganisms present in the normal soil has decreased and their rate of occurrence has shifted. While 100 million bacteria per gram are active in normal soil, they have been reduced to a few thousand due to acidification, while the proportion of soil fungi and toxin production has increased, and many of them are phytopathogenic. The reduced bacterial life cannot replace the necessary ultramicro, micro and macro elements, so they will be missing from the crop.

However, bacterial counts are reduced not only by pesticides and acidification, but also by a lack of carbohydrate and cellulose in the soil. Since both the stalk and the straw are burnt to the feet, cellulose does not enter the soil; cellulose-degrading bacteria cannot hydrolyze sugars, but these are essential for the life of other bacteria.

Spontaneous improvements are not expected as, for example, the production and use of ammonium nitrate is financially encouraged.

The solution to the problem can only be expected from biological control. That is why attempts have been made to replace the microorganisms that are missing from the soil, so that cellulose-degrading bacteria and rhizobium are already used in agriculture.

One of the most important biogenic elements in life is nitrogen, which is present in the atmosphere in unlimited quantities. Nitrogen, however, is one of the most passive elements and can only be commercially manufactured in a usable form at high energy costs.

Bacteria are also capable of absorbing elemental nitrogen. 'Elements capable of binding elemental nitrogen can be divided into two groups, one of which is a free-living microorganism which binds nitrogen in the presence of oxygen (eg Azotobacter.) Or in the absence of oxygen (eg Clostridium species). The second group consists of microorganisms which, independently, but together with the host plant, under the symbiosis of its host plant, carry out their vital functions, including nitrogen sequestration. These include rhizobium species living in the tubers of leguminous plants and also symbiont blue-green algae species. ”(István Pais,“ The role of micronutrients in agriculture ”, agricultural publishing, Budapest 1980, p. 126). Azotobacteria, when bound to nitrogen in the presence of oxygen, synthesize useful and valuable organic materials. They grow well even at high nitrate concentrations (0.1%) '

and using 1 gmannite carbon source to fix 16-18 mg of nitrogen.

Nitrogen fixation is an enzymatic process, the details of which are still unclear. Nitrogen trapping by enzymes can only be a distant goal of industrial biology; maybe it will come to fruition sometime, but by then you can't help the ground anymore.

According to the state of the art, only mass cultivation of microorganisms and their introduction into soil can be the basis of biological nitrogen fixation.

Among the nitrogen-binding microorganisms, Rhizobiumoka is already cultivated in Hungary and introduced into the soil as inoculants. Although this was a step towards biological protection, the field of application of this vaccine is, unfortunately, narrow, because on the one hand, Rhizobium is only a modest number of soil bacteria and, on the other hand, it is expected to produce butterflies.

The other large group capable of absorbing nitrogen, Azotobacter, is not bound to a single plant family and should therefore be primarily experimented with. There were also attempts in this direction, but they failed; in the form of the vaccine, the bacteria introduced into the soil did not reproduce properly and had no effect. In the absence of tangible results, experts have finally ruled out the need for nitrogen-producing bacteria ("Azotobacter inoculations ... if our soils are well maintained by appropriate agrotechnical procedures, do not need to be populated with a small amount of inoculant relative to their mass." Soil Biology ”, Akadémiai Kiadó, Budapest 1954, p. 1126).

Despite these limitations and prejudices, it was an object of the present invention to provide a bacterial inoculum that binds nitrogen to the soil and thereby significantly improves crop yields. A further object of the invention is to restore the soil pH to the soil and to promote soil regeneration with appropriate nutrients.

In our experiments with Azotobacterekke 'we discovered the cause of the ineffectiveness of earlier Azotobacter vaccines: the Azotobacter vaccines used so far contained only cells, biomass isolated from the culture broth. In most cases, these cells could not reproduce in the soil without nutrients. This led us to the idea that a much better inoculum could be obtained by not isolating the biomass from the culture broth at the end of the fermentation, but concentrating the culture broth as it is, with its salts, organic compounds, metabolites, or drying it to a powder. The substances in the broth concentrate thus provide the bacteria with the necessary living conditions.

We have also experimented with Rhizobium and have come to the real surprise: Rhizobium - when fermented in sterile, nitrogen-free medium to maintain cell growth, proliferation, build new cell bodies, is forced to fix the nitrogen in the air and synthesize proteins, as it can only receive nitrogen. With that, that other one fell

There are 188,434 accepted views that Rhizobium can only fix nitrogen in the air in the root tubers of butterfly plants. This greatly increased the applicability of Rhizobium.

Obtainable according to the invention therefore Azotobactereket soil inoculum ⁵ and - depending on the intended use - include Rhizobiumokat optionally also together with other additives suitable for nutrition and optionally biologically active.

According to the invention of the National Institute of Public health management ¹⁰ Collection of Microorganisms National No. 00238. strain Azotobacter chroococcum deposited under no. The Rhizobium strain deposited under the name of. ¹⁵

The present invention therefore relates to a process for the preparation of a bacterial soil inoculum. Further feature of the invention that are cultured at about ²⁰ to 28 ° C for 24-80 hours recessed OKI 00239 Azotobacter chroococcum marked strain of nitrogen-free liquid culture medium, and then concentrated or dried to a powder and this concentrated or powder of culture broth

- where appropriate, vitamins, growth-regulating substances and trace elements, and ²⁵

- optionally adding culture broth or concentrate or drying powder thereof of the Rhizobium strain OKI 00238 fermented under the above conditions.

The cultivation is preferably carried out in ³⁰ large volume edények-. The activity of the fermentation broth is measured by determining the hormones and the nitrogen content of the medium, which is initially nitrogen free. At the end of the fermentation, the cell number is about 2.4 to 2.8 billion / ml. The broth szá- about ³⁵ razanyaga. It contains 20% protein, which is also an indicator of the bacterial effectiveness.

The culture broth obtained after completion of the fermentation is evaporated or dried to a powder. Evaporation in vacuo to temperatures of about 35 ° C is carried out, preliminary ⁴⁰ preferably 1/5 of its original volume - the culture broth is concentrated to 1/10. Drying is carried out at low temperature to prevent the microorganisms from being damaged. In case of spray drying, the inlet temperature is preferably 50-60 ° C, an exit temperature ^{of 45} 130-140 'C. If a vortex dryer is used, the drying temperature is preferably about 35 ° C.

Biologically active substances such as vitamin B, gibberellin, heteroauxin may be added to the resulting vaccine, although Azotobacter itself synthesizes such substances (e.g., 3-6 µg / ml heteroauxin and 10-20 pg / ml during 40-50 hours culture). ml of gibberellin). The vaccine is removed and stored in a cool place protected from light. 55

The following examples further illustrate the process of the present invention. The composition of the media used in the examples is as follows:

az

Káliumdihidrofoszfát 0.3 g Foszkál 0.3 g Citric acid 0.2 g magnesium Sulfate 0.3 g potassium Sulphate 0.2 g NaCI 0.4 g

Vas II chlorides 0.05 g Calcium carbonate (precipitated) 6.0 g Sacharose 20, C g Trace Element Solution 1.0 ml Nycnelemoldat: Boric acid 2.0 g cobalt sulfate 0.2 g ammonium molybdate 5.0 g potassium iodide 0.5 g zinc Sulphate 0.2 g aluminum Sulphate 0.3 g Sodium umbromids 0.5 g AZ II Káliumdihidrofoszfát 0.2 g Foszkál 0.3 g Citric acid 0.2 g magnesium Sulfate 0.3 g potassium Sulphate 0.2 g NaCI 0.4 g Vas (II) Chlorides 0.05 g Calcium carbonate (precipitated) 7.0 g Molasses 40, Cg Phylamalt 10.0 g Trace element solution (as 1.0 ml AZ-I) AZ III Components of the medium as 20, C g

AZ-I but Agar agar gives 1000 ml tap water, pH self-leveling, 6.5 ad 1000 ml tap water ad 1000 ml tap water pH self-adjusting, 6.5 ad 1000 ml medium

/. example

Propagation of Azotobacter chroococcum

500 ml Erlenmeyer flasks containing 100-100 ml of AZ-I medium were inoculated with the strain maintained in the lyophilized state. The medium was previously sterilized in an autoclave at 120 ° C for 30 minutes. The flasks were shaken for 48 hours at 28 ° C on a flat shaker at 300 min ^-1 . The prepared fermentation broths are suitable for inoculation, spreading and lyophilization.

Example 2

Inoculation of Azotobacter chroococcum

AZ-ΠΙ medium was used for plating, and its composition corresponds to AZ-I, except that it contains 2% agar to solidify the medium. The liquified medium was filled into Petri dishes and sterilized as in Example 1. The frozen medium was inoculated and placed in a 28 'C thermostat. After 48 hours, colonies were evaluated. Separate the appropriate colonies and inoculate another shake flask.

Example 3

100 L of AZ-I medium were sterilized at 120 ° C for 45 minutes and then inoculated with the inoculum prepared in Example 2 using a 0.5% v / v seed culture. The culture broth is aerated with 1 L / L air and incubated at 28 ° C for 48 hours.

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Example 4

Preparation of inoculum

2000 liters of AZ-II medium was sterilized at 120 ° C for 45 minutes and then inoculated with 100 liters of the vaccine prepared in Example 3. The culture broth is incubated with air at 1 liter / liter for 1 minute at 28 ° C for 36 hours.

Example 5

Producing fermentation

000 L of AZ-II medium was sterilized at 120 ° C for 45 minutes. The medium cooled to 28 ° C was inoculated with the inoculum prepared according to Example 4. The pH (as in the previous examples) is about 6.5 and self-adjusting and does not change during fermentation. The culture was aerated with 1 liter / liter air per minute. The progress of the fermentation and the suitability of the culture broth are checked by microscopic cell counting, total nitrogen determination by Kjeldal and determination of biological activity by Brian's method (P. W. Brian, H.G. Hemming, Natura 189, 758/1961 /). The number of cells at 60 hours is 2.4-2.8 billion / ml.

The culture medium is concentrated to 1/7 of its volume under reduced pressure at 35 ° C to reduce its volume. The concentrate has a bacterial count of about 12 billion / ml, a protein content of 2.08-2.1 and a nitrogen content of 0.332-0.336%.

Example 6

Cultivation of Rhizobium

Rhizobium is depicted in Figures 1-5. is cultured in an analogous manner to Example 1A in nitrogen-free medium. For the first fermentation, AZ-I medium was used, which was transferred to AZ-II medium after 48 hours. The efficiency of the incubation is examined after 40-48 hours. If a large number of mosaic-like cells are observed in the fermentation broth, the germ number is ΙΟ'θ-ΙΟ ¹¹ .

The resulting Rhizobium culture broth is mixed with 10-20% of the Azotobacter preparation and the mixture is homogenized.

The soil grafts prepared and packaged as described above are concentrates. When used, the concentrated concentrate is used at a 100-fold dilution and the powder formulation at a 1000-fold dilution. Diluted soil graft should be applied to the seedbed when sowing. Usually 10 liters of the concentrate and 1.5 kg of the powder composition are calculated per hectare.

"Bacterial fertilizer" accelerates the development of plants, yields several weeks earlier, yields and crude protein levels increase significantly. Due to its protein content, the soil inoculum enriches the soil with proteins and buffers its pH to a near neutral value. Not only the nitrogenase enzymes are responsible for the various beneficial effects, but also the biostimulants, vitamin B-1 content and buffer capacity of the soil inoculum.

The soil inoculum produced according to the invention does not contain harmful or toxic substances which cannot be mixed with dressing agents and other chemicals. It is used for the rapid decontamination of our highly chemized environment, as it can reduce the use of nitrogen fertilizers.

The following experiments are presented to illustrate the effect of the soil inoculum.

I. Treatment of Lupine with Azotobacter Concentrate

The concentrate prepared in Example 5 was applied to the seed bed at 100-fold dilution and then seeded. The effect of the drug on the plants was already visible at 6-8 weeks of age; the experimental boards showed a stronger improvement compared to the control. The numerical results are shown in the table below:

Treatment Plant- height cm 1 stem average weight ounces Parcellatermés kg ° / /She control 80,50 0.99 0.89 100 handled 98.00 1.14 1.20 135

II. Treatment of sunflower with Azotobacter concentrate

The experiment was carried out on the secondary sunflower cultures of Rákóczi MgTSz in Solti, as already described for lupine. At the beginning of flowering, the amount of macro and microelements taken up by the plants was examined. The following result was obtained:

N SHE/ ' She P g / kg K g / kg ca g / kg mg g / kg handled 3.9 4.6 52 22.4 7.4 untreated 3.7 4.0 25 22.0 6.8

The high potassium uptake is striking.

The yield and intestinal content of the fruit is illustrated by the following formula: handled control yield q / ha 26.99 21.62 dry matter content,% 92 92 crude protein,% 21.5 19.1 raw fat,% 38.3 35.8

Crude protein and fat data refer to the dry matter of the crop. The additional yield was 5.37 q / ha.

The present invention has the following main advantages:

- its application produces significant additional yields,

- it enables the use of fertilizers to be reduced, thereby reducing pollution of the environment,

- having regard to the fact that it contains substances necessary for the growth of soil microbes, it provides an opportunity for soil regeneration,

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- the inoculum buffers the soil pH in a neutral direction,

- shortens cultivation cycles to ensure that the crops are ripe.

Claims (4)

1. A method of soil bacterial vaccine preparation, characterized in that the culturing OKI 00239 marked Azo- ^one recessed tobacter strain chroococcum nitrogen-free liquid culture medium at about 28 ° C for 24-80 hours, and then concentrated or dried to a powder and the pulp and the culture broth and to powder ¹ vitamins, growth regulators and trace elements, where appropriate, and - optionally adding culture medium or a concentrate thereof or a powder obtained by drying of Rhizobium strain OKI 00238 fermented under the above conditions. The method of claim 1, wherein the culture broth of Azotobacter is concentrated under reduced pressure to a volume of 1/5 to 1/10 of its volume. 3. A process according to claim 1 or 2, characterized in that in the preparation of a combined seed stock containing Azotobacter and Rhizobium, the concentrates of the two broths are mixed in a ratio of 10: 1 to 5: 1. 4. A method of increasing agricultural yields and improving the soil, characterized in that the method of claim 1-4. The soil slurry concentrate or powder produced according to any one of claims 1 to 4 is diluted with water before sowing into the seedbed.