CS273622B2 - Mixture for ice crystals' nuclei formation suitable as hail-storm protection - Google Patents

Abstract

The above composition provides ice crystal germs and thus leads to the freezing of droplets in super-cold clouds. It contains as the active ingredient the bacterial mass obtained from cultures of Pseudomonas syringae, Erwinia herbicola or Pseudomonas fluorescens or the mixtures or the cell membranes or super natant material of this cultures. The compositions prepared with liquid or solid carrier substances are injected by means of an aircraft or other method into the cloud layer where they result in the freezing of a large number of fine water particles even before a small number of large lumps of ice form in the absence of crystal germs.

Description

(57) (Cooking refers to an ice crystal seed composition to protect against hail. This mixture may initiate freezing of water droplets in supercooled clouds.

Obsahuje as active substance, it contains a bacterial mass obtained from cultures of Pseudomonas syringae, Erwinia herbicola or Pseudomonas fluorescens, or a mixture thereof, or cell membranes isolated or supernatant thereof. Mixtures prepared with liquid or solid carriers can be assisted by aircraft or otherwise conveyed to a cloud layer where many fine water particles are frozen before they can form large pieces of ice due to the small amount of crystal nuclei.

(13) B2 5 (51) Int. C 12 C 12 Cl. N 1/20 // Rj38

CS 273622 32

The invention relates to a composition suitable for protection against hail, which causes the formation of ice crystals and crystallization centers,

3e the well-known fact that hail causes considerable damage in economic life. Therefore, artificial weathering methods are used increasingly in which the hail protection is introduced! into the atmosphere of a certain inorganic substance. Silver iodide and lead iodide (DT-OS No. 25 38 861) are most commonly used for this purpose.

The clouds you are in! they are often in a colloidly unstable state in the atmosphere.

The water droplets forming the clouds are supercooled, find it! ee also in liquid state at temperatures below freezing.

In natural clouds it is generally observed that the droplets themselves at temperatures in the range! -10 to -15 ° C do not freeze yet. The basis for this is that the freezing process can only start at a certain level of internal energy; In other words; From the energy point of view, pure water droplets formed by condensation can freeze only considerably below freezing point.

The freezing process is facilitated by the presence of foreign particles. However, these particles are present in the atmosphere only at a concentration much lower than the concentration at which atmospheric water vapor condenses. The essence of microphysical influence lies directly in the fact that the missing natural nuclei of crystals are replaced by artificial means.

When few seed crystals are present, only few ice crystals are formed in critical areas of the clouds, and these cause hail.

The efficiency of man-made and cloud-embedded ice crystals is largely dependent on their so-called activity threshold temperature. This threshold temperature is the highest temperature at which it exists! the particles may still induce a phase change of the water droplets. The best inorganic substance for the formation of ice crystals is silver iodide, the temperature of which is -4.5 ° C. The lead temperature of lead iodide used in Hungary to prevent hail is approximately -7 ° C.

With the help of particles sprayed into the atmosphere, the damage caused by hail can be reduced, as these particles will increase the number of condensation germs, and therefore only small grains of non-hazardous ice are produced. These ice grains melt through the atmosphere during their fall and reach the earth only in the form of rain.

The freezing, i.e. the crystallizing properties of the most commonly used inorganic compounds is that the crystal lattice of these substances is largely similar to the crystal lattice of ice. The corresponding lattice constants of ice crystals and silver iodide crystals differ by about 1 to 2%. The difference between the lattice constants of ice and lead iodine is somewhat greater; nevertheless the lead temperature of lead iodide is also below

5 ° C. A considerable difference between the two compounds also lies in the solubility in water; the silver iodine permeability is 0. 10 g / 100 ml, the solubility of lead iodide is. 10 ⁴ g / 100 ml. Lead Oodld is used in Mačfersku in the amount of about 2000 kg per year.

This means an average contamination of lead iodide of 200 to 250 g / ha, since the compound gets into the soil with rainwater and is then incorporated into plant and animal organisms.

There are also environmental pollution problems associated with silver iodide. The utility of this compound is further limited by the fact that the substance degrades under the influence of light.

The use of inorganic compounds has the following: disadvantages;

- pollute the environment,

- their activity threshold temperature is very low and their full spectrum of activity decreases over a wide range of minus temperatures (the spectrum of silver iodide starts at -4.5 ° C and continues down to -15 and -20 ° C),

- the cost of production and the cost of use are too high.

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a crystalline substance for ice formation which is:

CS 273S22 B2 did not pollute the environment as it would be degraded in a short time by natural means and whose threshold temperature and activity spectrum would be around 0 ° C,

Previously, it has been found that certain organic substances are also suitable as artificial nucleation agents. Therefore, not only inorganic compounds, but also organic substances such as artificial crystal nuclei such as methaldehyde, floroglucin and the like have been experimented.

In the 1970s, it was found that at least a portion of the natural, nucleated crystals of the atmosphere, consist of organic substances of biological origin and come from decomposed vegetation and marine plankton.

In nature, microorganisms Pseudomonas syringae, Erwinia herbicola and Pseudomonas fluorescens occur, which cause frost damage to plants as the causative agent of the formation of ice germs / 0. Baceteriol, 154/1, 359-355 / 1985 //. Therefore, the investigations have been directed to reduce this effect by cloning (European Patent Application Publication No. 138,426).

It has now been found, and surprisingly, that bacterial mass obtained from cultures of Pseudomonas syringae, Erwinia herbicola and Pseudomonas fluorescens or mixtures thereof, or isolated cell membrane cultures or supernatant of these cultures, is excellently suitable as an active ingredient for ice crystal forming compositions.

Liquid compositions made from the active ingredients obtained by fermentation can be dispersed by spraying into the atmosphere, effectively containing solid preparations can be used as pyrotechnic mixtures.

Dispersion of the liquid compositions can be carried out on the ground or from an aircraft by means of a generator. Scattered on the ground is somewhat more expensive. Particles scattered into the atmosphere move in the vertical airflow that often occurs up to the plane of the clouds.

This method has the disadvantage that it can only be used under certain meteorological or geographic conditions. Helping the aircraft is considerably simpler, the particles can disperse in the appropriate concentration to the right place.

Solid formulations can be delivered to the desired sites of use using pyrotechnic cartridges applied from aircraft.

The composition according to the invention has the following advantages over known solutions:

- they do not pollute the environment but are degraded by microbiological degradation,

- are generally more potent than the known inorganic compounds used,

- their threshold temperature is about -3 ° C, the spectrum of activity is up to -10 ° C (silver iodide: -4,5 to -20 ° C, lead iodide -7,5 to -20 ° C),

the specific manufacturing cost of the composition is substantially lower than the known substances so far produced.

The microorganisms used in the sense of the invention are known to be deposited in the National Colleotion of Plant Pathogenic Bacteria, Herpanden, UK under the following numbers: Pseudomonas syringae pv. Syringe van Halí N.C.P.P.B. 2,507;

Erwinia herbicola / Lohnia / dye N.C.P.P.B. 2 281;

Pseudomonas fluorescens migula N.C.P.P.B. 1 598.

In order to produce the active ingredient of the composition according to the invention, Pseudomonas syringae, Erwinia herbicola, Pseudomonas fluorescens or mixtures of these bacteria are inoculated at 10-10 ° g / ml of nutrient solution which has been sterilized in a manner known per se. The culture is fermented for 24 to 36 hours at 20 to 30 ° C. Then the bacterial mass is separated from the excess liquid. The bacterial mass may be diluted with 50 to 100% by weight of a buffer solution having a concentration of 0.1 to 2% by weight, preferably a solution of dihydrogen phosphate pH 6.5 to 7, and then subjected to ultrasonic treatment. The disrupted cells are dehydrated, preferably using lyophilization. To disrupt cells, the bacterial mass can also be mixed with a glucose solution at a concentration of 10-80% by weight, and the active substance can then be separated by centrifugation or chromatography. Finally, the bacterial mass can also be sonicated and centrifuged at 20,000 to 40,000 g to separate disrupted cells. The cell membranes thus obtained are washed and then separated from the wash liquid.

The composition according to the invention contains a preferred bacterial mass in a concentration of 0.01 to 60% by weight and the remainder to 100% constitutes the required amount of liquid or solid carriers such as 0.1 to 1% (w / w) sodium chloride solution in water, silica , zeolite or cellulose acetate solution.

For culture of the bacteria, a nutrient medium of known composition or nutrient media with special additives may be used.

The invention is illustrated by the following examples.

Example 1

Two liters of nutrient solution (DXFCO Nutrient, pH = 7.1) is sterilized for 30 minutes for 3 minutes in an autoclave at 110 ° C and then inoculated under sterile conditions with 10 / ml Pseudomonas syringae. The culture is left on a shaker at 28 ° C for 35 hours.

After this time, the number of bacteria rises to ΙΟ 10 to 10 ^ / ml. Sterile air is passed through the culture to supply oxygen. The finished culture is then placed in an ice bath for 24 hours and centrifuged at 0 ° C for 20 minutes at 400 rpm. The separated bacterial mass (25 g / liter of culture) is taken up in 25 ml of 20 mM phosphate buffer (potassium dihydrogen phosphate, pH 7.0). Both the bacterial mass and the cell-free supernatant are used to produce the ice crystal seed composition of the invention.

Example 2

The procedure is as described in Example 1, except that the nutrient medium is inoculated with 10 ⁴ / ml Erwinia herbicola.

Example 3

The procedure is as described in Example 1, except that the nutrient medium is inoculated with Pseudomonas fluorescens.

Example 4

The bacterial mass produced by the procedure of Example 1 is sonicated / machine power *. 250 V //. The treatment time was 20 seconds, followed by a 20 second pause, and sonicated again for 20 seconds. This treatment is carried out a total of ten times. In doing so, the vessel is externally cooled with ice water, or? heating the material can be harmful to organic substances. The suspension containing the disrupted cells is poured into a round flask and is cooled in liquid air in a New Brunswik apparatus and lyophilized at +4 ° C and 2.6 kPa.

A dilution series ranging from 0.05 to 3 mg / liter is prepared from the lyophilisate with 10 mM phosphate buffer. The samples as well as 0.5 ml of buffer were added to the cells of an ependorf centrifuge. The cells are then cooled at a cooling rate of 5 ° C / hour from 0 ° C to -20 ° C. All members of the culture row of the culture of the invention freeze uniformly between -4.0 and -4.3 ° C. From this it can be concluded that the ultrasonic treated bacterial mass has an effect on the formation of crystals.

Example 5

The bacterial mass prepared according to Example 1 was diluted 1: 1 with a 2 molar glucose solution at room temperature and homogenized for 25 to 30 minutes. The solid substance is centrifuged and the centrifugate is applied to a column packed with DEA Sephadex A50 (a cross-linked polysaccharide which carries diethyl-2-hydroxy-propyl-aminoethyl groups as functional groups). Operate at room temperature. The centrifuge flows through the columns for 2 hours. The column is then washed with ten times the column volume

10 mM Tris-acetate buffer (tris- (hydroxymethyl) -aminomethaneacetate, pH 6.5). Finally, the active substance elutes from the column with 200 ml of 0.1 M and 1.0 M sodium chloride solution each, collecting 6 ml fractions. The dry matter content of all fractions was adjusted to 1 mg / ml. Thereafter, as described in Example 4 of the reconstitution series and prepared by freezing experiments as described in Example 4, the Oako control serves a sodium chloride solution whose concentration corresponds to the sodium chloride content of the test solutions. Test solutions freeze at temperatures ranging from -5.8 to -6.0 ° C, while controls at temperatures ranging from -9.8 to -12.3 ° C. The experiment carried out shows that the substance treated with the glucose solution helps to form crystals.

Example 6

The procedure is as described in Example 4, but with the bacterial mass produced according to Example 2. The results of the freezing procedures are identical to those described in Example 4.

Example 7

The procedure is as described in Example 5, but using the bacterial mass prepared according to Example 3. The results of the freezing experiments are identical to those described in Example 5.

Example Θ

Cell membranes are isolated from the bacterial mass obtained in Example 1 to determine whether cell membranes have an effect on crystal nucleation or not. The mass isolated from 3 liters of culture liquid is suspended in 80 ml of 10 nM phosphate buffer (pH 7.0) containing 0.14% by weight mercaptoathanol. Thereafter, the cells are sonicated for 30 minutes (instrument power: 250 w). After a 1 minute break, the sonic treatment is repeated for a total of five times. The excitement is about 70%. After disruption, the undigested cells are separated by centrifugation at 1500 g, the supernatant is centrifuged at 0 ° C for 30 minutes at 30,000 g. The pellet containing cell membranes is washed twice with the above phosphate buffer, then centrifuged and finally suspended in the buffer. After determining the dry matter content, a dilution series was prepared from the substance as described in Example 4. Each member of the series was frozen under the conditions described in Example 4 at a temperature in the range of -4.0 to -4.3 ° C. The 10 mM phosphate buffer used as a control freezes at -10.8 ° C

Example 9

The liquid supernatant obtained by centrifuging the bacterial mass prepared according to Example 1 is lyophilized as described herein. The white powder obtained is used in the preparation of the dilution series according to the procedure of Example 4. All the members of the rejection series are frozen at a temperature of -4.2 ° C. An oak control is used with the culture medium as well as the culture supernatant of Pseudomonas syringae ice (this manipulated Psudomonas causes no freezing damage); Controls are freezing at -10.2 ° C or -10.3 ° C. The experiment demonstrates that the supernatant also contains substances that form the seeds of ice crystals.

Example 10.

The culture supernatant obtained in Example 2 was diluted 3-fold with distilled water three times and then stirred at room temperature for 2 hours with DEAE Sephadex A 50 anion exchange resin. pH = 6.5 /. The column was eluted with 200 ml of 0.1 M and 1.0 M sodium chloride solution each, collecting 5 ml fractions. Test solutions freeze at temperatures ranging from -5.3 to -6 ° C, while the corresponding control solutions freeze. containing only sodium chloride, freeze at temperatures ranging from -9.8 to -12.3 ° C.

Example 11

Claims (3)

The wet bacterial mass obtained by the procedure of Example 1 is mixed with a sodium chloride solution to form a sprayable preparation. To this end, 0.1 kg of bacterial mass is mixed with 1 kg of 0.1% sodium chloride solution. EXAMPLE 12 0.5 kg of the bacterial mass obtained according to Example 2 are mixed with 0.1 kg of 0.1% sodium chloride solution in a mixer. To this mixture was added 0.5 kg of Aerosil (SiO 2), homogenized and finally dried at room temperature. The preparation may be dispersed into the atmosphere by a pyrotechnic route. EXAMPLE 13 The procedure described in Example 12 was followed, but using 0.7 kg of synthetic zeolite as solid filler. This preparation can also be dispersed into the atmosphere by pyrotechnic means. Example 14 The procedure described in Example 11 is carried out but the bacterial mass is mixed CLAIMS 1. An ice-crystal formation composition suitable for hail protection, based on ice-crystal-forming bacteria cultures, characterized in that it consists of a bacterial mass obtained from cultures of Pseudomonas syringae, Erwinia herbicola or Pseudomonas fluorescens, or mixtures thereof, or containing isolated cell membranes or supernatant of these cultures, 2. A composition according to claim 1 comprising a bacterial mass from cultures of Pseudomonas syringae pv. Syringe van Halí N, C, P, P, B, 2507, - Erwinia herbicola / Lohnis / dye N.C.P.P.B, 2281 or Pseudomonas Fluorescens migula N.C.P.P.B, 1598 or mixtures thereof, or cell membranes isolated from these cultures or supernatant from these cultures, 2 kg of an aqueous solution of 50% by weight of cellulose acetate. Example 15 From the bacterial mass isolated by the method of Example 1, solutions of concentrations of 0.1, 0.2 and 1.0 g / liter, respectively, are prepared with 0.1% sodium chloride solution. , 2 mm. The freezing properties of the droplets are studied in a diffusion chamber. In this chamber, the droplets are applied to surfaces cooled with peltier elements and treated with silicone grease. The surface temperature, which substantially corresponds to the droplet temperature, due to the considerable weight difference, is measured with a platinum resistance thermometer and read on a digital display with an accuracy of 0.1 ° C. The rate of cooling of the chamber and thus of the droplets can be controlled. In the temperature range important for the measurement, namely below 0 ° C, the cooling rate can be regulated in the range of 1 to 10 ° C 5 ° C / min » The temperature _t50 , i.e. the temperature at which half the droplets are frozen, is measured. This temperature is for three samples of different concentrations of -3.2 ° C, -3.6 ° C and -1.5 ° C. From a solution with a concentration of 0.1 g / l, droplets of 70 to 80 µm are formed. Their temperature t ^ ₀ is -4 ° C. Droplets of 3 to 4 mm diameter, formed from the same solution, have a temperature tgQ of -1.2 ° C, □ 0.1% sodium chloride solution in distilled water is used as a control g / l Half of droplets with a diameter of 4 mm formed from this solution, heat freezes at -12 ^D I C this process starts freezing at -10 ° C and quenched at -14 ° C. Thus, the compositions of the invention cause a melting point increase of about 10 ° C. Example 16 A solution of 0.1 g / liter is prepared from the bacterial mass prepared according to the procedure of Example 1 using 0.1% sodium chloride solution. A 0.1 g / liter dispersion is prepared from silver iodide and 0.1% sodium chloride solution. The freeze-thaw conditions are tested in the diffusion chamber of Example 15. The cooling rate is below 0 ° C in the temperature range. ° C / min. Droplets with an average size of 1.2 mm fall on the cooling surfaces. Temperatures _tg0 in this case were -3.2 ° C for the composition of the invention and -4.9 ° C for silver iodide. The experiment has shown that the threshold temperature of the composition of the invention lies closer to 0 ° C, that is, the supercooled composition suppresses P β 6 D H E T OF THE INVENTION 3. A composition according to claim 1, comprising from 0.01 to 60% by weight of bacterial mass, cell membranes or culture supernatant, and the remainder up to 100% by weight constituting the required amount of liquid or solid carriers, such as from 0.1 to 60%. 1% by weight, sodium chloride solution in water, or silica, zeolite or cellulose acetate solution.