HU192370B - Composition suitable for protection against hail and comprising ice crystal forming substance - 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

FIELD OF THE INVENTION The present invention relates to a composition comprising an ice crystallizing agent produced by fermentation which is useful for controlling hail.

It is a well-known fact that hail causes significant damage to economic life.

As a result, the method of introducing inorganic materials into the air as a means of controlling the weather artificially is increasingly used.

Silver iodide and lead iodide are most commonly used for this purpose.

Dyen's procedure is described in Federal Patent Application Laid-Open No. 2,538,861.

Very often, clouds in the air are colloidally unstable.

This means that the water droplets that make up the cloud are "overcooled", that is, they remain liquid (overcooled) even at temperatures below 0 ° C.

It is generally observed in natural clouds that the droplets do not freeze even at temperatures below -10 ° C to -15 ° C. The reason for this is that the start of the freezing process is provided only by a certain level of internal energy; in other words, the freezing of pure cloud droplets formed by condensation, for energetic reasons, occurs only well below 0 °.

However, the process of freezing is facilitated by the presence of foreign particles. However, these particles are present in the atmosphere at orders of magnitude lower than those at which the condensation of atmospheric water vapor occurs. The essence of microphysical interventions is to artificially replace the missing, natural frost nuclei, which, due to their rarity, lead to the appearance of only a few ice crystals in the critical regions of the clouds, thereby causing the formation of ice particles.

The effectiveness of artificially generated and cloud-deposited ice-forming seeds depends to a great extent on their so-called. activity threshold temperature. This is the highest temperature at which a given particle can initiate a phase change in water droplets. The best inorganic ice-forming core is silver iodide with an activity temperature of 4.5 ° C. The threshold temperature of lead iodide used in the hail removal in Hungary is close to -7 ° C.

Inorganic compound particles introduced into the atmosphere are capable of preventing harmful hail by increasing the number of ice-forming cores and thus producing only small, harmless ice particles. These small particles then melt through the air layer and fall to the soil surface in the form of rain.

The most frequently used inorganic reagents, their particles have a "freezing", i.e. crystallizing properties, due to their high degree of similarity to the crystal lattice of ice. The difference between the corresponding lattice constants between ice and silver iodide is only 1-2%. The difference between the lead iodide and the ice lattice constants is slightly greater: Accordingly, the activity threshold temperature of the latter is already around -7 ° C. The essential difference between the two reagents is the degree of solubility in water:

silver iodide 8.10 · g and lead iodide 4.10 ⁴ grams / 100 cm ³

About 500 to 1000 kg of this reagent is used annually in Hungary, and even today around 2000 kg. This amount represents an average impurity of lead iodide of 200-250 grams per hectare after this substance returns to the soil with rainwater and is absorbed into plant and animal organisms.

The use of silver iodide has also raised environmental issues and inhibits its applicability by light degradation.

Common disadvantages of using inorganic reagents include:

- They pollute the environment.

- Ice crystal-forming activity threshold temperature is low and their total activity spectrum in the minus temperature range is wide band.

(The activity spectrum of silver iodide starts at -4.5 ° C and ends at -15, -20 ° C.)

- The cost of producing and applying the materials is high.

It is our aim to provide an ice crystal nucleating agent which does not pollute the environment because it is naturally degraded in a short period of time, and which also has a threshold temperature of 0 ° C and even its full activity spectrum.

It has already been recognized that certain organic materials are also suitable as artificial nucleating agents.

(French Patent Publication No. 2 484 774).

Therefore, in addition to inorganic materials, there has been a long history of attempts to use various organic compounds as artificial ice cores. Dyen materials eg. metaldehyde, dimethyl sulfoxide and urea.

It was also discovered in the seventies that at least some of the natural atmospheric ice crystal nuclei are organic material of biological origin, which is derived from degradable vegetation, marine plankton.

Naturally occurring bacteria, Pseudomonas Syringae, Erwinia herbicola and Pseudomonas fluorescens, and as ice nucleation agents, cause frost damage to plants. (J. Bacteriology 1985.164 / 1) pp. 359-66.)

Because these bacteria have hitherto acted as micro-organisms that have been harmful in nature, researchers have sought to reduce or prevent this effect by cloning. (European Patent Publication No. 138,426.)

It has been found that when Pseudomonsa syringae, Erwinia herbicola and Pseudomonas fluorescens, or mixtures thereof, are cultured under culture conditions, the resulting bacterial mass, or the digestate, can be used as an active ingredient in an ice crystal nucleation composition which can be used in the air to combat hail.

The liquid composition of the active ingredient obtained by fermentation can be sprayed into the atmosphere and the solid composition containing the active ingredient may be introduced into the atmosphere as a pyrotechnic mixture.

Dispersion of the liquid composition in the air can be accomplished by generators placed on the ground or in airplanes. The former solution involves less costly particulates released into the atmosphere by frequent vertical flows.

192,370 reach the cloud levels. The disadvantage of this method is that it can only be used in certain weather and geographical conditions. However, with the help of airplane generators, material particles are significantly lighter than required. administered at the appropriate concentration.

The solid formulation can be delivered to the desired airspace using airborne pyrotechnic cartridges.

An advantage of the composition of the invention over the prior art is that

- it does not pollute the environment and is microbiologically degradable on the soil surface,

- outperforms known inorganic materials,

- an activity threshold temperature starting at -3 ° C and an activity spectrum up to -10 ° C - lead iodide at -7,5 ° C, silver iodide starting at -4 ° C and -15 ° C - ^an opposite end in 20 C activity spectrum,

- the unit cost of producing the composition is much lower, art? of known materials.

The microorganisms used in the present invention are known; They shall be deposited and numbered as follows:

Pseudomonas syringae pv. syringae Van Hall N.C.P.P.

B. (G.B.) 2,507; Erwinia herbicola (Lohnis) dye N.C. P.P.B. 2,281; Pseudomonas fluorescens migraine N.C.P.P.B. 1598 (National Cellection Plant Pathogenic Bacteria)

Active ingredient composition of this invention are prepared by the known method are inoculated with 10 to 10 ⁶ Pseudomonas syringae, Erwinia herbicola and Pseudomonas fluorescens bacterium or mixture of bacteria in this medium was sterilized, and then the inoculated culture medium at 20-30 ° C for 24-36 hours shake and then separate the cell-containing mass from the supernatant.

Optionally, the bacterial mass thus obtained is diluted with 50 to 100% by weight of a 0.1 to 2% by weight buffer solution, preferably pH 6.5 to 7, preferably with alkali dihydrogen phosphate, and ultrasonically disrupted and preferably disrupted cells. Dried by lyophilization or by adding 10 to 80% by weight glucose solution to the mass and separating the digested bacterial mass by centrifugation or chromatography.

Alternatively, the bacterial mass may be sonicated and then centrifuged at 20,000 to 40,000 g to isolate intact cells and cell membranes. The cell membrane thus obtained is washed and then separated from the washing fluid.

The composition according to the invention comprises from 0.001 to 95% by weight of the active ingredient and 100% by weight of a liquid carrier, preferably 0.1 to 1% by weight of an alkali chloride solution or 0.1 to 2% by weight of an alkaline hydrogen phosphate solution, or a solid support, preferably a diatomaceous earth, silica gel or zeolite or a polymer, preferably cellulose acetate or sodium polyacrylate.

Bacteria can be grown using media of known composition or media containing special additives.

The following examples illustrate the composition of the invention and the process for preparing the active ingredient.

Example 1 liter of medium dextrose monohydrate for 30 minutes at 110 ° C in an autoclave sterilized, and then inoculated with No. 10 ³ Pseudomonas syringae bacteria under sterile conditions.

The mixture was then shaken in a water bath for 24-36 hours at 28 ° C. Then the number of bacteria increases to 10 * ⁶ -10 ^{2 3} .

Oxygen supply is provided by supplying sterile air. The resulting culture was placed in a 0 ° C (ice) bath for 24 hours. Cells were separated from the medium at 0 ° C by centrifugation at 400 rpm for 20 minutes.

The sedimented bacterial mass (25 g / l of medium) is taken up in 25 cm ^{3 of} 20 M ¹⁰ phosphate buffer (KH ₂ PO ₄ ; ph 7.0) at ⁶ kg.

Both the bacterium mass and the bacterial-free supernatant are used to prepare an ice crystal nucleating composition.

Example 2

In each case, the same procedure was followed as in Example 1, except that 10 * Erwinia herbicola bacteria were inoculated into the culture medium, not Pseudomonas syringae.

Example 3

All proceed as in Example 1 with the exception that 10 µl of Pseudomonas fluorescens bacteria were inoculated into the medium, not Pseudomonas syringae.

Example 4

The bacterial mass of Example 1 was subjected to ultrasonic cell lysis. The power of the ultrasound device used was 250 W, the treatment lasted for 20 seconds, which was repeated ten times with 20 second breaks.

Heat the solution (which may cause damage to the organic matter) by preventing the solution from warming up with an outside ice bath. The suspension containing the fully digested cells is then poured into a round-bottom flask and frozen in liquid air, lyophilized at + 4 ° C under a vacuum of 2.6 kPa on a New Brunswick apparatus.

The lyophilizate is prepared a dilution series from 0.05 mg / cm ³ range, applied solvent 10 M.10 phosphate in ⁶ kg (KH ₂ PO _4). Weigh 0.5 cm ^{3 of} the samples as well as the buffer into Eperdorf centrifuge tubes. The tubes are then cooled from 0 ° C to -20 ° C using a temperature control system at a cooling rate of 5 ° C / h. It was determined that all members of the dilution series were uniformly frozen between -4.0 and 4.3 c, i.e., the ultrasound bacterial mass produced by fermentation had a crystalline nucleating property.

Example 5

The bacterial mass prepared in Example 1 was mixed with 2M glucose in a 1: 1 ratio at room temperature for 25-30 minutes. The mass was then centrifuged and the supernatant was DEAE Sephadex A 50 ion-31

192,370 replace it with a column filled with resin. Sephadex is a crosslinked polysaccharide containing a diethyl-2-hydroxypropylaminoethyl functional group. The flow is at room temperature and takes 2 hours. Subsequently, the column was subjected to a 10-fold column volume of 10 M 10 * ⁶ kg of TRIS acetate [(tris-hydroxymethyl) aminomethane acetate; pH 6.5] wash buffer. The active ingredient was cleaved from the column with 200-200 cm 3 of 0.1-1.0 M NaCl, collecting 6 cm ³ fractions.

The active substance content of each fraction was adjusted to 1 mg / cm ³ , and a dilution series of Example 4 was prepared therefrom and a freezing experiment was performed as described in Example 4. As a control, a NaCl solution was used at a concentration equal to the NaCl concentration of the solutions of the invention. The solutions of the invention are frozen to -5.8, -6.0 ° C temperature range, while the corresponding controls between -9.8 and -12.3 ^V C temperature. This experiment proves that the material obtained as a result of glucose shock has a crystalline nucleating property.

Example 6

Example 4 is repeated but the bacterial mass of Example 2 is used as starting material.

The result of the freezing experiment is the same as in Example 4.

Example 7

The supernatant obtained after centrifugation of the bacterial mass prepared in Example 1 was lyophilized as described in Example 2. The final white powder was measured and diluted! line was prepared as described in Example 4. The dilution! All members of rows 2 to 4 were frozen at 4.2 ° C.

As a control, culture medium and a culture of Pseudomonas syringae ice lyophilized to sit ₁ _ úszóját used.

The former -l0,2 ^e C, while the latter is frozen at -103'C. The experiment shows that the supernatant contains an ice crystal nucleating agent.

Example 10

The supernatant of the bacterial mass obtained in Example 2 was twice diluted with three times distilled water and then mixed with a DEAE Sephadex A 50 anion exchanger at room temperature for 2 hours with a magnetic stirrer. The adsorbed material is then loaded onto a chromatography column and the column is washed with 10 volumes of 10 M 10 * kg TRIS acetate pH 6.5 buffer. The fractionation was carried out with 200-200 ml of 0.1-1.0 M NaCl solution, collecting 6 cm ³ fractions. The active substance content of each fraction was measured and the fractions were subjected to a freezing experiment. The fractions of the invention were frozen between -5.3 and -6 ° C. The freezing of the corresponding control solutions containing only NaCl was between -9.8 and -12.3 ^e C.

rál rn;

stem down:

m

Á zé se ra zc zi a

k <

0

Ci b:

power:

5i z

e;

Example 5 is repeated, but starting from 30 bacterial masses of Example 3.

The result of the freezing experiment is the same as in Example 5.

Example 8

From the bacterial mass produced in Example 1, the cell membrane of the bacterium is isolated to determine if it has a crystalline nucleating effect.

The mass separated from the ³ L culture was resuspended in 80 cm ^{3 of} 10 M 10 * kg phosphate buffer (KH ₃ PO ₄ ; ph 7.0) in the presence of 0.14% mercaptoethanol.

The cells were then subjected to ultrasound treatment for 30 seconds in 15 repetitions, with a 1 minute interval between treatments. The power of the device is 25 W.

The excavation was 70% efficient. Then, the unbroken cells are sedimented by centrifugation at 1500 g and the supernatant centrifuged at 30,000 g for ^an 0 C for 40 minutes. The cell membrane pellet was washed twice with the above phosphate buffer and centrifuged. Finally, the pellet washed in this way is suspended in phosphate buffer, the active ingredient content is determined, and the dilution series described in Example 4 is prepared. Dilution under the freezing conditions described in Example 4! Each of the rows was frozen at -40.0 to -4.3'C as opposed to 10 M.10 ** kg phosphate buffer used as a control. which froze at -10.8 ° C.

Example 11

A sprayable preparation of the wet bacterial mass of Example 1 was prepared. In a stirrer, 0.01 - kg of the bacterial mass of Example 1, separated from the supernatant, was mixed with 1 kg of 0.1% NaCl.

The liquid mixture is used for spraying.

Example 12

In a mixer, 0.5 kg of the bacterial mass of Example 2 is mixed with 0.1 kg of 0.1% NaCl solution and then 0.5 kg of Aerosil (SiO ₂ ) is added. The mixture was homogenized and dried at room temperature.

Pyrotechnically applied composition,

Example 13

All proceed as in Example 12, but using 0.7 kg of synthetic zeolite instead of 03 kg SiO3. After homogenization and drying at room temperature, a pyrotechnically applicable composition is obtained.

Example 14

All in all. The procedure is as in Example 1, but the bacterial mass is mixed with 2 kg of a 50% aqueous solution of cellulose acetate.

Example 15

Example 9

From the bacterial mass prepared in Example 1, 0.1 with 60% NaCl solution 0.1; 03 and 1.0 g / l concentrate-41

Prepare a solution of 192.370 rations and form droplets with a mean diameter of $ 1 mm.

Their freezing properties are studied in a diffusion chamber.

The drops in the chamber are placed on a surface cooled with Peltier elements and coated with silicone grease. The surface temperature, which is essentially the same as the droplet temperature due to the significant difference in weight, is measured with a platinum resistance thermometer. Read the temperature on the digital display to one decimal place. The rate of cooling of the chamber and thus of the droplets can be controlled. The cooling rate ranges from 1-3 degrees / min. Below 0 ° C for us.

Droplets of the above size are placed on the cooling surface. The droplets had a 50% freezing point (the temperature at which half of the droplets were frozen).

From a solution of 0.1 g / l, droplets with a diameter of 70 to 80 are formed. Its 50% freezing point is 4 ° C. Then 0.1 g / l forming droplets having an average diameter of 3-4 mm kocentrációjú solution thereof in 50% by freezing hőmérséldete -l, 2 ^e C

As a control, 4 mm diameter droplets were formed from distilled water containing 0.1 g / l / NaC. Their freezing started at -lOv and ended at -14 ° C. The 50% freezing point was -12 ° C.

It can be seen that the composition of the invention produces a freeze point rise of about 10 ° C in distilled water.

Example 16

From the bacterial mass obtained in Example 10, a 0.1 g / l solution is prepared in 0.1% NaCl solution. A dispersion of silver iodide and 0.1 wt% NaCl was prepared at a concentration of 0.1 g / l. Their freezing properties are investigated in the diffusion chamber described in Example 15.

The cooling rate used is 2 degrees / min in the range below 0 ° C.

Drops with an average diameter of 1.2 mm were placed on the cooling surface. The droplets have a 50% freezing point of -3.2 ° C for minΊ0 W containing the composition of the invention. For the silver iodide sample, this temperature is -4.9 ° C.

It thus appears that the activity temperature of the present composition is capable of forming kristálygócokat proximal higher, i.e., the temperature 0 C ^this temperature and can thus megakadá15 lyozni system subcooling successfully.

Claims (2)

CLAIMS 1. A composition comprising an ice crystalline active agent for controlling hail, characterized in that the cell mass obtained by fermentation of Pseudomonas syringea, Erwinia herbicola or Pseudomonas fluorescens in an amount of from 0.001 to 95% by weight is determined by cellular cell lysis by physical or chemical means. the membrane or the supernatant obtained by fermentation and the liquid support in an amount of 100% by weight, preferably 0.1 to 1% by weight of an alkali metal chloride solution or 0.1 to 2% by weight of an alkali metal dihydrogen phosphate solution or solid support, preferably silica, silica gel or zeolite or polymer, Preferably, it contains cellulose acetate. Composition according to claim 1, characterized in that it contains 0.1% by weight of a sodium chloride solution as a liquid carrier.