SU615871A3 - Method of obtaining methan-oxidizing bacteria - Google Patents

Description

This invention relates to technical microbiology, namely to the issues of microbiological synthesis of proteins from methane, and concerns a method for producing methane-oxidizing bacteria capable of converting methane into dietary protein. There is a known method for producing methane-oxidizing bacteria, according to which soil samples are introduced in a liquid mineral nutrient medium containing dibasic potassium phosphate, magnesium sulphate in the amount of O, 2 g / l and traces of ferric chloride. The cultivation was carried out in the presence of a mixture of methane and air, taken in a 1: 1 ratio. The resulting culture is subcultured on a mineral agar medium, followed by cultivation and release of a bacterial culture of TOURA l. The aim of the invention is to simplify the process and obtain a bacterial culture with high activity. For this purpose, according to the proposed method, a soil sample is used, separated from the bottom sediment of the border biotope, and 1 ml of additional solution is added to the liquid mineral nutrient medium (per 1 liter) prepared by mixing 18 liters of distilled water with the following components (ggami, g: lithium chloride 0.5; copper water sulphate l.Oj semi-sodium zinc sulphate l.Oj boric acid 11.0} ammonium sulphate 1.0} hydrate chloris tin tin cerium sulfate nickel 1, O; four chelate; manganese chloride 7.0} hexahydrate cobalt nitrate 1.0} titanium oxide 1.0} potassium iodide 0.5 and potassium bromide 0.5. the cultivation process, the medium with a mixture of methane and air, in a ratio of 1: 1 (by volume). For the bottom sediment of the border biotope used, it is characteristic that there are no conditions for the metabolic process of the methane-oxidizing bacteria strain due to the lack of it; oxygen in the solution of mineral nutrients. For the preparation of a liquid mineral nutrient medium, two-mixed potassium phosphate, magnesium sulfate in an amount of 0.2 g / l and traces of ferric chloride, as well as 1 ml of a solution containing 18 p distilled water, g, are used per 1 liter, g: lithium chloride 0.5; Copper sulfate 1.0%, Zinc sulfate 1.0; Boric acid 11.0 | Ammonium sulfate 1.0; tin chloride hydrate O, 5i nickel sulphate 1.0 1.0 | four-water manganed chloride 7.0} cobalt hexahydrate 1.0 | titanium oxide 1.0, potassium iodide 0.5 and potassium bromide 0.5. The cultivated culture is sown (macaqs are transferred) to the nutrient medium-mineral agar and are subsequently cultivated on it. Then, single cells from the formed colonies are isolated and cultivated on a nutrient solution consisting of mineral substances in the presence of a mixture of methane and air in a 1: 1 volume draining. Subsequently, pure bacterial cultures are isolated. Example 1. To release methane of oxidizing bacteria, take 1 g of sample no. 4 from bottom sediment near the coast of Shenze, in the area of the Max Planck Institute of Limnology, and bring this sample into the liquid mineral nutrient medium described above. The resulting mixture is cultivated in a 200 ml manther at 27 ° C in a gaseous medium consisting of one side of methane and one volume part of air. After three days, dense bacterial overgrowth, detected by a microscope, occurred on a nutrient medium. The resulting culture was subcultured on a mineral-agar medium and for five days they continued the cultivation process in an environment of methane and air, taken in a precarious relationship, until microscopic colonies visible under the microscope were formed on the surface. From the resulting population of bacteria, single cells are isolated in droplets of the nutrient medium. They are treated with the indicated mixture of methane and air. After five days, pure cultures of bacteria (strain M 102) were formed from the single cells in the droplets of the single cell solution. They are isolated in a known manner. It is established that from 1 weight, h. methane was formed. 0,8-0,9 weight.h. bacterial culture, Example 2. In order to characterize the culture obtained in Example 1 | of the compounds listed in table. 1, substrates are prepared as 1% solutions in a mineral nutrient medium. The obtained substrate inoculated the test culture — a strain of methane-oxidizing bacteria. After 3.7 and 21 days from the start of the culture, the test was performed. The results are shown in table. At the same time designate signs; no use of substrate + weak use i very noticeable use.

Carbohydrates: Glucose

fructose galactose mannose ribose

±

arabinose

lactose

maltose

sugar for

starch

cellulose

Alcohols:

methanol

ethanol

propenol

butanol

isoamyl

benzene

Aldehydes:

formaldehyde

adaldehyde benzaldehyde Fatty acids: formic acid propionic acid

Amino Acids: Glycine

Cystine

Alannn Phenylalanine

everyday substrates: indole formation

Table continuation

±

+ +

+ +

+ f

test for methylrot catalase

okskeyra

Me Conker Brotf.

(bouillon)

litmus milk liquefied gelatin

From the data in the table of type UO, out of all the tested nutrient media, the fatty acids were significantly decomposed (were used) and a number of media were consumed weakly, in particular, from alcohol-ampho- methanol. This indicates the specificity of the test culture to only one type, the carbon source of methane.

Example 3. Preparing substrate solutions by adding organic compounds at a concentration of 1% to a liquid mineral nutrient medium of the specified type;

The test culture strain M-102 was seeded into the obtained substrate solution and cultured at pH 7, temperature in a fermenter upon shaking.

For comparison, the same culture is sown on a similar nutrient medium, but without organic additives, and the cultivation is carried out in the presence of a mixture of methane and air, taken in the ratio of i: l.

Samples are taken after 3.5 and 7 days. As a result, it was found that the subject ShTamm M-102 is specific for methane.

Example 4. The strain M-1O2 was tested for color samples. The results are shown below:

Sample (test) Coloring:

Gram Negative

Acidity Negative

controversial

Flagella Negative

capsules Negative

Continued table;

+ i + +

Thus, it can be concluded

that there are no acceptors in the test culture.

Example 5. The methane oxidation activity of the strains is determined. For this purpose, 1 l of a liquid mineral medium of the specified type is introduced into a 5 liter fermenter and the test culture is inoculated at 27 ° C in the presence of a mixture of methanol and air in a 1: 1 ratio by volume. The yield of the culture obtained after 5 days was 1.5 g, which, based on the methane consumed, is 0.8 weight parts. culture on 1 weight.h. methane.

EXAMPLE 6 The medium seeded according to Example 5 was cultured in a 5 liter fermenter with shaking under the presence of a mixture of natural gas and air in a 1: 1 ratio by volume.

The concentration of the culture obtained after 5 days was 1.5 g of dry matter.

The culture is separated from the culture medium and its composition is determined. It is established that by 70% it consists (on a dry basis) of protein, the rest is carbohydrates, lipids, and the constituent parts of the cell walls.

The yield of the culture in terms of methane was 0.8 weight.h. the dry mass of bacteria from 1 wt.h, natural gas. On breathing consumed 2O% gas. This corresponds to the highest yield ever achieved.

Claims (1)

Example 7. The experiment is similar to Example 6, but cultivation is carried out in a mixture of methane and air consisting of 59 vol. air and 41% by volume of methane. The mixture contained 11.8 o6.% Oxygen (i.e., the mixture was outside the limits of the vertical hazard). In this case, all other safety measures were provided for when working with gas in the room (supply of communications with safety valves for overpressure, removal of a fermentor from the main room to a special one, installation of control and signaling devices, etc.). During cultivation, methane and air are fed sequentially, in dotted intervals, depending on the yield (at a yield of 28 g / l of dry matter of biomass, the duration of the gas supply is 4 min., The exposure interval is 12 min. subjected to continuous dialysis, removing growth retarding products. Biomass yield was 28 g / l of njimating medium. The examples given confirm the efficiency of obtaining methane-oxidizing bacteria by the proposed method. Formula of the invention Method of obtaining meta-oxide bacteria, which includes the introduction of soil samples into a liquid mineral nutrient medium containing two-substituted potassium phosphate, magnesium sulphate in the amount of 0.2 g / l and traces of ferric chloride, cultivating it in a mixture of methane and air, taken in a 1: 1 ratio , reseeding the obtained culture on a mineral-agar medium with subsequent cultivation on this mixture and isolation of a bacterial culture, characterized in that, in order to simplify the process and to obtain a bacterial culture that has a high The soil extracted from the bottom sediment in the boundary biotope is used, in addition to the liquid mineral nutrient medium, additionally clearly from 1 liter of nutrient medium per liter of solution containing 18 l of distilled water from the following components, g: lithium chloride O , 5, anhydrous copper sulfate 1, Oh, zinc sulfate zinc sulfate 1.0, boric acid 11.0, ammonium sulfate 1.0, tin chloride hydrate O, 5, nickel sulfate water sulfate 1, Oh, four-water manganese chloride 7, 0, cobalt hexahydrate 1, O, titanium oxide 1, O, potassium iodide 0.5 and methyl bromide s O 5. Sources of information taken in Attention 1F expertise: 1. Microbiologists, vol. 38, no. 2, 1969, p. 251-257.