UA102337U - COMPOSITION OF NUTRITIONAL ENVIRONMENT FOR THE PRODUCTION OF SURFACE-ACTIVE SUBSTANCES BY OXOXIDATING MICRO-ORGANISMS - Google Patents

Abstract

The composition of the nutrient medium for the production of surfactants by oil-oxidizing microorganisms consists of mineral and organic substances, and as mineral components used, (g / l of distilled water): KHRO1,5; NaHPO3.0; NaCl-5.0; NHCl-1.0; and as an organic component glucose in the amount of 2.0 g / l.

Description

The useful model belongs to microbiology, industrial biotechnology and is intended for the production by non-pathogenic microorganisms with high oil-oxidizing capacity of microbial surfactants (surfactants), which can be used for ecologically safe cleaning of the environment from chronic oil pollution, as the basis of biodegradable detergents and bioemulsifiers in the pharmaceutical industry, in agriculture, to create new environmentally friendly technologies in the oil industry, etc.

Biological surfactants (bio-surfactants) are no less effective than synthetic analogues, as they not only have a wide spectrum of functional activity, but also have a number of advantages, such as non-toxicity, stability of physicochemical properties in a wide range of temperatures, pH values of the environment and its salinity The most important advantage of biosurfactants is biodegradability, which makes them quite promising for the creation of environmentally safe technologies related to cleaning the environment from oil pollution. Methods of elimination of chronic oil pollution, based on the decomposition of oil products by microorganisms, are recognized as one of the most safe and effective. Bacterial degradation is limited by the hydrophobic nature of hydrocarbons and their insolubility in water. An effective solution to this problem is possible in the case of production of bio-surfactants by oil-oxidizing bacteria. They are capable of dispersing petroleum products or hydrophobizing cell surfaces, which increases the efficiency of bacterial contact with hydrocarbons and at the same time accelerates the process of oxidation of petroleum products.

Representatives of the genus are known among a wide range of microorganisms-producers of PAS

Rzeidotopav, namely: R. ayegidiposa, which form biosurfactants on a nutrient medium in the presence of growth substrates - liquid or solid paraffins IarayeI-Mamzhdotsa A.M.

Snagasiegiganop oi Ptatpoiiryi roodiseyi Bu Rzeidotopakh aegidiposis isoiaie B520 / A.M. Arabic-

Mamzhdotsa, M.M. Aboshmaga, M.A.N. Nazzoshpa // Arri. Viospet. Vioyesppoi - 2009. - Mine. 157. - R. 329-345). Rhamnolipids synthesized by P. aegidipoza belong to effective biosurfactants, which are used to remove hydrophobic substances from contaminated soils.

K.5.M. Vaitap Rgodisiip, sNagasiyegigayop apa arriisayop oi Biozypasiapi / Ranapati K.5.M.

Waptap, Edmjaga SakKre // Vioyespppoiodu. - 2008. - Mine. 7 (2). - R. 360-370.|. Although bacteria of the genus P. aegidipoza produce surfactants with low values of surface tension (30-32 mN/m),

With intense emulsifying activity, a low value of the critical concentration of micelles (5-65 mg/l) and a high affinity for hydrophobic organic molecules

IViozinastapy Rhodysiyup ru Rzeytopav from 5 years. іїот 50іЙ Ovipa M/neu az Satop 5о0ігсе / |Ргауеезн

V.M., Zopiuatbu A.V., Magiarrap S. ei ai. // Meme MoiK Zsiepse Choshgpai. - 2011. - Mine. 4(4). - R. 99-1034, but they belong to pathogenic microorganisms (Cygneum bacillus), and therefore they cannot be used in the processes of cleaning the environment from oil pollution.

The search for oil-oxidizing microorganisms that are not pathogenic and capable of producing biosurfactants in a specially selected nutrient environment remains an urgent task of ecobiotechnology.

In many works, various variations were considered with the selection of components for the preparation of an optimal nutrient medium either only for the purpose of isolating biosurfactants in their pure form, or for the purpose of preparing a surface-active biological preparation based on it, composed only of a monoculture, which has an oil-oxidizing effect relative to certain petroleum hydrocarbons (Patent of Ukraine for an invention Mo 71792А dated 15.12.2004, Byul. Mo 12, 2004;

OBbauogi 0.5. Oedgadayop oi Nuagosagrop5 apa Biozypnasiapi rgodisiup Bu Rzeydotopaz zr. vigaip

I RI / O.5. Obrauotgii, M.O Pogi, 5.A Aderizoue, S.O. Oueihiro., A.E. Otoiauo, O.O. Atypa // MMopa

Most common Mistobiiodu apa Viotesppoiodu. - 2009. - Mine. 25, I5!ce 9. - R. 1615-1623. |.

The closest analogue to the useful model in terms of the obtained result, which is achieved, is the Patent of Ukraine for the invention Mo 10467А dated 12.25.1996, Bull. Mo 4/1996 (prototype), in which the Rzepdotopav5 5r RB-17 strain cultivated in a nutrient medium is used to obtain environmentally safe extracellular surfactants (g/l of distilled water): MaMOz - 3.0; K"HRO:xxZNgO - 2.0; KNeRO" - 1.5; Ma5Oxx7NgO - 0.5;

MazSeN'O"; - 5.0; HebOghH2O - 0.0005; SasSi" - 0.05; carbon source - 20.0. Cultivation is carried out in aerated conditions (200 rpm) on a nutrient medium of the above composition with a pH of 6.8-7.2 at a temperature of 28-30"C. The following are used as a source of carbon nutrition: ethanol, glycerin, glucose, sunflower oil, petroleum. During cultivation (3-5 days), the value of the surface tension of bacterial suspensions decreased from "73 mJ/m: (for distilled water) to 30.0 mJ/m7 for the culture liquid (the resulting product). The output of extracellular rhamnolipid bio - surfactant was 2.3-9.5 g/l, biopolymer (polysaccharide of alginate 60 nature) - 4.0-6.0 g/l.

The main disadvantage of the prototype is: the complexity of the technological process of obtaining bacterial suspensions with improved surface-active properties, i.e., the production of bio-surfactant takes place under aeration conditions (cultivated on a circular rocker for 3-5 days). When a significant amount of organic substances, for example, alcohols, are introduced into an aqueous solution, the surface tension of the solutions themselves, even in the absence of microorganisms, significantly decreases (to 28-35 mJ/mg) - this correction is not taken into account.

The task of a useful model is to select the composition of the nutrient medium for the production of extracellular surface-active substances by oil-oxidizing microorganisms under the condition of a small amount of mineral components and consumption of the organic component, which will allow to reduce the influence of the nutrient medium in the absence of microorganisms (control) on the revealed improved surface-active properties of bacterial suspensions with taking into account the amendment to control when simplifying the technology (without additional aeration) of bio-surfactant production.

The task is solved by the composition of the nutrient medium for the production of surface-active substances by oil-oxidizing microorganisms, consisting of mineral and organic substances, which differs in that the mineral components are used (g/l of distilled water): KNeRoOx - 1.5; MagNRO" - 3.0; Mass - 5.0; MNASI- 1.0; and as an organic component: glucose in the amount of 2.0 g/l.

Cultivation of microorganisms is carried out under aerobic conditions in nutrient medium Mo 1 (PS-Y), containing (g/l): КНгРОх - 1.5; MagNRO:» - 3; Mass - 5; MNASI - 1; glucose - 2; peptone - 10; yeast extract - 5; and in nutrient medium Mo 2 (PS-II) containing (g/l): KNegROx - 1.5;

MagNRO: - 3; Mass - 5; MNASI - 1; glucose - 2; with a pH of 7.0-7.2 at a temperature of 28-30 "C. So, glucose is used as carbon nutrition, nitrogen sources are ammonium salt and peptone, carbon and nitrogen sources are yeast extract (nutrient medium Mo 1 with peptone and yeast extract - PS-YI, with an enriched nitrogen content compared to carbon) or ammonium salt (Mo 2 nutrient medium without peptone and yeast extract - PS-YI, with a low nitrogen content compared to carbon). Cultivation time - 3-5 days.

The ability of oil-oxidizing microorganisms to produce biosurfactants is judged by indirect signs - by the decrease in surface tension (07, mJ/m7) of liquid cultures of bacteria relative to distilled water (c(NgO), mJ/m") and nutrient medium in the absence of

From microorganisms (c(PS-Y) and c(PS-II), mJ/m") and by the emulsifying ability of biosurfactants. In order to determine the type of biosurfactants - extracellular or cell-bound, bacterial cells are separated by centrifugation (6500 rpm /min for 20 min) and measure the surface tension of the supernatants.

The value of the surface tension (b, mJ/m-) is measured by the method of detachment of the plate from the surface of the liquid (Wilhelm's method) with an accuracy of x 0.5 mJ/m2 at a temperature of 25 76.

Research on the ability of bacterial cultures to produce bio-surfactant in a specially selected nutrient medium was conducted at the Biotechnological Scientific and Educational Center of the Odessa National University named after I.Y. Mechnikova.

As research objects, non-pathogenic strains of bacteria of the genus Rzendotopav were used, which were previously isolated and selected, and are currently kept in the collection of microorganisms of the Department of Microbiology, Virology and Biotechnology of the Odessa National University named after I. Mechnikov: R. Piogezsepe OMYOZ28, R. taporniya OMIZ29 , R. serasia

OMYZ27. The strains of R. PMogezsepe OMIZ328 and R. tayorniiia OMIZ29 were isolated from the marine environment, and R. serasiaa OMOZ3Z27 - from soil contaminated with petroleum products. Previous laboratory studies have shown | Oil-oxidizing activity of some strains of bacteria of the genus Rzeidotopaz / Gudzenko T.V. etc. // Microbiology and biotechnology. - 2013. - Mo 4. - P. 72-80, that the strains listed above have a fairly high oil-oxidizing capacity relative to crude oil, which is significantly enhanced when using a consortium of these strains R. PIogezsepv5

OMYiIZ28, R. tapuronia OMOZ29, R. serasia OMOZ327, taken in a volume ratio of 1:1:1.

The results of the tensiometric study of surface-active properties of liquid cultures of bacteria (KB) of the genus Rzeidotopaz and their supernatants (C) are presented in the table.

Table ) o(PO-1) - o", zd ||| ly ra ve | ooo

Strain OMyZ28 OoMmyZ29 "ser i ) extract (PS HER o(PO-II) - o, zdo 001 am levi) hoyu luzo) va o o o(NgO) 25-72.0 mJ/mg; K(NgO) - 0 ,2028

As can be seen from the data in the table, when studying the surface properties of bacterial cultures and their supernatants, it is necessary to take into account the correction for the nutrient medium due to the absence of microorganisms - in the presence of a significant amount of organic substances, the surface tension of their aqueous solutions also decreases: o(PS-I) - 51.2 mJ /me; o(PS-II) - 67.9 mJ/m-.

Bacteria of the genus Rzeidotopah produce mainly extracellular bio-surfactants, which is evidenced by the improved surface-active properties of their supernatants obtained after separation of bacterial cells by centrifugation. The emulsifying properties of the supernatants, assessed by the emulsification index (E24, 95) relative to sunflower oil, are high (60 Ob).

The use of a specially selected nutrient medium PS-II for non-pathogenic bacteria of the genus Rhaenydotopav allows to reduce the number of chemical reagents from eight according to the prototype to five, to simplify the technology of obtaining microbial surfactants (the stage of additional aeration during cultivation, which lasts 5 days according to the prototype, is excluded). In the proposed useful model, compared to the prototype, the correction for the nutrient medium due to the absence of microorganisms is taken into account. The effectiveness of the use of bio-surfactants produced by oil-oxidizing microorganisms (R. Yanogezsepe OMIZ28, R. serasia OMOZ27, R. tapornia

OMIZ329) for intensification of biotechnological purification (at 75-90 95) of water and soil from petroleum products.

Claims (1)

USEFUL MODEL FORMULA The composition of the nutrient medium for the production of surface-active substances by oil-oxidizing microorganisms, consisting of mineral and organic substances, which differs in that the following mineral components are used (g/l of distilled water): KNeRoOx - 1.5; MagNRO:x - 3.0; Mass - 5.0; MNASI - 1.0; and as an organic component - glucose in the amount of 2.0 g/l.