RU2692629C1 - Bacterial strain pseudomonas azotoformans for bioconversion of hydrocarbons from oil-contaminated and oil-product-contaminated water to a biodiesel source - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology. Strain of bacteria Pseudomonas azotoformans 1Y-2014, capable of biodegradation of solid paraffins and polycyclic aromatic compounds in water, liquid slimes contaminated with oil and oil products with simultaneous transformation of hydrocarbon xenobiotics into biomass of lipid metabolites of biodiesel sources is deposited in IBPM RAS under registration number VKM B-3254D.EFFECT: bacterial strain Pseudomonas azotoformans VKM B-3254D increases degree of purification of soil and water, liquid sludge from oil and oil products under conditions of low temperatures.1 cl, 4 tbl, 2 ex, 2 dwg

Description

The strain of bacteria Pseudomonas azotoformans for the bioconversion of hydrocarbons from polluted with oil and oil products in the source of biodiesel

The invention relates to the field of biotechnology and relates to obtaining a new strain of bacteria Pseudomonas azotoformans VKM B-3254D, designed to enhance the biodegradation of solid paraffins and polycyclic aromatic compounds in soil, water, liquid sludge contaminated with oil and oil products at low temperatures and for the transformation of hydrocarbon xenobiotics in biomass of lipid metabolites used as a source of biofuel - biodiesel.

A known strain of Pseudomonas putida 36, which is capable of biodegradation of oil and oil products [Dyadechko VN On the biological reclamation of oil-contaminated sandy soils of the Middle Ob / V.N. Dyadechko, L.E. Tolstokorova, S.N. Gashev, M.N. Gasheva, A.V. Soromotin, E.B. Zhdanova // Soil Science. - 1990. - № 9. - p. 148-151].

A known strain of Bacillus species 739 [USSR Author's Certificate No. 1743019, 1989] and the bacterial preparation Bacispecin derived from it [RF Patent No. 2 077 397, publ. 1997], used for the reclamation of soils polluted by oil and oil products.

A known method of purification of water and soil from oil, petroleum products and polymer additives in the drilling fluid, including the introduction of microorganisms, characterized in that the microorganisms use the association of bacterial strains Rhodococcus erythropolis BKM Ac-1339D, Bacillus subtilis BKM B-1742D and Pseudomonas putida B-17KD RF patent №2 093 478, publ. 10/20/1997].

Known consortium of microorganisms-destructors: Alcaligenes sp. G04, Microclimate 9, used for the purification of natural and waste waters, soils and soil grounds from oil, oil products and residual oil contamination [RF Patent No., 2 376 084, publ. 12/20/2009].

Known consortium of strains of microorganisms Acinetobacter sp. VKM B-2753D and Ochrobactrum sp. VKM B-2754D, possessing nitrogenase activity. The consortium is capable of fixing atmospheric nitrogen and has a high utilizing capacity with respect to oil and oil products with their high content in the substrate [RF Patent № 2 553 540, publ. 06/20/2015].

A known method of processing oil sludge [RF Patent №2 198 747, publ. 2000], which consists in the fact that the oil sludge is mixed with microorganisms and biostimulant. To do this, clean soil is added to oil sludge and sawdust in a mass ratio of 1: 2: 1; the bacterial strain Bacillus sp. Is used as a microorganism. VNIISHM 132, and as a biostimulant - protein feed additive "Biotrin", after the incubation period of microorganisms (at least 50 days), additional tillage with biostimulator is carried out.

There is a method of neutralization of soils from petroleum products [RF Patent №2 075 447, publ. 1997], in which the collection of contaminated soil, the separation and processing of sludge cultured biological strains of aerobic and anaerobic microorganisms.

The main declared abilities of strains and consortia of microorganisms, in the submitted patents, are the purification of soil, sewage, soil and sludge collector from oil products with the loss of the energy resource (without the release of secondary raw materials).

The closest to the claimed technical essence is the microbiological method of increasing the content of light and oil fractions in oil, oil products and other hydrocarbon raw materials with simultaneous isomerization of the gasoline fraction [RF Patent № 2 405 825, 10.12.2010]. The proposed invention allows to improve the quality of the oil by increasing the content of light and oil hydrocarbon fractions, reducing the content of dark fractions, fuel oil and coke, which in turn provides an improvement in the quality and performance characteristics of the resulting oil fractions.

Thus, in these patents the stated properties of known strains and consortia of microorganisms — the transformation of hydrocarbon xenobiotics into biomass of lipid metabolites was not found.

 The objective of the invention is to obtain a strain of Pseudomonas azotoformans, providing activation of the biodegradation of solid paraffins and polycyclic aromatic compounds in the soil, water, liquid sludge contaminated with oil and oil products with simultaneous transformation of hydrocarbon xenobiotics to biomass of lipid metabolites used as a source of biofuel - biology bioprocessors used as biofuels - bio biological xenobiotics used as sources of biofuel

This is the technical result.

To achieve a technical result, the accumulative culture of Pseudomonas azotoformans on the medium of the following composition was obtained from oil-polluted soils of the Usinsky district of the Komi Republic: per 1000 ml of water: 30 g, NaNO ₃ - 3.0 g; KH ₂ PO ₄ - 1.0 g; KCl - 0.5 g; MgSO ₄ × 5H ₂ O — 0.5 g; 15-25 ° С, 3-5 days under conditions of liquid phase fermentation.

The strain is characterized by the following morphological, cultural and physiological characteristics:

Gram-negative straight moving sticks with polar flagella.

Colonies on meat-peptone agar, Chapek-Doksa and Ashby mediums are small, round-shaped with smooth edges, convex, translucent,

On the IPA, the colonies are yellowish, on Čapek-Doksa and Ashby are whitish with a yellowish tinge.

The consistency of the mucous.

It releases a diffusing fluorescent yellow-green pigment on the environment for 3-4 days, on the Absha medium - on the seventh.

Aerobe, a facultative anaerob, is capable of denitrification.

Lactose-negative, does not grow at 37 ° C. The catalase test is positive.

The preferred culture medium is meat-peptone agar.

The data of molecular genetic analysis:

The level of sequence similarity of the fragment of the 16S rRNA gene of strain 1Y-2014 with the sequences of the closest types of strains

The level of sequence similarity of the fragment of the gyrB gene and the protein sequence of strain 1Y-2014 with the sequences of the closest types of strains

In the classification of microorganisms by pathogenicity groups, the Sanitary and Epidemiological Rules SP 1.3.2322-08 dated May 1, 2008, “Safety of work with microorganisms of the III – IV groups of pathogenicity (danger) and pathogens of parasitic diseases”, this species (genus) does not appear.

The storage mode of the strain - long-term storage in a lyophilized form in tightly sealed glass ampoules. Short-term storage (for the preparation of biomass with targeted use) - periodic reseeding - 1 time in 2 months with the storage of grown pure culture on oblique agar medium of the following composition: per 1000 ml of water: sucrose - 30 g, NaNO ₃ - 3.0 g; KH ₂ PO ₄ - 1.0 g; KCl - 0.5 g; MgSO ₄ × 5H ₂ O — 0.5 g; microbiological agar - 20.0 g; in closed tubes in a refrigerator at a temperature not higher than +6 and not lower than + 1 ° С.

The oil-oxidizing ability of the strain Pseudomonas azotoformans VKM B-3254D and the ability of the strain to utilize oil products in lipid metabolites was studied in laboratory experiments with a model oil-polluted medium. The results are shown in the examples below.

Example 1. The strain Pseudomonas azotoformans VKM B-3254D was tested for changes in the content of petroleum products and PAHs under conditions of liquid-phase cultivation on апapek’s medium enriched with oil.

In flasks with a capacity of 250 ml, non-agarized Czapek medium was prepared without sucrose, of the following composition: per 1000 ml of water - NaNO ₃ - 3.0 g; KH ₂ PO ₄ - 1.0 g; KCl - 0.5 g; MgSO ₄ × 5H ₂ O - 0.5 g, sterilized. Added oil of 0.5 g and 5 ml of accumulated strain. Cultured for 7 days at + 23 ° C and at + 6 ° C, 220 rpm. As a control, the content of petroleum products, PAH, in the environment of čapek with the addition of oil without biomass of the strain was determined.

After seven days, the oil was removed from the fermentation flasks by extraction with hexane and the hydrocarbon fractions were analyzed. The separation of the extracts was performed by the method of column chromatography. On the column with aluminum oxide, the fraction of n-alkanes and polycyclic aromatic compounds (fraction F1) was separated from the polar compounds. Next, on a column of silica gel, the fraction F1 was divided into fractions of n-alkanes (F2) and polycyclic aromatic compounds (F3) [D. Saturated hydrocarbons in background and polluted soils of the Pre-Urals / D.N. Gabov, V.A. Beznosikov, B.M. Kondratenok, I.V. Gruzdev // Soil Science. - 2010. - № 10. - P. 1–7].

The basis for determining PAHs in soil is the M 03-04-2002 method — measuring the mass fraction of benzo [a] pyrene in soil samples, bottom sediments and solid waste by high performance liquid chromatography using the Fluorat-02 analyzer as a fluorimetric detector [ Gabov D.N. Patterns of formation of polycyclic aromatic hydrocarbons in the soils of the Northern and Middle Taiga / D.N. Gabov, V.A. Beznosikov, B.M. Kondratenok, E.V. Yakovlev // Soil Science. - 2008. - № 11. - p. 1334–1343].

A visual inspection of the flasks before and after cultivation of the Pseudomonas azotoformans VKM B-3254D strain in the oil-polluted biomass at a temperature of + 6 ° C recorded emulsification of petroleum hydrocarbons (as evidenced by a color change) compared to the flask in which the oil-polluted medium was not treated with a strain (Control ) (Fig.1, 2).

In Fig. 1. The beginning of the experiment: a) Control, b) A variant with the addition of biomass of the strain Pseudomonas azotoformans VKM B-3254D.

In Fig. 2. Experiment after 7 days: a) Control, b) Option with the addition of biomass of the strain Pseudomonas azotoformans VKM B-3254D.

The cleaning efficiency was confirmed by a decrease in the concentration of petroleum products in samples after exposure for 7 days both at room temperature and at a temperature of + 6 ° C (Table 1).

Table 1. Changes in the concentration of petroleum products (mg) in samples after exposure for 7 days

Experience option Mass of oil products in the sample after exposure at room temperature, mg The mass of oil in the sample after exposure at a temperature of + 6 ° C, mg Control 300.0 182.0 Strain Pseudomonas azotoformans VKM B-3254D 195.0 172.0

The introduction of the culture of the test strain after seven days of incubation under normal conditions showed a significant decrease in the amount of 3-4-nuclear PAHs, whereas there were no statistically significant changes in the amount of 5-6-nuclear PAHs (Table 2).

Table 2. The number of substances PAH (µmol) in the experience in normal conditions for 7 days

PAH Control Strain Pseudomonas azotoformans VKM B-3254D fluoren 4992 1166 phenanthrene 644 240 anthracene 10.67 3.78 pyrene 106.44 15.04 benzo [a] anthracene 19,12 3.07 benz [b] fluoranthene 29.96 10.83 benzo [a] pyrene 2.74 3.11 dibenz [a, h] anthracene 4.78 12.09 benzo [ghi] perylene 1.70 0.00 PAH amount 5811.97 1454.96

As a result of incubation under conditions of + 6 ° C, preferential decomposition of phenanthrene was noted, insignificant accumulation of benz [b] fluoranthene and benz [ghi] perylene, as well as elimination from the benzo [a] pyrene system, which has high carcinogenic activity. No other statistically significant changes in the content of polyarenes were noted (Table 3).

Table 3. The amount of substance PAH (µmol) in the experiment for 7 days when exposed at a temperature of + 6 °

PAH Control Strain Pseudomonas azotoformans VKM B-3254D fluoren 72 72 phenanthrene 239 61 anthracene 5.06 7.47 pyrene 16.44 14.85 benzo [a] anthracene 6.71 8.55 benz [b] fluoranthene ten nineteen benzo [a] pyrene 6.98 0.00 dibenz [a, h] anthracene 0.00 0.00 benzo [ghi] perylene 7.90 15.11 PAH amount 365,10 198.31

Example 2. The strain Pseudomonas azotoformans VKM B-3254D was tested for changes in the fatty acid composition under cultivation conditions in the апapek medium, enriched with oil. In 250 ml flasks, non-agarized Czapek medium was prepared without sucrose, of the following composition: per 1000 ml of water - NaNO ₃ - 3.0 g; KH ₂ PO ₄ - 1.0 g; KCl - 0.5 g; MgSO ₄ × 5H ₂ O - 0.5 g, sterilized. Added oil of 0.5 g and 5 ml of accumulated strain. Cultured for 7 days in an incubator at 24 degrees and at 220 rpm. As a control, the content of fatty acids in the accumulated biomass of the strain Pseudomonas azotoformans VKM B-3254D on the апapek medium without added oil was determined, as well as the content of fatty acids in the апapek medium with the addition of oil only (Table 4). Fatty acids were isolated by the method of Sinyak and co-authors [Copyright certificate № 542932 USSR, G 01 N 1/28, 1977, Byul. No. 2.].

Table 4. Mass fraction of fatty acids for complex 1U – 2014,%

Acids Wednesday + strain Pseudomonas azotoformans VKM B-3254D Wednesday + oil + strain Pseudomonas azotoformans VKM B-3254D Control (oil) From 12: 0 0.0 2.6 6.4 From 14: 0 0.0 10.3 one- From 16: 0 75.0 52.6 48.2 From 16: 1 4.2 0.0 - From 18: 0 16.7 19.2 8.2 C 18: 1 (11) 0.0 3.8 1.8 From 18: 2 cis 4.2 11.5 16.4

Name of acids: C12: 00 - lauric; C14: 0 - myristic; C16: 0 - palmitic; C16: 1 - palmitoleic; C18: 0 - stearin; C18: 1 (11) –Weight; C18: 2 - linoleic;

± ∆ is the confidence interval of the absolute error at P = 0.95

The fatty acid composition of the lipids of the Pseudomonas azotoformans VKM B-3254D strain was carried out using high-performance liquid chromatography (Smartline, Knauer, Germany) and gas chromatography (Kristall 2000 M gas chromatograph with flame ionization detector. The identification of lipids was carried out by chromatography-mass method). spectrometry using a Finnigon Trase DSQ Thermo-Electron instrument (USA). These modern methods of analysis provide qualitative and quantitative determination of fatty acids even at low concentrations of limiting components.

In the absence of petroleum products, the main synthesis of the fat metabolites of the complex (strain Pseudomonas azotoformans VKM B-3254D) falls on saturated fatty acids: C16: 00-palmitic (75%) and C18: 00 stearic (16.7%); limiting acids are unsaturated C16: 1 - palmitoleic and C18: 02 linoleic, the mass fraction of which was 4.2%.

With the utilization of petroleum products, the metabolism of the complex changes (1Y 2014). The synthesis of new acids, such as C12: 00 - lauric, C14: 00 - myristic and C18: 01 - oleic, begins. The synthesis of linolenic acid C18: 02 has doubled.

The synthesis of lauric and myristic acids is associated with the need for bacteria to emulsify petroleum products, as a result of which an increase in the surface of bacteria’s contact with oil and an improvement in its utilization occurs. Lauric acid has antimicrobial and antibacterial properties, thus, microorganisms sterilize the environment in which they are located.

Biofuel - biodiesel consists of a mixture of fatty acid esters [Atadashi, I.M. High quality biodiesel and its diesel engine application: A review / I.M. Atadashi, M.K. Aroua, A. Abdul Aziz // Renewable Sustainable Energy Rev. - 2010. - V. 14. - P. 1999-2008]. The main characteristic of modern diesel engines is the cetane number from 45 to 55. The standard fuel is characterized by the cetane number 48-51, and the fuel of the highest quality (premium) has the cetane number 51-55. According to Russian standards, cetane number of summer and winter diesel fuel should be at least 48 units [GOST 305-82. Diesel fuel; GOST R 52709]. The content of fatty acids indicates the volume fraction of cetane. When calculating the characteristics of biodiesel [http://brteam.ir/analysis/#id02] on the basis of the composition and content of individual fatty acids (Table 4), it was revealed that the Pseudomonas azotoformans VKM B-3254D strain is able to transform hydrocarbon xenobiotics into lipid metabolism biomass- biodiesel sources.

Claims (1)

The strain of bacteria Pseudomonas azotoformans VKM B-3254D used for bioconversion of hydrocarbons from polluted with oil and oil products of water into the source of biodiesel.

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