RU2531232C1 - STRAIN OF BACTERIA Rhodococcus rhodochrous - DESTRUCTOR OF PETROLEUM HYDROCARBONS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: bacterial strain Rhodococcus rhodochrous is deposited in the Russian Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms RAS under the registration number RCM Ac-2018 D. The destruction with this strain within 7 days of crude oil is up 100%, oil sludge - up to 98%, black oil - up to 96%.

EFFECT: strain has high destructive activity against petroleum hydrocarbons included in oil sludge, as well as against crude oil and black oil.

1 dwg, 1 tbl, 3 ex

Description

The invention relates to biotechnology, in particular to microbiology, as well as to ecology. It concerns the production of a new culture of bacteria that is effective in cleaning natural objects contaminated with oil and oil products, detoxifying hazardous oil-containing wastes and providing a decrease in the concentration of hydrocarbons, including heavy hydrocarbon fractions.

Despite a significant amount of work devoted to the microbiological disposal of oil waste and the bioremediation of hydrocarbon-contaminated lands, this problem remains open for new solutions. At the same time, the biological utilization of oil sludge, a hazardous oil-containing waste having a high content of biologically and chemically stable heavy hydrocarbon fractions, is especially acute.

A known bacterial strain Mycobacterium flavescens EX-91 VKPM B-6000 (RF patent No. 2053296, IPC (6) C12N 1/20, C02F 3/34, C12R 1:32, C12N 1/20, publ. 1996). This strain showed an effective destruction of oil, fuel oil, diesel fuel and kerosene by 30; 43; 45 and 81%, respectively, at a temperature of 10 ° C.

A known bacterial strain Rhodococcus species 56-D CCM V-61 B (Central collection of microorganisms of the state concern "Biological product") used to purify water and soil from oil and oil products (RF patent No. 2101352, IPC (6) C12N 1/20, C02F 3/34, B09C 1/10, C12R 1:01, publ. 1998). This strain carries out purification from oil, fuel oil, diesel fuel, kerosene at a temperature of 12-30 ° C for 7-14 days.

Known bacterial strains of Rhodococcus erythropolis for the decomposition of oil and oil products (RF patent No. 2257409, IPC (7) C12N 1/20, B09C 1/10, C02F 3/34, C12N 1/20, C12R 1:01, publ. 2005; patent RF №2257410 IPC (7) C12N 1/20, B09C 1/10, C02F 3/34, C12N 1/20, C12R 1:01, publ. 2005). The strains were deposited in the All-Russian collection of industrial microorganisms under the numbers VKPM As-1668 and VKPM As-1667, respectively. Strains shows a high efficiency of destruction in relation to oil. The destruction of chronic oil pollution by strains of Rhodococcus erythropolis VKPM Ac-1668 and Rhodococcus erythropolis VKPM Ac-1667 was 70.7% and 71.7% at 30 days of the experiment, respectively.

The disadvantage of all of the above strains is the lack of ability to process heavy petroleum hydrocarbons that are part of the sludge.

The closest analogue is the strain Rhodococus globerulis N-42 for the decomposition of oil and oil products, deposited in the All-Russian collection of industrial microorganisms under the number VKPM AC-1641 (RF patent No. 2300561, C12N 1/20 (2006.01), C02F 3/34 (2006.01), B09C 1/10 (2006.01), C12R 1/00 (2006.01), publ. 2007). For the strain, the ability to reduce the initial concentration of hydrocarbons by 81.3% in oil-contaminated soil is shown for two months. In the soil with chronic oil pollution, the destruction of hydrocarbons within one month was 80.7%.

The disadvantage of this strain is the low efficiency of reducing the concentration of hydrocarbons in the purification of chronic oil pollution represented by biologically stable hydrocarbons (similar to oil sludge).

The technical result of the present invention is to obtain a bacterial strain exhibiting high destruction activity against petroleum hydrocarbons that are part of oil sludge, as well as against oil and fuel oil.

To achieve a technical result, it is proposed to use a strain of bacteria Rhodococcus rhodochrous KR 32 isolated from oil-contaminated soil in the Krasnodar Territory. The strain is deposited in the All-Russian Collection of Microorganisms (VKM) of the Institute of Biochemistry and Physiology of Microorganisms of the Russian Academy of Sciences (Pushchino) under registration number VKM Ac-2018 D.

The identification of the strain was carried out on the basis of the study of its cultural, morphological and physiological and biochemical characteristics. The resulting strain is characterized by the following morphological, cultural, chemotaxonomic and physiological characters, allowing to determine its taxonomic affiliation. On meat-peptone agar (MPA) forms rounded, bright red colonies with a diameter of 1-2 mm (cultivation for 3 days, at 25 ° C), on meat-peptone broth under the same conditions, without shaking - turbidity, sediment. At the age of 15 hours, the culture is represented by gram-positive coryneform bacilli with a size of 0.7 × 2-8 μm, no branching was noted. At the age of 4 days, cells shorten to 1 μm, i.e. There is a pronounced developmental cycle of cocci-coli-cocci. Mobility was not observed in any of the developmental phases. Meso-diaminopimelic acid, arabinose and galactose (type IV cell wall) are found in the hydrolysates of whole cells. Methyl mycolates, by mobility during thin layer chromatography, correspond to nocardomicolic acids. Contains dihydrogenated menaquinones with eight isoprenoid units. Strict aerob. It grows at temperatures from 4 ° C to 37 ° C. Catalase and oxide positive. Uses as the sole source of carbon and energy: citrate, malate, succinate, propionate, pyruvate, glycerol; does not use the following compounds as the sole source of carbon and energy: lactate, fumarate, oxalate, adipate (sodium salts). Hydrolyzes: urea, tween-80; does not hydrolyze: starch, cellulose. Grows in the presence of 8% NaCl.

Based on cultural-morphological and physiological-biochemical characteristics, as well as on the basis of studying the sequence of 16S rRNA genes, the taxonomic affiliation of the strain was determined as Rhodococcus rhodochrous.

The maintenance of the strain is carried out on a dense agarized nutrient medium - nutrient agar (PA) of the following composition (g / l):

- pancreatic hydrolyzate of fish meal - 24.0;

- sodium chloride - 4.0;

- microbiological agar-agar - 12.0 ± 2.0;

- distilled water - 1 l.

Maintaining the strain in a test tube with beveled PA at a temperature of 4 ° C is carried out for no more than 1-2 months. After this period, reseeding to a fresh environment is required.

The strain is stored for a long period (years) by lyophilization of cell biomass on a lyophilic drying apparatus such as LS-1000 (or equivalent), followed by hermetic sealing of the lyophilisate in glass ampoules. Storage of lyophilized culture at (4-6) ° C. The strain tolerates freezing of crude biomass in a solution of glycerol, followed by storage at -70 ° C.

To obtain a strain, a 2-5 ml ampoule containing lyophilically dried on a LS-1000 freeze dryer the biomass of the strain Rhodococcus rhodochrous KR 32 VKM Ac-2018 D is sterilely opened, filled with a sterile syringe or Pasteur pipette with sterile saline. The resulting suspension is streaked on nutrient agar to obtain isolated bacterial colonies. The coincidence of the cultural and morphological properties of the obtained isolate with those of Rhodococcus rhodochrous KR 32 VKM Ac-2018 D.

Obtaining the biomass of the cells of the strain is carried out by deep cultivation on a liquid mineral medium of the following composition (g / l):

- KNO ₃ - 4;

- Na ₂ HPO ₄ × 12H ₂ O - 1.4;

- KH ₂ PO ₄ - 0.6;

- MgSO ₄ × 7H ₂ O - 0.8;

- standard solution of trace elements (10 elements) - 1 ml;

- distilled water - 1 l.

This medium is used to produce biomass in small volumes under laboratory conditions (up to 8 liters of liquid culture). If it is necessary to obtain large volumes (more than 8 liters of the initial liquid culture), a clarified extract from complex mineral fertilizers was used as a nutrient medium.

As the sole source of carbon and energy, sucrose, or vegetable oil, or hydrocarbons in concentrations of 0.5-2% by weight is added to the medium. As equipment, it is possible to use rocking flasks, bioreactors of various capacities and designs, which provide mixing and aeration of the medium in the required volume.

Cultivation on rocking flasks is carried out in vessels with a volume of 0.1-1.0 l at a platform speed of 100-200 rpm and a temperature of 20-30 ° C. The cultivation period is 4-7 days.

Cultivation on a cultivation apparatus AK-210 with a capacity of 10 l (filling factor up to 0.8) produced by the Institute of Biological Instrumentation of the Russian Academy of Sciences is carried out at a rotor speed of 160-280 rpm, a temperature of 25-30 ° C. The cultivation period is 2-3 days.

Cultivation in a fermentation module with a capacity of 100 l (fill factor 0.85) of integrated biotechnological equipment OKA-01-100T manufactured by the Institute of Biological Instrumentation of the Russian Academy of Sciences is carried out at rotor speeds of 140-250 rpm, temperature 25-30 ° C. The cultivation period is 3-4 days.

As a result, it is possible to obtain a culture with a biomass concentration of cells of the strain Rhodococcus rhodochrous KR 32 VKM Ac-2018 D up to 3 g / l (absolutely dry weight) and a living cell titer up to 5 × 109 CFU (colony forming units) / ml.

The destruction activity of the strain Rhodococcus rhodochrous KR 32 VKM Ac-2018 D is reflected in the ability to assimilate individual alkanes (C ₅ H ₁₂ , C ₆ H ₁₄ , C ₇ H ₁₆ , C ₈ H ₁₈ , C ₉ H ₂₀ , C ₁₀ H ₂₂ , C ₁₁ H ₂₄ , C ₁₂ H ₂₆ C ₁₃ H ₂₆ , C ₁₄ H ₃₀ , C ₁₅ H ₃₂ , C ₁₆ H ₃₄ , C ₁₇ H ₃₆ , C ₁₈ H ₃₈ , as well as paraffin - a mixture of higher alkanes C ₁₈ -C ₃₅ ) as the only source of carbon and energy during cultivation on an agarized mineral nutrient medium (a nutrient medium was used to produce biomass under deep cultivation in laboratory conditions, with the addition of 1.8% agar, individual alkanes were added and in cups in an amount of 1% immediately before solidification of the agar, cultivation was carried out at 10 ° C, 30 ° C, and 37 ° C for 7 days, the sowing was carried out using a microbiological loop, the inoculum was grown on a mineral medium with oil).

The practical applicability of the strain is associated with its ability to carry out effective destruction of Russian oil, heating oil and various oil sludge, differing in composition, including the content of heavy hydrocarbon fractions.

Laboratory tests conducted at the Kuban Medical Academy showed the pathogenicity of the strain. The tests were carried out in an acute experiment on white mice. Observation of the animals was carried out for 30 days after infection. The strain does not have toxic or toxigenic effects.

The drawing shows graphs of reducing the concentration of petroleum products in a mixture of oil sludge with soil. Graph 1 - decrease in concentration during processing of Rhodococcus rhodochrous KR 32 BKM Ac-2018 D, graph 2 - control.

Example 1. The destruction of the strain Rhodococcus rhodochrous KR 32 BKM Ac-2018 D of petroleum hydrocarbons that are part of the sludge, as well as oil and fuel oil was investigated in the following experiments. A liquid mineral medium was prepared to obtain biomass by deep cultivation in the laboratory. The medium was poured into 50 ml into 200 ml vials, sterilized by autoclaving for 30 min at 1 atm.

Oil-containing wastes (oil sludge) and commercial oil products were used as a substrate. Their composition, reflecting the content of heavy fractions, is shown in table 1. Oil-containing wastes (oil sludge) were autoclaved for 20 min at 0.5 atm, introduced into the medium in an amount of 0.5 g before inoculation. The medium was inoculated with a suspension of previously grown cells of the strain Rhodococcus rhodochrous KR-32 BKM Ac-2018 D in a mineral medium. The inoculum dose was 10 ⁶ cells / ml.

The bottles were placed on a rocking chair and cultivated at a temperature of 30 ° C for 7 days. The results were evaluated by the residual concentration of hydrocarbons in the medium, determined by the gravimetric method. As a control, flasks with the corresponding hydrocarbons were used, where the strain was not added. As can be seen from table 1, the proposed strain is able to grow on heavy petroleum products with a high content of aromatic and, including polycyclic compounds. A large decrease in the concentration of hydrocarbons during the destruction of a wide range of biologically sustainable oil wastes (oil sludge) and marketable oil products (oil, fuel oil) suggests that the strain Rhodococcus rhodochrous KR 32 BKM Ac-2018 D is able to utilize heavy oil fractions.

Table 1 The destruction of the strain Rhodococcus rhodochrous KR 32 VKM AC-2018 D of petroleum hydrocarbons of various sludges and marketable petroleum products Substrate The content of biologically stable components of hydrocarbon nature,% Utilization,% 3 days 7 days the control Methanonaphthenoaromatic fraction Paraffins Resins benzene / alcohol benzene Asphaltenes Oil sludge 1 84.58 1.05 6.25 8.12 78 97 9 Oil sludge 2 65.19 15,52 9.39 9.9 83 95 8 Oil sludge 3 90.64 0 2.08 / 1.71 5.93 91 98 7 Oil sludge 4 73.45 13.59 7.39 5.57 82 97 6 Oil sludge 5 80.24 12.68 4.6 2.48 85 98 8 Oil sludge 6 81.51 1.01 5.09 / 5.58 6.81 78 97 5 Oil sludge 7 36 6.71 3.61 / 2.62 10.38 76 95 3 Crude oil 79.43 0.21 5.31 / 7.04 8.01 79 one hundred 21 Fuel oil M-40 70.66 11.27 11.52 / 4.39 2.16 87 96 17

Example 2. The destruction of the strain Rhodococcus rhodochrous KR 32 VKM Ac-2018 D of petroleum hydrocarbons that are part of a mixture of oil sludge with soil, was investigated in laboratory conditions. For this, 300 g of oil sludge-soil mixture was added to 2 glasses per 1000 ml with a petroleum product concentration of 61 g / kg, 2 ml of strain culture, Rhodococcus rhodochrous KR 32 VKM Ac-2018 D with a cell concentration of 10 ⁸ CFU / ml, was added to one. The strain was not added to the second glass. Water was added to both glasses to 60% of the total moisture capacity of the soil mixture, as well as ammophos in an amount of 0.1 g in each glass. The cleaned soil was regularly mixed, moisture and the concentration of nutrients were measured and maintained at the initial level. The results were evaluated by the residual concentration of petroleum hydrocarbons, determined by the gravimetric method. Bioprocessing was repeated after 15 days of the experiment. After 30 days of purification, the degree of destruction of petroleum hydrocarbons at their initial concentration of 61 g / kg was 85%. In a glass, where the strain was not added, the decrease in concentration did not exceed 12%.

Example 3. The destruction of the strain Rhodococcus rhodochrous KR 32 BKM Ac-2018 D of the petroleum hydrocarbons that make up the mixture of oil sludge with soil was investigated in the field. Tests of the drug were carried out on the territory of the facility containing oily waste. Oil sludge mixed with soil was placed at a specially equipped training ground. In the selected area, the waste was treated with a suspension of cells of the strain Rhodococcus rhodochrous KR 32 BKM Ac-2018 D at a concentration of 10 ⁷ CFU / ml, at the rate of 10 l / ton. As a control, the same plot was prepared where the strain was not added. Ammophos was added to both sites at the rate of 300 g / ton; humidification and mixing were performed as necessary. Ammophosome treatments, wetting and loosening were repeated 3 times a month. Biological treatment was repeated after 20 days of the experiment. The results were evaluated by measuring the concentration of petroleum hydrocarbons by the gravimetric method. The drawing shows a decrease in the concentration of petroleum products in the processed waste (graph 1) and control (graph 2). As a result of treatment with the drug, the concentration of petroleum products decreased from 93.45 to 12.10 g / kg in 48 days (destruction was 87.05%), which shows the possibility and efficiency of using the strain to reduce the concentration of petroleum hydrocarbons in oil sludge.

Thus, the results obtained indicate that with the help of the strain Rhodococcus rhodochrous KR 32 BKM Ac-2018 D, effective destruction of oil hydrocarbons is achieved during biological treatment and detoxification of hazardous oil-containing wastes, including oil sludge, and restoration of oil-contaminated objects.

Claims (1)

The strain Rhodococcus rhodochrous BKM Ac-2018 D is a destructor of petroleum hydrocarbons.