LT4794B - The strain trichoderma harzianum vnb-16 destroying fuel oil, process for preparing thereof and use thereof - Google Patents

Abstract

The invention is in the field of microbiology, biotechnology, namely - the microorganism strains that destroy carbohydrates; their production methods; it can be used in cleaning oil polluted ground, for diminishing its toxic effect on the soil structure and the restoration of the soil's biological properties. The strain Trichoderma harzianum VNB-16 (deposited in the Institute of Botany, Biodestructor Research Laboratory (LT), collection number IBL: F-431) is an oil destructing strain that remains active in the soil after introduction, effects analogically phytopathogenic microorganisms and decreases toxicity absorbed by plants from soil.

Description

The present invention relates to the fields of microbiology, biotechnology, namely, strains of microorganisms which decompose hydrocarbons and can be used for cleaning soil contaminated with petroleum products.

Known strain BKM Ac-1339D from Rhodococcus erythropolis, described in aut.

1. SU 1 805 097, C02F 3/34, in the specification. This strain is used to clean water and soil from oil and petroleum products.

The strain deficiency is the ability of bacteria to degrade soluble hydrocarbons. It is not characterized by its ability to cut fuel oil. In addition, bacteria incorporated into the soil microorganism community are more difficult to survive in the environment of other microorganisms.

Known micromycete Candida guillermondii BKM Y-241, described in patent RU 2 007 372, C02F 3/34. E02B 15/04. in the description. This strain is used to purify water and soil from oil and petroleum product contamination.

The disadvantage of this strain is its inability to split fuel oil. Micromycetes do not improve soil structure, they are more difficult to reproduce in the natural communities of soil microorganisms.

The closest to the strain proposed for its action is the strain Actinomucor elegans TC405 described in U.S. Patent 4,415,662. This strain is a biodestructor of petroleum and petroleum products.

Strain of this material ⁵ drawback - of petroleum products split water, not improving the soil structure and does not have antagonist properties phytopathogenic microorganisms.

The object of the invention is to isolate a strain characterized by the ability to break petroleum hydrocarbons, serving as fuel oil. enabling accelerated utilization of petroleum products in soil, restoring its structure and biological activity.

The proposed strain of microorganism was isolated from the area of Vilnius Petroleum Base contaminated with sand saturated with industrial lubricants. The method of isolation comprises preparing an aqueous suspension of soil; inoculation of the prepared slurry on agarized brewing must and modified Chapeck media using 1% fuel oil as carbon source. Such media are used for the detection of oil-oxidizing micromycete strains, for purification of the culture and for isolation of the monosporous culture. The micromycete was grown in a thermostat at 26 ± 2 ° C for 7 days, the strain identified by cultures and morphological characteristics using the M.A. Rifai classification.

The strain is deposited in the Microorganism Collection of the Biodestructors Research Laboratory, Botanical Institute, Collecting Number IBL: F-431.

The proposed strain Trichoderma harzianum VNB-16, when grown on different media, has the following characteristics:

Morphological properties

Agarized medium of beer must. Colonies are growing fast. Colonies reach a diameter of 50 mm after 3 days and 90 mm after 5 days. Colony surface is dark green, mycelium dense, rather tall, friable with many cotton-like fronds. The lower half of the colony is colorless. Culture smell specific.

Agarized Chapek mineral medium. Colonies grow slower - 35 days after 7 days and 7 after

- 90 mm in diameter. Colonies light greenish with white and greenish fluffy micelles. The lower half of the colony is yellowish.

Saburo-dextrose agarized medium. Colonies grow rapidly, reaching 45 in diameter after 5 days and 5 90 mm in diameter. The micelle is very dense, growing in concentric rings, the edge of the colony

- yellowish, with a greenish center. Bottom half of the colony orange.

Hyphae sepate, branched, lvg-walled, colorless, 1.5-10.0 μ in diameter. Lateral branches usually · in groups of 2-3.

Fialides are short, tubular, 4.5-8.0 x 2.5-3.5 μ, usually in groups of 5, but can be solitary.

The conidia are small, spherical or short oval, smooth-walled, green in color at the tops of the phialids, forming spherical fake heads. The average size of conidia is 2.8-3.2x-2.5-2.8 μ.

Physiological properties

Carbon sources: The strain has good uptake of glucose, sucrose, galactose, xylose, carboxymethylcellulose. Organic acid salts, starch, glycerol are less absorbed.

Nitrogen Sources: The strain has good uptake of ammonium salts, NaNO ₃ , peptone, asparagine. Poorly absorbed casein.

The strain grows well on grains of natural substrates such as plant waste. Aerobic. Growth temperature 15-36'C, optimum 26-30 ° C. Grows in medium of acidity -H 4-7.

The strain has enzymatic activity: it produces proteases, cellulases. »

No synthesis of tyrosinase, laccase.

Antagonistic properties

The strain has high antibiotic activity against some phytopathogenic fungi, e.g., Rhizocionia solani, Fusarium momliforme, F. oxysporum, F. solani, Aliernaria alternata. However, it is not an antagonist of petroleum hydrocarbon-degrading bacteria and micromycetes-yeast, and even stimulates the growth of the latter (Candida lipolytica, Rhodotorula lactosa, Rhodotorula sp., Etc.).

The ability of the strain to oxidize petroleum hydrocarbons was evaluated by growth on agarized Chapek medium with 1% diesel fuel and fuel oil. Diameter (mm) of colonies of micromycete strains after 7 days of cultivation on Chapeck's medium and Chapek's petroleum-containing medium are given in Table 1.

&

table

A species of micromycet The strain number Chapeko medium Chapeck's medium with fuel oil Chapeck's medium with diesel fuel Trichoderma harzicmum VNB-16 90 90 90 Penicillium verruculosum MN-1 32 12th 23rd

On Chapek mineral medium with 1% fuel oil strain Trichoderma harzianum, colonies grow rapidly to reach a diameter of 90 mm after 7 days. Colonies are thin, mycelium low, weakly producing green pigment, colonies gray with white fronds.

The strain, when growing in fuel oil with mineral oil, uses a variety of phosphorus sources: bone meal, superphosphate, Kemira-Horti 2 fertilizer (NPK 6:12:24). The diameter (mm) of the proposed Trichoderma harzianum strain VNB-16 after 7 days on agarized media containing various fertilizers and fuel oil is shown in Table 2.

table

Control (KH ₂ PO ₄ ) Bone meal Superphosphate Kemira-Horti 2 Phosphorus source concentration, g / l 1 0.8 1.6 2.4 0.5 l 1.5 0.4 0.75 1.1 Colony diameter, mm 43 90 90 90 52 48 45 45 50 42

Kemira fertilizers and 1 g / l superphosphate additive have the best effect on the formation of the air mycelium when growing in fuel oil.

In a liquid medium with Kemira fertilizers and a mixture of fuel oil and diesel (1: 1 ratio), the strain offered grows well and the fuel oil splits into small flakes within 7 days. The biomass gain is presented in Table 3.

table

Variant Biomass in g / 50 ml Control (KH ₂ PO ₄ - 1.0 g / l) 0.040 Kemira 0.40 g / L 0.027 Kemira 0.75 g / l 0.056

The soil of the vegetable garden (400 g) is placed in vegetative dishes, to which 50 ml of fuel oil degraded by microorganisms are added. Infectious suspension of strain proposed 1 χ 10 ⁶ - 1x10 ⁷ start / g. Lupine is planted in the soil. After 6 weeks of cultivation, microbiological analysis showed that the micromycete survived and developed in fuel oil-contaminated soil. The hydrocarbon content of the studied soil decreased from 2388 to 370 mg / l during this period, ie by 6.5 times.

The summary of the above data indicates that the proposed strain Trichoderma harziamim VNB-16 has the ability to break down fuel oil and can be used to accelerate the recovery of petroleum products and to restore soil quality.

example

Cultivation of the proposed Trichoderma harziamim VNB-16 strain in liquid mineral medium by adding petroleum hydrocarbons as the sole carbon source.

Produce liquid nutrient mineral medium in the following composition (g / 1):

NH4NO3 1.5 kh ₂ after ₄ 1.0

KCl 0.5

MgSO ₄ x 7H ₂ O 0.5

FeSO ₄ x 7H ₂ O 0.001

Distilled water 1,000 ml

Adds 1% fuel oil or naphthalene as the sole carbon source. Transfer 50 ml into 500 ml shake flasks. The medium is sterilized by autoclaving for 30 min.

ό at 0.5 atm. Inoculate the cooled medium with 2 ml of a 7-day-old spore suspension of Trichoderma harzianum. The strain is incubated on shakers for 7-20 days at 26 + 2 ° C. After 7 days of cultivation, the fuel oil breaks down into fine flakes, and after 20 days only a light brown liquid remains.

The amount of petroleum product broken down during strain cultivation at 7 and 20 days is shown in Table 4.

table

Oil Product Home hydrocarbon content mg / l00 ml 7 days 20 days Balance Broken Balance Broken Naphthalene 719 406.4 43.5% 60.5 91.5% Fuel oil 715 644.2 10.0% - -

From the data presented, it appears that the proposed strain Trichoderma harzianum VNB-16 breaks down a wide range of hydrocarbons: the hydrocarbons that make up the heavy petroleum fractions naphthalene and fuel oil.

example

Use of the proposed strain Trichoderma harzianum VNB-16 for composting of oil products in soil.

For substrate contamination, micromycete biomass is grown in the laboratory under sterile conditions. After 6-7 days of culture, prepare a micromycete spore suspension on agarized beer must medium at 26 ± 2 ° C. The conidia are washed with sterile saline and the resulting suspension is diluted to maintain a fungal stock titer of 1 x ^{10 6} - 1 x 10 ⁷ / l ml. The prepared suspension is used to contaminate contaminated soil.

The soil contaminated with petroleum products is rolled up, mixed with nitrogen and phosphorus fertilizers, sprinkled or sprayed with a micromycete spore suspension and mixed well. Inoculated micromycete substrate is kept under natural conditions (at 10-30 ° C, maintaining optimum moisture of compostable soil)

- 3 months with periodic monitoring of micromycete development and oil product change in the cleaning soil.

By priming as described above, compared to a control sample that did not inoculate Trichoderma harzianum VNB-16, after 30 days. fuel oil decreased by 85%. The consistency of the soil has changed. It got cleaner, lost the smell of oil.

Example 3

Utilization of the above described oil-based primed soil for plant cultivation - final soil remediation.

Oil-cleaned soil is not toxic to higher plants. Seed germination in fuel oil contaminated soil without micromycete was 50% for Trichoderma harzianum VNB-16 and 80% for Trichoderma harzianum VNB-16. The lupine green mass gain in 2 weeks - 0.86 and 1.46 g / ex, respectively.

Rounded soil can be used for plant cultivation and for the complete restoration of soil structure and its biological properties, since the introduced microorganism Trichoderma harzianum VNB-16 stays active in the soil, antagonizes phytopathogenic microorganisms and reduces the toxicity of oil-contaminated soil to plants.

Claims (5)

DEFINITION OF INVENTION 1. Strain Trichoderma harzianum VNB-16 (deposited at the Institute of Botany Institute for Biodestructors Research (LT), Collection No. IBL: F-431) is a petroleum-degrading strain that antagonizes phytopathogenic microorganisms and reduces the toxicity of oil-contaminated soil to plants. 2. The strain Trichoderma harzianum VNB-16 is intended for use in the cleaning and restoration of oil-contaminated soil. 3. A process for the preparation of a strain of Trichoderma harzianum VNB-16 according to claim 1, wherein the prepared suspension of the oil-contaminated soil is seeded on agarized brewing must and modified Chapek medium with 1% fuel oil as oil decomposer. For detection of micromycete strains, purification of their culture and isolation of the monosporic culture, the micromycete is grown in a thermostat at 26 ± 2 ° C for 7 days and the strain is identified by cultures and morphology using the MA Reef classification. 4. The method of using the strain Trichoderma harzianum VNB-16 according to claims 1 and 2 for cleaning the soil contaminated with fuel oil according to claim 3, characterized in that the microorganism biomass is cultivated in culture media, followed by preparation of an aqueous micromycete biomass suspension. is about 10 ^? -10 ⁷ / l ml, the pre-prepared substrate is inoculated with a micromycete suspension by watering or spraying said suspension, then the substrate is mixed to maintain moisture suitable for the strain to proliferate and remain active in the substrate-primer. The use of soil in a rounded manner according to claim 4 for growing plants and for the final restoration of soil structure and its biological properties.