RU2614263C1 - Trametes hirsuta strain - producer of ethanol - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: Trametes hirsuta MT-24.24 basidiomycete strain has the ability to produce ethanol. Trametes hirsuta basidiomycete strain is deposited in the Russian National Collection of Industrial Microorganisms under accession number VKPM F-1288 and can be used in medicine and food industry.

EFFECT: invention allows to obtain high quality alcohol containing no impurities such as acetone and isopropanol.

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Description

The invention relates to biotechnology and relates to a strain of a basidiomycete fungus (basidiomycete) Trametes hirsuta producing ethyl alcohol from carbohydrates, cellulosic and lignocellulosic substrates. Microbiologically produced ethanol can be used as a fuel component for internal combustion engines, in medicine and the food industry.

A known strain of basidiomycete Phlebia sp. MKFC40001, capable of producing ethyl alcohol from sawdust with a yield of 43.9% of the theoretically possible, after 20 days of incubation under semi-anaerobic conditions. However, the use of this strain predetermines the need for pretreatment of the lignocellulosic substrate under aerobic conditions for 8 weeks (US 9145568, 2015).

Also known is the Phanerochaete chrysosporium strain producing ethyl alcohol from lignocellulosic substrates (crushed grain, corn straw) by solid-phase fermentation under semi-anaerobic conditions (US 8309328, 2012). Moreover, this strain shows the ability to produce, along with ethyl alcohol, compounds such as acetone and isopropanol, which leads to the desired product of insufficient quality and the need for additional purification.

Closest to the invention is a strain of Trametes suaveolens, capable of producing up to 7.7 g / l of ethyl alcohol from glucose in 18 days of incubation under semi-anaerobic conditions. (K. Okamoto et al. Production of ethanol by the white-rot basidiomycetes Peniophora cinerea and Trametes suaveolens // Biotechnology letters, 2010, Vol. 32, No. 7, p. 909-913). Thus, when using this strain, a sufficiently long cultivation time is necessary. In addition, this strain is capable of producing alcohol only from carbohydrate raw materials, so the range of raw materials used is limited.

The objective of the invention is to obtain a new effective strain - producer of ethyl alcohol from glucose, cellulosic and lignocellulosic raw materials.

The task is achieved by creating a strain of basmetiomycete Trametes hirsuta VKPM F-1288 - a producer of ethyl alcohol from glucose, cellulose and lignocellulosic raw materials.

The technical result consists in creating a strain capable of producing ethyl alcohol from glucose, cellulosic and lignocellulosic raw materials (substrates). This reduces the time of cultivation. In addition, the described strain allows to obtain high-quality alcohol that does not contain impurities such as acetone and isopropanol.

A new strain of Trametes hirsuta is isolated from a natural fruiting body found on dead Betula pendula wood. Pieces of the fungus cut from the middle of the fruit body were transferred under aseptic conditions to a selective solid nutrient medium (wort agar) containing 30 mg / l ampicillin and 4 mg / l benomyl. The grown mycelium was identified as a pure basidial culture by the presence of buckles on the mycelium. The taxonomic affiliation of the described strain to the species Trametes hirsuta was confirmed by the molecular genetic method. The strain of the basidiomycete Trametes hirsuta is deposited in the All-Russian Collection of Industrial Microorganisms and has registration number VKPM F-1288.

Characteristics of the strain Trametes hirsuta.

The isolated strain belongs to the class Basidiomicetes, subclass Agaricomycetidae, order Polyporales, family Polyporaceae, genus Trametes, species hirsuta. The species T. hirsuta is not pathogenic for humans and does not form toxic metabolites at any stage of morphogenesis.

Cultivation conditions: wort agar, temperature 26-28 ° C, cultivation time is 5-7 days.

Storage conditions: in test tubes with wort agar at 4-6 ° C for 12-18 months without reseeding.

Macromorphological signs:

On wort agar, the colony is rounded with a rhizoid margin, slightly expressed concentrically zonal, fuzzes around the inoculum, the profile is elevated. Aerial white mycelium, substrate mycelium colorless, the reverse side of the colony is not stained. The colony structure is cottony, slightly rough, and dense. With aging, the mycelium becomes leathery, dense.

Microscopic signs:

Hyphae of aerial mycelium up to 3 microns in diameter, branched, short. On the mycelium there are large strongly convex buckles of a stable form.

The mycelium Trametes hirsuta (hereinafter T. hirsuta), grown under submerged cultivation, has the ability to produce ethyl alcohol.

Vegetative mycelium of basidiomycete is grown in submerged culture under aseptic aerobic conditions. Cultivation is carried out in rocking flasks with a volume of 750 ml on an orbital rocking chair at a temperature of 28 ° C. The medium for submerged cultivation contains glucose 10-30 g / l, soy flour 5-15 g / l, potassium dihydrogen phosphate 2-3 g / l, magnesium sulfate 0.2-0.3 g / l and tap water. The pH of the medium is adjusted to 5.5-6.2. The nutrient medium is autoclaved at a temperature of 121 ° C for 30 minutes. The duration of the process of obtaining liquid seed mycelium is 4 days, fermentation - 4 days.

Strain T. hirsuta is able to ferment carbohydrates (e.g. glucose) and cellulosic and lignocellulosic substrates (e.g. carboxymethyl cellulose sodium salt, microcrystalline cellulose, straw) under anaerobic conditions.

Below are examples illustrating the invention, but not limiting it.

Example 1. Obtaining immersed mycelium of T. hirsuta.

Immersed cultivation of the strain T. hirsuta is carried out on an orbital shaker with a rotation speed of 250 rpm at a temperature of 28 ° C in rocking flasks with a volume of 750 ml containing 100 ml of culture medium of the following composition: glucose 20 g / l, soy flour 10 g / l, potassium dihydrogen phosphate 2.5 g / l and magnesium sulfate 0.25 g / l. The pH of the medium is adjusted to 5.5-6.2. The process of submerged cultivation is carried out in two stages. At the first stage, liquid seed is obtained; at the second stage, fermentation is carried out.

For the preparation of inoculum, a culture grown in test tubes on wort agar is used. Pieces of agar with a size of not more than 3 × 3 mm in an amount of 30-40 pieces are aseptically introduced into rocking flasks containing 100 ml of culture medium and cultured for 4 days. Then, 10 ml of seed in aseptic conditions is introduced into rocking flasks containing 100 ml of culture medium and cultured for 4 days. The yield of air-dried biomass is 5.8 g / l of nutrient medium on the 4th day of fermentation.

Example 2. Obtaining immersed mycelium of T. hirsuta.

Obtaining immersed mycelium of T. hirsuta is carried out according to example 1, but glucose, soy flour, potassium dihydrogen phosphate and magnesium sulfate are added to the nutrient medium in an amount of 10 g / l, 5 g / l, 3 g / l and 0.3 g / l, respectively. The yield of air-dried biomass is 4.2 g / l of nutrient medium on the 4th day of fermentation.

Example 3. Obtaining immersed mycelium T. hirsuta.

Obtaining immersed mycelium of T. hirsuta is carried out according to example 1, but glucose, soy flour, potassium dihydrogen phosphate and magnesium sulfate are added to the nutrient medium in an amount of 30 g / l, 15 g / l, 2 g / l and 0.2 g / l, respectively. The yield of air-dried biomass is 4.1 g / l of nutrient medium on the 4th day of fermentation.

Example 4. Obtaining ethyl alcohol from glucose.

An immersed culture of T. hirsuta is prepared according to Example 1. The residual glucose concentration in the culture fluid is determined by the DNS method. A sterile glucose solution at a concentration of 20 g / l is aseptically introduced into the rocking flasks with the submerged culture. The flasks are closed with sterile hydraulic locks and thermostatically controlled at 25 ° C in a dark place. Sampling to determine the concentration of ethyl alcohol and glucose in the culture fluid is performed every 24 hours. Determination of the concentration of ethyl alcohol is carried out by gas chromatography. The data obtained on the study of the dynamics of accumulation of the target product and the loss of the original substrate using the described strain of Trametes hirsuta are summarized in table.

As follows from the data presented, the maximum concentration of ethyl alcohol in the culture fluid is 9.5 g / l on the 7th day of fermentation. Thus, the cultivation of ethanol using the described strain in comparison with the use of the known strain can increase the yield of alcohol and reduce the time of cultivation.

Example 5. Obtaining ethyl alcohol from cellulosic and lignocellulosic raw materials (substrates).

As substrates used: carboxymethyl cellulose sodium salt in an amount of 20 g / l, microcrystalline cellulose brand Avicel in an amount of 20 g / l, crushed straw with a particle size of 2-4 mm in an amount of 30 g / l. The substrates are placed in 750 ml rocking flasks at the indicated concentrations, 10 ml of tap water are added. The flasks are autoclaved at a temperature of 121 ° C for 30 minutes.

An immersed culture of T. hirsuta is prepared according to Example 1. 90 ml of the immersed culture are placed in sterile flasks with substrates, closed with sterile water locks, and a pale pink solution of potassium permanganate is used as an additional sterilizing substance in the water locks. The flasks are thermostated at 25 ° C for 11 days.

Determination of the concentration of ethyl alcohol is carried out by gas chromatography. The concentration of ethyl alcohol in the culture fluid after 7 days of fermentation of sodium carboxymethyl cellulose is 3 g / l, microcrystalline cellulose 2.6 g / l, crushed straw 2.6 g / l.

The resulting alcohol does not contain such impurities as acetone and isopropanol.

Thus, the described strain allows the production of ethanol of improved quality from a wider range of raw materials used by a simpler technology.

Claims (1)

The strain of the basidiomycete Trametes hirsuta VKPM F-1288 is a producer of ethyl alcohol.