RU2061751C1 - Strain of yeast candida ethanolica - a producer of biomass - Google Patents

Abstract

FIELD: microbiological industry. SUBSTANCE: thermotolerant strain of yeast Candida ethanolica BKM Y-2752 D - a producer of biomass based on ethanol-containing raw shows high growth rate (0.46-0.63 h^-1). Strain is able to develop at 30-43 C without addition of additional growth factors to medium and provides the decrease of consumption indices at high temperature of cultivation by organic substrate and cooling. Strain is deposited in All-Union Collection of Industrial microorganisms at number BKM Y - 2752 D. Strain does not show pathogenicity but able to competition. Strain of yeast Candida ethanolica BKM Y-2752 D can be used in biotechnological manufacture on the basis of ethanol-containing raw and in development of perspective technology associated with biomass processing. Strain is used for yeast biomass production. EFFECT: increased yield of yeast strain. 6 tbl

Description

 The invention relates to the field of biotechnological production and represents a new strain of yeast used for the production of biomass by growing it on nutrient media containing ethanol or ethanol-containing substrates as a carbon source.

 The production efficiency of yeast biomass is determined primarily by the activity of the cultured producer strain: its high growth rate and high substrate yield.

 A production-valuable feature of a strain is its thermal tolerance and ability to develop, providing the required quality of the resulting biomass without reducing productivity when changing in a wide range of growing temperature, since in industrial conditions there are often fluctuations in the process parameters for this parameter, usually leading to a decrease in the activity of the production culture .

 These signs, along with competitiveness with respect to yeast of other species in unsterile production, as well as the sanitary and hygienic properties of the strain / pathogenicity, the MPC value, etc. /, determine the feasibility of its use in industry.

 For the production of biomass on environments with ethanol and ethanol-containing substrates, various strains of yeast of the genus Candida / 1,2 / are used.

 The task was to select a yeast strain, characterized by expanding the temperature range of cultivation without reducing productivity and the true protein content in biomass, as well as increasing competitiveness under production conditions. The technical result that can be obtained using this strain is the possibility of conducting the fermentation process in a wide temperature range while increasing the productivity of the process and ensuring the dominance of the producer, as well as expanding the stock of ethanolassimilating cultures of biomass producers. The inventive Strain VKM U-2752 is isolated from a population of industrial culture of a pilot plant of the Bashkir BHK. Barnett Y.A. was used as a determinant on the basis of which the strain was identified. Payhe R.W. Jarrow D. Jeests. Characteristics and identification, Cambridge University Press, 125.1983.

 The strain is stored in the All-Union Collection of Microorganisms under the number VKM U-2752.

Morphological features:
the cells in the liquid glucose-peptone medium on the 3rd day are oval, elongated / 3-4 / x / 7-15 / mm, single, in short chains;
pseudomycelium after 7-6 days / culture on glass on morphological agar / well developed, tree-branching;
stroke / after 3 weeks / on wort-agar smooth, shiny lobed edge, cream color;
colonies on wort agar after 3 days are slightly convex, smooth, shiny, the edge is even, the center is slightly raised, the color is grayish-white;
ascospore, ballistospore, telestospor does not form.

 Physiological signs.

 Assimilates: D-glucose, ethanol, glycerin / slow /, lactic, succinic, citric / weak /, gluconic / weak / acid.

 Does not assimilate: D-galactose, sucrose, maltose, lactose, cellobiose, trehalose, melibiosis, raffinase, melicytosis inulin, starch, D-xylose, L-arabinose, D-arabinose, D-ribose, L-ramnose, erythritis, erythritis, erythritis dulcite, D-mannitol, sorbitol, methyl glucoside, inositol, xylitol, arabitol, glucuronic acid, methanol.

 Ferment glucose / slowly /. It does not ferment galactose, sucrose, maltose, lactose, raffinose, melibiosis, trehalose.

 Growth on hydrocarbons is absent.

It grows on a medium without vitamins at a maximum temperature of 43 ^o C.

The most active growth is observed in the temperature range of cultivation 30-42 ^o C. The maximum growth rate under different cultivation conditions reached 0.25-0.63 h ^-1 , the maximum biomass yield / 97% / containing 45% of the true protein is provided at a temperature medium 39 ^o C and a dilution rate of 0.45 h ^-1 . An almost complete absence of cyanide resistance was noted.

 The strain is non-pathogenic and competitive.

 The inventive strain of Candida ethanolica VKM U-2752 compared with the selected prototype relates to a different type of yeast Candida and has a number of features.

 Morphological differences.

 The cells are larger / 3-4 / x / 7-15 / microns than yeast C, valida (2.0-6.2) x (3.0-2.0) microns. The colonies are slightly convex, smooth, shiny, the edges are even, the center is slightly raised, the color is grayish-white in contrast to the known strain, which is flat, velvety or slightly deflated, or smooth with a smooth edge, or irregular in shape, whitish-gray.

 The difference in physiological and technological characteristics.

The inventive strain is characterized by a greater degree of thermal tolerance, has a wider cultivation temperature range of -30-43 ^o C. The maximum growth rate / 0.63 h ^-1 / exceeds the growth rate of the prototype strain by 20%, while the latter is inferior to the claimed yeast strain by the maximum achieved a yield of 7%, which determines the advantages of the new strain in productivity.

 The absence of alternative pathways for substrate oxidation in the VKM U-2752 strain declares a higher ethanol utilization efficiency.

A significant difference between the proposed strain VKM U-2752 is also the ability to actively grow almost without changing the growth rate / 0.56-0.63 h ^-1 / in a wide range of cultivation temperature from 32 to 40 ^o C, which determines the great competitiveness of the strain during prolonged cultivation in non-aseptic production conditions. The use of a strain belonging to the species C. ethanolica, previously not used in industrial production, expands the stock of ethanol-assimilating biomass producing crops.

 Strain C. ethanolica BHM U-2752 was tested under different cultivation conditions under the conditions of pH-stat, chemostat and different population densities in comparison with the prototype strain and other industrial strains.

Example 1. Periodic cultivation of ethanolassimilating strain C. ethanolica VKM U-2752 was carried out in flasks on a shaker at an ambient temperature of 30 to 42 ^o With an interval of 2 ^o . Yeast was grown on standard mineral medium-9M containing 1% / vol. / ethanol. Mineral composition of the medium: NH ₄ H ₂ PO ₄ -10 g / l, K ₂ HPO ₄ -10 g / l, MgSO ₄ • 7 H ₂ O-0.7 g / l. FeSO ₄ • 7 H ₂ O-12.5 mg / l, MnSO ₄ • 7 H ₂ O-12.5 mg / l, ZnSO ₄ • 7 H ₂ O- -12.5 mg / l, NaCl-6, 3 mg / l. The increase in biomass / g / l ASV / was evaluated after 24 hours from the start of the process.

 The growth activity of thermotolerant ethanol-assimilating strains of VKM U-2752 at different temperatures in flasks on a rocking chair is shown in Table 1.

 The possibility of cultivating the VKM U-2752 strain in a wide range of medium temperature during periodic cultivation was shown.

Example No. 2. Continuous cultivation of the yeast strain C.ethanolica VKM U-2752 was carried out under sterile conditions in the pH-auxostat mode using the Ankum fermenter with automatic temperature control / 39 ^o C /, pH / 4.0 /, dissolved oxygen / pO ₂ 20% of saturation /, ethanol supply / 1.5% /, mineral medium / g / l /: KH ₂ PO ₄ -1.5, NH ₄ Cl-1,0; MgSO ₄ • 7 H ₂ O-0.45 and trace elements / mg / l /: FeSO4 FeSO ₄ 4 • 6 H ₂ O-36; ZnSO ₄ • 7 H ₂ O-18; CuSO ₄ • 5 H ₂ O-7.55; MnSO ₄ • 5 H ₂ O-15; COCl ₂ • 6 H ₂ O-1; CaCl • 2 H ₂ O-1. The results of assessing the maximum growth rate of the investigated strain are presented in table 2.

Example 3. Continuous cultivation of the yeast C. ethanolica VKM U-2752 was carried out under conditions similar to example 2 at cultivation temperatures from 30 to 44 ^o C with an interval of 2 ^{o C. The} characteristics of the maximum specific strain growth rate at different temperatures are presented in table 3.

 A high degree of thermal tolerance of the VKM U-2752 strain with continuous cultivation was confirmed.

Example No. 4. Continuous cultivation of the yeast C.ethanolica VKM U-2752 was carried out under sterile conditions in a chemostat mode. In the process of cultivation used a mineral medium similar to that described in example 2 and containing 1% / vol./ ethanol. The cultivation of the yeast strain C.ethanolica BKM U-2752 was carried out at a temperature of 39 ^o C. Characteristics of the growth of the studied yeast and the quality of the biomass are presented in table 4.

 Example 5. The biomass of yeast With ethanolica VKM U-2752, obtained by cultivation in a chemostatic mode / example 4 /, was analyzed for amino acid content. The amino acid composition of the yeast protein C.ethanolica BKM Y -2752 is given in table 5.

 The usefulness of the VKM U-2752 yeast protein by amino acid composition was shown.

 Example 6. Periodic cultivation of thermotolerant ethanol-assimilating yeast C. ethanolica VKM U-2572 was carried out in an Ankum fermenter with a working volume of 12 l.

The cultivation of yeast at a temperature of 38 ± 1 ^o C and pH 4.0+ 0.2 was carried out on a mineral medium containing mg / l: K ₂ SO ₄ -304,0; MgSO • 7 H ₂ O-154.0; FeSO ₄ 7 H ₂ O-15.6; ZnSO ₄ • 7 H ₂ O-8.8; MnSO ₄ • 5 H ₂ O-8.0 and H ₃ PO ₄ -0.24 ml / L with the addition of 2.0% / vol. / Ethanol.

 During the growth, the respiration rate of the cells of the studied yeast was measured by the amperometric method using the Ur-7 polarograph.

 To determine the cyanide resistance of yeast cultures when measuring the respiration rate of the cells, potassium cyanide acid / I μg / was added as an inhibitor to a buffer solution containing potassium phosphate / pH 6.5 / and glucose / 1% /. Yeast cyanide resistance was expressed by comparing the respiration rate of cells with and without the addition of an inhibitor. The results are presented in table.6.

 The absence of alternative pathways for the oxidation of ethanol in the BKM U-2572 strain was established, which explains its high productivity in comparison with the known one due to an increase in the efficiency of substrate use.

 Thus, the proposed invention "the strain of yeast Candida ethanolica MKM U-2752-producer of biomass will increase production efficiency by expanding the temperature range of cultivation, ensuring the dominance of the strain, while increasing productivity in biomass and protein. TTT1 TTT2 TTT3 TTT4 TTT5 TTT6

Claims (1)

 Candida ethanolica BKM U-2752 yeast strain is a biomass producer.

Images