RU2391402C2 - Biocatalyst for producing ethanol from pentose - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and is meant for increasing efficiency of biotechnical production of ethanol from cellulose-containing material. The biocatalyst for producing ethanol from pentose is immobilised miscella of microfungus capable of fermenting pentose into ethanol, contained in a cryogel based on polyvinyl alcohol, with the following ratio of initial components in wt %: miscella of microfungus - 7.5-20.0, polyvinyl alcohol - 2.5-5.0 and water - up to 100. The biocatalyst can be reused to produce ethanol from pentose in periodic conditions.

EFFECT: biocatalyst is characterised by semi-inactivation period of up to 36 days, provides rate of conversion of pentose to ethanol of up to 0,34 g/l/h and output of the desired product of up to 50% from the deposited substrate.

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Description

The invention relates to biotechnology, specifically to biocatalysts based on cells of mycelial fungi included in the matrix of a polymer gel carrier, catalyzing the conversion of pentoses to ethyl alcohol, and is intended to increase the efficiency of biotechnological production of ethanol from cellulose-containing raw materials.

In modern industrial processes, ethanol is obtained microbiologically by fermentation of glucose using yeast cells as biocatalysts. As a source of glucose, chemical or enzymatic hydrolysates of starch or cellulose-containing products, agricultural or industrial wastes are used. In addition to glucose, they contain up to 25 g / l pentose (xylose, arabinose and ribose), not fermented by the traditionally used in industrial alcoholic races of yeast. To increase the economic attractiveness of the process of producing ethanol from cellulose-containing raw materials, it is advisable to use biocatalysts that provide ethanol from pentoses.

As such biocatalysts, natural strains of mycelial fungi of the genera Rhizopus Aspergillus, Fuzarium and Mucor are used, which are able to ferment various sugars, including pentoses, into ethanol [Olsson L., Hahn-Hägerdal B. (1996) Fermentation of lignoellulosic hydrolysates foranol . // Enzyme Microb. Technol., V.18, p. 312-331]. Biocatalysts, which are a developed metabolically active fungal mycelium, are obtained by culturing spores in rich nutrient media under aerobic conditions, followed by their use to produce ethanol from pentoses under anaerobic conditions. As a source of fermentable sugars, both solutions of individual pentoses and media containing pentoses as part of complex mixtures of sugars, in particular chemical and enzymatic hydrolysates of cellulose-containing raw materials [Millati R., Edebo L., Taherzadeh M.J., can be used. (2005) Performance of Rhizopus, Rhizomucor, and Mucor in ethanol production from glucose, xylose, and wood hydrolyzates. // Enz. Microb. Technol., V.36, p. 294-300].

The effectiveness of biocatalysts designed for the conversion of pentoses to ethanol is conveniently compared by the following indicators:

- the duration of the conversion process (h);

- ethanol yield based on the starting substrate (%);

- the productivity of the process or the rate of conversion (fermentation), defined as the concentration of the target product, ethanol, referred to the duration of the conversion process (g / l / h).

Known biocatalyst for the production of ethanol from pentoses, which is the mycelium of the fungus Aspergillus terreus [Pushalkar S., Rao KK (1998) Short communication: Ethanol fermentation by a cellulolytic fungus Aspergillus terreus. II World J. Microbiol. Biotechn., V.14, p. 289-291]. To obtain a biocatalyst, 10 ⁸ spores / ml are introduced into a nutrient medium containing 1% glucose and a set of salts necessary for cell growth, then cells are cultured under aerobic conditions at 28 ° C with constant stirring for 24 hours. The obtained biocatalyst under anaerobic conditions provides ethanol yield from xylose of 5.6% in 144 hours, and from arabinose - 8% in 120 hours. The productivity of the conversion process (fermentation rate) is 0.02 and 0.03 g / l / h, respectively. The main disadvantage of this biocatalyst is the extremely low productivity of the process that it provides.

Known biocatalyst based on cells of the mycelial fungus Mucor indicus for the production of ethanol from pentoses present in the medium as the sole source of fermentable sugars, or as part of sugar mixtures in acid wood hydrolysates [Sues A., Millati R., Edebo L., Taherzadeh MJ (2005) Ethanol production from hexoses, pentoses, and dilute-acid hydrolyzate by Mucor indicus. 11 FEMS Yeast Research, V.5, p.669-676]. To obtain a biocatalyst, 1 ml of a spore suspension with a concentration of (5-6) × 10 ⁶ spores / ml is added to 150 ml of a nutrient medium containing 33 g / l of glucose and the necessary set of salts and organic components. Cells are cultured until a metabolically active mycelium is formed at 30 ° C under aerobic conditions with constant stirring for 24 hours. When converting individual pentoses, the ethanol yield is: from xylose 18% in 88 hours, from arabinose 15% in 93 hours, which corresponds to the speed fermentation - 0,068 or 0,053 g / l / h, respectively. The yield of ethanol in the conversion of xylose, which is present in the composition of acid hydrolysates of coniferous wood waste, is 13.3%, and the fermentation rate is 0.025 g / l / h. The disadvantages of the biocatalyst are the low speed of the fermentation of pentoses and the relatively low yields of the target product.

The use of a similar catalyst obtained under conditions of elevated temperature (37 ° C) while controlling the pH of the medium (5.0 ± 0.7) allows to increase the yield of the target product to 22% of the introduced substrate, and the fermentation rate to 0.11 g / l / h [Millati R., Edebo L., Taherzadeh M.J. (2005) Performance of Rhizopus, Rhizomucor, and Mucor in ethanol production from glucose, xylose, and wood hydrolyzates.// Enz. Microb. TechnoL, V. 36, p. 294-300]. However, the use of elevated temperatures and the need to control pH technologically and economically complicate the process. An increase in temperature reduces the productivity of the biocatalyst under the action of accumulating ethanol.

Biocatalysts are known, which are cells of the mycelial fungi Rhizopus javanicus or Rhizopus oryzae, designed to produce ethanol from xylose [Skory CD, Freer SN, Bothast RJ (1997) Screening for ethanol-producing filamentous fungi. // Biotechn. Lett., V.19, p.203-206]. To obtain a biocatalyst, 10 ⁸ spores are added to 20 ml of culture medium containing 0.3% yeast extract, 0.5% peptone, 0.3% malt extract and 50 g / l xylose. Cells are cultured until a metabolically active mycelium is formed under aerobic conditions with constant stirring for 24 hours. Ethanol yield is 23.4% (Rhizopus javanicus) and 21.6% (Rhizopus oryzae) of the introduced substrate, and the fermentation rate is 0, respectively. 16 or 0.15 g / l / h.

A common drawback of all the described biocatalysts is that they are all designed for single use. The inability to reuse is associated with a rapid deterioration of the initial characteristics of the biocatalyst under the influence of ethanol accumulating in the medium and due to a decrease in the pH of the medium due to the dissolution of CO ₂ released during fermentation and the accumulation of by-products, in particular acetic acid and other organic acids. The inability to reuse the biocatalyst creates the problem of the constant build-up of fresh biomass and the disposal of waste cells and makes the process economically inefficient.

Improving the characteristics of the biocatalyst and increasing its attractiveness for use in industrial processes allows its production in an immobilized form.

Immobilization allows you to increase the resistance of mycelial fungal cells in relation to the negative effects of the above factors and increase the time during which the biocatalyst remains active.

Immobilized cell-based biocatalysts have greater technological and economic attractiveness due to the possibility of their repeated use, simplification of the procedure for separating cell biomass from the culture medium, and eliminating the need to utilize spent biomass after each working cycle.

Known biocatalyst for producing ethanol from xylose, which is a cell of the mycelial fungus Mucor circinelloides ATCC 1216 B, distributed in the matrix of the protein polymer poly-D-lysine [Lubbehusen TL, Nielsen. J., Mclntyre M. (2004) Aerobic and anaerobic ethanol production by Mucor circinelloides during submerged growth.//Appl. Microbiol. Biotechnol, V. 63, p. 543-548]. To obtain a biocatalyst in a cell, which consists of two coverslips (26 mm × 76 mm), separated by a thin gasket and placed in a metal case with holes for supplying the substrate and draining the culture fluid, pour 1 ml of a 0.1% polymer solution of poly- D-lysine with the properties of polycationite. Further, spores are introduced into the cell to their final concentration of 50-80 spores in the cell. Spore immobilization is based on the adsorption of spores on glass due to multiple electrostatic interactions between negatively charged chemical groups located on the surface of spores and a positively charged protein polymer covering the glass. Then, a nutrient medium containing 20 g / l xylose, 4.83 g / l gelatinized rice flour, 60 g / l nicotinic acid, 60 g / l thiamine, 60 g / l Na ₂ HPO is pumped through the cell at a speed of 3 ml / h ₄ 30 g / l KH ₂ PO ₄ 5 g / l NaCl, 10 g / l (NH ₄ ) Cl. Cell cultivation is carried out at a pH of about 5.0 at 28 ° C under aerobic conditions for 40 hours before the formation of a metabolically active mycelium. Such a biocatalyst provides 0.08 g / l ethanol for 40 hours at a conversion of 20 g / l xylose. The yield of the target product is 0.4% of the introduced substrate, and the fermentation rate is 0.002 g / l / h. On arabinose, the activity of the biocatalyst has not been studied. Unlike the given analogues, this catalyst can be used in a flow reactor, that is, repeatedly in the volume of the same reactor.

This technical solution, as the closest to the claimed type of biocatalyst, which is immobilized cells of a microscopic fungus that can ferment pentoses into ethanol, is taken as a prototype.

The main disadvantage of the biocatalyst adopted for the prototype is its low productivity, due to the following reasons:

1. The applied method of immobilization allows you to initially enter into the biocatalyst a very low concentration of spores distributed over the surface of the carrier. The number of active immobilized cells of the biocatalyst, which determines its productivity, is limited by the geometric dimensions of the reactor, since immobilization is carried out due to electrostatic interactions of spores with a polymer distributed on the surface of glass plates. As a result, the possibility of increasing the productivity of the process is limited by the possibility of increasing the geometric dimensions of the reactor. This circumstance makes it difficult to scale the process.

2. The pH dependence of the strength of the electrostatic interaction between immobilized cells and polylysine in the biocatalyst. When the pH shifts to the acidic region as a result of the accumulation of metabolites and partial dissolution of the CO ₂ formed during fermentation, the strength of the electrostatic complex decreases, part of the immobilized cells leaves the biocatalyst, which leads to a decrease in its productivity. The need to maintain the pH at a constant level complicates the technology and increases the cost of obtaining the target product.

3. The functioning of the biocatalyst is designed only for aerobic conditions in which the bulk of the consumed substrate is used by the cells to accumulate biomass. The possibility of the functioning of this biocatalyst under anaerobic conditions, which are known to be most favorable for reducing the cell growth rate and the accumulation of the target product [Schlegel G. General Microbiology. // M .: Mir, 1987, 567 pp.], The authors of the development is not provided.

The objective of the invention is to obtain a biocatalyst based on immobilized cells of mycelial fungi for the production of ethanol from pentoses, which long retains high productivity and is suitable for repeated use in a batch process.

The problem is solved in that the biocatalyst for producing ethanol from pentoses contains the mycelium biomass of a microscopic fungus capable of fermenting pentoses into ethanol included in the gel matrix of the polymer carrier, while the biocatalyst contains cryogel of polyvinyl alcohol as a gel matrix in the following ratio of components (wt. .%):

microscopic fungus mycelium biomass - 7.5 ÷ 20.0;

polyvinyl alcohol - 2.5 ÷ 5.0;

water - up to 100.

As the biomass of the mycelium of a microscopic fungus, the mycelium biomass of any microscopic fungi capable of producing ethanol from pentoses can be used.

PVA cryogel formed during freezing-thawing of an aqueous solution of polyvinyl alcohol is characterized by good performance characteristics, as well as high porosity of the resulting gel matrix, which provides easy diffusion of substrates and products [Lozinsky V.I. A new family of macroporous and supermacroporous materials for biotechnological purposes - polymer cryogels. // Proceedings of the RAS. Ser. Chemical, 2008, No. 5, p.1-18]. PVA cryogel is chemically stable at low pH values, as well as in the presence of ethanol and other microbial metabolites that accumulate during the production of ethanol.

The immobilization of mycelial hyphae in the volume of the PVA cryogel leads to their spatial separation by the porous structure of the matrix, which, unlike the prototype, allows the creation of high concentrations of cells uniformly distributed and fixed in the carrier in the reactor volume, and thereby increase the rate of ethanol accumulation in the culture medium. Such a spatial distribution of cells creates improved conditions for metabolism, which provides an increase in the yield of the target product and increase the productivity of the process.

The inventive biocatalyst provides high productivity of the process for a long time, the half-inactivation period is from 17 to 36 days, depending on the culture.

The convenience of separating the immobilized biomass from the culture medium in combination with the ability to maintain high productivity for a long time allows the inventive biocatalyst to be used repeatedly under batch conditions to produce ethanol from pentoses.

Quantitative ratios in which the components are part of the inventive biocatalyst are found experimentally.

The pore size in the forming matrix depends on the amount of PVA in it, which is determined by its concentration in the initial mixture with spores, which must be frozen and thawed. When the PVA content is less than 2.5 wt.%, Too large pores are formed through which spores are washed out from the carrier, accompanied by a decrease in the productivity of the biocatalyst and a decrease in its mechanical strength. When the PVA content in the biocatalyst is higher than 5.0 wt.%, The viscosity of its initial solutions in suspension with spores increases, which complicates the uniform dispersion of spores, and also leads to a noticeable decrease in pore size. The latter circumstance leads to a deterioration in mass transfer conditions inside the biocatalyst and a decrease in its productivity.

The lower limit of the concentration of mycelium biomass in the claimed composition of the biocatalyst is determined by the fact that at a concentration below 7.5 wt.% The resulting biocatalyst has insufficient productivity, and the upper limit of the concentration of biomass is determined by the fact that when the mycelium accumulates in the pores of the polymer matrix over 20.0 wt. .% there is clogging of the pores with cells of the metabolically active mycelium, worsening the conditions of mass transfer inside the biocatalyst granules and reducing the effectiveness of the biocatalyst. Thus, exceeding the specified upper limit of the concentration of cells is impractical.

The inventive biocatalyst is prepared as follows: a suspension of spores of the mycelial fungus is added to an aqueous PVA solution containing the calculated amount of polymer, mixed until a homogeneous mass is obtained, which is then used to form a PVA cryogel with simultaneous inclusion of spores in its porous structure by freezing this mass and keeping it in a frozen state and subsequent thawing. To obtain a biocatalyst of the claimed composition, the obtained PVA cryogel is placed in a nutrient medium and cultivation is carried out under aerobic conditions to germinate spores inside the pores of the polymer matrix and form a metabolically active mycelium capable of producing ethanol from pentoses under anaerobic conditions.

The cultivation of immobilized spores is carried out in standard nutrient media containing the components necessary for the growth of mycelium throughout the volume of the polymer matrix [S. Semenov Laboratory media for actinomycetes and fungi. Directory. // M .: Agropromizdat, 1990, 240 pp.]. Spore material for an immobilized biocatalyst is obtained using known biotechnological techniques in accordance with the particular characteristics of the culture. After the accumulation of spores, they are washed away from the surface of dense nutrient media using traditional microbiological methods used to obtain a suspension of spores in aqueous solutions [Workshop on Microbiology. (Ed. By Netrusov A.I.). // M.: Publ. Academy, 2005, 608 pp.]. The cultivation of spores included in the polymer matrix, in order to build up mycelium inside the pores of the carrier and form a biocatalyst, is carried out under aerobic conditions at a temperature optimal for the cultivation of the corresponding microscopic fungus culture. The duration of cultivation is determined by the above limit concentrations of mycelial biomass accumulating in the granules. The accumulation of mycelial biomass is controlled by increasing the solids content of the biocatalyst using the standard method of bringing the sample to constant weight.

In contrast to the prototype, the inventive biocatalyst can be given any convenient shape, in particular the shape of spherical granules, irregularly shaped particles, gel blocks, sheets, etc. For this, the spore suspension in the polymer solution is either frozen in the appropriate form or equipment intended for this purpose is used (extruders, granulators, etc.). The possibility of varying the shape of the biocatalyst technically and technologically simplifies its practical application and scaling.

Obtaining ethanol using the inventive biocatalyst is carried out in a standard way, namely, as a result of the cultivation of immobilized cells under anaerobic conditions in reactors with liquid nutrient media containing pentoses. Sugar consumption and ethanol accumulation in the medium are controlled by known methods (chromatographic and spectrophotometric) using standard analytical equipment.

The inventive biocatalyst for producing ethanol from pentoses, which is a microscopic fungus mycelium capable of producing ethanol and their pentoses, is included in the polyvinyl alcohol cryogel matrix, when the claimed ratio of components is new and provides a technical result consisting in increasing the productivity of the process for producing ethanol from pentoses (increasing the conversion rate of pentoses to ethanol) and increasing the yield of the target product, ethanol. An additional technical result is that the inventive biocatalyst provides high productivity of the conversion process for a long time, which allows it to be used repeatedly.

Figure 1 presents the dynamics of the accumulation of ethanol in periodic processes of fermentation of pentoses with repeated use of the inventive biocatalyst based on cells of various mycelial fungi in various environments containing pentoses:

(▲) based on Aspergillus terreus F-1025 mycelial fungus cells to produce ethanol from xylose

(■) based on cells of the mycelial fungus Mucor circinelloides F-1262 to produce ethanol from arabinose

(●) based on the cells of the mycelial fungus Fuzarium oxisporum F-931 to obtain ethanol from pentoses that are part of the corn stalk enzymatic hydrolyzate.

The following examples illustrate the possibility of practical implementation of the proposed technical solution.

Example 1. Biocatalyst based on cells of the mycelial fungus Aspergillus terreus F-1025 to obtain ethanol from xylose

To obtain a biocatalyst, to 30 g of an 11% aqueous solution of polyvinyl alcohol add 3 ml of a suspension of spores of Aspergillus terreus F-1025 filamentous fungus with a concentration of 5 × 10 ⁶ cells / ml and mix until a homogeneous mass is obtained, which is then used to form using cryogranulation installation [RF Patent No. 2036095] spherical granules with a diameter of 1.5 ± 0.2 mm The granules are frozen at -20 ° C, kept in a frozen state for 16 hours, then thawed at 8 ° C. The obtained granules of polyvinyl alcohol cryogel (33 g) with the mycelial fungus spores included in it are placed in an aerobic reactor with 1 l of medium containing glucose (120 g / l), yeast extract (5 g / l), (NH ₄ ) ₂ SO ₄ (3.0 g / l), MgSO ₄ × 7H ₂ O (0.25 g / l), ZnSO ₄ × 7H ₂ O (0.25 g / l); NaCl (5.0 g / L), K ₃ HPO ₄ (3.0 g / L), and they are cultured at 28 ° C for 48 hours to germinate spores and accumulate mycelial biomass in the pores of the carrier. Thus formed biocatalyst with a total mass of 88.5 g has the following composition (wt.%): Mycelium biomass - 10.0; polyvinyl alcohol - 3.7; water - up to 100.

The obtained biocatalyst is placed in a batch anaerobic reactor (1 L) filled with medium (pH 5.0) containing xylose (45 g / L) as well as the main fermentable substrate, and also MgSO ₄ × 7H ₂ O salt (0.25 g / l); (NH ₄ ) ₂ SO ₄ (3.0 g / l); ZnSO ₄ × 7H ₂ O (0.25 g / L); NaCl (5.0 g / L); K ₂ HPO ₄ (3.0 g / l). The fermentation process is carried out at 30 ° C without stirring. Within 80 h, 20 g / l of ethanol was obtained, the yield of the target product was 44.4% of the introduced substrate, the fermentation rate was 0.25 g / l / h.

Example 2. Biocatalyst based on cells of the mycelial fungus Mucor circinelloides F-1262 to obtain ethanol from arabinose

To obtain a biocatalyst, 50 ml of a 12% aqueous solution of polyvinyl alcohol are added 5 ml of a suspension of spores of mycelial fungus Mucor circinelloides F-1262 with a concentration of 1 × 10 ⁵ cells / ml and mixed until a homogeneous mass is obtained, which is then poured into 96-well immunological flat bottom plates (0.25 ml per well) to form polyvinyl alcohol cryogel granules. The granules are frozen at -15 ° C, kept in a frozen state for 20 hours, then thawed at 4 ° C. The obtained polyvinyl alcohol cryogel granules (55 g) with the mycelial fungus spores included in it are placed in an aerobic reactor with 1 l of glucose-containing medium (100 g / l), yeast extract (8 g / l), (NH ₄ ) ₂ SO ₄ (3.5 g / l), MgSO ₄ × 7H ₂ O (0.2 g / l), ZnSO ₄ × 7H ₂ O (0.2 g / l); NaCl (3.0 g / L), K ₂ HPO ₄ (3.0 g / L), and they are cultured at 30 ° C for 62 hours to germinate spores and accumulate mycelial biomass in the pores of the carrier. Thus formed biocatalyst with a total mass of 200 g has the following composition (wt.%): Mycelium biomass - 14.5; polyvinyl alcohol - 3.0; water - up to 100.

To obtain alcohol, the biocatalyst is placed in a batch anaerobic reactor (1 L) filled with medium (pH 5.0) containing arabinose (45 g / L) as the main fermentable substrate, as well as MgSO ₄ × 7H ₂ O salt (0, 25 g / l); (NH ₄ ) ₂ SO ₄ (3.0 g / l); ZnSO ₄ × 7H ₂ O (0.25 g / L); NaCl (5.0 g / l), K ₂ HPO ₄ (3.0 g / l) and carry out the fermentation process at 28 ° C without stirring. Within 46 h, 15.8 g / l of ethanol was obtained, the yield of the target product was 35% of the introduced substrate, the fermentation rate was 0.34 g / l / h.

Example 3. Biocatalyst based on cells of the mycelial fungus Rhizopus oryzae F-814 to obtain ethanol from ribose

To obtain a biocatalyst, to 10 g of a 12.5% aqueous solution of polyvinyl alcohol add 5 ml of a suspension of spores of mycelial fungus Rhizopus oryzae F-814 with a concentration of 1 × 10 ⁶ cells / ml and mix until a homogeneous mass is obtained, which is then poured into 96- well immunological plates with a spherical bottom (0.3 ml per well) to form polyvinyl alcohol cryogel granules. The granules are frozen at -15 ° C, kept in a frozen state for 14 hours, then thawed at room temperature. The resulting polyvinyl alcohol cryogel granules (15 g) with the mycelial fungus spores included in it are placed in an aerobic reactor with 300 ml of glucose-containing medium (100 g / l), yeast extract (3 g / l), (NH ₄ ) ₂ SO ₄ (3.0 g / L), MgSO ₄ × 7H ₂ O (0.2 g / L), ZnSO ₄ × 7H ₂ O (0.2 g / L); NaCl (1.0 g / l), K ₂ HPO ₄ (3.0 g / l), and they are cultured at 28 ° C for 36 hours to germinate spores and accumulate mycelial biomass in the pores of the carrier. Thus formed biocatalyst with a total mass of 50 g has the following composition (wt.%): Mycelium biomass - 20.0; polyvinyl alcohol - 2.5; water - up to 100.

The obtained biocatalyst is placed in a batch anaerobic reactor (1 L) filled with medium (pH 5.0) containing ribose (45 g / L) as the main fermentable substrate, as well as MgSO ₄ × 7H ₂ O salt (0.2 g / l); (NH ₄ ) ₂ SO ₄ (2.5 g / l); ZnSO ₄ × 7H ₂ O (0.2 g / L); NaCl (3.0 g / L); K ₂ HPO ₄ (3.0 g / l) and carry out the fermentation process at 28 ° C without stirring. Within 63 h, 12.6 g / l of ethanol was obtained, the yield of the target product was 28% of the applied substrate, the fermentation rate was 0.20 g / l / h.

Example 4. Biocatalyst based on cells of the mycelial fungus Fuzarium oxisporum F-931 to obtain ethanol from pentoses that are part of the enzymatic hydrolyzate of corn stalks

To obtain a biocatalyst, to 100 g of a 10% aqueous solution of polyvinyl alcohol add 1 ml of a suspension of spores of mycelial fungus Fuzarium oxisporum F-931 with a concentration of 1 × 10 ⁸ cells / ml and mix until a homogeneous mass is obtained, which is then poured with an even layer with a thickness of 0. 3 cm on a baking sheet, which is placed in a freezer at -13 ° C, where it is kept frozen for 18 hours, thawing is carried out at 4 ° C. A cryogel of polyvinyl alcohol is obtained in the form of a sheet material (100 g) with the spores of the mycelial fungus included in it, which is crushed into granules of arbitrary shape with a linear size of no more than 2.5 mm using a screw extruder and a cutter. The granules obtained are placed in an aerobic reactor with 1 l of medium containing malt wort (100 g / l), (NH ₄ ) ₂ SO ₄ (3.0 g / l), MgSO ₄ × 7H ₂ O (0.25 g / l ), ZnSO ₄ × 7H ₂ O (0.1 g / L); NaCl (2.0 g / l), K ₂ HPO ₄ (3.0 g / l), and they are cultured at 28 ° C for 52 hours to germinate spores and accumulate mycelial biomass in the pores of the carrier. Thus formed biocatalyst with a total mass of 200 g has the following composition (wt.%): Mycelium biomass - 7.5; polyvinyl alcohol - 5.0; water - up to 100.

The obtained biocatalyst is placed in a batch anaerobic reactor (1 l) filled with medium (pH 4.5), which is an enzymatic hydrolyzate of corn stalks containing pentoses (xylose 7.9 g / l and arabinose 0.1 g / l) and hexoses (glucose 2.4 g / l and galactose 0.6 g / l), and carry out the fermentation process at 30 ° C without stirring. Within 24 hours, 5.5 g / l of ethanol was obtained from all sugars present in the medium, while the yield of the target product from consumed pentoses was 50%, the fermentation rate was 0.23 g / l / h.

Example 5. Biocatalyst based on cells of the mycelial fungus Aspergillus terreus F-2036 to obtain ethanol from pentoses that are part of the beet pulp enzymatic hydrolyzate

To obtain a biocatalyst, 100 ml of a 13% aqueous solution of polyvinyl alcohol add 15 ml of a suspension of spores of Aspergillus terreus F-2036 mycelium fungus with a concentration of 2 × 10 ⁷ cells / ml and mix until a homogeneous mass is obtained, which is then poured into 96-well immunological dies with a spherical bottom (0.15 ml per well) to form polyvinyl alcohol cryogel granules. The granules are frozen at -20 ° C, kept them frozen for 10 hours, then thawed at 4 ° C. The obtained polyvinyl alcohol cryogel granules (115 g) with the mycelial fungus spores included in it are placed in an aerobic reactor with 1 l of medium containing glucose (120 g / l), yeast extract (8 g / l), (NH ₄ ) ₂ SO ₄ (3.0 g / l), MgSO ₄ × 7H ₂ O (0.25 g / l), ZnSO ₄ × 7H ₂ O (0.25 g / l); NaCl (2.0 g / l), K ₂ HPO ₄ (3.0 g / l), and they are cultured at 30 ° C for 32 hours to germinate spores and accumulate mycelial biomass in the pores of the carrier. Thus formed biocatalyst with a total weight of 320 g has the following composition (wt.%): Mycelium biomass - 12.8; polyvinyl alcohol - 4.1; water - up to 100.

The obtained biocatalyst is placed in a batch anaerobic reactor (1 l) filled with medium (pH 4.65), which is an enzymatic hydrolyzate of unclarified beet pulp fibers containing pentoses (arabinose 10.4 g / l and xylose 0.2 g / l) and hexoses (glucose 13.0 g / l and fructose 2.2 g / l), and carry out the fermentation process at 28 ° C without stirring. Within 42 hours, 12.2 g / l of ethanol was obtained from all sugars present in the medium, while the yield of the target product from consumed pentoses was 43.4%, the fermentation rate was 0.29 g / l / h.

Example 6. An example of repeated use of the inventive biocatalyst to obtain ethanol from pentoses in a batch process.

With repeated use of the inventive biocatalyst in a batch process after completion of one cycle, carried out under the conditions described in the corresponding Examples, the culture fluid containing the target product is poured through a mesh filter installed in the reactor, after which a new portion of the substrate-containing solution is added to the reactor and the next cycle is carried out in similar conditions.

The drawing shows the dependence of the final concentration of ethanol accumulating in the medium at the end of each working cycle, carried out as described in Examples 1 (▲), 2 (■) and 4 (●), with repeated use of the biocatalyst in the periodic process of fermentation of pentoses. The presented data confirm the possibility of repeated use of the inventive biocatalyst to obtain ethanol from pentoses in a batch process, and also allow you to determine the half-inactivation period of the biocatalysts shown in Examples 1, 2 and 4, which is 869 hours, 810 hours and 405 hours, respectively.

The above examples show that the inventive biocatalyst can be used to produce ethanol both once and repeatedly in batch processes for the fermentation of various pentoses (xylose, arabinose, ribose), taken individually or in mixtures with other sugars, while it provides an increase in speed conversions up to 0.34 g / l / h and the yield of the target product up to 50% of the applied substrate.

Thus, the proposed technical solution can be used to increase the efficiency of biotechnological production of ethanol from cellulose-containing raw materials.

Claims (1)

Biocatalyst for producing ethanol from pentoses, characterized in that it contains immobilized mycelium of a microscopic fungus capable of fermenting pentoses into ethanol, incorporated into a polyvinyl alcohol-based cryogel, in the following ratio, wt.%:
microscopic fungus mycelium 7.5 ÷ 20.0 polyvinyl alcohol 2.5 ÷ 5.0 water up to 100

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