RU2354135C2 - Method of processing plant raw material wastes - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: liquid-phase fermentation is carried out using thermophylic fungus strain Myceliophthora termophila F-2109. Hydrolysis of plant raw material of liquid-phase derived during liquid-phase fermentation and containing ferments providing degradation of starch, cellulose and lignin is carried out. Solid state fermentation is carried out in the layer of product mixture of liquid-phase fermentation and enzymatic hydrolysis of plant raw material not exceeding 15 mm. The processing is carried out by batch replacement of finished product with fresh medium.

EFFECT: fungus biomass is compatible with digestive tract microflora of animals, increase of plant raw material biodegradation.

1 dwg, 1 ex

Description

The invention relates to the technology of enzymatic hydrolysis of waste from the forest, woodworking and agricultural industries. More specifically, the production of feed additives from dihydroquercetin production wastes by the action of enzymes that degrade biopolymers such as lignin, cellulose and starch, and form soluble sugars. The invention can be used in the manufacture of a wide range of feed additives by enriching them with microbial synthesis products using a hydrolyzate and in research practice. Soluble sugars obtained by hydrolysis of lignin, cellulose and starch can be used for the production of medicines, food and feed protein preparations, alcohol for energy purposes.

Known methods of processing plant materials, which in fact the impact can be divided into four types: physical, mechanical, chemical and biological. Each of these methods individually contributes to the partial degradation of the complex structure of these biopolymers, which significantly increases the availability of processed raw materials for enzymatic hydrolysis.

Industrial methods of acid hydrolysis of wood to glucose are known, but they are accompanied by partial degradation of glucose itself and the formation of toxic impurities, which must be freed. To carry out this process requires expensive, energy intensive and corrosion resistant equipment.

The most promising is the use of enzymes that degrade lignin, cellulose and starch, which are used in the alcohol, brewing, food industries, bakery, feed production and others, where the raw materials are plant materials or plant processing waste. The use of these enzymes increases the yield of target products and allows you to approach the creation of non-waste technologies. The unresolved problem is that these biopolymers are very complex substrates for the action of enzymes and producers are not well understood that would be fully economically viable and satisfy the needs of industries using plant materials.

The known technology of processing plant materials in the process of solid-phase cultivation of higher basidiomycetes, such as Tyromyces lacteus and Goriolus hirsutus (I.M. Gracheva, A.Yu. Krivova. "Technology of enzyme preparations", NPO "ELEVAR", 2000, p. 315-317).

Mushrooms grew and accumulated biomass due to the breakdown of cellulose, hemidellulose, pectin and lignin. Culture on a solid substrate synthesized the whole complex of necessary enzymes. It was found that the degree of enzymatic degradation of these biopolymers was as follows: hemicelluloses decomposed, degraded with the growth of xylotrophs (white rot mushrooms) by 58%, pectin substances - by 94%, cellulose - 53%. Lignin destruction of plant substrates was 40-45%. At the same time, due to the growth of fungi in the solid substrate, the content of protein and easily digestible carbohydrates increased. The degradation of the substrate by fungi began after the completion of active growth. The most active process proceeded within 15 days.

Known technology is implemented according to the traditional scheme of operations. Plant materials are loaded into a container. The container is sealed and sterilized with sharp steam at a temperature of 120 ° C for 1 hour. After sterilization, the container is cooled to ambient temperature and an inoculum culture of microorganisms producing enzymes degrading lignin, cellulose and starch is introduced into a solid substrate. After sowing, the fungus is cultured under the specified conditions of humidity, temperature, aeration and process time. The performance of solid-phase processes directly depends on the optimization of mass transfer parameters and maintaining the humidity of the working environment.

Thus, an increase in humidity disturbs the loose texture of the medium, mushroom mycelium grows only from the surface, and a zone with a low oxygen content is formed inside, growth in which is impossible, which leads to a significant decrease in the production of fermentation products due to incomplete use of the substrate.

In addition, in these zones toxic products of biomass autolysis accumulate, which leads to a significant decrease in the quality of the target products.

Another disadvantage of the known process is the duration and high energy intensity associated with difficulties in controlling and providing aseptic conditions for carrying out the solid-phase process, which significantly increases the cost of manufactured products.

The high energy costs of carrying out solid-phase fermentation are also associated with periodic conditions for the implementation of the process, the profitability of which can be achieved only by using large-capacity technological equipment, large production areas and laborious manual labor to service the preparatory stages of the process, which significantly reduces the competitiveness of the equipment used.

The well-known "Biotechnological method of complex processing of waste products of palm oil production" (RF patent N 2161415, А23К 1/00, 2001.01.10 - prototype).

The method consists in the fact that solid waste is subjected to acid hydrolysis. Hydrolyzed solid waste is mixed with liquid waste. Inoculation is carried out by the biomass of the bacterial strain Acinetobacter baumanni M-1. Liquid-phase fermentation is carried out at a pH of 6.5-7.5 and a temperature of 35-45 ° C. The resulting microbial suspension is separated. Post-treatment of the liquid stream is carried out. Condensed biomass is subjected to heat treatment at 90 ° C for 1 hour. Then dried and get fodder protein. Solid waste and solid fraction formed after liquid-phase fermentation are subjected to solid-phase fermentation at a pH of 6.5-7.5 and a temperature of 35-45 ° C. Get the finished product - compost. The method ensures the purity of palm oil production and allows to obtain marketable products in the form of feed protein and compost.

The disadvantages of this method include acid hydrolysis of solid waste, which, as you know, is accompanied by partial degradation of sugars and the accumulation of toxic impurities, which requires additional procedures for the purification of the obtained substances before their use in the liquid phase fermentation process.

When carrying out solid-phase fermentation in the specified method, solid waste that is not processed and seeded by extraneous microflora from palm oil production is used as a nutrient medium, and solid waste inseminated by microflora from liquid-phase fermentation with a low concentration of microorganisms as a seed culture is used, which leads to a significant increase solid-phase fermentation time (as indicated in the example, 12 days) and, accordingly, significant energy consumption.

Using the bacterial culture Acinetobacter baumanni M-1 to obtain feed protein does not provide sufficient compatibility of mushroom biomass with microflora of the gastrointestinal tract of animals.

A significant temporary difference in the processes of hydrolysis, liquid-phase and solid-phase fermentation and the implementation of post-treatment of intermediate products do not allow to automate the process, which reduces the competitiveness of the known technology.

The technical result of the invention is to obtain a processing product suitable as a feed additive, reducing the processing time of plant materials, increasing the depth of degradation of the raw materials and the productivity of the method.

This result is achieved by the fact that the method of processing waste plant materials involves liquid-phase fermentation using a thermophilic strain of the fungus Myceliophthora termophila F-2109, hydrolysis of plant materials with a liquid-phase fraction obtained by liquid-phase fermentation, and containing enzymes that degrade starch, cellulose and lignin, and solid phase fermentation carried out on a mixture of liquid-phase fermentation products and enzymatic hydrolysis of plant materials in a mixture layer not exceeding 15 mm, at processing is carried out under conditions of batch replacement target fresh medium product.

As a result of the implementation of the method of the present invention, significant advantages have been identified over known analogues, namely:

- the use of a thermophilic strain of the fungus Myceliophthora termophila F-2109 [Institute of Biochemistry and Physiology of Microorganisms. All-Russian Collection of Microorganisms, Myceliophthora termophila (Apinis 1963) van Oorschot 1977, F-2109 ← INMI, VKM F-2109 ← TUB, WFPL 264A. (WFPL 264A). Obtained as: Sporotrichum termophilum. Synonyms: Sporotrichum termophilum Apinis 1963 (Wednesday 11, 45 C, L-1, C-5, B-4, K-1)], isolated from the rumen of ruminants, allowed to solve the problem of compatibility of fungal biomass with microflora of the gastrointestinal tract of animals and the high temperature of cultivation of the fungus and enzymatic hydrolysis of plant materials significantly increased the aseptic reliability of the processes and the quality of the produced feed additives;

- the use of products obtained from enzymatic hydrolysis and cultivation of fungi, significantly intensified enzymatic reactions at all stages of the process under study and thereby reduce the total processing time of plant materials;

- carrying out solid-phase fermentation on a hydrolyzed substrate has allowed a multiple reduction of the process time and thereby significantly increase its productivity;

- periodic batch replacement of the target product with fresh nutrient medium in the continuous process of enzymatic reactions has significantly reduced the cost of manufactured products, increased the degradation depth of the complex structure of lignin, cellulose and starch to glucose and to ensure the competitiveness of the new technology.

The technological scheme of the process of processing plant waste material is schematically shown in the drawing, which shows a container 1 with a nutrient medium, a flask 2 with a sowing crop, a sealed container 3 equipped with a heat-exchange jacket and a bubbler aerator (liquid-phase cultivation apparatus), a container 4 for vegetable raw materials, a sieve 5 with holes 1 mm (fraction separator), a sealed container 6 with a heat-exchange jacket (hydrolysis apparatus), autoclave 7 (solid-phase apparatus).

In the process of processing plant materials, the cultivation of microorganisms - producers of biomass and target enzymes, was carried out on the well-known nutrient medium Chapek, in which an additional 30% of sawdust was added.

The producer used the thermophilic strain of the fungus Myceliophthora termophila F-2109 isolated from the rumen of ruminants, which was grown in flask 2 on a shaker at a temperature of 45 ° C for 1.5 days.

Liquid-phase cultivation of mushrooms was carried out in apparatus 3, equipped with a heat-exchange jacket and a bubbler type aerator at a temperature of 50 ° C and an air flow rate of 3 l / min.

Enzymatic hydrolysis of sawdust was carried out in apparatus 6, equipped with a heat-exchange jacket, at a temperature maintained in the range of 50-65 ° C.

The products of liquid-phase cultivation of mushrooms were filtered through a sieve 5 with 1 mm openings.

Solid phase fermentation was carried out in an autoclave 7, in which a sieve 5 with fermented sawdust enriched with biomass of mushrooms was installed. Solid phase fermentation was carried out at a temperature of 50 ° C without forced ventilation.

Drying of the products of processing (biodegradation) of plant materials was carried out on wire racks placed in an oven at a temperature of 90 ° C. All operations to implement the method were performed manually.

Example. A method of processing (biodegradation) of plant materials was performed as follows. Before the process of biodegradation of plant materials begins, 1.5 l of nutrient medium, composition (g / l) were introduced into apparatus 3 from tank 1:

NH ₄ NO ₃ - 3 g / l; CaCO ₃ - 0.5 g / l; NaH ₂ PO ₄ · 3H ₂ O - 3 g / l; MgSO ₄ · 7H ₂ O - 0.5 g / l; FeSO ₄ · 7H ₂ O — 0.01 g / l; KCl - 0.5 g / l; corn extract - 20 ml; starch - 10 g / l; sucrose - 30 g / l, sawdust (waste from the production of dihydroquercetin) in a volume of 0.5 DM ³ .

A solid substrate (a sawdust of Siberian larch - waste from the production of dihydroquercetin) weighing 2.7 kg was introduced into apparatus 6 with a volume of 3 dm ³ from a tank 4, after which apparatuses 3 and 6 were sterilized in an autoclave at a temperature of 120 ° С for 1 hour. After cooling apparatuses 3 and 6 to a temperature of 50 ° C, a 1.5-day inoculum of the fungus Myceliophthora termophila F-2109 in a volume of 200 ml was grown in apparatus 3 with a volume of 3 dm ³ , grown on a Chapek medium in a flask on a rocking chair, at a temperature of 45 ° C .

The cultivation of the fungus in apparatus 3 was carried out at a flow rate of aeration air of 3 l / min and a temperature of 50 ° C for 2 days, and in apparatus 6, the temperature was maintained in the range of 50-65 ° C.

After 2 days, 1 L of cultivation volume was drained from apparatus 3, which was passed through a sieve 5, resulting in a liquid fraction containing a solution of enzymes degrading starch, lignin and cellulose, and a solid fraction containing fermented sawdust and mushroom biomass. Then, 1 liter of fresh nutrient medium was introduced into apparatus 3.

The obtained liquid fraction containing the enzyme solution was introduced into the apparatus 6 for the enzymatic hydrolysis of sawdust, and the fermented sawdust enriched with mushroom biomass was placed on a sieve 5 with a thin layer. A sieve 5 was installed in an autoclave 7, in which a temperature of 50 ° C was maintained. Solid phase fermentation was carried out for 12 hours in an ambient atmosphere (without forced ventilation), which allowed at least five generations of fungal growth under optimal conditions with complete consumption by the fungi of residual nutrients from the fermented substrate.

After a 12-hour solid-phase cultivation of mushrooms, the sieve 5 was removed from the autoclave 7, and the produced product was dried to constant weight. Further, the process of degradation of plant materials was performed according to the algorithm:

- one liter of cultivation volume was drained from apparatus 3 into sieve 5;

- in the apparatus 3 of the tank 1 made one liter of fresh nutrient medium;

- on sieve 5, the merged volume was divided into solid and liquid fractions;

- hydrolyzed sawdust was removed from apparatus 6;

- sawdust weighing 2.7 kg and 1 liter of enzyme solution were introduced into apparatus 6 from tank 4;

- hydrolyzed sawdust from apparatus 6 was mixed with sawdust enriched with biomass of mushrooms obtained after fractional separation on a sieve 5, and placed with a layer height of 15 mm;

- a sieve 5 was installed in an autoclave 7, in which the 12-hour solid-phase fermentation process was repeated.

The indicated algorithm was repeated 10 times (5 days of a continuous process), while 27 kg of sawdust were processed and 27.5 kg of a fermented product enriched with readily available carbohydrates and biomass of mushrooms were obtained, and the productivity of processing (biodegradation) of plant materials using a 3 dm hydrolysis apparatus ³ was 5.4 kg / day.

Claims (1)

A method of processing waste plant materials, involving liquid-phase fermentation using a thermophilic strain of fungus Myceliophthora termophila F-2109, hydrolysis of plant materials with a liquid-phase fraction obtained by liquid-phase fermentation and containing enzymes that degrade starch, cellulose and lignin, and carry out solid-phase fermentation liquid-phase fermentation and enzymatic hydrolysis of plant materials in a mixture layer not exceeding 15 mm, while processing is carried out under conditions x portioned fresh medium replacement target product.