CS209042B1 - A method for pretreating dry biomass of microorganisms for extraction - Google Patents

Abstract

The invention relates to the technology of production of protein products by microbiological method, in particular the technology of extraction purification of biomass of microorganisms from residual lipids or residual hydrocarbons and lipids. More precisely, the invention relates to preliminary treatment of dry biomass of microorganisms for extraction. The method consists in that the dry biomass is treated with a wetting mixture, which consists of at least one organic solvent and water, with expulsion of organic solvents from the wetting mixture, which is carried out at a pressure reduction *from atmospheric pressure to a residual pressure of 0.05 to 0.01 MPa. The invention can find application in the production of food and feed proteins.

Description

The invention relates to the technology of production of protein products by microbiological method, in particular the technology of extraction purification of biomass of microorganisms from residual lipids or residual hydrocarbons and lipids (the composition of residual substances depends on the nutrient medium used in the production of biomass). More precisely, the invention relates to a method of pre-treatment of dry biomass of microorganisms for extraction (the water content in dry biomass does not exceed 30% by weight).

Nowadays, methods are known _for the extraction of hydrocarbons and lipids from the biomass of microorganisms based on their extraction with organic solvents.

The efficiency of extraction of hydrocarbons and lipids from microbial biomass with organic solvents is largely determined by the internal structure of the biomass particles, taking into account the large diffusion resistance ^caused by the solid phase. To improve the particle structure, the solid phase is pre-treated for extraction.

At present, there are two main directions in the technology of extractive purification of dry biomass of microorganisms:’ extractive purification of biomass using mechanical pre-treatment of moisture and temperature of biomass for extraction and using gasoline as an extraction agent;

Extractive purification of powdered biomass using a multicomponent extraction agent.

The implementation of the first direction is associated with the granulation of biomass and the subsequent flattening of the granulate (the form of "petals"). For example, the method of extracting lipids from dry biomass of microorganisms cultivated on petroleum hydrocarbons according to the USSR Patent No. 207 309 consists in the fact that before extraction, dry powdered biomass is rolled in a series of passes through multi-pass rolling mills (where it is ground), the ground material is moistened, subjected to heat treatment and granulation, after which "petals" are formed from the granulate. After the above-mentioned preliminary treatment of biomass, lipids are extracted from the "petals" by using gasoline as an extraction agent. However, the direct grinding of dry biomass does not lead to any significant improvement in the internal structure of biomass particles due to the low coefficients of friction between the biomass and the surface of the rollers. In the method of extractive purification of biomass according to USSR Patent No. 461,641, the biomass, unlike the above-described procedure, is moistened before grinding, which improves the preliminary treatment of the biomass for extraction.

However, the described methods have significant drawbacks. Their use requires rather complicated and energy-intensive equipment (heating pans, rolling mills, granulation equipment). In addition, it is not possible to use highly effective water-containing extraction agents, since the “petals” of the biomass are disrupted in this case.

! The technology of extractive purification of powdered biomass using a multi-component extraction agent (second direction) allows to eliminate the mentioned shortcomings. In this case, the preliminary treatment of biomass for extraction is carried out by treating dry powdered biomass with a wetting mixture consisting of organic solvents and water.

For example, the method of extractive purification of dry biomass of microorganisms cultivated on petroleum hydrocarbons according to USSR copyright certificate No. 581,700 involves processing the biomass with a wetting mixture consisting of water, isopropyl alcohol and hexane in an amount of 5 to 60% by weight, 30 to 90% by weight, or 5 to 40% by weight, extraction being carried out with a mixture of the same solvents used in an amount of 0.5 to 10% by weight, 8 to 80% by weight, or 10 to 90% by weight.

In addition, a method of pre-treatment of dry biomass of microorganisms cultivated on petroleum hydrocarbons for extraction is known (USSR copyright certificate No. 591,501), which is carried out as follows:

Dry powdered biomass with an average particle size of 30 to 80 μm and a moisture content of 4 to 6% is suspended under intensive stirring in a non-polar solvent, for example in extraction gasoline, hexane, petroleum ether or in a mixture of the above non-polar solvents with a polar solvent, for example ethanol, isopropanol or any other polar solvent. In addition, the suspension can be carried out in a miscella (an extraction agent in which the extraction agents are dissolved). The weight ratio of the solid to the liquid phase when suspending the biomass can be 1:3 to 1:10. Water, saturated water vapor or a humidifying mixture of solvents including water is added to the suspended biomass until the moisture content in the biomass reaches 15 to 50% by weight and at the same time the temperature of the biomass reaches 45 to 65 °C. The suspension time, starting with the supply of water, saturated water vapor or a humidifying mixture, is 10 to 15 minutes.

Biomass, which has lyophilic properties, is adsorbed on the surface of the dispersed water droplets and is thus moistened. The biomass particles swell and form loose flocculent aggregates, which allow easy access of the extraction agent inside the pores of the particles. Further processing during extraction of such pre-treated biomass with various extraction agents yields a high-quality product containing a maximum of 0.1% residual hydrocarbons and a maximum of j 1 to 2% lipids. i

However, the described methods of pre-treatment of microbial biomass for extraction (USSR copyright certificate No. 581.700 and 591.501) have significant shortcomings.

The starting biomass, which is subjected to extraction purification after drying in a spray dryer, contains about 2 kg of air per 1 ton of biomass. Air makes it difficult for the solvent to penetrate into the pores of the solid particles, which leads to a decrease in the degree of extraction of the extractable material (lipids and hydrocarbons). The presence of air in the biomass also contributes to the flotation of the biomass particles in the solvent, which leads to an increased removal of the solid phase with the miscella from the extraction device; as a result, the performance of the device decreases.

Another disadvantage of the described method of pre-treatment of biomass for extraction is the need to introduce an excess amount of water (about 0.8 tons of water per 1 ton of biomass) into the solvent, solvent mixture or miscella, which is due to the uneven contact of water droplets with biomass particles. The introduction of a large amount of water complicates the process of solvent regeneration, while heat consumption is higher, and the quality of biomass is lower.

The aim of the invention is to eliminate the aforementioned shortcomings.

The invention has set itself the basic task of developing a method for pre-treatment of dry biomass of microorganisms, based on the treatment of dry biomass with a wetting mixture composed of at least one organic solvent and water, which would allow the creation of a biomass structure that would ensure an increase in the degree of extraction of the extractable material from the biomass, reduce the entrainment of biomass together with the miscella from the extraction device, as well as intensify the humidification of the biomass.

This problem is solved by proposing a method for pre-treating dry biomass of microorganisms for extraction, based on the treatment of dry biomass with a wetting mixture consisting of at least one organic solvent and water, wherein the treatment of dry biomass with a wetting mixture is carried out according to the invention with the expulsion of the organic solvent from the wetting mixture, carried out by reducing the pressure from atmospheric to residual (var 0.05 to 0.01 MPa.

The invention proposes the following variants for carrying out the above processes (processing of dry biomass and expulsion of organic solvent):

— the processing of dry biomass with a wetting mixture and the expulsion of organic solvents from the wetting mixture take place sequentially;

— processing of dry biomass with a wetting mixture is carried out by reducing the pressure from atmospheric to a residual pressure of 0.05 to 0.01 MPa while simultaneously expelling organic solvents from the wetting mixture. f

The method according to the invention ensures uniform humidification of biomass with a minimum (up to 5%) water content in the humidifying mixture. This can be deduced from the fact that during vacuum stripping, mainly the organic solvent evaporates from the humidifying mixture and the mixture is enriched with water, ensuring intensification of the humidification of biomass.

During vacuum distillation of organic solvents, air is intensively removed from the interior of the pores of biomass particles and additional channels and pores are formed for its removal to the surface of the particles together with a mixture of extracting substances in the gas-liquid phase, which ensures the formation of a biomass structure that is favorable for the extraction of hydrocarbons and lipids. Removing air from biomass during evacuation improves the access of the solvent (during extraction) to the interior of the pores, which allows to increase the degree of extraction of hydrocarbons and lipids. This also improves the precipitation of solid particles in the solvent < and thus reduces the removal of biomass together with the miscella from the extraction device.

The method of pre-treating the biomass of microorganisms for extraction according to the invention is carried out as follows.

Biomass of microorganisms (yeast, fungi, bacteria), which is cultivated on various nutrient media, for example on the basis of petroleum hydrocarbons or ethanol or methanol or natural gas, is dried to a residual moisture content of maximum 30% by weight, preferably 5 to 10% by weight. The average particle size of the biomass is 70 to 200 μπι. Then the biomass is processed with intensive mixing at a temperature of 10 to 50 ° C with a wetting mixture. The wetting mixture can be single-phase or even two-phase and can be a mixture of polar, non-polar organic solvent and water or non-polar organic solvent and water or polar organic solvent and water. The following compositions can be mentioned as examples of wetting mixtures: ; hexane-isopropanol-water, gasoline-ethanol-water, gasoline-water, hexane-water, ethanol-water, isorpolanol-water.

The water content in a three-component wetting system is usually 1 to 5% by weight, the non-polar solvent 40 to 80% by weight, the remainder being a polar organic solvent. In a two-component wetting mixture, the non-polar organic solvent and polar organic solvent content is 50 to 95% by weight, the remainder being water. The weight ratio of the liquid and solid phases, i.e. the wetting mixture and biomass, is 2:1 to 4:1. ,

Water can be introduced, with intensive stirring, into a previously obtained suspension consisting of biomass and the above-mentioned organic solvents, or into a suspension consisting of biomass, the above-mentioned organic solvents and a certain amount of water.

The biomass is treated with the wetting mixture within 10 to 20 minutes, expelling the organic solvent vapors and reducing the pressure from atmospheric to a residual pressure of 0.05 to 0.01 MPa.

In this process, organic solvents are expelled down to a residual content in the biomass of a maximum of 1% by weight.

The treatment of dry biomass with a wetting mixture and the expulsion of organic solvents from the wetting mixture can be carried out according to the invention as follows:

— the treatment of dry biomass with a wetting mixture and the expulsion of organic solvents from the wetting mixture may be carried out one after the other. In the above case, the said treatment with the wetting mixture is carried out at atmospheric air pressure and the expulsion — with a reduction in pressure from atmospheric to a residual pressure of 0.05 to 0.01 MPa;

— processing of dry biomass with a humidifying mixture and expulsion of organic solvents from the humidifying mixture is carried out simultaneously with reduced air pressure

209042 __ from atmospheric to residual pressure 0.05 to 0.01 MPa.

The biomass pretreated by the method according to the invention is then subjected to extractive purification with organic solvents or mixtures thereof by any known method.

For a better understanding of the invention, the following specific examples are given. The results of the extraction of extractables (lipids or hydrocarbons and lipids) from biomass after its preliminary treatment for extraction are demonstrated in the table following the examples.

Example 1 I

Biomass of powdered yeast Candida ^{quiliermondii} , cultivated on purified liquid paraffins, with a particle size of 70 to 200 pm and a residual moisture of 8.1% by weight, with a total content of extractive substances (hydrocarbons and lipids) of 3.6% by weight, is treated in an amount of 120 g, at a temperature of 50 °C and with stirring, for 10 minutes with a mixture of gasoline and ethanol in a ratio (% by weight) of 80:20. Water is added to the obtained suspension in an amount of 2'°/γ, based on the weight of the mixture of the above salts within 10 minutes. As a result, a suspension of biomass is obtained in the growth mixture at a weight ratio of the liquid and solid phases of 2:1. Evacuation and expulsion of organic solvents from the growth mixture is carried out with the addition of water. The residual pressure is 0.05 ^- to 0.04 MPa. The residual content of organic solvents in the mass after expulsion is about 1% by weight.'''' · ¹

After processing the biomass with a wetting mixture and removing organic solvents, the suspension is filtered, and then the biomass is subjected to a three-stage

SO2 cleaning at a temperature of 50 °C and a duration of ^1st stage of 20 min. A mixture of the following composition (% by weight) is used as an extraction agent: gasoline 80, ethanol 19, water 1. The weight ratio of extraction agents and biomass is 2:1.

At each stage, the suspension is filtered and the total content of extractables (hydrocarbons and lipids) in the mixture is analyzed.

Example 2

The experimental conditions and methodology are similar to those in Example 1. The difference is that the pressure is reduced from atmospheric to a residual pressure of 0.03 to 0.02 MPa.

Example 3; The experimental conditions and methodology are similar to those in Example 2. The difference is that yeast strains cultivated on petroleum distillates are used. The initial total content of extractives (hydrocarbons and lipids) in the biomass is 22.3% by weight.

Example 4

The experimental conditions and methodology are similar to those described in Example 1. The difference is that the biomass of dry powdery fungi Bl. Trisporo is used, which were cultivated on an ethanol-based nutrient medium. The moisture content of the biomass is 5% by weight. The initial content of extractive substances (lipids) in the biomass is 18% by weight. The pressure is reduced from atmospheric to a residual pressure of 0.02 to 0.01 MPa.

Example 5

L 1 The experimental conditions and methodology are similar to those of example 1. The difference is that the biomass of dry powdered yeast Rhodoto| rula aurantica is used, which was cultivated on a nutrient medium based on ethanol. The moisture content of the biomass is 2.9% | by weight. The initial content of extractive substances (EpídA) in the biomass is 39.3% by weight. A mixture consisting of 60% by weight, ethanol and 40% by weight. water is used as a wetting mixture. The pressure is reduced from atmospheric to a residual pressure of 0.05 to 0.03

MPa.

PHUad 6

The experimental conditions and methodology are similar to those in Example 1. The difference is that the treatment of dry biomass with a wetting mixture at atmospheric air pressure and the expulsion of organic solvents from the wetting mixture is carried out 4 minutes after the said treatment with a pressure reduction from atmospheric to a residual pressure of 0.05 to 0.04 MPa.

i ¹ i ·

Example 7

The experimental conditions and methodology are similar to those in example 1. The difference is that the processing of dry biomass with a wetting mixture and the expulsion of organic solvents from the mixture are carried out by reducing the pressure from atmospheric to a residual pressure of <0.03 to 0.01 MPa. - - - :

·'' : ·>·'· · ... , ¹ .'. ' Example 8

The experimental conditions and methodology are similar to those in Example 1. The difference is that the processing of dry biomass with a wetting mixture is carried out at a temperature of 15 °C. The aforementioned processing and expulsion of organic solvents from the wetting mixture is carried out under pressure reduction from atmospheric to a residual pressure of 0.02 to 0.01 MPa.

Example 9 (check)

The experimental conditions and methodology are similar to those in Example 1, with the exception that the treatment of biomass with the wetting mixture is carried out at atmospheric air pressure and without the expulsion of organic solvents (according to a known method).

The structure of the biomass obtained as a result of the preliminary treatment of the biomass for extraction according to [ Examples 1 to 9, was evaluated according to the degree of total extraction of extractive substances, which was achieved in the three-stage extraction purification. ________ ___

The table shows comparative results of extraction of extractive substances from biomass that was pre-treated for extraction according to the invention (Examples 1 to 8) and the known method (Example 9).

Table

Number attached Composition of the humectant mixture ¹ % by weight. Residual pressure MPa Composition of the extraction agent % wt. Extraction degree of extractive substances according to extraction degrees, % by weight 1st degree 2nd degree 3rd degree Total extraction degree of extractives, % by weight. 1 ' 2...... ' _T ,. . 4 5 '...... 6 7 8 1 gasoline-80 · ' ethanol-20 water, which is added to the suspension in an amount of 2% by weight of the listed organic solvents 0.05-0.04 gasoline-80 ethanol-19 51.2 9.1 2.3 62.6 2 99 0.03-0.02 _99 63.1 8.3 4.9 76.3 3 _5» 99 45.0 30.5 12.4 87.9 4 5 ethanol-60 0.02-0.01 >» 61.2 12.1 5.0 78.3 6 water 40 similar to 0.05-0.03 99 45.8 9.7 5.7 59.2 in example 1 0.05-0.04 99 51.3 10.3 5.8 67.4 7 similar to example 1 0.03-0.01. gasoline-80 ethanol-19 water 1 69.1 9.0 3.1 81.2 8 similar to example 1 0.05-0.04 gasoline-80 ethanol-19 water 1 48.2 8.3 2.9 . 59.4 9 (control) 9» 1 atmospheric air pressure without expelling organic solvents 99 41.5 10.5 3.3 52.3

As is evident from the table, the humidification of biomass ! with the expulsion of organic solvents from the humidification mixture significantly increases the extraction degree of extractable substances (lipids or hydrocarbons and lipids) compared to the known method. After the extraction cleaning, the miscella contains practically no solid particles, which ! indicates that the biomass is not carried out together ! with the miscella from the extraction device.

Claims (2)

SUBJECT A process for pretreating both dry biomass and microorganisms for extraction, wherein the dry matter is dried up ^. ; the biomass treats the humidifying mixture, which also consists of at least one organic; solvent and water, characterized in that the treatment of the dry biomass with the humidifying mixture is carried out while expelling the organic solvents from the | a humidifying mixture which is carried out under reduced pressure from atmospheric to a residual pressure of 0.05 to 0.01 MPa. OF THE INVENTION .................................................. .. \ 2. The process of claim 1, wherein the dry biomass is treated successively with the humidifying mixture and expelled organic solvents from the humidifying mixture. 3. A process according to claim 1, characterized in that ^one processing dry biomass humidifier effected at the pressure reduction from atmospheric pressure to a residual from 0.05 to 0.01 MPa, and simultaneous expulsion of organic solvents zvhléovací mixture.