RU2044773C1 - Method for fermentation of carbohydrate-containing mediums with the help of bacteria which produce butanol, acetone, ethanol and/or isopropanol and device for its realization - Google Patents

Abstract

FIELD: fermentation. SUBSTANCE: method is carried out in two steps. The first step involves cultivation bacteria by mixing at pH 4.0-5.5 and/or at 30-40 C, rate of stream being 0.080.45 h^-1. At the second step bacteria are immobilized on porous carrier at ratio 1:10-1:11 or desired product is isolated by microfiltration. The second step is carried out at 27-40 C and pH 3.0-5.0, rate of stream being 0.14-0.4 h^-1. Caked glass having open pores or pumice or other material having high SiO₂-content and pores 20-200 μm in size are used as said carrier. Ratio of said carrier and volume of fermented medium is 1: 5-1:1.6, moisture capacity of said carrier being 0.35 ml/cm³. Desired product is isolated by extraction with higher alcohols or higher fatty acids, thus prepared phase being removed to the first and/or the second step of fermentation. Desired product also may be isolated by diffuse evaporation at 30-90 C through membrane of silicone rubber, nitrogen or hydrogen or carbon oxide, or their mixture. Gas distillation at 30-90 C also may be used to produce desired product, mentioned above gases being used. Device for realization of proposed method comprises two reactors being connected consequently. The former reactor is autoclave which has a mixer. The latter reactor has device for producing directed stream of circulation of medium and filler with immobilized microorganisms. Said reactor is connected with outside contour for isolation of desired product. EFFECT: improves efficiency of the method.

Description

 The invention relates to a method for fermentation of carbohydrate-containing media using bacteria producing butanol, acetone, ethanol and / or isopropanol, which consists of at least two stages, and in the first stage, the bacteria multiply continuously, and in the second, continuously or periodically, the target product is formed.

 In the known method (1), there are two usual stages of cultivation, and changing the flow rate or using phosphate control, the process is set so that in the first stage (propagation stage) the concentration of bacteria is high and remains constant, i.e. to establish dynamic equilibrium (stationary state) and already part of the substrate is processed into butanol, acetone and / or ethanol. At the second stage, in this case, the highest possible yield of the desired fermentation products should be achieved in the shortest possible time. If the duct velocity in the first stage is too high, more cells will be carried out than they are formed. If the flow rate is too low, cell growth may stop.

 This method has the disadvantage that it does not provide, on the one hand, obtaining sufficiently high yields, and on the other hand, the stability of the continuous production of culture for a long time.

 The purpose of the invention is the creation of a method by which for a long time it would be possible to stably obtain a high yield and which would allow the use of starting materials containing not only hexoses.

 The goal is achieved in that the bacteria in the second stage are immobilized on a carrier.

The proposed method in terms of flow velocity is very flexible, since viable bacteria in the second stage are retained on the carrier due to adhesion. Moreover, taking into account the process, the flow rate in the first stage can be chosen so that at this stage almost exclusively the formation of bacteria occurs, which would then be partially transferred by the stream to the second stage. As a result, a large population of bacteria is created in the second stage, since viable bacteria bind to the material of the carrier, and dead bacteria are washed out with the medium enriched in the target product. Thus, at the first stage, it is possible to work with such flow rates at which the concentration of the products formed does not yet exceed the toxic limit, therefore, the bacterial culture is not significantly damaged. It is preferable to use sintered glass with open pores, pumice or other similar material with a high SiO ₂ content and an open-porous structure with a pore size of 20-200, preferably 5-100 μm, and a moisture capacity of 0.35 or more ml / cm ³ as a carrier. In this case, the ratio between the volume of the carrier material and the total working volume should be in the range 1:10 1: 1.1, preferably 1: 5 1: 1.6, due to which a particularly high fermentation efficiency is achieved (in particular, if in the first stage while stirring the contents of the reactor, the flow rate is 0.08-0.45, preferably 0.1-0.38 h ^-1 , pH 4.0-5.5, preferably 4.3-5.1, most preferably 4.5 -5 and / or the temperature of 30-40, preferably 33-38 ^C, and in the second stage the flow rate is 0,14-0,4, preferably 0.25-0.35 h ^-1, pH 3,0-5 , 0, prefer flax 4.0-4.8, most preferably 4.2-4.5, and / or the temperature of 27-40, preferably 30-37 ^C, most preferably 32-35 ° ^C).

In another embodiment of the proposed method, it is possible to divert the medium from the stage of formation of the target product, to isolate the product by extraction, preferably in a known manner, with higher alcohols or higher fatty acids, and return the resulting depleted phase to the stage of product formation and / or the first stage . Due to this, in the second stage, the concentration of the product is maintained at a low level, therefore bacteria are not harmed. As a result, the formation of products occurs all the time at a high speed and this process is maintained for a very long time. It is possible to further carry out the separation of the product without exerting a negative effect on the fermentation stage. Can be formed in the second stage the products withdrawn from the medium (preferably continuously) by pervaporation, using for this purpose a perfusion membrane, in particular silicone rubber, and the process is carried out at 30-90, preferably at 40-80 ^C. Reduction of the vapor pressure with side diffusion evaporation is achieved using a carrier gas, for example N ₂ , H ₂ , CO ₂ , a mixture of these gases, a fermentation gas or using vacuum. However, it is possible to separate the desired products (preferably continuously) in the second stage and by stripping with gas, using, for example, N ₂ , H ₂ , CO ₂ , a mixture of these gases or fermentation gas. The volume ratio between the flow of liquid and gas is 0.05: 1 1.6: 1 and the process is carried out at a temperature of 30-90, preferably 40-80 ^about C.

 All these methods of separation of the target products, along with the possibility of using non-traditional sugars, allow the use of more concentrated sugar solutions, ensure the achievement of higher productivity and a stable process for a long time.

 To regulate the process with particularly high accuracy, you can change the amount of the supplied nutrient solution and / or sugar solution depending on the content of the target product in the second stage and thereby establish exactly the concentration of the nutrient solution or sugar, which is necessary for the optimal process.

 In the proposed device, a bioreactor, preferably a bioreactor such as an autoclave with a stirrer, and a reactor loaded with material containing microorganisms, preferably a fixed-bed reactor, can be connected in series. In this case, the fixed-bed reactor is connected to the external circuit for the continuous separation of the target product. Thanks to this combination of reactors, it is very easy to control the growth rate of the microorganisms used and the formation of the target product in the second stage.

 In this case, in the fixed-bed reactor, one or more guide devices can be mounted to ensure circulation inside the reactor itself. Due to this circulation, the concentration of the nutrient medium and the product becomes approximately the same at all points of the reactor. To ensure circulation in the reactor, local gas inlet devices or guides for directional inlet of the circulating medium flow can be mounted. In the case of introducing gas, circulation is achieved due to the airlift effect, and in the case of introducing a directed fluid flow due to its corresponding velocity.

 In the external circuit, a perdiffusion module can be mounted to separate the products formed. This module may include a membrane, in particular of silicone rubber. In this case, the most favorable conditions are provided for the isolation of the target product from the medium. In this case, when the flow is carried out relative to the membrane by a cross current, the deposition of microorganisms on it is prevented. And finally, a countercurrent stripping column can be mounted to separate the target product in the external circuit. Moreover, the choice of one or another method of separation of the target product depends on the nature of the starting material used, the nature of the resulting product and the microorganisms used.

 At the first stage of the proposed method, such a duct speed is established at which bacteria multiply and acid formation occurs at this stage, but no noticeable product formation occurs. At this stage of growth, bacteria exhibit good adhesive properties, due to which, when moving to the second stage, they are immobilized on the material of the carrier. If the flow rate is too low, cell growth will occur in the first stage, and product formation can occur at the same time, as a result of which the bacteria lose their ability to adsorb on the carrier material.

 Instead of immobilization in the second stage of the method, cells can be separated from the medium by microfiltration, preferably cross-precision microfiltration, and then returned to the process. As a result, the same effect is achieved as with immobilization.

 In addition, a high concentration of the target product in the first stage (at a low flow rate) leads to degeneration of bacteria, resulting in a reverse transition from product formation to acid formation, which, as practice has shown, is irreversible. If the duct speed is too high, bacteria are washed out in the first stage, as a result of which after a while the number of bacteria in the second stage will be already insufficient.

 Therefore, the flow rate should be set depending on the specific conditions.

 Figure 1 shows a process diagram in which the products formed are separated by diffusion evaporation; figure 2 is a diagram by which the separation of products is carried out by extraction; figure 3 is a diagram in which the product from the medium is isolated by distillation.

 Numeral 1 denotes a collection from which the nutrient solution is piped 2 through pump 3 to the first stage, which is designed as a bioreactor such as an autoclave with an agitator 4. From the bioreactor, the nutrient substrate enriched with biomass is piped 5 through pump 6 to the second stage, which is designed as a fixed bed reactor 7. A pipe 8 exits from this reactor, through which the product-enriched medium is discharged from the reactor by means of a pump 9.

 The product-enriched medium (Fig. 1) passes through a heat exchanger 10 in which it is heated by the heat of the return product already separated from the medium. The heated product-enriched medium is passed through a pipe 11 to a heater 12 and from there to a perdiffusion module 13. The perdiffusion module 13 has a membrane 14, on one side of which a medium is passed, and on the other, a carrier gas. Through the membrane, the product passes from the medium to the gas, which is introduced into the module 13 through the pipe 15. The medium purified from the product is sent through the pipe 16 to the heat exchanger 10 via the pump 17 and is returned to the fixed-bed reactor through the pipe 18. Excess medium, practically containing no product, is discharged through line 19. A part of the medium (shown in dashed line in Fig. 1) is sent through line 20 to a bioreactor such as an autoclave with a mixer using a pump 21. The product-enriched gas from the perdiffusion module 13 is supplied through a pipe 22 to a cooling device 23, in which the product is condensed and discharged through a pipe 24. The gas separated from the product through a pipe 25 is returned to the perdiffusion module 13.

 The product-enriched product discharged from the fixed-bed reactor (Fig. 2) is sent through a pipe 8 to a heater 26, from which it enters a mixer 27. An extractant is supplied to this mixer through a pipe 28, and a mixture of the extractant and the product-enriched medium is sent through a pipe 29 to the separator 30. In the separator 30, the product-containing extractant and the medium purified from it are separated. At the same time, the product-enriched extractant is discharged through pipeline 31, and the medium purified from it is discharged through pipeline 32. The medium purified from the product is then returned through pipeline 33 to the reactor with a fixed bed, and its excess is discharged through pipeline 34. As shown in dashed figure 2 In a line, the product-free medium may also return to the first stage via line 35 via pump 36. The separation of the extractant and the product can be carried out in the usual way, for example by distillation, etc.

 The product-enriched medium that is discharged through a pipe 8 using a pump 9 (Fig. 3) is again sent to a heat exchanger 10, from which it is supplied through a pipe 37 to a heater 38 and from there through a pipe 39 to a stripping column 40. In this column, the product-enriched medium interacts with a countercurrent gas that is supplied to the column through line 41 via pump 42. The product enriched gas through line 43 is sent to a cooling device 44, in which the product is condensed and discharged through line 45. Not containing The product-containing gas is returned to the stripping column using a pump 42. The product-free medium is sent via line 46 via a pump 47 to a heat exchanger 10, in which the still-heated medium exchanges heat with fresh medium coming from a fixed bed reactor 7. From of the heat exchanger 10, the product-free medium enters the reactor with a fixed bed 7. And in this case, an excess of medium is removed from the system via line 48. The pipe 49 and the pump 50 shown by the dashed line also serve to return free second medium to the first stage of the product, namely the bioreactor autoclave type with an agitator 4.

PRI me R 1. As a nutrient medium, the following composition is used, g / l: Xylose (anhydrous) 60 KN ₂ PO ₄ 1 K ₂ NRA ₄ 1 (NH ₄ ) ₂ SO ₄ 2 MgSO ₄ ˙ 7H ₂ O 0.1 NaCl 0.01 Na ₂ MoO ₄ ˙ 2H ₂ O 0.01 CaCl ₂ 0.01 MnSO ₄ ˙ H ₂ O 0.015 FeSO ₄ ˙ 7H ₂ O 0.015 Biotin (mg / l) 0.1 Thiamine chloride 0.002 n- aminobenzoic acid 0.002 Na ₂ S ₂ O ₄ 0.035 Resazurin 0.001 Yeast extract 0.5 Na ₄ OOCCH ₃ 2.2
As fermenting organisms, Clostridium acetobutylicum ATCC 824 is used, which is taken in the form of an appropriate culture before introducing it into the process.

The nutrient medium from the collection 1 is fed into the bioreactor in the form of an autoclave with a stirrer 4 at a speed of 0.3 h ^-1 . The amount of inoculum in this case is approximately 10% of the contents of the bioreactor. Fermentation in the bioreactor is carried out at 37 ^about C and a pH of 5.1. By choosing a specific flow rate, the density of cells in the bioreactor remains approximately constant, and new cells resulting from the growth of bacteria are fed via line 5 to the fixed bed reactor 7, where they are retained on the support material due to adhesion. Sintered glass with open pores, the volume of which is 50% of the total working volume, is used as a carrier. Fermentation temperature in a fixed bed reactor 7 is 33 ^{° C} and pH 4.3. The total flow rate at both stages is 0.15 h ^-1 . The contents of the reactor in the fixed bed reactor 7 are purged with nitrogen, due to which the circulation is carried out thanks to the guides mounted in it.

 Performance and output are given in table 1.

PRI me R 2. Use a nutrient solution of the same composition and the same inoculum, and the process in a bioreactor such as an autoclave with a stirrer and in a reactor with a fixed bed is carried out in the same conditions. The fixed bed reactor is connected to an external circuit in which the products formed are discharged continuously or periodically. Derived from a fixed bed reactor enriched product fluid is heated to a temperature of about 40 ^{° C} and sent to a stripping column to which the countercurrent thereto vvoditcya nitrogen icpolzuyuschiycya in kachectve gas stripping. The volumetric ratio between liquid and gas flows is 0.35: 1. The liquid, practically containing no products, after cooling to the fermentation temperature, partly returns to the fixed bed reactor 7, moreover, its smaller part can also be returned to the autoclave-type bioreactor with stirrer 4.

 Excess fluid containing no product is discharged from the system. Product enriched gas from the stripping column is passed through a cooling device in which products can condense and discharge. The obtained stationary values are given in table.2.

 As can be seen from the table. 1,2, the productivity and the degree of steam sugar in the case of the method in accordance with example 2, i.e. with continuous product withdrawal in the external circuit, significantly higher. This result also occurs when, as shown by the above experiments, fermentation is carried out for a longer time, more than 500 hours.

Claims (9)

1. The method of fermentation of carbohydrate-containing media using bacteria producing butanol, acetone, ethanol and / or isopropanol, providing for the fermentation of the medium at a pH of 4-5.5 and a temperature of 27-40 ^o C in two stages, the first of which carry out continuous bacterial growth and on the second - the formation periodically or continuously of the target product, the supply of a nutrient solution and / or sugar solution depending on the content of the target product in the second stage, followed by isolation of the target product, characterized in that in the second stage b the bacteria are immobilized on a porous carrier with a ratio of the volume of the carrier and the volume of the fermented medium 1:10 1: 1.1 or separated by microfiltration from the fermented medium and returned to the process, while in the first stage the fermentation is carried out with stirring the medium, the flow rate of 0.08 0, 45 h ^{- 1} , pH 4.0 5.5 and / or a temperature of 30 40 ^o C, and the second at a flow rate of 0.14-0.4 h ^{- 1} , pH 3.0-5.0 and / or temperature 27-40 ^o C. 2. The method according to claim 1, characterized in that sintered glass with open pores, or pumice, or other material with a high SiO ₂ content with pore sizes of 20-200 μm and a moisture capacity of at least 0.35 ml / is used as a carrier. cm ³ .  3. The method according to claims 1 and 2, characterized in that the ratio of the volume of the carrier and the volume of the fermented medium is 1: 5-1: 1.6.  4. The method according to claims 1 to 3, characterized in that a carrier with pore sizes of 50-100 microns is used.  5. The method according to claims 1 to 4, characterized in that the selection of the target product is carried out by extraction with higher alcohols or higher fatty acids, the resulting phase is returned to the first and / or second stage of fermentation. 6. The method according to PP.1-5, characterized in that the selection of the target product is carried out by diffusion evaporation through the membrane at a temperature of 30
90 ^o C, while reducing the vapor pressure from the side of the diffusion evaporator is carried out using nitrogen, or hydrogen, or carbon dioxide, or a mixture of these gases, or a fermentation gas, or vacuum. 7. The method according to claims 1 to 4, characterized in that the selection of the target product is carried out by gas distillation at 30-90 ^o C, while the gas used is nitrogen, hydrogen, carbon monoxide, a mixture of these gases or fermentation gas in a ratio of medium and gas 0.05: 1 1.6 1.  8. A device for fermenting carbohydrate-containing media using bacteria producing butanol, acetone, ethanol and / or isopropanol, containing two reactors connected in series, the first of which contains a stirrer, and the second a device for creating a directed flow of a circulating medium, and the second reactor can be communicated with an external circuit for separation of the target product from the fermented medium, characterized in that the first reactor during the process is an autoclave, and in the second reactor it is stationary ohm or fluidized bed filler with microorganisms immobilized on it.  9. The device according to claim 8, characterized in that the external circuit for isolating the target product from the fermented medium contains a stripping column or a perdiffusion module with a membrane, for example, of silicone material.

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