RU2375454C1 - Method of producing organic solvents, mainly butanol - Google Patents

Abstract

FIELD: chemistry. ^ SUBSTANCE: method of producing organic solvents involves periodical reduction of pressure in a fermenter during fermentation of carbohydrate-containing material and simultaneous distillation of organic solvents and gases formed during fermentation. Fermentation is carried out on a culture medium using Clostridium acetobutilicum bacteria. ^ EFFECT: increased output of butanol and allows for varying ratio of desired products during biosynthesis; total output of solvent is 8,5 g/l/day. ^ 6 cl, 2 dwg, 3 ex

Description

The invention relates to the microbiological industry and for the production of organic solvents - butanol, acetone and ethanol by biosynthesis of carbohydrate-containing materials.

The most valuable of these solvents is butanol.

Butanol is an expensive organic solvent, it is widely used in the manufacture of nitro-lacquers and oil varnishes in the production of complex solvents, synthetic rubber and silk, and in the extraction of pharmaceuticals, it is used as a raw material for the production of almost all plastics and their solvents, therefore, it constantly has on the market of chemical reagents and substances high demand.

Organic solvents can also be used as biofuels.

Currently, in connection with the advent of new economical production technologies, interest in butanol obtained by microbiological methods and its use as biofuel has sharply increased. At the same time, butanol has a number of advantages over ethanol. Compared to ethanol, butanol can be mixed in higher proportions with gasoline and can be used in existing car engines without modifying the air-fuel mixture formation system. Butanol releases more energy per cycle than ethanol or methanol, and about 10% more than gasoline.

Butyl alcohol (butanol) C ₄ H ₉ OH is a colorless liquid with a characteristic odor of fusel oil. Known normal primary butyl alcohol CH ₃ (CH ₂ ) 3 OH, normal secondary butyl alcohol CH ₃ CH ₂ CH ₂ (OH) CH ₃ , isobutyl alcohol (CH ₃ ) 2CHCH ₂ OH, trimethylcarbinol (CH ₃ ) 3COH.

In industry, butanol is produced by propylene oxosynthesis using nickel-cobalt catalysts at 130-150 ° C and a pressure of 20-35 MPa.

Butanol began to be produced in the 10s of the XX century by a microbiological method using bacteria of the species Clostridium acetobutylicum. The raw material for production was glucose from sugarcane, beets, corn, wheat, cassava.

It is known that bacteria of the species Clostridium acetobutylicum, when fermenting various carbohydrates, synthesize three target products simultaneously: butanol, acetone and ethanol, the percentage of which is approximately 60:30:10 (respectively). This ratio is not strictly constant and can vary in the direction of increasing the yield of a particular fermentation product. Moreover, the composition of the raw materials used for fermentation largely affects the yield of solvents and the proportion of the target products. So, when cultivated on flour media, various strains of Clostridium acetobutylicum synthesize a total amount of solvents of 18-19 g / l, including 11-12 g / l and 4-5 g / l of acetone. In addition, on media from flour, 1.5-2.5 g / l of ethanol is synthesized (Korneeva O.S. Zherebtsov N.A. et al. Role of amylolytic enzymes Clostridium acetobutylicum in solvent biosynthesis. Biotechnology, 1986, No. 3 pg. 133-136).

A known method of increasing the yield of solvents during the periodic cultivation of bacteria of the species Clostridium acetobutylicum on flour media by preliminary liquefaction of starch with a recycled mash containing active amylolytic enzymes. At the same time, the total yield of solvents reaches 19 g / l, and the fermentation time is reduced as. SU 160485, 12.21.88.

The above methods have all the disadvantages of batch processes.

Known detachable-topping method for producing butanol, used on an industrial scale, which partially compensated for the disadvantages of the periodic process of butanol biosynthesis. With this method, part of the volume of the culture medium is constantly removed from the fermenter and an equal volume of the medium is added. The selected portion of the culture medium is sent for rectification. Obviously, in this case, the sugars contained in the selected portion are irretrievably lost. In addition, in the classical version of acetone-butyl fermentation with a 4% level of available glucose in the culture medium, fermentation ceases after 72 hours due to the "consumption" of available glucose by microorganisms (in the case when no additional nutrients were added, except for the original ones). Moreover, the introduction of additional nutrition does not increase the duration of fermentation due to the fact that by 72 hours in the culture medium, a concentration of solvents is reached that is toxic to the producing microorganism. A well-known fact is that in the process of biosynthesis of alcohols with the accumulation of butanol, ethanol and acetone, there is a decrease in the activity of biosynthetic microorganisms, because alcohols are inhibitors of the process of alcoholic fermentation. In order to eliminate this, butanol and other organic compounds are constantly removed from the process.

A known fermentation method for producing butanol and other organic solvents using two different strains of bacteria, with a constant removal of the resulting organic compounds. The invention describes a process for the production of solvents, mainly butanol, from sugar, proceeding in two stages, for each of which different microorganisms are responsible. The first stage is the production of butyric acid from a solution of carbohydrates by the first microorganism, the second stage is the production of butanol from butyric acid by the second microorganism, and then the isolation of butanol. For the stage of acidogenesis, strains of anaerobic bacteria such as: Clostridium tyrobutyricum, C. thermobutyricum, C. butyricum, C. cadaveros, C. cellobioparum, C. cochlearium, C. felsineum, C. pasteurianum, C. roseum, C. rubrum can be used C. sporogenes. For the stage of solventogenesis, strains of the following microorganisms are most often used: Clostridium acetobutylicum, C. beijerinkii, C. aurantibutyricum and C. tetanomorphum. In this invention, the main producer of butyric acid is C. thermobutyricum, C. tyrobutyricum, and the producer of butanol is C.acetobutylicum or a similar bacterium in the solvent-based growth phase. The process is as follows: a sterile solution of carbohydrates in the first reactor is converted into butyric acid using the first microbe; butyric acid, after sterilizing or otherwise removing the first microbe, is sent to the second reactor for conversion to butanol; butanol and other organic compounds are continuously removed from the second reactor by activated sorbents, membranes or by distillation (US No. 5753474, 1988).

A known method of fermentation of carbohydrate-containing media using bacteria that produce butanol, acetone, ethanol and / or isopropanol, consisting 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. The products formed in the second stage are removed from the medium (preferably continuously) by diffusion evaporation using for this purpose a perfusion membrane, in particular from silicone rubber (RU 2044773, 1989).

The main disadvantages of the described methods are associated with the difficulties of isolating organic solvents from the culture medium, since this is the most energy-intensive and expensive link in the technology for their preparation. Today, there are many known methods for the separation of alcohols from a heterogeneous medium: distillation - distillation through the vapor phase, see "Kasatkin AG The main processes and apparatuses of chemical technology, 8th ed., M., 1971 "; separation on membranes see “Hwang S.-T., Kammermeyer K. Membrane separation processes. M .: Chemistry, 1981 "or" Shaposhnik V.A. Membrane electrochemistry // Soros Educational Journal. 1999. No. 2. S.71-77 "; sorbent separation “Zolotov Yu.A., Kuz'min N.M. Preconcentraion in Inorganic Analysis. Amsterdam: Elsevier, 1990; freezing "Popes L. Concentration by freezing, M., 1982".

The disadvantages of all known methods of separation is the high energy intensity and the need for relatively frequent replacement or restoration of the initial properties of functional elements - membranes or solid sorbents.

Currently, the main directions of research in the field of organic solvents and biofuels in the UK, USA, Germany, China and Japan are the construction of new strains of acetone-butyl bacteria by genetic engineering methods and the development of new technological methods for isolating the final target products.

Improvements of well-known domestic technologies in acetone-butyl production relate, as a rule, to the creation of new strains that increase the yield of target products, mainly butanol, as the most valuable of them.

Closest to the proposed is a continuous method for producing organic solvents, in particular acetone-butanol-ethanol (ABE), including the preparation of carbohydrate-containing raw materials, fermenting it in a fermenter on a nutrient medium using bacteria producing butanol, acetone and ethanol, distillation of organic solvents and gases formed during the fermentation, and replenishment with solutions of carbohydrates and mineral salts (US 2005089979, 2005-04-28).

All of the above methods, including the latter, have disadvantages inherent in the known processes for isolating the target products — high energy intensity and the need to restore the original properties — membranes or solid sorbents, and, in addition, are characterized by a low yield of target products, in particular butanol. None of the known sources describe the possibility of changing the ratio of the fractional yield of organic solvents (acetone-butanol-ethanol) in order to increase the yield of butanol, as the most valuable of them, although it is known that the ratio of the fractional yield of organic solvents is slightly changed can be used for cultivation of various nutrient media. It is also possible to construct new strains of acetone-butyl bacteria by genetic engineering methods.

An object of the present invention is to increase the yield of butanol by creating optimal conditions for the biosynthesis of solvents while reducing energy consumption at the stage of butanol separation.

To solve this problem, a method for producing organic solvents has been proposed, including the preparation of a carbohydrate-containing raw material, fermenting it in a fermenter on a nutrient medium using bacteria producing butanol, acetone and ethanol, distilling off organic solvents and gases formed during the fermentation, and feeding with carbohydrate and mineral solutions salts, in which the biosynthesis process is carried out with a periodic decrease in pressure in the fermenter during the fermentation of carbohydrate-containing raw materials and simultaneous distillation of organic their solvents and gases formed during fermentation.

The pressure decrease begins when the concentration of organic solvents in the fermenter is reached, approaching toxic for the producing culture, optimally - when the concentration of butanol in the culture medium is reached - 8-9 g / l and continue until the concentration of butanol in the culture medium is reached - 5-2 g / l while the pressure in the fermenter during the distillation is 0.90-0.94 kg / cm ² .

As a bacterium producing butanol, acetone and ethanol, it is preferable to use a bacterium of the species Clostridium acetobutylicum, although others, for example Clostridium beijerinckii, can be used.

Known methods for producing ethyl alcohol, in which the biosynthesis process is carried out continuously simultaneously with the distillation of the resulting alcohol-vapor and carbon dioxide under reduced pressure. RF 2230788, 2001, Eurasian patent 000876, from 1999

However, the authors are not aware of the sources of information that would describe the use of this technique in the production of butanol, possibly because the boiling point of butanol is much higher (117 ° C) than the boiling point of ethanol (76 ° C) and water.

Numerous studies conducted by the authors have shown that if during the biosynthesis of organic solvents a periodic pressure decrease is carried out and the gases formed during fermentation are removed, then the change in the concentrations of the resulting organic solvents is quasi-cyclic, i.e. the process is quasi-cyclical.

The term quasi-cyclic (quasi ... from Latin quasi - something like, as if, as if, an integral part of complex words corresponding in meaning to the words: “supposedly”, “imaginary”, “false.” The Great Soviet Encyclopedia) was used by the authors in connection with with the fact that, in fact, the biosynthesis process can be carried out continuously and can last almost arbitrarily long.

During the studies, the authors unexpectedly discovered that during the period of lowering the pressure, the synthesis of solvents sharply intensifies. A possible explanation for this is the effect of variable pressure on biological systems, which entails a change in the microenvironment of cell membranes, leading to disruption of the processes of transfer of substances through the membranes, a change in the rates of enzymatic processes in the cell, and the emergence and development of reparative reactions accompanied by new syntheses (V. Akopyan ., Korzhevenko G.N., Shangin-Berezovsky G.N. Hidden reserve of growth and development of living systems.Vestnik of Agricultural Science, 1988, No. 4, (380), pp. 96-105).

Studies have also shown that the concentration of sugars in the process of lowering the pressure does not practically change, but in the intervals decreases. It can be assumed that in the intervals between pressure drops there is an accumulation in the culture of acid precursors (butyric and acetic). Then, in the process of lowering pressure, there is an enhanced processing of acid precursors into solvents. This may be due primarily to the redistribution of part of the culture from acid-producing morphology to solvent producers. It is important to note that the ratio of solvents formed during pressure reduction (the highest yield of butanol) remains the same for the next 20 hours after the previous pressure decrease, which means that the intensification of the biosynthesis of solvents during pressure reduction does not stop even after it normalization.

To ensure that the process proceeds as described above, when the concentration of organic solvents (mainly butanol) in the culture medium is increased to values that are close to toxic for the producing culture, the pressure in the fermenter above the surface of the liquid is reduced to a level that ensures evaporation of organic solvents from the liquid medium, removal and condensation of their vapors. When a negative, relative to atmospheric, pressure is created above the surface of the liquid medium in the fermenter, the vapor pressure of the solvents above the surface drops, and the rate of evaporation from the surface of the liquid medium is accelerated in proportion to the degree of pressure decrease. Consequently, the removal of solvent vapors and gaseous metabolites from the space above the surface of the liquid medium accelerates their evacuation from the volume. An additional intensification of the evacuation process is caused by numerous gas bubbles (CO ₂ , H ₂ ) generated during biosynthesis that float to the surface, which significantly increases the liquid / gas interface area, on which organic solvent molecules with high surface activity are concentrated due to surface sorption .

The selected gaseous medium is sent to the refrigeration unit, where the solvents condense and flow into the storage volume. Removal of organic solvents continues until their concentration (mainly butanol) in the culture fluid decreases to values that do not affect biosynthesis but are sufficient to reduce the level of extraneous microflora, i.e. to values that ensure the safety of the main process.

After this, the removal of solvent vapors is temporarily stopped, and nutrients are introduced into the culture medium in an amount that compensates for the loss of these substances due to their utilization by producing microorganisms in the previous period. Biosynthesis continues, the content of organic solvents, mainly butanol) in the medium increases and the cycle repeats. Nutrition with essential nutrients can be carried out continuously.

The cycle repetition rate depends on the upper (at which the pressure begins to decrease) and lower (at which the evacuation is stopped) levels of butanol in the medium, the evacuation rate of vaporous products, and the evaporation area.

Varying the above parameters, it is possible to obtain target products with different proportions of their outputs; you can increase the yield of butanol up to 70-90% compared with 60% in the known methods.

The evaporation area can be increased, for example, by increasing the intensity of “cold” boiling under reduced pressure, creating waves on the surface, and spraying the medium with special devices.

The proposed method for producing organic solvents, mainly butanol, can be carried out as follows.

A carbohydrate-mineral mixture prepared in the standard way (a solution of mineral salts, a necessary set of vitamins and one of the following carbon sources: glucose, mannose, xylose, galactose, fermentolizate of non-food plant polysaccharides containing glucose, mannose, xylose, arabinose, separated from wood residues, is introduced into the fermenter (lignin), or whey lactose, at a concentration of 2-4%); then the inoculum of bacteria Clostridium acetobutylicum, var. elyzaveticus with a cell density of 1-2 billion / ml. 30 minutes after sowing, intensive evolution of fermentation gases begins, after 5-6 hours - synthesis of organic acids and 10-12 hours - intensive synthesis of organic solvents, the speed of which reaches a maximum by 28-36 hours. At this time, the concentration of bacterial cells in the fermenter is also maximum - 3 · 10 ⁹ cells / ml suspension. At the same time, they begin to lower the pressure in the fermenter to remove solvents from the culture medium to the level of 5-2 g / l and simultaneously feed a solution of carbohydrates and mineral salts into the fermenter, maintaining the concentration of carbohydrates in the fermenter in the range of 0.8-1.2%, while periodically removing solvents from the fermenter and part of the suspension, so that the flow rate was in the range of 0.025-0.035 h ^-1 . The process continues in continuous mode for 500 hours, although there are practically no restrictions on the time of cultivation and synthesis of solvents.

The invention is illustrated by examples that are not restrictive.

Example 1. The periodic method of the biosynthesis of organic solvents.

2.5 l of 4% glucose solution, 120 g of flour, 300 ml of inoculum consisting of flour (40 g / l), water and bacteria Clostridium acetobutylicum CK 425, VKPM B-4786 registration number, are introduced into a pre-sterilized fermenter with a working volume of 3 l density of 1-2 billion / ml. Acetone-butanol-ethanol fermentation is carried out at a temperature of 37 ° C. Fermentation is continued for 72 hours. After 72 hours, the fermentation process is stopped, the pressure in the fermenter is reduced by 3 hours to a value of 0.94 kg / cm ² and condensation of the distilled vapors gives an average of 250 ml of a solution containing 5% butanol, 1.5% acetone and 0.5% ethanol. The average productivity of the process is 3 g / l / day.

Example 2. A method of implementing the process of biosynthesis of organic solvents in accordance with the present invention.

In a pre-sterilized fermenter with a working volume of 3 l, 2.5 l of a 4% glucose solution is added, 120 g of flour, 300 ml of inoculum consisting of flour (40 g / l), water and bacteria Clostridium acetobutylicum are added, registration number VKM B-1787 with a density 1-2 billion / ml. Continuous acetone-butanol-ethanol fermentation is carried out at a temperature of 37 ° C. The duration of the process is at least a week. When the concentration of butanol in the medium is 9 g / l, the pressure in the fermenter is reduced by 3 hours to - 0.92 kg / cm. An average of 250 ml of a solution containing 6.5% butanol, 2% acetone and 0.75% ethanol is obtained by condensation of the distilled vapors (solvent ratio 70: 21: 9). As soon as the concentration of butanol in the medium reaches 2 g / l, the pressure in the fermenter is increased to atmospheric. The pressure reduction procedure is repeated each time when the concentration of butanol is 9 g / l. After removal of butanol and associated gases, a nutrient medium containing 50 g of glucose is introduced into the fermenter. Once every three days, 28 g of yeast autolysate is added to the nutrient medium. The productivity of the process is on average 8.5 g / l / day.

Example 3. The method of implementing the biosynthesis of organic solvents in accordance with the present invention.

In a pre-sterilized fermenter with a working volume of 3 l, 2.5 l of a 4% glucose solution is added, 120 g of flour, 300 ml of inoculum consisting of flour (40 g / l), water and bacteria Clostridium beijerinckii are added, registration number KM MSU No. 101 with density 1-2 billion / ml. Acetone-butanol-ethanol fermentation is carried out at a temperature of 37 ° C. The duration of the process is at least a week. The productivity of the process is on average 4 g / l / day. When the concentration of butanol in the medium is 9 g / l, the pressure in the fermenter is reduced by 0.5 hours to - 0.94 kg / cm ² . By condensation of the distilled vapors, 50-60 ml of a solution is obtained, which exfoliates upon aging. The top layer (5-10 ml) contains 70-75% butanol, 2-4% acetone and 0.1-1% ethanol. The lower layer contains 5-7% butanol, 0.5-1.5% acetone and 0.1-1% ethanol. Since the solubility of butanol in the liquid is 6.7%, this makes it possible to separate the upper layer of 75-80% butanol from water by simple decantation, which further eliminates the costly operations of isolating the target product and significantly reduces energy costs. As soon as the concentration of butanol in the medium reaches 5 g / l, the pressure in the fermenter is increased to atmospheric. The pressure reduction procedure is repeated each time when the concentration of butanol in the fermenter reaches 9 g / l. After evacuation of butanol at low pressure, a nutrient medium containing 50 g of glucose is introduced into the fermenter. Once every three days, 28 g of yeast autolysate is added to the nutrient medium. The productivity of the process is on average 10 g / l / day.

In the presented drawings, figures 1 and 2 show a graph of the dependence of the yield of the target products and the nature of the process from the time of fermentation. Figure 1 illustrates a standard biosynthesis process, figure 2 - the proposed method.

Thus, the proposed biosynthesis process allows combining the processes of fermentation and simultaneous distillation of organic solvents from the fermenter while maintaining the viability and fermentation activity of the cells of the producing bacterium.

The advantage of the proposed technical solution is to increase the yield of butanol by creating optimal conditions for the biosynthesis process while reducing energy consumption at the isolation stage.

From the analysis of the data obtained, it follows that the biosynthesis process in this case proceeds quasi-cyclically, which is achieved by periodically lowering the pressure in the fermenter during the fermentation of the carbohydrate-containing raw materials and at the same time distilling off the organic solvents and gases generated during the fermentation. Such a process is devoid of the disadvantages of both continuous and batch processes.

Claims (6)

1. The method of producing organic solvents, including the preparation of carbohydrate-containing raw materials, fermenting it in a fermenter on a nutrient medium using bacteria producing butanol, acetone and ethanol, distillation of organic solvents and gases formed during the fermentation, and feeding with carbohydrate and mineral salts solutions, characterized the fact that the process is conducted with a periodic decrease in pressure in the fermenter during the fermentation of carbohydrate-containing raw materials and the simultaneous distillation of organic solvents and gases, schihsya in the process of digestion. 2. The method according to claim 1, characterized in that the pressure reduction begins when the level of butanol concentration in the fermenter is reached, approaching toxic for the producing culture. 3. The method according to claim 2, characterized in that the pressure decrease in the fermenter is started upon reaching a concentration of butanol in the culture medium of 8-9 g / L. 4. The method according to claim 3, characterized in that the pressure decrease in the fermenter is continued until the concentration of butanol in the culture medium reaches 5-2 g / l. 5. The method according to claim 1, characterized in that the pressure in the fermenter during the distillation is 0.90-0.94 kg / cm ² . 6. The method according to claim 1, characterized in that the bacterium producing butanol, acetone and ethanol is a bacterium of the genus Clostridium acetobutylicum.

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