CN108841912B - Process for preparing and separating bio-butanol and bio-ethanol - Google Patents
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Abstract
The invention belongs to the technical field of fermentation, and discloses a process for preparing and separating bio-butanol and bio-ethanol, which comprises the following steps: step 1) treating straws, step 2) preparing a fermentation medium, step 3) producing alcohol, and step 4) separating alcohol and preparing protein powder. The process can simultaneously obtain the biological butanol, the biological ethanol and the mycoprotein powder, and improves the additional value.
Description
Technical Field
The invention belongs to the technical field of fermentation, and particularly relates to a process for preparing and separating bio-butanol and bio-ethanol.
Background
Butanol is a colorless liquid, has a vinous flavor, is miscible with ethanol, diethyl ether and various other organic solvents, and vapor forms an explosive mixture with air with an explosion limit of 1.45-11.25 (vol.). The plasticizer is mainly used for manufacturing n-butyl ester plasticizers of phthalic acid, aliphatic dibasic acid and phosphoric acid, is widely used in various plastic and rubber products, and is also a raw material for preparing butyraldehyde, butyric acid, butylamine, butyl lactate and the like in organic synthesis. Biobutanol is a similar biofuel to bioethanol. The raw materials and the production process are similar to those of bioethanol, but the vapor pressure of the biobutanol is low, the tolerance to impurity water is high when the biobutanol is mixed with gasoline, the corrosivity is low, and compared with the existing biofuel, the bio-ethanol gasoline can reach a higher mixing ratio with the gasoline without modifying vehicles.
Butanol can be prepared by a fermentation process similar to ethanol. However, butanol is much more expensive to produce than ethanol, i.e., larger facilities for evaporation, heating, cooling, etc., are required to produce butanol, and the capital cost is higher. Therefore, the key to realizing the commercialization of the biological butanol is to improve the conversion rate of the raw material into butanol and accelerate the conversion process. Depending on the development of efficient biocatalysts and optimization of the design of the production process. Like ethanol, the traditional production method of biobutanol also consumes a large amount of agricultural products, and therefore, a new technology for producing butanol by using various biobased wastes is being researched to solve the problem of competing for grains with people. The traditional preparation method of the biological butanol is to prepare the biological butanol by fermenting corn, wheat, soybean and other grain grains as raw materials. However, the production of biofuel from grains as raw materials cannot meet the social requirements and endangers the safety of grains. Researchers have indicated that even if all corn and soybeans grown in the united states are used to produce bioenergy, they can only meet 12% of the gasoline demand and 6% of the diesel demand, respectively, in the united states society. Corn and soybeans are the first to meet food, feed and other economic requirements and cannot be used to produce biofuels. Experts show that the production of biobutanol from non-grain crops as raw materials is the direction of future development, and the future energy industry may expect to use crop cellulose, such as cereal straw, to produce biobutanol. Currently, the biobutanol industry is still in its infancy. In recent years, as the price of petroleum is rising, many enterprises with strategic eyes participate in the research of the biological butanol.
Bioethanol refers to the conversion of various biomasses into fuel alcohol by fermentation of microorganisms. It can be used alone or mixed with gasoline to prepare ethanol gasoline as automobile fuel. The majority of the fuel ethanol produced industrially at present is prepared from food crops, and has scale limitation and unsustainability in the long term. The second generation of biofuel ethanol using lignocellulose as raw material is the key to determine the large-scale replacement of petroleum in the future.
Chinese patent "CN 102796692A" discloses a method for genetically modifying clostridium acetobutylicum, which can more efficiently utilize xylose and arabinose, and solvents such as butanol, ethanol, acetone and the like with higher concentration. Chinese patent "CN 103409470A" discloses a method for producing butanol, ethanol and acetone by inoculating acetone butanol clostridium and then yeast, and performing mixed fermentation.
Corn stalks belong to agricultural wastes, are mostly discarded or burned at will, but the burning causes environmental pollution, is forbidden by national regulations, and at present, a plurality of enterprises are researching utilization methods of the corn stalks. Because the cellulose, hemicellulose and lignin in the original corn straws have compact and complex structures, the cellulose is difficult to be hydrolyzed into monosaccharide by enzyme, and how to process the corn straws to be beneficial to the fermentation of bacterial strains to produce ethanol is the research direction of people. The fermentation of butanol using biomass such as lignocellulose has become a hot spot in current research. It is a breakthrough to select the alcohol-producing bacteria for modification and compatibility to improve the alcohol yield. The applicant made further research on the basis that previous research on a method for producing the bio-butanol and the bio-ethanol by fermenting the corn straws serving as the main raw material obtains the mixed alcohol by fermenting the straws serving as the main raw material.
Disclosure of Invention
On the basis of the previous work, the invention provides a process for preparing and separating bio-butanol and bio-ethanol.
The invention is realized by the following technical scheme:
a process for the preparation and separation of bio-butanol and bio-ethanol comprising the steps of: step 1) treating straws, step 2) preparing a fermentation medium, step 3) producing alcohol, and step 4) separating alcohol and preparing protein powder.
Further, the method comprises the following steps:
step 1) straw treatment: firstly, pulverizing corn stalks to be within 5cm, placing under the conditions of pressure of 1.5-2MPa and residence time of 10-15min to carry out steam explosion pretreatment, and then carrying out explosion; sieving corn straw subjected to steam explosion treatment with a 50-mesh sieve, collecting undersize, adding the undersize into water 1.5-2 times of the weight of the corn straw, heating to 60 ℃, stirring for 90min at 100rpm under the condition of heat preservation, heating to 121 ℃, preserving heat for 10min, and naturally cooling to room temperature to obtain a culture solution;
step 2) preparing a fermentation medium: mixing the trichoderma koningii seed liquid and the aspergillus niger seed liquid, inoculating the mixture into a culture solution according to the inoculation amount of 6-8%, culturing for 96 hours, controlling the stirring speed to be 100rpm during culturing, controlling the culturing temperature to be 32 ℃, and controlling the pH value in the culturing process to be 4-5 by feeding ammonia water; after the culture is finished, carrying out ultrasonic treatment, adjusting the temperature to 55 ℃, adding lysozyme, and carrying out enzymolysis for 12 hours under the heat preservation condition; then inactivating enzyme at 121 deg.C for 5min, and naturally cooling to room temperature to obtain fermentation culture medium;
step 3) alcohol production: inoculating the clostridium acetobutylicum seed solution into an anaerobic fermentation tank containing a fermentation culture medium for culturing at the temperature of 30-32 ℃ for 24-36h, then inoculating the neurospora crassa seed solution, continuing culturing for 48-72h, taking the fermentation broth after the culture is finished, centrifuging at 8000rpm for 10min, and collecting the upper-layer liquid and thallus precipitate;
step 4) separating alcohol and preparing protein powder: distilling the upper layer liquid under reduced pressure to obtain a crude product, and then rectifying and dehydrating to obtain the bio-butanol and the bio-ethanol; washing the thallus precipitate with water, drying at 80 deg.c and crushing to obtain protein powder.
Preferably, the first and second electrodes are formed of a metal,
the density of the Trichoderma koningii seed liquid is 1 multiplied by 108cfu/mL。
Preferably, the first and second electrodes are formed of a metal,
the density of the Aspergillus niger seed liquid is 1 multiplied by 108cfu/mL。
Preferably, the first and second electrodes are formed of a metal,
the ultrasonic treatment time is 40min, and the ultrasonic frequency is 30 KHz.
Preferably, the first and second electrodes are formed of a metal,
the addition amount of the lysozyme is 2 ten thousand U: 1L of the solution.
Preferably, the first and second electrodes are formed of a metal,
the inoculation amount of the clostridium acetobutylicum seed liquid is 5-7%.
Preferably, the first and second electrodes are formed of a metal,
the inoculation amount of the neurospora crassa seed liquid is 8-10%.
Preferably, the first and second electrodes are formed of a metal,
the trichoderma koningii seed liquid and the aspergillus niger seed liquid are mixed according to the volume ratio of 2-3: 1-2.
The specific strains selected by the embodiment of the invention are Trichoderma koningii ATCC18649, Aspergillus niger ATCC1015, Clostridium acetobutylicum ATCC824 and Neurospora crassa ATCC 44317; seed solutions of the respective strains can be obtained by conventional culture methods described in textbooks or literatures.
Compared with the prior art, the invention has the advantages that the invention mainly comprises but is not limited to the following aspects:
according to the invention, the mixed solvent obtained by fermentation is subjected to distillation separation treatment, and mycoprotein is processed, so that the industrial added value is improved;
in the process for preparing the fermentation medium, the ultrasonic-assisted lysozyme is adopted to carry out wall breaking treatment on the thalli, so that the wall breaking effect is good, and the influence of an organic solvent on the growth of subsequent strains is avoided;
the steam explosion pretreatment can partially degrade the hemicellulose and the lignin, destroy the wrapping effect of the lignin and the hemicellulose on the cellulose, destroy the crystallization area of the cellulose, increase the porosity and the inner surface area of the raw material, and be more beneficial to the subsequent enzyme hydrolysis of the cellulose.
The invention carries out crushing and blasting treatment on the agricultural waste straws, can provide normal growth conditions for trichoderma koningii and aspergillus niger, has relatively low raw materials and can reduce the enterprise cost; corn straws are treated by trichoderma koningii and aspergillus niger, and then thalli are subjected to enzymolysis to obtain a culture medium containing reducing sugar and mycoprotein, so that the culture medium is used for the anaerobic alcohol production of subsequent strains, the fermentation efficiency is improved, agricultural wastes are utilized, and the cost is reduced. According to the invention, the cavitation of ultrasonic waves is utilized to generate local high pressure and high temperature, the bacterial cells are impacted to cause cell deformation and rupture, the lysozyme is utilized to assist in wall breaking and dissolving to obtain the mycoprotein, the concentration of the prepared mycoprotein liquid is high, the method is suitable for anaerobic fermentation, and the cost is reduced.
The endoglucanase produced by Trichoderma koningii fermentation has high activity, but the produced cellulase has the defect of low activity of beta-glucosidase generally, and Aspergillus niger has high capacity of producing the beta-glucosidase and low capacity of producing endoglucanase and exoglucanase. The activity of the filter paper enzyme and the activity of the beta-glucosidase are both greatly improved, and the yield of reducing sugar in fermentation liquor is correspondingly improved. Cellulose is hydrolyzed to generate hexose mainly comprising glucose, hemicellulose is hydrolyzed to obtain pentose mainly comprising xylose, but the pentose is difficult to be utilized by microorganisms to produce alcohol, clostridium acetobutylicum capable of utilizing glucose is selected to produce butanol and ethanol, and neurospora crassa is adopted to produce the alcohol by utilizing the xylose; the first inoculation of the clostridium acetobutylicum can utilize glucose fermentation to produce alcohol, but cannot utilize xylose, when the glucose is reduced to a certain concentration, the clostridium acetobutylicum is already a dominant flora, neurospora crassa is inoculated, the clostridium acetobutylicum can utilize high-concentration xylose to produce ethanol, and cannot compete with the clostridium acetobutylicum to utilize glucose, and the clostridium acetobutylicum can be fermented in a synergistic manner to jointly produce butanol and ethanol.
Drawings
FIG. 1: enzyme-producing activity of different strains;
FIG. 2: influence of different strain fermentation modes on the alcohol yield;
FIG. 3: influence of the mixed fermentation time on the amount of alcohol produced.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
A process for the preparation and separation of bio-butanol and bio-ethanol comprising the steps of:
firstly, pulverizing corn stalks to be within 5cm, then placing the corn stalks under the conditions of 1.5MPa of pressure and 15min of residence time for steam explosion pretreatment, and then carrying out explosion; sieving corn straw subjected to steam explosion treatment with a 50-mesh sieve, collecting undersize, adding the undersize into 2 times of water by weight, heating to 60 ℃, stirring at 100rpm under the condition of heat preservation for 90min, heating to 121 ℃, preserving heat for 10min, and naturally cooling to room temperature to obtain a culture solution;
subjecting Trichoderma koningii seed solution (density of 1 × 10)8cfu/mL) and Aspergillus niger seed solution (density 1X 10)8cfu/mL) is mixed according to the volume ratio of 2:1, then the mixture is inoculated into culture solution according to the inoculation amount of 6-8%, the culture is carried out for 96 hours, the stirring speed is controlled to be 100rpm during the culture, the culture temperature is 32 ℃, and the pH value in the culture process is controlled to be 5 by feeding ammonia water; after the culture is finished, carrying out ultrasonic treatment for 40min, wherein the ultrasonic frequency is 30KHz, the temperature is adjusted to 55 ℃, lysozyme is added, and the addition amount of the lysozyme is 2 ten thousand U: 1L of solution, and carrying out enzymolysis for 12 hours under the condition of heat preservation; then inactivating enzyme at 121 deg.C for 5min, and naturally cooling to room temperature to obtain fermentation culture medium;
mixing seed solution of Clostridium acetobutylicum (density of 3 × 10)8cfu/mL) was inoculated into an anaerobic fermenter containing a fermentation medium at a temperature of 32 ℃ for 36 hours in an inoculum size of 7% (by volume), and then inoculated with a Neurospora crassa seed solution (density of 3X 10)8cfu/mL), the inoculation amount is 10% (volume ratio); continuously culturing for 72h, after fermentation is finished, taking the fermentation liquor, centrifuging for 10min at 8000rpm, collecting upper-layer liquid and thallus precipitate, and then detecting the content of butanol and ethanol in the upper-layer liquid by gas chromatography; obtaining a crude product through reduced pressure distillation, and then obtaining the bio-butanol and the bio-ethanol through rectification and dehydration processes; washing the thallus precipitate with water, drying at 80 deg.c and crushing to obtain protein powder.
Example 2
The method for producing the bio-butanol and the bio-ethanol by fermenting the corn straws as the main raw material comprises the following steps:
firstly, crushing the corn stalks to be within 5cm, then placing the crushed corn stalks under the conditions of 2MPa of pressure and 10min of residence time for steam explosion pretreatment, and then carrying out explosion; sieving corn straws subjected to steam explosion treatment by a 50-mesh sieve, collecting undersize, adding the undersize into water with the weight of 1.5 times of that of the corn straws, heating to 60 ℃, stirring for 90min at 100rpm under the condition of heat preservation, heating to 121 ℃, preserving heat for 10min, and naturally cooling to room temperature to obtain a culture solution;
subjecting Trichoderma koningii seed solution (density of 1 × 10)8cfu/mL) and Aspergillus niger seed solution (density 1X 10)8cfu/mL) is mixed according to the volume ratio of 3:2, then the mixture is inoculated into culture solution according to the inoculation amount of 6-8%, the culture is carried out for 96 hours, the stirring speed is controlled to be 100rpm during the culture, the culture temperature is 32 ℃, and the pH value in the culture process is controlled to be 4.5 by feeding ammonia water; after the culture is finished, carrying out ultrasonic treatment for 40min, wherein the ultrasonic frequency is 30KHz, the temperature is adjusted to 55 ℃, lysozyme is added, and the addition amount of the lysozyme is 2 ten thousand U: 1L of solution, and carrying out enzymolysis for 12 hours under the condition of heat preservation; then inactivating enzyme at 121 deg.C for 5min, and naturally cooling to room temperature to obtain fermentation culture medium;
mixing seed solution of Clostridium acetobutylicum (density of 2 × 10)8cfu/mL) was inoculated into an anaerobic fermenter containing a fermentation medium at a temperature of 30 ℃ for 24 hours in an inoculum size of 7% (by volume), and then inoculated with a Neurospora crassa seed solution (density of 2X 10)8cfu/mL), the inoculation amount is 10% (volume ratio); continuously culturing for 72h, after fermentation is finished, taking the fermentation liquor, centrifuging for 10min at 8000rpm, collecting upper-layer liquid and thallus precipitate, and then detecting the content of butanol and ethanol in the upper-layer liquid by gas chromatography; obtaining a crude product through reduced pressure distillation, and then obtaining the bio-butanol and the bio-ethanol through rectification and dehydration processes; and washing the thallus precipitate with water, drying at 80 ℃, and finally crushing to obtain the protein powder.
Example 3
The change of the main components of the corn straw by the blasting is shown in the table 1:
TABLE 1
| Index (I) | Before blasting | After blasting |
| Hemicellulose and cellulose% | 61.2 | 48.9 |
| Lignin% | 22.7 | 15.6 |
| Oligo- |
0 | 6.9 |
| Cello- |
0 | 3.1 |
And (4) conclusion: the blasting causes the cell wall of the straw to be damaged, and part of hemicellulose and cellulose are degraded and dissolved out, so that the subsequent enzymolysis of cellulose by cellulase is facilitated; the crystallinity and polymerization degree of cellulose are reduced in the blasting pretreatment process, and the hemicellulose is degraded into xylose and the like through self-hydrolysis and can be used as a carbon source of a strain.
Example 4
Taking example 1 as an example, the influence of the way of treating straws by the single strain and the composite strain on the enzyme production activity is detected, the specific enzyme production activity is shown in fig. 1, the filter paper enzyme produced by trichoderma koningii fermentation has high activity, but the produced cellulase has the defect of low activity of beta-glucosidase, aspergillus niger has high ability of producing beta-glucosidase and low ability of producing filter paper enzyme, the two strains are subjected to synergistic fermentation on straws, the activities of the filter paper enzyme and the beta-glucosidase are greatly improved, and the enzyme activities can reach 357U/ml and 395U/ml respectively.
The main components of the fermentation medium of the invention are shown in table 2:
TABLE 2
| Components | The hexasaccharide content is g/l | The content of pentasaccharide is g/l | Protein content g/l |
| Trichoderma koningii | 51.8 | 33.4 | 17.1 |
| Aspergillus niger | 39.2 | 26.8 | 13.9 |
| Mixed fermentation | 73.6 | 45.7 | 21.4 |
As shown in Table 2, the fermentation medium of the invention is rich in hexaose and pentaose, has a protein content of more than 20g/L, and can be used as a carbon source and a carbon source for anaerobic fermentation of Clostridium acetobutylicum and Neurospora crassa.
Example 5
Influence of different strain fermentation alcohol production modes on alcohol production quantity:
using example 1 as the experimental group, a control group was set, wherein control group 1: only clostridium acetobutylicum is adopted, and the fermentation time is 108 h; control group 2: only neurospora crassa is adopted, and the fermentation time is 108 h; control group 3: two strains are inoculated at the same time, and the fermentation time is 108 h. As shown in fig. 2, the experimental group had the highest butanol content and the control group 3 had the highest ethanol content, but the butanol yield was low, probably because neurospora crassa easily competed with clostridium acetobutylicum for producing a hexose carbon source, which is not favorable for clostridium acetobutylicum producing butanol; the difference between the ethanol yield of the experimental group and the ethanol yield of the control group 3 is not obvious, and the ethanol production performance of the experimental group is optimal by integrating the fermentation yields of butanol and ethanol. As shown in figure 3, butanol and ethanol are both obviously increased along with the increase of the mixed fermentation time, after 24 hours, the butanol amplification is obviously reduced, the ethanol amplification is obvious, and the fermentation time is selected from 48 to 72 hours, so that the best alcohol production performance is achieved.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (6)
1. A process for the preparation and separation of bio-butanol and bio-ethanol, said process comprising the steps of:
step 1) straw treatment: firstly, pulverizing corn stalks to be within 5cm, placing under the conditions of pressure of 1.5-2MPa and residence time of 10-15min to carry out steam explosion pretreatment, and then carrying out explosion; sieving corn straw subjected to steam explosion treatment with a 50-mesh sieve, collecting undersize, adding the undersize into water 1.5-2 times of the weight of the corn straw, heating to 60 ℃, stirring for 90min at 100rpm under the condition of heat preservation, heating to 121 ℃, preserving heat for 10min, and naturally cooling to room temperature to obtain a culture solution;
step 2) preparing a fermentation medium: mixing the Trichoderma koningii seed liquid and the Aspergillus niger seed liquid according to the volume ratio of 2-3:1-2, then inoculating the mixture into a culture solution according to the inoculation amount of 6-8%, culturing for 96h, controlling the stirring speed to be 100rpm during culturing, controlling the culturing temperature to be 32 ℃, and controlling the pH value in the culturing process to be 4-5 by feeding ammonia water; after the culture is finished, carrying out ultrasonic treatment, adjusting the temperature to 55 ℃, adding lysozyme, and carrying out enzymolysis for 12 hours under the heat preservation condition; then inactivating enzyme at 121 deg.C for 5min, and naturally cooling to room temperature to obtain fermentation culture medium; the addition amount of the lysozyme is 2 ten thousand U: 1L of the solution;
step 3) alcohol production: inoculating the clostridium acetobutylicum seed solution into an anaerobic fermentation tank containing a fermentation culture medium for culturing at the temperature of 30-32 ℃ for 24-36h, then inoculating the neurospora crassa seed solution, continuing culturing for 48-72h, taking the fermentation broth after the culture is finished, centrifuging at 8000rpm for 10min, and collecting the upper-layer liquid and thallus precipitate;
step 4) separating alcohol and preparing protein powder: distilling the upper layer liquid under reduced pressure to obtain a crude product, and then rectifying and dehydrating to obtain the bio-butanol and the bio-ethanol; washing the thallus precipitate with water, drying at 80 deg.C, and pulverizing to obtain protein powder.
2. The method according to claim 1, wherein the density of the Trichoderma koningii seed solution is 1 x 108cfu/mL。
3. The method according to claim 1, wherein the Aspergillus niger seed solution has a density of 1 x 108cfu/mL。
4. The method according to claim 1, wherein the ultrasonic treatment time is 40min, and the ultrasonic frequency is 30 KHz.
5. The method of claim 1, wherein the inoculum size of the clostridium acetobutylicum seed solution is 5-7%.
6. The method of claim 1, wherein the Neurospora crassa seed liquid is inoculated in an amount of 8-10%.
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| CN101492700B (en) * | 2009-03-05 | 2012-07-04 | 王建设 | Intensive processing method for stalk articles or agricultural castoff |
| CN103409470B (en) * | 2013-05-15 | 2017-10-17 | 中国科学院广州能源研究所 | A kind of method using the mixed sugar segmentation containing pentose and hexose, mixed fungus fermentation production ethanol, butanol and acetone |
| FR3013732B1 (en) * | 2013-11-22 | 2016-10-21 | Ifp Energies Now | ENDOGLUCANASTIC VARIANTS WITH IMPROVED ACTIVITY AND USES THEREOF |
| CN106834361A (en) * | 2017-03-24 | 2017-06-13 | 黑龙江中丹建业生物能源有限公司 | The method that stalk produces cellulosic ethanol |
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