CN115537431A - Method for producing ethanol by fermenting corn fiber treated by citric acid and corn fiber-based ethanol product - Google Patents
Method for producing ethanol by fermenting corn fiber treated by citric acid and corn fiber-based ethanol product Download PDFInfo
- Publication number
- CN115537431A CN115537431A CN202110741035.0A CN202110741035A CN115537431A CN 115537431 A CN115537431 A CN 115537431A CN 202110741035 A CN202110741035 A CN 202110741035A CN 115537431 A CN115537431 A CN 115537431A
- Authority
- CN
- China
- Prior art keywords
- corn fiber
- gas
- saccharification
- fermentation
- citric acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
- C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for producing cellulosic ethanol by fermenting corn fiber by using citric acid as a catalyst, which comprises the following steps: the method comprises the following steps of (1) effectively mixing solid corn fiber and a small amount of low-concentration citric acid solution in a helical ribbon type stirring reactor, pretreating at high temperature and high pressure, and performing low-strength structural damage on the corn fiber by using citric acid to hydrolyze hemicellulose of the corn fiber and make the cellulose suitable for enzymolysis saccharification and generate a low-level inhibitor at the same time; the sequence of the biorefinery process is adjusted into a pretreatment-saccharification-detoxification-fermentation process, and the pretreated corn fiber material is subjected to enzymatic saccharification, biological detoxification and ethanol fermentation; the inactivation of the biological detoxified strain is completed by secondary enzymolysis and saccharification. The saccharification, detoxification, inactivation and fermentation can be carried out in the same reactor, so that high fermentation indexes are obtained, material loss, wastewater discharge, operation cost and contamination risk are greatly reduced, and the space and process steps of the reactor are saved.
Description
Technical Field
The invention relates to the field of biorefinery, in particular to a method for pretreating corn fibers and producing cellulose-based ethanol by using citric acid as a catalyst.
Background
Corn fiber, which is used as the outer pericarp of the corn kernels, is a byproduct of the corn starch production process by corn wet milling. It is composed of corn seed coat and a small amount of starch attached to the inside, which contains higher content of hemicellulose, and its arabinoxylan can reach 30-50% (w/w) of dry weight. Compared with other agricultural biomass-derived raw materials, the corn fiber is not required to be collected from farmlands, but is directly obtained from factories for producing corn starch, and after being output from wet grinding equipment, any additional processing processes such as cleaning and the like are not required, so that the corn fiber is a more convenient and easily available lignocellulose raw material with wide sources.
At present, the process of producing ethanol by using corn fiber as a raw material mainly has the following obstacles: the hemicellulose content of corn fiber is very high, and only partial degradation can be realized in a pretreatment step, while complete degradation of the corn fiber needs to be completed in a subsequent enzymatic saccharification step. The acetic acid generated by the acetyl in the hemicellulose during the degradation process must be reduced to a low level by a biological detoxification mode to realize high-yield enzymolysis and ethanol fermentation. Because the common biological refining detoxification step is after pretreatment and before enzymatic saccharification, acetic acid generated by hemicellulose in corn fiber in the enzymatic saccharification process cannot be removed in a detoxification manner, so that a large amount of acetic acid is accumulated and ethanol fermentation fails.
The method for producing cellulose-based ethanol by using corn fiber as a raw material has the defects of low technical index and conversion rate, high pollution emission and the like, so that the establishment of a method for realizing the efficient conversion of the corn fiber is necessary.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is already known to a person skilled in the art.
Disclosure of Invention
In view of the technical problems existing in the production of cellulosic ethanol by the conversion of the corn fiber, the invention provides a method for pretreating the corn fiber by using citric acid as an acid catalyst and producing ethanol by using the corn fiber. Then carrying out ethanol fermentation. The whole processes of saccharification in situ, liquid biological detoxification and fermentation only need to be carried out in the same fermentation tank, and the period can be reduced to 144h.
The invention mainly achieves the aim through the following technical scheme, and provides a method for producing ethanol by fermenting corn fiber treated by citric acid, which comprises the following steps:
dilute acid pretreatment: mixing the corn fiber and a citric acid solution, controlling the temperature to be 150-180 ℃ for reaction, then adjusting the pH to 4.8-5.0, and carrying out pre-saccharification, biological detoxification, secondary saccharification and ethanol fermentation.
According to some embodiments of the invention, the method comprises the steps of:
(1) Dilute acid pretreatment: mixing the corn fiber and a citric acid solution, and controlling the temperature to be 150-180 ℃ for reaction; then adjusting the pH value to 4.8-5.0;
(2) Pre-saccharification: carrying out enzymolysis on the corn fiber obtained in the step (1) in an aqueous phase environment under the action of cellulase;
(3) Biological detoxification: inoculating a detoxification strain into the mixture obtained after the pre-saccharification in the step (2) for biological detoxification treatment;
(4) And (3) secondary saccharification: heating the mixture subjected to biological detoxification in the step (3) to 45-55 ℃, preserving the heat for more than 12 hours, and then cooling to obtain corn fiber hydrolysate;
(5) Ethanol fermentation: inoculating fermentation strains into the corn fiber hydrolysate, and performing ethanol fermentation.
Further, (4) a secondary saccharification process: and (4) heating the mixture subjected to biological detoxification in the step (3) to 45-55 ℃, preserving the heat for more than 12 hours to inactivate the detoxified strains, and then cooling to obtain the corn fiber hydrolysate.
As a preferred embodiment of the present invention, steps (2) to (5) are carried out in the same reactor with stirring and temperature control functions, for example, a fermenter. Namely, the processes of enzymolysis saccharification, liquid biological detoxification and biological fermentation are completed in situ in sequence, so that the risk of bacterial contamination in the process of transferring the tank is avoided.
The reaction time in the dilute acid pretreatment process in the step (1) is more than 2min, further 2-5 min, further 2-4 min.
Stirring is carried out in the dilute acid pretreatment process in the step (1), and the stirring speed is 30-60 rpm.
Further, the temperature is controlled to be 160-170 ℃ in the dilute acid pretreatment process in the step (1).
In the step (1), the ratio of the mass (g) of the citric acid obtained after the citric acid solution is purified to the dry weight (g) of the corn fiber is (2-6): 100.
In the invention, citric acid is used as a milder acid catalyst, so that the concentration of the inhibitor after pretreatment can be reduced; in addition, the weak acidity can raise the pH of the system compared with that of strong acid, and can reduce Ca (OH) greatly in the neutralization process 2 The usage amount of the fermentation liquid reduces the influence on the subsequent ethanol fermentation. In addition, as stirring is usually required in the fermentation process, the citric acid is used to avoid more calcium salt particles, reduce the influence on cells of the ethanol fermentation strain and avoid the influence on the yield of ethanol.
Neutralizing agents used to adjust the pH in step (1) include, but are not limited to, calcium hydroxide, sodium hydroxide, or combinations thereof.
The solid content of the pre-saccharification system comprising corn fiber, cellulase and water in the step (2) is 20-40% (w/v).
In the step (2), the dosage of the cellulase per gram of the corn fiber (DM) is 5-20 mg.
The temperature is controlled to be 40-60 ℃ in the pre-saccharification process in the step (2), and the time is 6-24 h.
And performing pre-saccharification treatment to obtain a hydrolysate with high sugar concentration, wherein furfural, 5-hydroxymethylfurfural and high-concentration acetic acid exist in the hydrolysate in a free form. Then inoculating inhibitor biodegradation strains, performing the biological detoxification process of the inhibitor in a liquid state and aerobic form, simultaneously ensuring that fermentable sugar in the inhibitor is not excessively consumed as much as possible, and finally converting all furfural, 5-hydroxymethylfurfural and acetic acid in hydrolysate into carbon dioxide and water.
The dilute acid pretreatment process of step (1) inevitably produces inhibitors that inhibit the enzymatic activity, cell growth and metabolic activity of the fermenting microorganism. The inhibitor includes but is not limited to furfural, 5-hydroxymethyl furfural and acetic acid.
The detoxified strain in step (3) may be any strain capable of metabolizing the inhibitor as defined above.
Further, the detoxified strain in step (3) includes, but is not limited to, any one of ramaria resinifera and paecilomyces variotii. Further, the detoxified strain in the step (3) is Paecilomyces variotii FN89, and the preservation number of the strain is CGMCC17665. The preservation date is 5, 8 and 2019, and the preservation address is the China general microbiological culture Collection center. It has strong degradation capability to the inhibitor, and preferentially utilizes the inhibitor in the hydrolysate system and then utilizes the fermentable sugar.
The inoculation amount in the step (3) is 5 to 20 percent, and further 7 to 18 percent.
Ventilating with ventilation amount of 0.1-10 vvm during detoxification. The temperature of the detoxification process is 20-37 ℃, the stirring speed is 300-700 rpm, and the time is 24-52 h.
And (4) basically and completely removing the inhibitor in the system through the detoxification treatment in the step (3).
In order to inactivate Paecilomyces variotii FN89, temperature rise-heat preservation treatment is carried out after detoxification treatment, and in the process, the inventor unexpectedly finds that the process is beneficial to further saccharification of corn fiber, prolongs the saccharification reaction process, and produces more fermentation sugar from fiber raw materials.
And (5) in the step (4), aerating is not carried out in the secondary saccharification process.
The final temperature of the temperature reduction in the step (4) is 25-35 ℃.
The fermentation strain in the step (5) is Saccharomyces cerevisiae, and further is Saccharomyces cerevisiae Z100. The preservation number is CGMCC 17734. The preservation date is No. 2019, 05 and 08, and the preservation address is China general microbiological culture Collection center.
The inoculation amount of the fermentation strain in the step (5) is 7-15% (v/v).
The temperature of the fermentation process in the step (5) is 25-35 ℃, the stirring speed is 150-300rpm, the pH value is controlled to be 5-6, and the fermentation time is 60-144 h.
The concentration of ethanol in the fermentation liquid in the step (5) is more than 5%, and further more than 6.5%. The discharge amount of wastewater was 9.9kg/kg ethanol. Are all similar to the related indexes of ethanol fermented by corn raw materials.
The invention also provides a corn fiber-based ethanol product prepared by any one of the methods.
Compared with the prior art, the invention has the following positive effects:
the method has the advantages that the pre-saccharification is carried out firstly, then the liquid biodegradation of the inhibitor is carried out, and the inhibitor in the corn fiber after the pretreatment, especially the high-content acetic acid, can be completely degraded; secondly, mild citric acid is adopted to replace strong acid as a pretreatment catalyst of the corn fiber, so that the generation of inhibitors and the biodegradation strength of the inhibitors are reduced; thirdly, the added secondary saccharification process inactivates the biological detoxification strains, prolongs the saccharification time and improves the initial concentration of fermentable sugar; finally, saccharification, inhibitor biodegradation and fermentation are carried out in the same reactor, so that the risk of bacteria contamination can be effectively reduced.
Detailed Description
The following examples are intended to describe the present invention in further detail, but the present invention is not limited to the scope defined by the examples.
DM in the present invention refers to the dry weight of corn fiber.
The composition of the synthetic medium in the examples is as follows: 20g/L glucose, 2g/L potassium dihydrogen phosphate, 1g/L ammonium sulfate, 1g/L yeast extract and 1g/L magnesium sulfate heptahydrate.
Examples YPD medium composition was as follows: 20g/L peptone, 20g/L glucose and 10g/L yeast extract.
Example 1
Dilute acid pretreatment: adding 1200g (DM) of corn fiber and a citric acid solution into a 20-L reactor, wherein the solid-to-liquid ratio of the DM to the citric acid solution is 2:1 (w/w), stirring and mixing uniformly. Wherein, the ratio of the mass (g) of the added citric acid solution after being purified to the mass (DM, g) of the corn fiber is 4. The reaction conditions of the dilute acid pretreatment process are as follows: the temperature is controlled at 165 +/-1 ℃, the time is 2min, and the stirring speed is 50rpm. And after the dilute acid pretreatment reaction is finished, discharging, wherein the corn fiber is in a wet state, and the solid content is about 50% (w/w).
Pre-saccharification: the pre-saccharification process is carried out in a 5-L fermentation tank, and the corn fiber is pre-saccharified in a water phase environment under the action of cellulase. The pre-saccharification conditions are as follows: the pH was adjusted to pH4.8 with a solids content of 25% (w/v), the cellulase dosage per gram of dilute acid pretreated corn fiber (DM) was 5mg, the temperature was controlled at 50 deg.C, and the mixture was stirred at 200rpm for 12h, during which time 20wt% calcium hydroxide solution was added at any time to control the pH at 4.8.
As a large amount of acetyl is released along with the progress of saccharification in the pre-saccharification process, the pH value is reduced due to the increase of the concentration of acetic acid in the saccharification liquid, and therefore, alkali is supplemented at any time in the pre-saccharification process so as to stabilize the pH value of the system.
Preparing seed liquid for biodegradation process: the Paecilomyces variotii FN89 in the frozen tube is firstly cultured on a synthetic medium plate for 72h for activation, and the temperature is 37 ℃; then, transferring the spores on the flat plate to a synthetic culture medium inclined plane by using an inoculating loop, and culturing for 72h at 37 ℃; the spores were then washed off the slant with 0.05% (w/v) Tween 80, transferred to 100mL of synthetic medium in 500mL Erlenmeyer flasks at 1% (v/v), and incubated at 37 ℃ for 18h at 300rpm as seed solution for the biodegradation process.
Biological detoxification: inoculating the detoxified strain Paecilomyces variotii FN89 into the mixture obtained after pre-saccharification according to the inoculation amount of 10% (v/v), wherein the stirring speed is 500rpm, the ventilation amount is 1vvm, the temperature is 37 ℃ and the time is 36h in the biological detoxification process.
And (3) secondary saccharification: and after biological detoxification is finished, continuously raising the temperature to 50 ℃ for 12 hours, and then cooling to 30 ℃ to obtain the corn fiber hydrolysate for fermentation.
Preparing seed liquid for fermentation: transferring the saccharomyces cerevisiae Z100 preserved in the freezing storage tube into a 100mL conical flask filled with 30mL YPD medium, and culturing at 30 ℃ for 12h for activation; inoculating 10% (v/v) of the inoculum into corn fiber hydrolysate with solid content (w/w) of 5%, and culturing at 30 deg.C and 200rpm for 12h to obtain first-stage seed solution. And (3) transferring the primary seed liquid into corn fiber hydrolysate with the solid content (w/w) of 10% according to the inoculation amount of 10% (v/v), and culturing at 200rpm and 30 ℃ for 16h to obtain the saccharomyces cerevisiae Z100 secondary seed liquid.
Ethanol fermentation: inoculating the saccharomyces cerevisiae Z100 secondary seed liquid into the corn fiber hydrolysate at an inoculation amount of 10% (v/v). The temperature in the fermentation process is 30 ℃, the stirring speed is 200rpm, the pH value is 5.5, and the fermentation time is 72 hours. The pH value is adjusted by 5mol/L sodium hydroxide solution.
Compositional measurements were performed on the dilute acid pretreated corn fiber of example 1 to verify the effective disruption of the corn fiber structure by citric acid pretreatment. The measurement method used is referred to a two-step acidolysis method published by the us renewable energy laboratory. The two-step acidolysis method comprises the following steps: sulfuric acid is used, the concentration of the sulfuric acid is 72wt%, the dosage of the sulfuric acid is 1ml/0.1g of corn fiber samples (DM), the first-step acidolysis temperature is 25 ℃, and the time is 40 minutes; then 28ml of deionized water is added, the mixture is uniformly mixed, then the temperature is raised to 120 ℃, the second step of acidolysis is carried out, the temperature of the second step of acidolysis is controlled to be 120 ℃, the time is 1 hour, the acidolysis is completed, and the obtained sample is used for analyzing the content of the components. The test results are shown in Table 1.
TABLE 1 compositional analysis of virgin corn fiber and citric acid pretreated corn fiber
Example 2
Dilute acid pretreatment: adding 1500g (DM) of corn fiber and a citric acid solution into a 20-L pretreatment reactor, wherein the solid-to-liquid ratio of the DM to the citric acid solution is 2:1 (w/w), stirring and mixing uniformly. Wherein the ratio of the mass (g) of the added citric acid solution after being purified to the mass (DM, g) of the corn fiber is 6. The reaction conditions in the dilute acid pretreatment process are as follows: the temperature is controlled at 175 +/-1 ℃, the time is 5min, and the stirring speed is 50rpm. And after the dilute acid pretreatment reaction is finished, discharging, wherein the corn fiber is in a wet state, and the solid content is about 50% (w/w).
Pre-saccharification: the pre-saccharification process is carried out in a 5-L fermentation tank, and the corn fiber is pre-saccharified in a water phase environment under the action of cellulase. The pre-saccharification conditions are as follows: the pH is adjusted to 4.8, the solid content is 25% (w/v), the dosage of cellulase per gram of dilute acid pretreated corn fiber (DM) is 5mg, the temperature is controlled to be 50 ℃, the stirring is carried out at 200rpm for 12h, and the pH is controlled to be 4.8 by supplementing 30wt% of calcium hydroxide solution at any time during the stirring.
Preparing seed liquid for biodegradation process: the Paecilomyces variotii FN89 in the frozen tube is firstly cultured on a synthetic medium plate for 72h for activation, and the temperature is 37 ℃; then, transferring the spores on the synthetic medium flat plate to a synthetic medium inclined plane by using an inoculating loop, and culturing for 72 hours at 37 ℃; the spores were then washed from the slant with 0.05% (w/v) Tween 80, transferred to 100mL of synthetic medium in 500mL Erlenmeyer flasks at 1% (v/v), and incubated at 37 ℃ for 18h at 300rpm as seed solution for the biodegradation process.
Biological detoxification: inoculating the detoxified strain Paecilomyces variotii FN89 into the mixture obtained after pre-saccharification according to the inoculation amount of 5% (v/v), wherein the stirring speed is 500rpm, the ventilation amount is 2vvm, the temperature is 37 ℃ and the time is 48h in the biological detoxification process.
And (3) secondary saccharification: after biological detoxification is finished, the temperature is raised to 50 ℃ and maintained for 12h, and then the temperature is reduced to 30 ℃ to obtain corn fiber hydrolysate for fermentation.
Preparing seed liquid for fermentation: transferring the saccharomyces cerevisiae Z100 preserved in the freezing storage tube into a 100mL conical flask filled with 30mL YPD medium, and culturing at 30 ℃ for 12h for activation; inoculating 10% (v/v) of the inoculum into corn fiber hydrolysate with solid content (w/w) of 5%, and culturing at 30 deg.C and 200rpm for 12h to obtain first-stage seed solution. And (3) transferring the primary seed liquid into corn fiber hydrolysate with the solid content (w/w) of 10% according to the inoculation amount of 10% (v/v), and culturing at the temperature of 200rpm and 30 ℃ for 16h to obtain the saccharomyces cerevisiae Z100 secondary seed liquid.
Ethanol fermentation: and inoculating the saccharomyces cerevisiae Z100 secondary seed liquid into the corn fiber hydrolysate in an inoculation amount of 10% (v/v). The temperature in the fermentation process is 30 ℃, the stirring speed is 200rpm, the pH value is controlled at 5.5, and the fermentation time is 72 hours. The pH value is adjusted by 5mol/L sodium hydroxide solution.
Comparative example 1
Dilute acid pretreatment: adding 1200g (DM) of corn fiber and a citric acid solution into a 20-L reactor, wherein the solid-to-liquid ratio of the DM to the citric acid solution is 2:1 (w/w), stirring and mixing uniformly. Wherein, the ratio of the mass (g) of the added citric acid solution after being purified to the mass (DM, g) of the corn fiber is 4. The reaction conditions of the dilute acid pretreatment process are as follows: the temperature is controlled at 165 +/-1 ℃, the time is 2min, and the stirring speed is 50rpm. And after the dilute acid pretreatment reaction is finished, discharging, wherein the corn fiber is in a wet state, and the solid content is about 50% (w/w).
Adjusting the pH of dilute acid pretreated corn fiber: the pH of the corn fiber after diluted acid pretreatment is adjusted to 4.8-5.0 by using a calcium hydroxide solution with the concentration of 20% (w/w) for the subsequent biological detoxification process.
Preparation of seeds for the biodegradation process: the Paecilomyces variotii FN89 in the frozen tube is firstly cultured on a synthetic medium plate for 72h for activation, and the temperature is 37 ℃; then, transferring the spores on the flat plate to a synthetic culture medium inclined plane by using an inoculating loop, and culturing for 72h at 37 ℃; uniformly coating a flat plate full of Paecilomyces variotii FN89 spores on the surface of 200g of the corn fiber material in a mass ratio of 1:10, placing the seeds in an incubator to be cultured for 72 hours to be used as detoxified seeds.
Solid biological detoxification: mixing the detoxified seeds in a proportion of 1:10 is added to the surface of the corn fiber raw material at the temperature of 37 ℃ for 48 hours.
Pre-saccharification: pre-saccharifying the corn fiber after biological detoxification, wherein the pre-saccharifying process is carried out in a 5-L fermentation tank. The pre-saccharification conditions are as follows: the solid content was 25% (w/v), the cellulase amount per gram of corn fiber (DM) was 5mg, and the mixture was stirred at 200rpm at 50 ℃ for 12 hours, during which time 30wt% calcium hydroxide solution was added at any time to control the pH at 4.8.
Preparing seed liquid for fermentation: transferring the saccharomyces cerevisiae Z100 preserved in the freezing storage tube into a 100mL conical flask filled with 30mL YPD medium, and culturing at 30 ℃ for 12h for activation; inoculating 10% (v/v) of the inoculum into corn fiber hydrolysate with solid content (w/w) of 5%, and culturing at 200rpm and 30 deg.C for 12h to obtain primary seed solution. And (3) transferring the primary seeds into corn fiber hydrolysate with the solid content (w/w) of 10% according to the inoculation amount of 10% (v/v), and culturing for 16h at 200rpm and 30 ℃ to obtain a saccharomyces cerevisiae Z100 secondary seed solution.
Ethanol fermentation: inoculating the saccharomyces cerevisiae Z100 secondary seed liquid into the corn fiber hydrolysate in an inoculation amount of 10% (v/v). The condition temperature in the fermentation process is 30 ℃, the stirring speed is 200rpm, the pH value is controlled at 5.5, and the fermentation time is 72 hours. During the fermentation, 5mol/L sodium hydroxide solution is used for adjusting the pH value.
Comparative example 2
Dilute acid pretreatment: adding 1200g (DM) of corn fiber and a citric acid solution into a 20L reactor, wherein the solid-to-liquid ratio of the DM to the citric acid solution is 2:1 (w/w), stirring and mixing uniformly. Wherein, the ratio of the mass (g) of the added citric acid solution after being purified to the mass (DM, g) of the corn fiber is 4. The reaction conditions of the dilute acid pretreatment process are as follows: the temperature is controlled at 165 + -1 deg.C, the time is 2min, and the stirring speed is 50rpm. And after the dilute acid pretreatment reaction is finished, discharging, wherein the corn fiber after the dilute acid pretreatment is in a wet state, and the solid content is about 50% (w/w). Adjusting the pH value of the corn fiber pretreated by dilute acid to be 4.8, and then carrying out pre-saccharification.
Pre-saccharification: the pre-saccharification process is carried out in a 5-L fermenter. The pre-saccharification conditions are as follows: the solid content is 25% (w/v), the dosage of the cellulase for each gram of the pretreated corn fiber (DM) is 5mg, the temperature is controlled to be 50 ℃, the stirring is carried out for 12 hours at 200rpm, and 20wt% of calcium hydroxide solution is supplemented at any time during the stirring to control the pH to be 4.8.
Preparing seed liquid for biodegradation process: the Paecilomyces variotii FN89 in the frozen tube is firstly cultured on a synthetic medium plate for 72h for activation, and the temperature is 37 ℃; then, transferring the spores on the flat plate to a synthetic culture medium inclined plane by using an inoculating loop, and culturing for 72h at 37 ℃; the spores were then washed off the slant with 0.05% (w/v) Tween 80, transferred to 100mL of synthetic medium in 500mL Erlenmeyer flasks at 1% (v/v), and incubated at 37 ℃ for 18h at 300rpm as seed solution for the biodegradation process.
Biological detoxification: inoculating the detoxicated strain Paecilomyces variotii FN89 into the mixture obtained after pre-saccharification at an inoculation amount of 10% (v/v), wherein the stirring speed in the biological detoxification process is 500rpm, the ventilation amount is 1vvm, the temperature is 37 ℃, and the time is 48h.
Preparing seed liquid for fermentation: transferring the saccharomyces cerevisiae Z100 preserved in the freezing storage tube into a 100mL conical flask filled with 30mL YPD medium, and culturing at 30 ℃ for 12h for activation; inoculating 10% (v/v) of the inoculum into corn fiber hydrolysate with solid content (w/w) of 5%, and culturing at 200rpm and 30 deg.C for 12h to obtain first-stage seed solution. And (3) transferring the primary seed liquid into corn fiber hydrolysate with the solid content (w/w) of 10% according to the inoculation amount of 10% (v/v), and culturing at 200rpm and 30 ℃ for 16h to obtain the saccharomyces cerevisiae Z100 secondary seed liquid.
Ethanol fermentation: inoculating the saccharomyces cerevisiae Z100 secondary seed liquid into the corn fiber hydrolysate in an inoculation amount of 10% (v/v). The temperature in the fermentation process is 30 ℃, the stirring speed is 200rpm, the pH value is controlled at 5.5, and the fermentation time is 72 hours. The pH was adjusted by 5mol/L sodium hydroxide solution.
TABLE 1 test results of ethanol concentration in fermentation broth
| Concentration of ethanol in fermentation broth | |
| Example 1 | 70.2g/L |
| Example 2 | 57.2g/L |
| Comparative example 1 | 31.6g/L |
| Comparative example 2 | 50.5g/L |
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the concept of the present invention, and these modifications and decorations should also be regarded as being within the protection scope of the present invention.
Claims (10)
1. A method for producing ethanol by fermenting corn fiber treated by citric acid comprises the following steps:
dilute acid pretreatment: mixing the corn fiber and a citric acid solution, controlling the temperature to be 150-180 ℃ for reaction, then adjusting the pH to 4.8-5.0, and carrying out pre-saccharification, biological detoxification, secondary saccharification and ethanol fermentation.
2. The method of claim 1, comprising the steps of:
(1) Dilute acid pretreatment: mixing corn fiber and citric acid solution, and controlling the temperature to be 150-180 ℃ for reaction; then adjusting the pH value to 4.8-5.0;
(2) Pre-saccharification: carrying out enzymolysis on the corn fiber obtained in the step (1) in an aqueous phase environment under the action of cellulase;
(3) Biological detoxification: inoculating a detoxification strain into the mixture obtained after the pre-saccharification in the step (2) for biological detoxification treatment;
(4) And (3) secondary saccharification: heating the mixture subjected to biological detoxification in the step (3) to 45-55 ℃, preserving the heat for more than 12h, and then cooling to obtain corn fiber hydrolysate;
(5) Ethanol fermentation: inoculating fermentation strain into corn fiber hydrolysate, and performing ethanol fermentation.
3. The method according to claim 2, wherein the steps (2) to (5) are carried out in the same reactor, and the reactor has stirring and temperature control functions; further, the reactor is a fermenter.
4. The method according to claim 1 or 2, wherein the reaction time in the dilute acid pretreatment process is more than 2 min; and/or the presence of a gas in the gas,
stirring in the dilute acid pretreatment process at the stirring speed of 30-60 rpm; and/or the presence of a gas in the gas,
controlling the temperature to be 160-170 ℃ in the dilute acid pretreatment process; and/or the presence of a gas in the atmosphere,
the ratio of the mass of the citric acid after the citric acid solution is purified to the dry weight of the corn fiber is (2-6) to 100; and/or the presence of a gas in the gas,
neutralizing agents used to adjust the pH include, but are not limited to, calcium hydroxide, sodium hydroxide, or combinations thereof.
5. The method of claim 2, wherein the pre-saccharification system comprising corn fiber, cellulase and water in step (2) has a solid content of 20-40% (w/v); and/or the presence of a gas in the atmosphere,
in the step (2), the dosage of the cellulase per gram of the corn fiber (DM) is 5-20 mg; and/or the presence of a gas in the gas,
the temperature is controlled to be 40-60 ℃ in the pre-saccharification process in the step (2), and the time is 6-24 h.
6. The method of claim 2, wherein the detoxified bacterial species in step (3) are strains capable of metabolizing inhibitors including, but not limited to, furfural, 5-hydroxymethylfurfural, acetic acid; and/or the presence of a gas in the gas,
the detoxification strain in the step (3) comprises any one of but not limited to Sclerotinia resinifera and Paecilomyces variotii, further, the Paecilomyces variotii FN89 is Paecilomyces variotii, and the preservation number of the strain is CGMCC 17665; and/or the presence of a gas in the atmosphere,
the inoculation amount in the step (3) is 5-20 percent, and further 7-18 percent; and/or the presence of a gas in the atmosphere,
ventilating in the biological detoxification process in the step (3), wherein the ventilation volume is 0.1-10 vvm; and/or the presence of a gas in the gas,
the temperature is controlled to be 20-37 ℃ in the biological detoxification treatment process in the step (3), the stirring speed is 300-700 rpm, and the time is 24-52 hours.
7. The method of claim 2, wherein the secondary saccharification process in step (4) is not aerated; and/or the presence of a gas in the atmosphere,
the final temperature of the temperature reduction in the step (4) is 25-35 ℃.
8. The method according to claim 2, wherein the fermentation strain in step (5) is Saccharomyces cerevisiae, further Saccharomyces cerevisiae Z100 with a collection number of CGMCC 17734; and/or the presence of a gas in the gas,
the inoculation amount of the fermentation strain in the step (5) is 7-15% (v/v); and/or the presence of a gas in the atmosphere,
in the step (5), the temperature is controlled to be 25-35 ℃ in the fermentation process, and the stirring speed is 150-300 rpm; and/or the presence of a gas in the atmosphere,
controlling the pH value of the fermentation process in the step (5) to be 5-6; and/or the presence of a gas in the gas,
the fermentation time in the step (5) is 60-144 h.
9. The method according to claim 1 or 2, wherein the concentration of ethanol in the fermentation broth is 5% or more, further 6.5% or more.
10. A corn fiber-based ethanol, characterized by being prepared by the method of any one of claims 1 to 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110741035.0A CN115537431A (en) | 2021-06-30 | 2021-06-30 | Method for producing ethanol by fermenting corn fiber treated by citric acid and corn fiber-based ethanol product |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110741035.0A CN115537431A (en) | 2021-06-30 | 2021-06-30 | Method for producing ethanol by fermenting corn fiber treated by citric acid and corn fiber-based ethanol product |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115537431A true CN115537431A (en) | 2022-12-30 |
Family
ID=84723042
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110741035.0A Pending CN115537431A (en) | 2021-06-30 | 2021-06-30 | Method for producing ethanol by fermenting corn fiber treated by citric acid and corn fiber-based ethanol product |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115537431A (en) |
-
2021
- 2021-06-30 CN CN202110741035.0A patent/CN115537431A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11753658B2 (en) | Pichia stipitis strain and cultures and uses of the same | |
| CN104774876B (en) | A kind of method of lignocellulose biomass comprehensive utilization | |
| CN102985550A (en) | Compositions and methods for fermentation of biomass | |
| CN113046278B (en) | Composite microbial agent for fermenting and degrading cellulose in straws | |
| JP5711873B2 (en) | Simultaneous saccharification and fermentation of cellulosic materials | |
| US20220315886A1 (en) | Methods for propagating microorganisms for fermentation & related methods & systems | |
| Pradechboon et al. | Alkali pretreatment and enzymatic saccharification of blue-green alga Nostochopsis lobatus for bioethanol production | |
| CN112852649B (en) | High-temperature-resistant saccharomyces cerevisiae strain for producing cellulosic ethanol and fermentation application thereof | |
| KR101075602B1 (en) | Mutant Strain of Brettanomyces custersii and Method of Ethanol Production Using the Same | |
| CN115537431A (en) | Method for producing ethanol by fermenting corn fiber treated by citric acid and corn fiber-based ethanol product | |
| CN112941120B (en) | Method for producing microbial oil by using VFAs and lignocellulose raw materials | |
| Bajpai et al. | Kinetics of ethanol production by immobilized Kluyveromyces marxianus cells at varying sugar concentrations of Jerusalem artichoke juice | |
| JP5317262B2 (en) | Production method of ethanol by fungi | |
| CN111690707B (en) | Method and production system for preparing humus and artificial aged pit mud by double-round fermentation of waste vinasse | |
| JP6322187B2 (en) | Ethanol production method from cellulosic biomass | |
| CN112159762B (en) | Fusarium oxysporum SWH-3 and method for producing diosgenin by fermentation of fusarium oxysporum SWH-3 | |
| CN212741403U (en) | Lost grain double-round fermentation humus preparation production system | |
| CN113215203B (en) | Method for producing ethanol by co-fermentation saccharomycetes through expansion culture and fermentation | |
| CN115678920B (en) | Method for fermenting ethanol by high-solid-content cellulose | |
| CN112662710B (en) | Method for producing L-lactic acid by continuous fermentation of lignocellulose | |
| CN117904218A (en) | Method for producing lactic acid | |
| CN110438172B (en) | Method for producing grease by co-utilizing starch and lignocellulose raw materials | |
| CN106520846A (en) | Improved lignocellulose biomass hydrothermal pretreatment method | |
| CN105950694B (en) | Novel process for co-producing wheat polysaccharide and active peptide by using alcohol fermentation waste liquid | |
| CN118109524A (en) | Method for producing biological butanol by fermenting lignocellulose raw material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |