CN109161566B

CN109161566B - Method for producing butyric acid by using whole components of corncobs

Info

Publication number: CN109161566B
Application number: CN201811144501.1A
Authority: CN
Inventors: 江凌; 郑文秀; 赵倩如; 黄和; 朱丽英
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2018-09-29
Filing date: 2018-09-29
Publication date: 2021-07-20
Anticipated expiration: 2038-09-29
Also published as: CN109161566A

Abstract

The invention discloses a method for producing butyric acid by using full components of corncobs. Using gamma-valerolactone and 1, 3-dimethyl imidazole dimethyl phosphate ([ Mmim ] DMP) as solvents to pretreat the corncobs, using water as an anti-solvent to precipitate the corncobs, performing suction filtration and separation to obtain residues, using the dried corncob residues as substrates, and performing enzymatic hydrolysis on cellulase to obtain fermentable sugars; the lignin extracted from the pretreatment solvent is precipitated by acid and is used for preparing the macroporous calcium alginate-lignin microspheres for embedding the clostridium tyrobutyricum, so that the butyric acid is produced by fermenting the macroporous calcium alginate-lignin microspheres by utilizing fermentable sugar, and the comprehensive utilization of all components of the corncobs is realized. According to the invention, the butyric acid is produced by effectively utilizing the full-component fermentation of the corncobs, the combined use of [ Mmim ] DMP and gamma-valerolactone greatly improves the pretreatment effect, enhances the enzymolysis efficiency of the corncobs, obtains high-yield fermentable sugar, and the yield of butyric acid is improved compared with that of free cells by embedding clostridium tyrobutyricum in macroporous calcium alginate-lignin microspheres.

Description

Method for producing butyric acid by using whole components of corncobs

Technical Field

The invention relates to the technical field of comprehensive utilization of biomass resource corncobs, in particular to a method for producing butyric acid by using all components of corncobs.

Background

Corn is one of international food crops and also a commodity which can be traded. In China, corn is widely planted, with a yield of about 2.2 million tons in 2016. The sweet corn industry is expected to produce about 4000 million tons of corn cob by-product each year, depending on the ratio between the corn kernels and the corn cobs. In south africa, corn is the major export commodity used primarily to produce second generation corn products such as corn chips, corn meal, and glucose. Because of the large amount of corn consumed each year, corncobs have become one of the largest agricultural wastes in the south africa, and the annual output is about 900 ten thousand tons, which accounts for nearly 20% of the residual amount of corn. In the past, most of corncobs are burnt as fuels and discarded as waste or used as fertilizers, which not only causes huge resource waste, but also causes serious environmental pollution and influences ecological balance. To reduce human dependence on non-renewable resources, many researchers are increasingly focusing on the utilization of agricultural biomass to produce biofuels and high value-added chemicals. Lignocellulosic biomass is an ideal feedstock because it is renewable and available in large quantities around the world. Corncobs are a practical lignocellulosic biomass feedstock with the advantages of low lignin and high carbohydrate content. Therefore, it has been developed and utilized by the biorefinery industry of China for the production of xylose, xylo-oligosaccharides and biofermentation products such as xylitol, ethanol, butanediol and furfural, etc.

Before the corn cob is comprehensively utilized, the raw material must be pretreated, because the high crystallinity of the cellulose and the existence of lignin make enzymes difficult to enter the biomass and cause low yield of fermentable sugar, so that the resistance structure of the lignocellulose is damaged through pretreatment, and the separation of three major components is realized. Biomass pretreatment is usually carried out by several methods, including physical methods such as steam explosion, ammonia fiber explosion, microwave treatment or pyrolysis; chemical methods, such as concentrated acid hydrolysis, dilute acid hydrolysis, alkaline treatment or organic solvent treatment; there are also biological methods. Ionic liquids are increasingly considered to be the most promising new solvents for lignocellulose pretreatment. Ionic liquids are salts with melting points below 100 ℃ and have a wide range of physicochemical properties, such as low volatility, high thermal stability, non-flammability, unique dissolution characteristics, and are not harmful to the environment and human health. By selecting proper anion and cation components, the physical and chemical properties of the combined ionic liquid are greatly different. 1, 3-dimethyl imidazole dimethyl phosphate ([ Mmim ] DMP) is an environment-friendly solvent, can be used for pretreatment of corncobs, dissolves lignocellulose, and has biocompatibility with cellulase. However, pre-treating biomass using only a single component is relatively wasteful, as ionic liquids are still too expensive for many commercial applications. The biomass-derived solvent gamma-valerolactone is proved to be capable of promoting mild pretreatment of the lignocellulose biomass, the gamma-valerolactone is an ideal solvent capable of sustainable development and can be used for producing energy and carbon-based consumer goods, the gamma-valerolactone is added into the [ Mmim ] DMP to be used as a cosolvent, mass transfer resistance can be overcome, and the [ Mmim ] DMP is assisted to be improved to dissolve lignocellulose, so that the lignocellulose biomass is subjected to mild and effective pretreatment.

After the corncobs are pretreated, the corncobs can be subjected to enzymolysis and saccharification to obtain fermentable sugars, the main components of the fermentable sugars are glucose and xylose, lignin extracted in the pretreatment process is also a resource, in order to utilize the lignin, clostridium tyrobutyricum is a dominant strain which can simultaneously utilize the glucose and the xylose as carbon sources to produce butyric acid, and the clostridium tyrobutyricum is embedded by utilizing the lignin, sodium alginate and calcium carbonate to form microspheres so as to ferment and produce the butyric acid. Thereby realizing the comprehensive utilization of the whole components of the corncobs.

Disclosure of Invention

The invention aims to develop a process for producing butyric acid by using full components of corncobs, improve biomass conversion through enzymatic hydrolysis, improve the productivity of fermentable sugar, and prepare microspheres embedding clostridium tyrobutyricum by using extracted lignin so as to produce butyric acid by using the fermentable sugar.

The purpose of the invention is realized by the following technical scheme:

the process for producing butyric acid by using the whole components of corncobs comprises the following specific steps:

(1) washing and drying corncobs, and crushing;

(2) under the protection of nitrogen atmosphere, mixing the corncob powder with 1, 3-dimethyl imidazole dimethyl phosphate ([ Mmim ] DMP) and gamma-valerolactone for pretreatment, then cooling to room temperature, adding deionized water as an anti-solvent, stirring for reaction to precipitate the corncob powder, and performing suction filtration and separation to obtain corncob residues and filtrate;

(3) washing the corncob residues with deionized water, drying, mixing with an acetate buffer solution, adding cellulase for enzymolysis, boiling the solution after the reaction is finished to quench the enzymatic reaction, and centrifuging the boiled solution to obtain a fermentable sugar solution with glucose and xylose as main components;

(4) adding acid into the filtrate obtained in the step (2) to adjust the pH value, precipitating lignin in the filtrate at low temperature, washing the lignin precipitate with deionized water and drying;

(5) resuspending clostridium tyrobutyricum in a mixed solution containing sodium alginate, lignin and calcium carbonate, dropwise adding the mixture into a calcium chloride solution stirred by magnetic force by using an injector, and embedding the clostridium tyrobutyricum by an extrusion method;

(6) and (3) fermenting the microspheres embedded with the clostridium tyrobutyricum by using the fermentable sugar solution as a carbon source to produce butyric acid.

In the method, in the step (1), the crushed particle size of the corncob is 80-120 meshes.

In the step (2), the corncob powder and the 1, 3-dimethyl imidazole dimethyl phosphate are mixed according to the mass ratio of 1: 6.5-1: 19.5, then 10-16 wt% of gamma-valerolactone is added, and the mixture is heated and stirred at 90-110 ℃ for 3-4 h.

In the step (2), plasma water anti-solvent with the same volume is added into the blending solution of the pretreated corncob powder, the 1, 3-dimethyl imidazole dimethyl phosphate and the gamma-valerolactone.

In the step (3), the corncob residues are mixed with an acetate buffer solution according to the substrate concentration of 50 g/L; the acetate buffer solution was 50 mM, pH4.8, and then subjected to enzymatic hydrolysis at 50 ℃ and 150 rpm. Cellulase was added at 45 FPU/g dry corn cob residue.

In the step (4), hydrochloric acid is added to slowly acidify until the pH is adjusted to 2.0, and then the lignin is refrigerated and precipitated at 4 ℃.

In the step (5), the mixed solution contains sodium alginate with the mass fraction of 2%, lignin with the mass fraction of 0.5% and calcium carbonate with the mass fraction of 5%; the concentration of the calcium chloride solution is 11 mg/mL.

In the step (6)The composition of the fermentation medium was as follows: 50 g/L of fermentable sugar liquid, 5 g/L of yeast powder, 5 g/L of peptone, 6 g/L of NaCl, (NH)₄)₂SO₄3 g/L，K₂HPO₄1.5 g/L, MgSO₄·7H₂O 0.6 g/L，FeSO₄·7H₂O0.03 g/L, L-cysteine hydrochloride 0.3 g/L, resazurin 0.1%, pH 6.0.

The method can realize the production of butyric acid by using the whole components of the corncobs, the gamma-valerolactone and the [ Mmim ] DMP are used together for pretreating the lignocellulose corncobs, the [ Mmim ] DMP is used as an environment-friendly ionic liquid and is green and renewable, the gamma-valerolactone is used as a cosolvent and is a sustainable production resource, and the two methods are combined with a pretreatment process and are environment-friendly and accord with the green sustainable development principle. The pretreatment of the corncob powder by combining gamma-valerolactone and [ Mmim ] DMP can efficiently destroy the structure of lignocellulose, degrade and extract lignin, provide advantages for subsequent enzymatic reaction, add cellulase into the corncob residue separated after pretreatment for enzymolysis to obtain fermentable sugar solution with high yield, precipitate lignin components from the separated filtrate by using acid, and use the lignin components for embedding clostridium tyrobutyricum.

Drawings

FIG. 1 is a flow chart of the method of the present invention.

Detailed Description

The technical scheme of the invention is further explained by combining the description of the attached drawings and the detailed description.

The medium composition of clostridium tyrobutyricum in the examples was: 30 g/L glucose, 5 g/L yeast powder, 5 g/L peptone, 6 g/L NaCl, (NH)₄)₂SO₄3 g/L，K₂HPO₄ 1.5 g/L，MgSO₄·7H₂O 0.6 g/L，FeSO₄·7H₂O0.03 g/L, L-cysteine hydrochloride 0.3 g/L, resazurin 0.1%, pH 6.0.

The composition of the fermentation medium was as follows: 50 g/L of fermentable sugar liquid, 5 g/L of yeast powder, 5 g/L of peptone, 6 g/L of NaCl, (NH)₄)₂SO₄3 g/L，K₂HPO₄1.5 g/L, MgSO₄·7H₂O 0.6 g/L，FeSO₄·7H₂O0.03 g/L, L-cysteine hydrochloride 0.3 g/L, resazurin 0.1%, pH 6.0.

Example 1

As shown in fig. 1, the method of the present invention comprises the steps of:

(1) washing and drying corncobs, and then crushing the corncobs to obtain the corncobs with the particle size of 80-120 meshes;

(2) pretreatment:

0.3 g of corncob powder was taken, added to 5 mL of a mixture of [ Mmim ] DMP and 16 wt% of gamma-valerolactone (16 wt% means the mass of gamma-valerolactone/([ Mmim ] DMP + gamma-valerolactone total mass)), and heated at 90 ℃ under nitrogen atmosphere in a 50 mL three-necked round-bottomed flask and reacted for 4h with magnetic stirring at 1000 rpm. After the reaction was complete, the reaction mixture was diluted with the same volume of deionized water, which was used as an anti-solvent to precipitate the pretreated corn cob powder. The resulting cob residue and filtrate were separated by suction filtration.

(3) The cob residue was repeatedly washed with deionized water to remove possible [ Mmim ] DMP and gamma-valerolactone residues and dried at 65 ℃ for 10 hours to give a pretreated cob powder.

0.25g of the pretreated corncob powder was placed in a 25 mL Erlenmeyer flask, and 5 mL of 50 mM acetate buffer (pH 4.8), 45 FPU/g cellulase (purchased from Ningxia & Debi Co.) was added thereto, and the enzymatic reaction was carried out for 72 hours at 150rpm in a constant temperature water bath shaker at 50 ℃. After the completion of the reaction, the enzymatic reaction was quenched by boiling the enzymatic solution for 5 minutes, and centrifuged at 3000 Xg for 5 min to measure the sugar concentration by HPLC. The glucose yield was 94.9% and the xylose yield was 53.3% calculated from the cellulose and hemicellulose content of the corncobs before pretreatment.

(4) The filtrate was acidified slowly to pH 2.0 by addition of 4M HCl and stored in a refrigerator at 4 ℃ for 48 hours to precipitate lignin. The lignin precipitate was washed with deionized water and dried for further use.

(5) Preparation of microspheres

Boiling the culture medium of Clostridium tyrobutyricum to removeOxygen, autoclaved at 115 ℃ for 30 min, carbon source and nitrogen source separately sterilized. After inoculating 5% of the bacterial solution to the medium, the medium was cultured at 37 ℃ for 48 hours. After the third generation activation, the third generation seed culture medium is used as the mother solution for the next cultivation. The strain was cultured to the logarithmic phase using the same culturing procedure, after which the strain was centrifuged at 3000 Xg for 10 min to collect the cells, washed with 0.1M phosphate buffer (pH 7.4) with the cell concentration controlled at 10 g/L, resuspended in 20 mL of a mixed solution containing sodium alginate at a mass fraction of 2%, lignin at 0.5% and calcium carbonate at 5%, and dropped into 100 mL of magnetically stirred 11 mg/mL CaCl with a 10 mL syringe₂Preparing microspheres embedding clostridium tyrobutyricum in the aqueous solution, stirring for reaction for 30 min, and incubating the microspheres at 4 ℃ for 4 h. Then washed with 0.5M HCl until no more bubbles are formed, washed with phosphate buffer and stored in sterile peptone solution at 4 ℃ until use.

(6) Production of butyric acid by fermentation

The fermentation was carried out in a 5L fermenter containing 2L of fermentation medium. The fermenter was sterilized at 115 ℃ for 30 min before inoculation. Inoculating 10 wt% of microspheres into a fermentation tank to start fermentation, and introducing N₂Anaerobic conditions were maintained, with 5% inoculum of free cells as a control, incubated at 37 ℃ for 48 hours by addition of 6M NaOH and 3M H₂SO₄The pH was controlled on-line at 6.0. Fermentation was performed in single batch and repeated batch mode. In the repeated batch mode, the immobilized cells were grown continuously batch by batch, and when the fermentable sugar concentration decreased to 0, fresh sterile carbon source was added to the medium to start the fermentation of a new batch. After 10 times of repeated batch fermentation, the yield of butyric acid produced by the embedded clostridium tyrobutyricum fermentation is 22.5 g/L, the yield is 0.45 g/g, the free cells are only 22.2 g/L, the yield is 0.44 g/g, and the butyric acid produced by the embedded clostridium tyrobutyricum is purer.

Example 2

(2) pretreatment:

0.3 g of corncob powder was added to a mixture of 10 mL of [ Mmim ] DMP and 14 wt% gamma-valerolactone and heated at 90 ℃ in a 50 mL three-necked round-bottomed flask under nitrogen and reacted for 3 h with magnetic stirring at 1000 rpm. After the reaction was complete, the reaction mixture was diluted with the same volume of deionized water, which was used as an anti-solvent to precipitate the pretreated corn cob powder. The resulting cob residue and filtrate were separated by suction filtration.

0.25g of the pretreated corncob powder was placed in a 25 mL Erlenmeyer flask, and 5 mL of 50 mM acetate buffer (pH 4.8), 45 FPU/g cellulase (purchased from Ningxia & Debi Co.) was added thereto, and the enzymatic reaction was carried out for 72 hours at 150rpm in a constant temperature water bath shaker at 50 ℃. After the completion of the reaction, the enzymatic reaction was quenched by boiling the enzymatic solution for 5 minutes, and centrifuged at 3000 Xg for 5 min to measure the sugar concentration by HPLC. The glucose yield was 89.0% and the xylose yield was 44.7% calculated from the cellulose and hemicellulose content of the corncobs before pretreatment.

(5) Preparation of microspheres

The Clostridium tyrobutyricum medium was boiled to remove oxygen, autoclaved at 115 ℃ for 30 min, and the carbon source and nitrogen source were separately sterilized. After inoculating 5% of the bacterial solution to the medium, the medium was cultured at 37 ℃ for 48 hours. After the third generation activation, the third generation seed culture medium is used as the mother solution for the next cultivation. The strain was cultured to the logarithmic phase using the same culturing procedure, after which the strain was centrifuged at 3000 Xg for 10 min to collect the cells, washed with 0.1M phosphate buffer (pH 7.4) with the cell concentration controlled at 10 g/L, resuspended in 20 mL of a mixed solution containing sodium alginate at a mass fraction of 2%, lignin at 0.5% and calcium carbonate at 5%, and dropped into 100 mL of magnetically stirred 11 mg/mL CaCl with a 10 mL syringe₂Preparing microspheres embedding clostridium tyrobutyricum in the aqueous solution, stirring for reaction for 30 min, and incubating the microspheres at 4 ℃ for 4 h. Then washed with 0.5M HCl until no more bubbles are formed,then washing with phosphate buffer solution, and storing in sterile peptone solution at 4 ℃ for later use.

(6) Production of butyric acid by fermentation

The fermentation was carried out in a 5L fermenter containing 2L of fermentation medium. The fermenter was sterilized at 115 ℃ for 30 min before inoculation. Inoculating 10 wt% of microspheres into a fermentation tank to start fermentation, and introducing N₂Anaerobic conditions were maintained, with 5% inoculum of free cells as a control, incubated at 37 ℃ for 48 hours by addition of 6M NaOH and 3M H₂SO₄The pH was controlled on-line at 6.0. Fermentation was performed in single batch and repeated batch mode. In the repeated batch mode, the immobilized cells were grown continuously batch by batch, and when the fermentable sugar concentration decreased to 0, fresh sterile carbon source was added to the medium to start the fermentation of a new batch. After 10 times of repeated batch fermentation, the yield of butyric acid produced by fermentation of embedded clostridium tyrobutyricum is 15.3 g/L and 0.43 g/g, the yield of free cells is only 14.7 g/L, and the yield is 0.42 g/g.

Example 3

(2) pretreatment:

0.3 g of corncob powder was added to a mixture of 15 mL of [ Mmim ] DMP and 10 wt% of gamma-valerolactone and heated at 110 ℃ in a 50 mL three-necked round-bottomed flask under nitrogen atmosphere and reacted for 3 h with magnetic stirring at 1000 rpm. After the reaction was complete, the reaction mixture was diluted with the same volume of deionized water, which was used as an anti-solvent to precipitate the pretreated corn cob powder. The resulting cob residue and filtrate were separated by suction filtration.

0.25g of the pretreated corncob powder was placed in a 25 mL Erlenmeyer flask, and 5 mL of 50 mM acetate buffer (pH 4.8), 45 FPU/g cellulase (purchased from Ningxia & Debi Co.) was added thereto, and the enzymatic reaction was carried out for 72 hours at 150rpm in a constant temperature water bath shaker at 50 ℃. After the completion of the reaction, the enzymatic reaction was quenched by boiling the enzymatic solution for 5 minutes, and centrifuged at 3000 Xg for 5 min to measure the sugar concentration by HPLC. The glucose yield was calculated to be 82.2% and the xylose yield was calculated to be 42.7% based on the cellulose and hemicellulose content of the corncobs prior to pretreatment.

(5) Preparation of microspheres

The Clostridium tyrobutyricum medium was boiled to remove oxygen, autoclaved at 115 ℃ for 30 min, and the carbon source and nitrogen source were separately sterilized. After inoculating 5% of the bacterial solution to the medium, the medium was cultured at 37 ℃ for 48 hours. After the third generation activation, the third generation seed culture medium is used as the mother solution for the next cultivation. The strain was cultured to the logarithmic phase using the same culturing procedure, after which the strain was centrifuged at 3000 Xg for 10 min to collect the cells, washed with 0.1M phosphate buffer (pH 7.4) with the cell concentration controlled at 10 g/L, resuspended in 20 mL of a mixed solution containing sodium alginate at a mass fraction of 2%, lignin at 0.5% and calcium carbonate at 5%, and dropped into 100 mL of magnetically stirred 11 mg/mL CaCl with a 10 mL syringe₂Preparing microspheres embedding clostridium tyrobutyricum in the aqueous solution, stirring for reaction for 30 min, and incubating the microspheres at 4 ℃ for 4 h. Then washed with 0.5M HCl until no more bubbles are formed, washed with phosphate buffer and stored in sterile peptone solution at 4 ℃ until use.

(6) Production of butyric acid by fermentation

The fermentation was carried out in a 5L fermenter containing 2L of fermentation medium. The fermenter was sterilized at 115 ℃ for 30 min before inoculation. Inoculating 10 wt% of microspheres into a fermentation tank to start fermentation, and introducing N₂Anaerobic conditions were maintained, with 5% inoculum of free cells as a control, incubated at 37 ℃ for 48 hours by addition of 6M NaOH and 3M H₂SO₄The pH was controlled on-line at 6.0. Fermentation was performed in single batch and repeated batch mode. In the repeated batch mode, the immobilized cells were grown continuously batch by batch, and when the fermentable sugar concentration decreased to 0, fresh sterile carbon source was added to the medium to start the fermentation of a new batch. Repeating the steps for 10 timesThe yield of butyric acid produced by fermentation of the embedded clostridium tyrobutyricum is 12.8 g/L and 0.40 g/g, and the yield of free cells is only 12.2 g/L and 0.38 g/g.

Claims

1. A method for producing butyric acid by using the whole components of corncobs is characterized by comprising the following steps:

(1) washing and drying corncobs, and crushing;

(2) under the protection of nitrogen atmosphere, mixing the corncob powder with 1, 3-dimethyl imidazole dimethyl phosphate according to the mass ratio of 1: 6.5-1: 19.5, then adding gamma-valerolactone accounting for 10-16 wt% of the total mass of the 1, 3-dimethyl imidazole dimethyl phosphate and the gamma-valerolactone, heating and stirring at 90-110 ℃ for 3-4 h, then cooling to room temperature, adding deionized water as an anti-solvent, stirring for reaction to precipitate the corncob powder, and performing suction filtration and separation to obtain corncob residues and filtrate;

2. The method as claimed in claim 1, wherein the corncob is pulverized to have a particle size of 80-120 mesh in the step (1).

3. The method according to claim 1, wherein in the step (2), a plasma water anti-solvent is added into a blending solution of the pretreated corncob powder, the 1, 3-dimethyl imidazole dimethyl phosphate and the gamma-valerolactone in an equal volume.

4. The method according to claim 1, wherein in the step (3), the corncob residue is mixed with an acetate buffer at a substrate concentration of 50 g/L; the acetate buffer concentration was 50 mM, pH 4.8.

5. The method according to claim 1, wherein in the step (3), the enzymolysis is carried out at 150rpm and 50 ℃.

6. The method of claim 1, wherein in step (3), the cellulase is added in an amount of 45 FPU/g dry corncob residue.

7. The method according to claim 1, wherein in the step (4), the lignin is precipitated by adding hydrochloric acid and slowly acidifying until the pH is adjusted to 2.0, and then refrigerating at 4 ℃.

8. The method as claimed in claim 1, wherein in the step (5), the mixed solution contains 2% of sodium alginate, 0.5% of lignin and 5% of calcium carbonate by mass fraction; the concentration of the calcium chloride solution is 11 mg/mL.

9. The method according to claim 1, wherein in the step (6), the composition of the fermentation medium is as follows: 50 g/L of fermentable sugar liquid, 5 g/L of yeast powder, 5 g/L of peptone, 6 g/L of NaCl, (NH)₄)₂SO₄3 g/L，K₂HPO₄1.5 g/L, MgSO₄·7H₂O 0.6 g/L，FeSO₄·7H₂O0.03 g/L, L-cysteine hydrochloride 0.3 g/L, resazurin 0.1%, pH 6.0.