CN102586342A - Method for lowering fermentation inhibitor from source - Google Patents

Abstract

The invention discloses a method for reducing fermentation inhibitors derived from lignocellulose from the source. The method combines steam explosion technology and air classification technology and applies it to the pretreatment of lignocellulosic raw materials. The two-stage steam explosion carding process effectively pretreats the characteristics of each tissue of the fiber raw material selectively, which can not only ensure that the fiber tissue achieves a better pretreatment effect, but also avoid excessive degradation of the parenchyma, thereby improving the quality of the fiber raw material. At the same time, it can effectively control the occurrence of side reactions, that is, reduce the content of inhibitors, save the introduction of detoxification unit operation, and simplify the process. In addition, in this process, the separation and selective pretreatment of fiber cells and parenchyma cells provide a new way for the hierarchical and multi-stage conversion of lignocellulosic raw materials and the utilization of biomass rights, and the two-stage steam explosion carding process also has It helps the rehydration of fiber raw materials evenly, reduces the steam explosion conditions of the second-stage materials and reduces the content of raw materials, and improves the utilization rate of raw materials.

Description

A method for reducing fermentation inhibitors from the source

technical field

The invention relates to a method for reducing fermentation inhibitors derived from lignocellulose, in particular to a pretreatment method with steam explosion technology as the core.

Background technique

The structure and composition of lignocellulose are complex. Among them, the cellulose composed mainly of glucose groups has a dense structure and is difficult to hydrolyze, while the hemicellulose composed of various heterosaccharide groups has a loose structure and can be hydrolyzed into single sugar. A variety of lignocellulosic raw material pretreatment methods are currently being studied, such as acid hydrolysis, steam explosion, ammonia fiber explosion, and wet oxidation. Various pretreatment processes produce a variety of compounds, the types and contents of which vary with the properties of cellulose raw materials and pretreatment conditions, and mainly form three types of microbial growth inhibitors: 1) Weak acids: acetic acid, formic acid, acetylpropane Acid etc. Acetic acid is produced by deacetylation of hemicellulose. Formic acid and levulinic acid are degradation products of 5-hydroxymethylfurfural (HMF, 5-Hydroxymethylfurfural). At the same time, formic acid can also be produced by the degradation of furfural in an acidic environment; 2) furan aldehydes: Mainly furfural (Furfural) and HMF, which are produced by dehydration of pentose and hexose in acidic environment respectively; 3) Phenolic compounds: mainly formed by lignin degradation.

Steam explosion technology is considered to be one of the most promising and cost-effective techniques for plant fiber pretreatment. The raw material is heated to 180-235°C with steam, and the pressure is maintained for a certain period of time. When it is suddenly decompressed and sprayed, the volume increases sharply, and its solid material structure is destroyed by the action of mechanical force. Several advantages of steam explosion can be summarized as follows: (1) It can be applied to various plant biomass, and the pretreatment conditions are easy to adjust and control. (2) The three components of hemicellulose, lignin and cellulose will be separated in three different processes, namely water-soluble components, alkali-soluble components and alkali-insoluble components. (3) The enzymatic conversion rate of cellulose can reach the theoretical maximum. (4) The lignin after steam treatment can still be used for conversion of other chemical products. (5) The sugar produced by hemicellulose can be fully utilized and converted into liquid fuel. (6) The fermentation inhibitors produced in the steam explosion process can be greatly reduced by controlling the steam explosion conditions. This pretreatment method is applicable to, and is playing an increasing role in, various plant biomasses such as hardwoods, softwoods, agricultural wastes such as bagasse, wheat straw, straw, corn stover and other non-cellulosic feedstocks. However, the biggest disadvantage of steam explosion pretreatment technology is that it still produces some fermentation inhibitors with less content than other pretreatment technologies.

Inhibition of these inhibitors on subsequent fermentation microorganisms has become one of the major bottlenecks in lignocellulose bioprocessing. Necessary measures to deal with inhibitors shall be taken to reduce or eliminate their inhibitory effects. Various methods have been tried to detoxify the lignocellulose hydrolyzate before fermentation, including biological, physical and chemical methods and combinations of different measures. Biological methods include detoxification of the hydrolyzate using specific enzymes (such as laccase) or microorganisms. Laccase is a class of copper-containing oxidoreductases, which specifically perform one-electron oxidation on phenolic substrate molecules to generate corresponding active free radicals, and the active intermediates are then transformed into dimers, oligomers and polymers. thing. In this process, the oxidative polymerization reaction of small molecular weight phenolic compounds occurs, and high molecular weight compounds with low toxicity are generated, thereby reducing the toxicity of phenolic compounds in lignocellulose pretreatment liquid. Mussatto et al. treated the hydrolyzate with a mutant strain of Saccharomyces cerevisiae that could only use acetic acid to reduce the concentration of acetic acid from 6.8 g/L to 0.4 g/L. Physical methods include vacuum drying and concentration, cooking, activated carbon adsorption, ion exchange adsorption and solvent extraction, etc. Vacuum concentration and cooking can greatly reduce volatile inhibitors, and ion exchange and solvent extraction can effectively reduce the content of acetic acid, furan aldehyde and phenolic compounds The chemical method includes utilizing various alkalis (NH4OH, NaOH, Ca(OH)2, etc.) and excess lime method to treat the hydrolyzate, wherein NH4OH can effectively remove furan aldehydes, and excessive lime treatment can effectively improve the single sugar utilization. In addition, washing pretreated raw materials is also a simple detoxification process. Combining two or more methods can achieve better detoxification effect.

However, the detoxification step will undoubtedly increase the fermentation cost of bio-based products, make the process more complicated, and also lead to the loss of a part of fermentable sugars, so the detoxification process before fermentation must be minimized during the fermentation process. Feasible methods include breeding highly resistant strains to improve their inherent tolerance, or optimizing the hydrolysis process from the source to control the production of inhibitors and reduce their content. Lignocellulosic raw materials have complex, heterogeneous and multi-level structures. In terms of chemical composition, the chemical composition of lignocellulosic raw materials includes cellulose, hemicellulose and lignin. From the perspective of cell composition, it includes fibrous fibroblasts and miscellaneous cells (including ducts, parenchyma cells, epidermal cells, etc.). Fibroblasts are the most important and basic cells in plant fiber raw materials, and are the supporting tissues of plant raw materials. Fibroblasts generally have well-developed secondary walls (that is, thicker). Another important type of cells in plant materials is parenchyma cells, which play a role in storing nutrients in plant growth. Parenchyma cells are characterized by large lumens, thin walls, and short lengths. In view of the structural and morphological differences of the two types of cells, it can be seen that the pretreatment conditions required by the two types of cells are different. Fibrous cells have a high degree of lignification of the cell wall and a dense structure, which has a large mass and heat transfer resistance during the heating process and is not easily torn; parenchyma cells have thin walls and large cavities, which are not only conducive to mass and heat transfer, but also conducive to water vapor Flashing physically tears it apart. In different raw materials, the content and structure of these two types of cells are different, which essentially determines the difference in the processing conditions they require. Therefore, the treatment conditions can be optimized for different tissue cells, so as to achieve the best hydrolysis effect and minimize the degree of side effects.

The two-stage steam explosion combing process developed by the present invention is different from the traditional two-stage steam explosion process. Here, it refers to the use of milder steam explosion conditions to carry out the first stage steam explosion, and the first stage material is separated by an air classification device. Grading to obtain parenchyma and fibrous tissue, and then the fibrous tissue is subjected to the second stage of steam explosion under suitable conditions. The two-stage steam explosion carding process can effectively perform selective pretreatment according to the characteristics of each tissue, which can not only ensure that the fiber tissue achieves a better pretreatment effect, but also avoid excessive degradation of the parenchyma tissue, thereby improving the quality of fiber raw materials. At the same time, it can effectively control the occurrence of side reactions, that is, reduce the content of inhibitors, save the introduction of detoxification unit operation, and simplify the process. In addition, in this process, the separation and selective pretreatment of fiber cells and parenchyma cells provide a new way for the hierarchical and multi-stage conversion of lignocellulosic raw materials and the utilization of biomass rights, and the two-stage steam explosion carding process also has It helps the rehydration of fiber raw materials evenly, reduces the steam explosion conditions of the second-stage materials and reduces the content of raw materials, and improves the utilization rate of raw materials.

Contents of the invention

The purpose of the present invention is to aim at the production of fermentation inhibitors in the current lignocellulose pretreatment process has become one of the main bottlenecks in the lignocellulose bioprocessing process, and the commonly used detoxification process not only increases the fermentation cost of bio-based products, but also makes The process is more complicated, and it also leads to the loss of part of the fermentable sugar. A new two-stage steam explosion carding process has been developed. The two-stage steam explosion carding process here is different from the traditional two-stage steam explosion process, which means that the first stage steam explosion is carried out under milder steam explosion conditions, and the first stage materials are classified by an air classification device to obtain Parenchyma and fibrous tissue, and then the fibrous tissue is subjected to the second stage of steam explosion under suitable conditions.

Technical scheme of the present invention is as follows:

The novel two-stage steam explosion carding process for reducing the content of fermentation inhibitors derived from lignocellulose from the source provided by the present invention comprises the following steps: 1) at a pressure of 0.5 to 1.5MP

a, steam explosion conditions of 1 to 10 minutes are used to carry out the first-stage steam explosion treatment on straw raw materials; 2) classify the first-stage steam-exploded materials through an airflow classification device to obtain parenchyma and fibrous tissue; 3) put The fibrous tissue obtained by carding is subjected to the second stage of steam explosion treatment under the condition of a pressure of 0.5-1.5MPa and a time of 1-10min; 4) the first stage of steam explosion, carding and the second stage of steam explosion The material is subjected to enzymolysis and fermentation; 5) The moisture content of the straw raw material, the steam-exploded material in the first stage, the parenchyma and fibrous tissue after carding, the steam-exploded material in the second stage, the inhibitor content in the washing liquid, Enzymolysis rate and conversion rate of fermentation products.

In the present invention, the enzymolysis conditions are that the solid-to-liquid ratio of steam-exploded straw and pH 4.850mM citric acid buffer is 1:10-1:30, the amount of enzyme added is 15IU/g-110/g substrate, at 50°C, Enzymolysis in a water-bath shaker at 150rpm for 24-120h. Then, centrifuge and filter to obtain the enzymatic hydrolyzate.

The beneficial effects of the present invention are as follows: (1) The two-stage steam explosion carding process effectively requires selective pretreatment according to the characteristic requirements of each tissue, which can ensure that the fibrous tissue achieves a better pretreatment effect and avoid thinning Excessive degradation of the wall tissue, thereby increasing the enzymatic hydrolysis rate of fiber raw materials, can effectively control the occurrence of side reactions, that is, reduce the content of inhibitors, save the introduction of detoxification unit operations, and simplify the process. (2) In this process, the separation and selective pretreatment of fibroblasts and parenchyma cells provide a new way for the hierarchical and multi-stage conversion of lignocellulosic raw materials and the utilization of biomass rights. (3) The second-stage steam explosion carding process helps to rehydrate the fiber raw materials evenly, reduces the steam explosion conditions of the second-stage materials and reduces the content of raw materials, and improves the utilization rate of raw materials.

Detailed ways

The present invention will be further described below by embodiment.

The cellulase preparation used in the embodiment of the present invention was purchased from Xiasheng Company, 110FPAIU/ml, 1200IU xylanase/ml.

Example 1: Weigh 200g of dry corn stalks and put them into a steam explosion tank, and then pass water vapor into it after airtight to make the tank pressure rise to 1.1MPa rapidly, and after maintaining this pressure for 4 minutes, release the pressure quickly to obtain the first stage Steam-exploded straw. Take an appropriate amount of the first-stage steam-exploded straw, and comb it on a centrifugal airflow classification device to obtain parenchyma and fibrous tissue respectively. Weigh an appropriate amount of combed fibrous tissue, put it into a steam explosion tank, and then inject water vapor after sealing it to make the tank pressure rise to 1.2MPa rapidly. After maintaining this pressure for 4 minutes, release the pressure quickly to obtain the second stage Steam-exploded straw. The pretreatment conditions were 1.2MPa, 8min, and no carding as the pretreatment control group.

Weigh 1g (dry base) of steam-exploded straw in the first stage, steam-exploded straw after carding, steam-exploded straw in the second stage and the steam-exploded straw in the control group respectively, dissolve them in 20ml of water and extract at 70°C for 2 hours, and take the supernatant The content of furfural and hydroxymethylfurfural (HMF) in the hydrolyzate was measured by high performance liquid phase (Agilent 1200HPLC, USA), and the content of soluble lignin in the hydrolyzate was measured by a UV spectrophotometer at 280nm. The sum of the contents of furfural, hydroxymethylfurfural (HMF) and soluble lignin is the inhibitor content in the hydrolyzate.

Weigh 10g of the first stage of steam-exploded straw, combed steam-exploded straw, second-stage steam-exploded straw and pretreatment control group steam-exploded straw, add 200ml pH 4.850mM citric acid buffer solution, add 0.5ml cellulose Enzyme, hydrolyze at 50°C, 150rpm in a water-bath shaker for 48h. The content of reducing sugar in the enzymatic hydrolyzate was measured by DNS method. Then, centrifuge and filter to obtain the enzymatic hydrolyzate. Take 20ml of enzymolysis solution, add water to 40ml, add 0.24g of potassium dihydrogen phosphate, 0.4g of peptone, add glucose until the initial sugar concentration in the enzymolysis solution is 25g/L, add 2mL of serratiamarcescens seed solution, adjust the pH to 5, and Anaerobic fermentation at 37°C for 48h. Fermentation control group using only glucose as carbon source. The content of 2,3-butanediol was determined by HPLC. The final equivalent inhibitor content is the sum of the content in the carded parenchyma and the content of the material after the second steam explosion, and the content of 2,3-butanediol can be calculated in the same way.

The results showed that the inhibitor content of the experimental group was 33% lower than that of the pretreatment control group, and the 2,3-butanediol content of the experimental group was higher than that of the 2,3-butanediol fermentation control with sugar as the carbon source. The 2,3-butanediol content of the pretreatment control group was 66.7% lower than that of the 2,3-butanediol fermentation control group with only sugar as carbon source.

Example 2: Weigh 200g of dry corn stalks and put them into a steam explosion tank, and then pass water vapor into it after airtight to make the tank pressure rise to 1.2MPa rapidly, and after maintaining this pressure for 4 minutes, release the pressure quickly to obtain the first stage Steam-exploded straw. Take an appropriate amount of the first-stage steam-exploded straw, and comb it on a centrifugal airflow classification device to obtain parenchyma and fibrous tissue respectively. Weigh an appropriate amount of combed fibrous tissue, put it into a steam explosion tank, and then inject water vapor after sealing it to make the tank pressure rise to 1.2MPa rapidly. After maintaining this pressure for 4 minutes, release the pressure quickly to obtain the second stage Steam-exploded straw. The pretreatment conditions were 1.2MPa, 8min, and no carding as the pretreatment control group.

Weigh 1g (dry base) of steam-exploded straw in the first stage, steam-exploded straw after carding, steam-exploded straw in the second stage and the steam-exploded straw in the control group respectively, dissolve them in 20ml of water and extract at 70°C for 2 hours, and take the supernatant The content of furfural and hydroxymethylfurfural (HMF) in the hydrolyzate was measured by high performance liquid phase (Agilent 1200HPLC, USA), and the content of soluble lignin in the hydrolyzate was measured by a UV spectrophotometer at 280nm. The sum of the contents of furfural, hydroxymethylfurfural (HMF) and soluble lignin is the inhibitor content in the hydrolyzate.

Weigh 10g of the first stage of steam-exploded straw, combed steam-exploded straw, second-stage steam-exploded straw and pretreatment control group steam-exploded straw, add 200ml pH 4.850mM citric acid buffer solution, add 0.5ml cellulose Enzyme, hydrolyze at 50°C, 150rpm in a water-bath shaker for 48h. The content of reducing sugar in the enzymatic hydrolyzate was measured by DNS method. Then, centrifuge and filter to obtain the enzymatic hydrolyzate. Take 20ml of enzymolysis solution, add water to 40ml, add 0.24g of potassium dihydrogen phosphate, 0.4g of peptone, add glucose until the initial sugar concentration in the enzymolysis solution is 25g/L, add 2mL of serratiamarcescens seed solution, adjust the pH to 5, and Anaerobic fermentation at 37°C for 48h. Fermentation control group using only glucose as carbon source. The content of 2,3-butanediol was determined by HPLC. The final equivalent inhibitor content is the sum of the content in the carded parenchyma and the content of the material after the second steam explosion, and the content of 2,3-butanediol can be calculated in the same way.

The results show that the inhibitor content of the experimental group is 27% lower than that of the pretreatment control group, and the 2,3-butanediol content of the experimental group is higher than that of the 2,3-butanediol fermentation control with sugar as the carbon source. The 2,3-butanediol content of the pretreatment control group was 66.5% lower than that of the 2,3-butanediol fermentation control group with only sugar as carbon source.

Example 3: Weigh 200g of dry corn stalks and put them into a steam explosion tank, and then inject water vapor after airtight to make the tank pressure rise rapidly to 1.1MPa, and after maintaining this pressure for 4 minutes, release the pressure quickly, thereby obtaining the first stage Steam-exploded straw. Take an appropriate amount of the first-stage steam-exploded straw, and comb it on a centrifugal airflow classification device to obtain parenchyma and fibrous tissue respectively. Weigh an appropriate amount of combed fibrous tissue, put it into a steam explosion tank, and then inject water vapor after sealing it to make the tank pressure rise to 1.2MPa rapidly. After maintaining this pressure for 4 minutes, release the pressure quickly to obtain the second stage Steam-exploded straw. The pretreatment conditions were 1.2MPa, 8min, and no carding as the pretreatment control group.

Weigh 1g (dry base) of steam-exploded straw in the first stage, steam-exploded straw after carding, steam-exploded straw in the second stage and the steam-exploded straw in the control group respectively, dissolve them in 20ml of water and extract at 70°C for 2 hours, and take the supernatant The content of furfural and hydroxymethylfurfural (HMF) in the hydrolyzate was measured by high performance liquid phase (Agilent 1200HPLC, USA), and the content of soluble lignin in the hydrolyzate was measured by a UV spectrophotometer at 280nm. The sum of the contents of furfural, hydroxymethylfurfural (HMF) and soluble lignin is the inhibitor content in the hydrolyzate.

Weigh 10g of the first stage of steam-exploded straw, combed steam-exploded straw, second-stage steam-exploded straw and pretreatment control group steam-exploded straw, add 200ml pH 4.850mM citric acid buffer solution, add 0.5ml cellulose Enzyme, hydrolyze at 50°C, 150rpm in a water-bath shaker for 48h. The content of reducing sugar in the enzymatic hydrolyzate was measured by DNS method. Then, centrifuge and filter to obtain the enzymatic hydrolyzate. Take 40ml of enzymolysis solution, add 0.2g of potassium dihydrogen phosphate, 0.08g of ammonium sulfate, 0.016g of magnesium sulfate, 0.008g of calcium chloride, and 0.08g of yeast powder. Glucose was added until the initial sugar concentration in the enzymolysis solution was 25g/L, 2mL Angel yeast seed solution was added, the pH was adjusted to 5.5, and anaerobic fermentation was carried out at 32°C for 48h. Fermentation control group using only glucose as carbon source. The ethanol content was determined by HPLC. The final equivalent inhibitor content is the sum of the content in the carded parenchyma and the content of the material after the second steam explosion, and the ethanol content can be calculated in the same way.

The results showed that the inhibitor content of the experimental group was 33% lower than that of the pretreated control group. The ethanol content of the experimental group was 13.8% higher than that of the pretreatment control group, and 22.4% and 33% lower than that of the ethanol fermentation control group with sugar as carbon source, respectively.

Claims (4)

1. A method for reducing the fermentation inhibitor of lignocellulose source from the source, comprising the following steps: 1) Carry out the first-stage steam explosion treatment to the straw raw material; 2) Combing the first steam explosion material to obtain parenchyma and fibrous tissue; 3) Carry out the second stage of steam explosion treatment to the fibrous tissue obtained by carding; 4) Carry out enzymatic hydrolysis and fermentation to the materials after the steam explosion in the first stage, carding and the steam explosion in the second stage respectively; 5) Determination of the moisture content of the straw raw material, the steam-exploded material in the first stage, the parenchyma and fibrous tissue after carding, and the steam-exploded material in the second stage, the content of inhibitors in the washing liquid, and the content of fermentation products. 2. The method according to claim 1, wherein the first stage steam explosion treatment of step 1) is to carry out 1～10min under the steam pressure of 0.5～1.5MPa in the steam explosion tank. 3. The method according to claim 1, wherein the second stage steam explosion treatment of step 3) is to carry out 1～10min under the steam pressure of 0.5～1.5MPa in the steam explosion tank. 4. The method according to claim 1, wherein the enzymolysis condition in step 4) is: steam-exploded stalks and pH 4.8 and a concentration of 50mM citric acid buffer have a solid-to-liquid ratio of 1: 10 to 1: 30, adding The amount of enzyme is 15IU/g-110/g substrate, and the enzyme is hydrolyzed in a shaker at 50°C and 100-200rpm for 24-120h. Then, centrifuge to obtain the enzymatic hydrolyzate.