WO2018072472A1

WO2018072472A1 - Method for reducing inhibiting effect of byproducts in lignocellulose alkaline pretreatment liquid and preparation of cellulosic ethanol based on the method

Info

Publication number: WO2018072472A1
Application number: PCT/CN2017/091475
Authority: WO
Inventors: 刘泽寰; 林蒋海; 黄清; 吕晓静; 傅菁; 李帅
Original assignee: 广东启智生物科技有限公司
Priority date: 2016-10-21
Filing date: 2017-07-03
Publication date: 2018-04-26
Also published as: CN106544370A; CN106544370B

Abstract

A method for reducing an inhibiting effect of byproducts in lignocellulose alkaline pretreatment liquid. The method is achieved by treating lignocellulose alkaline pretreatment liquid by means of glucose oxidase without changing a reaction container and comprises the following steps: S1, adding lignocellulose to alkali liquor for treatment to obtain pretreatment liquid; and S2, adding glucose oxidase to the pretreatment liquid obtained in step S2 for reaction.

Description

Method for reducing by-product inhibition effect in lignocellulosic alkali pretreatment liquid and preparing cellulose ethanol based on the method

Technical field

The invention relates to the field of biochemical engineering and fermentation engineering, in particular to a method for reducing the inhibitory effect of by-products in a lignocellulosic alkali pretreatment liquid and preparing ethanol based on the method.

Background technique

Lignocellulose is an ideal raw material for replacing fossil energy because of its wide source, rich content and renewable. The production of bioethanol, biobutanol, biosynthesis and other alternative energy sources from lignocellulose has become a research hotspot in the world. Since ethanol can be directly used in existing engines in various proportions and can be produced by microbial fermentation, the production of fuel ethanol from lignocellulose has become one of the research focuses.

The lignocellulose that can be used as a raw material for producing fuel ethanol is mainly agricultural and forestry waste, such as crop straw, bagasse, wood processing waste, and the like. As the largest renewable energy on the earth, lignocellulose accounts for about 90% of the world's annual biomass production. Lignocellulose is mainly composed of three high polymers of cellulose (40% to 50%), hemicellulose (20% to 30%) and lignin (20% to 28%) by covalent and non-covalent combination. . In the lignocellulose in the natural state, cellulose is generally embedded in a polymer formed of lignin, hemicellulose, or the like to form a complex network structure. This structure plays an active role in maintaining plant morphology and resisting foreign invasion, but since the substrate of cellulase is a non-crystalline single-chain cellulose molecule, the structure of natural lignocellulose becomes an obstacle in the process of cellulose degradation. . Therefore, the lignocellulosic feedstock must undergo a pretreatment step to remove the cellulose-encapsulated lignin and hemicellulose while opening the crystalline region of the cellulose, thereby exposing the cellulose to cellulase for hydrolysis and saccharification.

Considering the ease of operation of various methods and economic cost factors, dilute acid and alkali treatment are currently the most concerned treatment methods, but both pretreatment methods lead to different levels of three main components of lignocellulose. Degradation and production of a series of chemical reactions, resulting in a variety of by-products, and these by-products often inhibit the enzymatic hydrolysis and fermentation process, limiting its promotion and application. Therefore, exploring the method of eliminating the inhibition of enzymatic hydrolysis and fermentation by by-products is a research hotspot in the field of lignocellulose pretreatment at home and abroad.

Most of the by-product inhibitors produced during the pretreatment of lignocellulose are produced by the decomposition of hemicellulose and lignin. They are mainly classified into three categories: i) furan derivatives derived from pentose and hexose, including furfural. (furfural) and 5-hydroxymethylfurfural (5-HMF); ii) organic acids (also known as fatty acids, also known as fatty acids), including hemicellulose-derived acetic acid and further degradation by furfural or hydroxymethylfurfural Forming formic acid, levulinic acid, etc.; iii) lignin-derived phenols and aromatic compounds. Studies have shown that furfural and hydroxymethylfurfural mainly affect the redox balance of fermentation process and inhibit the activity of glycolytic enzyme; organic acid mainly inhibits yeast growth and ethanol production; phenolic compounds affect cell membrane permeability. Affect the growth and fermentation of yeast. Microorganisms such as yeast can metabolize organic acids, furfural, and HMF, and convert them into less toxic compounds, so they are relatively less toxic, but they still have an inhibitory effect in the presence of high concentrations.

The phenolic compound has a much lower toxicity than the by-products derived from cellulose and hemicellulose, although the concentration is low. For example, ferulic acid produces a concentration inhibitory effect on yeast that is two orders of magnitude lower than the required concentration of formic acid, acetic acid, and levulinic acid (1 mM vs 100 mM), while coniferylaldehyde requires only 0.1 mM. Yeast produces inhibition.

In order to solve the problem of by-product inhibition, it is usually necessary to use a detoxification step to detoxify the pretreated raw materials. Common methods of detoxification include physical methods, chemical methods, and biological methods. By vacuum evaporation, more than 98% of furfural and part of acetic acid in the wood hydrolyzate can be removed; activated carbon adsorption can remove 95% of the phenolic compound; and 60% of acetic acid in the corn stover hydrolysate is removed by membrane adsorption. Over-liming is a common chemical detoxification method that removes most of the furfural in the lignocellulosic hydrolysate; however, it causes about 10% loss of fermentable sugar due to adsorption. A major disadvantage of most physical or chemical detoxification methods is the need for additional processing steps, which not only increases the complexity of the process, but also introduces additional consumption of reagents and energy. The biological method uses microorganisms such as laccase and peroxidase or white rot fungi to degrade the phenolic compounds in the hydrolyzate by oxidation, without adding new equipment and operations. However, the current biological method takes a long time and the conditions are harsh. widely used.

In this context, in order to improve the process of converting lignocellulose into ethanol, especially for the disadvantages of cumbersome, time-consuming, waste water and energy consumption of the current detoxification process, the present invention proposes a novel biological detoxification of the lignocellulosic alkali pretreatment liquid. The method requires only one step of enzymatic hydrolysis to complete the detoxification and eliminate the inhibition of the fermentation ability of by-products on cellulase and yeast.

Summary of the invention

In view of the above, an object of the present invention is to overcome the deficiencies of the prior art, and to provide a method for reducing the by-product inhibition effect in a lignocellulosic alkali pretreatment liquid and a process for preparing cellulosic ethanol based on the method.

In order to solve the above technical problem, the present invention is implemented by the following scheme:

Method for reducing by-product inhibition effect in lignocellulosic subtraction pretreatment liquid, using glucose oxygen The enzyme treatment of lignocellulosic alkali pretreatment liquid. The alkaline pretreatment liquid pretreats the lignocellulose for the lye used in the prior art to remove the cellulose-coated lignin and hemicellulose, and opens the crystalline region of the cellulose to expose the cellulose. The cellulase is hydrolyzed and saccharified. As mentioned in the background, lignocellulose is subjected to an alkali treatment to produce various by-products which have an inhibitory effect on subsequent cellulase enzymatic hydrolysis and yeast fermentation, and the present invention innovatively adds a glucose oxidase (GOD) pair. The biological detoxification of the pretreatment liquid not only degrades the phenolic compounds in the pretreatment liquid, but also metabolizes most of the by-products such as organic acids, furfural, hydroxymethylfurfural, and greatly reduces the content of by-products. The invention has simple process, and only needs to add glucose oxidase to react after treating the lignocellulose by the alkali method, thereby completeing detoxification and eliminating inhibition of cellulase enzymatic hydrolysis and yeast fermentation by by-products.

Specifically, the following steps are included:

S1: the lignocellulose is added to the lye to be processed to obtain a pretreatment liquid; the lye includes, but not limited to, sodium hydroxide, potassium hydroxide, sodium ethoxide, potassium ethoxide, etc., which can be selected according to the prior art; The conditions of the dosage ratio, temperature, time and the like involved in the treatment are also selected according to the prior art;

S2: Glucose oxidase is added to the pretreatment liquid in the step S2 to carry out the reaction.

In the above step S2, the pretreatment liquid is cooled to room temperature and the pH is adjusted to 4.5 to 5.5 before the glucose oxidase is added to the pretreatment liquid. The alkali-treated pretreatment liquid has a higher temperature and a higher alkalinity, and inhibits the activity of glucose oxidase. Therefore, it is necessary to adjust the temperature and acidity and alkalinity to a reasonable range to provide a more suitable environment.

Further, in step S2, the pH is adjusted using concentrated phosphoric acid. Glucose oxidase can degrade the phenolic compounds in the pretreatment liquid, but the phenolic compound is only one of the by-products in the pretreatment liquid, and also contains a large amount of organic acids, furfural, hydroxymethylfurfural and the like. Although in the subsequent microbial fermentation process such as yeast, it can metabolize organic acids, furfural, hydroxymethylfurfural, etc., but when the concentration of organic acid, furfural, hydroxymethylfurfural, etc. is high, microorganisms such as yeast cannot completely metabolize them, and then Fermentation inhibition. The inventors have found that the pH adjustment of concentrated phosphoric acid has a better pH buffer capacity relative to sulfuric acid, which is beneficial to maintain pH stability, thereby facilitating the maintenance of glucose oxidase and subsequent cellulase activity, and facilitating organic Acid, furfural, hydroxymethylfurfural and other degradation and metabolism, to the greatest extent eliminate the inhibition effect of this part of by-products; compared to dilute phosphoric acid, the amount is small, can reduce the volume as much as possible, thereby improving the subsequent cellulose enzymatic hydrolysis The concentration of fermentable sugar can save energy in the ethanol distillation process.

Further, in step S2, the mixed glucose oxidase and the pretreatment liquid are reacted for 24 to 48 hours. The ratio of glucose oxidase to pretreatment liquid, reaction temperature, stirring speed, etc. can be selected according to a limited number of tests, and it is because of the use of glucose oxidase to treat the pretreatment liquid in a short time. Degradation of by-products is accomplished to the greatest extent (24 to 48 h).

A method for producing ethanol using lignocellulose, comprising the following steps:

i) lignocellulosic alkali pretreatment and detoxification: using the above method for reducing the inhibitory effect of by-products in the lignin alkali pretreatment liquid;

Ii) Cellulase enzymatic hydrolysis: adding cellulase to the system in step i) for enzymatic hydrolysis;

Iii) Microbial fermentation: The system in step ii) is inoculated with Saccharomyces cerevisiae for fermentation.

In step ii), the enzyme is hydrolyzed for 60 to 80 hours.

Ferment in step iii) for 20 to 30 hours.

The amount of cellulase and Saccharomyces cerevisiae, the temperature of the reaction process, the stirring speed, etc. can be selected according to the prior art, and after detoxification in the pretreatment of lignocellulose, the by-products of the cellulase are reduced. The inhibition of fermentation and fermentation of Saccharomyces cerevisiae accelerates the speed of enzymatic hydrolysis and fermentation, so that the purpose of enzymatic hydrolysis and fermentation can be achieved in a short time; in addition, due to the detoxification treatment using glucose oxidase, no special removal is required. The product and the washing step, compared with the existing lignocellulose production process of ethanol, the invention has the advantages of simple operation, simple equipment demand, low energy consumption and water consumption.

Compared with the prior art, the present invention has the following beneficial effects: the present invention firstly applies glucose oxidase (GOD) to a lignocellulosic alkali pretreatment liquid for biological detoxification, and removes by-products from the pretreatment liquid to cellulose. Enzyme and inhibition of S. cerevisiae. The invention utilizes the detoxification ability of glucose oxidase to construct a kind of inhibition effect of by-products can be largely eliminated by adding a certain amount of glucose oxidase after alkali pretreatment without replacing the reaction container. The same reaction system realizes all processes of lignocellulose pretreatment, detoxification, enzymatic hydrolysis and fermentation to produce ethanol. Compared with the existing commonly used methods such as water elution toxicity and chemical detoxification, the process is simple and environmentally friendly. The low water consumption, easy automation, and easy expansion of the reaction scale help to promote the industrial application of lignocellulosic ethanol.

DRAWINGS

Figure 1 is a flow chart for producing ethanol from lignocellulose;

Figure 2 is a data diagram of Embodiment 1;

Figure 3 is a data diagram of Embodiment 2;

Figure 4 is a data diagram of Embodiment 3;

Figure 5 is a data plot of Comparative Example 1.

detailed description

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described below in conjunction with the accompanying drawings.

Example 1

GOD treatment of 1% NaOH pretreated bagasse and fermentation to produce ethanol

S1: accurately weigh 2.5g of bagasse, add 1% NaOH solution with a liquid-solid ratio of 10:1 (substrate concentration 10% (w/v)), treat bagasse at 121 °C for 1 h, and cool at room temperature. Treatment liquid

S2: under sterile conditions, adding commercially available concentrated phosphoric acid to the pretreatment liquid, adjusting the pH to about 5, then adding 10 U GOD, 30 ° C, detoxification reaction at 200 rpm for 24 h;

S3: under aseptic conditions, the cellulase is added to the above pretreatment liquid, the ratio of the enzyme addition amount to the substrate is fixed to 6 mg enzyme / g substrate, and then placed in a constant temperature shaker, hydrolyzed at 45 ° C, 160 rpm for 72 h; Sampling and measuring the enzymatic hydrolysis of bagasse during the hydrolysis process;

S4: then adding peptone to a final concentration of 20 g/L, and adding the pre-activated Saccharomyces cerevisiae suspension to the hydrolysate in step S3 in an amount of 20 g wet weight/L, 30 Fermentation was carried out at °C, 200 rpm for 48 h, and the fermentation process was sampled to detect the ethanol fermentation.

This example was repeated three times, and the sample treated without GOD was used as a control group, and the enzymatic hydrolysis of bagasse and ethanol yield were detected by high performance liquid chromatography. The results are shown in Fig. 2 (hollow GOD-treated sample, solid control group) . Due to the low concentration of NaOH used in the pretreatment, the by-products produced have no obvious inhibition on the fermentation of cellulose hydrolase and Saccharomyces cerevisiae; glucose (16.6g/L) and ethanol (7.1g/L) can be obtained without GOD treatment. Generated, but the addition of GOD can increase the yield of glucose (20.6g / L) and ethanol (8.3g / L) to some extent.

Example 2

GOD treatment of 2% NaOH pretreated bagasse and fermentation to produce ethanol

S1: accurately weigh 2.5g of bagasse, add 2% NaOH solution with a liquid-solid ratio of 10:1 (substrate concentration 10% (w/v)), treat bagasse at 121 °C for 1h, and cool at room temperature. Treatment liquid

S2: under sterile conditions, adding commercially available concentrated phosphoric acid to the pretreatment liquid, adjusting the pH to about 5, then adding 15 U GOD, 30 ° C, detoxification reaction at 200 rpm for 24 h;

This example was repeated three times, and the sample which was not treated with GOD was used as a control group, and was examined by high performance liquid chromatography. The enzymatic hydrolysis of sugarcane bagasse and ethanol production were measured. The results are shown in Fig. 3 (hollow GOD-treated samples, solid as a control group). Without the treatment of GOD, the inhibition of cellulose hydrolase and Saccharomyces cerevisiae fermentation was more obvious due to the increase of NaOH concentration. The concentration of glucose was only 2.2g/L after 48h of enzymatic hydrolysis, and the production of ethanol was not detected. After GOD treatment, the activity of cellulase was restored. After 48 h of enzymatic hydrolysis, the glucose concentration reached 25.2 g/L, and the final ethanol concentration reached 9.9 g/L.

Example 3

GOD treatment of 3% NaOH pretreated bagasse and fermentation to produce ethanol

S1: accurately weigh 2.5g of bagasse, add 3% NaOH solution with a liquid-solid ratio of 10:1 (substrate concentration 10% (w/v)), treat bagasse at 121 °C for 1h, and cool at room temperature. Treatment liquid

S2: under sterile conditions, adding commercially available concentrated phosphoric acid to the pretreatment liquid, adjusting the pH to about 5, then adding 25 U GOD, 30 ° C, detoxification reaction at 200 rpm for 24 h;

This example was repeated three times, and the sample treated without GOD was used as a control group, and the enzymatic hydrolysis of bagasse and ethanol production were detected by high performance liquid chromatography. The results are shown in Fig. 4 (hollow GOD-treated sample, solid as a control group) . Without the treatment of GOD, the by-product produced completely inhibited the fermentation of cellulose hydrolase and Saccharomyces cerevisiae due to the further increase of NaOH concentration. The formation of glucose was not detected at all, and the production of ethanol was not detected. After GOD treatment, cellulose was produced. The activity of the enzyme was restored. After 48 hours of enzymatic hydrolysis, the glucose concentration reached 25.9 g/L, and the final ethanol concentration reached 10.3 g/L.

Comparative example 1

This comparative example is similar to Example 3 except that in step S3, 40 U and 100 U laccase were used instead of GOD, respectively. This example was repeated three times, and the sample treated without laccase was used as a control group to use a high-efficiency liquid.

Chromatographic detection

The enzymatic hydrolysis of sugarcane bagasse and ethanol yield were as shown in Fig. 5 (the hollow 100 U laccase-treated sample, the cross-over 40 U laccase-treated sample, and the solid control group). Due to the high concentration of NaOH, the by-products produced completely inhibited the fermentation of cellulose hydrolase and Saccharomyces cerevisiae, and the formation of glucose was not detected at all, and the production of ethanol was not detected. Even after 40 U of laccase treatment, there was no improvement in inhibition. ,still However, glucose and ethanol were not detected; after 100 U laccase treatment, the activity of cellulase was recovered. After 48 h of enzymatic hydrolysis, the glucose concentration was 17.4 g/L, and the final ethanol concentration reached 7.9 g/L. Both glucose and ethanol production were less than the results of the 25U GOD treatment.

The above embodiments are only specific implementations of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims

A method for reducing the inhibitory effect of by-products in a lignocellulosic alkali pretreatment liquid, characterized in that the lignocellulosic alkali pretreatment liquid is treated with glucose oxidase.
The method for reducing the by-product inhibition effect of the lignocellulosic alkali pretreatment liquid according to claim 1, which comprises the following steps:

S1: the lignocellulose is added to the alkali solution for treatment;

S2: Glucose oxidase is added to the pretreatment liquid in the step S2 to carry out the reaction.
The method for reducing the by-product inhibitory effect in the lignocellulosic alkali pretreatment liquid according to claim 2, wherein in step S2, the pretreatment liquid is cooled to room temperature and the pH is adjusted to 4.5 before the glucose oxidase is added. -5.5.
The method for reducing the by-product inhibitory effect in the lignocellulosic alkali pretreatment liquid according to claim 3, wherein in step S2, pH adjustment is carried out using concentrated phosphoric acid.
The method for reducing the by-product inhibitory effect in the lignocellulosic alkali pretreatment liquid according to claim 4, wherein in step S2, the mixed glucose oxidase and the pretreatment liquid are reacted for 24 to 48 hours.
Use of the method of any one of claims 1 to 5 in ethanol production.
A method for producing ethanol using lignocellulose, comprising the steps of:

i) lignocellulosic alkali pretreatment and detoxification: treatment by the method according to any one of claims 1 to 5;

Ii) Cellulase enzymatic hydrolysis: adding cellulase to the system in step i) for enzymatic hydrolysis;

Iii) Microbial fermentation: The system in step ii) is inoculated with Saccharomyces cerevisiae for fermentation.
The method for producing ethanol using lignocellulose according to claim 7, wherein the enzyme is hydrolyzed in step ii) for 60 to 80 hours.
The method for producing ethanol using lignocellulose according to claim 8, wherein the fermentation in step iii) is carried out for 20 to 30 hours.