Monitoring method for measuring decomposition process of lignocellulose in straw by microorganisms

Abstract

The invention discloses a monitoring method for measuring the decomposition process of lignocellulose in straw by microorganisms, and belongs to the technical field of organic matter decomposition, and the monitoring method comprises the following steps: (1) extracting water-soluble substances from the straw in the microorganism pretreatment process or fermentation process; (2) performing enzyme digestion treatment on the first solid; (3) measuring the content of total sugar in the collected solution; and (4) measuring the relationship between the heat yield of the straw culture and the total sugar by using an isothermal microcalorimetric method. According to the method, monitoring of microorganisms on the decomposition process of lignocellulose in straw is measured through a calorimetric method, the content of various saccharides such as total reducing sugar, total sugar and monosaccharide in an extraction solution is measured, the content of various saccharides in the pretreatment and / or fermentation process is known, the lignocellulose decomposition condition is known, and the content of the lignocellulose in the straw is obtained. And analyzing the relationship among the biological heat, various sugars and total soluble sugars, and directly determining the decomposition state of the microorganisms on the lignocellulose through the relationship.

Description

Technical Field

[0001] The invention relates to the technical field of organic matter decomposition, in particular to a monitoring method for measuring the decomposition process of lignocellulose in straw by microorganisms. Background Art

[0002] The lignocellulose in the straw can be decomposed into polysaccharide components during the biological pretreatment process, and then the polysaccharide components are converted into small molecular water-soluble sugars to serve as the basis for the subsequent production of bioenergy and various useful chemicals. In the prior art, non-biological methods are often used for pretreatment, including physical pretreatment and chemical pretreatment. However, these non-biological pretreatment methods will produce inhibitory by-products during the treatment process, which is not conducive to the subsequent production of energy and / or chemicals. Biological pretreatment is an environmentally friendly technology that combines mild reaction conditions, enzyme hydrolysis specificity, avoidance of inhibition, and has the advantages of less by-products and low energy consumption requirements.

[0003] Therefore, providing a method for monitoring the decomposition process of lignocellulose in straw using high-calorie measuring microorganisms is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0004] In view of this, in order to understand the decomposition process and decomposition efficiency of lignocellulose by microorganisms in the pretreatment stage and / or solid waste fermentation treatment stage, and to guide the microbial resource utilization process, the present invention adopts a non-biological invasive method to achieve process monitoring of microbial treatment by measuring biological heat, and combines the content of water-soluble sugars (including various monosaccharides, disaccharides, oligosaccharides and polysaccharides) in the measurement process to understand the heat in the pretreatment process and the content of water-soluble sugars in the fermentation process, and characterizes the productivity of sugars in the pretreatment and solid waste fermentation processes.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A monitoring method for measuring the decomposition process of lignocellulose in straw by microorganisms comprises the following steps:

[0007] (1) Extraction of water-soluble substances from straw during microbial pretreatment or fermentation:

[0008] Inoculating microorganisms into a calorimeter bottle containing straw powder, placing the calorimeter bottle in an independent measurement channel of an isothermal microcalorimeter for pretreatment cultivation, wherein the working temperature and pretreatment time are set according to the growth conditions of the inoculated microorganisms, and after the microorganism pretreatment is completed, adding citrate-phosphate buffer to the calorimeter bottle, and then centrifuging to obtain a first leachate and a first solid;

[0009] (2) subjecting the first solid to enzymatic digestion treatment:

[0010] The first solid is put into a ball mill and shaken, and the shaken first solid is evenly divided into two parts, and sodium citrate buffer is added to the first part of the first solid, and then the mixture is shaken evenly in a shaker and centrifuged to extract the water-soluble sugar component to obtain a second leachate and a second solid;

[0011] Adding ligninase and cellulase to the second portion of the first solid, and adding sodium citrate buffer to perform enzymolysis reaction, centrifuging after the enzymolysis is completed, collecting the supernatant to obtain a third leachate for analyzing the content of the product after enzymolysis, the easily bioavailable sugar is the sum of the sugars in the first leachate and the second leachate, and the total bioavailable sugar is the sum of the sugars in the first leachate and the third leachate;

[0012] (3) Determine the total sugar content in the collected solution:

[0013] The total sugar content was determined photometrically after acid hydrolysis of the aqueous extract of the solid matrix using the phenol-sulfuric acid method;

[0014] Among them, the phenol-sulfuric acid method was based on "Determination of total sugar content in chicory root by phenol-sulfuric acid method" by Sun Yun, Liu Jie, Liu Jiajia, etc.; the operation was performed according to the scheme in the reference document.

[0015] (4) Isothermal microcalorimetry to measure the relationship between heat production and total sugar in straw culture:

[0016] An isothermal calorimeter was used to measure the heat production rate of the culture during straw pretreatment, and the relationship between the heat production of the straw culture and the total sugar in each part of the extract was obtained. The optimal conditions for producing the maximum bioavailable sugar and total sugar were obtained, thereby optimizing the pretreatment and fermentation process and technology of lignocellulose.

[0017] Furthermore, the calorimeter bottle in step (1) is a polypropylene test tube, and the calorimeter bottle is equipped with a ventilated PTFE membrane screw cap.

[0018] Furthermore, the pretreatment culture temperature in step (1) is 26-28°C, and the pretreatment culture time is 30-40 days.

[0019] Furthermore, in step (1), the volume ratio of the microorganism to the straw powder is 2 ml: 1 g.

[0020] Furthermore, the microorganisms in the present invention are conventional microorganisms used for straw pretreatment and can be purchased from a strain storage library;

[0021] Preferably, the microorganism is Phanerochaete chrysosporium, purchased from China General Microbiological Culture Collection Center, numbered CGMCC 3.7212, but the present invention does not involve biological preservation.

[0022] Alternatively, the microorganism is Gloeophyllum trabeum.

[0023] Further, the concentration of the citrate-phosphate buffer in step (1) is 0.1 mol / L;

[0024] Furthermore, the volume ratio of the citrate-phosphate buffer to the straw powder is 20-30 ml: 1 g.

[0025] Furthermore, in step (2), the oscillation frequency of the first solid in the ball mill is 55-60 times / s, and the oscillation time is 10-15 min.

[0026] The shaking speed of the shaker is 180 rpm, the temperature is 35° C., and the time is 20 h.

[0027] Further, in step (2), the concentration of sodium citrate buffer in the first portion of the first solid is 0.1 mol / L, and the amount added is 40-60 ml / g of the first solid;

[0028] The concentration of sodium citrate buffer in the second portion of the first solid is 0.1 mol / L, and the added amount is 40-60 ml / g of the first solid.

[0029] Furthermore, in step (2), the mixed addition amount of the lignin enzyme and the cellulase is 40-60 μl / g of the first solid.

[0030] The mass ratio of the lignin enzyme to the cellulase is 1:8-9.

[0031] Furthermore, in step (2), the centrifugation temperature is 4° C., the centrifugation rate is 6500-8000 rpm, and the centrifugation time is 10-20 min.

[0032] The present invention monitors the decomposition process of lignocellulose in straw by microorganisms by measuring the calorimetric method, and combines the measurement of the contents of various sugars such as total reducing sugar, total sugar, monosaccharide, etc. in the extraction solution to understand the contents of various sugars during pretreatment and / or fermentation, and the decomposition of lignocellulose, and analyzes the relationship between bioheat and various sugars and total soluble sugars, and directly clarifies the decomposition state of lignocellulose by microorganisms through this relationship. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1

[0035] A monitoring method for measuring the decomposition process of lignocellulose in straw by microorganisms:

[0036] (1) Extraction of water-soluble substances from straw during microbial pretreatment or fermentation:

[0037] In order to measure the caloric data of microorganisms during the pretreatment process or fermentation process, the microorganisms are inoculated into a calorimeter bottle containing straw powder. The calorimeter bottle adopts a polypropylene test tube and is equipped with a vented PTFE membrane screw cap. The calorimeter bottle is placed in an independent measurement channel of the isothermal microcalorimeter, with an operating temperature of 26-28°C and a pretreatment of 30-40 days. After the microbial pretreatment (fermentation) time is over, 10-15ml0.1mol / L citrate-phosphate buffer (pH7.0) is added to extract water-soluble compounds from the solid material, and centrifuged to obtain the first leachate and the first solid. If the first leachate is collected and not immediately processed or measured, it needs to be stored at -20°C.

[0038] 1 ml of Phanerochaete chrysosporium was inoculated to pre-treat rice straw to decompose the lignocellulose in the rice straw. The rice straw needed to be treated before inoculation with microorganisms, and the rice straw needed to be air-dried and crushed to a particle size of less than 5 mm. The mass of the air-dried husk straw was 0.5 g.

[0039] (2) Enzymatic digestion of the first solid

[0040] The first solid was placed in a ball mill and shaken at 55 times / s for 10 minutes, then dried at room temperature and stored away from light, and the evenly mixed first solid was equally divided into two parts, 10-15 ml of 0.1 mol / L sodium citrate buffer was added to the first part of the first solid, and shaken and cultured on a shaker at 180 rpm and 35°C for 20 hours, and then centrifuged at 4°C and 6500 rpm for 10 minutes to extract the water-soluble sugar component to obtain a second leachate and a second solid.

[0041] 10-15 μl of lignin enzyme and cellulase (Accellerase series cellulase developed by DuPont) were added to the second first solid, and enzymatic hydrolysis was carried out in 10-15 ml of 0.1 mol / L sodium citrate buffer. After the enzymatic hydrolysis was completed, centrifuged at 4°C and 6500 rpm for 10 min, and the supernatant was collected to obtain a third extract for analyzing the product content after enzymatic hydrolysis.

[0042] Among them, the easily bioavailable sugar is the sum of the sugars in the first leachate and the second leachate, and the total bioavailable sugar is the sum of the sugars in the first leachate and the third leachate. In this embodiment, the analysis is mainly based on the total bioavailable sugar.

[0043] (3) Determination of the total sugar content in the collected solution

[0044] The total sugar content was determined photometrically after acid hydrolysis of the aqueous extract of the solid matrix using the phenol-sulfuric acid method.

[0045] In this embodiment, the total sugar content of the first leachate after acid hydrolysis was measured based on the phenol-sulfuric acid method to be 25.8 mg, the total sugar content of the second leachate to be 6.3 mg, and the total sugar content of the third leachate to be 33.5 mg. The content of readily bioavailable sugars was calculated to be 76.8 mg / g, and the content of total bioavailable sugars was 185.6 mg / g.

[0046] At the same time, the control group was set to be treated without adding microorganisms, and the remaining steps were the same as the steps of this embodiment. The total sugar content of the first extract was 0, the total sugar content of the second extract was 1.2 mg, and the total sugar content of the third extract was 28.5. The content of easily bioavailable sugars was calculated to be 4.8 mg / g, and the content of total bioavailable sugars was 114 mg / g.

[0047] (4) Isothermal microcalorimetry to measure the relationship between heat production and total sugar in straw culture

[0048] The heat production rate of straw culture was measured using an isothermal calorimeter, and the heat signal was evaluated using data processing software such as orgin to understand the metabolic heat yield coefficient (Yo / x) and metabolic heat-related parameters. The metabolic heat yield coefficient can be used to investigate the possible affinity relationship with the amount of sugar in the water-extractable sugar investigated, and ultimately obtain the optimal conditions for producing the maximum bioavailable sugar and total amount of sugar, thereby optimizing the pretreatment and fermentation process of lignocellulose.

[0049] In this example, the relationship between total bioavailable sugars was analyzed by determining the metabolic heat production coefficient of the strain growing on rice husk straw. The value of Yo / x was obtained using data analysis software, and the relationship between Yo / x and the content of easily bioavailable sugars and total bioavailable sugars was analyzed. In this example, Yo / x was calculated to be 115 kJ / g.

[0050] The method of the present invention can be used for monitoring the fermentation process of rice husk straw by microorganisms during the pretreatment process. Sampling can be performed multiple times during the whole pretreatment process, and the measurement and calculation of the above method can be performed to obtain the changes of the sugars that are easy to be bioavailable and the total bioavailable sugars over time in multiple time periods. Furthermore, the bioheat is further measured to obtain the metabolic heat yield coefficient (Yo / x), and the corresponding metabolic heat yield coefficient (Yo / x) and the sugar content are subjected to curve fitting. Through the fitting curve, the relationship between the metabolic heat production coefficient and the total sugar is obtained, and the corresponding metabolic heat production coefficient and the pretreatment time are determined through the appropriate total sugar content, and then it is determined under what conditions the calorimeter bottle reaches the optimal pretreatment condition, so as to optimize and determine the pretreatment time required for the experimental process.

[0051] Example 2

[0052] The same technical scheme as in Example 1 was adopted, except that 1 ml of Gloeophyllum trabeum was inoculated in step (1). The results showed that the total sugar content of the first leaching solution after acid hydrolysis was 23.6 mg based on the phenol-sulfuric acid method, the total sugar content of the second leaching solution was 8.4 mg, and the total sugar content of the third leaching solution was 36.8 mg. The content of easily bioavailable sugars was calculated to be 80.8 mg / g, and the content of total bioavailable sugars was 194.4 mg / g. Yo / x=416 kJ / g was calculated in this example.

[0053] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A monitoring method for measuring the decomposition process of lignocellulose in straw by microorganisms, characterized in that: The following steps are involved: (1) Extraction of water-soluble substances from straw during microbial pretreatment or fermentation: Inoculating microorganisms into a calorimeter bottle containing straw powder, placing the calorimeter bottle in an independent measurement channel of an isothermal microcalorimeter for pretreatment cultivation, wherein the working temperature and pretreatment time are set according to the growth conditions of the inoculated microorganisms, and after the microorganism pretreatment is completed, adding citrate-phosphate buffer to the calorimeter bottle, and then centrifuging to obtain a first leachate and a first solid; (2) subjecting the first solid to enzymatic digestion treatment: 1) placing the first solid in a ball mill and shaking it, dividing the first solid after shaking evenly into two equal parts, adding sodium citrate buffer to the first part of the first solid, shaking evenly on a shaker, and then centrifuging to extract the water-soluble sugar component to obtain a second leachate and a second solid; 2) adding lignin enzyme and cellulase to the second portion of the first solid, and adding sodium citrate buffer to carry out enzymolysis reaction, centrifuging after the enzymolysis is completed, collecting the supernatant to obtain a third leachate, which is used to analyze the content of the product after enzymolysis, the easily bioavailable sugar is the sum of the sugars in the first leachate and the second leachate, and the total bioavailable sugar is the sum of the sugars in the first leachate and the third leachate; (3) Determine the total sugar content in the collected solution: The total sugar content was determined photometrically after acid hydrolysis of the aqueous extract of the solid matrix using the phenol-sulfuric acid method; (4) Isothermal microcalorimetry to measure the relationship between heat production and total sugar in straw culture: An isothermal calorimeter was used to measure the heat production rate of the culture during straw pretreatment, and the relationship between the heat production of the straw culture and the total sugar in each part of the extract was obtained. The optimal conditions for producing the maximum bioavailable sugar and total sugar were obtained, thereby optimizing the pretreatment and fermentation process and technology of lignocellulose.

2. A monitoring method for measuring the decomposition process of lignocellulose in straw by microorganisms according to claim 1, characterized in that: The calorimeter bottle in step (1) is a polypropylene test tube, and the calorimeter bottle is equipped with a vented PTFE membrane screw cap.

3. A monitoring method for measuring the decomposition process of lignocellulose in straw by microorganisms according to claim 1, characterized in that: The pretreatment culture temperature in step (1) is 26-28° C., and the pretreatment culture time is 30-40 days.

4. A monitoring method for measuring the decomposition process of lignocellulose in straw by microorganisms according to claim 1, characterized in that: The mass ratio of the volume of the microorganism to the straw powder in step (1) is 2 ml: 1 g.

5. The method for monitoring the decomposition process of lignocellulose in straw by microorganisms according to claim 1, characterized in that: The concentration of the citrate-phosphate buffer in step (1) is 0.1 mol / L; The volume ratio of the citrate-phosphate buffer to the straw powder is 20-30 ml: 1 g.

6. A monitoring method for measuring the decomposition process of lignocellulose in straw by microorganisms according to claim 1, characterized in that: In step (2), the oscillation frequency of the first solid in the ball mill is 55-60 times / s, and the oscillation time is 10-15 min. The shaking speed of the shaker is 180 rpm, the temperature is 35° C., and the time is 20 h.

7. A monitoring method for measuring the decomposition process of lignocellulose in straw by microorganisms according to claim 1, characterized in that: In step (2), the concentration of sodium citrate buffer in the first portion of the first solid is 0.1 mol / L, and the amount added is 40-60 ml / g of the first solid; The concentration of sodium citrate buffer in the second portion of the first solid is 0.1 mol / L, and the added amount is 40-60 ml / g of the first solid.

8. The method for monitoring the decomposition process of lignocellulose in straw by microorganisms according to claim 1, characterized in that: The mixed addition amount of the lignin enzyme and the cellulase in step (2) is 40-60 μl / g of the first solid.

9. A monitoring method for measuring the decomposition process of lignocellulose in straw by microorganisms according to claim 8, characterized in that: The mass ratio of the lignin enzyme to the cellulase is 1:8-9.

10. The method for monitoring the decomposition process of lignocellulose in straw by microorganisms according to claim 1, characterized in that: The centrifugation temperature in step (2) is 4°C, the centrifugation rate is 6500-8000 rpm, and the centrifugation time is 10-20 min.